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the biorefinery term pertains the production of biofuels , bioenergy , and valuable chemicals from renewable biomass sources and aims to substitute petroleum refineries which produce several fuels and products from petroleum . biorefinery is the continuous processing of biomass into a range of profitable products ( food , feed , materials , and chemicals ) and energy ( fuels , power , and heat ) . this conception is similar to that used within the petrochemical industry , but renewable biomass feedstocks are used instead of using oil as the feedstock . renewable is a wide - ranging term , which could include any organic matter that becomes available on a continuous basis . this could include grasses , energy crops , agricultural feeds , or organic waste streams from animals and plants . grass is one of the promising energy crops for biogas production in some eu countries , where grass is covering a wide areas of agricultural land with high yields compared to other crops in europe . the biorefinery products ( i.e. , fuels , therapeutics , food additives , or secondary chemicals ) can be obtained using thermal , chemical , mechanical , enzymatic or microbial processes . biorefinery is targeting the separation of all the added value from the biomass feedstock , with little or no waste . this will lower the total environmental impact , besides improving the economics so that these processes can contend with the petrochemical industry ( figure 1 ) . using biomass in a biorefinery concept instead of oil for producing energy and chemicals . uniformity of feedstock represents one of the common factors between the classic petrochemical refinery process and biorefinery . biomass feedstocks could have a remarkable geographic and seasonal variations ( ranging from simple sugars to complex polysaccharides such as starch , cellulose , and hemicellulose , as well as more complex sources such as lignin , triglycerides , lipids , and proteins ) . this variation could be viewed as a disadvantage , due to the variation of the consistency and yield of the end products . on the other hand , complexity could be a desirable trait in order to obtain a more expansive range of products , although it needs cautious optimization in relation to the input material . moreover , one of the major disadvantages of biorefinery , compared to petroleum refinery , is in the miscellany of technologies required to obtain the end products . these include plant breeding and genetics , mechanical processes , sub- or supercritical fluid extractions , thermal treatments , chemical treatments , concurrent thermal and chemical treatment , enzymatic digestion , and biotransformations using microorganisms . biorefineries are classified based on their system components , that is , platforms , products , feedstocks , and conversion processes . platforms determine the complexity of the system in which they represent intermediates that link biorefinery systems and their processes ( i.e. , c5/c6 sugars , syngas , and biogas ) . products may be energy , that is , bioethanol and biodiesel , or valuable chemicals ( building blocks ) , that is , organic acids . feedstocks can come from edible crops , agricultural residues , forestry residues and industrial or domestic wastes ( bark , straw , paper mill black liquor , used cooking oils etc . ) . these are thermochemical ( e.g. , pyrolysis ) , biochemical ( e.g. , fermentation ) , mechanical ( e.g. , size reduction ) , and ( bio)chemical ( e.g. , esterification ) . the array of the most common terms that describe biorefineries are illustrated in table 1 . crop based biorefineries have gained tremendous interest in recent years , several pilot - scale systems have been built and currently a lot of research is going on building full scale systems . as with any other technology , apart from the main products in this case ethanol or lactic acid , several side products and wastes are also generated . the main focus of research so far has been on treatment of these wastes . also , most of the scientific literature is focused on treatment aspect only , but the focus has moved from treatment to valorization very recently . coal displaced the biomass fuels , considering wood , as the main energy source during the industrial revolution . since then a steady migration toward fossil fuels has continued , moving further away from biomass , not only for energy but also for sources of chemicals used to make everyday items . an outstanding pattern of this is furfural which can be produced from oat hulls . until 1960s , dupont produced nylon from biologically derived furfural ; however , nowadays it is being produced from fossil resources . the price of such fossil - fuel sources will be affected negatively in the near future due to the depletion of these oil reserves . industry is therefore now being encouraged to take a more inventive approach , looking ahead of oil and identifying biobased systems as valuable stockroom of crucial chemical building blocks . these chemicals are the basics of our modern lifestyle and can be found in products as miscellaneous as foods and fabrics . cereal grains have been a primary human food source since thousands of years ago , being one of the most vital source of calories for a large sector of the world population . the nature of grains produced worldwide relies on different factors , mainly being economic , cultural , and environmental . the availability of water and temperature are most likely the important environmental factors that determine the region in which these crops can be grown . the estimate for world cereal consumption in 2012/2013 has been increased to reach 2335 million tons ( mainly use of maize ) , 5 million tons higher than reported in the first half of 2012 . the existing cereal biorefinery uses dry cereals such as wheat , rice , and maize as raw materials . currently , some of these crops , that is , maize and wheat , are used to produce biofuels . maize starch is the major source for ethanol production in the u.s . , where wheat feedstock is used for ethanol production in canada , australia , and in some european countries . all of these crops are the conventional ones used for the production of first - generation biofuels . second - generation biofuels are produced from lignocellulosic crops , such as fast - growing trees and permanent grasses or from straw residues . great interest has been directed toward producing second - generation ethanol , since the above - mentioned crops can be cultivated in secondary lands without the need to interfere with the fertilized lands generally used for food production . these crops can be cultivated in the food - unsuitable poor soils , with relatively low amounts of water , but their productivities are low and a lot of effort is needed to elevate their yields . wide areas of land are required to cultivate the crops for biofuels production , together with irrigation and use of fertilizers , which may destroy wildlife habitats , as well as affect indigenous and rural poor communities around the world . instead the byproducts of these crops should be considered as a source of these biofuels . the cereal grains used for human food are milled to eliminate the bran and germ , in order to meet consumers sensory expectations . grains are striped by this process of key nutrients valuable to human health , that is , phenolics , dietary fiber , minerals , and vitamins , which also needs to be studied for their potential as feedstocks . building economical cereal - based biorefineries as a replacement for petroleum refineries would require considerable improvements in existing cereal processing strategies by applying more proficient processing of cereal grains for the production of a range of value - added products , that is , biochemicals and bioenergy . the main schematic industrial processing steps of some cereals are illustrated in figure 2 . wheat grains are separated into their constituents ( bran , germ , and endosperm ) by the milling process steps , in which wheat is ground into flour . before milling , the wheat is passed into conditioning bins to produce uniform moisture content throughout the grain . the bran and germ are separated from the endosperm by a sequence of breaking and grinding processes and subsequently the endosperm is reduced to form a uniform particle size by separating operation . schematic diagram of the main industrial processing of cereals illustrating products ( green shade ) and some byproducts ( red shade ) . the cleaned grains for milling are first conditioned , after which barley is washed - out with diluted sulfur dioxide . barley is then smashed through a pearling process to separate surface layers of the grain . the pearl barley is polished and flakes are made from them by steaming , flaking , and drying . furthermore , barley could be processed by malting , in which barley kernels are allowed to grow by first steeping the grains , which are then transferred from the steep tank to the germination vessel . germination is completed upon the full modification of the endosperm , after which the process is terminated by drying ( kilning process ) . the kilning brittle rootlets byproduct are removed to be used as a high protein feed ( 75% ) . also , milling is a crucial step in postproduction of rice . this process starts with the removal of the hard protective husk surrounding the grain , which is done by passing the rice through two spinning rubber roles and the resulted rice grain is packaged as brown rice healthy for body functions . the following step includes usage of fine milling to separate germ and bran layers from the grain and expose the white starch pulp , known as white rice . corn is processed by a dry milling process ( degerming ) in which hull , germ , and endosperm are separated prior to milling . this process starts by drying and cleaning the corn , which is then conditioned to 20% moisture . the majority of the outer bran , germ , and tip cap are removed from the moist grains , leaving the endosperm , which is then dried , cooled , and sieved . corn can also be processed by corn wet - milling , in which the precleaned grains are transferred to large steep tanks where they are soaked in a dilute sulfur dioxide solution . the corn germ is removed from the water soaked kernel , in which the germ is processed to recover the oil , and the remaining corn germ meal portion is collected for feed use . the corn nucleus is sieved to separate the bran leaving the starch and gluten protein . the gluten protein is concentrated and dried to form corn gluten meal with 60% protein feed . similar to corn , sorghum grains are processed either by dry or wet milling processes . additionally , sorghum could be milled for juice extraction by first being conditioned , then squeezed for juice extraction and produce bagasse byproduct which could be used as an excellent supplementation fiber for livestock or converted to a fuel . the filtered juice is precipitated and then the supernatant is evaporated and surface coagulated materials are removed to obtain syrup . nowadays , there is much attention for universal pollution along with growing production cost , and limited availability of raw materials leading to a special highlight on the importance of recovery , recycling , and valorization of food processing byproducts . the amount of biodegradable wastes drained to landfills in eu countries in 2020 is estimated to increase by 35% of its level in 1995 . the european manufacturers of processed foods are obliged to meet the terms of eu environmental policy concerned with utilization or disposal of byproducts and the huge amounts of aqueous and solid wastes produced . although these wastes represent serious economic and environmental challenges , they contain extensive amounts of potentially reusable materials and energy . one of the most contributing wastes to these challenges is cereal byproducts with biochemical oxygen demand ( bod ) and chemical oxygen demand ( cod ) levels that can reach 3.5 10 5 10 and 10 1.5 10 mg / l . generally , as a result of the change in ratio of fibrous and nonfibrous carbohydrates , the energy values for the parent grains are higher than that of mill - feeds . in contrast , the protein contents for the parent grains are commonly lower than that of mill - feeds . average percentage content of bioactive compounds of some cereal industrial processing byproducts ; wheat bran , rice husk , sweet sorghum bagasse , corn dry distillers grain , corn cob , and brewer s spent grains . general terms , referring to the cereal milling byproducts , are used to depict related components for several grains , whereas , some of these terms are used exclusively to describe a byproduct of one grain . one of the most important milling byproducts is bran which includes the coarse outer shell of the seed with small amounts of flour and higher content of fiber and protein . the embryo of the cereal seed adds up to form the germ meal which is a byproduct rich in lipids and protein . after removal of the germ and the starch endosperm during wet milling , the primary substance remaining is the protein rich gluten . moreover , grain screenings is a byproduct which contains a mixture of chaff , dust , broken grains , weed seeds , and all separated materials during cleaning and processing . the nutritional value of grain screenings varies with the relative proportion of each of the components . grain screenings are produced from all types of milled cereals with a minimum of 70% grains and maximum of 6.5% ash . grain hulls are the outer covering of the grain seed , commonly originating from oat and rice milling . grain hulls are high in crude fiber and relatively low in energy and protein , whereas rice hulls are high in silica . also , one of the major byproducts from malting industry is the barley malt sprouts which consist of roots , sprouts , and malt hulls . malt sprouts are considered as a protein source with minimum crude protein content of 24% . the oat milling byproduct is oat meal , consisting of lower quality oat meal portions with maximum crude fiber content of 4% . the production of flour is accompanied by some byproducts such as middlings ( abbreviated as midds ) which contain bran , shorts , germ , flour , and tailings and mostly originate from rye and wheat industrial processing . the maximum levels of fiber for rye and wheat middlings are 8.5% and 9.5% , respectively . the main rice milling byproduct is rice polishing , which is a good source of thiamin and crude fat and relatively a poor source of crude fiber . also , rice bran is another byproduct which has a high oil content and is a good source of b - complex vitamins , protein , and amino acids . the frequent corn byproducts of dry milling are corn flour , corn bran , and hominy feed , whereas the common byproducts of corn wet milling are liquefied corn product , starch molasses , germ meal , gluten , hydrolyzed corn protein , and condensed fermented corn extractives . also , sorghum milling can produce some byproducts , that is , gluten and grits . the efficient and economic disposal of cereal processing byproducts is the main concern of industrial society , in order to fulfill the environmental regulations that regard them as wastes destined to be drained off in most cases . this trend may be due to the lack of information for different economic biomolecules availability and technical processes for extracting them , besides the lack of commercial knowledge concerning customer requirements . the value addition and adequate utilization of cereal processing byproducts is very important for the valorization technology of such byproducts . moreover , if the valorization know - how is available for certain industrial byproduct in laboratory or pilot scale , the efficient scaling - up of this technique is limited by the standardization of the production process on the industrial scale . current sugar - based platform biorefineries mainly focus on the valorization of cellulose and hemicelluloses , while lignin is generally considered as a low - value residue . this may be due to the complexity and polydispersity of lignin compared to sugars normally released as uniform monomeric carbohydrates , which limit lignin wide - scale use in biorefineries . besides , recovery of lignin from their product streams is difficult , with different composition of lignin contained by the tissues of individual plants and according to its source . the efficient utilization of lignocellulosic materials requires its pretreatment prior to the enzymatic hydrolysis and fermentation , which results in opening up the cell wall structure by disturbing the lignin / hemicelluloses complex and exposing the cellulose to enzymatic hydrolysis . the pretreatment process is considered the most expensive step in the valorization of lignocellulosic byproducts , where it can contribute to about 30% of the total cost . due to this problem , several methods of pretreatment processes have been investigated which include alkaline or acid hydrolysis , milling and grinding , gas treatment by ozone or sulfur dioxide , steam explosion , organic solvent treatment , liquid hot water , wet oxidation , ammonia fiber explosion , biological treatments , cellulose solvent pretreatment , and ionic liquid . on the other hand , some of these methods , that is , acid hydrolysis and steam explosion , produce some byproducts like furfural and hydroxyl methyl furfural ( hmf ) which negatively affect the fermentation process , and this requires a separate detoxification step , for example , ion exchange , addition of activated charcoal , and over liming , which in turn increase the overall process cost . also , the expenditures are negatively affected by the power requirements for achieving the optimum reaction conditions for some pretreatments and costly downstream processing to remove extraneous chemicals that are added to the biomass . moreover , the use of expensive industrial equipments are necessary to avoid corrosion normally associated with acid hydrolysis . all of this reflect the need for integrated technologies for efficient treatment and the maximal recovery of energy from lignocellulosic byproducts . alternatively , the use of untreated lignocellulosic biomass as substrate cause several problems , that is , substrate agglutination and clogging , in addition to microbial degradation resistance . the substrate uniformity is one of the most significant factors that influence the energy poise and the total economics in the valorization process of lignocellulosic biomass . with high solid content , the sugar and resulting product concentrations will increase , which leads to a significant reduction in capital and production costs due to the compact equipment size and low energy consumption during heating , cooling , and distillation . on the other side , these high - solid loadings generate environmental problems due to the absence of free water in the pretreated material and difficulties to handle the slurry . also , some cereal processing byproducts , that is , liquid stillage , must be dried because of their short shelf life which extends the durability , but increases the overall economics of the valorized product . valorization is the transformation of byproducts to alternative fuels , energy and other useful chemicals , with specific attention for sustainability and environmental objectives . generally , cereal based biorefinery substrates are chemically ( i.e. , naoh ) or hydrothermally ( i.e. , steam explosion ) and/or enzymatically ( i.e. , cellulase ) pretreated before fermentation which could be through submerged , solid state , dark or photo fermentation . submerged fermentation ( smf ) is any aqueous fermentation process occurring in the presence of liquid substrate , whereas , solid - state fermentation ( ssf ) is any fermentation process occurring in the absence or near - absence of free water by employing a natural substrate as a solid support . dark ( df ) and photo ( pf ) fermentation is the bacterial conversion of organic substrate to biohydrogen through a complex process involving biochemical reactions , with the difference that pf is held by a diverse group of photosynthetic bacteria in the presence of light . the most recent valorization strategies for cereal based biorefinery wastes are illustrated in tables 2 and 3 and detailed in the following sections . productivity is based on volume of product per weight of raw waste or volume per volume of culture . productivity is based on volume of product per weight of raw waste or volume per volume of culture . organic acids and their derivatives are widely used in food , pharmaceutical , leather , and textile industries . however , the high production cost becomes a limitation for their applications in more fields . therefore , much attention has been paid to searching for cheaper sources for the fermentative production of organic acids . lactic acid ( la ) is one of the most important organic acids extensively used in pharmaceutical , food , chemical , textile , leather , polymer , and cosmetic industries . the replacement of the costly pure raw materials usually used for la production by agro - industrial residues can provide an attractive substitute because of their low prices . these agro - industrial residues include brewer s spent grain ( bsg ) enzymatic hydrolyzate which was incorporated into culture medium , with glucose as carbon source , for the production of la by lactobacillus delbrueckii.(48 ) with this mixture , a yield of 0.99 g / g of bsg was obtained after 60 h of fermentation . this yield dropped to 0.45 g / g when wheat bran acidic hydrolyzate was used as the nitrogen source for la production by lactobacillus rhamnosus.(51 ) also , corn steep liquor ( csl ) was investigated as a sole and low cost nitrogen source in addition to some components to replace yeast extract for the cost - effective production of la . g / l was achieved in shake - flask scale , whereas the concentration of la in a bioreactor was 110 rice bran is one of the most abundant agro - industrial byproducts that was investigated in several studies for the production of la . in two different trials , l. delbrueckii(49 ) and l. rhamnosus(53 ) were used to produce la from enzymatically hydrolyzed rice bran which was used as carbon and nutrient source . l. rhamnosus ( 56 kg / m ) was superior to l. delbrueckii ( 9 kg / m ) and was 1.5 times higher in la production than the glucose and yeast extract traditional medium due to the high nitrogen content and the presence of rice bran oil rich in fatty acids which is an important factor for la production . citric acid ( ca ) fermentation by aspergillus niger is one of the world s major industrial microbial processes . the same fungus was used to evaluate the economic ca production from the untreated bsg agro - industrial waste by solid state fermentation ( ssf ) . the resulted ca yield was 11.8 g / kg after 120 h of fermentation . among the vital organic acids is succinic acid ( sa ) , which is conventionally manufactured from petrochemicals through expensive processes . higher economic benefit could be achieved with the use of agro - industries byproducts as potential resources to produce petrochemically derived products . two types of wheat milling byproducts were enzymatically pretreated and used to produce sa by actinobacillus succinogenes . the pretreatment process was achieved through integrated biorefinery concept by using some wheat milling byproducts ( i.e. , bran ) as a substrate in ssf for aspergillus strains to produce glucoamylase and protease enzymes , which are used to pretreat wheat gluten or wheat bran . also a. succinogenes was anaerobically used for sa production from acid pretreated corn fiber byproduct and 35.5 microorganisms are the most important and convenient sources for the production of commercial enzymes . they can be overexpressed under suitable growth conditions to produce abundant quantities of enzymes . with the initiation of a new era in biotechnology , the amylase enzyme family came forward with lot of industrial applications such as brewing , bread making , pharmacy , starch processing , paper , and textile industries . the untreated bsg was used for the production of -amylase by bacillus sp . in a submerged fermentation system . the highest tested concentration of bsg ( 5% , w / v ) resulted in a 5-fold enhancement in the enzyme production . another wheat milling byproduct , called gruel , was used by kammoun et al . as sole carbon source for the production of -amylase from aspergillus oryzae . the experimental conditions revealed an enhanced enzyme production of 151.1 u / ml . also , ssf was applied for the production of this enzyme using paddy rice processing byproduct and the highest enzyme production was 211.5 u / gds ( units per gram dried solid ) . cellulases are another example of important industrial enzymes that are capable to degrade the most abundant cellulose biopolymer . a high yield ( 159.1 u / g ) of -glucosidase was achieved from rice bran - based ssf by the penicillium citrinum fungus . optimized the conditions of cellulase production by penicillium decumbens in ssf using rice bran as the substrate and obtained 5.76 iu / g , whereas lee et al . used rice bran in combination with yeast extract to produce the same enzyme in smf by bacillus subtilis and 196.8 u / ml was obtained . different agro - industrial residues were used for the production of this vital enzyme such as enzymatically treated wheat bran which exhibited 1.1 u / ml of the cellulase enzyme under smf by aspergillus flavus , or untreated corn cob which produced 5.25 iu / ml under smf by trichoderma reesei.(66 ) proteases are one of the most important categories of industrial enzymes , and the application of alkaline proteases has increased remarkably in a range of industrial processes including food , detergents , silk , and leather . romero et al . aimed at developing a process for protease production by bacillus licheniformis using corn distiller s dried grains with solubles ( cddgs ) , a bioethanol industry byproduct , as the sole carbon / nitrogen source . the maximum protease production was 0.16 u / ml after 55 h of fermentation . laccases are abundant in white - rot fungi , which are the only living organisms able to degrade whole wood components , particularly , the genus trametes which is the most efficient lignin degraders . the potential of the common brewing industry byproduct , barley bran , as a support - substrate for laccase production by trametes versicolor under ssf conditions was evaluated by rodrguez couto . laccase activity was enhanced by 13-fold in relation to inert support cultures with initial ammonium concentration of 0.2 vanillin is used as flavoring agent in food industry , as intermediate in the herbicides industry , drugs or antifoaming agents , as component of household products such as floor polishes and air fresheners . also it is used as food preservative because of its antimicrobial and antioxidant properties . the precursor of vanillin is ferulic acid which can be released from agricultural residues by physicochemical and/or enzymatic treatments . di gioia et al . explored the possibility of obtaining vanillin from the bioconversion of ferulic acid derived from enzymatically hydrolyzed wheat bran by an engineered e. coli strain . a vanillin concentration of 0.09 g / l was obtained , due to a high percent of reduction to vanillyl alcohol . another trial was performed by torres et al . , in which corn cob was hydrolyzed by alkaline to obtain 1171 mg / l ferulic acid that was used as a medium for vanillin bioproduction by the engineered e. coli . a maximum vanillin concentration of 239 mg / l was obtained after 22 h. a different technology of converting ferulic acid , from the rice bran oil byproduct , into vanillin was developed by a combination of the fungal strains aspergillus niger and pycnoporus cinnabarinus , in which the filtrate of a. niger culture was concentrated and vanillic acid in the filtrate was fermented into vanillin by p. cinnabarinus.(74 ) the concentration of vanillin reached 2.8 g / l . biopolymers are important materials having applications in several industrial sectors like pharmaceutical , food and cosmetic industries . they can be categorized to polyesters ( i.e. , polyhydroxyalkanoates ) , polyalcohols ( i.e. , xylitol ) and polysaccharides ( i.e. , pullulan ) . the biotechnological method of producing such polymers has high interest as an alternative to the chemical method because of the little energy required and specificity . however , the cost of these polymers is preventing their usage on a large scale . . therefore , researchers are trying to use various cheap carbon sources , including agro - industrial byproducts . one of the initial trials was conducted by mussatto and roberto to evaluate the applicability of acidic hydrolyzate bsg as culture medium for xylitol production by candida guilliermondii , and obtained a xylitol yield of 0.78 g / g . recently , the production of pullulan from aureobasidium pullulans was tested using csl as nutrient along with 15% ( w / v ) glucose as carbon source . g / l ) , which accounted for 18% increase compared with glucose . also , production of polyhydroxyalkanoate ( pha ) by sinorhizobium meliloti using enzymatic hydrolyzate of rice bran was tested . the pha contents increased with the increase in fermentation period from 24 to 96 h , with a maximum production of 3.6 valorization options could be extended to the pharmaceutical field for the bioproduction of antibiotics . the rifamycins are a group of antibiotics that are synthesized either artificially or naturally by the bacterium amycolatopsis . was investigated under ssf using different agro - industrial byproducts ( corn husk , corn cobs , and wheat bran ) . corn husk was the most suitable substrate ( 1.95 g / kg ) with 4-fold higher production than wheat bran and corn cobs . as a result of growing environmental attentiveness and the prominence placed on an ecological society in agreement with the global environment , biosurfactants of microbial origin have recently been recommended to substitute chemically synthesized surface active agents . low - cost media based on animal fat and csl combined with glucose , yeast extract , urea , and other inorganic nitrogen sources were evaluated for the production of biosurfactants by the yeast candida lipolytica.(82 ) only 2.5% of the csl remained after six days and 2.2 g / l of crude biosurfactant was produced , causing a maximum reduction in surface tension from 50 to 28 mn / m . hydrogen gas is an eco - friendly fuel with high energy content ( 122 kj / g ) . unlike fossil fuels emissions , only water vapor is released when hydrogen gas is used which contributes to reducing greenhouse effects significantly . although h2 has many obvious advantages , it remains a problem with storage and transportation . pressurised hydrogen gas takes a great deal of volume compared with other fuels like for example , gasoline that with equal energy content , needs about 30 times less volume at 100 bar gas pressure . due to the high explositivity there are also obvious safety concerns with the use of pressurized or liquefied hydrogen in vehicles as well as additional energy use for pressurizing or liquefication . pf by purple nonsulfur ( pns ) bacteria , like rhodobacter species , is among biohydrogen production methods that has attracted distinctive consideration . this is due to the fact that these photosynthetic bacteria have a number of advantages such as performing anoxygenic photosynthesis to produce hydrogen without oxygen , absence of hydrogen gas inhibition due to nitrogenase enzyme , utilization of a wide variety of substrates for hydrogen production and conferring different growth modes with respect to energy and carbon requirements . df is more effective than photo because of the sensitiveness of pf and requirement of low concentration substrates . biomass containing starch and cellulose , that is , agro - industrial byproducts , establish rich and consistent raw materials for biohydrogen production . an integrated biohydrogen refinery was suggested by several researchers , in which pretreated barley malt byproduct is utilized to produce biohydrogen by rhodobacter sphaeroides . used the obtained biohydrogen ( 1.07 l / kg ) as a fuel for the production of electricity through fuel cell , while kars and ceylan coproduced biohydrogen ( 0.4 l / l ) and 5-aminolevulinic acid ( 67.4 m ) using the same byproduct and microorganism . moreover , wheat starch was investigated using fed - batch operation for biohydrogen production by combined dark and light fermentation . the optimum dark / light ratio was /2 yielding the highest cumulative hydrogen ( 65.2 cm / g ) . also , alkaline - treated wheat milling byproduct was fermented with sewage sludge in different fermentation modes ( batch , semicontinuous and continuous ) . the maximum hydrogen yields of 64 cm / g of wheat feed dry weight were produced in batch mode . the anaerobic bacteria caldicellulosiruptor saccharolyticus was investigated for the production of biohydrogen from the alkaline pretreated sweet sorghum bagasse ( ssb ) , and a maximal volumetric hydrogen production rate of 10.6 mmol / lh was obtained . bioethanol produced from low - cost biomass is considered as an attractive substitute to fossil fuels to minimize dependence on oil and reduce carbon dioxide emissions . saccharomyces cerevisiae cells have average theoretical and practical ethanol yields of 0.5 and 0.4 g / g of glucose , respectively . currently , the main biomass for bioethanol is starch - rich feedstock in which high yields of glucose are rapidly obtained by enzymatic hydrolysis . substrate is one of the key costs in ethanol production . putting this aim into focus , many studies tried to investigate the possibility of using agro - industrial byproducts as the main substrate for bioethanol production . wheat bran is among these byproducts , in which it was used for bioethanol production by saccharomyces species after being hydrolyzed by acid or hydrothermal followed by enzymatic treatment . under these conditions , g / l ) when acidic hydrolyzed wheat stillage ( main residue from the starch - to - ethanol fermentation process ) was fermented by zymomonas mobiliz.(100 ) also , s. cerevisiae was investigated for the production of bioethanol using ssb pretreated with hydrothermal or acid treatment followed by enzymatic hydrolysis in submerged fermentation . g / l due to the usage of sweet sorghum juice mixed with the pretreated and dewatered bagasse . on the other hand , ssb without any pretreatment was used to immobilize s. cerevisiae through ssf for the production of bioethanol . the ethanol productivity of the immobilized cells was 2.24 times higher than the free cells , with an ethanol yield that reached 4.9 g / g . the ability to ferment ssb is not restricted only to bacterial cells , but it could be performed with fungal cells as well . goshadrou et al . studied the production of ethanol from the pretreated ssb by a zygomycetes fungus mucor hiemalis . the bagasse was treated with sodium hydroxide , prior to enzymatic hydrolysis by commercial cellulase and -glucosidase enzymes . a 24 h fermentation on the pretreated bagasse resulted in about 81% of the corresponding theoretical ethanol yield . the effect of different pretreatments and microbial strains on ssb for bioethanol production was investigated . it was found that maximum production of bioethanol was obtained ( 209.2 mg / g ) after 24 h fermentation by issatchenkia orientalis with dry bagasse previously treated with hcl and enzymes as a substrate . moreover , s. cerevisiae with acid / enzyme pretreated rice hull as a substrate was used for bioethanol production , and 0.11 g / g of rice hull was obtained with 84% of dissolved sugars converted to bioethanol . this yeast was also combined with candida shehatae to ferment the acidic hydrolyzed rice hull to ethanol . the pretreatment of rice hull by the wet air oxidation ( wao ) method was inspected by banerjee et al . the remained cellulose was around 92% , while the recovered lignin was 20% , showing oxidation of the majority of lignin . the high cellulose content and minor residual lignin in the solid fraction would greatly enable subsequent enzymatic hydrolysis , with improved ethanol yields from rice hull . different pretreatments with various corn milling byproducts were evaluated for the production of bioethanol . corn fiber was hydrolyzed by dilute sulfuric acid , and subsequently fermented by e. coli to produce 44 g / l of bioethanol . coupling a pervaporation membrane unit to the hydrolyzate fed - batch fermentation maintained the ethanol production below 25 g / l , with sugar utilized completely , for 5 days , which circumvented the toxicity effect of high ethanol concentration on cells viability . furthermore , the yeast strain pichia guilliermondii was anaerobically adapted to hydrolyzed corn cob and used for ethanol production without any detoxification or external nutrient supplementation . studied the feasibility of substituting yeast extract by the low cost csl to produce ethanol using clostridium strain . / l ) was produced after 360 h of fermentation in a 7.5 l bioreactor . butanol is a carbon branched chain primary alcohol that could be an alternative fuel to ethanol due to its superior properties . butanol is a metabolite of acetone / butanol / ethanol ( abe ) fermentation by clostridium species . abe fermentation has major hindrances , mainly the high cost of substrates and recovery . in order to overcome this shortcoming , the objective of several studies was to evaluate the economical use of pretreated agro - industrial byproducts to produce biobutanol . qureshi et al . compared acidic to enzymatic pretreatment of corn fiber byproduct subsequently fermented to biobutanol by clostridium beijerinckii . g / l , when a mutant of c. beijerinckii with considerable inhibitor - tolerance was used for butanol production from nondetoxified acidic corn fiber hydrolyzate . not only for the detoxification of the treated byproduct , but also for butanol separation from its fermentation broth . anaerobic biomethanation can be used as an alternative potential treatment for valorization of biodegradable solid waste . some of these organic solid wastes , that is , lignocellulosic byproducts have a limited biodegradability despite the high cod content . therefore , several studies were conducted to enhance the biomethanation process of such byproducts . for example , barley waste was pretreated by alkaline before it was anaerobically fermented by activated sludge . this pretreatment reflected positively on the production of methane , which increased to 222 ml / g of volatile solids . also , corn stillage was evaluated for anaerobic biogas production by biomethanator biomass after pretreatment with alkaline , and 763 ml / g of volatile solids was obtained . regaining biochemical energy available in agricultural biomass is one approach that could be used to counterweight the world s current fossil fuel consumption . agricultural crop byproducts , that is , cellulose and lignin , contain great amounts of biochemical energy . the potential for direct transformation of lignocellulosic biomass residues to electricity in a microbial fuel cell ( mfc ) was explored by several authors . gregoire and becker developed a new single chamber solid - substrate mfc to hold the cellulose hydrolysis and fermentation processes . untreated corn cob pellets were used as a fuel for the cell , which continuously produced electricity for more than 60 days . bioaugmentation with geobacter metallireducens improved mfc performance to generate a maximum power density of 230 mw / m . the acidic / enzymatic hydrolyzate of corn stover was used as a substrate for a mixture of electrogenic culture in one chamber mfc to produce bioelectricity , and 1180 mw / m was obtained . also the performance of mfcs was evaluated while treating rice mill wastewater by anaerobic sludge . maximum cod removal efficiency of 96.5% and lignin removal of 84% were obtained . maximum sustainable volumetric power ( 2.3 w / m ) was achieved with 100 external resistance . lignin is one of the key components of lignocellulosic biomass together with cellulose and hemicellulose , representing about 435% of most biomass feedstock . lignins in cereals are guayacyl - syringyl - p - hydroxyphenyl with some structural units derived from trans - p - coumaryl alcohol . the lignin is separated by either dissolution using solvents , that is , alkali , organosolv and milled wood ) or separation of insoluble lignin by acid hydrolysis of cellulose and hemicellulose , that is , klason and hydrolytic . this is clear in the klason procedure that provides quantitative separation of altered lignin . kraft lignins ( i.e. , lignoboost ) are accompanied by structural changes during the pulping process resulting in high content of free phenolic groups and c c linkages . this review contributes to knowledge about the future application of carbon rich agro - industrial byproducts for their value addition to biological chemicals and energy . it also offers economic and environmental benefits over traditional ways used to dispose off agro - industrial residues . massive amounts of agricultural biomass are burnt in an open environment resulting in the release of harmful gases , which is not a sustainable method . it is clear that the utilization of agro - industrial byproducts as fermentation substrates require hydrolysis pretreatment due to the presence of complex polysaccharides in such biomass . chemical hydrolysis uses up chemicals and produces chemically destructive effluents , whereas enzymatic hydrolysis requires optimization to achieve the best combination of enzymes for each feedstock and can not quickly adapt to variable feedstock composition . on the contrary , hydrothermal hydrolysis is an eco - friendly method , even though it consumes energy . although biomass may be cheap , the costs of processing technologies can be high . these technologies include not only pretreatment of biomass , but enzymatic saccharification of the pretreated biomass , and fermentation of the hexose and pentose sugars released by hydrolysis and saccharification . this can be achieved by increasing the efficiency of biorefinery in order to save resources and integrate in the existing industrial facilities , with eye on configurations that are eco - friendly . therefore , prior to the successful application of this process on large - scale setups , consideration must be focused on the improvement of techniques for the hydrolysis of lignocellulosic biomass that result in higher sugar yields . enzymatic hydrolysis requires further improvements especially in the terms of enzyme separation and reutilization after biomass treatment . also , the complete characterization of the agro - industrial byproducts is required , in which it could be useful for the production of new microbial valorized products . the genetically modified microorganisms could be used to ferment c5 sugars from hemicellulose , which is not normally assimilated by the natural microbial strains .
the growth of the biobased economy will lead to an increase in new biorefinery activities . all biorefineries face the regular challenges of efficiently and economically treating their effluent to be compatible with local discharge requirements and to minimize net water consumption . the amount of wastes resulting from biorefineries industry is exponentially growing . the valorization of such wastes has drawn considerable attention with respect to resources with an observable economic and environmental concern . this has been a promising field which shows great prospective toward byproduct usage and increasing value obtained from the biorefinery . however , full - scale realization of biorefinery wastes valorization is not straightforward because several microbiological , technological and economic challenges need to be resolved . in this review we considered valorization options for cereals based biorefineries wastes while identifying their challenges and exploring the opportunities for future process .
Introduction Biorefineries Cereal Biorefinery Cereal Biorefinery Waste Valorization The Lignin Enigma Conclusions
moreover , one of the major disadvantages of biorefinery , compared to petroleum refinery , is in the miscellany of technologies required to obtain the end products . platforms determine the complexity of the system in which they represent intermediates that link biorefinery systems and their processes ( i.e. crop based biorefineries have gained tremendous interest in recent years , several pilot - scale systems have been built and currently a lot of research is going on building full scale systems . as with any other technology , apart from the main products in this case ethanol or lactic acid , several side products and wastes are also generated . cereal grains have been a primary human food source since thousands of years ago , being one of the most vital source of calories for a large sector of the world population . great interest has been directed toward producing second - generation ethanol , since the above - mentioned crops can be cultivated in secondary lands without the need to interfere with the fertilized lands generally used for food production . germination is completed upon the full modification of the endosperm , after which the process is terminated by drying ( kilning process ) . the majority of the outer bran , germ , and tip cap are removed from the moist grains , leaving the endosperm , which is then dried , cooled , and sieved . the amount of biodegradable wastes drained to landfills in eu countries in 2020 is estimated to increase by 35% of its level in 1995 . although these wastes represent serious economic and environmental challenges , they contain extensive amounts of potentially reusable materials and energy . also , one of the major byproducts from malting industry is the barley malt sprouts which consist of roots , sprouts , and malt hulls . the efficient and economic disposal of cereal processing byproducts is the main concern of industrial society , in order to fulfill the environmental regulations that regard them as wastes destined to be drained off in most cases . the value addition and adequate utilization of cereal processing byproducts is very important for the valorization technology of such byproducts . moreover , if the valorization know - how is available for certain industrial byproduct in laboratory or pilot scale , the efficient scaling - up of this technique is limited by the standardization of the production process on the industrial scale . current sugar - based platform biorefineries mainly focus on the valorization of cellulose and hemicelluloses , while lignin is generally considered as a low - value residue . the pretreatment process is considered the most expensive step in the valorization of lignocellulosic byproducts , where it can contribute to about 30% of the total cost . the substrate uniformity is one of the most significant factors that influence the energy poise and the total economics in the valorization process of lignocellulosic biomass . valorization is the transformation of byproducts to alternative fuels , energy and other useful chemicals , with specific attention for sustainability and environmental objectives . a maximum vanillin concentration of 239 mg / l was obtained after 22 h. a different technology of converting ferulic acid , from the rice bran oil byproduct , into vanillin was developed by a combination of the fungal strains aspergillus niger and pycnoporus cinnabarinus , in which the filtrate of a. niger culture was concentrated and vanillic acid in the filtrate was fermented into vanillin by p. the pha contents increased with the increase in fermentation period from 24 to 96 h , with a maximum production of 3.6 valorization options could be extended to the pharmaceutical field for the bioproduction of antibiotics . this is due to the fact that these photosynthetic bacteria have a number of advantages such as performing anoxygenic photosynthesis to produce hydrogen without oxygen , absence of hydrogen gas inhibition due to nitrogenase enzyme , utilization of a wide variety of substrates for hydrogen production and conferring different growth modes with respect to energy and carbon requirements . the ethanol productivity of the immobilized cells was 2.24 times higher than the free cells , with an ethanol yield that reached 4.9 g / g . lignin is one of the key components of lignocellulosic biomass together with cellulose and hemicellulose , representing about 435% of most biomass feedstock . it also offers economic and environmental benefits over traditional ways used to dispose off agro - industrial residues .
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radiation therapy ( rt ) with or without concurrent chemotherapy is an established definitive treatment modality for both early and locally advanced stage nasopharyngeal cancer ( npc ) . although it allows favorable oncologic outcomes , there is a wide range of response to rt , highlighting the need to identify patients who might have radiation resistance and high risk of recurrence . thus , earlier prediction of rt response and consequent long - term prognosis might be a critical issue for tailoring subsequent treatment to increase the chance of a cure in an individual patient . the tnm staging system is the most commonly accepted staging method for head and neck cancer , and it aids clinicians in determining prognosis and in selecting the most optimal therapeutic modality . the tnm staging system is considered the most important prognostic factor ; however , because it is usually based on the size and/or extent of the primary tumor and/or metastatic lymph node , it sometimes does not correlate with actual tumor burden . although this uni- or bi - dimensional measurement is an important factor , three - dimensional volumetric data have become another important factor that should be considered , particularly when using non - surgical therapies , such as rt or chemotherapy . many studies have demonstrated the prognostic value of tumor volume in various cancers , and there is mounting evidence that pretreatment tumor volume and/or residual tumor volume have prognostic value [ 3 - 6 ] . however , few studies investigating changes in tumor volume during rt as a prognostic factor have been reported [ 7 - 10 ] . we previously reported on the prognostic impact of tumor volume reduction rate ( tvrr ) measured during adaptive rt for oropharyngeal cancer . in regard to the nasopharynx subsite of head and neck cancer , however , tvrr as a prognostic factor has not been reported yet , thus its prognostic role remains uncertain . the aim of this study was therefore to examine the volumetric parameters measured before and during definitive rt to determine possible prognostic implications in npc patients . to the best of our knowledge , the present study is the first to report on the value of tvrr as an independent prognostic factor in npc . we reviewed the medical records of consecutive npc patients treated with definitive rt with or without concurrent chemotherapy at samsung medical center between march 2006 and february 2013 . to be eligible for the current study , patients were required to have ( 1 ) histologically confirmed carcinoma of the nasopharynx , ( 2 ) no distant metastasis at the time of initial diagnosis , ( 3 ) completed more than 90% of the planned rt course without significant interruption , and ( 4 ) available rt plans . the exclusion criteria were ( 1 ) other non - squamous cell carcinoma histologic types such as lymphoma and salivary type carcinoma , ( 2 ) patients whose primary tumor or neck node was surgically removed or who received induction chemotherapy before rt , and ( 3 ) immediate follow - up loss after completion of rt . complete medical history taking and physical examination , direct flexible fiberoptic endoscopic examination , computed tomography ( ct ) scans and magnetic resonance image of the head - and - neck region , and whole - body f - fluorodeoxyglucose positron emission tomography ( pet ) with ct were performed in all patients . each patient underwent ct simulation in the supine position , immobilized by a thermoplastic mask , with a mouthpiece to keep the mouth open . simulation ct images were obtained at 2.5- to 3.75-mm slice intervals with intravenous contrast enhancement . a second ct simulation was performed in all patients in order to generate an adaptive re - plan after delivery of the median 14 fractions ( 12 to 17 fractions ) . all sets of acquired simulation ct images were imported into the pinnacle3 treatment planning system ( ver . 9.2 , royal phillips electronics , miami , fl ) , and the gross tumor volumes ( gtvs ) of the primary tumor and the metastatic lymph nodes were manually contoured . the delineation of gtv was based on both clinical examination findings as well as all available diagnostic images . rt techniques were either 3-dimensional conformal rt ( 3d - crt ) using 4 mv and/or 10 mv photons from linear accelerator or intensity - modulated radiation therapy ( imrt ) using 6 mv photons from helical tomotherapy . the clinical target volume ( ctv ) was arbitrarily subdivided into two risk levels : high risk ctv encompassed the immediately adjacent regions to the gtvs and the lymph nodes considered at equivocal risk of metastasis based on size , shape , and pet uptake ; and low risk ctv covered the apparently uninvolved lymphatics and more than one station away from the nodal gtv . the prescribed radiation dose was differently according to the rt technique . when using 3d - crt technique , the doses to the gtv , high risk ctv and low risk ctv were 70 gy in 35 fractions , 54 gy in 27 fractions , and 36 gy in 18 fractions , respectively , with 2 gy per fraction . when using imrt technique , simultaneous integrated boost ( sib ) was adapted , and the dose schedules changed during the study period . in earlier cases , the fraction sizes were 2.2 gy to the gtv and 2.0 gy to the ctv throughout the rt course . in later cases , the fraction sizes to the gtv and ctv were 2.2 gy and 2.0 gy during the first 18 fractions , and then 2.4 gy and 2.0 gy during the following 12 fractions , respectively . as a result , the actually delivered doses to the gtv , high risk and low risk ctvs were 66.0 or 68.4 gy in 30 fractions , 60 gy in 30 fractions , and 36 gy in 18 fractions , respectively . the schemes of the adaptive re - plans and measurements of the gtvs along the rt courses are illustrated in fig . 1 . in accordance with institutional guidelines , patients with locally advanced disease such as clinical t3 or t4 tumors and/or lymph node metastasis received concurrent chemotherapy with rt , unless they had no contraindication to addition of chemotherapy . three weeks after completion of rt , adjuvant chemotherapy ( 1 to 3 cycles of cisplatin+5-fluorouracil ) the primary tumor and the metastatic lymph nodes were delineated on both the pre- and mid - rt simulation ct images , and 3-dimensional tumor volumes were calculated on the rt planning system . tvrr was defined as the percent ( % ) reduction of the gtv in relation to the pre - rt gtv , where tvrr=(pre - rt gtv mid - rt gtv)/pre - rt gtv . all patients were asked to visit for follow - up evaluation of disease status on a regular basis . the first evaluation was scheduled at one month of rt completion , and then every 3 months for the first 2 years , every 6 months until 5 years , and annually thereafter . on each visit , a thorough physical examination was performed together with imaging studies alternating between ct of the head - and - neck region and pet - ct . the overall survival ( os ) duration was measured from the date of rt initiation to the date of death or last follow - up visit , and progression - free survival ( pfs ) from the date of rt initiation to the date of first recurrence of disease of any type or the last clinical follow - up . the rates of os and pfs were calculated using kaplan - meier estimates with the log - rank test for univariate comparison . the relationship between the volumetric parameters and clinical outcome was assessed using mann - whitney u test . prognostic factor analyses between tumor volume parameters were performed as both continuous and categorical variables to determine which independently affected prognosis . the optimal thresholds of the continuous variables were determined to be those producing the highest accuracy . the discriminatory performances of the volumetric data were assessed by receiver operating characteristic ( roc ) analysis . the log - rank test was used for validation of the determined cut - off values . cox proportional hazard regression analysis was used to determine the implications of the potential prognosticators . the statistical tests were two - sided , and a p - value of < 0.05 was considered statistically significant . factors with p - value of < 0.2 in univariate analysis were included in the multivariate analyses . variable risk was expressed as a hazard ratio ( hr ) with a corresponding 95% confidence interval ( 95% ci ) . we reviewed the medical records of consecutive npc patients treated with definitive rt with or without concurrent chemotherapy at samsung medical center between march 2006 and february 2013 . to be eligible for the current study , patients were required to have ( 1 ) histologically confirmed carcinoma of the nasopharynx , ( 2 ) no distant metastasis at the time of initial diagnosis , ( 3 ) completed more than 90% of the planned rt course without significant interruption , and ( 4 ) available rt plans . the exclusion criteria were ( 1 ) other non - squamous cell carcinoma histologic types such as lymphoma and salivary type carcinoma , ( 2 ) patients whose primary tumor or neck node was surgically removed or who received induction chemotherapy before rt , and ( 3 ) immediate follow - up loss after completion of rt . complete medical history taking and physical examination , direct flexible fiberoptic endoscopic examination , computed tomography ( ct ) scans and magnetic resonance image of the head - and - neck region , and whole - body f - fluorodeoxyglucose positron emission tomography ( pet ) with ct were performed in all patients . each patient underwent ct simulation in the supine position , immobilized by a thermoplastic mask , with a mouthpiece to keep the mouth open . simulation ct images were obtained at 2.5- to 3.75-mm slice intervals with intravenous contrast enhancement . a second ct simulation was performed in all patients in order to generate an adaptive re - plan after delivery of the median 14 fractions ( 12 to 17 fractions ) . all sets of acquired simulation ct images were imported into the pinnacle3 treatment planning system ( ver . 9.2 , royal phillips electronics , miami , fl ) , and the gross tumor volumes ( gtvs ) of the primary tumor and the metastatic lymph nodes were manually contoured . the delineation of gtv was based on both clinical examination findings as well as all available diagnostic images . rt techniques were either 3-dimensional conformal rt ( 3d - crt ) using 4 mv and/or 10 mv photons from linear accelerator or intensity - modulated radiation therapy ( imrt ) using 6 mv photons from helical tomotherapy . the clinical target volume ( ctv ) was arbitrarily subdivided into two risk levels : high risk ctv encompassed the immediately adjacent regions to the gtvs and the lymph nodes considered at equivocal risk of metastasis based on size , shape , and pet uptake ; and low risk ctv covered the apparently uninvolved lymphatics and more than one station away from the nodal gtv . the prescribed radiation dose was differently according to the rt technique . when using 3d - crt technique , the doses to the gtv , high risk ctv and low risk ctv were 70 gy in 35 fractions , 54 gy in 27 fractions , and 36 gy in 18 fractions , respectively , with 2 gy per fraction . when using imrt technique , simultaneous integrated boost ( sib ) was adapted , and the dose schedules changed during the study period . in earlier cases , the fraction sizes were 2.2 gy to the gtv and 2.0 gy to the ctv throughout the rt course . in later cases , the fraction sizes to the gtv and ctv were 2.2 gy and 2.0 gy during the first 18 fractions , and then 2.4 gy and 2.0 gy during the following 12 fractions , respectively . as a result , the actually delivered doses to the gtv , high risk and low risk ctvs were 66.0 or 68.4 gy in 30 fractions , 60 gy in 30 fractions , and 36 gy in 18 fractions , respectively . the schemes of the adaptive re - plans and measurements of the gtvs along the rt courses are illustrated in fig . in accordance with institutional guidelines , patients with locally advanced disease such as clinical t3 or t4 tumors and/or lymph node metastasis received concurrent chemotherapy with rt , unless they had no contraindication to addition of chemotherapy . three weeks after completion of rt , adjuvant chemotherapy ( 1 to 3 cycles of cisplatin+5-fluorouracil ) was optionally administered to some npc patients . the primary tumor and the metastatic lymph nodes were delineated on both the pre- and mid - rt simulation ct images , and 3-dimensional tumor volumes were calculated on the rt planning system . tvrr was defined as the percent ( % ) reduction of the gtv in relation to the pre - rt gtv , where tvrr=(pre - rt gtv mid - rt gtv)/pre - rt gtv . all patients were asked to visit for follow - up evaluation of disease status on a regular basis . the first evaluation was scheduled at one month of rt completion , and then every 3 months for the first 2 years , every 6 months until 5 years , and annually thereafter . on each visit , a thorough physical examination was performed together with imaging studies alternating between ct of the head - and - neck region and pet - ct . the overall survival ( os ) duration was measured from the date of rt initiation to the date of death or last follow - up visit , and progression - free survival ( pfs ) from the date of rt initiation to the date of first recurrence of disease of any type or the last clinical follow - up . the rates of os and pfs were calculated using kaplan - meier estimates with the log - rank test for univariate comparison . the relationship between the volumetric parameters and clinical outcome was assessed using mann - whitney u test . prognostic factor analyses between tumor volume parameters were performed as both continuous and categorical variables to determine which independently affected prognosis . the optimal thresholds of the continuous variables were determined to be those producing the highest accuracy . the discriminatory performances of the volumetric data were assessed by receiver operating characteristic ( roc ) analysis . the log - rank test was used for validation of the determined cut - off values . cox proportional hazard regression analysis was used to determine the implications of the potential prognosticators . the statistical tests were two - sided , and a p - value of < 0.05 was considered statistically significant . factors with p - value of < 0.2 in univariate analysis were included in the multivariate analyses . variable risk was expressed as a hazard ratio ( hr ) with a corresponding 95% confidence interval ( 95% ci ) . the median age was 51 years , ranging from 17 to 86 years , and 94.3% of the patients were male . approximately half of the pretreatment clinical t stage was ct1 ( 77 patients , 44.7% ) , but the majority of patients ( 130 patients , 81.8% ) had metastatic lymph nodes on initial diagnosis . the distribution of tnm stage based on the american joint committee on cancer ( ajcc ) seventh edition was stage i in 15 patients ( 9.4% ) , ii in 39 ( 34.5% ) , iii in 70 ( 44.0% ) , and iva / b in 35 ( 22.0% ) . the median total radiation dose was 68.4 gy ( 66 to 72.4 gy ) , and 113 patients ( 71.1% ) received imrt . a cisplatin - based regimen was administered concurrently to all patients : cisplatin 100 mg / m every 3 weeks was administered to 111 patients ( 69.8% ) ; weekly cisplatin 20 mg / m to 23 ( 14.5% ) ; and cisplatin plus docetaxel 20 mg / m every 3 weeks to one ( 0.6% ) , respectively . after completion of rt , adjuvant chemotherapy with cisplatin plus 5-fluorouracil was administered to 34.6% of patients . the median follow - up duration for survivors was 41.5 months ( range , 11.2 to 91.8 months ) . treatment failure of any type was observed in 43 patients ( 27.0% ) , and 13 patients died during the follow - up period . as expected , patients with more advanced tnm stage had worse os and pfs : os rates at 5 years were 100% in stages i and ii , 84.7% in stage iii , and 82% in stage iv ( p=0.045 ) , respectively . five - year pfs rates were 100% in stage i , 77% in stage ii , 64.0% in stage iii , and 58.2% in stage iv disease ( p=0.022 ) . the mean volumes of pre - rt and mid - rt gtvs were 45.9 cm ( range , 1.5 to 185.3 cm ) and 26.7 cm ( range , 1.0 to 113.8 cm ) , respectively . the mean tvrr relative to pre - rt baseline was 41.9% , ranging from 87% to 78% . tvrr did not correlate with the pre - rt gtv ( spearman s correlation coefficient , 0.041 ; p=0.511 ) . in correlation analyses between tnm stage and volumetric parameters , the meanstandard deviation ( sd ) of pre - rt gtv according to each stage was as follows : 12.66.3 cm in stage i , 29.422.5 cm in stage ii , 50.231.0 cm in stage iii , and 70.041.5 cm in stage iv ( spearman correlation coefficient , 0.562 ; p < 0.001 ) . the meansd of mid - rt gtv according to each stage was 7.14.6 cm in stage i , 13.78.6 cm in stage ii , 29.420.0 cm in stage iii , and 44.028.3 cm in stage iv ( spearman correlation coefficient , 0.583 ; p < 0.001 ) . tvrr showed a marginally significant correlation with clinical stages : 46.8%25.1% , 39.1%25.9% , 30.7%24.8% , and 30.8%21.7% , respectively ( spearman correlation coefficient , 0.138 ; p=0.082 ) . the results of the correlation analyses of the volumetric parameters according to recurrence status are shown in table 2 . patients without recurrence tended to show lower pre- and mid - rt gtvs and higher tvrr compared to patients with recurrence . as continuous variables , all volumetric parameters showed statistical significance in univariate analysis for pfs ( pre - rt gtv [ hr , 1.011 ; 95% ci , 1.004 to 1.018 ; p=0.03 ] , mid - rt gtv [ hr , 1.024 ; 95% ci , 1.013 to 1.035 ; p < 0.001 ] , and tvrr [ hr , 7.514 ; 95% ci , 2.777 to 20.329 ; p < 0.001 ] ) , but only tvrr displayed prognostic significance in multivariate analysis ( hr , 5.949 ; 95% ci , 1.090 to 32.467 ; p=0.038 ) . in roc analysis , a tvrr cut - off value of 35% showed the maximal discriminative power ( sensitivity , 66.7% ; specificity , 71.2% ) . when dichotomized by various tvrr cut - off values , the pfs rate for the higher tvrr group was higher than that of the lower tvrr group ( fig . the pfs rate of patients with tvrr > 35% was 79.2% , while that of those with tvrr 35% was 53.2% ( fig . results of univariate and multivariate analyses of the impacts of patient- and treatment - related factors on pfs are shown in table 3 . in multivariate analysis , tvrr was identified as an independently significant prognostic factor for pfs when adjusted for clinical factors ( hr , 2.877 ; 95% ci , 1.555 to 5.326 ; p=0.001 ) . the median age was 51 years , ranging from 17 to 86 years , and 94.3% of the patients were male . approximately half of the pretreatment clinical t stage was ct1 ( 77 patients , 44.7% ) , but the majority of patients ( 130 patients , 81.8% ) had metastatic lymph nodes on initial diagnosis . the distribution of tnm stage based on the american joint committee on cancer ( ajcc ) seventh edition was stage i in 15 patients ( 9.4% ) , ii in 39 ( 34.5% ) , iii in 70 ( 44.0% ) , and iva / b in 35 ( 22.0% ) . the median total radiation dose was 68.4 gy ( 66 to 72.4 gy ) , and 113 patients ( 71.1% ) received imrt . a cisplatin - based regimen was administered concurrently to all patients : cisplatin 100 mg / m every 3 weeks was administered to 111 patients ( 69.8% ) ; weekly cisplatin 20 mg / m to 23 ( 14.5% ) ; and cisplatin plus docetaxel 20 mg / m every 3 weeks to one ( 0.6% ) , respectively . after completion of rt , adjuvant chemotherapy with cisplatin plus 5-fluorouracil was administered to 34.6% of patients . the median follow - up duration for survivors was 41.5 months ( range , 11.2 to 91.8 months ) . treatment failure of any type was observed in 43 patients ( 27.0% ) , and 13 patients died during the follow - up period . as expected , patients with more advanced tnm stage had worse os and pfs : os rates at 5 years were 100% in stages i and ii , 84.7% in stage iii , and 82% in stage iv ( p=0.045 ) , respectively . five - year pfs rates were 100% in stage i , 77% in stage ii , 64.0% in stage iii , and 58.2% in stage iv disease ( p=0.022 ) . the mean volumes of pre - rt and mid - rt gtvs were 45.9 cm ( range , 1.5 to 185.3 cm ) and 26.7 cm ( range , 1.0 to 113.8 cm ) , respectively . the mean tvrr relative to pre - rt baseline was 41.9% , ranging from 87% to 78% . tvrr did not correlate with the pre - rt gtv ( spearman s correlation coefficient , 0.041 ; p=0.511 ) . in correlation analyses between tnm stage and volumetric parameters , the meanstandard deviation ( sd ) of pre - rt gtv according to each stage was as follows : 12.66.3 cm in stage i , 29.422.5 cm in stage ii , 50.231.0 cm in stage iii , and 70.041.5 cm in stage iv ( spearman correlation coefficient , 0.562 ; p < 0.001 ) . the meansd of mid - rt gtv according to each stage was 7.14.6 cm in stage i , 13.78.6 cm in stage ii , 29.420.0 cm in stage iii , and 44.028.3 cm in stage iv ( spearman correlation coefficient , 0.583 ; p < 0.001 ) . tvrr showed a marginally significant correlation with clinical stages : 46.8%25.1% , 39.1%25.9% , 30.7%24.8% , and 30.8%21.7% , respectively ( spearman correlation coefficient , 0.138 ; p=0.082 ) . the results of the correlation analyses of the volumetric parameters according to recurrence status are shown in table 2 . patients without recurrence tended to show lower pre- and mid - rt gtvs and higher tvrr compared to patients with recurrence . as continuous variables , all volumetric parameters showed statistical significance in univariate analysis for pfs ( pre - rt gtv [ hr , 1.011 ; 95% ci , 1.004 to 1.018 ; p=0.03 ] , mid - rt gtv [ hr , 1.024 ; 95% ci , 1.013 to 1.035 ; p < 0.001 ] , and tvrr [ hr , 7.514 ; 95% ci , 2.777 to 20.329 ; p < 0.001 ] ) , but only tvrr displayed prognostic significance in multivariate analysis ( hr , 5.949 ; 95% ci , 1.090 to 32.467 ; p=0.038 ) . in roc analysis , a tvrr cut - off value of 35% showed the maximal discriminative power ( sensitivity , 66.7% ; specificity , 71.2% ) . when dichotomized by various tvrr cut - off values , the pfs rate for the higher tvrr group was higher than that of the lower tvrr group ( fig . the pfs rate of patients with tvrr > 35% was 79.2% , while that of those with tvrr 35% was 53.2% ( fig . results of univariate and multivariate analyses of the impacts of patient- and treatment - related factors on pfs are shown in table 3 . in multivariate analysis , tvrr was identified as an independently significant prognostic factor for pfs when adjusted for clinical factors ( hr , 2.877 ; 95% ci , 1.555 to 5.326 ; p=0.001 ) . in this study , we investigated the usefulness of tvrr measured from simulation ct before and during adaptive rt in patients with npc . the most significant result of the present study was that tvrr was an independent prognostic factor in terms of predicting pfs in npc patients treated with definitive rt with or without concurrent chemotherapy . tvrr during the rt course has recently emerged as a prognostic factor in various malignancies including head and neck cancers . in a previous study , we reported on the prognostic impact of tvrr measured during definitive rt on loco - regional control . with a 35% cut - off value of tvrr , the 3-year loco - regional control rate was 94.4% in patients with higher tvrr , but 72.4% in those with less tvrr . yang et al . also demonstrated that tvrr was a predictor for local control in patients with oro- and hypopharyngeal cancer treated with imrt . hoeben et al . , who studied f - fluorothymidine pet - ct before and during the course of rt for head and neck squamous cell carcinoma , reported that gtv decrease above median ( 31% ) was associated with significantly better 3-year loco - regional control and disease - free survival . our results were consistent with those reported in these studies in that tvrr also influenced prognosis in npc . the other main finding from the present study was that pre- and mid - rt absolute tumor volumes had less impact on clinical outcomes than tvrr . several studies have reported association of pre - treatment tumor volume with the probability of tumor control and survival in patients with head - and - neck cancer arising in the subglottic larynx , the hypopharynx , the oropharynx , and the nasopharynx . the present study also showed that pre- and mid - rt gtv were statistically significant in univariate analysis . after adjustment for other volumetric parameters , however , these factors lost their prognostic significance ( table 3 ) . based on this result , we would speculate that tvrr has a greater prognostic value than absolute tumor volume . in addition to the prognostic role of tvrr , it could provide additional discriminative efficacy in risk group stratification during the course of definitive rt . adaptive re - planning during the rt course is an emerging issue being routinely incorporated into clinical practice at many institutions . variable degrees of volumetric and geometric changes are commonly observed during the rt course in treatment of head and neck cancer . many radiation oncologists recommend repeated ct simulation and subsequent re - planning during the rt course , as the adaptive rt may provide more optimal dose delivery to the target while decreasing the normal tissue toxicities [ 16 - 18 ] , and can even improve local control and quality of life . accordingly , adaptive rt has become essential in treatment of head - and - neck cancer patients . in the current clinical setting , volumetric parameters , which could be easily acquired , tvrr may have more than just prognostic value , and it could aid clinicians in the individualization of therapeutic strategy . most tumor responses are evaluated after completion of a definitive rt course , when it might be too late to commence an appropriate therapeutic modification . by predicting recurrence before the completion of definitive treatment an escalation of total radiation dose to a higher level than initially planned might be one example . it can be , more or less , quite easily implemented by using imrt that employs the sib technique . a second example is the indicator used in selection of candidates for intensified adjuvant chemotherapy . all of these modifications will hopefully contribute to improving clinical outcomes by early identification of patients who might not benefit from the initial treatment plan . we generated rt plans , either conventionally fractionated 3d - crt or dose - painting imrt , using the shrinking field technique . the initial rt plan was designed for 36 gy to the low risk ctv , and the subsequent plan was designed without further irradiation to this region . for this reason , the second simulation ct was performed in the third week after the initiation of rt at authors institution . one question that has arisen from the current study involves the optimal timing of the second simulation ct during the rt course . the nodal and primary tumor gtvs were reported to shrink by 1.2%-3% per treatment day on average , but their regression did not occur at a constant rate . the largest tumor volume changes occurred after the second week of the rt course and appeared to be significant after 3 - 4 weeks of rt , and could have a potential dosimetric impact when highly conformal rt techniques were used . in addition , wang et al . reported that re - planning for npc patients before the 25th fraction during imrt helps to ensure an adequate dose to the target volumes , as well as safely limited doses to the critical normal structures . a speculative conclusion that could be drawn from these studies is that the appropriate timing of the second ct , either for effective adaptive intervention or for assessment of tumor regression , might be longer than 3 weeks after the start of rt . however , if adaptive re - planning is performed in the late phase of the rt course , this advantage might decrease , as there might remain only a few fractions of rt . in the current study , most re - planning ct scans of patients were obtained between 13 to 15 fractions according to the institutional policy . it appears that this timing is quite reasonable in achieving the benefits of adaptive re - planning . second , epstein - barr virus ( ebv ) status , which is known to have prognostic importance in npc patients , was not fully addressed . again , because of the limitations of the retrospective data , tests identifying ebv were conducted in only 26 patients ( 16.4% ) . finally , there is a possibility of inter- and/or intra - physician variation in gtv measurements . despite these limitations , the discriminatory performance of the volumetric parameters could be utilized as an indicator for tailoring therapy on an individual patient basis . the current study shows that tvrr measured during the rt course is a proven independent prognostic factor predicting pfs . tvrr can be easily calculated during the process of adaptive rt by simply comparing the pre- and mid - rt simulation ct images . tvrr can provide not only prognostic information , but can also be a useful indicator for tailoring treatment strategies before the completion of definitive rt .
purposethe purpose of this study is to evaluate the prognostic significance of the tumor volume reduction rate ( tvrr ) measured during adaptive definitive radiation therapy ( rt ) for nasopharyngeal cancer ( npc).materials and methodswe reviewed the rt records of 159 npc patients treated with definitive rt with or without concurrent chemotherapy between january 2006 and february 2013 . adaptive re - planning was performed in all patients at the third week of rt . the pre- and mid - rt gross tumor volumes ( gtvs ) of the primary tumor and the metastatic lymph nodes were measured and analyzed for prognostic implications.resultsafter a median follow - up period of 41.5 months ( range , 11.2 to 91.8 months ) for survivors , there were 43 treatment failures . the overall survival and progression - free survival ( pfs ) rates at 5 years were 89.6% and 69.7% , respectively . the mean pre - rt gtv , mid - rt gtv , and tvrr were 45.9 cm3 ( range , 1.5 to 185.3 cm3 ) , 26.7 cm3 ( 1.0 to 113.8 cm3 ) , and 41.9% ( range , 87% to 78% ) , respectively . patients without recurrence had higher tvrr than those with recurrence ( 44.3% in the no recurrence group vs. 34.0% in the recurrence group , p=0.004 ) , and those with tvrr > 35% achieved a significantly higher rate of pfs at 5 years ( 79.2% in tvrr > 35% vs. 53.2% in tvrr 35% ; p < 0.001 ) . in multivariate analysis , tvrr was a significant factor affecting pfs ( hazard ratio , 2.877 ; 95% confidence interval , 1.555 to 5.326 ; p=0.001).conclusiontvrr proved to be a significant prognostic factor in npc patients treated with definitive rt , and could be used as a potential indicator for early therapeutic modification during the rt course .
Introduction Materials and Methods 1. Patient selection 2. Treatment scheme 3. Measurement of tumor volume and TVRR 4. Post-treatment follow-up 5. Statistical analysis Results 1. Patients and treatment characteristics 2. Survival outcome 3. Tumor volumetric parameters 4. TVRR as a prognostic factor Discussion Conclusion
we reviewed the medical records of consecutive npc patients treated with definitive rt with or without concurrent chemotherapy at samsung medical center between march 2006 and february 2013 . 9.2 , royal phillips electronics , miami , fl ) , and the gross tumor volumes ( gtvs ) of the primary tumor and the metastatic lymph nodes were manually contoured . three weeks after completion of rt , adjuvant chemotherapy ( 1 to 3 cycles of cisplatin+5-fluorouracil ) the primary tumor and the metastatic lymph nodes were delineated on both the pre- and mid - rt simulation ct images , and 3-dimensional tumor volumes were calculated on the rt planning system . the overall survival ( os ) duration was measured from the date of rt initiation to the date of death or last follow - up visit , and progression - free survival ( pfs ) from the date of rt initiation to the date of first recurrence of disease of any type or the last clinical follow - up . we reviewed the medical records of consecutive npc patients treated with definitive rt with or without concurrent chemotherapy at samsung medical center between march 2006 and february 2013 . 9.2 , royal phillips electronics , miami , fl ) , and the gross tumor volumes ( gtvs ) of the primary tumor and the metastatic lymph nodes were manually contoured . the primary tumor and the metastatic lymph nodes were delineated on both the pre- and mid - rt simulation ct images , and 3-dimensional tumor volumes were calculated on the rt planning system . the overall survival ( os ) duration was measured from the date of rt initiation to the date of death or last follow - up visit , and progression - free survival ( pfs ) from the date of rt initiation to the date of first recurrence of disease of any type or the last clinical follow - up . the median follow - up duration for survivors was 41.5 months ( range , 11.2 to 91.8 months ) . the mean volumes of pre - rt and mid - rt gtvs were 45.9 cm ( range , 1.5 to 185.3 cm ) and 26.7 cm ( range , 1.0 to 113.8 cm ) , respectively . as continuous variables , all volumetric parameters showed statistical significance in univariate analysis for pfs ( pre - rt gtv [ hr , 1.011 ; 95% ci , 1.004 to 1.018 ; p=0.03 ] , mid - rt gtv [ hr , 1.024 ; 95% ci , 1.013 to 1.035 ; p < 0.001 ] , and tvrr [ hr , 7.514 ; 95% ci , 2.777 to 20.329 ; p < 0.001 ] ) , but only tvrr displayed prognostic significance in multivariate analysis ( hr , 5.949 ; 95% ci , 1.090 to 32.467 ; p=0.038 ) . in multivariate analysis , tvrr was identified as an independently significant prognostic factor for pfs when adjusted for clinical factors ( hr , 2.877 ; 95% ci , 1.555 to 5.326 ; p=0.001 ) . the median follow - up duration for survivors was 41.5 months ( range , 11.2 to 91.8 months ) . the mean volumes of pre - rt and mid - rt gtvs were 45.9 cm ( range , 1.5 to 185.3 cm ) and 26.7 cm ( range , 1.0 to 113.8 cm ) , respectively . as continuous variables , all volumetric parameters showed statistical significance in univariate analysis for pfs ( pre - rt gtv [ hr , 1.011 ; 95% ci , 1.004 to 1.018 ; p=0.03 ] , mid - rt gtv [ hr , 1.024 ; 95% ci , 1.013 to 1.035 ; p < 0.001 ] , and tvrr [ hr , 7.514 ; 95% ci , 2.777 to 20.329 ; p < 0.001 ] ) , but only tvrr displayed prognostic significance in multivariate analysis ( hr , 5.949 ; 95% ci , 1.090 to 32.467 ; p=0.038 ) . in multivariate analysis , tvrr was identified as an independently significant prognostic factor for pfs when adjusted for clinical factors ( hr , 2.877 ; 95% ci , 1.555 to 5.326 ; p=0.001 ) . the most significant result of the present study was that tvrr was an independent prognostic factor in terms of predicting pfs in npc patients treated with definitive rt with or without concurrent chemotherapy . the largest tumor volume changes occurred after the second week of the rt course and appeared to be significant after 3 - 4 weeks of rt , and could have a potential dosimetric impact when highly conformal rt techniques were used .
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atrial fibrillation ( af ) is a common arrhythmia , and its prevalence is increasing as the population ages and more individuals survive with cardiovascular disease . af is the major cause of ischemic stroke and is moderately associated with increased mortality from stroke , heart failure , and cardiovascular disease . the use of anticoagulants is limited , however , due to poor patient compliance and increased risks for bleeding complications . considering these factors contributing to an insufficient success rate for stroke prevention , other treatment strategies are urgently needed to reduce the clinical and economic burden of af . rhythm control strategies have been proposed as a potential means to decrease stroke risk in af patients . anti - arrhythmic drug treatment is the standard approach to patients with af in the clinic , but adverse events commonly contribute to poor adherence . mostly , the efficacy of antiarrhythmic drug therapy ( adt ) is limited for there are high rates of af recurrence even when adt is used . , catheter ablation ( ca ) is currently more successful in maintaining sinus rhythm than antiarrhythmic drugs and has been proven superior to adt in selected patients . whether ca reduces af related stroke and death rates , however , has not been systematically reviewed . in the current study , we performed a meta - analysis of randomized controlled trials to evaluate stroke and death rates caused by af after ablation compared to drug therapy . we conducted a systematic literature search of pubmed and the cochrane central register of controlled trials covering the period from january 1990 to december 2014 by using the following search terms : randomized , ablation , atrial fibrillation , and studies were included in this meta - analysis if they met the following criteria : ( 1 ) had a random study design ; ( 2 ) the intervention group included patients ongoing catheter ablation compared with patients received antiarrhythmic drug therapy ; and ( 3 ) the endpoint was non - procedure related thromboembolic stroke / transient ischemic attack . as catheter ablation is an invasive procedure with attendant potential risks , including procedural stroke , we recorded the dates of the all - cause and procedure - related events . we excluded trials if catheter ablation was used in both treatment groups and if surgery for af was used . the study was performed according to the quorom statement for high - quality meta - analyses . information was recorded as follows : last name of the first author , year of publication , length of follow - up , mean age of study participants , sample size of each treatment , and number of end points in the intervention and control arms . in addition , left atrium size , left ventricular ejection fraction ( lvef ) , coronary artery disease ( cad ) , hypertension , chads2 score , anticoagulation time , and number of patient crossover to the ca arm in the adt arm was also recorded . we reviewed article titles and abstracts from the initial search and excluded those that did not meet the inclusion criteria . if the same population was studied in more than one study , we included the study with the longest follow - up time . the primary end point was major thromboembolic events that were not considered to be related to the treatment ( including stroke / transient ischemic attacks ) , and the secondary end point was all - cause mortality caused by af itself . the measure of treatment effect for the end point was reported by risk difference with 95% ci . fixed - effect models were used to calculate the combined risk difference and in sensitivity analyses . potential publication bias was assessed using the begg 's funnel plot and egger 's regression test . meta - regression models on stroke risk between the two groups using mean age in each study and a sensitivity analysis comparing risk of stroke by per protocol was performed to assess whether these factors would have affected the results . the effect of different follow - up periods among different trials on the heterogeneity of the pooled results was assessed by repeating the analysis using the number of strokes per 1000 patient - year . power analyses of individual studies and meta - analyses were all conducted by the software power and sample size calculation . all reported p values were two - sided , and p < 0.05 was determined as statistically significant . we conducted a systematic literature search of pubmed and the cochrane central register of controlled trials covering the period from january 1990 to december 2014 by using the following search terms : randomized , ablation , atrial fibrillation , and studies were included in this meta - analysis if they met the following criteria : ( 1 ) had a random study design ; ( 2 ) the intervention group included patients ongoing catheter ablation compared with patients received antiarrhythmic drug therapy ; and ( 3 ) the endpoint was non - procedure related thromboembolic stroke / transient ischemic attack . as catheter ablation is an invasive procedure with attendant potential risks , including procedural stroke , we recorded the dates of the all - cause and procedure - related events . we excluded trials if catheter ablation was used in both treatment groups and if surgery for af was used . the study was performed according to the quorom statement for high - quality meta - analyses . information was recorded as follows : last name of the first author , year of publication , length of follow - up , mean age of study participants , sample size of each treatment , and number of end points in the intervention and control arms . in addition , left atrium size , left ventricular ejection fraction ( lvef ) , coronary artery disease ( cad ) , hypertension , chads2 score , anticoagulation time , and number of patient crossover to the ca arm in the adt arm was also recorded . we reviewed article titles and abstracts from the initial search and excluded those that did not meet the inclusion criteria . if the same population was studied in more than one study , we included the study with the longest follow - up time . the primary end point was major thromboembolic events that were not considered to be related to the treatment ( including stroke / transient ischemic attacks ) , and the secondary end point was all - cause mortality caused by af itself . the measure of treatment effect for the end point was reported by risk difference with 95% ci . fixed - effect models were used to calculate the combined risk difference and in sensitivity analyses . potential publication bias was assessed using the begg 's funnel plot and egger 's regression test . meta - regression models on stroke risk between the two groups using mean age in each study and a sensitivity analysis comparing risk of stroke by per protocol was performed to assess whether these factors would have affected the results . the effect of different follow - up periods among different trials on the heterogeneity of the pooled results was assessed by repeating the analysis using the number of strokes per 1000 patient - year . power analyses of individual studies and meta - analyses were all conducted by the software power and sample size calculation . all reported p values were two - sided , and p < 0.05 was determined as statistically significant . thirteen randomized trials with 1952 patients were included in this review. the 13 trials were published between 2003 and 2014 . two trials were conducted at one center, whereas the rest of the trials were multicenter . one trial included patients with chronic af , while four trials included patients with paroxysmal and persistent af. , all of these trials compared pulmonary vein isolation with antiarrhythmic drug therapy . table 1 & 2 provides the baseline characteristics of the included studies . in most trials , the patients were followed for 12 months . there were no differences seen in patient characteristics . the mean age of enrolled patients was 57 9 years for ca patients and 57 11 years for adt patients . the mean left atrial diameter among patients in ca group was 41.6 5.7 mm , and in adt group was 41.9 5.9 mm . many patients had a history of hypertension , although few had significant structural heart disease . the average value of chads2 score , mentioned in four studies , were comparable ( 0.6 0.8 for ca patients and 0.7 0.8 for adt patients ) . as the data show , mean chads2 score , lvef , left atrium size , and the percentages of coronary artery disease , diabetes were comparable between the two groups . previous embolic events included transient ischemic events , stroke , pulmonary embolism , deep vein thrombosis and other peripheral embolism . in the antiarrhythmic therapy arm , drugs included mainly class i and class iii antiarrhythmic agents , either single or combination use , with the antiarrhythmic drug restricted to amiodarone in two of the trials . , in the catheter ablation group , no one had received antiarrhythmic drug therapy before enrollment in three trials . , , in the remaining trials , ca was used in patients after at least one antiarrhythmic drug regimen had failed . the ablation technique was pulmonary vein ablation combined with ablation of linear lesions in the left and right atria , ostia of the pulmonary veins , and cavotricuspid isthmus . the use of additional lesion ablations outside the pulmonary vein region was left to the discretion of the operator . oral anticoagulation ( international normalized ratio between 2 and 3 ) was required for at least three weeks before ablation in each trial , with a period ranging from 1 to 12 months after ca . in two studies , the period of anticoagulation use after ca was not noted . adt : antiarrhythmic drug therapy ; ca : catheter ablation ; nr : not reported ; tia : transient ischemic attack . the chads2 score is a measure of stroke risk in patients with atrial fibrillation , with scores ranging from 0 to 6 . congestive heart failure , hypertension , an age of 75 years or older , and diabetes mellitus were each assigned 1 point , and previous stroke or tia was assigned 2 points . adt : antiarrhythmic drug therapy ; at : minimum anticoagulation time after ablation or antiarrhythmic drug therapy ; ca : catheter ablation ; cad : coronary artery disease ; lad : left atrial diameter ; lvef : left ventricular ejection fraction ; htn : hypertension ; nr : not reported ; pee : previous embolic events ; shd : structural heart disease ; tia : transient ischemic attack . before the study finished , 445 of a total 855 drug - treated patients crossed over to a ca procedure after failure or intolerance to adt . a total of 23 in the ca arm and 26 in the adt arm had previous embolic events . ten patients were lost follow - up : four in the ablation arm and six in the aad arm . a total of seven patients in the group that underwent ca ( 0.64% ) and two patients in the adt group ( 0.23% ) had stroke or transient ischemic attack in the 13 trials . there was no difference in the rate of stroke or transient ischemic attack between the af ablation and adt therapy groups ( figure 2 ) . little evidence of heterogeneity was observed ( i = 0 , p = 0.981 ) , indicating the studies were very well - matched . the probability of potential publication bias existing among the 13 studies was estimated by begg 's funnel plots ( figure 3 ) and egger 's regression test ( p = 0.981 with 95%ci : 1.030 to 1.054 ) . meta - regression models showed there was no statistical evidence for heterogeneity due to mean age ( p = 0.95 ) . we performed a sensitivity analysis comparing risk of stroke by per protocol , no significant difference was shown between the two groups [ risk differences ( rd ) : 0.003 , 95%ci : 0.006 to 0.012 , p = 0.475 ] . a sensitivity analysis according to the follow - up years in the trials ( trial follow up > 24 months vs. < 24 months ) , also did not reveal any influence of follow - up years on trial results ( table 3 ) . similarly , no change was found in the effects of ca on stroke compared with adt ( 4.6 vs. 1.5 per 1000 patient - years in stroke rates ; rd : 0.002 , 95% ci : 0.004 to 0.008 ) ( figure 4 ) . the power of the meta - analysis with respect to stroke was 25.1% , using the risk of the medical therapy arm reported in this study . if there is a 50% reduction risk , 20,443 ca subjects with one control per case would be needed . a total of 13 deaths were reported ( five in the ablation arm and eight in the anti - arrhythmic drug arm ) . there was no difference in death rates between the ablation and anti - arrhythmic therapy groups ( figure 5 ) . the risk difference of deaths in all trials between patients randomized to ablation and those randomized to adt was 0.004 ( 95%ci : 0.014 to 0.006 , p = 0.472 ) , with no evidence for heterogeneity ( i = 0 , p = 0.953 ) . heterogeneity chi - squared = 4.15 ( d.f . = 12 ) , p = 0.981 ; i - squared ( variation in rd attributable to heterogeneity ) = 0.0 ; test of rd = 0 , z = 0.72 , p = 0.470 . adt : antiarrhythmic drug therapy ; ca : catheter ablation ; rd : risk differences . the risk difference for each study was plotted against se of rd . thirteen randomized trials with 1952 patients were included in this review. the 13 trials were published between 2003 and 2014 . two trials were conducted at one center, whereas the rest of the trials were multicenter . one trial included patients with chronic af , while four trials included patients with paroxysmal and persistent af. , all of these trials compared pulmonary vein isolation with antiarrhythmic drug therapy . table 1 & 2 provides the baseline characteristics of the included studies . in most trials , the patients were followed for 12 months . there were no differences seen in patient characteristics . the mean age of enrolled patients was 57 9 years for ca patients and 57 11 years for adt patients . the mean left atrial diameter among patients in ca group was 41.6 5.7 mm , and in adt group was 41.9 5.9 mm . many patients had a history of hypertension , although few had significant structural heart disease . the average value of chads2 score , mentioned in four studies , were comparable ( 0.6 0.8 for ca patients and 0.7 0.8 for adt patients ) . as the data show , mean chads2 score , lvef , left atrium size , and the percentages of coronary artery disease , diabetes were comparable between the two groups . previous embolic events included transient ischemic events , stroke , pulmonary embolism , deep vein thrombosis and other peripheral embolism . in the antiarrhythmic therapy arm , drugs included mainly class i and class iii antiarrhythmic agents , either single or combination use , with the antiarrhythmic drug restricted to amiodarone in two of the trials . , in the catheter ablation group , no one had received antiarrhythmic drug therapy before enrollment in three trials . , , in the remaining trials , ca was used in patients after at least one antiarrhythmic drug regimen had failed . the ablation technique was pulmonary vein ablation combined with ablation of linear lesions in the left and right atria , ostia of the pulmonary veins , and cavotricuspid isthmus . the use of additional lesion ablations outside the pulmonary vein region was left to the discretion of the operator . oral anticoagulation ( international normalized ratio between 2 and 3 ) was required for at least three weeks before ablation in each trial , with a period ranging from 1 to 12 months after ca . in two studies , the period of anticoagulation adt : antiarrhythmic drug therapy ; ca : catheter ablation ; nr : not reported ; tia : transient ischemic attack . the chads2 score is a measure of stroke risk in patients with atrial fibrillation , with scores ranging from 0 to 6 . congestive heart failure , hypertension , an age of 75 years or older , and diabetes mellitus were each assigned 1 point , and previous stroke or tia was assigned 2 points . adt : antiarrhythmic drug therapy ; at : minimum anticoagulation time after ablation or antiarrhythmic drug therapy ; ca : catheter ablation ; cad : coronary artery disease ; lad : left atrial diameter ; lvef : left ventricular ejection fraction ; htn : hypertension ; nr : not reported ; pee : previous embolic events ; shd : structural heart disease ; tia : transient ischemic attack . before the study finished , 445 of a total 855 drug - treated patients crossed over to a ca procedure after failure or intolerance to adt . a total of 23 in the ca arm and 26 in the adt arm had previous embolic events . ten patients were lost follow - up : four in the ablation arm and six in the aad arm . a total of seven patients in the group that underwent ca ( 0.64% ) and two patients in the adt group ( 0.23% ) had stroke or transient ischemic attack in the 13 trials . there was no difference in the rate of stroke or transient ischemic attack between the af ablation and adt therapy groups ( figure 2 ) . little evidence of heterogeneity was observed ( i = 0 , p = 0.981 ) , indicating the studies were very well - matched . the probability of potential publication bias existing among the 13 studies was estimated by begg 's funnel plots ( figure 3 ) and egger 's regression test ( p = 0.981 with 95%ci : 1.030 to 1.054 ) . meta - regression models showed there was no statistical evidence for heterogeneity due to mean age ( p = 0.95 ) . we performed a sensitivity analysis comparing risk of stroke by per protocol , no significant difference was shown between the two groups [ risk differences ( rd ) : 0.003 , 95%ci : 0.006 to 0.012 , p = 0.475 ] . a sensitivity analysis according to the follow - up years in the trials ( trial follow up > 24 months vs. < 24 months ) , also did not reveal any influence of follow - up years on trial results ( table 3 ) . similarly , no change was found in the effects of ca on stroke compared with adt ( 4.6 vs. 1.5 per 1000 patient - years in stroke rates ; rd : 0.002 , 95% ci : 0.004 to 0.008 ) ( figure 4 ) . the power of the meta - analysis with respect to stroke was 25.1% , using the risk of the medical therapy arm reported in this study . if there is a 50% reduction risk , 20,443 ca subjects with one control per case would be needed . a total of 13 deaths were reported ( five in the ablation arm and eight in the anti - arrhythmic drug arm ) . there was no difference in death rates between the ablation and anti - arrhythmic therapy groups ( figure 5 ) . the risk difference of deaths in all trials between patients randomized to ablation and those randomized to adt was 0.004 ( 95%ci : 0.014 to 0.006 , p = 0.472 ) , with no evidence for heterogeneity ( i = 0 , p = 0.953 ) . = 12 ) , p = 0.981 ; i - squared ( variation in rd attributable to heterogeneity ) = 0.0 ; test of rd = 0 , z = 0.72 , p = 0.470 . adt : antiarrhythmic drug therapy ; ca : catheter ablation ; rd : risk differences . the risk difference for each study was plotted against se of rd . the goal of this study was to conduct a meta - analysis to determine whether af ablation reduces the long - term risk of stroke compared to anti - arrhythmic drug therapy in randomized controlled trials . to our knowledge , this is to date the first meta - analysis to compare ca to adt for the risk of stroke and mortality induced by af . several observational research studies have suggested that af ablation strategy may associate with lower stroke and death rates compared to drug therapy. it also has been shown that the patients had long - term stroke rates similar to patients without atrial fibrillation in several studies . , one previous meta - analysis of eight randomized trials reported no significant difference between the two arms for death or stroke , although , all these studies recorded all - cause events . clearly , there are risks of stroke associated with ablation or drugs , the dilemma whether ca could reduce stroke rate of af was still unsolved . adt : antiarrhythmic drug therapy ; ca : catheter ablation ; rd : risk differences . = 12 ) , p = 0.953 ; i - squared ( variation in rd attributable to heterogeneity ) = 0.0 ; test of rd = 0 , z = 0.72 , p = 0.472 . adt : antiarrhythmic drug therapy ; ca : catheter ablation ; rd : risk differences . the potential risk of stroke in patients with af in the two groups with like age , sex , hypertension , vascular disease , and prior stroke or tia are matched well , as shown on following tables . stroke is an uncommon complication of ablation or drugs , while still having great impact on our rare end events . to find out the direct effect of ablation on stroke risk of af , we recorded events directly induced by af itself , and excluded events that were due to the procedure or drugs used . the major finding was that the two groups had similar rates of stroke and death , indicating homogeneous responses . our results provide insight into rhythm control treatment strategies , including ablative interventions that may not reduce the stroke rate on the basis that catheter ablation was more effective than drug therapy in maintaining sinus rhythm . in this meta - analysis , these findings indicate that there is not enough evidence on the relative effects between catheter ablation and anti - arrhythmic drugs . because stroke and death were rare study events , the analysis included studies which differed in the types of af , different definition of procedure - related or device - related adverse events , previous embolism events , definition for procedure - related events , the use of adt in the ca arm , and the time to anticoagulation before and after ablation . the patients included in the trials were mostly younger and may not reflect that typical age seen in patients with af . of note , there was minimal evidence of structural heart disease , the left atrial diameter was < 50 mm , a mean chad2s scores < 1 , and there was well - preserved systolic function with normal ejection fraction ( lvef > 50% ) in this cohort . thus , our results may not apply to other populations of patients , including very elderly patients or patients with underlying structural heart disease or more severe heart disease . these strengths include the absence of heterogeneity between the groups and the low probability of publication bias . despite these strengths , there are a few limitations that should be considered . most importantly , 445 of the total 855 drug - treated patients ( 52% ) crossed over to a ca procedure after failure or intolerance to adt before the end of the follow - up , and the significant crossover rate in the adt arm may contribute to the results that both groups had similar stroke rates . second , the total sample size of this meta - analysis may have been insufficient to detect significant differences between groups . more definitive evidence on the effect of ablation on af on the clinical end - points of stroke and mortality will be provided by several on - going multi - center rcts . , finally , there was a relatively short follow - up period of 12 months or less with few outcome data beyond one year . the follow - up period is a possible contributor to the lack of statistical difference observed . indeed , in one study , the stroke / tia regression models showed statistically significant different rates of events between cohorts that emerged after the first year of follow - up . a more prolonged follow - up period in a randomized trial may reveal differences not observed in the current study . in conclusion , our analysis revealed similar rates of stroke or transient ischemic attack in patients treated by catheter ablation or anti - arrhythmic drugs for atrial fibrillation . while catheter ablation did not reduce the rates of stroke or death compared to drug therapy , a large - scale clinical research trial to confirm these findings is warranted . these strengths include the absence of heterogeneity between the groups and the low probability of publication bias . despite these strengths , there are a few limitations that should be considered . most importantly , 445 of the total 855 drug - treated patients ( 52% ) crossed over to a ca procedure after failure or intolerance to adt before the end of the follow - up , and the significant crossover rate in the adt arm may contribute to the results that both groups had similar stroke rates . second , the total sample size of this meta - analysis may have been insufficient to detect significant differences between groups . more definitive evidence on the effect of ablation on af on the clinical end - points of stroke and mortality will be provided by several on - going multi - center rcts . , finally , there was a relatively short follow - up period of 12 months or less with few outcome data beyond one year . the follow - up period is a possible contributor to the lack of statistical difference observed . indeed , in one study , the stroke / tia regression models showed statistically significant different rates of events between cohorts that emerged after the first year of follow - up . a more prolonged follow - up period in a randomized trial in conclusion , our analysis revealed similar rates of stroke or transient ischemic attack in patients treated by catheter ablation or anti - arrhythmic drugs for atrial fibrillation . while catheter ablation did not reduce the rates of stroke or death compared to drug therapy , a large - scale clinical research trial to confirm these findings is warranted .
backgroundatrial fibrillation ( af ) is an independent risk factor for ischemic stroke and is associated with increased risk of death . randomized studies suggest improved quality of life for patients with af after successful catheter ablation compared to antiarrhythmic drug therapy . the value of ablation in long - term risk of ischemic stroke , however , has not been assessed . we conducted a meta - analysis to determine whether af ablation reduces the long - term risk of stroke compared to antiarrhythmic drug therapy in randomized controlled trials.methods & resultspubmed and the cochrane central register were searched for randomized trials from january 1990 to december 2014 comparing af catheter ablation to drug therapy . the results are reported as risk differences ( rds ) and 95% ci . thirteen trials were analyzed with 1097 patients treated by catheter ablation and 855 patients received antiarrhythmic drug therapy . overall , seven patients ( 0.64% ) in the catheter ablation group had ischemic stroke or transient ischemic attacks vs. two patients ( 0.23% ) in the drug therapy group . no difference was shown in the rate of stroke or transient ischemic attack between ablation and drug therapy ( rd : 0.003 , 95% ci : 0.006 to 0.012 , p = 0.470 ) , and no evidence of heterogeneity was observed ( i2 = 0 , p = 0.981 ) . no potential publication bias was found . there was also no difference in mortality between the two groups ( rd : 0.004 , 95% ci : 0.014 to 0.006 , p = 0.472).conclusionsthis meta - analysis of randomized controlled trials showed similar rates of ischemic stroke or transient ischemic attack and death in af patients undergoing catheter ablation compared to drug therapy . a larger prospective randomized trial to confirm this finding is warranted .
Introduction Methods Search strategy Selection criteria Data extraction Statistical analysis Results Study characteristics Drugs and technology Follow-up and withdrawals End points Discussion Study limitations Conclusions
in the current study , we performed a meta - analysis of randomized controlled trials to evaluate stroke and death rates caused by af after ablation compared to drug therapy . we conducted a systematic literature search of pubmed and the cochrane central register of controlled trials covering the period from january 1990 to december 2014 by using the following search terms : randomized , ablation , atrial fibrillation , and studies were included in this meta - analysis if they met the following criteria : ( 1 ) had a random study design ; ( 2 ) the intervention group included patients ongoing catheter ablation compared with patients received antiarrhythmic drug therapy ; and ( 3 ) the endpoint was non - procedure related thromboembolic stroke / transient ischemic attack . we conducted a systematic literature search of pubmed and the cochrane central register of controlled trials covering the period from january 1990 to december 2014 by using the following search terms : randomized , ablation , atrial fibrillation , and studies were included in this meta - analysis if they met the following criteria : ( 1 ) had a random study design ; ( 2 ) the intervention group included patients ongoing catheter ablation compared with patients received antiarrhythmic drug therapy ; and ( 3 ) the endpoint was non - procedure related thromboembolic stroke / transient ischemic attack . a total of seven patients in the group that underwent ca ( 0.64% ) and two patients in the adt group ( 0.23% ) had stroke or transient ischemic attack in the 13 trials . there was no difference in the rate of stroke or transient ischemic attack between the af ablation and adt therapy groups ( figure 2 ) . little evidence of heterogeneity was observed ( i = 0 , p = 0.981 ) , indicating the studies were very well - matched . we performed a sensitivity analysis comparing risk of stroke by per protocol , no significant difference was shown between the two groups [ risk differences ( rd ) : 0.003 , 95%ci : 0.006 to 0.012 , p = 0.475 ] . similarly , no change was found in the effects of ca on stroke compared with adt ( 4.6 vs. 1.5 per 1000 patient - years in stroke rates ; rd : 0.002 , 95% ci : 0.004 to 0.008 ) ( figure 4 ) . the risk difference of deaths in all trials between patients randomized to ablation and those randomized to adt was 0.004 ( 95%ci : 0.014 to 0.006 , p = 0.472 ) , with no evidence for heterogeneity ( i = 0 , p = 0.953 ) . a total of seven patients in the group that underwent ca ( 0.64% ) and two patients in the adt group ( 0.23% ) had stroke or transient ischemic attack in the 13 trials . there was no difference in the rate of stroke or transient ischemic attack between the af ablation and adt therapy groups ( figure 2 ) . little evidence of heterogeneity was observed ( i = 0 , p = 0.981 ) , indicating the studies were very well - matched . we performed a sensitivity analysis comparing risk of stroke by per protocol , no significant difference was shown between the two groups [ risk differences ( rd ) : 0.003 , 95%ci : 0.006 to 0.012 , p = 0.475 ] . similarly , no change was found in the effects of ca on stroke compared with adt ( 4.6 vs. 1.5 per 1000 patient - years in stroke rates ; rd : 0.002 , 95% ci : 0.004 to 0.008 ) ( figure 4 ) . the risk difference of deaths in all trials between patients randomized to ablation and those randomized to adt was 0.004 ( 95%ci : 0.014 to 0.006 , p = 0.472 ) , with no evidence for heterogeneity ( i = 0 , p = 0.953 ) . the goal of this study was to conduct a meta - analysis to determine whether af ablation reduces the long - term risk of stroke compared to anti - arrhythmic drug therapy in randomized controlled trials . in conclusion , our analysis revealed similar rates of stroke or transient ischemic attack in patients treated by catheter ablation or anti - arrhythmic drugs for atrial fibrillation . while catheter ablation did not reduce the rates of stroke or death compared to drug therapy , a large - scale clinical research trial to confirm these findings is warranted . a more prolonged follow - up period in a randomized trial in conclusion , our analysis revealed similar rates of stroke or transient ischemic attack in patients treated by catheter ablation or anti - arrhythmic drugs for atrial fibrillation . while catheter ablation did not reduce the rates of stroke or death compared to drug therapy , a large - scale clinical research trial to confirm these findings is warranted .
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human genomic dna is maintained in intimate contact with proteins that confer the structural integrity of chromosomes . other proteins associate intermittently with dna to affect its repair , replication , and transcription . these reactions are favored under oxidative conditions , and the production of covalent dna protein cross - links ( dpcs ) is enhanced by exposure of cells to diverse agents including chemical oxidants , ionizing radiation ( ir ) , ultraviolet radiation ( uv ) , reactive aldehydes , or chemotherapeutic drugs ( reviewed in ref ( 1 ) ) . sequestration of proteins required for dna repair , replication , or transcription is likely to impair these important functions . in addition , dpcs are large dna adducts that block dna replication and physically impede dna - related processes . the thiopurine 6-thioguanine ( 6-tg ) is among the therapeutic agents that promote dpc formation . 6-thioguanine nucleotides , the end product of the metabolism of 6-tg and of the anticancer immunosuppressants azathioprine and 6-mercaptopurine ( 6-mp ) , are substrates for incorporation into dna . additionally , exposure of cultured human cells to 6-tg or 6-mp depletes their antioxidant defenses and increases steady - state levels of reactive oxygen species ( ros ) . patients treated with thiopurines experience skin photosensitivity and have a significantly increased risk of developing skin cancer . dna 6-tg can act both as a type i and type ii uva photosensitizer ( reviewed in ref ( 9 ) ) . in the type i mode , extremely reactive purine radical cations or purine thiyl radicals are generated following uva activation of dna 6-tg . as a type ii sensitizer , dna 6-tg interacts with uva in the presence of molecular oxygen to generate singlet oxygen ( o2 ) , a form of ros that is particularly damaging to proteins . these include oxidized forms of dna 6-tg ( guanine sulfinate ( g ) and guanine sulfonate ( g ) ) and guanine ( 8-oxo-7,8-dihydroguanine ) as well as dna single- and double - strand breaks and dna interstrand cross - links ( icls ) . the combination of dna 6-tg and uva also induces protein damage in the form of carbonyls and oxidized thiols . it causes oxidation - related cross - linking between the subunits of multiprotein complexes including the pcna , ku , rpa , and mcm27 dna replication / repair complexes and between dna and proteins . importantly , protein damage induced by 6-tg+uva is associated with a significant attenuation of dna repair capacity . ciprofloxacin is a member of the fluoroquinolone family of antibiotics that are uva photosensitizers . protein damage by ciprofloxacin+uva also includes oxidation and cross - linking between subunits of dna replication and repair complexes and is associated with impaired dna repair . to our knowledge , the possible we previously demonstrated the formation of heat- and reducing agent - resistant dpcs between oligonucleotides containing oxidized 6-tg ( g ) and the amino or thiol groups of oligopeptides . the same study also presented preliminary evidence for the formation of dpcs in vivo in cultured human cells treated with 6-tg and exposed to low doses of uva radiation . immunoblotting identified dna repair proteins among the cross - linked species from cells treated with 6-tg and uva . specifically , the pcna dna repair / replication protein that is known to be susceptible to oxidation was identified along with msh2 and xpa , essential components of the dna mismatch repair and nucleotide excision repair pathways , respectively . the presence of these important dna repair factors in dpcs suggested that the obligatory , albeit transient association of dna repair proteins with dna might make them particularly vulnerable to inactivation by dna cross - linking . we have developed a sensitive and statistically rigorous approach to identifying cross - linked proteins . based on stable isotope labeling with amino acids in cell culture ( silac ) and mass spectrometry ( ms ) , the method is generally applicable to dna damaging treatments . here we describe the application of this proteomics - based technique to analyze in detail dpc formation by 6-tg treatment and by the uva activation of dna 6-tg in human cells . we also report the application of the same approach to examine dpc induction by uva activation of ciprofloxacin , a representative of a family of non dna - embedded uva photosensitizers . ccrf - cem cells were routinely grown in rpmi 1640 medium ( thermo fisher ) supplemented with 10% dialyzed fetal calf serum . for silac , growth medium was supplemented with a combination of either 100 mg per liter of light ( n , c ) or heavy ( n , c ) lysine and arginine ( ck isotopes ) . following growth for 7 d in this medium , full labeling of proteins was confirmed by ms ( data not shown ) . the drug ( 3 mm ) was included in the medium for 6 h prior to and during growth in 6-tg . dna was extracted , and dna 6-tg incorporation was quantified as described previously . cells were uva irradiated in phosphate buffered saline using a uvh 253 lamp ( uv light technology limited ) with maximum emission at 365 nm and a dose rate of 0.1 kj m s. neither photosensitizer treatment nor uva irradiation reduced cell viability , whereas photosensitizer+uva combinations were highly lethal . ros were determined by facs using cm - h2dcfda ( invitrogen ) as previously described . following treatment , 10 isotopically labeled control / treated cells were mixed and nuclei prepared by resuspension in 200 l of 10 mm tris - hcl ph 7.4 , 2.5 mm mgcl2 , 0.5% np40 , 1 mm dithiothreitol ( dtt ) and were harvested by centrifugation . chromatin was released from the nuclear pellet by resuspension in 25 mm na phosphate , ph 7.4 , 5 mm mgcl2 , 500 mm nacl , 0.5% triton , 1 mm edta , 1 mm dtt , 10% glycerol plus protease inhibitors . the chromatin pellet was washed three times by resuspension in the same buffer and then sheared by sequential passage through 19 g , 25 g , and 27 g needles ( 20 each ) . sheared chromatin samples containing 10 g of dna were applied to a hybond - n membrane using a slot blot apparatus ( ge healthcare ) . dna was cross - linked to the membrane by uvc irradiation from a stratalinker ( stratagene ) and was then washed extensively with 8 m urea ( sigma - aldrich ) and water . membrane - bound proteins were reduced with 10 mm dtt at 50 c for 30 min and alkylated by treatment with 55 mm iodoacetamide for 30 min at room temperature in the dark . the alkylation reaction was stopped by incubation with 10 mm dtt for 10 min at room temperature . following three washes with 10 mm triethylammonium bicarbonate ( teab ) , the proteins were digested by immersing the membrane in trypsin ( 12.5 ng/l ) overnight at 37 c . dtt , iodoacetamide , and trypsin were all prepared in 10 mm teab . for ms analysis of total cell lysates , whole - cell extracts were prepared with ripa buffer ( 50 mm tris - hcl ph 7.5 , 150 mm nacl , 0.1% sds , 0.5% na deoxycholate , 1% triton , and protease inhibitors ) . twenty micrograms of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) and stained with colloidal coomassie ( instant blue , expedeon ) . gel bands were excised and trypsin digested using a perkinelmer janus liquid handling system . tryptic peptides were analyzed by liquid chromatography - mass spectrometry ( lc ms ) using an ultimate 3000 uhplc system connected to either a q - exactive or orbitrap velos pro mass spectrometer ( thermo fisher scientific ) and acquired in data - dependent mode . the data were searched against human uniprot ( uniprot kb2012_08 taxonomy human 9606 canonical with contaminants 20 120 921 ) using the andromeda search engine and maxquant ( version 1.3.0.5 ) . for maxquant , a false discovery rate of 0.1% the data were uploaded into perseus version 1.4.0.11 ( maxquant ) for statistical analyses . chromatin extracts ( 20 g of protein ) were separated on 10% polyacrylamide gels ( invitrogen ) and transferred to hybond - n membranes . following washing with urea and water , membranes were probed with antibodies against msh2 , msh6 , pcna ( santa cruz ) , and rpa70 ( abcam ) . growth of ccrf - cem cells in the presence of 6-tg ( 0.30.9 m ) resulted in the thiopurine replacing around 0.050.6% of dna guanine . when dna was prepared from a standard number of 6-tg treated cells by the wizard ( promega ) extraction protocol , the amount recovered declined in a 6-tg concentration - dependent manner . irradiation of cells containing dna 6-tg with a modest dose of uva ( 50 kj / m ) further exacerbated dna losses . the effects of 6-tg and uva were synergistic , and uva alone had no detectable effect on dna recovery . the wizard extraction protocol involves a protein precipitation step prior to dna harvesting and the inclusion of a proteinase k digestion step prior to dna precipitation restored quantitative dna yields ( supplementary figure s1a ) . the reduced dna recovery from cells treated with 6-tg or 6-tg+uva was dependent on the presence of 6-tg in dna . the wizard dna purification protocol yielded quantitative dna recovery from 6-tg treated gm03467 lesch - nyhan cells without the inclusion of the protease digestion step ( data not shown ) . these cells do not express hypoxanthine - guanine phosphoribosyltransferase and can not scavenge 6-tg for incorporation into dna . in addition , quantitative dna yields were achieved without the additional protease digestion step if 6-tg incorporation into dna was prevented by treatment of ccrf - cem cells with 6-tg in the presence of hydroxyurea ( supplementary figure 1a ) . facs analysis ( supplementary figure 1b ) confirmed that exclusion of 6-tg from dna also reduced uva - induced ros levels . their dependence on protease digestion suggested that dna yields were reduced by cross - linking of dna to protein . this possibility was investigated further using chromatin from treated cells . to selectively enrich for proteins covalently attached to dna , chromatin extracted from 6-tg+uva treated ccrf - cem cells was washed extensively with high salt ( 500 mm nacl ) to deplete noncovalently associated proteins . sheared , salt - washed chromatin was applied to a hybond - n membrane that was then sequentially washed with 8 m urea and water . staining with syproruby and sybr green confirmed that extensive urea washing removed all detectable membrane - associated proteins from untreated chromatin while having no noticeable impact on the amount of bound dna . subsequent probing of the filter with a panel of antibodies confirmed that uva induced a 6-tg dose - dependent increase in the amount of the xpa , pcna , msh2 , and rpa70 ( the 70 kda subunit of the rpa single strand dna binding complex ) dna repair / replication proteins associated with the washed membrane ( figure 1a , b ) . ( a ) sheared , salt - washed chromatin from ccrf - cem cells that were untreated or treated with 6-tg and 50 kj / m uva as indicated was applied to hybond - n membranes . membrane - associated protein and dna was visualized by staining with sypro ruby and sybr green . . sheared chromatin from untreated ccrf - cem cells or cells treated with uva , 6-tg or 6-tg+uva was applied to hybond - n membranes . following extensive washing with water and 8 m urea , membranes were probed with antibodies as indicated . the observation that dna replication / repair proteins are enriched in hybond - n membrane - bound chromatin prompted us to undertake a comprehensive analysis of the proteome associated with hybond - n - bound dna to provide an unbiased screen for proteins involved in dpcs . the protocol is outlined in figure 2 , panel a. outline of silac analysis . ( a ) ccrf - cem cells were labeled with heavy or light arginine and lysine isotopes . half the cells were treated with 0.9 m 6-tg for 24 h and the other half left untreated . half of each of these two cultures was then irradiated with 50 kj / m uva . cells were mixed in 1:1 ratios for preparation of chromatin or whole cell extracts ( ripa ) as indicated . chromatin extracts were applied to a hybond - n membrane that was water and 8 m urea washed prior to in situ trypsin digestion and ms analysis . ripa extracts were subjected to short sds - page and in - gel trypsin digestion prior to ms analysis . briefly , ccrf - cem cells labeled with heavy or light isotopes of arginine and lysine were treated with 6-tg ( 0.9 m ) . half of each culture was then irradiated with uva ( 50 kj / m ) . chromatin was prepared from a total of 16 1:1 mixes of heavy and light isotope labeled cells that had been treated with 6-tg , uva , 6-tg+uva , or left untreated . the compositions of these mixes are shown in figure 2 , panel b. high salt - washed chromatin mixtures were loaded onto a hybond - n membrane that was extensively washed with water and 8 m urea . remaining membrane - associated proteins a total of 2611 proteins were identified in two independent experiments ( forward and reverse labeling analyses ) of hybond - n membrane bound chromatin from uva , 6-tg , or 6-tg+uva treated ccrf - cem cells ( supporting information ) . because the analysis was carried out with chromatin prepared from mixtures of heavy and light labeled cells , changes in the log2 heavy / light ( h : l ) protein ratios reveal an enrichment of proteins associated with membrane - bound dna . supplementary figure s2a presents the protein distribution for mix 1 in which the chromatin applied to the membrane was prepared from mixtures of untreated heavy- and untreated light - labeled cells . the tight symmetrical clustering of log2 h : l ratios around the zero value in the histogram confirms the expected equal representation of heavy and light labeled proteins . because > 99.5% of log2 values for these untreated cells lie between 1 and + 1 , in the subsequent analysis of treated chromatin we considered values that fall outside this range ( representing > 2-fold enrichment ) to be significant treatment - related changes that reflect dpc formation . comparison of mixtures of heavy 6-tg - treated / light untreated cells and heavy untreated / light 6-tg - treated cells ( mixes 3 and 9 ) revealed the effects of 6-tg treatment . supplementary figure s2b , c shows that 6-tg treatment shifted the membrane - associated protein distribution in the direction of the label in the treated cells . in the scatter plot ( figure 3a ) , 6-tg - induced asymmetry in log2 h : l ratios results in the majority of the data points occupying the lower right quadrant , a shift consistent with dpc formation . the effect was small , however , and only reached significance for approximately 10% ( 7 and 16% in two determinations ) of the detected proteins , which indicated that 6-tg - induces a low level of dna protein cross - linking . ( a ) the effect of 6-tg treatment ( 6-tg vs untreated ) . mix 3 versus mix 9 ( from figure 2b ) . ( d ) the additional effect of uva on 6-tg treated cells ( 6-tg+uva vs 6-tg ) . the calculated absolute values for log2 h : l ratio shown confirm that 6-tg+uva induces a significant change . they also validate synergy between 6-tg and uva . ( e ) heat map of log2 h : l ratio intensities of identified proteins . mix numbers refer to those in figure 2 , panel b. mixes of h- and l - labeled cells that received the same treatment are shown in bold . the moderate uva dose we used ( 50 kj / m ) did not induce detectable dna protein cross - linking , and the log2 h : l ratios for the comparison of mixes 2 and 5 ( figure 3b ) that addresses the effect of uva remain tightly clustered around the origin of the scatter plot . analysis of mixes 4 and 13 ( figure 3c ) revealed that the combination of 6-tg and uva caused extensive dna protein cross - linking . by comparing mixes 12 and 15 , we specifically examined the effect of uva on cells treated with 6-tg . in the absence of synergy between 6-tg and uva , the log2 h : l ratios would cluster around the origin of the scatter plot as they do for samples from cells treated with uva alone . figure 3 , panel d and supplementary figure s2d , e confirm that uva induces extensive dpc formation in cells treated with 6-tg . dpc induction by 6-tg , uva , and combined 6-tg+uva is summarized in the heat map in figure 3 , panel e. hierarchical cluster analysis reveals a family of proteins ( arrowed ) that appeared to be largely unaffected by any of the treatments and represent a set of false positives . subsequent analysis of the cross - linking profiles of these proteins ( see below ) confirmed their absence of susceptibility to cross - linking by either 6-tg or 6-tg+uva . in summary , silac analysis demonstrates that 6-tg induces a low level of dpcs in ccrf - cem cells . it also reveals that 6-tg and uva combine synergistically to cause extensive dpc formation . the ms analysis revealed changes in log2 h : l ratios that indicate decreases in protein yield from cells treated with 6-tg and 6-tg+uva relative to that from untreated cells ( the upper left quadrant of the scatter plots in figure 3 ) . while these changes are consistent with dpc formation , we considered two alternative mechanisms that might contribute to this protein underrepresentation . if the inhibition is sufficiently severe , it could reduce overall cellular protein content . an alternative ( and not exclusive ) possibility is that the acknowledged insolubility of oxidized proteins might contribute to diminished protein recovery . to investigate these eventualities , we compared the hybond - n membrane - bound proteome with proteins in unfractionated extracts . cultures of isotopically labeled ccrf - cem cells that had been treated with uva+6-tg , 6-tg alone , or uva alone were combined with an equal number of untreated reverse labeled cells , and the mixture was divided into two equal parts . chromatin extracted from one aliquot of cells was bound to a hybond - n membrane and processed for ms as described above . the remaining cells were used to prepare a dna - free whole cell extract using a standard ( ripa ) extraction procedure . trypsin digests of these whole cell extracts were compared with those of the corresponding hybond - n proteins . figure 4 , panels a and b show the effect of 6-tg treatment ( mixes 3 and 9 ) . scatter plots of log2 silac h : l ratios comparing the effects of treatments on chromatin and ripa ( whole cell ) extracts . mean values for log2 h : l ratios ripa extract mixes are presented on each panel . the vertical shifts in log2 h : l ratios from the origin of the scatter plot in the direction of the label of the chromatin from 6-tg - treated cells confirm that 6-tg induces dpcs . the small changes in mean log2 h : l ratio for the ripa extract proteins ( mix 3 = 0.31 ; mix 9 = 0.15 ) indicate that yields are largely unaffected by 6-tg treatment . in contrast , ripa extracts are enriched for proteins from untreated cells relative to those from cells treated with 6-tg+uva ( figure 4c , d ) yielding significant changes in mean log2 h : l values ( mix 4 = 1.15 ; mix 13 = 0.81 ) . as expected , uva did not affect protein recovery ( mean log2 h : l ratios 0.19 and 0.16 for mixes 2 and 5 , respectively ) . since neither 6-tg nor uva alone significantly influenced protein recovery , the changes in protein abundance in extracts from cells treated with 6-tg+uva can be ascribed to their combined effect . it follows that interference with transcription / translation during prolonged ( 24 h ) 6-tg treatment does not have a significant impact on protein yield . we conclude that the diminished protein recovery from 6-tg+uva treated cells most likely reflects depletion due to protein dna cross - linking allied to losses resulting from precipitation of proteins oxidized by 6-tg+uva . the observation that treatment with 6-tg+uva causes a measurable reduction in protein recovery indicates that silac analysis may slightly underestimate the extent of dpc formation by this combination . to identify proteins that were most susceptible to dna cross - linking , we used r combined with ggplot2 to determine and visualize the 95th ( for heavy - labeled treated cells ) and fifth ( for light - labeled treated cells ) percentile values of log2 h : l ratio changes for each of the 16 analyses . these values are shown in figure 5 , panel a along with a plot that provides a graphic representation of the predicted maximal effect of uva , 6-tg , or 6-tg+uva on dna protein cross - linking . cross - linking profile of ccrf - cem chromatin proteins most vulnerable to dpc formation . ( a ) 95th ( treated heavy - labeled ) or 5th ( treated light - labeled ) percentile values for log2 h : l ratios are presented and plotted for each of the 16 comparisons . ( b ) the cross - linking profile of the 200 proteins that best fit the profile in panel a is shown superimposed on the profile for all 2611 detected proteins ( in gray ) . ( c ) protein ontology of the best fit 200 proteins in panel b , as specified by uniprot . ( d ) cross - linking profiles of the 114 potential false positive proteins ( figure 3e ) . we used the perseus program ( maxquant ) to identify 200 proteins that best fit this profile . in figure 5 , panel b , the cross - linking profile of these proteins ( red ) is superimposed on that of all other identified proteins ( gray ) . figure 5 , panels a and b confirm that both 6-tg and 6-tg+uva induce cross - linking . of the 200 selected proteins , 192 are either predominantly nuclear or have been detected in the nucleus ( uniprot / genecards ) . around one - third of these proteins are involved in gene expression , and dna repair / replication comprises the next largest category ( 16% , figure 5c ) . dna and rna binding proteins are equally represented ( figure 5c ) and together account for about half of the 200 selected . since 6-tg is incorporated extensively into rna and uva cross - links protein to rna containing a photoreactive purine analog , some of these rna binding proteins may be present as rna - protein cross - links . direct measurements ( data not shown ) indicated that rna accounted for < 2% of the total nucleic acid in our chromatin preparations . we therefore rule out a significant contribution from rna - protein cross - links , and we conclude that these rna processing proteins are most likely present in dpcs . the cross - linking profiles for the 114 proteins identified as potential false positives by cluster analysis ( figure 3e ) are shown in figure 5 , panel d and their categorization in figure 5 , panel e. cross - linking analysis confirms that none of the treatments increased their representation in dpcs . these include particularly high abundance leading to high background and lower probability of detecting treatment - related dpc formation , a high affinity for dna , or strong direct binding to the hybond - n membrane independently of dna . consistent with these possibilities , histones ( see below ) and the highly abundant intermediate filament protein vimentin ( pi = 5 ) were among the 114 false positives . the intimate association of dna repair and replication proteins with dna is expected to increase their vulnerability to dna cross - linking . among 179 dna repair proteins ( http://sciencepark.mdanderson.org/labs/wood/dna_repair_genes.html ) , most matched the expected profile for 6-tg and 6-tg+uva dependent cross - linking ( figure 6a ) . analysis of the 30 best fits to the generic cross - link profile ( supplementary table s1 ) is shown in figure 6 , panel b. a similar analysis of whole cell ( ripa ) extracts of mixes 14 ( figure 2b ) indicated that most of these dna repair proteins were underrepresented to some degree in cells treated with 6-tg+uva ( figure 6c ) . this observation is consistent with a significant depletion of dna repair proteins by cross - linking to dna . ( a ) cross - linking profiles for the 52 detected designated dna repair proteins . ( b ) cross - linking profiles for the 30 most vulnerable dna repair proteins . ( c ) log2 silac h : l ratio plots for whole cell ripa extracts for the 30 most vulnerable dna repair proteins indicating that the majority are significantly depleted . the cross - linking profiles for the 125 kda catalytic ( pold1 ) and the 50 kda pold3 auxiliary subunits ( figure 7a , c ) were good fits to the generic profile of figure 5 , panel a confirming their vulnerability to dna cross - linking . these profiles suggest that pold1 is susceptible to cross - linking in cells treated with either 6-tg alone or 6-tg+uva , whereas pold3-dna cross - linking appears to be predominantly photochemical and requires both 6-tg and uva . although the 66 kda pold2 subunit was among the chromatin proteins identified by ms analysis , its profile deviated significantly from the generic plot suggesting that it is less susceptible to cross - linking . cross - linking profiles for : ( a ) dna polymerase delta 125 kda , ( b ) 60 kda , and ( c ) 55 kda subunits ; ( d ) mcm27 proteins ; ( e ) topoisomerases 1,2a and 2b . it comprises the mcm2-mcm7 proteins that assemble on dna as a circular hexamer to initiate replication . their cross - link profiles were essentially superimposable ( figure 7d ) indicating a shared vulnerability to cross - linking in cells treated with 6-tg or with 6-tg+uva . topoisomerases relieve supercoiling by cleaving dna ahead of the transcription or replication apparatus . this essential function is performed by the major human topoisomerases top1 and top2a , 2b . they were susceptible to dna cross - linking , and like pold3 , they also appeared to be particularly susceptible to photochemical cross - linking to dna containing 6-tg ( figure 7e ) . consistent with depletion due to dpc formation , the pold subunits , mcm proteins , and topoisomerases were all present in significantly reduced levels in whole cell extracts following 6-tg+uva treatment ( supplementary figure s3 ) . silac - hybond - n membrane binding was used to investigate dpc formation by uva - activated ciprofloxacin . treatment with 500 m ciprofloxacin and 50 kj / m uva induced significant dna protein cross - linking in ccrf - cem cells ( figure 8b ) as indicated by the clustering of most of the data for the comparison of mixes 2 and 3 in the lower right quadrant of the scatter plot . among the 2269 cross - linked proteins we identified , the representation of dna and rna binding proteins was closely similar to that generated by 6-tg+uva ( figure 8c ) . forty - one dna repair proteins ( http://sciencepark.mdanderson.org/labs/wood/dna_repair_genes.html ) were identified in ciprofloxacin+uva treated samples . of these , 29 fit the profile expected for dpc induction ( > 2-fold change ) ( supplementary table s1 , figure 8d ) . among ciprofloxacin+uva cross - linked dna repair proteins , there was a highly significant ( p < e ) overlap with dna repair proteins cross - linked by 6-tg+uva treatment ( figure 8e ) . hierarchical cluster analysis ( supplementary figure s4 ) confirmed ciprofloxacin+uva induced dpc formation and again revealed a group of candidate false - positive proteins . the cross - linking profiles of the 68 candidates confirmed that they were not present in treatment - related dpcs ( data not shown ) . histones were highly represented in this group , and there was significant overlap with false positives identified following 6-tg+uva treatment ( supplementary figure s4 ) . heavy- or light - labeled ccrf - cem cells were treated with 500 m ciprofloxacin ( cip ) for 1 h and uva ( 50 kj / m ) as indicated . salt - washed chromatin prepared from cells mixed in 1:1 ratios as indicated was applied to a hybond - n membrane . following washing with water and 8 m urea , ( c ) dna and rna binding proteins among cirofloxacin+uva ( green ) and 6-tg+uva ( blue ) . ( d ) cross - linking profiles for 29 dna repair proteins that best fit the most vulnerable profile . ( e ) overlap between 29 dna repair proteins cross - linked by ciprofloxacin+uva and the 30 cross - linked by 6-tg+uva . we have devised a silac and proteomics - based method to investigate dpc formation by photosensitizer / uva combinations and describe its application to human cells treated under conditions that mimic and amplify the clinical effects of photosensitizing medications . dpc induction by formaldehyde has been investigated by a similar approach that employs a modified chip technique . dna cross - linking by specific subsets of proteins has also been investigated using biotinylated double - stranded oligonucleotides , specific recognition sequences inserted into genomic dna in vivo , and immunoprecipitation of cross - linked proteins or trimethylated histones . many of these studies have employed silac or other labeling methods to quantify cross - linking . to our knowledge , selective enrichment of dpcs by hybond - n membrane binding has not previously been combined with silac - lc ms analysis . the approach identified more than 2000 cellular proteins that were cross - linked to dna by 6-tg , by 6-tg+uva , or by ciprofloxacin+uva . among the proteins most susceptible to cross - linking , most are nuclear and are involved in control of gene expression or dna repair / replication . this distribution is consistent with incorporated dna 6-tg or dna - bound or -intercalated ciprofloxacin acting as the predominant sources of the photochemical ros that drive dpc formation . one important aspect of our study is that it addresses cross - linking targeted to dna containing a reactive center ( 6-tg and 6-tg+uva ) as well as protein cross - linking to canonical dna constituents ( ciprofloxacin+uva ) . photoactivation of a site - specific dna- or rna - embedded thionucleobase , including 6-tg , has been used extensively to probe nucleic acid structure and protein nucleic acid cross - linking . uva - induced cross - linking of oligopeptides or purified proteins to 6-tg - containing oligonucleotides has also been used to model protein dna interactions in vitro , and we have presented preliminary evidence from 2d - dige and isopycnic density gradient analysis for dpc formation in cells treated with 6-tg and uva . the present study describes an unbiased and quantitative investigation into dpc formation in human cells by uva - activated photosensitizers . a modest level of dna substitution by 6-tg ( around 0.05% of dna guanine ) in ccrf - cem cells was sufficient to reduce dna recovery unless the dna was treated with a protease prior to precipitation during purification . protease - reversible dna losses during purification were exacerbated by exposure of the 6-tg - treated cells to 50 kj / m uva , a dose that had no detectable effect on dna recovery in the absence of prior 6-tg treatment . proteomic analysis confirmed the synergistic induction of dpcs by 6-tg and 50 kj / m uva radiation . 6-tg is an atypical photosensitizer because its incorporation into dna introduces highly reactive dna thiol groups through which dpcs may form preferentially . in contrast , dpcs induced by uva - activated ciprofloxacin , which is not incorporated into dna , must involve canonical dna components . our analysis revealed extensive dna protein cross - linking by ciprofloxacin+uva indicating that the approach is likely to be generally applicable to dpc analysis . thiopurines like 6-tg perturb the cellular redox balance and increase the concentrations of intracellular ros including superoxide anion ( o2 ) , h2o2 , and ultimately via metal - catalyzed reactions , the highly damaging hydroxyl radical ( oh ) , which is a possible source of dpcs . we have previously shown that icl formation in cells treated with 6-tg requires both ros and incorporated dna 6-tg . dpc induction shares these requirements , and dna - embedded 6-tg is a key participant in dna lesch - nyhan cells were invulnerable to dpc induction by either 6-tg alone or 6-tg+uva , and both treatments were without effect in ccrf - cem cells in which replication was inhibited to prevent the accumulation of dna 6-tg . on the basis of this requirement , we consider it unlikely that reactions between protein nucleophiles and oxidized dna guanine are a significant source of dpcs mediated by 6-tg or 6-tg+uva . oligopeptide cross - linking involving g , which is a good leaving group in nucleophilic substitution reactions , and peptide sh or nh2 groups . nucleophilic attack by proteins at dna g generated in the ros - rich environment is a possible mechanism for 6-tg - mediated dpc formation . cross - linking between a free protein nh2 group and a dna 6-tg radical cation or a thiyl radical generated by oxidation of dna 6-tg is a possible alternative reaction . ros generated during 6-tg treatment cause widespread protein oxidation , and dpcs may also form via the reaction of oxidized proteins with dna 6-tg . the more extensive photochemical cross - linking by 6-tg+uva most likely involves type ii photosensitization . like dna 6-tg since proteins are susceptible to o2-mediated oxidation , reactions between oxidized proteins and canonical dna constituents are likely to be a significant factor in dpc formation by 6-tg+uva and ciprofloxacin+uva . consistent with a common etiology , more than 75% of the dna repair proteins that were identified as highly vulnerable to cross - linking by ciprofloxacin+uva were among similar proteins identified in 6-tg+uva dpcs . members of the group containing the large catalytic pold1 subunit of dna polymerase and the mcm proteins were efficiently cross - linked by both 6-tg and 6-tg+uva , whereas cross - linking of proteins typified by the smaller pold3 and the topoisomerases appeared to be largely photochemical and depended on 6-tg+uva . these different cross - linking behaviors may reflect different cross - linking chemistries or may simply be related to the positioning of the proteins on dna . in the case of protein multimers such as dna pol or mcm27 that can also form intersubunit protein protein cross - links , their presence in dpcs might also reflect dna cross - linking of covalent protein transient dpc formation is a feature of many dna processing enzymes , and interference with the correct reversal of topoisomerase dna complexes is the basis of the therapeutic action of drugs such as camptothecin and etoposide . these specific enzyme - related dpcs are , however , atypical , and dpcs are likely to be structurally heterogeneous . most studies of the induction , processing , and biological effects of dpcs have used formaldehyde , a highly reactive molecule that causes protein damage , depletes cellular reduced glutathione levels , and induces icls as well as dpcs . the possible involvement of nucleotide excision repair , homologous recombination , and the proteasome in dpc reversal in human cells has been suggested but is not firmly established ( see ref ( 42 ) for review ) . fanconi anemia cells are extremely sensitive to formaldehyde , and evidence from mice and from chicken cells with defective aldehyde metabolism suggests a requirement for this dna repair pathway to repair aldehyde - induced dpcs . whether sprtn , the human homologue of putative dpc - specific proteases that has been identified in yeast and xenopus laevis , participates in the same or in a separate repair pathway remains to be determined . like formaldehyde , 6-tg+uva and ciprofloxacin+uva ( and most other treatments that cause dna protein cross - linking ) induce other dna lesions as well as dpcs and protein damage . this pleiotropy may have hampered attempts to define dpc repair pathways in human cells . among the putative systems for dpc repair , we identified essential ner , homologous recombinational repair , and fanconi pathway proteins as particularly susceptible to dna cross - linking . this observation raises the possibility that dpc repair might be compromised by depletion or oxidation of the repair proteins themselves . because the non - dpc damage induced by uva activated photosensitizers is unlikely to be the same as that caused by aldehydes , uva / photosensitizer combinations may be a useful addition to studies of dpc induction and repair in human cells .
long wavelength ultraviolet radiation ( uva , 320400 nm ) interacts with chromophores present in human cells to induce reactive oxygen species ( ros ) that damage both dna and proteins . ros levels are amplified , and the damaging effects of uva are exacerbated if the cells are irradiated in the presence of uva photosensitizers such as 6-thioguanine ( 6-tg ) , a strong uva chromophore that is extensively incorporated into the dna of dividing cells , or the fluoroquinolone antibiotic ciprofloxacin . both dna - embedded 6-tg and ciprofloxacin combine synergistically with uva to generate high levels of ros . importantly , the extensive protein damage induced by these photosensitizer+uva combinations inhibits dna repair . dna is maintained in intimate contact with the proteins that effect its replication , transcription , and repair , and dna protein cross - links ( dpcs ) are a recognized reaction product of ros . cross - linking of dna metabolizing proteins would compromise these processes by introducing physical blocks and by depleting active proteins . we describe a sensitive and statistically rigorous method to analyze dpcs in cultured human cells . application of this proteomics - based analysis to cells treated with 6-tg+uva and ciprofloxacin+uva identified proteins involved in dna repair , replication , and gene expression among those most vulnerable to cross - linking under oxidative conditions .
Introduction Materials and Methods Results Discussion
human genomic dna is maintained in intimate contact with proteins that confer the structural integrity of chromosomes . these reactions are favored under oxidative conditions , and the production of covalent dna protein cross - links ( dpcs ) is enhanced by exposure of cells to diverse agents including chemical oxidants , ionizing radiation ( ir ) , ultraviolet radiation ( uv ) , reactive aldehydes , or chemotherapeutic drugs ( reviewed in ref ( 1 ) ) . additionally , exposure of cultured human cells to 6-tg or 6-mp depletes their antioxidant defenses and increases steady - state levels of reactive oxygen species ( ros ) . as a type ii sensitizer , dna 6-tg interacts with uva in the presence of molecular oxygen to generate singlet oxygen ( o2 ) , a form of ros that is particularly damaging to proteins . the same study also presented preliminary evidence for the formation of dpcs in vivo in cultured human cells treated with 6-tg and exposed to low doses of uva radiation . the presence of these important dna repair factors in dpcs suggested that the obligatory , albeit transient association of dna repair proteins with dna might make them particularly vulnerable to inactivation by dna cross - linking . here we describe the application of this proteomics - based technique to analyze in detail dpc formation by 6-tg treatment and by the uva activation of dna 6-tg in human cells . the effect was small , however , and only reached significance for approximately 10% ( 7 and 16% in two determinations ) of the detected proteins , which indicated that 6-tg - induces a low level of dna protein cross - linking . the moderate uva dose we used ( 50 kj / m ) did not induce detectable dna protein cross - linking , and the log2 h : l ratios for the comparison of mixes 2 and 5 ( figure 3b ) that addresses the effect of uva remain tightly clustered around the origin of the scatter plot . in the absence of synergy between 6-tg and uva , the log2 h : l ratios would cluster around the origin of the scatter plot as they do for samples from cells treated with uva alone . these values are shown in figure 5 , panel a along with a plot that provides a graphic representation of the predicted maximal effect of uva , 6-tg , or 6-tg+uva on dna protein cross - linking . we have devised a silac and proteomics - based method to investigate dpc formation by photosensitizer / uva combinations and describe its application to human cells treated under conditions that mimic and amplify the clinical effects of photosensitizing medications . among the proteins most susceptible to cross - linking , most are nuclear and are involved in control of gene expression or dna repair / replication . uva - induced cross - linking of oligopeptides or purified proteins to 6-tg - containing oligonucleotides has also been used to model protein dna interactions in vitro , and we have presented preliminary evidence from 2d - dige and isopycnic density gradient analysis for dpc formation in cells treated with 6-tg and uva . dpc induction shares these requirements , and dna - embedded 6-tg is a key participant in dna lesch - nyhan cells were invulnerable to dpc induction by either 6-tg alone or 6-tg+uva , and both treatments were without effect in ccrf - cem cells in which replication was inhibited to prevent the accumulation of dna 6-tg . consistent with a common etiology , more than 75% of the dna repair proteins that were identified as highly vulnerable to cross - linking by ciprofloxacin+uva were among similar proteins identified in 6-tg+uva dpcs . in the case of protein multimers such as dna pol or mcm27 that can also form intersubunit protein protein cross - links , their presence in dpcs might also reflect dna cross - linking of covalent protein transient dpc formation is a feature of many dna processing enzymes , and interference with the correct reversal of topoisomerase dna complexes is the basis of the therapeutic action of drugs such as camptothecin and etoposide . like formaldehyde , 6-tg+uva and ciprofloxacin+uva ( and most other treatments that cause dna protein cross - linking ) induce other dna lesions as well as dpcs and protein damage .
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effective health initiatives in the nineteenth century and also most of the infrastructural measures in public health are signs of the recognition of such association between health and social status . in fact , most of the substantial reductions in mortality rates of infectious diseases , e.g. tuberculosis , happened before introduction of modern and effective medical treatments . to be exact , such declines emanated from improvements in people s living conditions and food baskets ( 1 ) . it is shown that from different determinants of health , social determinants account for approximately 50% of health differences ( 2 ) . constitution of world health organization ( who ) whose draft was written in 1946 also shows that along with taking challenges of medical care and effective treatments into account , who founders were also to tackle social roots of health problems . they believed that health is a complete physical , mental and social wellbeing and not only the absence of disease and infirmity ( 3 ) . although social health has been one of three staples of every definition of health , its concept bears no resemblance to physical and mental dimensions of health . its unique feature is that it can be thought of a society and an individual characteristic simultaneously . as russell put it , on one hand a society is healthy when everyone has equal opportunities and access to basic goods and services , something which leads to high performance of citizens ( 4 ) . in fact , law abiding , equality in wealth , public participation in decision making and social capital can be indicators of a healthy society ( 4 ) . on the other hand , individualistic social health points to wellbeing of an individual which is , in turn , related to the quality of living and interacting with other people and also quality of reactions to social institutions and conventions ( 4 ) . for the first time , belloc and breslow ( 1971 ) took a scientific and practical approach to study social health . they defined it as degree of members function in a society and then made up a social health index accordingly . they asked some questions about social , mental and physical dimensions of health to figure out members function ( 5 ) . couple of years later , cathy donald and colleagues ( 1978 ) proposed a new concept again . their reasoning was that , health is something beyond just reporting disease symptoms , rates and individual functional abilities . they believed that wellbeing is a matter apart from physical and mental health . according to their perception , social health is both a health basis and also a subject of that ( 6 ) . considering different definitions and approaches , it seems that social health can be defined in three ways : social health as a social dimension of an individual s health , along with physical and mental health , which is concerned with being in a relationship with society . a healthy society as pro - health social conditions and finally as a better social status generally that according to each society s situations , can have different objective meanings and examples . indices help us determine the degree of changes ( 7 ) . from different indices which are used to measure social health , the following can be mentioned ; fordham social health index , which measures some different variables in different age groups and also some variables regardless of age . they are child poverty , child abuse and infant mortality in childhood ; suicide , drug abuse , high school drop - out and teenage pregnancy in adolescence ; unemployment , income and health care coverage in adulthood ; poverty and life expectancy at 65 in old age and finally violent crime , alcohol - induced mortality , affordable house and income inequality in all ages ( 8) . ontario healthy communities coalition index that attributes these traits to a healthy population : clean and safe environment , peace , equity and social justice , adequate access to food , water , shelter , income , security , work and recreation , adequate access to health care , opportunity for learning and developing skills , strong and supportive relations and networks , supportive work environments for family and individual wellbeing , extensive participation of dwellers in decision making , cultural and spiritual heritage , varied and dynamic economy , protection of natural environment and responsible consumption of resources that assures their sustainability ( 9 ) . north carolina population health workgroup concept that believes a healthy population should enjoy security , affordable house ; accessible transportation system , job security , healthy and safe environment , sustainable ecosystem and accessible and prevention - focused health care ( 10 ) . and finally , healthy village concept of who eastern mediterranean office that includes clean and safe material environment , social coordination , openness to experiences , interactions and various relationships , protection and promotion of cultural and historical heritage , appropriate and accessible health care , economic variation and originality and sustainable utilization of available resources ( 11 ) . in iran , rafiey and colleagues for the first time conducted a delphi survey to construct a national concept for social health and then an acceptable index for it ( 12 ) . since rafiey and colleagues concept has no empiric backing , we aimed to put that in an empiric trial . present study is an ecologic and correlational study in which iran s provinces ( 30 provinces in 2007 ) were the study units . in rafiey and colleagues study ( delphi method ) , there were five rounds of comment and feedback between researchers and iranian social health experts . at first , 63 experts were chosen as delphi members . out of these initial participants , there were two criteria to choose participants : 1 ) scientific scholarship and reliability in health and social issues and 2 ) experience in health and social issues . the participants expertise ranged from economics , development , management and epidemiology to social medicine , psychiatry , social and clinical psychology and social work . the first category contained those traits on which most delphi participants agreed . according to these traits , a healthy society is a society in which 1 ) there is no one under poverty line , 2 ) there is no violence and 3 ) population growth is under control . however , whenever any difference could not be found in the first category , the second category can be added to concept of a healthy society ; thus , a healthy society is a society in which , in addition to existing aforementioned traits in first category , 4 ) there is no gender discrimination , 5 ) all of citizens are the same in the eyes of law ( in enforcing and being supported by law ) , 6 ) human rights treaty and other related treaties are established , 7 ) education is free and compulsory until secondary school , and is free thereafter , 8) there is universal access to health care , 9 ) security is assured , 10 ) liberty of conscience is granted , and 11 ) people are satisfied with their lives . the third category expresses further traits of an ideal healthy society ; a healthy society is a society in which , as well as the existence of previous traits , 12 ) there is universal insurance coverage , 13 ) there is equitable distribution of income ( everybody is given his / her rights and there is equal opportunity in income earning ) , 14 ) there is no unemployment , 15 ) there is no racial , ethnic and regional discrimination , 16 ) government is legitimate , 17 ) democracy is the only way of electing governors , and finally 18 ) governors are supervised democratically . in the present study , in order to provide an empirical confirmation for social health concept , 6 indicators were chosen from above 18 indicators . there were three criteria to choose these indicators ; inclusion of first category traits , availability and accessibility of required and reliable data in the country ( for all provinces ) , and convertibility to quantitative data . from this , in our study a healthy society is a society in which ; 1 ) nobody is under poverty line , 2 ) there is no violence , 3 ) population growth is under control , 4 ) education is free and compulsory until secondary school , and is free thereafter , 5 ) there is universal insurance coverage , and finally 6 ) there is no unemployment . in this study , required data for population growth was extracted from 2006 national census and also 2007 population estimates . violence data ( willful murder , assault & battery ) was drawn from iranian police force statistics . data for poverty ( proportion of food expenditure to non - food expenditure ) was taken from a national study on household budget in 2006 , published by iranian central bank . to better expression , more elaboration on the concept and definition of each indicator is provided . absolute poverty means not having access to standards of life i.e. adequate food , house and clothing . relative poverty means that a person is not able to reach a particular level of living standards which is being known as necessary and desired in the society . in the present study , proportion of food expenditure to non - food expenditure was used as a measure of poverty where the higher the share of food expenditure to non - food expenditure , the higher the degree of poverty ( 13 ) . 2 ) violence ; although it seems easy to define violence but like other social issues there is no any consensus on it . as corporal punishment is the simplest type of violence , early definitions were on corporal punishment ; violence is the expression of physical or verbal force against self or others , forceful action against one s will bearing hurt ( 14 ) . who has defined it as the intentional use of physical force or power , threatened or actual , against oneself , another person , or against a group or community which either leads to injury or has a high likelihood of injury , death , psychological harm , maldevelopment , or deprivation ( 15 ) . in the present study , by violence we mean willful murder , assault and battery which is defined as ; willful murder ; killing by will and authority so that a person deliberately , consciously and premeditatedly takes the lives of others whether the deed is typically deadly or not ( 16 ) . assault & battery ; causing maim and mayhem whether it is intentional or unintentional . if trauma does not result in bleeding even with inner fracture it would be assault ( 16 ) . 3 ) population growth ; population means a group of individuals who dwell in a specified area ( village , city , county , province or a country ) continually and normally in households which share a unique political stand and national and ethnic features ( 17 ) . natural growth rate ( ngr ) demonstrates increase or decrease in population number , exclusive of immigration , which can be computed only by raw mortality and morbidity rates ( formula 1 ) and represents that how many people will be added to population annually per 1000 person . formula : ngr = mortality - birthmean population1000 4 ) literacy : a literate person is somebody who can read and write a simple text in persian or in other languages , whether he / she has an official credential or not . in this study , literacy rate has been used to measure literacy status in provinces ( formula 2 ) . formula : literacy rate = number of literate personsnumber of6 and over six years old persons1000 5 ) insurance coverage : in the present study insurance coverage is the number of people who were under the coverage of social security organization ( including public , private , arbitrary , occupational drivers and special insurance ) at the end of 2006 . 6 ) unemployment : in our study an unemployed is somebody who does not have any paid job or salaried job and is not self - employed , is ready to work and is a job seeker ( has tried to find a paid job or salaried job or put up a self - employed job ) . unemployment rate is the proportion of the unemployed population to the active population ( 18 ) . this should be noted that due to some reasons we could not include gini index and assault & battery rates in our study . assault & battery rate for the reason that there was a reverse relationship between it and willful murder unexpectedly ( r= 0.248 , p= 0.09 ) . this is maybe because reports of assault & battery cases were not precise in some provinces . the reason for exclusion of gini index was that again contrary to expectations this index had no correlation with other indices ( for example with poverty ; r= 0.007 , p= 0.48 and with unemployment r=0.005 and p= 0.48 ) . range restriction was our interpretation of this issue i.e. whenever a variable has a narrow variance this variable has to be called a constant rather a variable . in order to prove the construct validity and obtain a social health index , an exploratory factor analysis was conducted on six indicators of population growth , willful murder , poverty , unemployment , insurance coverage and literacy . factor analysis investigates that whether a number of observed correlated variables are related to a smaller number of unobserved variables called factors . factors are latent constructs that influence observed variables and factor analysis discovers the nature of these constructs . as indicators had different units of measurement and in order to equalize the units , z scores was measured for each indicator : formula : z = x where x is the mean of sample , is the mean of population and is the standard deviation of population . and finally , to calculate the value of social health index in each province the following formula was used ; formula : ishie = zlit+zins.cov(zngr+zpov+zmurd+zunemp ) where ishie stands for empirical iranian social health index and z , lit , ins.cov , ngr , pov , murd and unemp denote the standardized rate , literacy , insurance coverage , natural growth rate , poverty and unemployment respectively . in our study , all of analyses were done in spss software ( version 17 ) . as table 1 shows tehran had the best status in the index of proportion of food expenditure to non - food expenditure ( poverty ) . it indicates that 80 percent of tehran s households income is spent on non - food expenditure and they already crossed basic needs level . also , as it was expected sistan & baluchistan had the highest rates of violence and poverty and lowest rates of literacy and insurance coverage . plus , hormozgan had the lowest rate of population natural growth . iran s provinces are ranked based on their social health index value in table 2 . as this table illustrates , tehran , semnan , esfahan , boushehr and mazandaran had the highest social health index values whereas sistan & baluchistan , lorestan , ilam , kohkiloyeh and kermanshah had the lowest social health index values . table 3 shows some information on capability of variables ( indicators ) in construction of factors . kaiser- meyer- olkin ( kmo ) measure of sampling adequacy tests the amount of variance in data which can be explained by factors bartlett s test of sphericity is used to test the hypothesis that the variables ( indicators ) in correlation matrix are correlated . in this test a p - value lower than 0.05 indicates that there are significant relationships between variables and it is possible to find out latent structures in data . considering values of kmo ( 0.66 ) and bartlett s test ( p - value<0.0001 ) in table ( 4 ) , our data had this capability to make up factors and reveal a latent construct that we call it social health . following the factor analysis , factors were extracted . as figure 1 illustrates , eigenvalue of two factors were more than 1 , something which means that these two factors could explain the highest amount of variance in data . as it can be seen , variables of natural growth rate , proportion of food to non - food expenditure , insurance coverage and literacy rate were loaded on the first factor , and variables of willful murder and unemployment rate were loaded on second factor . totally , these two factors explained 68.25 percent of social health variance ( 48.65 percent by the first factor and 19.60 percent by second one ) . the researchers named the first and second factors as diathesis and problem respectively . in the present study , we tried for the first time to investigate empirically the concept of social health in iran . according to factor analysis in our study , an iranian healthy society can be defined as a society that is stout with less deviance and problem , i.e. with more and more literate and insured people , controlled growth rate and less and less willful murder , poverty and unemployment . there are some differences and similarities between this concept and concepts of other studies . as it can be seen in following sentences , although the measures are somehow different but most of the indices of social health , used in other parts of the world , use indicators similar to our study : literacy and education : availability of learning opportunities ( ontario index ) ( 9 ) ; high school drop - out ( fordham index ) ( 8) ; insurance / health : adequate access to health care ( ontario index ) ( 9 ) ; prevention - focused and accessible health care ( north carolina index ) ( 10 ) ; accessibility and appropriateness of health care / access to basic health care ( eastern mediterranean index ) ( 11 ) ; infant mortality rate and health care coverage in adults ( fordham index ) ( 8) . poverty : adequate access to food , water , shelter , income , security , job and recreation by public ( ontario index ) ( 9 ) ; poverty in children and elderly ( fordham index ) ( 8) ; murder : security ( ontario , north carolina , eastern mediterranean ) ( 9 , 10 , 11 ) ; violent crime ( fordham index ) ( 8) . unemployment : adequate access to job by public ( ontario index ) ( 9 ) ; job for seekers ( north carolina ) ( 10 ) ; adult unemployment ( fordham index ) ( 8) . following substantial reduction in mortality rates in 1950s , iran had experienced a booming population growth . the number of its population had tripled in 30 years and mounted to 50 million in 1980s . now iran has one of the youngest populations in the world and is experiencing a transient fertility phase ( 19 ) . however , the importance of population growth in iran s social health index can have some policy implications . its fertility rate fell from 7.0 births per woman in 1979 to 1.9 births per woman in 2006 ( 21 ) . according to our index of social health , iran has to stick firmly with its current family planning programs to make a healthy society and any change in population policies should be made carefully . due to its young population , the employment has been one of the most challenging matters for iran in recent years . as our study also shows , the unemployment is a matter of focus for health experts too . we have to give more weight to employment if we are to improve social health . as statistics says 12.3% of iranian people are unemployed and this matter is worse in marginalized parts and groups of the country ( 22 ) . although the government has put its stamina on this matter , but the problem somehow remains . home business and easy start - up business are initiatives that is hoped to work in some ways . however , there are more investments needed . there is a strong relationship between poverty and sustainable development and social welfare in a country . there is no doubt that in a country with higher rates of poverty , it would be really hard to make improvements in other determinants of social health and consequently in social health . unemployment , illiteracy , uncontrolled population growth and crime are the inevitable companions of poverty . iran has had some plans to decrease its poverty rate and release more people off poverty trap . according to statistics , targeted subsidies plan that is being performed in iran is capable of making remarkable changes in peoples lives and improve poverty rates in iran . according to a well - known model ( ecologic model ) of violence , biological , social , cultural , economic and political factors all however , it is believed that social factors are the most important determinants of violence ( 25 ) . as it was revealed in our study violence has a strong association with health in its various dimensions ( physical , mental and social ) ( 26 ) . due to some cultural issues however , as violence is one of the iran s social health components , in the first place it might be better to devise some initiatives to get access to better violence data . there is no doubt that the more people of a country are literate , the better they are in obtaining and protecting health . program is referred as one of the effective ways to improve literacy worldwide . however , the literacy rate is still low in some regions and among some groups of iranian people , especially women . it might be the reason why experts chose the literacy as a component of social health in iran . according to data , 85% of iranian people are literate ( 23 ) . enrichment of home schooling and literacy movement initiatives in marginalized parts of the country , like rural regions , are of ways that can improve literacy rate in iran . about 10 percent of people in iran are under no insurance coverage ( 27 ) . it has been performed in rural settings from 2004 and has some fruitful and promising achievements ( 28 ) . it is now planned to extend to urban areas and cover all of iranian population . improvement of social health can be one of its results . from the above - stated matters , it can be drawn that social health concept is a dynamic and fluid concept . hence , in different societies depending on their achievements and situations and in different times there would be different concepts of social health . so , it can be suggested that there should be a redefinition of concept every 5 or 10 years in every country . indeed , we intended to include more indicators of social health on which experts agreed in rafiey and colleagues study . it seems that by entering more indicators into factor analysis , stronger empirical evidence will be provided . considering importance of poverty , violence , literacy , insurance coverage , population growth and unemployment in formation of a health society , these factors should be consistent parts of iran s social health policy agenda . above that , as most of these factors are beyond the health sector , a strong intersectoral collaboration is needed to achieve that healthy society . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .
background : as social health is a condition - driven , dynamic and fluid concept , it seems necessary to construct and obtain a national and relevant concept of it for every society . providing an empirical back up for iran s concept of social health was the aim of the present study.methods:this study is an ecologic study in which available data for 30 provinces of iran in 2007 were analyzed . in order to prove construct validity and obtain a social health index , an exploratory factor analysis was conducted on six indicators of population growth , willful murder , poverty , unemployment , insurance coverage and literacy.results:following the factor analysis , two factors of diathesis ( made up of high population growth , poverty , low insurance coverage and illiteracy ) and problem ( made up of unemployment and willful murder ) were extracted . the diathesis and problem explained 48.6 and 19.6% of social health variance respectively . from provinces , sistan & baluchistan had the highest rate of poverty and violence and the lowest rate of literacy and insurance coverage . in terms of social health index , tehran , semnan , isfahan , bushehr and mazandaran had the highest ranks while sistan and baluchistan , lurestan , kohkiloyeh and kermanshah occupied the lowest ones.conclusion:there are some differences and similarities between iranian concept of social health and that of other societies . however , a matter that makes our concept special and different is its attention to population . the increase in literacy rate and insurance coverage along with reduction of poverty , violence and unemployment rates can be the main intervention strategies to improve social health status in iran .
Introduction Materials and Methods Results Discussion Conclusion Ethical considerations
considering different definitions and approaches , it seems that social health can be defined in three ways : social health as a social dimension of an individual s health , along with physical and mental health , which is concerned with being in a relationship with society . in iran , rafiey and colleagues for the first time conducted a delphi survey to construct a national concept for social health and then an acceptable index for it ( 12 ) . present study is an ecologic and correlational study in which iran s provinces ( 30 provinces in 2007 ) were the study units . according to these traits , a healthy society is a society in which 1 ) there is no one under poverty line , 2 ) there is no violence and 3 ) population growth is under control . however , whenever any difference could not be found in the first category , the second category can be added to concept of a healthy society ; thus , a healthy society is a society in which , in addition to existing aforementioned traits in first category , 4 ) there is no gender discrimination , 5 ) all of citizens are the same in the eyes of law ( in enforcing and being supported by law ) , 6 ) human rights treaty and other related treaties are established , 7 ) education is free and compulsory until secondary school , and is free thereafter , 8) there is universal access to health care , 9 ) security is assured , 10 ) liberty of conscience is granted , and 11 ) people are satisfied with their lives . in the present study , in order to provide an empirical confirmation for social health concept , 6 indicators were chosen from above 18 indicators . formula : literacy rate = number of literate personsnumber of6 and over six years old persons1000 5 ) insurance coverage : in the present study insurance coverage is the number of people who were under the coverage of social security organization ( including public , private , arbitrary , occupational drivers and special insurance ) at the end of 2006 . in order to prove the construct validity and obtain a social health index , an exploratory factor analysis was conducted on six indicators of population growth , willful murder , poverty , unemployment , insurance coverage and literacy . and finally , to calculate the value of social health index in each province the following formula was used ; formula : ishie = zlit+zins.cov(zngr+zpov+zmurd+zunemp ) where ishie stands for empirical iranian social health index and z , lit , ins.cov , ngr , pov , murd and unemp denote the standardized rate , literacy , insurance coverage , natural growth rate , poverty and unemployment respectively . also , as it was expected sistan & baluchistan had the highest rates of violence and poverty and lowest rates of literacy and insurance coverage . plus , hormozgan had the lowest rate of population natural growth . as this table illustrates , tehran , semnan , esfahan , boushehr and mazandaran had the highest social health index values whereas sistan & baluchistan , lorestan , ilam , kohkiloyeh and kermanshah had the lowest social health index values . as it can be seen , variables of natural growth rate , proportion of food to non - food expenditure , insurance coverage and literacy rate were loaded on the first factor , and variables of willful murder and unemployment rate were loaded on second factor . in the present study , we tried for the first time to investigate empirically the concept of social health in iran . with more and more literate and insured people , controlled growth rate and less and less willful murder , poverty and unemployment . there are some differences and similarities between this concept and concepts of other studies . as it can be seen in following sentences , although the measures are somehow different but most of the indices of social health , used in other parts of the world , use indicators similar to our study : literacy and education : availability of learning opportunities ( ontario index ) ( 9 ) ; high school drop - out ( fordham index ) ( 8) ; insurance / health : adequate access to health care ( ontario index ) ( 9 ) ; prevention - focused and accessible health care ( north carolina index ) ( 10 ) ; accessibility and appropriateness of health care / access to basic health care ( eastern mediterranean index ) ( 11 ) ; infant mortality rate and health care coverage in adults ( fordham index ) ( 8) . however , the importance of population growth in iran s social health index can have some policy implications . there is no doubt that in a country with higher rates of poverty , it would be really hard to make improvements in other determinants of social health and consequently in social health . from the above - stated matters , it can be drawn that social health concept is a dynamic and fluid concept . considering importance of poverty , violence , literacy , insurance coverage , population growth and unemployment in formation of a health society , these factors should be consistent parts of iran s social health policy agenda .
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the exponential demand for energy is evidenced by dwindling fossil fuel supplies and record - high oil and gas prices due to global population growth and economic development ( fig . this energy shortage has significant implications to the future of our society for example , in order for 10 billion people to sustain their current lifestyle with their current energy consumption , we need a minimum of ten additional terawatts ( tws ) , an equivalent of 150 millions of barrels of oil per day ( 150 m boe / day ) , until the year 2050 . the energy crisis is further exacerbated by major concerns about global warming from greenhouse gas emissions due to increasing fossil fuel consumption [ 6 - 8 ] . ) at this large scale , solar energy seems to be the most viable choice to meet our clean energy demand . the sun continuously delivers to the earth 120,000 tw of energy , which dramatically exceeds our current rate of energy needs ( 13 tw ) . this implies that covering only 0.1% of the earth s surface with solar cells of 10% efficiency would satisfy our current energy needs ; however , the energy currently produced from sunlight remains less than 0.1% of the global energy demand ( fig . the major barrier for the large - scale use of solar energy is the high cost and inadequate efficiencies of existing solar cells . the best commercial solar cells based on single - crystal silicon are about 18% efficient . these conventional p - n junction cells , so - called first - generation devices , suffer from the high cost of manufacturing and installation . the second - generation devices consisting of cuingase2 ( cigs ) polycrystalline semiconductor thin films can reduce the price significantly , but it does not reduce the challenge to make their efficiencies more practical . now the third - generation solar cells , such as dye - sensitized solar cells ( dsscs ) , bulk heterojunction cells [ 17 - 19 ] , and organic cells , are promising for inexpensive and large - scale solar energy conversion ( table 1 ) ; however , laboratory dsscs based on cheap dye sensitization of oxide semiconductors are typically less than 10% efficient , and those based on even cheaper organic materials are 25% efficient . nanostructured semiconductors , organic - inorganic hybrid assemblies , and molecular assemblies present new opportunities to design such third - generation light energy conversion devices . considerable efforts have been devoted to the development of more efficient photoanode materials , such as ordered mesostructured materials and one - dimensional ( 1-d ) nanostructures ( nanowires , nanotubes , and nanorods ) [ 22 - 24 ] . examples of 1-d nanostructures include highly ordered tio2 nanotube arrays synthesized with anodization of ti foils , zno nanowire arrays synthesized with aqueous solutions , and carbon nanotube ( cnt ) and si nanowire arrays synthesized with chemical vapor deposition ( cvd ) [ 23 - 34 ] . bandgap - tunable semiconductor zero - dimensional ( 0-d ) nanomaterials , such as cds [ 35 - 37 ] , pbs , bi2s3 , cdse , and inp quantum dots ( qds ) , have demonstrated extraordinary optical and electronic properties that open up possibilities for revolutionary advances in photovoltaic ( pv ) devices . the combination of 1-d with 0-d nanostructures is attracting more interests from the solar cell community . assembly methods for these hybrid nanostructures include wet - chemistry processes through chemical functionalization [ 43 - 47 ] and dry routes through the electrostatic - force - directed assembly . in particular , 1-d nanostructures are promising for photovoltaic devices due to several performance and processing benefits , such as a direct path for charge transport and large surface areas for light harvest offered by the geometry of such nanostructures . for example , the mobility of electrons in 1-d nanostructures is typically several orders of magnitude higher than that in semiconductor nanoparticle films commonly used in dsscs ( table 2 , data from [ 23,50 - 54 ] ) . this review addresses some of the current research issues in the field of new solar cells based on 1-d semiconductor nanostructures , and hybrids of 1-d nanomaterials and dye molecules or qds . these solar cells include dsscs with tio2 nanotube or zno nanowire arrays as photoanodes in lieu of nanoparticle networks , solar cells with tunable - bandgap qds supported by wide - bandgap semiconductor nanotube / nanowire or cnt arrays , and solar cells with coaxial p - n junctions in vertically - aligned 1-d nanostructures or p - n junctions between the 1-d nanostructure and the substrate . we summarize recent advances and discuss the performance and properties of these innovative solar energy harvesting and conversion devices . comparison of electron mobilities ( cm v s ) of 1-d nanomaterials and nanoparticles field - effect and ; intrinsic mobility of a 300 nm long and 3.9 nm diameter single - walled carbon nanotube ( swcnt ) ; 830 nm wide si nanowires ; 20 nm wide ge nanowires ; 1617 m long and 130200 nm wide zno nanowire ; for particle size around 4 nm ; for 48 nm anatase tio2 nanocrystals ; it is 15 cm v s in single crystal tio2 a dye - sensitized solar cell ( dssc ) is a type of photoelectrochemical ( pec ) solar cell which has been studied extensively [ 55 - 57 ] . in a dssc , dye molecules are used to sensitize wide - bandgap semiconductors , such as tio2 and zno , which assist in separating electrons from photo - excited dye molecules . the visible light absorbed by dye molecules is more intensive than the uv light absorbed by wide - bandgap semiconductors for solar radiation , even if the energy of visible light is lower than that of uv light . the sensitization of wide - bandgap semiconductors by adsorbed monolayers of dye molecules began in the late 1960s with the work of gerishcer and memming . a conceptual and practical breakthrough occurred in the late 1980s when grtzel and coworkers started using high - surface - area semiconductors for dsscs [ 16,60 - 63 ] . a schematic representation of a dssc is shown in fig . its working principle is : ( 1 ) the incident photon is absorbed by the dye molecule adsorbed on the surface of nanocrystalline tio2 particles and an electron from the molecular ground state s is excited to an excited state s * ; ( 2 ) the excited electron of the dye is injected into the conduction band of the tio2 particles , leaving the dye molecule to an oxidized state s ; ( 3 ) the injected electron percolates through the porous nanocrystalline structure to the transparent conducting oxide ( tco ) layer of the glass substrate ( negative electrode or anode ) and finally through an external load to the counter electrode ( positive electrode or cathode ) ; ( 4 ) at the counter electrode , the electron is transferred to the triiodide ( ) in electrolyte to yield iodide ( i ) ; ( 5 ) the cycle is closed through reducing the oxidized dye by the iodide in the electrolyte . however , there is no net chemistry in terms of chemicals created or destroyed in the device , so the cell is regenerative . the working principle of a dye - sensitized nanostructure solar cell ( adapted from ref . ) the anodes of dsscs are typically constructed with a thin film ( ~10 m ) of wide bandgap semiconductor nanoparticles involving sno2 , zno , or tio2[68 - 74 ] . the nanoparticle film provides a large surface ( ~1000 times higher than the geometrical area of the electrode ) for absorption of light - harvesting molecules ( usually ruthenium - based dyes ) ; however , electrons are usually trapped by isolated nanoparticles , surface states , or defect states . the so - called trap effects slow down the electron transport through diffusion and limit the device efficiency , which has been proved by time - resolved photocurrent and photovoltage measurements and modeling studies . under full sunlight , the average injected electron may experience a million trapping events before either being collected by the electrode or recombining with an oxidizing species . for example , electron transport in crystalline wires is expected to be several orders of magnitude faster than percolation through a random polycrystalline network ( table 2 ) . the zno nanowire array of high surface area was synthesized using a simple two - step process in aqueous solutions . briefly , a 1015-nm - thick film of zno quantum dots was deposited onto fluorine - doped tin oxide ( fto ) conductive glass substrates by dip coating , and wires were then grown from these nuclei through thermal decomposition of a zinc complex . individual zno nanowire resistivity varied from 0.3 to 2.0 cm , with an electron concentration of 15 10 cm and mobility ( ) of 15 cm v s. using the einstein relation , d = kbt/e , an electron diffusivity dn = 0.050.5 cm s for a single nanowire can be estimated . compared with zno nanoparticles ( dn 10 cm s ) , the nanowire array anode can collect charge carriers much more effectively by introducing highly ordered architectures . law et al . showed that the injection process in nanowires is complete after ~5 ps , but continues for ~100 ps in the nanoparticles . the new cell exhibited a short - circuit current density jsc = 5.35.85 ma / cm , an open - circuit voltage voc = 0.610.71 v , a fill factor ff = 0.360.38 , and an efficiency = 1.21.5% under am1.5 sun illumination ( 100 3 mw / cm ) . the external quantum efficiency of these cells peaks at 4043% near the absorption maximum of the dye and is limited primarily by the relatively low dye loading of the nanowire film . analogous to zno nanowire arrays , well - aligned , self - organized tio2 nanotubes have been fabricated with the goal to improve electron transport pathways for solar energy conversion devices [ 24,29,30,81 - 83 ] . even though the efficiencies of these devices are not as high as cells fabricated with standard tio2 nanoparticles , respectable performance has been demonstrated . tio2 nanotube arrays allow direct charge transport along the length of the nanotube toward the electrode ; however , this assumes that the charge transport in mesoporous tio2 is limited by interparticle traps . the advantage of nanorod or nanotube arrays will not be apparent if the surface trapping limits the charge transport . comparison between these 1-d nanostructures and standard films fabricated from sintered nanoparticles may very well assist in elucidating the mechanism for electron transport in these materials . recently , highly ordered tio2 nanotube arrays were synthesized by anodic oxidation of titanium and have generated considerable scientific interest [ 24 - 32 ] . to fabricate nanotube devices , these nanopores initially have an amorphous structure , which can be transformed to anatase tio2 upon annealing to over 450 c . the porous film forms on the titanium foil and a compact titanium dioxide layer forms between the unoxidized titanium and the nanotubes during the heating process . reported high open - circuit voltages of up to 860 mv for this cell structure . their best cells reached efficiencies of over 4% under am1.5 sun ( iodide / triiodide - based cells ) . the nanotube devices display inhibited recombination characteristics with longer electron lifetimes , indicating fewer recombination centers in the nanotube film compared with a nanoparticle film . a disadvantage of using tio2 nanotube arrays for anode fabrication is that the device requires illumination from the back side ( through the pt cathode ) because the counter electrode fabricated from ti is opaque . this is not the optimal configuration for dsscs because the platinum counter electrode partially reflects light and the iodine in the electrolyte absorbs photons at lower wavelengths . therefore , the challenge is to achieve highly ordered tio2 nanotube arrays on fto substrates , especially nanotubes with increased film thickness . taking advantage of extremely high electron mobilities of single - walled cnts ( swcnts ) , brown et al . deposited tio2 nanoparticles on an swcnt network . when modified with a sensitizer such as ru(ii)(bpy)(dcbpy ) , the swcnt / tio2 film provided an unnoticeable influence on the charge injection from dye molecules into tio2 nanoparticles , but improved charge separation according to transient absorption and emission measurements . the rate of the back electron transfer between the oxidized sensitizer ( ru(iii ) ) and tio2 was slower in the presence of the swcnt scaffold . the incident photon to charge carrier efficiency ( ipce ) at all wavelengths was enhanced by a factor of ~1.4 as a result of introducing a swcnt scaffold in the mesoscopic tio2 film . this is due to the suppressed back electron transfer and the improved electron transport within the nanostructured tio2 film . however , the improvement in photocurrent generation was neutralized by a lower photovoltage , as the apparent fermi level of the tio2 and swcnt composite became more positive than that of pristine tio2 . the dye - sensitized swcnt / tio2 cell had = 0.13% , voc = 0.26 v , and jsc = 1.8 ma / cm . it is not surprising that the semiconductor nanotube / nanowire arrays are not always highly ordered , however . for instance , clumps of nanotubes and crack - like features in the films prepared by electrochemically anodizing titanium metal have been observed . the formation of clusters of bundled nanotubes in nominally oriented arrays could adversely affect the transport and recombination dynamics in tio2 films . clusters of bundled nanotubes could be produced during the anodization process or during the cleaning and evaporative drying process of the as - grown films through capillary forces of the liquid acting between the nanotubes . such capillary forces have the potential to not only bundle nanotubes but also to crack the film . removing liquids from the mesopores of the arrays by the supercritical co2 ( scco2 ) drying technique can yield bundle - free and crack - free nanotube films . compared with h2o / air - dried tio2 nanotube array film , zhu et al . found that the ethanol / scco2-dried films could prevent morphological disorders induced by capillary stress and enhance the total surface area of a film accessible to dye molecules by 23% . the electron transport was about twice as fast in the ethanol / scco2-dried film than in the h2o / air - dried film . the photoresponse of the dssc with ordered films exhibited jsc = 5.7 ma / cmvoc = 0.58 v , and ff = 0.56 to offer a solar conversion efficiency = 1.9% . in contrast , the dssc with less - ordered films showed a jsc of 4.9 ma / cmvoc of 0.60 v , and an ff of 0.53 to yield = 1.6% . in general , dsscs are relatively well developed in recent years with the following possible improvements . the growth of tio2 nanotube arrays on a transparent anode will be beneficial for the light absorption . a compact tio2 particle film between the fto anode and the electrolyte was recently proved to reduce charge recombination losses , which might be a valuable hint to design tio2 nanotube array for dsscs . use of bundle - free and crack - free 1-d nanostructure arrays is another principle for solar cell assembly . high aspect ratio nanotube / nanowire arrays are expected to load more dye molecules , but the dilemma is that tubes / wires longer than the diffusion length of electrons will degrade the efficiency of electron collection . recent work also includes the molecular engineering of suitable ruthenium compounds , which are known for their excellent stability . the use of solvent - free electrolytes such as ionic liquids has made striking advances during the past few years . these nonvolatile redox melts show great promise for use in outdoor photovoltaic systems and have been discussed in detail elsewhere . the combination of two or more nanostructure architectures provides another option to modulate the performance of light - harvesting devices [ 94 - 97 ] . as presented in the previous section , the electron transport across particles is susceptible to recombination loss at the grain boundaries and charge trapping in nanostructured semiconductor films prepared from particles . the use of nanotube / nanowire support to anchor light - harvesting assemblies ( e.g. , semiconductor particles and dye molecules ) provides a convenient way to capture photogenerated charges and transport them to electrodes . quantum - dot - sensitized solar cells ( qdsscs ) provide additional opportunities that are not available with dye - sensitized solar cells ( fig . first , the use of quantum dots in lieu of the dye molecules provides the ability to tune the optical absorption in the solar cell through selection of semiconductor material and particle size . second , qdsscs can potentially exploit the recently observed multiple electron - hole pair generation per photon to achieve higher efficiencies than that predicted by shockley and queisser . an array of zno nanowires , grown vertically from an fto / glass substrate and decorated with cdse quantum dots , serves as the photoanode . a second fto / glass substrate , coated with a 100 layer of pt , is the photocathode . the space between the two electrodes is filled with a liquid electrolyte and the cell is illuminated from the bottom ( reprinted with permission from ref . . a swcnt is an ideal channel for collecting and transporting charges across light - harvesting assemblies . significant progress has been achieved in synthesizing semiconductor - cnt composite films in recent years [ 102 - 109 ] . these earlier studies have mainly focused on establishing synthetic strategies and characterizing the composite systems , including cnts with tio2 , sno2 , cdse , and cds nanocrystals . most of the wet - chemistry strategies involve chemical functionalization of the cnt surface followed by the assembly of nanocrystals onto the cnts via covalent , noncovalent , or electrostatic interactions . swcnts were dispersed in tetrahydrofuran ( thf ) with the aid of tetraoctylammonium bromide ( toab ) . the adsorption of cd ions on the swcnt surface followed by reaction with s provides a simple and convenient method of preparing swcnt cds composites . the cds - swcnt composite is capable of generating a photocurrent from visible light with unusually high efficiencies , in which the luminescence of cds is quenched by swcnt . transient absorption experiments have confirmed the quick deactivation of excited cds on the swcnt surfaces , as the transient bleaching recovers in about 200 ps . the ability of the cds - swcnt nanocomposite system to undergo photoinduced charge separation opens up new ways to design light - harvesting assemblies . although the wet - chemistry methods work well with randomly dispersed cnts , they are not suitable for vertically aligned cnts . the aligned structure will be damaged in the wet processing because the upper ends of the neighboring nanotubes have been seen to bundle together and cause some nanotubes to lay down . have recently developed a material - independent dry route based on the electrostatic - force - directed assembly ( esfda ) to assemble aerosol nanocrystals onto cnts . in principle , the esfda technique works for both random cnts and aligned cnts without the need for chemical functionalization or other pretreatments of the cnts . in esfda , charged and nonagglomerated aerosol nanocrystals were produced from a mini - arc plasma source and then delivered to the electrically biased cnts in an inert carrier gas . the electric field near the cnt surface was enhanced significantly and the aerosol nanocrystals were attracted to the external surface of the cnts . with this technique , chen et al . have demonstrated the successful assembly of various nanocrystals , including single - component nanocrystals ( au , ag , and sno2 ) and multicomponent nanocrystals ( sno2 and ag ) , onto randomly dispersed multiwalled cnts ( mwcnts ) , swcnts , and vertically aligned mwcnts . the highly ordered charge transport channel is not limited to only cnts , but also to other semiconductor ( zno or tio2 ) 1-d nanostructures . quantum - dot - sensitized nanowire solar cell based on photosensitization of zno nanowires with cdse quantum dots has been demonstrated . a zno nanowire array can be grown directly onto transparent and conducting fto substrates from an aqueous solution of zn(no3)2 and methenamine between 80 and 95 c . cdse qds were assembled on the zno nanowires by using bifunctional molecules of the type x - r - y , where x and y are groups that bind to cdse ( x = sh ) and zno ( y = cooh ) , respectively . the photocurrent is generated from visible light by the excitation of electron - hole pairs in the cdse qds . the electrons are injected across the qd - nanowire interface into the zno , a process that is facilitated by the overlap between the electronic states in the qd and the zno conduction band . the morphology of the nanowires provides the photoinjected electrons with a direct electrical pathway to the photoanode . the zno nanowire - based qdsscs exhibited = 0.4% , voc = 0.50.6 v , jsc = 12 ma / cm , and a fill factor ff ~0.3 . tio2 is another important wide - bandgap semiconductor that is widely used in both dsscs and qdsscs . sun et al . reported an investigation on the cds qds sensitized tio2 nanotube array photoelectrodes and their performance in photoelectrochemical solar cells . the highly ordered tio2 nanotube films were synthesized by anodic oxidation in a nh4f organic electrolyte . the cds qds were deposited into the crystalline tio2 nanotubes by the sequential chemical bath deposition method . the cds - tio2 cells exhibited impressive = 4.15 % , voc = 1.27 v , jsc = 7.82 ma / cm , and ff = 0.578 under am1.5 illuminations . these results clearly demonstrated that significant improvement on the pec cell efficiency can be obtained via incorporating inorganic semiconductor qds into the tio2 nanotube array films . a number of active research areas could significantly contribute to the advancement of qdsscs . maximizing the overlap between the solar spectrum and the solar cell absorption spectrum through judiciously selecting tunable - bandgap qds the multiple exciton generation with uv photons demonstrated in qds is yet to be proved in solar cells . the challenge is to find ways to effectively collect the resulting excitons before they recombine since recombination occurs at a femtosecond time scale . assembling qds onto nanotubes / nanowires with wet - chemistry methods will likely damage the ordered arrays , and molecule linkers between qds and 1-d nanostructures are likely to be potential barriers for charge transfer . dry methods such as esfda could potentially achieve better electronic transfer between qds and 1-d nanostructures . use of fe or ni as catalysts in cvd growth of cnts constrains the material types of substrates and makes it difficult to achieve ohmic contacts between cnts and substrates . more understanding on the electronic transfer at the qds - nanotubes / nanowires heterojunction interface and cnts - substrate interface is imperative to further improve the performance of qdsscs . many researchers have been eager to explore carbon nanostructures such as swcnt assemblies for energy conversion devices because of their unique electrical and electronic properties , wide electrochemical stability window , and high surface area [ 118 - 120 ] . fullerenes , for example , exhibit rich photochemistry and act as an electron shuttle in photochemical solar cells . they also play an important role in improving the performance of organic photovoltaic cells . on the other hand , the exciton annihilation and charge separation processes have been characterized by transient absorption and emission measurements . for example , the photoresponse of cnt filaments was realized in early years from the elastic response of the aligned bundles between two metal electrodes . hot carrier luminescence from ambipolar cnt field - effect transistors ( fets ) has been monitored by avouris and coworkers . the relaxation of electrons and holes to the fundamental band edge occurs within 100 fs after photoexcitation . these early studies confirmed the ability of cnts to possess a band structure that can undergo electron - hole charge separation with visible light excitation . however , spatially confined charge carriers in the nanotube are bound by coulombic interactions with the bound pair referred to as an exciton [ 125 - 127 ] . a small fraction of the excitons are able to dissociate and form unbounded electron - hole ( e - h ) pairs . a key question is whether the photoinduced charge carriers generated in swcnts can be collected suitably for photocurrent generation , similar to the photovoltaic application of other semiconductors . double - walled carbon nanotubes ( dwcnts ) were also directly configured as energy conversion materials to fabricate thin film solar cells , with nanotubes serving as both photogeneration sites and a charge carriers collecting / transport layer . the solar cells consisted of a semi - transparent thin film of nanotubes conformally coated on an n - type crystalline silicon substrate to create high - density p - n heterojunctions between nanotubes and n - si to favor charge separation and extract electrons ( through n - si ) and holes ( through nanotubes ) . the p - type dwcnts were first formed as an ultrathin film on the water surface with the aid of ethanol , and then transferred to an n - si substrate . experiments have shown = 1.38% , voc = 0.5 v , jsc = 13.8 ma / cm , and ff = 19% under am1.5 illumination , proving that dwcnts - on - si is a potentially suitable configuration for solar cells . two key constraints for this type of solar cell are ( 1 ) the material must be sufficiently thick and pure to absorb most of the solar photons with energies above the material s bandgap ; and ( 2 ) the material must have a high minority carrier diffusion length to effectively collect the photogenerated charge carriers . one attractive method to improve the light absorption and charge carrier collection involves high aspect ratio cylindrical absorbers , such as 1-d nanowires . a preferred implementation includes the use of wires that are sufficiently long to absorb most of the incident light and have sufficiently small diameters to facilitate efficient radial collection of carriers , even for relatively impure absorber materials ( fig . methods are required ( 1 ) to prepare large area arrays of vertically aligned core - shell nanowires ; ( 2 ) to make electrical junctions to such wire arrays ; and ( 3 ) to make electrical contacts to the backsides of these devices . these challenges have been investigated by various means , including chemical vapor deposition ( cvd ) growth of wire arrays , etching of flat substrates to produce wire arrays , and creating conductive polymer electrical junctions with wires . schematic cross section of a radial p - n junction core - shell nanowire solar cell . the light gray area is n type ; the dark gray area is p type ( reused with permission from ref . . copyright 2005 , american institute of physics . ) in an earlier theoretical work by kayes et al . , a device physics model has been developed for radial p - n junction nanowire / nanorod solar cells , in which densely packed nanorods , each having a p - n junction in the radial direction , are oriented with the rod axis parallel to the incident light direction . the radial p - n junction nanorod geometry produces significant improvements in the efficiencies of cells made from materials that have diffusion lengths at least two orders of magnitude less than their optical thickness and low recombination in the depletion region ( for example , exciton lifetime > optimal cells have a radius approximately equal to the minority - electron diffusion length in the p - type core , and their doping levels must be high enough that a rod of such radius is not fully depleted . in silicon with very low diffusion lengths ( ln= 100 nm ) , extremely large efficiency gains ( from 1.5% to 11% ) are possible by exploiting the radial p - n junction nanorod geometry , provided that the trap density in the depletion region remains fixed at a relatively low level ( < ~310 cm ) . . experimentally showed that optimal efficiencies can be obtained when the si wires have a diameter comparable to the minority carrier diffusion length . smaller diameters increase the surface area , thereby increasing the surface and junction recombination with few accompanying improvements in carrier collection . based on the study of kayes et al . , tsakalakos et al . also estimated an efficiency of 1518% for si nanowire solar cells . according to the calculation , the lateral diffusion of minority carriers to the p - n junction , which is at most 50500 nm away , was proposed rather than many microns away as in bulk si solar cells . an array of si nanowires ( diameter = 189 30 nm , length ~16 m ) was fabricated with cvd . a current density of ~1.6 ma / cm for 1.8 cm cells was obtained , and a broad external quantum efficiency was measured with a maximum value of~12% at 690 nm . the optical reflectance of the silicon nanowire solar cells was reduced by one to two orders of magnitude compared with planar cells . the junction area of a cnts / si cell is no more than the surface area of the anode ( si substrate ) . therefore , rough architectures rather than planar ones are expected to improve the junction areas . semiconductor nanowire / nanorods with radial p - n junctions make it possible to orthogonalize the direction of light absorption and carrier collection , and thus can enable efficient carrier collection in optically thick nanowire arrays even when minority carrier diffusion lengths are shorter than the optical absorption length . although coaxial si nanowire / nanorod p - n junction solar cells have high theoretical efficiencies , the cost of cvd growth of si nanowires on si substrates is high . au as a catalyst used in the cvd growth of si nanowire arrays also reduces the lifetime of carriers in silicon due to the presence of au in the nanowire . identifying inexpensive catalysts such as the sun provides enormous potential in helping to resolve the growing demand for energy worldwide ; however , the high costs of implementing solar energy is a significant barrier compared with traditional energy sources , such as fossil fuels . the cost of a photovoltaic system is directly related to the low conversion efficiency , diluted energy density of solar radiation , and costly materials and fabrication process . during the past decade , the development of nanoscience and nanotechnology has launched new ways to design efficient solar cells . strategies have been developed to design nanostructure architectures of semiconductors , metals , and polymers for solar cells . theoretical and modeling studies have also helped to understand the optical and electrical processes of the photovoltaic conversion . the examples discussed in this review summarized how 1-d nanostructures , hybrids of 1-d nanomaterials / molecules , and qds could aid in dsscs , qdsscs , and conventional p - n junction solar cells . while the new generation of photovoltaic cells offers many opportunities , it also presents challenges such as in the following directions ( 1 ) ordered assemblies of two or more nanocomponents on electrode surfaces ; ( 2 ) new sensitizers or semiconductor systems that can harvest infrared photons ; and ( 3 ) multiple exciton generation in semiconductor qds . worldwide , solar power is the star attraction for venture capitalists due to its booming laboratory research and commercialization . although other forms of renewable energy can make significant contributions to current markets , sunlight is most available in the amount required to substitute completely for the energy quantities currently derived from hydrocarbons . this work was financially supported by the national science foundation through an ner grant ( cmmi-0609059 ) .
the current global energy problem can be attributed to insufficient fossil fuel supplies and excessive greenhouse gas emissions resulting from increasing fossil fuel consumption . the huge demand for clean energy potentially can be met by solar - to - electricity conversions . the large - scale use of solar energy is not occurring due to the high cost and inadequate efficiencies of existing solar cells . nanostructured materials have offered new opportunities to design more efficient solar cells , particularly one - dimensional ( 1-d ) nanomaterials for enhancing solar cell efficiencies . these 1-d nanostructures , including nanotubes , nanowires , and nanorods , offer significant opportunities to improve efficiencies of solar cells by facilitating photon absorption , electron transport , and electron collection ; however , tremendous challenges must be conquered before the large - scale commercialization of such cells . this review specifically focuses on the use of 1-d nanostructures for enhancing solar cell efficiencies . other nanostructured solar cells or solar cells based on bulk materials are not covered in this review . major topics addressed include dye - sensitized solar cells , quantum - dot - sensitized solar cells , and p - n junction solar cells .
Introduction Dye-Sensitized Solar Cells Quantum-Dot-Sensitized Solar Cells 1-D Nanostructure Solar Cells Conclusion Acknowledgment
the exponential demand for energy is evidenced by dwindling fossil fuel supplies and record - high oil and gas prices due to global population growth and economic development ( fig . the energy crisis is further exacerbated by major concerns about global warming from greenhouse gas emissions due to increasing fossil fuel consumption [ 6 - 8 ] . ) the major barrier for the large - scale use of solar energy is the high cost and inadequate efficiencies of existing solar cells . these conventional p - n junction cells , so - called first - generation devices , suffer from the high cost of manufacturing and installation . now the third - generation solar cells , such as dye - sensitized solar cells ( dsscs ) , bulk heterojunction cells [ 17 - 19 ] , and organic cells , are promising for inexpensive and large - scale solar energy conversion ( table 1 ) ; however , laboratory dsscs based on cheap dye sensitization of oxide semiconductors are typically less than 10% efficient , and those based on even cheaper organic materials are 25% efficient . considerable efforts have been devoted to the development of more efficient photoanode materials , such as ordered mesostructured materials and one - dimensional ( 1-d ) nanostructures ( nanowires , nanotubes , and nanorods ) [ 22 - 24 ] . bandgap - tunable semiconductor zero - dimensional ( 0-d ) nanomaterials , such as cds [ 35 - 37 ] , pbs , bi2s3 , cdse , and inp quantum dots ( qds ) , have demonstrated extraordinary optical and electronic properties that open up possibilities for revolutionary advances in photovoltaic ( pv ) devices . this review addresses some of the current research issues in the field of new solar cells based on 1-d semiconductor nanostructures , and hybrids of 1-d nanomaterials and dye molecules or qds . these solar cells include dsscs with tio2 nanotube or zno nanowire arrays as photoanodes in lieu of nanoparticle networks , solar cells with tunable - bandgap qds supported by wide - bandgap semiconductor nanotube / nanowire or cnt arrays , and solar cells with coaxial p - n junctions in vertically - aligned 1-d nanostructures or p - n junctions between the 1-d nanostructure and the substrate . comparison of electron mobilities ( cm v s ) of 1-d nanomaterials and nanoparticles field - effect and ; intrinsic mobility of a 300 nm long and 3.9 nm diameter single - walled carbon nanotube ( swcnt ) ; 830 nm wide si nanowires ; 20 nm wide ge nanowires ; 1617 m long and 130200 nm wide zno nanowire ; for particle size around 4 nm ; for 48 nm anatase tio2 nanocrystals ; it is 15 cm v s in single crystal tio2 a dye - sensitized solar cell ( dssc ) is a type of photoelectrochemical ( pec ) solar cell which has been studied extensively [ 55 - 57 ] . quantum - dot - sensitized solar cells ( qdsscs ) provide additional opportunities that are not available with dye - sensitized solar cells ( fig . quantum - dot - sensitized nanowire solar cell based on photosensitization of zno nanowires with cdse quantum dots has been demonstrated . , a device physics model has been developed for radial p - n junction nanowire / nanorod solar cells , in which densely packed nanorods , each having a p - n junction in the radial direction , are oriented with the rod axis parallel to the incident light direction . the radial p - n junction nanorod geometry produces significant improvements in the efficiencies of cells made from materials that have diffusion lengths at least two orders of magnitude less than their optical thickness and low recombination in the depletion region ( for example , exciton lifetime > optimal cells have a radius approximately equal to the minority - electron diffusion length in the p - type core , and their doping levels must be high enough that a rod of such radius is not fully depleted . according to the calculation , the lateral diffusion of minority carriers to the p - n junction , which is at most 50500 nm away , was proposed rather than many microns away as in bulk si solar cells . although coaxial si nanowire / nanorod p - n junction solar cells have high theoretical efficiencies , the cost of cvd growth of si nanowires on si substrates is high . identifying inexpensive catalysts such as the sun provides enormous potential in helping to resolve the growing demand for energy worldwide ; however , the high costs of implementing solar energy is a significant barrier compared with traditional energy sources , such as fossil fuels . the examples discussed in this review summarized how 1-d nanostructures , hybrids of 1-d nanomaterials / molecules , and qds could aid in dsscs , qdsscs , and conventional p - n junction solar cells .
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the fusion between the sperm and the egg marks the beginning of a new individual and ensuing development of either man or beast depends upon a number of additional cell fusion events ( reviewed by ogle et al . thus , mononuclear cytotrophoblasts fuse to form the syncytiotrophoblast layer of the placenta in man and certain other mammals . syncytiotrophoblasts control the exchange of gases , nutrients and waste products between the fetus and the mother , protect the fetus against the maternal immune system and are also responsible for the production of hormones , which like chorionic gonadotropin , regulate the continuation of pregnancy . in addition , in the developing individual , myoblasts fuse to form multinucleated skeletal muscle fibers , while cells of monocytic origin fuse to form osteoclasts , which participate in bone sculpturing and remodeling as well as in the regulation of serum calcium concentrations . it is well - known that skeletal muscle is regenerated through fusion of muscle fibers with satellite cells ( bischoff 1994 ) and that macrophages may fuse to form multinucleated giant cells with enhanced phagocytic capabilities in response to injury and antigenic challenges ( vignery 2005 ) . recent data indicate that additional cell fusions may contribute to tissue repair in the adult ( vassilopoulos et al . 2003 ; wang et al . 2003 ) . bone - marrow - derived ( bmd ) cells have been shown to fuse with hepatic cells , nerve cells and gastrointestinal cells and the theory has been put forward that such fusions may serve to repair damaged or corrupted cells ( alvarez - dolado et al . however , it is still debated whether such fusions are important to tissue repair and whether they engage bmd stem cells or more differentiated fusogenic cells like monocytes / macrophages ( reviewed by vignery 2005 ) . intriguingly , the accumulation of macrophages in injured organs may reflect not only the need to remove debris but also to repair compromised cells through heterotypic fusions ( vignery 2005 ) . moreover , in recent transplant experiments , irradiated mice showed evidence of fusions between crypt stem cells and donor bmd cells ( rizvi et al . 2006 ) . in thus transplanted mice , representatives of all cell lineages derived from intestinal crypt stem cells ( absorptive columnar , enteroendocrine , goblet and paneth cells ) showed expression of chromosomal markers characterizing donor bmd cells . this phenomenon appeared to depend upon tissue injury since expression of donor cell markers never was detected in non - irradiated mice ( rizvi et al . ( 1999 ) , who have demonstrated that cells phagocytosing apoptotic cells may acquire functional dna . moreover , a mechanism of cell cell invasion ( entosis ) was recently described and could potentially transfer dna between cells ( overholtzer et al . thus , viral as well as normal sequences from apoptotic cells have been found to be active also in phagocytosing cells ( holmgren et al . this observation challenges our understanding of apoptosis as an efficient way of clearing corrupted or alien dna . the viral genome encodes envelope ( env ) proteins , which bind to cell surface receptors and assist the virus in entering the cell . the infected cell commences to synthesize env proteins , which , upon insertion in the plasmalemma , engages receptor proteins in neighboring cells and initiate fusions . interestingly , modifications of the cytoplasmic tail of several env proteins appear to modify fusions . thus , proteolytic cleavage of the cytoplasmic tail regulates fusions and also cellular signaling events , such as tyrosine kinase activity , may be involved in regulation of virally induced cell fusions ( kubo et al . thus , a cell infected by a specific virus becomes resistant to infections with other viruses that bind to the same receptor . this phenomenon , which probably reflects receptor saturation , has led to the grouping of retroviruses into different subtypes , which bind to the same receptor . other types of viruses encode fusogenic proteins ( like influenza hemagglutinin ) , which do not bind to surface receptors , but which liberate a fusion peptide in the acidic milieu of endosomes and thus initiate viral host cell fusion following endocytic uptake of viral particles . both these proteins and the env proteins are referred to as class i viral fusion proteins and contain hydrophobic fusion peptides buried within their sequences . the cleaved peptides undergo conformational changes , resulting in hairpin - like , alpha - helical bundles , which bring the viral and cell membranes into close apposition and thereby facilitates fusion ( reviewed by chen and olson 2005 ) . in 1911 aichel suggested that cancer cells might spontaneously fuse with host cells to produce hybrids with supernumerary chromosomes that could evolve into cells of increased malignancy . subsequent studies have confirmed this prediction by demonstrating that cells with mixed phenotypes spontaneously appear in co - cultures of normal and malignant cells ( barski and cornefert 1962 ; busund et al . 1994 ) ( figs . 1 , 2 , 3 ) . additionally , transplanted tumor cells of animal ( busund et al . 1974a , b ) or human ( goldenberg et al . 1974 ; goldenberg and pavia 1982 ; mortensen et al . 2004 ) origin may fuse with and acquire phenotypic characteristics of normal host cells ( fig . , data suggested that the putative fusion partner was of macrophage or endothelial origin but , in most cases , definite identification was not achieved . in a few cases , it has also been demonstrated that the fused cells expressed genetic markers of both parental cell types ( goldenberg et al . 1974 ; mortensen et al . ( 2004 ) demonstrated that , initially after fusion , bi- or multinucleated cells formed and that such cells had the parental genomes seggregated in diffferent nuclei ( heterokaryons ) ( fig . subsequently , mitotic figures appeared showing an admixture of the parental chromosomes ( fig . eventually , cells with the parental genomes mixed in a single nucleus ( synkaryons ) were detected ( fig . 2k o ) . in agreement with the notion that synkaryons appear after mitotic divison of heterokaryons , 3 ) . a few recent studies present compelling evidence that cells with genetic characteristics of hybrid cells also may appear in human tumors ( andersen et al . 2007 ; chakraborty et al . thus , in two cases of renal carcinomas , arising in bone marrow transplant recipients , some tumor cells showed genetic markers characterizing the healthy donor ( chakraborty et al . moreover , in patients with multiple myelomas , osteoclasts have been shown to contain an admixture of nuclei , of which some possess tumor - specific chromosomal translocations while others are devoid of translocations ( andersen et al . finally , lymphoma - specific genetic abnormalities were described in endothelial cells in b cell lymphomas ( streubel et al . host cell hybrids did form in these patients , the combined results from studies in vitro and in vivo do present compelling evidence that cell fusions do occur in tumors ( reviewed by pawelek 2005 ) . co - culture of human breast cancer ( mcf-7 ) cells and human umbilical vein endothelial cells ( huvec ) . cells were stained for the cancer cell marker cytokeratin ( red , c ) , the endothelial cell marker vimentin ( green , b ) and dna ( bisbenzimide , blue , d ) . note in the merged image ( a ) one fused , multinucleated cell reacting for both cytokeratin and vimentin ( orange - yellow)fig . the cultures were subjected to fluorescent in situ hybridization ( fish ) with probes recognizing all bovine chromosomes ( green ; b , g , l ) and all human chromosomes ( red ; c , h , m ) and counter - stained for dna with dapi ( blue ; d , i , n ) and observed in differential interference contrast ( dic ; e , j , o ) . merged red - green images are shown in a , f , k. note that initially , binuclear cells ( heterokaryons ) form , having one bovine and one human nucleus ( a e ) . occasionally , tri- or multinuclear cells with different admixtures of bovine and human nuclei are also detected . at longer times after mixing , mitotic figures , containing an admixture of bovine and human chromosomes appear ( f j ) and , eventually , cells with a single nucleus , containing an admixture of the two genomes in mixture ( synkaryons ) are detected ( k o)fig . mixed culture of human breast cancer cells and bovine endothelial cells submitted to double fish as in fig . 2 ( a double fish ; b combined dapi and dic ) . low power micrograph showing a pair of synkaryons with the bovine and human genomes admixed in single nuclei ( arrows ) . in addition , nuclei hybridizing only for the bovine ( green ) or only for the human ( red ) genome occurfig . a , b section from lung of a nude mice injected with human breast cancer ( mda - mb-231 ) cells in the tail vein ( mortensen et al . the section underwent fish for the mouse genome ( red ) and the human genome ( green ) ( a ) and dna was counterstained with dapi ( b ; blue ) . note one nucleus in which the human and mouse genome co - localize ( arrow ) . c : similar section , stained with an antibody detecting human ( but not mouse ) p53 ( red ; p53 is mutated and overexpressed by the breast cancer cells ) , an antibody to beta - catenin ( to mark cell membranes ) and for dna with bisbenzimide ( blue ) . note a micrometastasis of human breast cancer cells having violet ( red + blue ) fluorescent nuclei . d section stained for human p53 ( red ) and the endothelial marker von willebrand factor ( green ) and dna ( blue ) . note a human cancer cell with a violet ( red + blue ) nucleus showing membrane - staining for von willebrand factor . since von willebrand factor is not normally expressed by the breast cancer cells , this image is suggestive of a fusion between a human breast cancer cell and a mouse endothelial cell . similar results were obtained using double fish for the human and mouse genome and imunofluorescent staining for von willebrand factor ( described by mortensen et al . co - culture of human breast cancer ( mcf-7 ) cells and human umbilical vein endothelial cells ( huvec ) . cells were stained for the cancer cell marker cytokeratin ( red , c ) , the endothelial cell marker vimentin ( green , b ) and dna ( bisbenzimide , blue , d ) . note in the merged image ( a ) one fused , multinucleated cell reacting for both cytokeratin and vimentin ( orange - yellow ) chromosomal markers and cell fusions . the cultures were subjected to fluorescent in situ hybridization ( fish ) with probes recognizing all bovine chromosomes ( green ; b , g , l ) and all human chromosomes ( red ; c , h , m ) and counter - stained for dna with dapi ( blue ; d , i , n ) and observed in differential interference contrast ( dic ; e , j , o ) . merged red - green images are shown in a , f , k. note that initially , binuclear cells ( heterokaryons ) form , having one bovine and one human nucleus ( a e ) . occasionally , tri- or multinuclear cells with different admixtures of bovine and human nuclei are also detected . at longer times after mixing , mitotic figures , containing an admixture of bovine and human chromosomes appear ( f j ) and , eventually , cells with a single nucleus , containing an admixture of the two genomes in mixture ( synkaryons ) are detected ( k o ) chromosomal markers and cell fusions . mixed culture of human breast cancer cells and bovine endothelial cells submitted to double fish as in fig . 2 ( a double fish ; b combined dapi and dic ) . low power micrograph showing a pair of synkaryons with the bovine and human genomes admixed in single nuclei ( arrows ) . in addition , nuclei hybridizing only for the bovine ( green ) or only for the human ( red ) genome occur cancer host cell fusion in vivo . a , b section from lung of a nude mice injected with human breast cancer ( mda - mb-231 ) cells in the tail vein ( mortensen et al . 2004 ) . the section underwent fish for the mouse genome ( red ) and the human genome ( green ) ( a ) and dna was counterstained with dapi ( b ; blue ) . note one nucleus in which the human and mouse genome co - localize ( arrow ) . c : similar section , stained with an antibody detecting human ( but not mouse ) p53 ( red ; p53 is mutated and overexpressed by the breast cancer cells ) , an antibody to beta - catenin ( to mark cell membranes ) and for dna with bisbenzimide ( blue ) . note a micrometastasis of human breast cancer cells having violet ( red + blue ) fluorescent nuclei . d section stained for human p53 ( red ) and the endothelial marker von willebrand factor ( green ) and dna ( blue ) . note a human cancer cell with a violet ( red + blue ) nucleus showing membrane - staining for von willebrand factor . since von willebrand factor is not normally expressed by the breast cancer cells , this image is suggestive of a fusion between a human breast cancer cell and a mouse endothelial cell . similar results were obtained using double fish for the human and mouse genome and imunofluorescent staining for von willebrand factor ( described by mortensen et al . host cell fusions is , of course , if they are relevant to the patient . in fact , there are two opposing views . the first is based on early experiments on fusions induced to occur between cancer cells and normal cells in culture . with few exceptions these studies were , in fact , seminal to the discovery of tumor suppressor genes ( reviewed by anderson and stanbridge 1993 ) . since tumor suppressor genes , like p53 and rb , frequently are inactivated in cancer cells , fusions would present cancer cells with unperturbed tumor suppressors from the normal fusion partner and consequently initiate cell cycle arrest or apoptosis . although , this certainly applied for the cell types studied in the contributions cited above , it may not be a general rule . thus , production of monoclonal antibodies depends upon the fact that it is possible to fuse antibody - producing spleen cells with myeloma cells to obtain hybridomas that retain the unlimited proliferative ability of the tumor cell partner and the antibody production of the normal cell ( kohler and milstein 1975 ) . in fact , several studies documented that some fusions may lead to cells of increased malignancy ( barski and cornefert 1962 ; busund et al . 2003 ; chakraborty et al . possibly , the genetic make - up of tumors may dictate the outcome of individual cancer host cell fusions . moreover , fusions lead to aneuploidy and chromosomal instability , which characterizes most cancers and may , by itself , stimulate carcinogenesis ( duelli et al . 2007 ) . cell fusion events must be extremely well controlled . due to their major importance to fertilization , placentation , muscle development , bone structure , calcium homeostasis and the immune defense system , additionally , the potential role of cell fusions as a repair mechanism and the role of cell fusions in cancer development and progression have further stimulated research in this field . in spite of this , much less is known about cell cell fusion mechanisms than is known about how intracellular membranes fuse through v- and t - snares . interestingly , engineered flipping of the v- and t - snare machinery has been shown to promote cell cell fusions ( weber et al . however , it is evident that this mechanism is not a physiological mediator of cell cell fusions . interestingly , v- and t - snares act similar to class i viral fusion proteins in that they form bundles of alpha - helices , which result in membrane apposition and fusion ( blumenthal et al . 2003 ; jahn et al . 2003 ) . of the many proteins , which to date have been shown ( or proposed ) to be involved in cell fusions in mammals , only the syncytin family appears to use a similar alpha - helical mechanism . that they do is not surprising in view of the fact that syncytins represent conserved endogenous retroviral env sequences . the founding family member , syncytin-1 , was discovered as a protein capable of mediating fusions between cytotrophoblasts into syncytiotrophoblasts ( blond et al . this capability may have contributed to a high degree of evolutionary conservation of the syncytin-1 sequence . syncytin-1 represents the env protein of the human endogenous retroviral ( herv ) w sequence , which entered the primate genome 2540 million years ago . in contrast , most other retroviral sequences inserted in our genome have been subject to inactivating changes and probably represent garbage sequences . molecular studies have shown that syncytin-1 ( env w ) shares a structure similar to class i viral fusion proteins , especially in the region of the n- and c - terminal heptad repeats ( nhr and chr ) , and shares a common fusion mechanism with these proteins ( chang et al . a synthetic peptide derived from the chr is also capable of inhibiting syncytin-1-mediated fusions by perturbing this mechanism ( chang et al . syncytin-1 binds to the d - type retroviral receptor asct-2 ( blond et al . 2000 ) and may use another neutral amino acid transporter ( asct-1 ) as an auxiliary receptor ( lavillette et al . syncytin antibodies , syncytin-1 downregulation through antisense oligonucleotides and the syncytin-1 chr peptide have been shown to inhibit fusions between trophoblast - derived cells ( blond et al . agents increasing cellular levels of camp or camp analogues have been shown to promote cytotrophoblast fusions in vitro ( keryer et al . 1998 ) and such agents are also known to elevate protein and mrna levels of syncytin-1 in isolated cytotrophoblasts ( frendo et al . also estradiol may regulate syncytin-1 expression ( carino et al . 2003 ) and the placenta - specific transcription factor gcma interacts with two upstream sites in the herv - w 5-long terminal repeat and stimulates syncytin-1 transcription ( yu et al . additionally , experimentally induced truncations in the cytoplasmic tail of syncytin-1 increases its fusogenicity ( drewlo et al . 2006 ) , similar to what has been observed for some virally derived env proteins ( reviewed by kubo et al . however , if modifications in the cytoplasmic tail of syncytin-1 are of physiological importance for regulating fusogenicity has yet to be demonstrated . a second syncytin - family member , syncytin-2 , syncytin-2 also represents a highly conserved endogenous retroviral envelope gene and is derived from the herv frd sequence ( blaise et al . immunocytochemical studies have localized syncytin-1 primarily to syncytiotrophoblasts as well as to cytotrophoblasts ( fig . 2004 ) , which may reflect differences in antibodies , fixation procedures and controls . a major point of concern is also the degree of cross - reactivity to the related sequences in syncytin-2 , which recently was localized to a subpopulation of cytotrophoblasts ( malassin et al . the syncytin-1 receptor protein asct-2 was recently localized to cytotrophoblasts ( hayward et al . 2007 ) . more studies on the exact distribution of syncytin expression in the placenta using syncytin-1 and -2 specific antibodies seem warranted in order to exactly localize where fusions are likely to occur . nuclei are lightly counterstained with haematoxylin human term placenta immunocytochemically stained with a syncytin-1 peptide antiserum . nuclei are lightly counterstained with haematoxylin trophoblast cell fusions are , however , not unique to the primate placenta . thus , also in the mouse placenta , trophoblasts fuse to form syncytiotrophoblasts but mice do , of course , not express herv sequences . amazingly , an in - silico search of the mouse genome unraveled the existence of two murine endogenous retroviral ( merv ) env genes , labeled syncytin - a and -b ( dupresssoir et al . these genes were also fusogenic and orthologous genes were present in additional species of muridae ( rats , gerbils , voles and hamsters ) and appear to have entered the rodent lineage some 20 million years ago ( dupresssoir et al . 2005 ) . they were expressed in the placenta and at least syncytin - a has been shown to be involved in the formation of syncytiotrophoblasts in mice ( gong et al . this represents an amazing example of parallel acquisition of retroviral genes of importance to reproduction in primates and rodents and poses the question whether also other species may have acquired similar viral genes of importance to cell fusions . the ability of syncytins to induce cell fusions may not be their only physiologic role . thus , recent studies have shown that while syncytin-2 and syncytin - b also possess immunosuppressive activity , syncytin-1 and syncytin - a do not ( mangeney et al . 2000 ) but subsequent studies have revealed the presence of syncytin-1 also in the brain ( antony et al . 2004 ) and in breast , colon and endometrial cancers ( bjerregaard et al . 2007 , larsen , talts , andersen , bjerregaard and larsson : work in progress ) . in fact , in all systems studied so far , from mating yeast to man , a bewildering array of mechanisms have been identified ( see chen and olson 2005 ; chen et al . molecules potentially involved in mammalian cell fusions include adam ( a disintegrin and metalloproteinase domain ) 12 ( meltrin alpha ) , which has been associated with myoblast and osteoclast cell fusions ( abe et al . 1999 ; in addition , adams 1 and 2 ( fertilins ) may be involved in sperm oocyte fusions , but do not seem indispensable for this function and , in man , the fertilin alpha gene is dysfunctional ( chen and olson 2005 ; cho et al . cd9 is also expressed by bewo trophoblast tumor ( choriocarcinoma ) cells and has been linked both to bewo invasiveness and to invasion during mouse embryo implantation ( hirano et al . 1999 ; liu et al . cd9 has , together with another tetraspanin family member , cd81 , also been linked to myoblast fusion and myotube maintenance ( tachibana and hemler 1999 ) . moreover , antibodies to either cd9 or cd81 have been shown to block fusions induced by mason pfizer monkey virus a d - type retrovirus ( duelli et al . 2005 ) . tetraspanins are known to organize other proteins into membrane microdomains and may link to the actin cytoskeleton via ewi ( glu - trp - ile ) and erm ( ezrin radixin moesin ) proteins ( hemler 2003 ; sala - valds et al . possibly , their role as organizers of other proteins into microdomains may play a role in their involvement with cell fusions ( zivyat et al . they do not express characteristics of fusogenic proteins like the syncytins , snares and class i viral envelope proteins , but are coexpressed with syncytin-1 , at least in bewo cells . a macrophage fusion receptor ( mfr , sirpalpha ) , resembling cd4the cell surface receptor for hiv has been identified in macrophages ( saginario et al . mfr , which belongs to the immunoglobulin superfamily , binds another member of this family , cd47 . cd47 is also structurally related to proteins expressed by vaccinia and variola viruses ( chen et al . whereas expression of mfr is restricted to myeloid cells and neurons , cd47 is ubiquitously expressed . mfr is transiently induced in macrophages at the onset of fusion while cd47 expression is constant . it has been hypothesized that cd47 initially binds to a long form of mfr to secure recognition and then switches to bind a shorter form to bring the plasma membranes closer ( 510 nm ) for fusion ( vignery 2005 ) . additionally , or alternatively , cd47 may promote calcium entry by forming a membrane pore ( reviewed in chen et al . also the hyaluronan receptor , cd44 , is induced transiently when macrophages start to fuse . mice lacking dc - stamp are osteopetrotic and lack multinucleated osteoclasts and giant cells ( yagi et al . 2005 ) . dc - stamp is a seven - transmembrane receptor , somewhat similar to the hiv co - receptor cxcr4 , but a ligand has yet to be identified . these and many more molecules , including integrins , vacuolar atpase and receptors and their ligands , as well as different signaling intermediates have been associated with cell fusions . with the exception of the syncytins , most of the mammalian molecules so far studied do not fulfill strict criteria for fusogens and several are dispensable for fusions ( reviewed by chen and olson 2005 ; chen et al . however , this may reflect molecular redundancy and does not definitely exclude that these molecules may participate in fusions . it is noteworthy that , in both man and mouse , two different syncytins are expressed and are both fusogenic . it is possible that , in the mammalian system , fusion requires a flotilla of molecules organized into membrane microdomains by proteins like cd9 , encompassing receptors / ligands , signaling entities , proteases and fusogens of retroviral origin that are capable of forming alpha - helical bundles , which bring membranes closer . syncytins and related retroviral envelope sequences may , in this connection , function both as fusogens and receptor ligands . however , it seems unlikely that as irreversible an event as a cell cell fusion should depend upon a single receptor ligand interaction . thus , both facilitatory and inhibitory factors are expected to be part of the flotilla . ( 2004 ) documented that human breast cancer cells fused with endothelial cells in culture . stimulated by studies showing that syncytin-1 was involved in cytotrophoblast cell fusions ( blond et al . 2000 ; mi et al . 2000 ) , we examined whether a similar mechanism could account for cancer endothelial cell fusions . expression of syncytin-1 was documented in the breast cancer cell lines examined ( mcf-7 and mda - mb-231 cells ; bjerregaard et al . 2006 , sk - br-3 cells : talts , bjerregaard and larsson : unpublished data ) . moreover , we found that both tumor cells and endothelial cells expressed the syncytin-1 receptor asct-2 . use of phosphorthioate - protected syncytin-1 antisense oligonucleotides downmodulated syncytin-1 expression as measured by either quantitative rt - pcr or western blotting and inhibited breast cancer endothelial cell fusions , whereas a scrambled oligonucleotide control was without effect . additionally , the syncytin-1 chr peptide , referred to above , also inhibited the fusions whereas a control peptide was without effect ( bjerregaard et al . however , neither the antisense nor the chr peptide experiments effected a total inhibition of cancer - endothelial cell fusions . there may be several reasons to this . thus , the antisense oligonucleotide did not totally downmodulate syncytin-1 levels and could therefore not be expected to decrease cell fusions to zero and the lack of total inhibition by the chr peptide could potentially be ascribed to proteolytic degradation . however , we also detected that the breast cancer cells produced syncytin-2 and can not exclude that also this molecule contributed to the cancer further experiments using shrna - directed downmodulation of both syncytin-1 and -2 as well as of additional putative fusogenic retroviral sequence are now underway to test this . we next examined two series of human breast cancer patients for tumoral expression of syncytin-1 using a polyclonal antiserum raised to a synthetic nonapeptide derived from the syncytin-1 sequence . in addition , tumors were also screened for expression of asct-2 using a peptide antiserum . preabsorption of the antisera with the corresponding peptides , but not with irrelevant peptides , abolished staining ( larsson et al . the results showed that 38% of all breast cancer samples showed detectable staining for syncytin and that endothelial cells expressed asct-2 ( fig . 6 ) . moreover , significant expression of asct-2 was detected also in many tumor cells ( fig . the degree of syncytin immunostaining was visually graded using coded specimens and statistical analysis showed that it correlated positively with disease - free survival of the patients ( larsson et al . 2007b ) . multivariate analysis included age dichotomized at 40 years , tumor size dichotomized at 20 mm , grade and adjuvant therapy and identified syncytin expression as an independent prognostic indicator of increased disease - free survival also when used as a continuous variable , syncytin expression emerged as a significant prognostic indicator for disease - free survival in the cox model ( p = 0.02 ) ( larsson et al . . 6human breast cancers immunocytochemically stained for syncytin-1 ( a ) or with antigen - preabsorbed antiserum ( b ) and for asct-2 ( c , e ) or with antigen - preabsorbed antiserum ( d ) . note presence of syncytin immunoreactivity in tumor cells and of asct-2 immunoreactivity in endothelial cells ( arrows ) and in tumor cells human breast cancers immunocytochemically stained for syncytin-1 ( a ) or with antigen - preabsorbed antiserum ( b ) and for asct-2 ( c , e ) or with antigen - preabsorbed antiserum ( d ) . note presence of syncytin immunoreactivity in tumor cells and of asct-2 immunoreactivity in endothelial cells ( arrows ) and in tumor cells the involvement of syncytin-1 in tumor cell fusion events was subsequently confirmed by strick et al . ( 2007 ) , working with endometrial carcinomas . in their study , downmodulation of synctytin-1 expression also inhibited fusions between endometrial tumor cells . in agreement with findings on placental cells this apparent discrepancy may possibly be ascribed to the fact that estrogen treatment upregulated tgfbeta , which interfered with syncytin - induced fusions . thus , estrogen did , in fact , stimulate cell fusions if tgfbeta was immunoneutralized . conversely , additions of tgfbeta 1 or 3 reduced fusions induced by camp - elevating agents . this effect was observed both in endometrial carcinoma cells and in trophoblast - derived cells . these results show that both camp and estrogens positively may regulate syncytin-1 expression in tumor cells and that the tgfbeta family may negatively regulate the fusogenic effects of syncytin-1 in both trophoblasts and cancer cells ( strick et al . 2007 ) . the role of syncytin-1 and cell fusions in cancer needs further study . thus , our data show that syncytin-1 is not the only fusogenic protein expressed by breast cancer cells and the results presented by strick et al . ( 2007 ) show that additional regulators , such as tgfbeta isoforms may be important modulators of cell fusions . so far , our breast cancer data indicate that syncytin-1 expression constitutes a positive prognostic factor . this is not the same as to say that cell fusions may be universally beneficial to cancers . first , syncytins may have additional effects within the tumor environment ( larsson et al . secondly , it seems likely that several factors within the cancer and its stroma ( inactivated tumor suppressor genes , activated oncogenes , expression of fusogens and of cd9 and cd81 as well as tgfbeta ) may act together to bring about a tumor profile that may be as diverse as the one demonstrated by the cell fusion experiments referred to above . interestingly , however , expression of cd9 has been analyzed in a number of tumors and seems , like syncytin expression , to predict a good prognosis ( funakoshi et al . we propose that syncytins may be fusogens of importance to both trophoblast and cancer cell fusions and that they possibly also may mediate additional cell fusion events , acting in concert with other molecules with both enhancing and inhibitory regulatory effects . cell fusion events must be extremely well controlled . due to their major importance to fertilization , placentation , muscle development , bone structure , calcium homeostasis and the immune defense system , additionally , the potential role of cell fusions as a repair mechanism and the role of cell fusions in cancer development and progression have further stimulated research in this field . in spite of this , much less is known about cell cell fusion mechanisms than is known about how intracellular membranes fuse through v- and t - snares . interestingly , engineered flipping of the v- and t - snare machinery has been shown to promote cell cell fusions ( weber et al . however , it is evident that this mechanism is not a physiological mediator of cell cell fusions . interestingly , v- and t - snares act similar to class i viral fusion proteins in that they form bundles of alpha - helices , which result in membrane apposition and fusion ( blumenthal et al . 2003 ; jahn et al . 2003 ) . of the many proteins , which to date have been shown ( or proposed ) to be involved in cell fusions in mammals , only the syncytin family appears to use a similar alpha - helical mechanism . that they do is not surprising in view of the fact that syncytins represent conserved endogenous retroviral env sequences . the founding family member , syncytin-1 , was discovered as a protein capable of mediating fusions between cytotrophoblasts into syncytiotrophoblasts ( blond et al . this capability may have contributed to a high degree of evolutionary conservation of the syncytin-1 sequence . syncytin-1 represents the env protein of the human endogenous retroviral ( herv ) w sequence , which entered the primate genome 2540 million years ago . in contrast , most other retroviral sequences inserted in our genome have been subject to inactivating changes and probably represent garbage sequences . molecular studies have shown that syncytin-1 ( env w ) shares a structure similar to class i viral fusion proteins , especially in the region of the n- and c - terminal heptad repeats ( nhr and chr ) , and shares a common fusion mechanism with these proteins ( chang et al . a synthetic peptide derived from the chr is also capable of inhibiting syncytin-1-mediated fusions by perturbing this mechanism ( chang et al . syncytin-1 binds to the d - type retroviral receptor asct-2 ( blond et al . 2000 ) and may use another neutral amino acid transporter ( asct-1 ) as an auxiliary receptor ( lavillette et al . syncytin antibodies , syncytin-1 downregulation through antisense oligonucleotides and the syncytin-1 chr peptide have been shown to inhibit fusions between trophoblast - derived cells ( blond et al . agents increasing cellular levels of camp or camp analogues have been shown to promote cytotrophoblast fusions in vitro ( keryer et al . 1998 ) and such agents are also known to elevate protein and mrna levels of syncytin-1 in isolated cytotrophoblasts ( frendo et al . also estradiol may regulate syncytin-1 expression ( carino et al . 2003 ) and the placenta - specific transcription factor gcma interacts with two upstream sites in the herv - w 5-long terminal repeat and stimulates syncytin-1 transcription ( yu et al . additionally , experimentally induced truncations in the cytoplasmic tail of syncytin-1 increases its fusogenicity ( drewlo et al . 2006 ) , similar to what has been observed for some virally derived env proteins ( reviewed by kubo et al . however , if modifications in the cytoplasmic tail of syncytin-1 are of physiological importance for regulating fusogenicity has yet to be demonstrated . a second syncytin - family member , syncytin-2 , syncytin-2 also represents a highly conserved endogenous retroviral envelope gene and is derived from the herv frd sequence ( blaise et al . immunocytochemical studies have localized syncytin-1 primarily to syncytiotrophoblasts as well as to cytotrophoblasts ( fig . 2004 ) , which may reflect differences in antibodies , fixation procedures and controls . a major point of concern is also the degree of cross - reactivity to the related sequences in syncytin-2 , which recently was localized to a subpopulation of cytotrophoblasts ( malassin et al . the syncytin-1 receptor protein asct-2 was recently localized to cytotrophoblasts ( hayward et al . 2007 ) . more studies on the exact distribution of syncytin expression in the placenta using syncytin-1 and -2 specific antibodies seem warranted in order to exactly localize where fusions are likely to occur . nuclei are lightly counterstained with haematoxylin human term placenta immunocytochemically stained with a syncytin-1 peptide antiserum . nuclei are lightly counterstained with haematoxylin trophoblast cell fusions are , however , not unique to the primate placenta . thus , also in the mouse placenta , trophoblasts fuse to form syncytiotrophoblasts but mice do , of course , not express herv sequences . amazingly , an in - silico search of the mouse genome unraveled the existence of two murine endogenous retroviral ( merv ) env genes , labeled syncytin - a and -b ( dupresssoir et al . these genes were also fusogenic and orthologous genes were present in additional species of muridae ( rats , gerbils , voles and hamsters ) and appear to have entered the rodent lineage some 20 million years ago ( dupresssoir et al . 2005 ) . they were expressed in the placenta and at least syncytin - a has been shown to be involved in the formation of syncytiotrophoblasts in mice ( gong et al . this represents an amazing example of parallel acquisition of retroviral genes of importance to reproduction in primates and rodents and poses the question whether also other species may have acquired similar viral genes of importance to cell fusions . the ability of syncytins to induce cell fusions may not be their only physiologic role . thus , recent studies have shown that while syncytin-2 and syncytin - b also possess immunosuppressive activity , syncytin-1 and syncytin - a do not ( mangeney et al . 2000 ) but subsequent studies have revealed the presence of syncytin-1 also in the brain ( antony et al . 2004 ) and in breast , colon and endometrial cancers ( bjerregaard et al . 2007 , larsen , talts , andersen , bjerregaard and larsson : work in progress ) . in fact , in all systems studied so far , from mating yeast to man , a bewildering array of mechanisms have been identified ( see chen and olson 2005 ; chen et al . molecules potentially involved in mammalian cell fusions include adam ( a disintegrin and metalloproteinase domain ) 12 ( meltrin alpha ) , which has been associated with myoblast and osteoclast cell fusions ( abe et al . 1999 ; in addition , adams 1 and 2 ( fertilins ) may be involved in sperm oocyte fusions , but do not seem indispensable for this function and , in man , the fertilin alpha gene is dysfunctional ( chen and olson 2005 ; cho et al . cd9 is also expressed by bewo trophoblast tumor ( choriocarcinoma ) cells and has been linked both to bewo invasiveness and to invasion during mouse embryo implantation ( hirano et al . 1999 ; liu et al . cd9 has , together with another tetraspanin family member , cd81 , also been linked to myoblast fusion and myotube maintenance ( tachibana and hemler 1999 ) . moreover , antibodies to either cd9 or cd81 have been shown to block fusions induced by mason pfizer monkey virus a d - type retrovirus ( duelli et al . 2005 ) . tetraspanins are known to organize other proteins into membrane microdomains and may link to the actin cytoskeleton via ewi ( glu - trp - ile ) and erm ( ezrin radixin moesin ) proteins ( hemler 2003 ; sala - valds et al . possibly , their role as organizers of other proteins into microdomains may play a role in their involvement with cell fusions ( zivyat et al . they do not express characteristics of fusogenic proteins like the syncytins , snares and class i viral envelope proteins , but are coexpressed with syncytin-1 , at least in bewo cells . a macrophage fusion receptor ( mfr , sirpalpha ) , resembling cd4the cell surface receptor for hiv has been identified in macrophages ( saginario et al . mfr , which belongs to the immunoglobulin superfamily , binds another member of this family , cd47 . cd47 is also structurally related to proteins expressed by vaccinia and variola viruses ( chen et al . whereas expression of mfr is restricted to myeloid cells and neurons , cd47 is ubiquitously expressed . mfr is transiently induced in macrophages at the onset of fusion while cd47 expression is constant . it has been hypothesized that cd47 initially binds to a long form of mfr to secure recognition and then switches to bind a shorter form to bring the plasma membranes closer ( 510 nm ) for fusion ( vignery 2005 ) . additionally , or alternatively , cd47 may promote calcium entry by forming a membrane pore ( reviewed in chen et al . also the hyaluronan receptor , cd44 , is induced transiently when macrophages start to fuse . mice lacking dc - stamp are osteopetrotic and lack multinucleated osteoclasts and giant cells ( yagi et al . 2005 ) . dc - stamp is a seven - transmembrane receptor , somewhat similar to the hiv co - receptor cxcr4 , but a ligand has yet to be identified . these and many more molecules , including integrins , vacuolar atpase and receptors and their ligands , as well as different signaling intermediates have been associated with cell fusions . with the exception of the syncytins , most of the mammalian molecules so far studied do not fulfill strict criteria for fusogens and several are dispensable for fusions ( reviewed by chen and olson 2005 ; chen et al . however , this may reflect molecular redundancy and does not definitely exclude that these molecules may participate in fusions . it is noteworthy that , in both man and mouse , two different syncytins are expressed and are both fusogenic . it is possible that , in the mammalian system , fusion requires a flotilla of molecules organized into membrane microdomains by proteins like cd9 , encompassing receptors / ligands , signaling entities , proteases and fusogens of retroviral origin that are capable of forming alpha - helical bundles , which bring membranes closer . syncytins and related retroviral envelope sequences may , in this connection , function both as fusogens and receptor ligands . however , it seems unlikely that as irreversible an event as a cell cell fusion should depend upon a single receptor ligand interaction . thus , both facilitatory and inhibitory factors are expected to be part of the flotilla . studies by mortensen et al . ( 2004 ) documented that human breast cancer cells fused with endothelial cells in culture . stimulated by studies showing that syncytin-1 was involved in cytotrophoblast cell fusions ( blond et al . 2000 ; mi et al . 2000 ) , we examined whether a similar mechanism could account for cancer endothelial cell fusions . expression of syncytin-1 was documented in the breast cancer cell lines examined ( mcf-7 and mda - mb-231 cells ; bjerregaard et al . 2006 , sk - br-3 cells : talts , bjerregaard and larsson : unpublished data ) . moreover , we found that both tumor cells and endothelial cells expressed the syncytin-1 receptor asct-2 . use of phosphorthioate - protected syncytin-1 antisense oligonucleotides downmodulated syncytin-1 expression as measured by either quantitative rt - pcr or western blotting and inhibited breast cancer endothelial cell fusions , whereas a scrambled oligonucleotide control was without effect . additionally , the syncytin-1 chr peptide , referred to above , also inhibited the fusions whereas a control peptide was without effect ( bjerregaard et al . however , neither the antisense nor the chr peptide experiments effected a total inhibition of cancer - endothelial cell fusions . thus , the antisense oligonucleotide did not totally downmodulate syncytin-1 levels and could therefore not be expected to decrease cell fusions to zero and the lack of total inhibition by the chr peptide could potentially be ascribed to proteolytic degradation . however , we also detected that the breast cancer cells produced syncytin-2 and can not exclude that also this molecule contributed to the cancer further experiments using shrna - directed downmodulation of both syncytin-1 and -2 as well as of additional putative fusogenic retroviral sequence are now underway to test this . we next examined two series of human breast cancer patients for tumoral expression of syncytin-1 using a polyclonal antiserum raised to a synthetic nonapeptide derived from the syncytin-1 sequence . in addition , tumors were also screened for expression of asct-2 using a peptide antiserum . preabsorption of the antisera with the corresponding peptides , but not with irrelevant peptides , abolished staining ( larsson et al . the results showed that 38% of all breast cancer samples showed detectable staining for syncytin and that endothelial cells expressed asct-2 ( fig . 6 ) . moreover , significant expression of asct-2 was detected also in many tumor cells ( fig . . the degree of syncytin immunostaining was visually graded using coded specimens and statistical analysis showed that it correlated positively with disease - free survival of the patients ( larsson et al . 2007b ) . multivariate analysis included age dichotomized at 40 years , tumor size dichotomized at 20 mm , grade and adjuvant therapy and identified syncytin expression as an independent prognostic indicator of increased disease - free survival also when used as a continuous variable , syncytin expression emerged as a significant prognostic indicator for disease - free survival in the cox model ( p = 0.02 ) ( larsson et al . 6human breast cancers immunocytochemically stained for syncytin-1 ( a ) or with antigen - preabsorbed antiserum ( b ) and for asct-2 ( c , e ) or with antigen - preabsorbed antiserum ( d ) . note presence of syncytin immunoreactivity in tumor cells and of asct-2 immunoreactivity in endothelial cells ( arrows ) and in tumor cells human breast cancers immunocytochemically stained for syncytin-1 ( a ) or with antigen - preabsorbed antiserum ( b ) and for asct-2 ( c , e ) or with antigen - preabsorbed antiserum ( d ) . note presence of syncytin immunoreactivity in tumor cells and of asct-2 immunoreactivity in endothelial cells ( arrows ) and in tumor cells the involvement of syncytin-1 in tumor cell fusion events was subsequently confirmed by strick et al . ( 2007 ) , working with endometrial carcinomas . in their study , downmodulation of synctytin-1 expression also inhibited fusions between endometrial tumor cells . in agreement with findings on placental cells ( vide supra ) , both camp elevating agents and estrogens upregulated syncytin-1 expression . however , only camp elevating agents stimulated cell fusions ( strick et al . this apparent discrepancy may possibly be ascribed to the fact that estrogen treatment upregulated tgfbeta , which interfered with syncytin - induced fusions . conversely , additions of tgfbeta 1 or 3 reduced fusions induced by camp - elevating agents . this effect was observed both in endometrial carcinoma cells and in trophoblast - derived cells . these results show that both camp and estrogens positively may regulate syncytin-1 expression in tumor cells and that the tgfbeta family may negatively regulate the fusogenic effects of syncytin-1 in both trophoblasts and cancer cells ( strick et al . 2007 ) . the role of syncytin-1 and cell fusions in cancer needs further study . thus , our data show that syncytin-1 is not the only fusogenic protein expressed by breast cancer cells and the results presented by strick et al . ( 2007 ) show that additional regulators , such as tgfbeta isoforms may be important modulators of cell fusions . so far , our breast cancer data indicate that syncytin-1 expression constitutes a positive prognostic factor . this is not the same as to say that cell fusions may be universally beneficial to cancers . first , syncytins may have additional effects within the tumor environment ( larsson et al . secondly , it seems likely that several factors within the cancer and its stroma ( inactivated tumor suppressor genes , activated oncogenes , expression of fusogens and of cd9 and cd81 as well as tgfbeta ) may act together to bring about a tumor profile that may be as diverse as the one demonstrated by the cell fusion experiments referred to above . interestingly , however , expression of cd9 has been analyzed in a number of tumors and seems , like syncytin expression , to predict a good prognosis ( funakoshi et al . we propose that syncytins may be fusogens of importance to both trophoblast and cancer cell fusions and that they possibly also may mediate additional cell fusion events , acting in concert with other molecules with both enhancing and inhibitory regulatory effects .
cell fusions are important to fertilization , placentation , development of skeletal muscle and bone , calcium homeostasis and the immune defense system . additionally , cell fusions participate in tissue repair and may be important to cancer development and progression . a large number of factors appear to regulate cell fusions , including receptors and ligands , membrane domain organizing proteins , proteases , signaling molecules and fusogenic proteins forming alpha - helical bundles that bring membranes close together . the syncytin family of proteins represent true fusogens and the founding member , syncytin-1 , has been documented to be involved in fusions between placental trophoblasts , between cancer cells and between cancer cells and host cells . we review the literature with emphasis on the syncytin family and propose that syncytins may represent universal fusogens in primates and rodents , which work together with a number of other proteins to regulate the cell fusion machinery .
Cell fusions in the developing mammal Cell fusions contribute to tissue repair Cell fusions during viral infections Cancerhost cell fusions Mechanisms behind cellcell fusions Fusions between normal cells Cancer cell fusions
the fusion between the sperm and the egg marks the beginning of a new individual and ensuing development of either man or beast depends upon a number of additional cell fusion events ( reviewed by ogle et al . however , it is still debated whether such fusions are important to tissue repair and whether they engage bmd stem cells or more differentiated fusogenic cells like monocytes / macrophages ( reviewed by vignery 2005 ) . the viral genome encodes envelope ( env ) proteins , which bind to cell surface receptors and assist the virus in entering the cell . the cleaved peptides undergo conformational changes , resulting in hairpin - like , alpha - helical bundles , which bring the viral and cell membranes into close apposition and thereby facilitates fusion ( reviewed by chen and olson 2005 ) . due to their major importance to fertilization , placentation , muscle development , bone structure , calcium homeostasis and the immune defense system , additionally , the potential role of cell fusions as a repair mechanism and the role of cell fusions in cancer development and progression have further stimulated research in this field . of the many proteins , which to date have been shown ( or proposed ) to be involved in cell fusions in mammals , only the syncytin family appears to use a similar alpha - helical mechanism . the founding family member , syncytin-1 , was discovered as a protein capable of mediating fusions between cytotrophoblasts into syncytiotrophoblasts ( blond et al . they were expressed in the placenta and at least syncytin - a has been shown to be involved in the formation of syncytiotrophoblasts in mice ( gong et al . molecules potentially involved in mammalian cell fusions include adam ( a disintegrin and metalloproteinase domain ) 12 ( meltrin alpha ) , which has been associated with myoblast and osteoclast cell fusions ( abe et al . it is possible that , in the mammalian system , fusion requires a flotilla of molecules organized into membrane microdomains by proteins like cd9 , encompassing receptors / ligands , signaling entities , proteases and fusogens of retroviral origin that are capable of forming alpha - helical bundles , which bring membranes closer . secondly , it seems likely that several factors within the cancer and its stroma ( inactivated tumor suppressor genes , activated oncogenes , expression of fusogens and of cd9 and cd81 as well as tgfbeta ) may act together to bring about a tumor profile that may be as diverse as the one demonstrated by the cell fusion experiments referred to above . we propose that syncytins may be fusogens of importance to both trophoblast and cancer cell fusions and that they possibly also may mediate additional cell fusion events , acting in concert with other molecules with both enhancing and inhibitory regulatory effects . due to their major importance to fertilization , placentation , muscle development , bone structure , calcium homeostasis and the immune defense system , additionally , the potential role of cell fusions as a repair mechanism and the role of cell fusions in cancer development and progression have further stimulated research in this field . of the many proteins , which to date have been shown ( or proposed ) to be involved in cell fusions in mammals , only the syncytin family appears to use a similar alpha - helical mechanism . the founding family member , syncytin-1 , was discovered as a protein capable of mediating fusions between cytotrophoblasts into syncytiotrophoblasts ( blond et al . molecules potentially involved in mammalian cell fusions include adam ( a disintegrin and metalloproteinase domain ) 12 ( meltrin alpha ) , which has been associated with myoblast and osteoclast cell fusions ( abe et al . these and many more molecules , including integrins , vacuolar atpase and receptors and their ligands , as well as different signaling intermediates have been associated with cell fusions . it is possible that , in the mammalian system , fusion requires a flotilla of molecules organized into membrane microdomains by proteins like cd9 , encompassing receptors / ligands , signaling entities , proteases and fusogens of retroviral origin that are capable of forming alpha - helical bundles , which bring membranes closer . secondly , it seems likely that several factors within the cancer and its stroma ( inactivated tumor suppressor genes , activated oncogenes , expression of fusogens and of cd9 and cd81 as well as tgfbeta ) may act together to bring about a tumor profile that may be as diverse as the one demonstrated by the cell fusion experiments referred to above . we propose that syncytins may be fusogens of importance to both trophoblast and cancer cell fusions and that they possibly also may mediate additional cell fusion events , acting in concert with other molecules with both enhancing and inhibitory regulatory effects .
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genome mapping has become an essential tool in order to identify and characterize the genetic basis of phenotypic traits of organisms as well as to detect single genes of interest ( e.g. disease related genes ) or to improve selective breeding . genome mapping also offers the possibility of comparative mapping , which brings valuable knowledge production potential about genome structure , function , and evolution . several mapping methods exist that have shaped the work of geneticists in the past decades . genetic linkage maps or radiation hybrid maps give a global view of genetic markers mapped to groups that in a best case scenario correspond to whole chromosomes [ 1 , 2 ] . while these approaches characterize a species genome with relatively low marker density ( about one marker per mbp genomic sequence ) , today mapping approaches that use large insert clone libraries such as bacs ( bacterial artificial chromosomes ) [ 3 , 4 ] have a higher impact , not only offering very dense maps but also revealing a part of the genome sequence and gene content . in the age of genomics mapped clones are of special value for the analysis of distinct genomic regions by a variety of molecular techniques . especially , the sequencing of large genomes takes profit from bac maps , as they enable the reconstruction of chromosomal sized sequences from draft whole genome shotgun assemblies or sequencing strategies that apply a hierarchical first map then sequence strategy . among the methodologies to construct bac maps , fingerprinting by restriction analysis [ 57 ] or hybridization of labeled marker sequences to bac colony filters , as well as pcr - based methods [ 9 , 10 ] , have been widely used for a variety of species to build de novo maps of overlapping bac clones . today , protocols for high - throughput sequencing of bac end sequences ( bac - es ) enable comparative mapping strategies that rely on sequence alignment to closely related species , which have been sequenced to completeness [ 11 , 12 ] . this strategy is based on automated pipelines for sequencing and bioinformatics that have been established worldwide during the last decade enabling the genome analysis of a wide range of species . genomic information of species spanning a wider range of evolutionary distance will contribute to the complex questions of duplication and divergence . furthermore , comparison between genomic - rich species and genomic - poor species has been a powerful tool for unravelling genes as well as specific genome regions of interest . in addition , comparative genomics is broadly used to identify and characterize functional regions in vertebrate genomes by the detection of evolutionary constrained sequences ( purifying selection on functional sequences ) , or for revealing directional selection operating on different parts of the genome . the present work together with work on the european seabass , dicentrarchus labrax , adds genomic information to much shorter distances . in addition , due to their interest for aquaculture these species have already piled up an important body of biological knowledge : they are dwelling in about the same geographic range with a relatively stable gradient of physicochemical parameters , but have distinct ecology , behaviour , sex determination systems , contrasting reproductive biology , differential stress tolerance to a set of stressors , differential tolerance to pathogens , physiology , and so forth . these are applicable to closely related species for identifying adaptive evolution and functional variation by fine scale comparisons of genes and genomic regions and by simultaneous investigation of sequence polymorphism and divergence . whereas the assignment of homologues groups between closely related species can be achieved already by low density maps [ 1517 ] rearrangements take place by processes such as translocation , inversions , duplications , and deletions . therefore , in addition to synteny mapping ( on the same chromosome ) , reconstructing homologous colinearity by comparative bac maps will enhance significantly the detection of new regions in the genome that are important for function as well as it will contribute to a better differentiation of lineage - specific constraint . to date the comparative bac mapping method is particularly suitable to map fish genomes , because several good quality draft reference fish genomes are already available in public databases for single or multispecies comparison . five teleost fish genomes have been nearly completely sequenced ( http://www.ensembl.org ) : the zebrafish ( danio rerio ( hamilton ) ) , the medaka ( oryzias latipes ( temminck and schlegel ) ) and the three - spined stickleback ( gasterosteus aculeatus l. ) belonging to the order of cypriniformes , beloniformes , and gasterosteiformes , respectively , as well as the two pufferfishes ( takifugu rubripes ( temminck and schlegel ) and tetraodon nigroviridis ( marion de proc ) ) belonging to the order of tetraodontiformes . sequencing of fish genomes is of particular interest as among vertebrates the ray - finned fishes ( actinopterygians ) are the most diverse groups comprising approximately 23,700 extant species . genome projects in teleosts were first focused on model species either due to their compact genome size ( pufferfishes ) , their importance in developmental research ( zebrafish ) , or as ecological model ( three - spined stickleback ) . the genome analysis of the three main model fish species , the zebrafish ( danio rerio ) , pufferfish ( tetraodon nigroviridis ) and medaka ( oryzias latipes ) , have shown that two rounds of genome duplications took place early in vertebrate evolution as well as a third round of genome duplication occurred in the ray - finned fish before the teleost radiation [ 2325 ] . the interest to study nonmodel fish species first was based either on their importance in evolution or on their high commercial impact . the latter enhanced the research in the two main species of commercial interest in the mediterranean , the gilthead sea bream sparus aurata l. and the european seabass dicentrarchus labrax l. the gilthead sea bream belongs to the order perciformes and sparidae family which comprises about 100 species of which 24 are described from the north / eastern atlantic and mediterranean coasts but also in the red sea and australia - new zealand . the family of sparidae also includes several species such as dentex dentex , pagrus pagrus , diplodus puntazzo which are important to mediterranean aquaculture or pagrus major which is routinely produced in japan . sparus aurata is one of the main target species for aquaculture and fisheries in europe with 133,000 tons aquaculture production in 2008 ( http://www.fao.org/fishery/culturedspecies/sparus_aurata/en ) . sea bream is a mass spawner having a 2 - 3 months sequential spawning of batches of eggs and a particular sex determination system that is characteristic also for other candidates in aquaculture . sea bream belongs to the protandrous hermaphrodite turning into females after the 2nd year [ 2628 ] . another characteristic behaviour of sea bream is cannibalism between juveniles of different size . in general sea bream we have recently published a comparative bac map of the european seabass , which is closely related to the gilthead sea bream ( both order of perciformes ) . the three - spined stickleback gasterosteus aculeatus genome was successfully used as reference genome in this regard . stickleback does not belong to the order perciformes , but is the most closely related genome to seabass among the currently available five sequenced teleost genomes . the comparative bac mapping has already shown that some stickleback chromosomes have a nearly complete synteny with those of seabass . here we present the results of comparative mapping of bac end sequences of the gilthead sea bream to the three - spine stickleback as well as to contigs of the european seabass genome project ( kuhl et al . the establishment of a dataset containing information about similarities , synteny relationships as well as colinearity will enhance significantly future approaches to identify and characterize new and novel functional genome regions . it also represents an important first step for gilthead sea bream whole - genome sequencing by second generation techniques as well as high - throughput snp detection analysis , precise mapping of ests of specific interest , the identification of members of gene families and the differentiation of orthologous from paralogous genes . the sparus aurata bac - library was constructed by amplicon express and was provided by the institute of marine biology and genetics of the hellenic centre for marine research ( imbg - hcmr ) . the total genome coverage of the library is 7 - 8-fold with an average insert size of 120 kbp per bac - clone . for template preparation , bac - clones were inoculated in 2 384 deep well plates containing 190 l of 2yt media and 12.5 mg / l chloramphenicol and cultivated for 18 hours at 37c with rigorous shaking at 1100 rpm in titramax 1000 incubators ( heidolph instruments ) . e. coli cells were centrifuged at 2750 g for 5 minutes ( rt ) and the supernatant was removed . the bacterial pellets were resolved in 20 l buffer 1 ( 50 mm tris hcl , 10 mm edta , ph 8 supplemented with 25 mg / l rnase ) . cell lysis was performed with 20 l of buffer 2 ( 0.2 m naoh , 1% sds ) for 5 minutes and for neutralization 20 l of buffer 3 ( 0.933 m kacetat , ph 4.8 ) was added . cell debris was separated from dna containing supernatant by centrifugation at 2750 g for 30 minutes ( rt ) . supernatants from both plates ( 40 l each ) were combined in a new 384 deep well plate for size selective dna precipitation with 38 l of a polyethylene - glycol 6000/2-propanol mixture ( 75% 2-propanol(v / v)/100 g / l peg-6000 ) and centrifuged at 2750 g for 30 minutes at 4c . supernatants were depleted by centrifugation of the inverted plates at 172 g for 1 minute ( rt ) . the remaining dna pellets were washed with 40 l of 70% ethanol ( v / v ) to remove residual salts and peg . most of the bac - dna template preparations were done by an automated process that was developed at the mpi for molecular genetics . bac - dna templates were end sequenced using abi bigdyev3.1 terminator chemistry and t7 or sp6 primers . after postsequencing cleanup by ethanol / natrium acetate ( naoac ) precipitation , sequence analysis was performed on abi3730xl capillary sequencers with 36 cm capillary arrays . processing of raw sequencing data was done by the phred basecaller , quality clipping , and vector - clipping by lucy . initially , bac end sequences ( bac - es ) were aligned to the stickleback genome using blastn with low stringency parameters ( -w 7 -q -1 -e 0.01 ) . the output tables were further screened for the best hits of bacs aligned with both ends that matched the same chromosome in the reference . a further screening step involved orientation ( ) and distance between paired bac ends ( insert size smaller than 300 kb ) . to improve the number of mapped bacs we did a multiple genome alignment approach . we aligned all assembled contigs from the advanced seabass genome project ( unpublished results , for a brief description see ) to the stickleback genome and rebuild the stickleback genome with the seabass contigs . we then performed the same alignment steps as above for the sea bream bacs on the virtual seabass chromosomes and transformed the hit coordinates back to the stickleback sequence . again read pairs were checked for consistency and alignments of both approaches were combined to result in an increased number of mapped bacs . for those bacs that mapped in both approaches we calculated the distance between the coordinates found by direct mapping and by mapping via seabass sequence , to check the placement error distribution induced by the coordinate transformation . if there were discrepancies between the same bacs in the two alignment approaches we considered those results that had better matches according to our consistency criteria . the start and end coordinates of the bac clones were converted to gff format and included in the ensembl genome browser for the stickleback genome ( http://www.ensembl.org/gasterosteus_aculeatus/info/index ) . bacs that form a minimal tiling path were chosen as described in and may be browsed independently of the other placed bacs . the number of bacs sequenced from both ends was 41,509 ( physical coverage ~5.5x-6.5x ) . bacs sequenced from one direction accounted for 5,485 ( only t7 dir./~0.7x-0.8x ) and for 3,965 ( only sp6 dir./~0.5x-0.6x ) . the average , the sequences were submitted to embl nucleotide database [ embl : fr502695-embl : fr595162 ] . mapping all sequences directly to the stickleback genome resulted in 70,425 hits ( 76.2%/just best hit was counted ) . further screening for paired bac ends matching to a single reference chromosome resulted in 19,776 hits ( 23.8% of paired end sequenced bacs ) . this indicates that many hits in the raw blast are false positives as expected due to the low stringency parameters used . after checking for read orientation and distance 17,184 hits ( 8,592 bacs/20.7% of paired bac es ) were consistently mapped . these bacs ( 1.16x1.3x physical genome coverage ) were already useful for building contigs of overlapping bac - clones , but these covered only 58.1% of the stickleback chromosomes . to further improve the mapping results we tried a multiple genome alignment approach . unpublished results ) were concatenated according to the order of their ortholog sequences in the stickleback genome . as seabass belongs to the order of perciformes , additionally , synteny of genes is more conserved than nucleotide sequence in distantly related teleosts , which legitimizes the use of the nucleotide sequence of a closer relative of sea bream ordered by a species that is not as close in the phylogeny . after aligning sea bream bac es to this sequence the hit coordinates this way results from directly mapping to stickleback and indirectly mapping to stickleback ( via orthologous seabass sequence ) can be combined . a total of 87,614 bac ends ( 94.8% ) had hits with low stringency blast in the multiple genome alignment approach . paired bac ends that mapped to the same reference chromosome accounted for 34,174 reads ( 41.2% of paired bac es ) . consistency checks of read pairs resulted in 29,392 ( 14,696 bacs/35.4% of paired bac es ) . the combination of both mappings resulted in 28,530 bac es ( 14,265 bacs ) with consistent mapping to the 21 stickleback chromosomes ( 1,488 bac es from direct and 27,042 from indirect mapping ) . the insert size distribution of mapped clones and its implications on genome size are shown in figure 1 . ( see excel sheet with mapped bac es in supplementary material available online at doi : 10.11552011329025 ] . ) as the transformation of the seabass sequence coordinates to coordinates on stickleback sequence introduces some placement error , we compared the coordinates of bac clones ( 7,646 ) that were consistently mapped in both mapping approaches . the median deviation of start and end between the two mapping strategies ( see figure 2 ) was 66 bp , the average deviation was 293 bp , and 96.7% of the mapped bacs had a deviation of less than 1,000 bp . as the placed bacs and their overlaps are much larger than these values , the fuzziness introduced by the seabass to stickleback coordinate transformation seems to be negligible . the minimal tiling path calculated from consistently placed bacs covered 73.5% of stickleback chromosomes with about 13,415 gene loci predicted ( 70.2% of 19,121 genes predicted in stickleback chromosome sequences ) . the largest bactig covers 2.15 mbp and n50 bactigs size is 337,253 bp . these sizes refer to the stickleback genome , as there is a large size difference between stickleback and sea bream the bactig sizes should be multiplied by a factor of 1.72 to estimate their sizes in sea bream . the mapped clones may be browsed alongside the stickleback genome and the ordered seabass bac clones at www.ensembl.org ( login : heiner7@hotmail.com/passw : breambacmap2010 ) . for a comparison of sea bream and seabass minimal tiling path clones mapped to stickleback chromosome vi and xxi , see figure 3 ( for all stickleback chromosomes see supplementary material . clones with inconsistent matches of both ends in terms of distance or orientation were screened manually for potential intra - chromosomal rearrangements . if an inconsistency was indicated by two or more independent bac clones it was considered a potential rearrangement . studying the genomes of important commercial species is of direct value to the efficiency and exploitation of the species . despite the fact that today access to other fish genomes , including the sister species of the gilthead sea bream the european seabass dicentrarchus labrax , is available , additional genome information is of importance concerning identification of species specific characteristics like disease resistance , salinity , and temperature tolerance by comparative genomics additionally , it will offer the possibility to use population genomics approaches combining interspecific divergence with intraspecific diversity for identifying adaptive variation , or comparative genomics for identification of small noncoding regions of functional importance . the power of such approaches is in general decreasing with increasing evolutionary distance , either because of intervening confounding effects and increased complexity or because of alignment problems . the identification of ultraconserved elements ( uces ) , similar genomic elements as well as conserved noncoding elements ( cnes ) have pin pointed to genomic regulatory blocks , developmental regulatory target genes ( s ) and phylogenetically relevant and functionally genes . comparative mapping of bac - es is a fast and efficient method to deduce genome maps of nonmodel organisms from sequenced genomes of model organisms due to conserved synteny . thus the tremendous efforts that have been undertaken for model organisms in the past decade pay out now for the genomics of farmed animals . we have recently shown for the european seabass that the stickleback genome is a good choice for comparative studies in fish belonging to the order of perciformes . in this study nucleotide alignments in the present study indicate that sea bream and stickleback may be more diverged than seabass and stickleback . the resulting mapping data represents a valuable tool for genomic research in this important aquaculture species . bac clones covering regions of interest may be easily chosen for advanced analysis using the ensembl genome browser tool . besides these benefits some conclusions can be drawn on genome size of sea bream , when comparing the distribution of distances between two corresponding bac ends mapped to the stickleback chromosomes and their real distance observed by pulse field gelelectrophoresis during bac library construction . to estimate an organism 's genome size flow cytometry of cells with stained nuclei has been widely applied . according to www.genomesize.com the resulting c - value found by this method for g. aculeatus is 0.58 pg of haploid dna content . in d. labrax the c - value accounts for 0.78 pg and in s. aurata it is even higher with 0.95 pg . if one relates the c - values to the lowest value observed in stickleback , the seabass genome should be 1.34-fold larger and the sea bream genome should be 1.64-fold larger than the stickleback genome . these values are in good concordance with values found by bac insert size distributions of the comparative mappings : s. aurata / g . aculeatus = 1.3 . nevertheless predicting the genome size by sequencing - based methods results in lower values for each genome compared to the size estimations of flow cytometry - based methods ( g. aculeatus 462 mbp versus 567 mbp ; d. labrax 601 mbp versus 763 mbp ; s. aurata 791 mbp versus 929 mbp ) . on the one hand , sequencing - based methods tend to underpredict genome sizes as some regions are hard to assemble and are later on missing in the published genome assemblies . on the other hand , flow cytometry might overestimate genome sizes due to including ongoing replication of dna in the measurements or some staining background of rna : rna or rna : dna hybrids . thus , we believe that the true genome size of the three teleosts lies in between the borders defined by the two approaches . the differences in genome - size among species may arise from insertion or deletion ( indel ) of large dna fragments and/or insertion or deletion of small dna sequences . given their frequency particularly the small deletions largely outnumber small insertions [ 37 , 38 ] , small indels have been recognised as major mechanisms responsible for genome - size evolution in eukaryotic organisms . several studies have also shown that the wide range of variation in genome size among eukaryotic organisms is correlated with the amount of repetitive elements in the genome [ 3941 ] . in fishes for example , the contribution of repetitive elements to genome - size difference between medaka and takifugu is estimated about 54% . although the above interpretations are coherent and in agreement with results previously reported in fishes on the forces driving the evolution of genome size , they are still speculative since they are not based on comparisons at nucleotide sequence level . further analysis of indels and repetitive elements are required to clarify the respective contribution to the differences in genome size between these species . this could be done when the genome of seabass and sea bream will be completely sequenced or in near future via a targeted analysis of large conserved sequence regions between the three species . the mapping of the bac inserts was done in a similar way as described here for the sea bream . when comparing both maps , the seabass map outperforms the sea bream map in most parameters like reference coverage ( 87.0% versus 73.5% ) , covered stickleback gene predictions ( 85.4% versus 70.2% ) , n50 bactig size ( 1.2 mbp versus 0.34 mbp ) , or total number of bactigs ( 588 versus 1,485 ) . differences in map quality may be based on the lower physical genome coverage and average insert size of the gilthead sea bream bac library than the european seabass bac library . the lower coverage of mapped bacs for sea bream complicates the detection of chromosomal rearrangements . nevertheless , we could identify a number of 202 potential intrachromosomal rearrangements between stickleback and sea bream that were spanned by two or more clones . this number is similar to what was found , when comparing stickleback to seabass ( 214 potential rearrangements , ) . seabass shares 114 of the 202 rearrangements with sea bream , thus the phylogenetic relationship between the three species seems to be reflected by the number of rearrangements . although the alignment coordinates of additional 1,402 sea bream bacs imply other large , scale intra - chromosomal rearrangements , their evidence is weak as they are covered by a single bac clone only . . nevertheless , inconsistently mapped bacs ( see supplementary material ) may be subject to further investigations ( e.g. , checking overlaps by pcr ) to proof further potential rearrangements . the comparative sea bream bac map described here is a valuable tool for researchers in the field . it allows the fast selection of bac clones that cover genes or genomic loci of interest by simple blast searches in the stickleback genome and picking one of the bacs covering the hit location . according to the coverage of the stickleback genome we estimate that about 75% of the sea bream genome is covered by the mapped clones . additional genes , which are not represented by mapped bacs , may be found by blast searches against the bac end sequences that comprise about 60.6 mbp of the sea bream genome , the largest set of genomic sequences published for sea bream so far . these data will be also of great value in future projects like a sea bream genome project and will allow scaffolding of wgs contigs or a sequencing strategy based on mapped clones , which will be especially useful , if short read sequencing technologies will be applied . thus , taking also into account the comparative seabass bac map , ordered large insert clones for the two main aquaculture species in the mediterranean are now available and may contribute to answer basic biological questions like what makes up the difference between hermaphrodite ( sea bream ) and temperature - driven sex differentiation ( seabass ) as well as aquaculture - related questions that aim at improved breeding stocks in both species .
this study presents the first comparative bac map of the gilthead sea bream ( sparus aurata ) , a highly valuated marine aquaculture fish species in the mediterranean . high - throughput end sequencing of a bac library yielded 92,468 reads ( 60.6 mbp ) . comparative mapping was achieved by anchoring bac end sequences to the three - spined stickleback ( gasterosteus aculeatus ) genome . bacs that were consistently ordered along the stickleback chromosomes accounted for 14,265 clones . a fraction of 5,249 bacs constituted a minimal tiling path that covers 73.5% of the stickleback chromosomes and 70.2% of the genes that have been annotated . the n50 size of 1,485 bactigs consisting of redundant bacs is 337,253 bp . the largest bactig covers 2.15 mbp in the stickleback genome . according to the insert size distribution of mapped bacs the sea bream genome is 1.71-fold larger than the stickleback genome . these results represent a valuable tool to researchers in the field and may support future projects to elucidate the whole sea bream genome .
1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions
today , protocols for high - throughput sequencing of bac end sequences ( bac - es ) enable comparative mapping strategies that rely on sequence alignment to closely related species , which have been sequenced to completeness [ 11 , 12 ] . five teleost fish genomes have been nearly completely sequenced ( http://www.ensembl.org ) : the zebrafish ( danio rerio ( hamilton ) ) , the medaka ( oryzias latipes ( temminck and schlegel ) ) and the three - spined stickleback ( gasterosteus aculeatus l. ) belonging to the order of cypriniformes , beloniformes , and gasterosteiformes , respectively , as well as the two pufferfishes ( takifugu rubripes ( temminck and schlegel ) and tetraodon nigroviridis ( marion de proc ) ) belonging to the order of tetraodontiformes . the latter enhanced the research in the two main species of commercial interest in the mediterranean , the gilthead sea bream sparus aurata l. and the european seabass dicentrarchus labrax l. the gilthead sea bream belongs to the order perciformes and sparidae family which comprises about 100 species of which 24 are described from the north / eastern atlantic and mediterranean coasts but also in the red sea and australia - new zealand . in general sea bream we have recently published a comparative bac map of the european seabass , which is closely related to the gilthead sea bream ( both order of perciformes ) . the three - spined stickleback gasterosteus aculeatus genome was successfully used as reference genome in this regard . here we present the results of comparative mapping of bac end sequences of the gilthead sea bream to the three - spine stickleback as well as to contigs of the european seabass genome project ( kuhl et al . we then performed the same alignment steps as above for the sea bream bacs on the virtual seabass chromosomes and transformed the hit coordinates back to the stickleback sequence . bacs that form a minimal tiling path were chosen as described in and may be browsed independently of the other placed bacs . the minimal tiling path calculated from consistently placed bacs covered 73.5% of stickleback chromosomes with about 13,415 gene loci predicted ( 70.2% of 19,121 genes predicted in stickleback chromosome sequences ) . the largest bactig covers 2.15 mbp and n50 bactigs size is 337,253 bp . besides these benefits some conclusions can be drawn on genome size of sea bream , when comparing the distribution of distances between two corresponding bac ends mapped to the stickleback chromosomes and their real distance observed by pulse field gelelectrophoresis during bac library construction . if one relates the c - values to the lowest value observed in stickleback , the seabass genome should be 1.34-fold larger and the sea bream genome should be 1.64-fold larger than the stickleback genome . when comparing both maps , the seabass map outperforms the sea bream map in most parameters like reference coverage ( 87.0% versus 73.5% ) , covered stickleback gene predictions ( 85.4% versus 70.2% ) , n50 bactig size ( 1.2 mbp versus 0.34 mbp ) , or total number of bactigs ( 588 versus 1,485 ) . differences in map quality may be based on the lower physical genome coverage and average insert size of the gilthead sea bream bac library than the european seabass bac library . the comparative sea bream bac map described here is a valuable tool for researchers in the field . according to the coverage of the stickleback genome we estimate that about 75% of the sea bream genome is covered by the mapped clones . additional genes , which are not represented by mapped bacs , may be found by blast searches against the bac end sequences that comprise about 60.6 mbp of the sea bream genome , the largest set of genomic sequences published for sea bream so far . thus , taking also into account the comparative seabass bac map , ordered large insert clones for the two main aquaculture species in the mediterranean are now available and may contribute to answer basic biological questions like what makes up the difference between hermaphrodite ( sea bream ) and temperature - driven sex differentiation ( seabass ) as well as aquaculture - related questions that aim at improved breeding stocks in both species .
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osteoporotic vertebral compression fracture ( ovcf ) is a common cause of pain and disability in the elderly population , affecting nearly one - quarter of individuals over the age of 50 during their lifetime . worldwide , there are approximately 1.4 million new ovcfs reported each year , with approximately 750 000 annually in the united states alone [ 13 ] . ovcfs often result in significant pain that often leads to decreased mobility , loss of independence , and subsequent loss of bone density , and have negative effects on the respiratory and digestive systems . the direct medical costs associated with ovcfs have been estimated at $ 13.8 billion annually in the us alone , and indirect costs in lost productivity , pain , and suffering are even greater . conservative management , including bed rest , painkillers , and bracing , may help to reduce pain over weeks or months ; however , in frail elderly patients , long periods of inactivity are associated with higher rates of pneumonia , decubitus ulcers , venous thromboembolism , and even death . invasive surgery is not the optimal treatment due to their usually old age and associated comorbidities . today , vertebral augmentation procedures , including percutaneous vertebroplasty ( pvp ) and percutaneous kyphoplasty ( pkp ) , are commonly used in the treatment of ovcfs [ 810 ] . pkp utilizes inflatable bone tamps to reduce fracture , restore vertebral anatomy , and control cement injection . since the first report of pvp in 1987 , the use of pvp and pkp for the treatment of ovcfs has been dramatically increased during the last 20 years . potency and effectiveness of these procedures recently , a us large claims database analysis showed that pkp was associated with a significant reduction in mortality compared to conservative treatment and pvp . pkp is now considered to be as minimally invasive and effective as conventional pvp for rapid pain relief in patients with painful ovcfs resistant to conservative treatment . in established osteoporosis , the definition of a vertebral fracture is usually based on the presence of a deformation of the vertebral body on lateral radiographs . early diagnosis of ovcfs is crucial and the detection of vertebral fractures is based primarily on the identification of vertebral collapse . however , this can be misleading in the presence of a fracture without radiologic collapse , and the definition of occult osteoporotic vertebral fractures ( oovfs ) has been established . patients highly suspected to have a fresh occult vertebral fracture , who have persistent pain and normal x - ray and ct images , should undergo additional radiologic tests , especially mri . mri is the most confirmative screening examination used to determine the presence of painful oovfs ; t2 and stir sequences of mri images show hyperintensity signal because of bone edema in the affected vertebra . oovfs are disabling causes of severe low back pain , requiring bed rest and increasing the risk of comorbidities . moreover , these patients usually require high doses of painkillers , especially for multiple oovfs , which can cause serious adverse effects . thus , it is essential and necessary to enhance the vertebral body with occult fracture to prevent further vertebral collapse . because the number oovfs patients was small and usually mixed with other ovcfs cases , clinical treatment of oovfs has been rarely discussed . few reports exist documenting the outcomes of vertebral augmentation procedure as an alternative to traditional conservative therapy for painful oovfs , most of which are limited to case reports and small series of patients [ 1719 ] . in this report , we present a series of 89 patients with painful oovfs treated with pkp , and we assess the radiological and clinical outcomes . the aim of the research was to assess the effectiveness and safety of pkp in the management of painful oovfs . the research was approved by ethics committee of the first affiliated hospital of soochow university ( suzhou , china ) . from january 2011 to december 2013 , 923 kyphoplasties were performed in 688 patients at our institution . among them , 89 patients ( 55 females and 34 males ) , aged 5679 years , had 1 or 2 oovfs , and underwent 142 pkp procedures . the mean time since onset of pain to the intervention was 1.25 months in all patients . bone mineral density was preoperatively measured as an indicator of osteoporosis with dual - energy x - ray absorptiometry in all cases . we only included the osteoporotic patients ( according to the world health organization ; these patients have a t - score 2.5 or lower , meaning a bone density that is 2.5 standard deviations below the mean of a 30-year - old man / woman ) . occult osteoporotic vertebral fractures ( oovfs ) were defined as vertebral fractures without radiographic measurable compression . the indications for pkp were oovfs with acute pain and not responding to nonoperative treatment , which included all types of medications , nerve blocks , exercises , and osteoporotic medications to strengthen the bone . patients included in our research underwent a plain x - ray , magnetic resonance imaging ( mri ) , and multislice computed tomography ( msct ) to evaluate the main cause of pain and to plan the treatment . mri was the most confirmative screening examination used to determine the presence of painful oovfs ; stir sequences of mri images show hyperintensity signal because of bone edema in the affected vertebra . patients with local infections , non - correctable coagulation disorders , and other systemic diseases were excluded from the treatment . patients received general anesthesia and were positioned prone on a radiolucent operation table . using image guidance x - rays , a small incision was made and a probe was placed into the vertebral space at the fracture site . the bone was drilled and a balloon ( kyphon inc . , sunnyvale , california ) was inserted on each side . the balloon was then inflated slowly for injecting bone cement . during the cement injection , radiographic assessment and clinical examinations were performed preoperatively , and at 1 month , 6 months , and 2 years after treatment . the anterior and middle vertebral heights were obtained from standing lateral radiographs for compromised and adjacent uncompromised control vertebrae . for each vertebra , the normal height of the compromised vertebrae was estimated from the mean of the measurements from the closest uncompromised vertebral cephalad and caudalad to the treated level . vertebral body height variations were calculated by the following method : ( fractured vertebral body height / normal vertebral body height ) 100% . back pain was measured using a visual analog scale ( vas scores ) with values from 0 to 10 , where 0 indicates no pain and 10 indicates the worst pain imaginable . oswestry disability index ( odi ) scores , which are used to assess functional capacity , were also documented , in which a lower percentage indicates a better health status . the odi has been shown to have high test - retest reliability and is the most commonly recommended condition - specific outcome measure in patients with acute and chronic low back pain . the sf-36 questionnaire is an in - depth and broad - ranging health - related quality of life evaluation consisting of 36 questions , where a higher score indicates a better health status . the results are categorized into 8 domains ; bodily pain ( bp ) , vitality ( vt ) , physical function ( pf ) , and social function ( sf ) have been shown to have high reliability and responsiveness in spinal injury patients and were selected for evaluation in our study [ 2224 ] . complications such as bleeding , infection , stroke , pulmonary embolism , and cardiac arrest were recorded . the rates of cement leakage outside the vertebral body were measured postoperatively using radiographs and ct scans . mean changes , including mean values and standard deviations , in anterior vertebral body height variation and middle vertebral body height variation , sf-36 score , vas and odi score , were evaluated and analyzed with spss software ( spss 19.0 , inc . , comparisons between different time points were performed using a paired student s t test and tested by the repeated - measures analysis of variance ( anova ) . the research was approved by ethics committee of the first affiliated hospital of soochow university ( suzhou , china ) . from january 2011 to december 2013 , 923 kyphoplasties were performed in 688 patients at our institution . among them , 89 patients ( 55 females and 34 males ) , aged 5679 years , had 1 or 2 oovfs , and underwent 142 pkp procedures . the mean time since onset of pain to the intervention was 1.25 months in all patients . bone mineral density was preoperatively measured as an indicator of osteoporosis with dual - energy x - ray absorptiometry in all cases . we only included the osteoporotic patients ( according to the world health organization ; these patients have a t - score 2.5 or lower , meaning a bone density that is 2.5 standard deviations below the mean of a 30-year - old man / woman ) . occult osteoporotic vertebral fractures ( oovfs ) were defined as vertebral fractures without radiographic measurable compression . the indications for pkp were oovfs with acute pain and not responding to nonoperative treatment , which included all types of medications , nerve blocks , exercises , and osteoporotic medications to strengthen the bone . patients included in our research underwent a plain x - ray , magnetic resonance imaging ( mri ) , and multislice computed tomography ( msct ) to evaluate the main cause of pain and to plan the treatment . mri was the most confirmative screening examination used to determine the presence of painful oovfs ; stir sequences of mri images show hyperintensity signal because of bone edema in the affected vertebra . patients with local infections , non - correctable coagulation disorders , and other systemic diseases were excluded from the treatment . patients received general anesthesia and were positioned prone on a radiolucent operation table . using image guidance x - rays , a small incision was made and a probe was placed into the vertebral space at the fracture site . the bone was drilled and a balloon ( kyphon inc . , sunnyvale , california ) was inserted on each side . the balloon was then inflated slowly for injecting bone cement . during the cement injection , radiographic assessment and clinical examinations were performed preoperatively , and at 1 month , 6 months , and 2 years after treatment . the anterior and middle vertebral heights were obtained from standing lateral radiographs for compromised and adjacent uncompromised control vertebrae . for each vertebra , the normal height of the compromised vertebrae was estimated from the mean of the measurements from the closest uncompromised vertebral cephalad and caudalad to the treated level . vertebral body height variations were calculated by the following method : ( fractured vertebral body height / normal vertebral body height ) 100% . back pain was measured using a visual analog scale ( vas scores ) with values from 0 to 10 , where 0 indicates no pain and 10 indicates the worst pain imaginable . oswestry disability index ( odi ) scores , which are used to assess functional capacity , were also documented , in which a lower percentage indicates a better health status . the odi has been shown to have high test - retest reliability and is the most commonly recommended condition - specific outcome measure in patients with acute and chronic low back pain . the sf-36 questionnaire is an in - depth and broad - ranging health - related quality of life evaluation consisting of 36 questions , where a higher score indicates a better health status . the results are categorized into 8 domains ; bodily pain ( bp ) , vitality ( vt ) , physical function ( pf ) , and social function ( sf ) have been shown to have high reliability and responsiveness in spinal injury patients and were selected for evaluation in our study [ 2224 ] . complications such as bleeding , infection , stroke , pulmonary embolism , and cardiac arrest were recorded . the rates of cement leakage outside the vertebral body were measured postoperatively using radiographs and ct scans . mean changes , including mean values and standard deviations , in anterior vertebral body height variation and middle vertebral body height variation , sf-36 score , vas and odi score , were evaluated and analyzed with spss software ( spss 19.0 , inc . , comparisons between different time points were performed using a paired student s t test and tested by the repeated - measures analysis of variance ( anova ) . significant improvements in sf-36 score , vas , and odi score were observed postoperatively and at 1 month , 6 months , and 2 years after treatment . five new adjacent vertebral fractures in 89 patients were detected during the 2-year follow - up period . the levels were t5 ( n=4 ) , t6 ( n=5 ) , t7 ( n=6 ) , t8 ( n=9 ) , t9 ( n=8 ) , t10 ( n=10 ) , t11 ( n=18 ) , t12 ( n=17 ) , l1 ( n=18 ) , l2 ( n=15 ) , l3 ( n=13 ) , l4 ( n=12 ) , and l5 ( n=7 ) ( figure 1 ) . the injected cement volume was 5.21.3 ml and the time of fluoroscopy was 10.51.4 minutes for each level . there were 50 cases ( 81 vertebral bodies ) done with bilateral access and 39 cases ( 61 vertebral bodies ) with unilateral access . a representative case of a 77-year - old female patient with l2 and l4 oovfs who received pkp treatment using unilateral access is shown in figure 2 . there were no treatment - related complications such as bleeding , infection , stroke , pulmonary embolism , and cardiac arrest . general properties of the patients are summarized in table 1 . on the basis of comparison of pre - operative and every postoperative data , anterior vertebral body height and middle vertebral body height were maintained in all cases . the mean anterior vertebral body height variation changed from 96.53.4% preoperatively to 97.22.5% postoperatively ( p>0.05 ) and maintained at 95.73.4% at 2 years postoperatively . the mean middle vertebral body height variation changed from 96.32.8% preoperatively to 97.93.1% postoperatively and maintained at 96.74.4% at 2 years postoperatively . statistically , no significant difference was detected at the immediate postoperative time and every follow - up assessment compared with the preoperative value . the mean vas score of these patients decreased from 8.31.2 preoperatively to 2.90.7 postoperatively and maintained at 3.01.2 at 2 years postoperatively , and the odi score decreased from 76.412.5 preoperatively to 26.75.6 postoperatively and maintained at 27.88.3 at 2-year postoperatively ( table 2 ) . the sf-36 scores for vitality ( vt ) , bodily pain ( bf ) , social functioning ( sf ) , and physical function ( pf ) all showed notable improvement ( p<0.05 ) ( table 3 ) . statistically significant differences were observed at the immediate postoperative time and each follow - up assessment compared with the pre - operative value . the postoperative ct images of the pkp - treated vertebrae indicated a cement extravasation in 12 vertebras , representing 8.45% of all treated levels . cement extravasation was located in the paravertebral soft tissues ( 6 leaks ) , vertebral venous plexus ( 2 leaks ) , and adjacent disk area ( 4 leaks ) . the levels were t5 ( n=4 ) , t6 ( n=5 ) , t7 ( n=6 ) , t8 ( n=9 ) , t9 ( n=8 ) , t10 ( n=10 ) , t11 ( n=18 ) , t12 ( n=17 ) , l1 ( n=18 ) , l2 ( n=15 ) , l3 ( n=13 ) , l4 ( n=12 ) , and l5 ( n=7 ) ( figure 1 ) . the injected cement volume was 5.21.3 ml and the time of fluoroscopy was 10.51.4 minutes for each level . there were 50 cases ( 81 vertebral bodies ) done with bilateral access and 39 cases ( 61 vertebral bodies ) with unilateral access . a representative case of a 77-year - old female patient with l2 and l4 oovfs who received pkp treatment using unilateral access is shown in figure 2 . there were no treatment - related complications such as bleeding , infection , stroke , pulmonary embolism , and cardiac arrest . on the basis of comparison of pre - operative and every postoperative data , anterior vertebral body height and middle vertebral body height were maintained in all cases . the mean anterior vertebral body height variation changed from 96.53.4% preoperatively to 97.22.5% postoperatively ( p>0.05 ) and maintained at 95.73.4% at 2 years postoperatively . the mean middle vertebral body height variation changed from 96.32.8% preoperatively to 97.93.1% postoperatively and maintained at 96.74.4% at 2 years postoperatively . statistically , no significant difference was detected at the immediate postoperative time and every follow - up assessment compared with the preoperative value . the mean vas score of these patients decreased from 8.31.2 preoperatively to 2.90.7 postoperatively and maintained at 3.01.2 at 2 years postoperatively , and the odi score decreased from 76.412.5 preoperatively to 26.75.6 postoperatively and maintained at 27.88.3 at 2-year postoperatively ( table 2 ) . the sf-36 scores for vitality ( vt ) , bodily pain ( bf ) , social functioning ( sf ) , and physical function ( pf ) all showed notable improvement ( p<0.05 ) ( table 3 ) . statistically significant differences were observed at the immediate postoperative time and each follow - up assessment compared with the pre - operative value . the postoperative ct images of the pkp - treated vertebrae indicated a cement extravasation in 12 vertebras , representing 8.45% of all treated levels . cement extravasation was located in the paravertebral soft tissues ( 6 leaks ) , vertebral venous plexus ( 2 leaks ) , and adjacent disk area ( 4 leaks ) . osteoporosis is a systemic bone disorder characterized by reduced bone mass and degradation of skeletal microarchitecture . osteoporotic vertebral compression fractures ( ovcfs ) are one of the most common fractures associated with osteoporosis . in addition to acute or chronic pain , ovcfs are also associated with progressive spinal deformity , impaired physical function , decreased quality of life , and increased mortality in this population . the direct medical costs associated with ovcfs have been estimated at $ 13.8 billion annually in the us alone , and indirect costs in lost productivity , pain , and suffering are even greater [ 2527 ] . conservative management includes analgesics , bed rest , external bracing , and a combination of these treatments . however , there are still limitations of these methods . long - term bed rest often leads to further demineralization and may predispose the patients to future ovcfs . as for medication of anti - inflammatory drugs and certain types of analgesics , it may be difficult for patients to tolerate the adverse effects . in frail elderly patients , long periods of inactivity are associated with higher rates of pneumonia , decubitus ulcers , venous thromboembolism , and even death . in case of failure of these non - surgical methods , until the beginning of the 1980s the only options left were surgical stabilization and spine straightening via dorsal instrumentation . however , there were limits to the application of this type of surgery , especially with patients in relatively poor general condition . pvp and pkp are 2 minimally invasive surgical procedures in which bone cement is injected into the fractured vertebral body . clinical results indicate both are effective in relieving back pain caused by the ovcfs , especially those that are refractory to conservative treatment . the main difference between the 2 procedures is that pkp involves the use of an inflatable bone tamp , in an attempt to create a cavity in the vertebral body prior to cement injection , while pvp does not [ 3133 ] . recently , a us large claims database analysis showed that pkp was associated with a significant reduction in mortality compared to conservative treatment and pvp . pkp is now considered to be as minimally invasive and effective as conventional pvp for rapid pain relief in patients with painful ovcfs resistant to conservative treatment . early diagnosis of ovcfs is important , and the detection of vertebral fractures is based primarily on the presence of a deformation of the vertebral body on lateral radiographs . occult osteoporotic vertebral fractures ( oovfs ) , which imply symptomatic vertebral fracture without measurable radiographic compression , can be misleading . the typical mri finding in acute compression fracture is hypointensity in t1-weighted image , heterogeneous intensity or hyperintensity on t2-weighted image , and hyperintensity on fat - suppressed t2-weighted image or on short - inversion time - inversion recovery image . in our study , numerous fractures would have been missed on standard radiographs , only being confirmed by mri . therefore , patients highly suspect of acute osteoporotic vertebral fracture , with acute symptomatic pain and normal x - ray and ct images , should undergo an additional imaging test , especially mri . histological study demonstrated trivial microtrabeculae fracture and end - plate fracture in the involved vertebral bodies . . showed that back pain was equally likely to occur with each type of vertebral fracture . lee et al . reported that there was little correlation between the degree of collapse of the vertebral body and the level of pain . oovfs are important causes of severe back pain , leading to chronic disability similar to that in patients with severe vertebral collapses . therefore , it seems essential and necessary to enhance vertebral bodies with oovfs to prevent possible vertebral collapse . clinicians may miss oovfs , which leads to delayed diagnosis or misdiagnosis . current treatment recommendations vary from analgesia to surgery . despite the benefits of vertebral augmentation procedures , reports discussing the vertebral augmentation procedure as an alternative to traditional medical therapy for the treatment of oovfs are scarce . there are several reports on the results of pvp and pkp in treating oovfs ; favorable results were reported , but , unfortunately , the literature data is mostly limited to case reports and small series of patients , and high - quality assessment methods are lacking . pham et al . reported that 21 cases of oovfs in 16 patients presented with a typical history of acute back pain with no substantial vertebral deformation on the initial plain radiographs . reported that 10 out of 95 ovcfs showing signal intensity changes on mri were difficult to identify on plain radiographs due to almost no collapse of the vertebral body . mao et al . reported results in 45 patients with oovfs who underwent pvp and pkp . they found notable pain alleviation and improved functional activity after pkp intervention and did not detect delayed collapse of the treated vertebral bodies . pereira et al . evaluated the clinical outcome and technical feasibility of percutaneous sacroplasty in treating insufficiency fractures of the sacrum . they performed 67 sacroplasty in 58 consecutive patients with intractable pain from osteoporotic fractures or from sacral tumors . the purpose of our study was to describe our experience and to assess the safety and effectiveness of pkp in patients with painful oovfs . in a consecutive series of 89 patients with painful oovfs , who had 142 levels of treatment in a 2-years follow - up period , we found pkp leading to a remarkable remission of pain and improvement of function . significant improvement in vas score , odi score , and sf-36 score was observed postoperatively and at each follow - up time . it is likely that pain relief is obtained through stabilization of the fracture by the cement . another explanation proposed is that the injected bone cement causes thermal necrosis and chemotoxicity of the intraosseous pain receptors . there have been numerous studies evaluating the anatomical distribution of ovcfs , which were consistently showing 2 prevalent peaks of vertebral fractures : the first in the mid - thoracic spine region ( t7/t8 ) and another in the thoraco - lumbar junction [ 3537 ] . vertebral fractures in the lower lumbar area , including l3 , l4 , and l5 level , are quite uncommon [ 3941 ] . our study revealed the peak of oovfs is the thoracolumbar junction area , representing 55% of overall spine fractures , which was consistent with ovcfs . it also shows that the lower lumbar vertebrae are frequently affected , representing 22.5% of overall spine fractures . it is likely that the lower lumbar area suffers less biomechanical stress , which could explain the temporary preservation of vertebral bodies weakened by a fracture . in these patients , oovfs are disabling causes of severe low back pain , and conservative treatments often fail . minimally invasive procedures , such as pkp and pvp , are effective treatment options that can result in rapid pain relief and rehabilitation , and prevent delayed vertebral collapse . the less frequent cement leakage with pkp is probably because this technique creates a cavity that allows for a more viscous cement to be injected under lower pressure . moreover , the expansion of the balloon compacts cancellous bone in the intravertebral cavity , which may also reduce the rate of cement leakage . in our collective , asymptomatic cement leak was seen in 8.45% of treated cases , notably lower than the documented average for ovcfs . this may be attributed to the relatively intact vertebral cortex and end - plate of the occult fractured vertebral body . in our experience , some leakage via the deficiency in the vertebral wall can be avoided by good operative technique . once cement leakage is detected , the operator may adjust the needle direction or stop the injection immediately . the injection process was stopped when the cement reached the margins of the vertebral body . based on these individual skills , no symptomatic cement extravasation occurred in our cases ; however , the significance of an asymptomatic leak remains unclear . it had been suggested that cement leaks into the adjacent intervertebral disc space affect the mechanical loading of either the disc or adjacent vertebrae , which might predispose the patient to an adjacent - level fracture [ 4446 ] . in our study , there were 5 new adjacent vertebral fractures during the follow - up period : 3 fractures were associated with cement leakage nearby and 2 fractures were related to sandwich vertebra . a sandwich vertebra is an intact vertebral body located between 2 previously cemented vertebras . because of double load shifts , the sandwich vertebra was prone to an increased risk of adjacent vertebral fracture however , adjacent - level fracture can also reflect systematic weakening of bone in the osteoporotic spine . we therefore believe this phenomenon may also represent the natural progression of osteoporosis in this series . hence , the long - term impacts of these asymptomatic cement leaks and sandwich vertebral fractures need further research . first , this study was performed retrospectively , which produces weaker evidence than a prospective study . second , only patients who underwent pkp were included in our study ; there was no control or alternative treatment , such as pvp . further additional prospective studies are needed to assess the effectiveness and safety of pkp in treatment of painful oovfs . to achieve earlier detection and diagnosis of painful oovfs , suspected patients with osteoporosis who have symptomatic pain and normal x - ray and ct images should undergo further mri testing . patients with incapacitating vertebral pain can be treated with , and benefit from , pkp .
backgroundthe definition of a vertebral fracture is usually based on the presence of a deformation of the vertebral body and this can be misleading in the presence of a fracture without radiologic collapse with the definition of occult osteoporotic vertebral fractures ( oovfs ) . stir sequence of mri images showing hyperintensity signal was the most confirmative screening examination used to determine the presence of painful oovfs . to date , clinical management of oovfs has been rarely discussed.material/methodsbetween 2011 and 2013 , 89 patients suffering from painful oovfs underwent 142 percutaneous balloon kyphoplasty ( pkp ) procedures . outcome data ( mean variation of anterior and middle vertebral body height , visual analog scale [ vas ] scores , oswestry disability index [ odi ] scores , and sf-36 scores ) were recorded preoperatively , postoperatively , and at 1 month , 6 months , and 2 year after treatment , to evaluate the results.resultswe successfully treated 89 patients ( 142 vertebral bodies ) with pkp . cement leakages were observed in 12 ( 8.45% ) treated vertebral bodies and there were 5 new adjacent vertebral fractures during the follow - up period . the mean variation of anterior and middle vertebral body height changed from 96.53.4% preoperatively to 97.22.5% postoperatively ( p>0.05 ) and from 96.32.8% preoperatively to 97.93.1% postoperatively ( p>0.05 ) , respectively . the mean vas scores were reduced significantly from pre - surgery to post - surgery ( 8.31.2 to 2.90.7 ; p<0.05 ) , as was the odi score ( 76.412.5 to 26.75.6 ; p<0.05 ) . the sf-36 scores , including bodily pain ( bf ) , vitality ( vt ) , physical function ( pf ) , and social functioning ( sf ) , all showed notable improvement ( p<0.05 ) . these variations were maintained during the 2-year follow - up period.conclusionspkp is a safe and effective method in the treatment of painful oovfs .
Background Material and Methods Patients Operative technique Radiographic and clinical assessment Statistical analysis Results Safety and efficacy Radiographic measurement Clinical evaluation Cement extravasation Discussion Conclusions
today , vertebral augmentation procedures , including percutaneous vertebroplasty ( pvp ) and percutaneous kyphoplasty ( pkp ) , are commonly used in the treatment of ovcfs [ 810 ] . in established osteoporosis , the definition of a vertebral fracture is usually based on the presence of a deformation of the vertebral body on lateral radiographs . however , this can be misleading in the presence of a fracture without radiologic collapse , and the definition of occult osteoporotic vertebral fractures ( oovfs ) has been established . mri is the most confirmative screening examination used to determine the presence of painful oovfs ; t2 and stir sequences of mri images show hyperintensity signal because of bone edema in the affected vertebra . mri was the most confirmative screening examination used to determine the presence of painful oovfs ; stir sequences of mri images show hyperintensity signal because of bone edema in the affected vertebra . during the cement injection , radiographic assessment and clinical examinations were performed preoperatively , and at 1 month , 6 months , and 2 years after treatment . the results are categorized into 8 domains ; bodily pain ( bp ) , vitality ( vt ) , physical function ( pf ) , and social function ( sf ) have been shown to have high reliability and responsiveness in spinal injury patients and were selected for evaluation in our study [ 2224 ] . mri was the most confirmative screening examination used to determine the presence of painful oovfs ; stir sequences of mri images show hyperintensity signal because of bone edema in the affected vertebra . during the cement injection , radiographic assessment and clinical examinations were performed preoperatively , and at 1 month , 6 months , and 2 years after treatment . the results are categorized into 8 domains ; bodily pain ( bp ) , vitality ( vt ) , physical function ( pf ) , and social function ( sf ) have been shown to have high reliability and responsiveness in spinal injury patients and were selected for evaluation in our study [ 2224 ] . significant improvements in sf-36 score , vas , and odi score were observed postoperatively and at 1 month , 6 months , and 2 years after treatment . five new adjacent vertebral fractures in 89 patients were detected during the 2-year follow - up period . the mean anterior vertebral body height variation changed from 96.53.4% preoperatively to 97.22.5% postoperatively ( p>0.05 ) and maintained at 95.73.4% at 2 years postoperatively . the mean middle vertebral body height variation changed from 96.32.8% preoperatively to 97.93.1% postoperatively and maintained at 96.74.4% at 2 years postoperatively . the mean vas score of these patients decreased from 8.31.2 preoperatively to 2.90.7 postoperatively and maintained at 3.01.2 at 2 years postoperatively , and the odi score decreased from 76.412.5 preoperatively to 26.75.6 postoperatively and maintained at 27.88.3 at 2-year postoperatively ( table 2 ) . the sf-36 scores for vitality ( vt ) , bodily pain ( bf ) , social functioning ( sf ) , and physical function ( pf ) all showed notable improvement ( p<0.05 ) ( table 3 ) . the mean anterior vertebral body height variation changed from 96.53.4% preoperatively to 97.22.5% postoperatively ( p>0.05 ) and maintained at 95.73.4% at 2 years postoperatively . the mean middle vertebral body height variation changed from 96.32.8% preoperatively to 97.93.1% postoperatively and maintained at 96.74.4% at 2 years postoperatively . the mean vas score of these patients decreased from 8.31.2 preoperatively to 2.90.7 postoperatively and maintained at 3.01.2 at 2 years postoperatively , and the odi score decreased from 76.412.5 preoperatively to 26.75.6 postoperatively and maintained at 27.88.3 at 2-year postoperatively ( table 2 ) . the sf-36 scores for vitality ( vt ) , bodily pain ( bf ) , social functioning ( sf ) , and physical function ( pf ) all showed notable improvement ( p<0.05 ) ( table 3 ) . early diagnosis of ovcfs is important , and the detection of vertebral fractures is based primarily on the presence of a deformation of the vertebral body on lateral radiographs . occult osteoporotic vertebral fractures ( oovfs ) , which imply symptomatic vertebral fracture without measurable radiographic compression , can be misleading . in our study , there were 5 new adjacent vertebral fractures during the follow - up period : 3 fractures were associated with cement leakage nearby and 2 fractures were related to sandwich vertebra .
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children experience rapid changes in their diets during early childhood at a time that they are also forming their eating habits . by the time children are 2 years old , picky eaters are typically characterized as consuming a narrow range of food , as well as rejecting several new and familiar food items . these behaviors can cause frustration , worry , or anxiety from the caregiver that the child is not consuming the appropriate nutrients needed for healthy growth . eating behaviors are shaped in a variety of ways , including what food the child is introduced to , the environment in which the food is served , and the way in which the food is prepared or typically consumed . strategies that caregivers use during mealtimes to encourage the child to eat can also affect their eating habits and behaviors . according to the us census bureau , 33% of children under age 5 are cared for in nonparental childcare arrangements for an average of 35 hours per week . the most popular form of nonparental childcare is center - based childcare ( cbcc ) , encompassing 67% of children in nonparental childcare arrangements . home - based childcare ( hbcc ) settings represent 30% of children in nonparental childcare arrangements . center - based childcare centers are a structured , school - like environment ; typically , they contain multiple classrooms comprising children of similar ages separated in each classroom with a set teacher to student ratio . home - based childcares are environments where children are cared for in the care provider s house . there are usually fewer children in hbcc than in cbcc settings , and typically , there is only 1 caregiver . home - based childcares have more freedom in terms of policies that need to be followed depending on their licensing status or involvement in federal reimbursement programs . although children are being cared for in hbcc and cbcc , they typically consume at least 1 meal . the academy of nutrition and dietetics ( formerly the american dietetic association ) recommends that children in part - time programs receive foods and beverages that provide at least one - third of the daily nutrient requirements , whereas those in full - term programs receive foods and beverages that meet at least one - half to two - thirds of daily nutrient needs . consequently , children are exposed to different eating environments and potentially different feeding strategies . however , most of the literature in this field focuses on parental feeding strategies , leaving a gap that focuses on how mealtime strategies used at different childcare locations may be varied from those at home . in addition , there is a gap in the literature that addresses how perceptions of child eating behavior , specifically picky eating , differ between caregivers . it is also unknown whether parents and childcare providers of the same child agree in their perceptions of child pickiness and whether pickiness perception has any impact on the mealtime strategies that are utilized . finally , little is known regarding the differences between hbcc and cbcc parents and providers , even though these are 2 of the most popular forms of nonparental childcare in the united states . therefore , the objectives of this study were to ( 1 ) compare perceptions of child pickiness between parents and childcare providers , ( 2 ) compare percent agreement in pickiness perception between the dyads of cbcc providers and parents and hbcc providers and parents , and ( 3 ) identify mealtime strategy utilization of each caregiver group ( ie , hbcc parents and hbcc providers ) based on pickiness perceptions . due to the differences in mealtime environments between the family home , hbcc , and cbcc , it was hypothesized that perceptions of child pickiness would differ among caregivers in these 3 locations . due to the considerable differences between hbcc and cbcc centers , especially in terms of structure and policies , it was further hypothesized that hbcc and cbcc caregivers would have different perceptions of child pickiness . finally , it was hypothesized that differing perceptions of child pickiness would affect the mealtime strategies that were utilized . this study was approved by the institutional review board at the university of illinois . parents and their families in champaign - urbana , il , a total of 27 families and 7 cbcc providers were recruited from the child development laboratory ( a cbcc on the university of illinois campus ) , and 25 families and 12 hbcc providers were recruited from hbcc centers . childcare providers were recruited via recruitment phone calls , mailed flyers , word of mouth , and advertising during local childcare provider workshops . participation requirements included having at least 1 child aged 3 - 5 years with no food allergies . the 3- to 5-year - old age range for the study was determined based on the literature findings that this age range is when picky eating behaviors peak . if families had 2 children in the age range of 3 - 5 years , they could enroll both children if desired . only 1 family from cbcc and 1 family from hbcc enrolled 2 children in the study ; all others enrolled only 1 child . this resulted in 26 parents from cbcc enrolled and 24 parents from hbcc enrolled in the study . two hbcc providers who were also mothers of children participating in the study were removed from the analysis . parents and teachers completed 2 surveys either online or in paper : the mealtime assessment survey ( mas ) and the parent / teacher mealtime strategies survey ( pms / tms ) . for all surveys , parents and childcare providers responded to questions using a 5-point likert scale with never , rarely , sometimes , often , or always response options . it was developed through a series of focus groups and conjoint analyses examining actions displayed by pe , nonpicky eater ( npe ) , and parents during feedings and adapted from questionnaires found in the literature regarding toddler mealtime behaviors . parent and teacher perceptions of the child s pickiness were determined via the question how often is your child / student a picky eater ? on the mas . responses were dichotomized to classify a child as pe ( always , often , and sometimes ) or an npe ( rarely and never ) . this method of dichotomization is well accepted and has been reported previously in the literature . questions on the tms were similar to those on the pms , although some questions were tailored for applicability to a childcare setting or were removed . to test the first objective , the mcnemar test for paired , binary data was used to determine differences in pickiness perceptions ( pe / npe ) between childcare providers and parents within each childcare setting . the chi - square test is not an appropriate test for these data because 2 perceptions per child were compared , thus violating the assumption of independence . for more detailed analysis that accounted for the clustering of responses within childcare setting , a multinomial cumulative logit model was used to explore the association of childcare setting ( cbcc vs hbcc ) , caregiver type ( parent vs provider ) , and their interaction on pickiness perception as measured using the full 5-point categorical scale . the proportional odds assumption was confirmed using the rao score test ( chi - square score statistic ) for testing equality of slope parameters . the resulting beta coefficient is interpreted as the increase in the log odds of higher perceived pickiness rating associated with a 1-unit change in a covariate after holding all other covariates as constant . to obtain the odds ratio ( or ) and the 95% confidence interval ( 95% ci ) , the exponent of the beta coefficient was determined . to test the second objective , percent agreement in pickiness perception between parents and childcare providers was determined based on whether or not the caregivers ( parent and childcare provider ) perceived the same child as a pe . if the 2 caregivers perceived the same child as a pe , their responses were recorded in the agreed category . across - childcare differences in percent agreement between parent - childcare provider pairs were compared using chi - square test . to test the third objective , the frequency of the use of mealtime strategies by the parents and the childcare providers was identified using chi - square test via child pickiness perception ( pe / npe ) that was dichotomized as previously described . all statistical analyses were performed using microsoft excel ( version 15.0.4727.1000 ; microsoft , redmond , wa , usa ) or statistical analysis software ( sas ) version 9.3 ( sas institute , cary , nc , usa ) . a 2-tailed , significance level of p < .05 was considered statistically significant . this study was approved by the institutional review board at the university of illinois . parents and their families in champaign - urbana , il , a total of 27 families and 7 cbcc providers were recruited from the child development laboratory ( a cbcc on the university of illinois campus ) , and 25 families and 12 hbcc providers were recruited from hbcc centers . childcare providers were recruited via recruitment phone calls , mailed flyers , word of mouth , and advertising during local childcare provider workshops . participation requirements included having at least 1 child aged 3 - 5 years with no food allergies . the 3- to 5-year - old age range for the study was determined based on the literature findings that this age range is when picky eating behaviors peak . if families had 2 children in the age range of 3 - 5 years , they could enroll both children if desired . only 1 family from cbcc and 1 family from hbcc enrolled 2 children in the study ; all others enrolled only 1 child . this resulted in 26 parents from cbcc enrolled and 24 parents from hbcc enrolled in the study . two hbcc providers who were also mothers of children participating in the study were removed from the analysis . parents and teachers completed 2 surveys either online or in paper : the mealtime assessment survey ( mas ) and the parent / teacher mealtime strategies survey ( pms / tms ) . for all surveys , parents and childcare providers responded to questions using a 5-point likert scale with never , rarely , sometimes , often , or always response options . it was developed through a series of focus groups and conjoint analyses examining actions displayed by pe , nonpicky eater ( npe ) , and parents during feedings and adapted from questionnaires found in the literature regarding toddler mealtime behaviors . parent and teacher perceptions of the child s pickiness were determined via the question how often is your child / student a picky eater ? on the mas . responses were dichotomized to classify a child as pe ( always , often , and sometimes ) or an npe ( rarely and never ) . this method of dichotomization is well accepted and has been reported previously in the literature . questions on the tms were similar to those on the pms , although some questions were tailored for applicability to a childcare setting or were removed . to test the first objective , the mcnemar test for paired , binary data was used to determine differences in pickiness perceptions ( pe / npe ) between childcare providers and parents within each childcare setting . the chi - square test is not an appropriate test for these data because 2 perceptions per child were compared , thus violating the assumption of independence . for more detailed analysis that accounted for the clustering of responses within childcare setting , a multinomial cumulative logit model was used to explore the association of childcare setting ( cbcc vs hbcc ) , caregiver type ( parent vs provider ) , and their interaction on pickiness perception as measured using the full 5-point categorical scale . the proportional odds assumption was confirmed using the rao score test ( chi - square score statistic ) for testing equality of slope parameters . the resulting beta coefficient is interpreted as the increase in the log odds of higher perceived pickiness rating associated with a 1-unit change in a covariate after holding all other covariates as constant . to obtain the odds ratio ( or ) and the 95% confidence interval ( 95% ci ) , the exponent of the beta coefficient was determined . to test the second objective , percent agreement in pickiness perception between parents and childcare providers was determined based on whether or not the caregivers ( parent and childcare provider ) perceived the same child as a pe . if the 2 caregivers perceived the same child as a pe , their responses were recorded in the agreed category . across - childcare differences in percent agreement between parent - childcare provider pairs the frequency of the use of mealtime strategies by the parents and the childcare providers was identified using chi - square test via child pickiness perception ( pe / npe ) that was dichotomized as previously described . all statistical analyses were performed using microsoft excel ( version 15.0.4727.1000 ; microsoft , redmond , wa , usa ) or statistical analysis software ( sas ) version 9.3 ( sas institute , cary , nc , usa ) . a 2-tailed , significance level of p < .05 was considered statistically significant . a total of 50 child , parent , and childcare provider triads participated in the study . there were approximately equal proportions of boys and girls ( 48% and 52% , respectively ) . 35% of the children were 3 years old , 40% were 4 years old , and 25% were 5 years old . most ( 72% ) of the parents in the study were women , 46% were between the age of 26 and 35 years , and 61% were white . most ( 47% ) childcare providers were between the age of 46 and 55 years and white . abbreviations : cbcc , center - based childcare ; hbcc , home - based childcare . the results from objective 1 showed that 56% of cbcc parents and 44% of cbcc providers perceived their child or student as a pe , whereas 57% percent of hbcc parents and 48% of hbcc providers perceived their child or student as being a pe ( figure 1 ) . although these proportions were not statistically significant within each childcare setting , using a multinomial cumulative logit model , the results showed that parents are about 1.4 times more likely than providers to rate the child as being more picky ( or , 1.4 ; 95% ci , 1.3 - 1.5 ) . in addition , hbcc parents and providers are also 1.4 times more likely to rate a child as being picky than cbcc parents and providers ( or , 1.4 ; 95% ci , 1.3 - 1.4 ) ( table 2 ) . differences in percent of caregivers who perceive the child as being a picky eater between cbcc providers ( n = 7 ) and parents ( n = 26 ) and hbcc providers ( n = 11 ) and parents ( n = 22 ) . proportions not significantly different within each childcare setting . multinomial cumulative logit regression model for the association of childcare type and caregiver with the outcome of increasing child pickiness perception . abbreviations : cbcc , center - based childcare ; ci , confidence interval ; hbcc , home - based childcare ; or , odds ratio . or of 1.4 for site indicates that hbcc parents and providers are 1.4 times more likely to perceive a child as being a pe . or of 1.4 for caregiver indicates that parents are 1.4 times more likely than childcare providers to perceive a child as being a pe . results from objective 2 , percent agreement in pickiness perception between hbcc and cbcc parents and childcare providers , showed that parents and providers do not agree in their perception of the same child s pickiness , supporting our hypothesis . center - based childcare parent / provider pairs significantly disagreed more than hbcc parent / provider pairs ; 41% of cbcc parent / provider pairs did not have the same perception of child pickiness compared with 26% of hbcc parent / provider pairs abbreviations : cbcc , center - based childcare ; hbcc , home - based childcare . significant at p < .05 using chi - square . in total , 41% of cbcc parents and teachers did not agree in their perception of the same child s pickiness as opposed to 26% of hbcc providers and parents not agreeing . results of objective 3 identified 2 strategies to be significantly different between cbcc parents who perceived their child as being picky compared with those parents who do not ( figure 2 ) . in contrast , no mealtime strategies were found to be significantly different when a cbcc provider perceived a child to be a pe vs when they did not . in other words , the mealtime strategies used by cbcc providers were consistent , regardless of their perception of a child s pickiness . association of mealtime strategies with child s pickiness perception ( pe [ n = 15 ] and npe [ n = 12 ] ) among center - based childcare parents . significance according to chi - square test ( * p < .05 ) . parents who perceived the child as a pe used both mealtime strategies more often than those who perceived the child as an npe . npe indicates nonpicky eater ; pe , picky eater . also , in objective 3 , 3 strategies were shown to be significantly different between hbcc parents who perceived their child to be a pe vs those who did not ( figure 3 ) . among hbcc providers , 2 strategies were found to be differently utilized based on differing perceptions of child pickiness ( figure 4 ) . association of mealtime strategies with child s pickiness perception ( pe [ n = 13 ] and npe [ n = 10 ] ) among home - based childcare parents . significance according to chi - square test ( * p < .05 , * * p < .01 ) . parents who perceived the child as a pe used all mealtime strategies more often than those who perceived the child as an npe . npe indicates nonpicky eater ; pe , picky eater . association of mealtime strategies with child s pickiness perception ( pe [ n = 11 ] and npe [ n = 12 ] ) among home - based childcare providers . significance according to chi - square test ( * p < .05 , * * p < .01 ) . parents who perceived the child as a pe used all mealtime strategies more often than those who perceived the child as an npe . the findings of this research showed that parents are more likely to identify their children as being pes than those children s care providers . it could be that parents are more sensitive to the child s eating behavior and are therefore more likely to perceive a child as being picky . because most parents worry about their child s growth , any indication of hesitance to eat from the child may lead the parent to perceive their child as a pe . on the contrary , childcare providers may not be as sensitive to their student s eating habits because they are caring for multiple children or focus on other objectives during mealtime . another theory as to why parents are more likely than childcare providers to rate their children as pes is that children may , in fact , display more picky eating behaviors at home than at childcare . children are likely aware that at home other food is available if they do not prefer what is served to them and are more inclined to reject or avoid that food until they are given an item they do enjoy . in addition , children are also aware of their parent s sensitivity to their eating habits and may know that if they avoid or refuse an item , they will be offered a different food . at childcare , especially cbcc , it is speculated that these options do not exist , therefore resulting in less pe behavior from the child . it was also found that between cbcc and hbcc caregivers ( both parents and childcare providers ) , hbcc caregivers are about 1.4 times more likely to rate a student as a pe . due to the nature of hbcc , in that it is typically a neighbor or friend of the parents , these caregivers may have similar ways of thinking or may talk more about the child s eating habits and , therefore , have a more similar perception of the child s pickiness than cbcc caregivers . in addition , the hbcc environment is more similar to the family home and there may not as many children as a typical cbcc , which lead children to act in a more similar manner during mealtimes in their hbcc location and their home vs cbcc children . similarly , hbcc parents and childcare providers were in greater agreement with each other in their perception of child pickiness than cbcc parents and providers . this may also reflect the fact that the hbcc environment is usually more similar to the home environment and cbccs . this similarity in environment could result in comparable behavior from the child and therefore similar perception of child pickiness . in addition , hbcc providers and parents oftentimes know one another on a personal level , which could result in sharing or coordination of mealtime strategies and therefore in more agreement in perceptions of child pickiness . regarding behavioral strategies around mealtimes , cbcc parents with perceived pes used a greater variety of mealtime strategies than parents with perceived npes , which confirmed our hypothesis . previous research found that using food as a contingency factor negatively affected a child s preference for that food . in contrast , parental modeling of positive eating behaviors has been shown to be effective in establishing healthy eating habits in children . parents with perceived pes may use more mealtime strategies than parents of perceived npes in efforts to improve food consumption in their child . center - based childcare providers did not change their mealtime strategies , regardless of their perception of child pickiness . strategies utilized by this group of caregivers were consistent , not only from student to student for the same childcare provider but also across childcare providers . this may be due to the policies and procedures surrounding mealtime that have been put into place by the cbcc that providers must follow . as with cbcc parents , hbcc parents with perceived pes were more likely to use different strategies , including rewards and making the meal into a game , to encourage eating . the consensus on whether the strategies of using foods / nonfoods as a reward for eating are beneficial are inconclusive . some studies have shown that these are negative strategies in that the child can develop dislike for the food that is being used as a means to receive the reward , whereas others show that using rewards can be motivating to children . the strategy of making a meal into a game to encourage eating may also be negative because it distracts the child from focusing on the meal and their satiety cues . similar to cbcc parents , these results indicate that parent perception of child pickiness does have an effect on mealtime strategy utilization . based on these results , hbcc parents with perceived pes are more likely to use ineffective strategies at mealtime in attempts to improve child eating . home - based childcare providers with perceived pes were found to use the strategy of spoon - feeding more than hbcc providers who did not perceive to have pes . children between the age of 3 and 5 years should have the ability to feed themselves , rendering this strategy inappropriate for children in this age range , but hbcc providers with perceived pes may not have this knowledge , and therefore , use this strategy . in addition , hbcc providers with perceived pes were found to offer a child a reward for eating more than providers who did not perceive to have pes . although a consensus on the benefits or consequences of using rewards for eating has not been reached , it may be that hbcc providers with perceived pes use this strategy because in a setting such as hbcc , this strategy is effective . although only 2 strategies were found to be different , these results indicate that pickiness perception does affect mealtime strategy utilization in hbcc providers . the findings from this study provide insight to not only elucidate differences regarding pickiness perception between parents and childcare providers and how that affects utilized mealtime strategies but also further our understanding regarding differences in mealtime between home and childcare overall . the caregivers with perceived pes , regardless of location , are more likely to use a variety of mealtime strategies , even potentially ineffective ones . these results can be shared with parents trying to decide which childcare to choose , be used as a basis for mealtime strategy interventions with parents and childcare providers , and aid educators when trying to create educational materials for parents or childcare providers on mealtimes . first , due to the difficulty of recruitment of this study population , our sample size is small . the study population is novel in that we enrolled pairs of parents and providers for the same child , meaning recruitment was 2-fold . not only did the childcare provider have to want to participate in the study , but so did the parents whose children attended the childcare . in addition , our study population included hbcc , a group that is not routinely investigated . contacting , locating , and building enough rapport with future research should focus on achieving a greater sample size in multiple states to validate findings . second , although these findings show that there are differences in pickiness perception among caregivers who care for the same child , we do not know the reason why . without understanding why differences in perceptions exist therefore , future research should focus on determining why parents and childcare providers of the same child have differing perceptions of child pickiness to develop appropriate feeding strategy interventions for caregivers based on their location .
picky eating is a problematic eating behavior caregivers may encounter with children under their care . a picky eater ( pe ) is typically characterized as consuming a narrow range of food , as well as rejecting several food items . much of the literature regarding pes involves parents , although use of nonparental childcare arrangements in the united states has increased in the past several decades . although data on parental mealtime strategies exist , little is known about how parent and childcare provider pickiness perceptions differ between types of childcare , such as center - based childcare ( cbcc ) and home - based childcare ( hbcc ) , or how these perceptions influence the mealtime strategies utilized . the objectives of this study were to ( 1 ) compare perceptions of child pickiness between parents and childcare providers , ( 2 ) compare percent agreement in pickiness perception between the dyads of cbcc parents and providers and hbcc parents and providers , and ( 3 ) identify mealtime strategy utilization based on pickiness perception . a total of 52 child , parent , and childcare provider triads participated in the study and completed the mealtime assessment survey and the parent / teacher mealtime strategy survey regarding the same child . results showed that parents are 1.4 times more likely than childcare providers to perceive a child as being picky , hbcc parents and providers are 1.4 times more likely to perceive a child as being picky than cbcc parents and providers , cbcc parents and providers disagree more in their perception of child pickiness than hbcc parents and providers ( 41% vs 26% ) , and finally , perception of child pickiness has a greater influence on mealtime strategies utilized by parents . these results can be used to focus intervention efforts aimed at improving child eating habits across the home and childcare location .
Introduction Methods Participants Measures Statistical analysis Results Discussion
by the time children are 2 years old , picky eaters are typically characterized as consuming a narrow range of food , as well as rejecting several new and familiar food items . the most popular form of nonparental childcare is center - based childcare ( cbcc ) , encompassing 67% of children in nonparental childcare arrangements . it is also unknown whether parents and childcare providers of the same child agree in their perceptions of child pickiness and whether pickiness perception has any impact on the mealtime strategies that are utilized . finally , little is known regarding the differences between hbcc and cbcc parents and providers , even though these are 2 of the most popular forms of nonparental childcare in the united states . therefore , the objectives of this study were to ( 1 ) compare perceptions of child pickiness between parents and childcare providers , ( 2 ) compare percent agreement in pickiness perception between the dyads of cbcc providers and parents and hbcc providers and parents , and ( 3 ) identify mealtime strategy utilization of each caregiver group ( ie , hbcc parents and hbcc providers ) based on pickiness perceptions . parents and teachers completed 2 surveys either online or in paper : the mealtime assessment survey ( mas ) and the parent / teacher mealtime strategies survey ( pms / tms ) . to test the second objective , percent agreement in pickiness perception between parents and childcare providers was determined based on whether or not the caregivers ( parent and childcare provider ) perceived the same child as a pe . parents and teachers completed 2 surveys either online or in paper : the mealtime assessment survey ( mas ) and the parent / teacher mealtime strategies survey ( pms / tms ) . to test the second objective , percent agreement in pickiness perception between parents and childcare providers was determined based on whether or not the caregivers ( parent and childcare provider ) perceived the same child as a pe . across - childcare differences in percent agreement between parent - childcare provider pairs the frequency of the use of mealtime strategies by the parents and the childcare providers was identified using chi - square test via child pickiness perception ( pe / npe ) that was dichotomized as previously described . a total of 50 child , parent , and childcare provider triads participated in the study . although these proportions were not statistically significant within each childcare setting , using a multinomial cumulative logit model , the results showed that parents are about 1.4 times more likely than providers to rate the child as being more picky ( or , 1.4 ; 95% ci , 1.3 - 1.5 ) . in addition , hbcc parents and providers are also 1.4 times more likely to rate a child as being picky than cbcc parents and providers ( or , 1.4 ; 95% ci , 1.3 - 1.4 ) ( table 2 ) . or of 1.4 for site indicates that hbcc parents and providers are 1.4 times more likely to perceive a child as being a pe . or of 1.4 for caregiver indicates that parents are 1.4 times more likely than childcare providers to perceive a child as being a pe . results from objective 2 , percent agreement in pickiness perception between hbcc and cbcc parents and childcare providers , showed that parents and providers do not agree in their perception of the same child s pickiness , supporting our hypothesis . center - based childcare parent / provider pairs significantly disagreed more than hbcc parent / provider pairs ; 41% of cbcc parent / provider pairs did not have the same perception of child pickiness compared with 26% of hbcc parent / provider pairs abbreviations : cbcc , center - based childcare ; hbcc , home - based childcare . in total , 41% of cbcc parents and teachers did not agree in their perception of the same child s pickiness as opposed to 26% of hbcc providers and parents not agreeing . it could be that parents are more sensitive to the child s eating behavior and are therefore more likely to perceive a child as being picky . it was also found that between cbcc and hbcc caregivers ( both parents and childcare providers ) , hbcc caregivers are about 1.4 times more likely to rate a student as a pe . similarly , hbcc parents and childcare providers were in greater agreement with each other in their perception of child pickiness than cbcc parents and providers . the findings from this study provide insight to not only elucidate differences regarding pickiness perception between parents and childcare providers and how that affects utilized mealtime strategies but also further our understanding regarding differences in mealtime between home and childcare overall . these results can be shared with parents trying to decide which childcare to choose , be used as a basis for mealtime strategy interventions with parents and childcare providers , and aid educators when trying to create educational materials for parents or childcare providers on mealtimes . without understanding why differences in perceptions exist therefore , future research should focus on determining why parents and childcare providers of the same child have differing perceptions of child pickiness to develop appropriate feeding strategy interventions for caregivers based on their location .
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the largest and most conspicuous exocrine system in the acariform mite suborders oribatida and astigmata is the paired opisthonotal glands ( syn . oil glands ) . these glands , according to a current and widely accepted hypothesis , evolved only once within ancient oribatida ( after the near - basal oribatid groups , palaeosomata and enarthronota , had split - off ) , and are considered characteristic of the four more - derived oribatid groups , parhyposomata , mixonomata , desmonomata , brachypylina , and also of astigmatid mites . the presence of opisthonotal glands not only constitutes a major argument for a proposed monophyletic unit of so - called glandulate oribatida , including astigmata ( norton 1998 ) , but also provides evidence for the evolutionary origin of astigmatid mites in an ancient opisthonotal gland - bearing oribatid group . in addition to the presence or absence of opisthonotal glands , the secretions of these glands provide phylogenetic information as they include a rich variety of hydrocarbons , terpenes , aromatics , and alkaloids in species - specific or group - specific patterns . these secretions , derived from a homologous gland system , allow the tracing of evolutionary lineages from near - basal to more - derived glandulate oribatida . apart from the well - studied opisthonotal gland secretions of astigmata ( kuwahara 2004 ) , the secretions of a selection of species of several major groups of glandulate oribatids have been investigated , including representatives of parhyposomata ( sakata and norton 2001 ) , a few species of mixonomata and desmonomata ( sakata et al . 2001 , 2005a , b ; shimano et al . 2002 ; sakata and norton 2003 ) , and also examples of brachypylina ( takada et al . 2005 ; saporito et al . 2007 ) . while parhyposomata produce phenols and naphthols , the so - called astigmatid compounds ( sensu sakata and norton 2001 ) evolved within ancient mixonomatans , and hence , are possibly found in all the above groups . these astigmatid compounds comprise a set of terpenes and aromatics , namely neral , geranial , neryl formate , 2-hydroxy-6-methyl - benzaldehyde , and -acaridial . the distribution of astigmatid compounds in oribatids is of special interest since these compounds not only provide chemical support for the evolutionary origin of astigmatid mites within ancient oribatida ( norton 1998 ) , but also define a presumably monophyletic subunit within glandulate oribatida , the astigmatid compounds - bearing oribatids ( raspotnig 2006 ) . several chemotaxonomic studies that examined these presumptive astigmatid compound - bearing groups have been undertaken and have yielded a fragmented picture of the distribution of astigmatid compounds . these compounds are difficult to detect in the more derived groups of desmonomata ( apart from trhypochthoniidae ) and generally in brachypylina , suggesting that , in these groups , astigmatid compounds have been prone to evolutionary reduction and replacement . in some cases , desmonomatan and brachypyline species are chemically dimorphic , with astigmatid compounds present only in juveniles and completely replaced in adults by novel compounds such as toxic alkaloids ( takada et al . in contrast , the full ( or nearly full ) set of astigmatid compounds seems to characterize highly derived mixonomatans , low to moderately derived representatives of desmonomata , and astigmata . specifically , these compounds have been detected in opisthonotal gland secretions of collohmannia gigantea ( raspotnig et al . 2001 ) , in all trhypochthoniidae so far investigated ( e.g. , sakata et al . 1995 , 2003 ) , and in astigmatid mites ( e.g. , kuwahara 2004 ) . however , there are large gaps in our knowledge of the distribution of astigmatid compounds : the most conspicuous gap being in the euphthiracaroidea , a speciose group of box mites whose opisthonotal gland chemistry has yet to be investigated . euphthiracaroids are considered a group of highly derived mixonomatans that , together with phthiracaroids , constitute a possible sister group of collohmannia . thus , euphthiracaroids are likely candidates for producing astigmatid compounds . the study of the chemistry of opisthonotal glands in euphthiracaroids is important not only to further our understanding of the evolution of astigmatid compounds , but also because this group is a crucial link for tracing a possible evolutionary lineage from oribatida to astigmata . with respect to the latter , some ( not all ! ) recent molecular studies ( e.g. , murell et al . 2005 ; domes et al . therefore , studying the opisthonotal gland chemistry may resolve this problem . in the present paper , we provide the first chemical analysis of the opisthonotal gland secretion of oribotritia berlesei , a large - sized oribatid mite from the near - basal euphthiracaroid family oribotritiidae . mites and mite extracts specimens of o. berlesei ( michael 1898 ) ( mixonomatan oribatida : euphthiracaroidea , oribotritiidae ) were collected from the litter and fermentation layer of a mixed forest near ferlach ( carinthia , austria ) . individuals were collected by hand or extracted from soil samples by using berlese - tullgren funnels . in all , 35 adults ( all in the course of one collection in september 2006 ) were used for chemical analyses , five adults were used for scanning electron microscopy , and 30 adults were transferred to petri dishes ( equipped with plaster of paris and dead wood ) and kept in the dark for a period of about 12 months either at room temperature ( one petri dish ) or at 10c ( second petri dish ) , respectively . individuals of both petri dishes laid eggs , and the offspring were reared to deutonymphal and tritonymphal instars . since immatures are burrowers in dead wood , pieces of dead wood ( provided in the rearing dishes ) were checked for immatures at weekly intervals . ten deutonymphs and eight tritonymphs were used in the chemical studies.extracts ( containing opisthonotal gland secretions ) of adults were prepared by immersing freshly collected , living , individuals in hexane ( one individual per 100 l ) for 30 min ( raspotnig et al . 2001 , 2005a , b ) . for juveniles , individual and pooled extracts ( containing two to six individuals ) were prepared similarly . mass spectrometry a trace gas chromatograph ( gc ) coupled to a voyager mass spectrometer ( ms ; both from thermo , vienna , austria ) and equipped with a zb-5ms fused silica capillary column ( 30 m 0.25 mm i.d . , 0.25 m film thickness , phenomenex , germany ) was used for the analyses . injection was splitless with helium ( at a constant flow rate of 1.5 ml min ) as a carrier gas . the column temperature was programmed from 50c ( held for 1 min ) to 200c at 10c min , and then to 300c at 15c min . the ion source of the mass spectrometer and the transfer line were kept at 150c and 310c , respectively . derivatization , syntheses , reference compounds , and other chemicals tri- , penta- , and heptadecane , as well as citral ( 60% geranial , 40% neral ) and selenium dioxide for the preparation of oxocitral ( see bellesia et al . for determination of double bonds in the heptadecadiene , dimethyl - disulfide ( dmds ) adducts were generated according to the method described by vincenti et al . 2005b ) was used as a natural source for 6,9-heptadecadiene and 8-heptadecene for chromatographic comparisons . in order to clarify further the structure of chrysomelidial , the o - methyl - oxime derivatives were prepared with methoxylamine hydrochloride ( mox.hcl ; 2% in pyridine ) : 200 l of mox was added to 50 l of extract and left at 70c for 1 h. products were cleaned in h2o , extracted with hexane , and analyzed . . secretions from eversible defense glands of larvae of the chrysomelid beetles , gastrophya viridula and plagiodera versicolora ( collected in the surroundings of graz , austria ) , known to contain natural ( 3s,8s)-chrysomelidial ( e.g. , pasteels et al . 1982 ) , were collected on small filter paper pieces , extracted in hexane ( one individual in 100 l ) , and used as sources for authentic reference compounds . hexane extracts of fresh leaves of actinidia polygama ( obtained from the botanical garden in tbingen , germany ) were taken as a natural source for ( 3r,8s)-dehydroiridodial ( yoshihara et al . scanning electron microscopy air - dried mites were mounted on aluminum stubs , sputtercoated ( agar sputtercoater , grpl , tulln , austria ) , and examined with a philips xl30 esem ( philips / fei , vienna , austria ) at high vacuum mode and 20 kv accelerating voltage . the main component in extracts , component c ( rt = 13.36 min ; ca . 45% of the total extract , based on peak areas ) had an ei fragmentation pattern resembling a methyl - cyclopentene monoterpene ( fig . 2a ) , with a molecular ion at m / z 166 ( 7 ) and characteristic fragments at m / z 151 ( 6 ) , 148 ( 46 ) , 138 ( 25 ) , 133 ( 9 ) , 123 ( 15 ) , 120 ( 12 ) , 119 ( 10 ) , 109 ( 49 ) , 108 ( 40 ) , 105 ( 30 ) , 95 ( 19 ) , 91 ( 27 ) , 81 ( 100 ) , 79 ( 78 ) , and 77 ( 38 ) . the spectrum was consistent with that of chrysomelidial from the literature ( e.g. , blum et al . 1978 ; oldham et al . 1996 ) and with a compound from the nist library ( 2-formyl-,3-dimethyl-2-cyclopentene-1-acetaldehyde ; cas no . 75332 - 42 - 2 : 2-methyl-5(1-methyl-2-oxoethyl)-1-cyclopentene-1-carbaldehyde ) . in order to prove the dialdehyde structure , we generated o - methyl - oxime derivatives , resulting in the elimination of peak c and the formation of two new peaks , c and c , with longer retention times ( rt = 14.92 and 15.11 min ) . the two compounds had identical mass spectra , with molecular ions at m / z 224 ( 4 ) , and fragment ions at m / z 193 ( 13 ) , 178 ( 22 ) , 146 ( 25 ) , 138 ( 100 ) , 107 ( 38 ) , 106 ( 95 ) , 91 ( 18 ) , 87 ( 21 ) , 79 ( 47 ) , and 77 ( 29 ) . this pattern indicated o - methyl - oximation of compound c , forming two isomeric dimethyloximes , which confirmed the proposed dialdehydic structure ( addition of two o - methyl - oxime groups to compound c = 166 plus 2 29 = new molecular ion at m / z 224 ) . peaks a ( tridecene ) , b ( tridecane ) , c1 ( epi - chrysomelidial = 3s,8s - chrysomelidial ) , d ( pentadecene ) , e ( pentadecane ) , f ( 6,9-heptadecadiene ) , g ( 8-heptadecene ) , h ( heptadecane ) , i ( tentatively , -springene ) , j ( tentatively , 9,17-octadecadienal)fig . b compound c1 , tentatively identified as epi - chrysomelidial = ( 3s,8r)-chrysomelidial . note the differences in relative intensities of ions at m / z 148 ( m h2o ) and m / z 138 ( m co ) . further diagnostic fragments are assigned to : m h2o co ( m / z 120 ) ; c7h9o ( m / z 109 ) , c7h7 ( m / z 91 ) , and c6h9 ( m / z 81 : base peak = the methyl - cyclopentene moiety ) typical mass chromatograms of extracts of oribotritia berlesei : a adult individual . b deutonymph . peaks a ( tridecene ) , b ( tridecane ) , c1 ( epi - chrysomelidial = 3s,8s - chrysomelidial ) , d ( pentadecene ) , e ( pentadecane ) , f ( 6,9-heptadecadiene ) , g ( 8-heptadecene ) , h ( heptadecane ) , i ( tentatively , -springene ) , j ( tentatively , 9,17-octadecadienal ) electron impact mass spectra of chrysomelidials in extracts of oribotria berlesei . b compound c1 , tentatively identified as epi - chrysomelidial = ( 3s,8r)-chrysomelidial . note the differences in relative intensities of ions at m / z 148 ( m h2o ) and m / z 138 ( m co ) . further diagnostic fragments are assigned to : m h2o co ( m / z 120 ) ; c7h9o ( m / z 109 ) , c7h7 ( m / z 91 ) , and c6h9 ( m / z 81 : base peak = the methyl - cyclopentene moiety ) for a comparison of compound c to authentic chrysomelidial and its characterization , we used ( 1 ) a synthetic mixture of chrysomelidial - stereoisomers , and ( 2 ) naturally occurring ( 3s,8s)-chrysomelidial . the synthetic mixture was assumed to contain four stereoisomers of chrysomelidial and dehydroiridodial ( according to bellesia et al . 1986 ) , namely two diastereomeric pairs of enantiomers , i.e. , ( 3s,8s)-chrysomelidial , ( 3r,8r)-dehydroiridodial , and ( 3s,8r)-chrysomelidial (= epi - chrysomelidial ) , ( 3r,8s)-dehydroiridodial . however , the mixture exhibited only two , poorly separable peaks at rt = 13.32 and 13.36 min , each peak corresponding to one pair of enantiomers , with single enantiomers not separable under the chromatographic conditions . the later - eluting peak of the mixture corresponded to compound c from the oribotritia extract , with respect to both retention time and ei fragmentation pattern . larval secretions of the leaf beetle gastrophysa viridula ( see methods and materials ) , contain mainly ( 3s,8s)-chrysomelidial ( original chrysomelidial ) . analysis of this secretion showed only one abundant component that eluted at rt = 13.36 min , corresponding to the later - eluting peak of the synthetic mixture and to compound c of the mite extracts . thus , the later - eluting peak of the synthetic mixture was assigned as either ( 3s,8s)-chrysomelidial or its enantiomer , ( 3r,8r)-dehydroiridodial . since dehydroiridodials are considered of plant origin only ( see discussion ) , compound c was tentatively identified as ( 3s,8s)-chrysomelidial . the mass spectrum of peak f ( rt = 16.36 min ) , the second most abundant compound in extracts ( about 25% of total ) , suggested a doubly unsaturated c17-hydrocarbon , based on a molecular ion at m / z 236 and a series of unsaturated hydrocarbon fragments . the dmds adduct of the heptadecadiene showed a molecular ion at m / z 362 and diagnostic ions at m / z 231 , 203 , 183 , 159 , 155 ( base peak ) , and 131 , consistent with 6,9-heptadecadiene ( see raspotnig et al . compound i ( rt = 18.70 min ; ca . 20% of the total ) showed a terpene fragmentation pattern with a molecular ion at m / z 272 ( 4 ) and further m / z of 257 ( 3 ) , 229 ( 4 ) , 203 ( 5 ) , 187 ( 10 ) , 161 ( 17 ) , 133 ( 31 ) , 120 ( 15 ) , 119 ( 17 ) , 107 ( 20 ) , 93 ( 50 ) , 81 ( 37 ) , 69 ( 100 ) , 55 ( 16 ) , 53 ( 17 ) , and 41 ( 59 ) . the compound was tentatively identified as -springene on the basis of mass spectral comparison to -springene spectra from literature and from the nist library . the remaining peaks a , b , d , e , g , and h , all of them minor components , appeared to be a series of hydrocarbons , with compounds b ( rt = 11.69 ) , e ( rt = 14.32 min ) , and h ( rt = 16.65 min ) exhibiting the spectra of saturated n - alkanes with molecular ions at m / z 184 , 212 , and 240 , respectively . the compounds were identified as tri- , penta- , and heptadecane by comparison of retention times to authentic standards . compounds a ( rt = 11.54 ) , d ( rt = 14.10 min ) , and g ( rt = 16.44 min ) had mass spectra of mono - unsaturated analogs , with molecular ions at m / z 182 , 210 , and 238 , corresponding to tri- , penta- , and heptadecene . the position of the double bonds was not determined because , due to their low amounts , the compounds were not recovered after dmds derivatization . the retention time of compound g was the same as that of 8-heptadecene ( from extracts of p. peltifer , see methods and materials ) . compound j ( rt = 19.27 min ) , also a minor component , had the following mass spectrum : m / z 264 ( 11 ) , 235 ( 3 ) , 221 ( 4 ) , 207 ( 4 ) , 165 ( 4 ) , 151 ( 7 ) , 137 ( 10 ) , 123 ( 10 ) , 109 ( 22 ) , 95 ( 61 ) , 81 ( 16 ) , 79 ( 52 ) , 67 ( 100 ) , 55 ( 38 ) , 54 ( 32 ) , 44 ( 32 ) , and 41 ( 69 ) . the compound was tentatively identified as 9,17-octadecadienal on the basis of these mass spectral data . the pattern of the ten compounds was consistently detected in 30 of the 35 individuals of adult o. berlesei ( fig . 1a ) , with relatively low inter - individual variation with respect to the relative abundance of components ( data not shown ) . extracts of juvenile o. berlesei extracts of juveniles ( exclusively deuto- and tritonymphs ) contained only two compounds in a 1:2 ratio ( peak c1 and c in fig . 1b ) : the major component c corresponded to tentatively identified ( 3s,8s)-chrysomelidial from the adult extracts , based on identical retention time and mass spectrum . the minor component c1 eluted earlier ( rt = 13.32 min ) but was not fully separable from ( 3s,8s)-chrysomelidial . the mass spectrum of compound c1 appeared to be similar to that of ( 3s,8s)-chrysomelidial ( fig . 2 ) ; one difference , however , was the greater intensity of the ion at m / z 138 in relation to the ion at m / z 148 [ c.f . , in ( 3s,8s)-chryomelidial the ion at m / z 148 is more intense ] . these data suggested the compound to be one of the other possible stereoisomers of chrysomelidial , most likely an epimeric diastereomer of ( 3s,8s)-chrysomelidial . moreover , the retention time and mass spectrum of compound c1 fully corresponded with those of the earlier - eluting peak of the synthetic chrysomelidial mixture , limiting the possibilities to epi - chrysomelidial ( 3s,8r - chrysomelidial ) and its enantiomer , ( 3r,8s)-dehydroiridodial . in addition , its retention time and mass spectrum matched those of plant - derived ( 3r,8s)-dehydroiridodial . thus , again assuming that dehydroiridodials are exclusively plant - derived compounds , compound c1 was tentatively identified as epi - chrysomelidial ( 3s,8r - chrysomelidial).from a total of eight extracts of juvenile o. berlesei investigated ( derived from 18 individuals ) , seven extracts consistently showed these two stereoisomers only ; the other extract ( an extract of a single individual ) apparently did not contain any compounds at all . potential secretory glands the most conspicuous exocrine gland system of o. berlesei is the paired opisthonotal glands that , in light microscopic observations , are visible through the cuticle of the notogaster as kidney - shaped sacs of considerable size ( maximal diameter about 0.5 mm : i.e. , ca . the glands open to the notogastral surface via one single flap - provided pore orifice on either side of the body ( fig . the flap , presumably an external closing mechanism , was found either in an opened ( fig . opisthonotal glands are present in adults and in all juvenile stages . fig . 3topography and external morphology of opisthonotal glands in oribotritia berlesei . a scanning electron micrograph ( sem ) of an adult individual , lateral view . d sem of the pore of another individual , orifice closed topography and external morphology of opisthonotal glands in oribotritia berlesei . a scanning electron micrograph ( sem ) of an adult individual , lateral view . occurrence of chrysomelidial even though iridoid monoterpenes are widely distributed in exocrine secretions of a diverse range of arthropods , chrysomelidial , so far , has only been reported from leaf beetle larvae ( chrysomelidae ) and from certain rove beetles ( e.g. , pasteels et al . originally , chrysomelidial was found as a major constituent of the larval eversible glands of the chrysomelids , gastrophysa cyanea ( blum et al . 1978 ) and plagiodera versicolora ( meinwald et al . 1977 ) , and its stereochemistry later elucidated by meinwald and jones ( 1978 ) . according to these studies , and to current knowledge , chrysomelidial in leaf beetles is a mixture of two diastereomers , ( 3s,8s - chrysomelidial (= the original chrysomelidial ) and ( 3s,8r)-chrysomelidial (= epi - chysomelidial ) , with the former being the predominant isomer in the majority of chrysomelidial - producing species ( e.g. , sugawara et al . in contrast , the remaining two possible stereoisomers ( there are four stereoisomers due to the two asymmetric carbon atoms at c3 and c8 ) are termed dehydroiridodials and are considered of plant origin only ( yoshihara et al . 1978 ; sugawara et al . 1979 ) . under the conditions used in this study , we were able to separate the diastereomers but not the respective enantiomers of chrysomelidial and dehydroiridodial . for instance , bellesia et al . ( 1986 ) described the chrysomelidials as a pair of enantiomers , whereas they are in fact diastereomers . in the present paper , ( 1979 ) for these compounds , but also use the more common nomenclature of bellesia et al . thus , assuming dehydroiridodials are found exclusively in plants , the compounds in o. berlesei extracts are ( 3s,8s)-chrysomelidial and ( 3s,8r)-chrysomelidial ( epi - chrysomelidial).our analysis of o. berlesei is not only the first example of the chemistry of exocrine glands of euphthiracaroidea but also the only report of chrysomelidials found outside the coleoptera . in o. berlesei , chrysomelidials most likely originate from the opisthonotal glands : these glands are quite large in o. berlesei , and thus probably capable of producing compounds in the amount detected . furthermore , some of the components , especially 6,9-heptadecadiene and some other hydrocarbons , found in the extract are characteristic constituents of opisthonotal glands of many oribatid ( and astigmatid ) mite species ( kuwahara 2004 ; raspotnig 2006 ) . functionally , at least in chrysomelidae , iridoid monoterpenes such as chrysomelidials are considered defensive or for microbial protection ; these functions are also generally attributed to opisthonotal glands of oribatida ( raspotnig et al . chemosystematic impact with respect to chemosystematic studies that use opisthonotal gland profiles of oribatida , the finding of chrysomelidials in o. berlesei is significant . in preliminary investigations , chrysomelidials have been found also in other species of euphthiracaroidea ( data unpublished ) , suggesting that this chemical class may be characteristic for species of oribotritiidae , possibly representing an important chemical synapomorphy of this family . also , two further compounds in extracts of o. berlesei , if our tentative identifications are correct , would be novel for oribatid and astigmatid mite opisthonotal gland secretions . the diterpene , -springene , which we tentatively identified as one of the three main components of the secretion , was originally found in secretions of springboks ( burger et al . 1978 ) , and has also been detected in exocrine secretions of further vertebrates , especially mammals and reptiles ( e.g. , waterhouse et al . 2003 ) , and invertebrates such as certain hymenopterans ( e.g. , howard et al . 2003 ; cruz - lopez et al . the other novel compound ( for oribatid and astigmatid mite opisthonotal gland secretions ) is the tentatively identified 9,17-octadecadienal , a minor constituent of the o. berlesei extract . both tentatively identified compounds , -springene and 9,17-octadecadienal , may be of chemosystematic value in oribatida . their unequivocal identification and possible distribution across other species of euphthiracaroidea remain to be studied . astigmatid compounds perhaps the most surprising result of our study is the lack of astigmatid compounds in the opisthonotal gland extract of o. berlesei . these compounds are common secretion constituents in mixonomata desmonomata and astigmata , and are regarded as characteristic of all groups above middle - derived mixonomatans . in collohmanniidae ( proposed close relatives of euphthiracaroids ) , astigmatid compounds , at least in some higher - derived desmonomatans and in brachypylines , may be reduced and replaced by other ( novel ) compounds in adult secretions of certain groups as in , for example , hermannia convexa ( raspotnig et al . 2005a ) and scheloribates ssp . thus , there is a chemical dimorphism between juveniles and adults , with juvenile secretions being distinguished by the plesiomorphic , astigmatid compounds - rich condition . this chemical dimorphism was tested in o. berlesei ; however , neither adults nor juveniles contained any astigmatid compounds . even though the lack of astigmatid compounds in o. berlesei may be explained by complete evolutionary reduction , this situation may suggest a possible polyphyletic origin of astigmatid compounds in opisthonotal glands in general . the lack of astigmatid compounds is even more surprising in light of the classification system proposed by haumann ( 1991 ) , in which oribotritiids are regarded as basal euphthiracaroids . astigmatid compounds - free opisthonotal gland secretions in adults and juveniles also occur in several groups of brachypylines ( raspotnig , unpublished data ) , but these groups are considered highly derived . the lack of astigmatid compounds in oribotritiidae could be reconciled by a systematic replacement of euphthiracaroids below the collohmanniidae , corresponding to a possible split from the oribatid stem - line before astigmatid compounds evolved . . the lack of astigmatid compounds may not necessarily be true for all oribotritiidae , and any extrapolation from data for o. berlesei remains speculative . our major aim for future studies is the search for astigmatid compounds in other representatives of euphthiracaroid families .
gas chromatographic mass spectrometric analyses of whole body extracts of oribotritia berlesei , a large - sized soil - dwelling oribatid mite , revealed a consistent chemical pattern of ten components , probably originating from the well - developed opisthonotal glands . the three major components of the extract were the iridoid monoterpene , ( 3s,8s)-chrysomelidial ( about 45% of the extract ) , the unsaturated hydrocarbon 6,9-heptadecadiene , and the diterpene -springene ( the latter two , each about 2025% of the extract ) . the remaining minor components ( together about 10% of the extract ) included a series of hydrocarbons ( tridecene , tridecane , pentadecene , pentadecane , 8-heptadecene , and heptadecane ) and the tentatively identified 9,17-octadecadienal . in contrast , analysis of juveniles showed only two compounds , namely a 2:1 mixture of ( 3s,8s)-chrysomelidial and its epimer , epi - chrysomelidial ( 3s,8r - chrysomelidial ) . unexpectedly , neither adult nor juvenile secretions contained the so - called astigmatid compounds , which are considered characteristic of secretions of oribatids above moderately derived mixonomata . the chrysomelidials , as well as -springene and octadecadienal , are newly identified compounds in the opisthonotal glands of oribatid mites and have chemotaxonomic potential for this group . this is the first instance of finding chrysomelidials outside the coleoptera .
Introduction Methods and Materials Results Discussion
apart from the well - studied opisthonotal gland secretions of astigmata ( kuwahara 2004 ) , the secretions of a selection of species of several major groups of glandulate oribatids have been investigated , including representatives of parhyposomata ( sakata and norton 2001 ) , a few species of mixonomata and desmonomata ( sakata et al . while parhyposomata produce phenols and naphthols , the so - called astigmatid compounds ( sensu sakata and norton 2001 ) evolved within ancient mixonomatans , and hence , are possibly found in all the above groups . in the present paper , we provide the first chemical analysis of the opisthonotal gland secretion of oribotritia berlesei , a large - sized oribatid mite from the near - basal euphthiracaroid family oribotritiidae . derivatization , syntheses , reference compounds , and other chemicals tri- , penta- , and heptadecane , as well as citral ( 60% geranial , 40% neral ) and selenium dioxide for the preparation of oxocitral ( see bellesia et al . peaks a ( tridecene ) , b ( tridecane ) , c1 ( epi - chrysomelidial = 3s,8s - chrysomelidial ) , d ( pentadecene ) , e ( pentadecane ) , f ( 6,9-heptadecadiene ) , g ( 8-heptadecene ) , h ( heptadecane ) , i ( tentatively , -springene ) , j ( tentatively , 9,17-octadecadienal ) electron impact mass spectra of chrysomelidials in extracts of oribotria berlesei . , ( 3s,8s)-chrysomelidial , ( 3r,8r)-dehydroiridodial , and ( 3s,8r)-chrysomelidial (= epi - chrysomelidial ) , ( 3r,8s)-dehydroiridodial . the remaining peaks a , b , d , e , g , and h , all of them minor components , appeared to be a series of hydrocarbons , with compounds b ( rt = 11.69 ) , e ( rt = 14.32 min ) , and h ( rt = 16.65 min ) exhibiting the spectra of saturated n - alkanes with molecular ions at m / z 184 , 212 , and 240 , respectively . moreover , the retention time and mass spectrum of compound c1 fully corresponded with those of the earlier - eluting peak of the synthetic chrysomelidial mixture , limiting the possibilities to epi - chrysomelidial ( 3s,8r - chrysomelidial ) and its enantiomer , ( 3r,8s)-dehydroiridodial . thus , again assuming that dehydroiridodials are exclusively plant - derived compounds , compound c1 was tentatively identified as epi - chrysomelidial ( 3s,8r - chrysomelidial).from a total of eight extracts of juvenile o. berlesei investigated ( derived from 18 individuals ) , seven extracts consistently showed these two stereoisomers only ; the other extract ( an extract of a single individual ) apparently did not contain any compounds at all . according to these studies , and to current knowledge , chrysomelidial in leaf beetles is a mixture of two diastereomers , ( 3s,8s - chrysomelidial (= the original chrysomelidial ) and ( 3s,8r)-chrysomelidial (= epi - chysomelidial ) , with the former being the predominant isomer in the majority of chrysomelidial - producing species ( e.g. thus , assuming dehydroiridodials are found exclusively in plants , the compounds in o. berlesei extracts are ( 3s,8s)-chrysomelidial and ( 3s,8r)-chrysomelidial ( epi - chrysomelidial).our analysis of o. berlesei is not only the first example of the chemistry of exocrine glands of euphthiracaroidea but also the only report of chrysomelidials found outside the coleoptera . in o. berlesei , chrysomelidials most likely originate from the opisthonotal glands : these glands are quite large in o. berlesei , and thus probably capable of producing compounds in the amount detected . the diterpene , -springene , which we tentatively identified as one of the three main components of the secretion , was originally found in secretions of springboks ( burger et al .
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trypanosoma brucei , trypanosoma cruzi and leishmania major are unicellular protozoa of considerable medical importance since they are the etiologic agents of sleeping sickness ( african trypanosomiasis ) , chagas disease ( american trypanosomiasis ) and leishmaniasis , respectively . the geographic range of these parasites is determined by their insect vectors : in the case of sleeping sickness , a blood sucking fly of the genus glossina , also known as the tsetse fly , for chagas disease , a reduviid bug known as the kissing bug and for leishmaniasis , a phlebotomine sandfly . while sleeping sickness occurs in sub - saharan africa , chagas disease is prevalent in latin america . leishmaniasis is considered to be endemic in 88 countries , 72 of which are developing countries in asia , south america and africa . together , these three parasitic diseases represent a huge burden since approximately 0.5 million people are infected with t. brucei , 10 million with t. cruzi and an estimated 12 million with different species of leishmania . in addition to the two human - infective subspecies , t. brucei gambiense and t. b. rhodesiense , other species and subspecies of african trypanosomes cause the disease known as nagana in domestic animals , imposing a further economic burden on several african countries . different forms of leishmaniasis are caused by at least 20 leishmanial species : cutaneous leishmaniasis , with an estimated 1.5 million cases , and visceral leishmaniasis , with about 500,000 new cases annually , are the most common . although control of the arthropod vectors of these diseases is an achievable goal and has been successful against the t. cruzi vector in parts of latin america , the alarming resurgence of sleeping sickness in africa and of leishmaniasis in parts of asia and latin america is a constant reminder of the need for better forms of chemotherapy and prevention of these diseases . more detailed information on african and american trypanosomiasis and the different forms of leishmaniasis , including vector distribution , disease control and treatment protocols can be found at http://apps.who.int / tdr/. trypanosoma brucei , t. cruzi and leishmania spp . are hemoflagellates of the family trypanosomatidae ( order kinetoplastida ) that is characterized by the presence of a single flagellum and one mitochondrion containing a unique organelle known as the kinetoplast which contains the mitochondrial dna ( simpson et al . , 2006 ) . each parasite has a complex life cycle that involves humans as one of their various hosts ( figure 1 ) . as some of the earliest divergent members of the eukaryotae ( haag et al . , 1998 ) , these parasites have peculiar aspects of gene expression , including polycistronic transcription of most of their genomes ( martnez - calvillo et al . , 2010 ) , rna polymerase i - mediated transcription of protein - coding genes ( gunzl et al . , 2003 ) , rna trans - splicing to generate mature , capped mrnas ( lebowitz et al . , 1993 ) and extensive rna editing to generate functional mrnas transcribed from mito - chondrial genes ( hajduk et al . , 1993 ) . apart from their medical relevance , these peculiar characteristics make these parasites very interesting models for studying genome evolution and other aspects of genome function . on the other hand , the early evolutionary divergence of these organisms has resulted in biochemical characteristics that are not common in higher eukaryotes , such as enzymes related to antioxidant metabolism ( olin - sandoval et al . , 2010 ) as well as sterol and glycosylphosphatidylinositol ( gpi ) biosynthesis ( lepesheva et al . , 2011 ; koeller and heise , 2011 ) that have been exploited as promising drug targets . genome sequencing of tri - tryp parasites began in the early 90s with the analyses of 518 expressed sequence tags ( ests ) generated from mrna isolated from bloodstream forms of t. b. rhodesiense ( el - sayed et al . , 1995 ) . shortly thereafter , a comparison between est and genomic sequences showed that sequencing random dna fragments was as efficient as est analyses for discovering new genes in the african trypanosome ( el - sayed and donelson , 1997 ) . in 1996 , an est analysis of cdna libraries constructed with mrna from l. major promastigotes was published ( levick et al . , 1996 ) , and the first est analysis of t. cruzi epimastigote forms was published in 1997 ( brando et al . , 1997 ) . during this period , pulsed - field gel electrophoretic analysis of chromosomes and the sequencing of large dna fragments from cosmid , bacterial artificial chromosome and yeast artificial chromosome libraries were also undertaken to generate physical maps of tri - tryp genomes ( blackwell and melville , 1999 ) . in 1999 , the sequence of a 257-kilobase region spanning almost the entire chromosome 1 of l. major revealed the unusual distribution of protein - coding genes that was later found to be characteristic of all tri - tryp genomes . the complete sequence of l. major chromosome 1 revealed 79 protein - coding genes , with the first 29 genes all encoded on one dna strand and the remaining 50 genes encoded on the opposite strand ( myler et al . , 1999 ) . the tri - tryp gene organization is reminiscent of bacterial operons , with protein coding genes densely packed within directional clusters in one strand separated by strand switch regions ( i.e. , changes in the coding strand ) ( figure 2 ) . experimental evidence suggests that transcription initiates bi - directionally between two divergent gene clusters ( martnez - calvillo et al . , 2003 , 2004 ) to produce polycistronic pre - mrnas that are subsequently processed . remarkably , with the exception of the spliced leader ( sl ) promoter , no promoter is recognized by rna polymerase ii and only a few transcription factors have been identified ( cribb and serra , 2009 ; cribb et al . , 2010 ) . even more surprisingly , although orthologs of all conserved components of the rna polymerase ii complex were identified in the tri - tryp genome ( ivens et al . , 2005 ) , the transcription of some trypanosomatid genes such as vsg ( variant surface glycoprotein ) and the procyclin genes of t. brucei , as well as several exogenous genes transfected into t. cruzi , are mediated by rna polymerase i once the polycistronic pre - mrna is produced , two coupled reactions ( trans - splicing and poly - adenylation ) result in mature monocistronic transcripts . trans - splicing means that every mature mrna has an identical capped sequence of 39 nucleotides , known at the spliced leader ( sl ) , at the 5 end ( liang et al . , 2003 ) . whilst no sequence consensus for polyadenylation or sl addition has been found , several studies have demonstrated that polypyrimidine - rich tracts located within intergenic regions guide sl addition and poly - adenylation , resulting in mature mrnas ( lebowitz et al . , 1993 ) intergenic sequences involved in the processing of t. cruzi , t. brucei and leishmania mrna have been thoroughly investigated by comparing mrna with genomic sequences , initially using est databases ( benz et al . , 2005 ; campos et al . , 2008 ; 2008 ) and , more recently , using high - throughput rna - sequencing ( rnaseq ) ( siegel et al . in addition to providing valuable information on the mechanisms of gene expression in these organisms , these analyses also yielded data that allowed the optimization of transfection vectors used to express foreign genes and genetic manipulation in trypanosomatids . comparative genomic analyses using the tri - tryp sequences have already provided interesting insights into the genetic and evolutionary bases of the distinct and shared lifestyles of these parasites . probably the most striking finding is that the three genomes display high levels of synteny and share a conserved set of ~6,200 genes , 94% of which are arranged in syntenic directional gene clusters ( el - sayed et al . , 2005a ) . alignment of the deduced protein sequences of the majority of the clusters of orthologous genes across the three organisms reveals an average 57% identity between t. cruzi and t. brucei and 44% identity between t. cruzi and l. major that reflected the expected phylogenetic relationships ( lukes et al . , 1997 ; the majority of species - specific genes occurs on non - syntenic chromosomes and consists of members of large surface antigen families . structural rnas , retroelements and gene family expansion are also often associated with breaks in the conservation of gene synteny ( el - sayed et al . , 2005a ) . multi - gene family expansions are generally species - specific and most pronounced in the t. cruzi genome . as discussed below , a number of t. cruzi multi - gene families encode surface proteins , such as trans - sialidases , mucin - associated surface proteins ( masp ) and mucins tcmuc and gp63 that likely play important roles in host - parasite interactions ( di noia et al . , 1995 ; vargas et al . , 2004 ; baida et al . , 2006 ; bartholomeu et al . , 2009 ) . based on their location in regions of synteny breaks these arrays may be subject to extensive rearrangements during the parasite s evolution and are thus directly associated with the specificities of each of the three parasitic diseases . chagas disease , caused by t. cruzi , is endemic in more than 20 latin american countries , where an estimated 10 million people are infected and the domiciliation of the triatomines exposes at least 90 million individuals to the risk of infection . with no vaccine or effective drug treatment available , the main strategy for control must rely on the prevention of transmission by the insect vectors and blood transfusions . the parasite proliferates in the midgut of several species of a triatomid hematophagous vector . after reaching the insect s hindgut , epimastigote forms differentiate into non - dividing , infective metacyclic trypomastigotes that are excreted in the insect s feces . trypomastigotes can infect a mammalian host by passing through mucous membranes or skin lesions during feeding by the insect . once inside the mammalian host , trypomastigotes invade different types of cells where they transform into proliferative intracellular amastigotes . after a number of cell divisions in the host cell cytoplasm , amastigotes differentiate into trypomastigotes that are released into the bloodstream after host cell rupture and , after being taken up by an insect during a blood meal , they start a new cycle ( brener , 1973 ) ( figure 1 ) . the highly heterogenous t. cruzi population consists of a large number of strains with distinct characteristics related to morphology , growth rate , parasitemia curves , virulence , pathogenicity , drug sensitivity , antigenic profile , metacyclogenesis and tissue tropism ( buscaglia and di noia , 2003 ) . despite the broad genetic diversity observed among different strains and isolates , early studies based on different genotyping strategies identified two major lineages in the parasite population , named t. cruzi i and t. cruzi ii ( souto et al . , 1996 ; momen 1999 ) . these divergent lineages occupy distinct ecological environments , namely , the sylvatic cycle ( t. cruzi i ) and the domestic cycle ( t. cruzi ii ) of chagas disease ( zingales et al . , 1998 ) , as well as further analyses led some authors to propose the sub - division of t. cruzi ii into five sub - groups : t. cruzi iia , iib , iic , iid and iie ( brisse et al . , 2000 ) . phylogenetic analyses of the t. cruzi strains became more confusing when additional data indicated the existence of not just two , but three major groups in the t. cruzi population , in addition to hybrid strains ( miles et al . , 1978 ; augusto - pinto et al . , 2003 ; de freitas et al . , 2006 ) . after intense debate , in 2009 an international consensus recognized the existence of six major strains , also known as discrete typing units ( dtus ) i vi ( zingales et al . , 2009 ) ( table 1 ) . since chagas disease spawns a variety of clinical forms , these studies are highly relevant : understanding the genetic variation among strains can potentially explain differences in disease pathogenesis , host preferences and , most importantly , provides essential information for the identification of new drug targets and good antigenic candidates for better diagnosis and vaccine development . for instance , t. cruzi ii strains and the hybrid strains belonging to t. cruzi v and vi are the predominant causes of human disease in south america ( zingales et al . , 2009 ) , whereas t. cruzi i strains are more abundant among wild hosts and vectors . although detailed analysis of the biological and molecular factors underlying t. cruzi population structure and the epidemiology of chagas disease are beyond the scope of this review , one must keep in mind that the genetic variability found in the t. cruzi population is an essential aspect to be considered when analyzing this parasites genome . cl brener , a clone derived from a hybrid t. cruzi strain belonging to t. cruzi vi , was chosen as a reference strain for the initial t. cruzi genome project . the hybrid nature of the cl brener clone became clear only after the genome sequencing had begun , when analyses of nuclear and mitochondrial sequences showed that this strain resulted from a fusion event that had occurred between ancient genotypes corresponding to strains belonging to t. cruzi ii and iii groups ( el - sayed et al . prior to this knowledge , the choice of the clone cl brener , initially classified as a member of sub - group iie , was based on five characteristics : ( 1 ) it was isolated from the domiciliary vector triatoma infestans , ( 2 ) its pattern of infectivity in mice was very well known , ( 3 ) it had preferential tropism for heart and muscle cells , ( 4 ) it showed a clear acute phase in accidentally infected humans , and ( 5 ) it was susceptible to drugs used to treat chagas disease ( zingales et al . , 1997 ) . in addition , several genomic studies had previously used this strain for karyotype analyses ( branche et al . , 2006 ) and the generation of physical maps and ests from all three stages of the parasite life cycle ( cano et al . , 1995 ; henriksson et al . , 1995 ; brando et al . , 1997 ; verdun et al . , 1998 ; the t. cruzi cl brener haploid genome , estimated to be 55 mb , was sequenced using the wgs ( whole genome shotgun ) strategy . because of its hybrid nature and the high level of allelic polymorphism , a 14x coverage , much higher than the usual 810x coverage , was required to distinguish the ambiguities derived from allelic variations from those produced by sequencing errors . in contrast to the other two tri - tryp genomes , the t. cruzi draft sequence ( el - sayed et al . , 2005b ) was published as an assembly of 5,489 scaffolds built by 8,740 contigs . four years later , based on synteny maps for the t. brucei chromosomes , weatherly et al . ( 2009 ) assembled the t. cruzi contigs and scaffolds initially in 11 pairs of homologous t. brucei - like chromosomes and , ultimately , in 41 t. cruzi chromosomes . since trypanosomatid chromosomes do not condensate during mitosis and are therefore not visualized in metaphasic cells the predicted number of t. cruzi chromosomes was based on studies of pulsed - field gel electrophoresis ( pfge ) analyses ( branche et al . , 2006 ) , which turned out to be similar to the number of assembled chromosomes . as mentioned above , the genome organization in t. cruzi is largely syntenic with the other tri - tryp ( t. brucei and l. major ) genomes , with most species - specific genes , such as surface protein gene families , occurring in internal and subtelomeric regions of non - syntenic chromosome ( el - sayed et al . , 2005a ) . because of its hybrid nature , the cl brener genome is represented by a redundant dataset since homologous regions displaying a high level of polymorphism were assembled separately , generating two set of contigs , each corresponding to one haplotype . to identify the two haplotypes , reads from the genome of the cloned esmeraldo strain , a member of t. cruzi ii , and representing one of the cl brener parental strain ( de freitas et al . thus , in the annotation data of the cl brener genome , the two haplotypes are referred to as esmeraldo - like or non - esmeraldo - like sequences ( aslett et al . , 2010 ) . the haploid cl brener genome has an estimated 12,000 genes . as with the other tri - tryps , the t. cruzi genes are organized in long polycistronic clusters that are transcribed by rna polymerase ii and processed into monocistronic mrnas that accumulate differentially during the various stages of the parasite life cycle . as indicated before , one of the main characteristics revealed by the complete sequence of the t. cruzi genome was the dramatic expansion of families encoding surface proteins ( el - sayed et al . , 2005a ) . compared to t. brucei and l. major , t. cruzi has the largest set of multi - gene families , perhaps because of its unique capacity to invade and multiply within different types of host cells . long terminal repeat ( ltr ) and non - ltr retroelements and other sub - telomeric also contribute to the large proportion of repetitive sequences ( 50% of the genome ) in this genome . the largest protein gene family encodes a group of surface proteins known as trans - sialidases ( ts ) , with 1,430 members . tss are surface molecules identified as virulent factors of t. cruzi that are responsible for transferring sialic acid from host sialogly - coconjugates to the terminal - galactose on t. cruzi mucins . mucin - associated surface proteins ( masp ) are the second largest t. cruzi gene family , with a total of 1,377 members . although masp sequences correspond to ~6% of the parasite diploid genome , they were only identified during annotation of the t. cruzi genome . masps are glycosylphosphatidylinositol ( gpi)-anchored surface proteins that are preferentially expressed in trypomastigotes ; these proteins are characterized by highly conserved n- and c - terminal domains and a strikingly variable and repetitive central region ( bartholomeu et al . , 2009 ) . together with the mucin and gp63 gene families , these four gene families account for ~17% of all protein - coding genes and are organized as dispersed clusters of tandem and interspersed repeats . other large families consist of the previously described rhs and dgf-1 genes whose functions are unknown and which , like the ts genes , occur mostly at subtelomeric locations . examples of other gene families with more than 10 members also present in the t. cruzi genome include glycosyltransferases , protein kinases and phosphatases , kinesins , amino acid transporters and helicases , in addition to several gene families encoding hypothetical proteins ( el - sayed et al . , 2005a ) . the collapse of nearly identical repeats in some gene families , such as the gene cluster encoding - and -tubulins , meant that not all copies of the family were included in the original genome assembly . 2007 ) described an analysis of the total genomic repetitive content of protein coding sequences and concluded that 18% of all protein coding sequences existed in 14 or more copies . in addition to the need to evade the host immune system , the existence of highly repetitive gene families in the t. cruzi genome in which a large number of gene copies can lead to the enhanced expression of various proteins may help to overcome a major problem in this genome , namely , the lack of strong promoters capable of generating high levels of mrna from single copy genes . it is also likely that many of the striking polymorphisms among t. cruzi isolates that are reflected in several epidemiological and pathological aspects of chagas disease are partly attributable to variability within regions containing gene families . whole genome comparisons of distinct t. cruzi lineages are beginning to improve our understanding of this question . soon after the cl brener genome was completed several groups began sequencing the genome of representative strains of other major t. cruzi lineages . as indicated above , the hybrid nature of the cl brener genome provided data for two genomes , with esmeraldo - like and non - esmeraldo contigs making it possible to distinguish information from t. cruzi ii and iii groups , respectively ( see table 1 ) . 2011 ) published a draft genome sequence of sylvio x10 , a strain belonging to t. cruzi i group , which is the predominant agent of chagas disease in central america and in the amazon . although rarely isolated from humans in endemic areas in southern countries of latin america where most cases of chagas disease with mega - syndromes occur , t. cruzi i strains are highly abundant among wild hosts and vectors ( zingales et al . thus , the distinct ecological niches occupied by t. cruzi i and ii strains , together with the fact these strains are highly divergent in terms of phylogenetic analysis , prompted franzn et al . ( 2011 ) to sequence the genome of a representative of t. cruzi i group and to undertake a comparative analysis with the cl brener genome . in agreement with previous analyses , the sylvio x10 genome was estimated to be ~44 mb in size , i.e. , smaller than the cl brener genome . indeed , smaller genomes seems to be a general feature of t. cruzi i strains ( branche et al . as expected , the architectures of the two genomes were very similar , with highly conserved syntenic regions corresponding to the gene - dense core of the coding regions organized for long polycistronic transcription . as with the cl brener genome , the presence of repetitive sequences meant that the sylvio x10 genome was represented as fragmented contigs . the technical difficulties associated with the assembly of repetitive sequences meant that only about 49% of the generated sylvio x10 sequence data was incorporated into contigs , leaving 710,109 reads that were not included in the assembly . consequently , the draft genome of sylvio x10 was assembled into 7,092 contigs , which is slightly less than the number of contigs reported for the draft genome of cl brener . the alignment of these contigs to both cl brener haplotypes showed that the mean nucleotide identity was greater between sylvio x10 and non - esmeraldo ( 98.2% ) than between sylvio x10 and esmeraldo ( 97.5% ) . this finding agrees with previous phylogenetic analyses indicating that sequences from t. cruzi i strains are more closely related to t. cruzi iii ( represented by the non - esmeraldo cl brener haplotype ) than to t. cruzi ii ( represented by the esmeraldo - like haplotype ) ( cerqueira et al . , 2008 ; ruvalcaba - trejo and sturm , 2011 ) . in contrast to the hybrid cl brener genome , for which the amount of heterozygosity in the core genome was estimated to be 5.5% ( el - sayed et al . , 2005a ) , the diploid sylvio x10 genome was homozygous ( < 0.08% heterozygosity ) . most importantly , analysis of the core gene content of cl brener and sylvio x10 revealed six open reading frames that were missing in the sylvio x10 genome . besides these six genes , estimations based on total sequence reads indicated that several multicopy gene families , including dgf , mucin , masp and gp63 contained substantially fewer genes in sylvio x10 than in cl brener . a 5.9 mb size difference between the sylvio x10/1 and cl brener genomes largely reflected the expansion of these gene families . however , the extent to which these genomic variations are related to strain differences in host preference and the ability to cause chagas disease remains to be determined . the advent of next - generation sequencing technologies has ushered in a new era in comparative sequencing by allowing the exploration of a wide range of evolutionary and pathological questions within the t. cruzi lineage . a consortium of laboratories funded by the national institutes of health / national institutes of allergy and infectious diseases ( niaid ) and the national human genome research institute ( nhgri ) is sequencing t. cruzi strains representative of each one of the six main groups , such as esmeraldo ( t. cruzi ii ) , 3869 ( t. cruzi iii ) , can iii ( t. cruzi iv ) , nrcl3 ( t. cruzi v ) and tula cl2 ( t. cruzi vi ) ( n. el - sayed , personal communication ) . our laboratory has been involved in the sequencing of another t. cruzi i strain ( dm28c ) and cl-14 , a non - virulent strain that belongs to the t. cruzi vi group ( s. teixeira , unpublished ) . in contrast to cl brener , balb / c mice injected with cl-14 trypomastigotes showed no parasitemia but developed high resistance against a lethal challenge with virulent trypomastigotes from the cl brener or y strains ( lima et al . , 1995 ) . our goal in this work is to use comparative analyses of the cl brener and cl-14 genomes to identify potential sequences that can restore the virulence of cl-14 and then test these in transfection protocols . in addition to investigations of the nuclear genome , several studies have examined the mitochondrial genome of kinetoplastids which contains a mass of concatenated dna known as kinetoplast dna ( kdna ) that is easily identified near the insertion of the flagellum ( brener , 1973 ) . in t. cruzi , kdna consists of a highly structured disk - shaped network of thousands of concatenated mini - circles 0.510 kb in size and dozens of concatenated maxicircles 2040 kb in size . whereas minicircle sequences are present exclusively in kinetoplastids , maxicircles are the homologues of mtdna molecules found in other eukaryotes ( lukes et al . , 1997 ) . ( 2006 ) described the complete sequences of maxicircle dnas corresponding to groups t. cruzi ii ( from sequences of the esmeraldo strain ) and iii ( from cl brener sequences ) . as with other trypanosomatid mitochondrial genes , sequence analyses showed that t. cruzi maxicircle dna contained frameshift errors in most of its genes that were corrected at the rna level by a complex u - insertion / deletion process known as rna editing ( hajduk et al . , 1993 ) . key elements of this repair process include grnas ( guide rnas ) which are encoded mainly by minicircles , although a few grna sequences are also present in maxicircles . the grnas hybridize to the 3 end of a target message and undertake direct u insertion and deletion by the so - called editosome machinery ( stuart and panigrahi , 2002 ) . the complete sequences of the 25 kb t. cruzi maxi - circles revealed 18 tightly clustered mitochondrial protein - coding genes and two rrna genes that were syntenic with previously sequenced maxicircles of t. brucei and leishmania tarentolae . fifteen of the 18 protein - coding genes were edited . outside the coding region , strain - specific repetitive regions and a variable region that was unique for each strain were identified ( westenberger et al . , 2006 ) . more recently , comparative analyses of the mitochondrial genomes of t. cruzi i , ii and iii were reported after ruvalcaba - trejo and sturm ( 2011 ) generated the sequence of the coding region of the maxicircle from sylvio x10 . in agreement with the nuclear genomic analysis , phylogenetic analysis of the maxicircle coding regions supported a close evolutionary relationship between t. cruzi i and iii . based on their mitochondrial dna analyses , these authors proposed a model in which an ancestral strain belonging to t. cruzi i provided the maxicircle for the progeny of a tci - tcii hybridization event that resulted in the generation of t. cruzi iii and t. cruzi iv strains . a subsequent back - cross hybridization between t. cruzi ii and t. cruzi iii strains resulted in the t. cruzi v and vi strains , such as cl brener , that carry the maxicircle from their t. cruzi iii ancestor . leishmania spp . are parasitic protozoa transmitted by the bites of phlebotomine sand flies that are endemic in tropical and subtropical regions worldwide . more than 20 species are responsible for a wide spectrum of diseases , known as leishmaniasis ( murray et al . , 2005 ) . parasites in this genus are classified into two subgenera according to the part of the sandfly gut where colonization and development occur : the subgenus leishmania ( leishmania ) consists of parasites with mid and foregut development , whereas the subgenus leishmania ( viannia ) consists of parasites that undergo hindgut development ( lainson et al . , 1977 ; bates , 2007 ) . depending on the species of leishmania , infection of humans may result in diverse clinical forms of leishmaniasis with symptoms ranging from self - healing cutaneous lesions ( l. major / l . infection by leishmania can also result in mucosal leishmaniasis ( mainly caused by l. braziliensis ) and diffuse cutaneous leishmaniasis ( mainly caused by l. in addition to the species of leishmania , other factors such as the genetic variability of the human host may determine the disease tropism and clinical manifestations in leishmaniasis ( blackwell et al . the world health organization ( who ) estimates that there are over two million new cases of leishmaniasis each year , with more than 360 million people at risk of contracting this disease in 88 countries on five continents ( asia , africa , europe , north america and south america ) ( www.who.int/tdrdiseases/leish ) . as part of their life cycle , leishmania spp . alternate between the alimentary tract of the sandfly vector , where they grow as extracellular flagellated promastigotes and differentiate into infective non - dividing metacyclic forms , and the phagolysosome of the vertebrate host macrophages , where they differentiate into aflagellated , replicative amastigotes ( figure 1 ) . there is no effective vaccine against leishmania and the available therapeutic arsenal is extremely limited ( mauel , 2002 ) . thus , completion of the genome sequences of several leishmania species ( ivens et al . , 2005 ; peacock et al . , 2007 ) represents a long awaited aspiration for groups involved in the discovery and development of new drugs and vaccine targets . leishmania major friedlin was chosen as the leishmania reference strain for the tri - tryp genome project . the l. major haploid genome ( ~32.8 mb ) is distributed among 36 relatively small chromosomes ranging from 0.28 to 2.8 mb in size ( wincker , 1996 ) and was sequenced after shotgun cloning of large dna fragments derived from chromosomal bands separated in agarose gels . prior to publication of the tri - tryp genome sequence , the complete sequences of chromosomes 1 and 3 from l. major were published ( myler et al . , 1999 ; worthey et al . , 2003 ) and an optical map of the entire genome was generated ( zhou et al . , 2004 ) . in 2007 , the complete genomes of two other leishmania species , l. infantum and l. braziliensis , were also described ( peacock et al . , 2007 ) . leishmania infantum , also known as l. chagasi in latin america , was chosen as the second leishmania species to have its genome sequenced on the basis of its virulence in animals , transmissibility in sandflies and adaptability to laboratory experimentation ( denise et al . , 2006 ) . this species is the causative agent of visceral leishmaniasis , the most serious form of the disease and frequently fatal if left untreated . the new world species l. braziliensis , within the subgenus l. ( viannia ) , is the third and most divergent species sequenced . the l. infantum and l. braziliensis genome sequences were obtained by the whole - genome shotgun approach with five- and six - fold coverage , respectively ( peacock et al . , 2007 ) . importantly , the three complete genomes are from strains that cause distinct types of leishmanial diseases , are adapted for maintenance and manipulation in the laboratory and are also frequently used in studies in vitro and in animal models of infection ( laurentino et al . , 2004 ; ivens et al . , 2005 ; denise et al . , 2006 ) . the complete sequences of all three leishmania genomes can be accessed in the tri - tryp database ; sequencing of the genome from a fourth species ( l. mexicana ) is in progress . surprisingly , comparative genomic studies of the three evolutionarily and geographically distinct species , l. major and l. infantum ( old world species ) and l. braziliensis ( new world species ) , showed very little divergence among the species in terms of genomic sequence and organization ( peacock et al . , 2007 ; lynn and mcmaster , 2008 ) , despite a divergence of 20100 million years within the leishmania genus ( lukes et al . , 1997 ) . the haploid genome of the three species has an estimated 8,300 genes , with more than 99% of them maintaining synteny in the three leishmania genomes ( peacock et al as with the other tri - tryps , the majority of leishmania genes are annotated as genes of unknown function . of the 8,300 genes , only 200 were identified as being differentially distributed between the three genomes . the l. braziliensis genome possesses 47 genes that are absent from the other two species , while 27 and 5 genes are specific for the l. infantum and l. major genomes , respectively . such high conservation in overall genome sequences and genome synteny indicates that the leishmania genome is highly stable and has not undergone large genomic rearrangements during speciation . this finding also suggests that only a few species - specific parasite genes may contribute to differential pathogenesis and tissue tropism ( peacock et al . thus far , the only gene for which there is experimental evidence indicating direct involvement in the differential tropism among leishmania diseases is the a2 locus . initially identified as an amastigote - specific gene family in l. donovani , a2 has been shown to play a major role in parasite virulence and visceralization ( zhang et al . , multiple copies of the a2 gene alternating with a distinct gene termed the a2rel gene are found in the genome of several species of the l. donovani group that is responsible for visceral diseases . although its precise function is still unknown , the product of the a2 gene , an endoplasmic reticulum protein with a large repetitive domain , may be related to the parasite stress response ( mccall and matlashewski , 2010 ) . in l. major , which does not cause visceral leishmaniasis , the a2 gene is a pseudogene and introduction of the l. donovani a2 gene into l. major enhanced the ability of l. major to survive in visceral organs of susceptible balb / c mice ( zhang et al . , 2003 ) . more recently , the expression of a2 in l. tarentolae , a lizard parasite that is not pathogenic in mammals , significantly increased the infectivity of this species and enhanced its ability to survive in the liver of balb / c mice ( mizbani et al . , 2011 ) . in addition to experiments suggesting a possible role for the a2 gene in the differential tropism of cutaneous and visceral leishmania parasites , the a2 gene has been used as a promising vaccine candidate and diagnostic antigen ( fernandes et al . , 2008 ) . another locus possibly involved in macrophage invasion and that varies considerably among the three leishmania genomes is the gp63 locus . also known as the major surface protease ( msp ) , gp63 constitutes a family of surface metalloproteases expressed in all trypanosomatids examined so far ( yao et al . , 2003 ) . gp63 sequences identified in the three leishmania genomes and in the t. cruzi and t. brucei genomes vary in their gene copy number , and this may have implications for differences in the disease phenotype ( voth et al . , 1998 ) . a similar conclusion may apply to the amastin multi - gene family that encodes a family of amastigote - specific , highly glycosylated hydrophobic surface proteins also present in t. cruzi but which has been greatly expanded in the genus leishmania ( teixeira et al . , 1995 ; interestingly , in the t. brucei genome , which does not have an intracellular stage , only two copies of a highly divergent amastin sequence are present ( jackson , 2010 ) . the identification of these species - specific genes represents an initial step towards the characterization of parasite factors that may determine the specificities of each type of parasitic infection . on the other hand , antigens common to all leishmania species could be used as potential vaccine candidates ( peacock et al . , 2007 ) . however , apart from differences in gene sequences , it is possible that the distinct clinical manifestations observed in the different parasitic diseases may be a consequence of differential gene expression that occurs throughout the various stages of life cycle in each parasite species . as discussed above , leishmania genes are arranged in the genome as directional gene clusters that resemble prokaryotic polycistronic transcription units ( martnez - calvillo et al . , 2004 ) . this type of gene organization and polycistronic transcription have profound implications on the regulation of gene expression , which must rely on post - transcriptional mechanisms ( boucher et al . , 2002 ; myung et al . , 2002 ; holzer et al . , 2006 ; leifso et al . , 2007 ) . since the dependency on promoter - based transcription initiation mechanisms for the control of mrna levels is greatly reduced , greater emphasis is placed on post - transcriptional regulatory mechanisms controlling mrna stability and translation , as well as protein turnover ( clayton , 2002 ) . despite significant differences in life stage morphology , biochemical properties and disease phenotypes , comparative gene expression studies have revealed surprisingly few differences in gene expression when mrna levels in different life - cycle stages or in the same stage but in different leishmania species were compared ( peacock et al . global interspecies analyses in l. major and l. infantum have shown that only 10%12% of differentially expressed genes are unique to each species ( rochette et al . , 2008 ; depledge et al . , 2009 ) . a more careful examination of the protein expression patterns and the elucidation of regulatory mechanisms will provide unique insights into this important aspect of the parasite s biology . this information will also improve our understanding of the host - parasite interaction and lead to the development of new strategies for contolling leishmaniasis . eukaryotic genomes contain an abundance of repeated dna and some of these repeated sequences are mobile elements . transposable elements ( tes ) are defined as dna sequences that are able to move from one location to another in the genome and have been identified in all organisms ( prokaryotic and eukaryotic ) examined so far . retroposons , also known as non - long - terminal - repeat ( ltr ) retrotransposons , are ubiquitous elements that transpose through an rna intermediate and are found in the genomes of most eukaryotes ( ivens et al . , 2005 ; peacock et al . , 2007 ) . trypanosoma brucei and t. cruzi contain long autonomous retroposons of the ingi clade ( tbingi and l1tc , respectively ) and short nonautonomous truncated versions ( tbrime and nartc , respectively ) , as well as degenerate ingi - related retroposons devoid of coding capacity ( dires ) that represent the most abundant transposable elements in these genomes ( < 3% of the nuclear genome ) . in contrast , l. major contains only remnants of extinct retroposons ( lmdires ) and short nonautonomous heterogenous elements ( lmsiders ) . signature known as lmsiders ( for short interspersed degenerate retroposons ) have also been identified in the genomes of l. major ( ivens et al . , 2005 ) , l. infantum and l. braziliensis ( peacock et al . , 2007 ) . unexpectedly , in l. braziliensis , the site - specific non - ltr retroposon slacs / czar , which is associated with tandemly repeated spliced leader sequences in an arrangement similar to that of the slacs or czar element in t. brucei or t. cruzi , respectively , is fully active ( aksoy et al . , 1987 ; villanueva et al . , 1991 however , in contrast to the african trypanosome genomes and similar to l. major ( ivens et al . , 2005 ) and l. infantum , no potentially active ingi - related retroposons were detected in the l. braziliensis genome ( bringaud et al . , 2009 ) . mobile elements are involved in creating mutations and genomic rearrangements and , in many eukaryotes , these effects can be regulated through an rna silencing mechanism such as rna interference ( rnai ) ( shi et al . , 2004b ; girard and hannon , 2008 ) . since first reported in 1998 ( fire et al . , 1998 ) , rnai has swept through all fields of eukaryotic biology and has proven to be a very useful tool for analyzing gene function in a variety of organisms in which the introduction or expression of short double - stranded rnas leads to the rapid destruction of cognate mrnas ( figure 4a ) ( ngo et al . , 1998 ) . this approach has a number of advantages : it is fast , requires very little sequence information , and reduces the expression of multiple gene copies , an action that is especially advantageous in asexual diploid organisms ( lacount et al . , soon after it was described in c. elegans , rnai was rapidly identified in t. brucei ( ngo et al . , 1998 ) . as discussed below , rnai has proven to be a powerful new tool for functional genomic studies in t. brucei . unexpectedly , experimental evidence as well as searches of genome databases quickly showed that rnai is absent in l. major and t. cruzi ( robinson and beverley , 2003 ; darocha et al . , 2004 ) . ( 2007 ) revealed that the genome of l. braziliensis retained key genes involved with rnai . these authors showed that l. braziliensis genome contains orthologs carrying domains characteristic of the dicer protein as well as genes with the typical argonaute domains paz and piwi , the latter containing conserved amino acid residues that are essential for functional tbago1 ( shi et al . , 2004a ) . in addition , an n - terminal rgg domain , present in tbago1 and shown to be essential for its association with polyribosomes , is also present in the l. braziliensis ago1 gene ( shi et al . , 2004a ) . ( 2010 ) demonstrated that the rnai pathway is functional in l. braziliensis and in other species within the leishmania subgenus viannia ( l. guyanensis and l. panamensis ) ( figure 4b ) . thus , this divergent species of leishmania appear to have retained not only the mechanisms for ( rnai)-mediated regulation but also potentially active retroposons ( peacock et al . , 2007 ) , which might have assisted to create the greater divergence within the l. braziliensis genome compared with the other leishmania species . for molecular parasitologists , these findings came as very good news since , as shown in t. brucei , the efficacy of rnai knockdown as a tool for systematic analysis of gene function is now also applicable in some species of leishmania . infection by t. brucei occurs after metacyclic trypomastigotes are injected into the bloodstream by the bite of a tsetse fly . in the mammalian host , the parasite differentiates into long slender trypomastigotes ( bloodstream form -bsf ) that divide , colonize the body fluids and can transform into a nonproliferative bloodstream form , also known as the short stump form . after a blood meal , trypomastigotes acquired by the insect vector differentiate into procyclic trypomastigotes in the gut , replicate and then migrate to salivary glands where they transform into metacyclic trypomastigotes ( matthews , 2005 ) ( figure 1 ) . the ability of the bsf to invade the central nervous system leads to the neurological manifestations associated with sleeping sickness . in contrast to t. cruzi and leishmania , t. brucei develops extracellularly throughout its entire life cycle . its direct exposure to a strong antibody response in the bloodstream requires a sophisticated immune evasion protocol , known as variant surface protein ( vsg ) switching ( pays et al . , 2007 ) . vsgs are bloodstream - specific surface proteins with a hypervariable n - terminal domain that is exposed extracellularly and a more conserved c - terminal domain buried in the parasite s surface coat ( van der ploeg et al . , 1982 ; since they are tightly packed at the surface , vsgs are able to shield other invariant surface proteins from attack by the immune system . the tactic for evasion is based on antigenic variation in which the expression of a specific vsg is replaced by another gene from a supply of almost thousand copies at a rate of approximately one event per 100 cell divisions ( van der ploeg et al . , 1982 ; turner and barry , 1989 ) . vsg genes are thus a family of t. brucei - specific genes whose monoallelic expression distinguishes african trypanosomes from the other two groups of pathogenic trypanosomatids ; the lack of antigenic variation in t. cruzi and leishmania spp . means that these species must evade the host s immune system by entering and multiplying inside host cells . after completion of the t. b. brucei genome , it was found that of the 9,068 predicted genes , including 904 pseudogenes , there were 1,700 t. brucei - specific genes , 806 of them ( or 9% of the whole genome ) corresponding to vsgs ( el - sayed et al . , 2005b ) . several vsg genes occur within hundreds of mini - chromosomes ( 50150 kb in size ) that are also part of the t. brucei genome . surprisingly , analyses of all vsg sequences showed that only 57 are fully functional genes and have all the recognizable features of a typical vsg ( berriman et al . , 2005 ) . it has been long known that vsg variability can be generated in t. brucei by creating mosaic genes that include pseudogenes ( roth et al . , 1989 ) . besides producing variability in vsg epitopes , these rearrangements may have the capacity to generate protein with new functions , as in the case of the sra gene discussed below ( vanhamme et al . , 2003 ) . as part of their life in the bloodstream , african trypanosomes that are pathogenic to humans have developed a mechanism to withstand other types of attack from the host immune system . in contrast to t. brucei brucei , which do not infect humans , t. b. gambiense and t. b. rhodesiense are resistant to trypanolytic factors ( tlf ) present in normal human serum ( nhs ) . tlf activity has been attributed to apolipoprotein l1 ( apol1 ) and haptoglobin ( hp)-related protein found in high density lipoprotein ( for a review , see wheeler , 2010 ) . nhs resistance in t. b. rhodesiense is conferred by a truncated vsg known as serum resistance associated ( sra ) protein which is located in endosomes and binds and neutralizes tlf ( de greef and hamers , 1994 ; de greef et al . , 1992 ; , 2003 ; prez - morga et al . , 2005 ; wheeler , 2010 ) . the killing of t. b. brucei requires the binding of tlf-1 and trafficking to the parasite acidic lysosome . it has been proposed that , in t. b. gambiense , changes in the receptor that binds tlf , as well as decreased expression of this gene , are responsible for the resistance of this t. brucei sub - species to attack by the human innate immune system ( kieft et al . , 2010 ) . since t. b. brucei is harmless to humans and t. b. gambiense is the most clinically relevant subspecies , jackson et al . ( 2010 ) sequenced the genome of t. b. gambiense using the whole - genome shotgun approach combined with bacterial artificial chromosome large insert sequencing . comparison of the sequences from a draft genome assembly of 281 contigs ( ~22.1 mb ) with the t. b. brucei genome data ( berriman et al . , 2005 ) revealed a very similar composition in terms of gene content , gene synteny and sequence identity ( 86.4% of t. b. gambiense coding sequences vary by < 1% from their orthologs in t. b. brucei ) . even though these subspecies behave differently in humans , there were very few differences in their genomes . one of these differences involved a gene encoding a putative iron - ascorbate oxidoreductase that is specific to a few t. b. brucei strains and is also absent in other trypanosomatids ( l. major and t. cruzi ) . thus , it seems that not only differences in gene content per se , but also individual single nucleotide polymorphisms ( indels ) and variations in gene expression , or a combination of these factors , may contribute to this phenotypic variation ( jackson et al . , 2010 ) . coordinated changes in gene expression are vital for trypanosomes since they must deal with rapid changes in their environment ( nutrient availability , temperature , host defenses , the presence of drug , etc . ) and be able to disseminate by alternating through different hosts . in addition , unique mechanisms for regulating gene expression are required since there is an almost complete absence of transcriptional control at the level of initiation . the lack of transcriptional regulatory elements , including a typical rna polymerase ii promoter , led to the conclusion that most factors involved in controlling gene expression act at the post - transcriptional level . by using approaches such as differential display , rna fingerprinting , differential screening of cdna libraries , random sequencing of cdna clones and dna microarrays , several groups have shown that significant changes in mrna levels occur during the life cycle of all tri - tryps ( teixeira et al . , 1995 ; el - sayed et al . , 1995 ; mathieu - daud et al . experimental evidence also indicates that , in addition to mrna stability , changes in polysomal mobilization constitute an important mechanism for regulating gene expression ( alves et al . , 2010 ) . with the advent of next generation sequencing technologies such as rnaseq , a global description of gene expression patterns in t. brucei has finally been achieved ( kolev et al . , 2010 ; nilsson et al . , 2010 ; siegel et al . , 2010 ; veitch et al . this approach has provided much more complete information about mrna structure and expression levels , including the patterns of spliced leader ( sl ) and poly - a additions , as well as alternative pre - mrna processing . moreover , these global gene expression analyses have confirmed that only two t. brucei genes ( poly - a polymerase and dna / rna helicase ) contain introns . 2010 ) showed that 2,500 alternative splicing events occur during processing of t. brucei genes , with a large number of these being regulated during the life cycle ( a total of 600 genes have transcripts with more than one trans - spliced variant ) . the alternative splicing reactions can alter the message dramatically , as shown by nilsson et al . ( 2010 ) and represented in figure 3 , with changes in the sl addition site leading to alterations in regulatory elements in the 5utr ( resulting in the modification of gene expression ) or the initiator aug ( generating a different n - terminus or even causing a complete change in the translated orf ) . aminoacyl trna synthetase transcripts are interesting examples of trans - spliced variants since differences in the enzyme n - terminus result in changes in the protein targeting signal : distinct mrnas produce proteins that are directed to mitochondria or remain in the cytoplasm ( nilsson et al . , 2010 ) . likewise , alternative splicing is a potential mechanism for dual localization of the t. cruzi lyt1 gene ( benabdellah et al . , 2007 ) . in addition to providing a mechanism for generating changes in the proteome , such flexibility in the selection of splicing sites is compatible with evidence that polycistronic transcription may also initiate at internal sites in gene clusters since correct processing of the 5 end of mrna can occur at various points in the primary transcript ( kolev et al . , 2010 ) . recently , chromatin immunoprecipitation in combination with conventional sanger sequencing ( respuela et al . , 2008 ) or next - generation sequencing technology ( siegel et al . , 2009 ; thomas et al . , 2009 ; wright et al . , 2010 ) has provided a much awaited analysis of the distribution patterns of modified histones throughout the t. brucei genome . as summarized in figure 3 , the divergent strand - switch regions ( ssr ) have been found to contain an enrichment of histone variants ( h2az and h2bv ) , acetylated histone 4 at lysine 10 ( h4k10ac ) , histone 3 acetylated at residues k9 and k14 and trimethylated at k4 , the bromodomain factor bdf3 , and the transcription factors trf4 and snap50 ( respuela et al . , 2008 ; siegel et al . , 2009 ; thomas et al . , 2009 ; wright et al . , 2010 ) . the histone variants h3v and h4v are present at transcription termination sites ( siegel et al . , 2009 ) . similar patterns of chromatin modifications from transcription start sites ( tss ) located at the beginning of polycistronic units were also found internally in the same polycistronic unit , suggesting the presence of internal tss , in agreement with rnaseq data ( siegel et al . , 2009 ; kolev et al . , 2010 ; the increasing number of published genomes and transcriptomic data , partly as a consequence of the introduction of new sequencing platforms , has created enormous challenges for the field of functional genomics . one of the most useful techniques that has been used to identify gene function in t. brucei is a combination of the inducible system mediated by t7 rna polymerase and the tetracy - cline repressor ( wirtz et al . , 1999 ) in combination with rnai gene knockdown . as indicated before , in contrast to t. cruzi and l. major , rnai is functional in t. brucei ( ngo et al . , 1998 ; robinson and beverley 2003 ; darocha et al . , 2004 ; lye et al . , 2010 ) . the first large scale functional genomics study using rnai was reported by morris et al . ( 2002 ) , who transfected t. brucei procyclic forms with a random rnai library . this library was generated by using a construct to inducibly express dsrna via two head - to - head tetracycline - regulated t7 rna polymerase promoters . by selecting parasites that expressed the dsrna but were unable to bind lectin , these authors identified clones with a reduced glycosylation of surface proteins and showed that these clones had lower expression of hexokinase ( morris et al . , 2002 ) . a few years later , the functions of 197 orfs from t. b. brucei chromosome i were tested by rnai . rnai - induced parasites were tested for growth , nuclear and kinetoplast abnormalities by dapi staining , and a pleio - tropic phenotype , morphology and motility . at least one of these phenotypes was found in 68 individual knockdowns ( subramaniam et al . , 2006 ) . in a similar approach , portman et al . ( 2009 ) identified novel components of the para - flagellar rod ( pfr ) structure by comparing proteomic profiles before and after ablating the expression of individual pfr proteins . the silencing of pfr1 and pfr15 was evaluated by two - dimensional difference gel electrophoresis , and the spots with a two - fold change in volume were subjected to tandem ms protein identification . proteomic analysis combined with rnai knockdown led to the identification of 30 proteins as potential pfr components , 20 of which were novel proteins . high - throughput cloning systems also enhance functional analyses . by using the gateway technology to create yeast two - hybrid vectors , eight non - redundant protein - protein interactions were detected among proteins with a pfr structure ( lacomble et al . , 2009 ) . these authors were able to construct a map showing the complex interaction of pfr proteins . the availability of efficient methods for genetic manipulation and for testing gene function through rnai knockdown has led to major advances in genomic , transcriptomic and proteomic analyses of t. brucei , and has made this species a model organism for studying basic aspects of trypanosomatid biology . more recently , with the discovery of functional rnai machinery in l. braziliensis and other leishmania species , gene function studies can now be conducted in this group and will soon provide valuable new information about leishmania - specific genes . however , as is becoming increasingly apparent from the data generated by comparative genomic analyses , each of the trypanosomatid species has its peculiarities . researchers thus face the challenge of developing new protocols specific for studying each parasite and its corresponding diseases . with our current knowledge of each tri - tryp disease and the new research methods that are being developed we can expect many new studies to emerge from hypotheses based on the tri - tryp genomic data . as a consequence of these new findings ,
in 2005 , draft sequences of the genomes of trypanosoma brucei , trypanosoma cruzi and leishmania major , also known as the tri - tryp genomes , were published . these protozoan parasites are the causative agents of three distinct insect - borne diseases , namely sleeping sickness , chagas disease and leishmaniasis , all with a worldwide distribution . despite the large estimated evolutionary distance among them , a conserved core of ~6,200 trypanosomatid genes was found among the tri - tryp genomes . extensive analysis of these genomic sequences has greatly increased our understanding of the biology of these parasites and their host - parasite interactions . in this article , we review the recent advances in the comparative genomics of these three species . this analysis also includes data on additional sequences derived from other trypanosmatid species , as well as recent data on gene expression and functional genomics . in addition to facilitating the identification of key parasite molecules that may provide a better understanding of these complex diseases , genome studies offer a rich source of new information that can be used to define potential new drug targets and vaccine candidates for controlling these parasitic infections .
Tri-Tryp Diseases and The Tri-Tryp Genomes The Genetic Diversity of T. Cruzi and the Genomes of Different Parasite Strains Comparative Genomics of Leishmania Species That Cause Distinct Forms of Leishmaniasis Functional Genomics of T. Brucei and Gene Expression Studies in a High Throughput Era
trypanosoma brucei , trypanosoma cruzi and leishmania major are unicellular protozoa of considerable medical importance since they are the etiologic agents of sleeping sickness ( african trypanosomiasis ) , chagas disease ( american trypanosomiasis ) and leishmaniasis , respectively . the geographic range of these parasites is determined by their insect vectors : in the case of sleeping sickness , a blood sucking fly of the genus glossina , also known as the tsetse fly , for chagas disease , a reduviid bug known as the kissing bug and for leishmaniasis , a phlebotomine sandfly . trypanosoma brucei , t. cruzi and leishmania spp . even more surprisingly , although orthologs of all conserved components of the rna polymerase ii complex were identified in the tri - tryp genome ( ivens et al . , 2005 ) , the transcription of some trypanosomatid genes such as vsg ( variant surface glycoprotein ) and the procyclin genes of t. brucei , as well as several exogenous genes transfected into t. cruzi , are mediated by rna polymerase i once the polycistronic pre - mrna is produced , two coupled reactions ( trans - splicing and poly - adenylation ) result in mature monocistronic transcripts . comparative genomic analyses using the tri - tryp sequences have already provided interesting insights into the genetic and evolutionary bases of the distinct and shared lifestyles of these parasites . since chagas disease spawns a variety of clinical forms , these studies are highly relevant : understanding the genetic variation among strains can potentially explain differences in disease pathogenesis , host preferences and , most importantly , provides essential information for the identification of new drug targets and good antigenic candidates for better diagnosis and vaccine development . in contrast to the other two tri - tryp genomes , the t. cruzi draft sequence ( el - sayed et al . in addition to the need to evade the host immune system , the existence of highly repetitive gene families in the t. cruzi genome in which a large number of gene copies can lead to the enhanced expression of various proteins may help to overcome a major problem in this genome , namely , the lack of strong promoters capable of generating high levels of mrna from single copy genes . leishmania major friedlin was chosen as the leishmania reference strain for the tri - tryp genome project . prior to publication of the tri - tryp genome sequence , the complete sequences of chromosomes 1 and 3 from l. major were published ( myler et al . leishmania infantum , also known as l. chagasi in latin america , was chosen as the second leishmania species to have its genome sequenced on the basis of its virulence in animals , transmissibility in sandflies and adaptability to laboratory experimentation ( denise et al . this species is the causative agent of visceral leishmaniasis , the most serious form of the disease and frequently fatal if left untreated . the complete sequences of all three leishmania genomes can be accessed in the tri - tryp database ; sequencing of the genome from a fourth species ( l. mexicana ) is in progress . this information will also improve our understanding of the host - parasite interaction and lead to the development of new strategies for contolling leishmaniasis . retroposons , also known as non - long - terminal - repeat ( ltr ) retrotransposons , are ubiquitous elements that transpose through an rna intermediate and are found in the genomes of most eukaryotes ( ivens et al . trypanosoma brucei and t. cruzi contain long autonomous retroposons of the ingi clade ( tbingi and l1tc , respectively ) and short nonautonomous truncated versions ( tbrime and nartc , respectively ) , as well as degenerate ingi - related retroposons devoid of coding capacity ( dires ) that represent the most abundant transposable elements in these genomes ( < 3% of the nuclear genome ) . signature known as lmsiders ( for short interspersed degenerate retroposons ) have also been identified in the genomes of l. major ( ivens et al . in the mammalian host , the parasite differentiates into long slender trypomastigotes ( bloodstream form -bsf ) that divide , colonize the body fluids and can transform into a nonproliferative bloodstream form , also known as the short stump form . in addition to providing a mechanism for generating changes in the proteome , such flexibility in the selection of splicing sites is compatible with evidence that polycistronic transcription may also initiate at internal sites in gene clusters since correct processing of the 5 end of mrna can occur at various points in the primary transcript ( kolev et al .
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cardiovascular disease ( cvd ) is the most prevalent cause of death in developed nations and it is increasing in prevalence in developing countries . while many factors contribute to the development of this disease in adults , such as , smoking , male gender , blood pressure , elevated cholesterol , diabetes , and renal failure , the mechanisms underlying cvd are still not fully understood [ 14 ] . one of the main problems in clinical practice is that the symptoms become evident late in the course of the disease . in fact , asymptomatic processes , that are associated with plaque formation , develop causing silent yet progressive tissue damage . if atheroma plaques finally rupture , highly thrombogenic material is released and an atherothrombotic event occurs . in this context , there is an urgent need to find out novel biomarkers of practical value for clinical intervention which , alone or combined with existing ones , allow cardiovascular risk prediction at individual level . currently , controversy exists regarding contribution of biomarkers to the information derived from conventional risk factors . when novel markers utility for predicting cvd was investigated in a wide cohort of more than 5000 individuals without cvd , the gains over conventional factors resulted to be minimal . however , positive outcomes are expected when high - risk populations are investigated ; thus , the risk level of selected patients , the chosen biomarkers to be investigated and other factors , such as , statistics , highly influence expected results . combination of multiple biomarkers in assessment of individual responses adds only moderately to standard risk factors . therefore , there is a substantial interest in the discovery and use of newer biomarkers , to complement the best existing ones and to identify persons who are at risk for the development of cardiovascular disease and who could be targeted for preventive measures . in particular , finding biomarkers that predict the risk of rupture will provide the opportunity to institute a preventive life style and permit timely pharmacological treatment . currently , the improvements in outpatient and inpatient care , diagnosis and biomarker discovery have reshaped the landscape of cvd . it is important to note that the new diagnostic methods currently available are based on noninvasive techniques that , although they present a number of benefits , may be limited in terms of specificity , sensitivity , availability and cost . -omics technologies has provided sensitive , fast and robust tools to analyze biomarkers in cvd . metabolites are small molecules that participate in general metabolic reactions and that are required for the maintenance , growth and normal function of a cell . the term metabolome , derived from the word genome , refers to the complete set of metabolites in an organism and its organelles [ 7 , 8 ] or the total complement of metabolites in a cell . in this way , metabolomics and metabonomics refer to the use of analytical methods to identify and quantify all metabolites in a biological system , as well as the monitoring of changes in the metabolome of a biofluid , cell culture or tissue sample following perturbation [ 8 , 10 ] . in parallel with genomics , transcriptomics and proteomics , application of metabolomic technologies to the study of cvd will increase our understanding of the pathophysiological processes involved and this should help us to identify potential biomarkers to develop new therapeutic strategies [ 11 , 12 ] . indeed , the identification and quantification of these low molecular weight molecules ( e.g. , lipids , amino acids , and sugars ) will define the phenotype of these diseases . from a clinical perspective , the study of metabolic changes that occur in response to different physiological processes will help establish the mechanisms underlying the disease . in terms of personalized medicine , pharmacometabonomic approaches can serve to predict the action of specific drugs in a particular individual based on the predose urinary metabolite profile . furthermore , the gut bacterial fauna influences drug efficacy , which could be deliberately modified to optimize the benefits and minimize adverse effects of a given treatment . in addition , this approach will help understanding how drugs act during patients ' recovery or how they influence outcome . there are several analytical strategies that can be used to analyse the metabolome , such as nuclear magnetic resonance ( nmr ) , fourier transformation infrared spectroscopy ( ft - ir ) [ 17 , 18 ] , and mass spectrometry ( ms ) coupled to separation techniques such as high performance liquid chromatography ( hplc ) , gas chromatography ( gc ) , or capillary electrophoresis ( ce ) . the combination of these different analytical techniques offers important advantages when analyzing the complete metabolome . high field h nmr is one of the preferred platforms for urine and plasma analysis [ 19 , 20 ] , as it is a nondestructive technique that does not require prior separation of the analytes and it provides detailed information on molecular structure . for example , the capacity to predict the occurrence of exercise - induced ischemia in patients with suspected cad was investigated by nmr blood analysis , demonstrating lactate , glucose , lipids , and long - chain fatty acids to be the main metabolites involved . xanthine and ascorbate were proposed as possible markers of plaque formation in an atherosclerotic mouse model and lipoprotein subclasses can now be analyzed by a commercial nmr - based protocol called nmr lipoprofile [ 23 , 24 ] . however , one of the main limitations of nmr is the poor sensitivity , although this can be improved enormously when it is combined with mass spectrometry . coupled to a separation technique , ms has recently been introduced into the metabolomics field and its use in such studies will constitute the main focus of this paper . indeed , gas chromatography / mass spectrometry ( gc - ms ) , liquid chromatography / mass spectrometry ( lc - ms ) and capillary electrophoresis / mass spectrometry ( ce - ms ) are the most powerful techniques for metabolite separation and analysis . gc - ms provides an extraordinary resolution , permitting the separation of structurally similar compounds that would otherwise be very difficult to separate by hplc . however , this technique requires the analyte to be volatile and thermally stable . in some cases , a chemical derivatization step is required prior to the chromatographic separation in order to render polar metabolites volatile . some of the metabolites best suited for gc - ms include fatty acids , organic acids , steroids , di - glycerides , sugars and sugar alcohols . for those metabolites that are not volatile and which can not be derivatized , thus , lc - ms can analyze a much wider range of chemical species ( polar and nonpolar metabolites ) with ample selectivity and sensitivity . apart from reversed phase chromatography ( rp - lc ) , which is widely used in metabolomics applications , hydrophilic interaction chromatography ( hilic ) is a complementary approach suitable for very polar metabolites ( nonvolatile ) . indeed , the metabolites suited to analysis by gc or hplc can be represented according to their polarity ( see scheme in figure 1 ) . similarly , capillary electrophoresis ( ce ) can be coupled to a mass spectrometer ( ms ) , with the particular advantage of improving the resolution of separation as narrower peaks than with lc are obtained . accordingly , different approaches have been described in combination with ion trap ( it ) , triple quadrupole ( qqq ) , time of flight ( tof ) , and q - tof instruments . the main advantage of qqq and q - tof instruments is that they provide the possibility of identifying the compounds by tandem ms / ms analyses . in order to obtain a full overview of the detectable molecules , electrospray ionization ( esi ) irrespective of the analytical approach used in metabolomics , particular care has to be taken in preparing the sample . bearing in mind that the typical half - lives of metabolic reactions in an organism are less than 1 s , it is important not to monitor metabolic changes extrinsic to the pathology or drug effect under study , producing misleading interpretations of the situation . evidence - based epidemiological studies have led to the discovery of well - established biomarkers . these studies now tend to be complemented by control - case investigation using a different methodology , based on two main stages : the discovery phase , resulting in a set of novel biomarkers candidates and the validation phase , where discovered potential biomarkers are further validated in a different cohort of samples . in this context , biological variation would be expected to be higher than the analytical variability and thus , it is essential to pay particular attention to : ( a ) the precise definition of a clinical phenotype ( in this sense , network - based analysis on associations among genes , proteins , metabolites , and environmental factors would be encouraged to increase sensitivity and selectivity of the diagnosis ) and ( b ) group matching in terms of sex , age , lifestyle , diet , or pharmacological treatment , otherwise attempts may fail in terms of disease prediction . for instance , gender and statins treatment strongly influence the findings in studies of cvd and when individuals with normal coronary arteries were compared with cvd patients , a > 99% confidence limit was only obtained for 6% of the predictions in the treated groups . technical reproducibility and sampling time are also critical to minimize external factors that will influence the results and their clinical relevance . ideally , snapshots of different conditions should be taken so that they can be quantitatively compared . if all these considerations are kept in mind , metabolomic research can set out to identify characteristic patterns that can be used for diagnostic purposes and risk prediction , substituting traditional , more expensive clinical approaches ( e.g. , angiography ) . in principle , metabolites can be measured in several body fluids or tissues , although plasma and urine are the most commonly used biological matrices in cardiovascular research due to their availability and clinical relevance as a source of potential biomarkers . almost all cells in the body communicate with the plasma , either directly or through different tissues and biological fluids , releasing at least part of their intracellular content . by contrast , urine is produced by renal filtration of the plasma and it is widely considered as one of the most important samples for diagnosis as it contains not only many plasma components but also the catabolic products of different metabolic pathways . sample pretreatment varies depending on the analytical platform chosen ( see the common strategies employed in figure 2 ) . metabolites from frozen tissue samples can be extracted and simultaneously fractionated by treating the ground tissue with mixtures of organic solvents , such that molecules are extracted in different fractions according to their polarity . if a biological fluid is the starting material ( urine , serum , plasma ) , metabolite fractions are usually obtained after proteins are removed by precipitation . the crude or diluted sample can then be injected directly , although matrix effects causing ion suppression should be expected . if analyzed by lc - ms(/ms ) , it may be desirable to preconcentrate ( e.g. , by lyophilisation ) or fractionate the sample prior to chromatographic separation . in case of gc - ms(/ms ) , preconcentration can be performed by solid phase microextraction ( spme ) with or without head space ( hs ) procedures , which are particularly useful when analysing volatile organic compounds ( vocs ) . for ce analysis three complementary approaches are used for metabolic research ( see figure 3 ) : metabolic fingerprinting , metabolic profiling , and metabolic footprinting . in the first case , and like proteomics strategies , an unbiased analysis is performed that is oriented towards defining clinically relevant differences rather than identifying all the molecules present in a sample . alternatively , metabolic profiling involves a preselection of a set of metabolites , or a specific class of compounds , that might participate in a targeted pathway . metabolic footprinting does not rely on the measurement of intracellular metabolites but rather , on monitoring those that are secreted or fail to be taken up by a cell or tissue [ 31 , 32 ] . below , we will discuss relevant findings from these approaches in cvd ( a compilation of the main studies is shown in table 1 ) . metabolic fingerprinting does not aim to identify the entire set of metabolites but rather to compare patterns or fingerprints of metabolites that change in response to a disease state , pharmacological therapies or environmental alterations , for example . a wide variety of biological matrices can be used for metabolic fingerprinting , such as urine , plasma / serum , tissues / cells and saliva . this approach can be used as a diagnostic tool to evaluate the disease state by comparing healthy controls and disease subjects , or to assay the success of a particular treatment ( prognosis / recovery ) . however , if we want to understand the mechanisms underlying a disease , qualitative and quantitative analyses are required . once a differential pattern is discovered , which provides information that can be considered as the pathological phenotype , further steps to identify the participating compounds ( qualitative ) and to determine the absolute amounts of metabolites that participate in the processes studied ( quantitative ) must be followed . this is not a trivial issue and prior to embarking on the task of discovering metabolic biomarkers , sufficiently sensitive and selective instruments and extensive compound libraries for metabolite identification should be available , while certain expertise in data analysis and interpretation will be necessary . one of the few metabolomic studies in the field of cvd involved a comparison of the metabolomic fingerprint obtained by gc - ms of plasma samples from non - st - segment elevation acute coronary syndrome ( nsteacs ) patients , stable atherosclerosis patients and healthy patients . citric acid , 4-hydroxyproline ( 4oh - pro ) , aspartic acid and fructose were found to decrease in nsteacs patients , whereas lactate , urea , glucose , and valine increased . both lactate and glucose are also involved in prediction of exercise - induced ischemia in patients with suspected cad . the decreased in 4oh - pro was especially interesting because circulating 4oh - pro is thought to prevent the binding of ldl to lipoprotein previously deposited in the vascular wall , as well as releasing already deposited ldl from the atherosclerotic lesions . it is also a component of collagen , which confers stability to the atherosclerotic plaque . the high resolution of ce - ms makes it a powerful technique to separate and analyse charged metabolites , although only a few metabolomic applications have been published to date . the isolation of polypeptide fraction from urine or plasma was analyzed by ce - ms and used to discriminate between coronary artery disease ( cad ) and non - cad patients with clinical symptoms and who had been subjected to coronary angiography . the stability of urine samples and their resistance to oxidation or precipitation reflect the advantages of this biological fluid for proteomic analysis . polypeptide profiling in urine is more reproducible than in plasma , with no significant loss of polypeptides over time when performing consecutive analyses over a 24-hour period , which also demonstrates the reproducibility of the ce - ms . in total , 200 of the most abundant polypeptides were detected and a set of 17 urinary polypeptides permitted cad and non - cad patients to be distinguished . among them , collagen -1 ( i and iii ) was augmented in cad samples , which was corroborated by their increased expression found in atherosclerotic plaques . metabolite profiling focuses on the analysis of a group of metabolites related to a specific metabolic pathway [ 38 , 39 ] . in this approach , technological advances have increased the number of metabolites that can be quantified simultaneously . moreover , the results of metabolic profiling are quantitatively independent of the technology used for data acquisition . metabolite profiling has been applied to cvd in order to identify and quantify metabolites that might serve as new biomarkers . a metabolite profile of peripheral blood from individuals undergoing planned myocardial infarction ( pmi ) has been established . serial blood from 36 patients were obtained before and at various intervals after pmi , and the changes in circulating levels of metabolites were identified by mass spectrometry - based metabolite profiling . most alterations produced by pmi were observed in the tricarboxylic acid cycle , in purine and pyrimidine catabolism , and in the pentose phosphate pathway . indeed , 7 metabolites were significantly affected immediately 10 minutes after the onset of myocardial injury ( p < .005 ) : alanine , aminoisobutyric acid , hypoxanthine , isoleucine / leucine , malonic acid , threonine , and trimethylamine n - oxide ( tmno ) . all these alterations were especially interesting as they were observed before any significant rise in the clinically available biomarkers in plasma ( ckmb and troponin t ) . after 60 minutes , six new metabolites had also changed significantly ( p < .005 ) : 1-methylhistamine , choline , inosine , serine , proline , and xanthine , with the later being a candidate of a marker for plaque formation in an atherosclerotic mouse model . the anatomic origin of the early metabolic changes observed was further explored in a subgroup of 13 patients by simultaneously comparing the metabolite levels obtained in samples from peripheral blood and from a catheter placed in the coronary sinus . a further 8 metabolites were transmyocardially enriched at least 1.3-fold 10 minutes after pmi ( taurine , ribose-5-phosphate , dcmp , lactic acid , amp , malic acid , glutamine and glutamic acid ) and once 60 minutes had passed , six additional metabolites augmented ( glycerol-3-phosphate , orotic acid , succinic acid , glycerate-2-phosphate , taurine and malic acid ) . plasma and urine samples from atherosclerotic and control rats have been compared by ultra fast liquid chromatography coupled to ion trap - time of flight ( it - tof ) mass spectrometry ( uflc / ms - it - tof ) . accordingly , 12 metabolites were identified as potential biomarkers in rat plasma and 8 metabolites in rat urine . the concentration of leucine , phenylalanine , tryptophan , acetylcarnitine , butyrylcarnitine , propionylcarnitine and spermine decreased in plasma , and 3-o - methyl - dopa , ethyl n2-acetyl - l - argininate , leucylproline , glucuronate , n(6)-(n - threonylcarbonyl)-adenosine and methyl - hippuric acid were diminished in the urine of atherosclerosis rats . conversely , ursodeoxycholic acid , chenodeoxycholic acid , lpc ( c16:0 ) , lpc ( c18:0 ) and lpc ( c18:1 ) increased in plasma and hippuric acid augmented in the urine from atherosclerosis rats . the alterations to these metabolites reflected the abnormal metabolism of phenylalanine , tryptophan , bile acids and amino acids . lysophosphatidylcholine ( lpc ) plays an important role in inflammation and cell proliferation , highlighting the relationship between lpc with the progress of atherosclerosis and other inflammatory diseases . the lipidomic profile of mice liver homogenates from cholesterol - free , low cholesterol and high cholesterol diets demonstrated the influence of dietary cholesterol intake and atherosclerosis . to obtain individual metabolite fingerprints , nearly 300 metabolites were measured in plasma samples by lc - ms / ms , including di- and tri - glycerides , phosphatidylcholines , lysophosphatidylcholines and cholesterol esters . when dietary cholesterol intake increased , the liver compensated for the elevation in plasma cholesterol by adjusting metabolic and transport processes related to lipid metabolism , which leads to an inflammatory , pro - atherosclerotic state . a cholesterol - free diet did not induce early atherosclerosis , while the low cholesterol diet only mildly induced early atherosclerosis . by contrast , intense early atherosclerosis was induced by the high cholesterol diet , in association with proinflammatory gene expression . indeed , a relationship appears to exist between cholesterol intake ( measured as cholesterol plasma levels ) and atherosclerotic lesion size . the lipidome of cell membranes and tissues has been studied by measuring the plasmalogens contained in rabbit and rat myocardial nuclei by esi - ms . plasmalogen is an ether lipid where the first position of glycerol binds a vinyl residue with the double bond next to the ether bond . the second carbon has a typical ester - linked fatty acid and the third carbon usually has a phospholipid head group , which can protect cells against the damaging effect of singlet oxygen . this seems to be the reason for the strong enrichment of plasmalogens found in the membrane of myocardial cells . metabolic changes associated to atherosclerosis have also been investigated through nmr and gc - ms metabolite profiling . there are clear biochemical explanations to these findings , and the alterations to these metabolites , which cause the final atherosclerotic lesion , can be related to different disorders . for instance , insulin resistance in diabetic patients increases the activity of transaminases , which are critical enzymes in amino acid metabolic pathways . hence , if the insulin response is deficient , these amino acid pathways will be altered , and many others metabolites will be affected such as glutamate , ketoglutarate , succynyl - coa , 4-oh - l - proline ( 4ohpro ) , 2-hydroxybutyrate , creatinine , pyruvate , oxaloacetate , malate , glycolate and 2,3,4-trihidroxybutirate . these effects could indicate damage to tissue at the intima artery walls . the myocardial metabolic response has been investigated in cad and left ventricular dysfunction ( lvd ) patients , both at baseline and following ischemia - reperfusion ( i / r ) . accordingly , glucose , lactate , free fatty acids , total ketones , 3-hydroxybutyrate , pyruvate , leucine / isoleucine and glutamate are present at lower concentrations in a preischemia state in the coronary sinus ( cs ) than in arterial samples ( reflecting myocardial uptake ) . by contrast , the alanine concentration is higher ( reflecting release ) . a principal components analysis ( pca ) shows several potentially important postoperative metabolic changes during the clinical course of the disease . ventricle dysfunction are associated with the global suppression of metabolic fuel uptake , and limited myocardial metabolic reserves and flexibility following global i / r stress is associated with cardiac surgery . the citric acid cycle plays an important role in oxidative phosphorylation and atp production in cardiomyocytes , and citric acid cycle intermediates are supplied by glycolysis and -oxidation of fatty acids . metabolomic profiling based on quantitative mass spectrometry was also used to study the heritability of premature coronary disease in 117 individuals unaffected by cad but with a family member affected . there was a string heritability of amino acid levels such as arginine , ornithine , alanine , proline , leucine / isoleucine , valine , glutamate / glutamine , phenylalanine and glycine , free fatty acids such as arachidonic , palmitic , linoleic and acylcarnitines . hence , it was concluded that metabolic changes associated with cad can be inherited and they are strongly related to age . this would indicate that metabolic processes could be controlled genetically , implying a correlation between genotype and phenotype in families with cad . more recently , a subset of 69 metabolites was shown to have diagnostic value , such that some derived factors showed discriminative capability for cad after pca . moreover , a signature composed of dicarboxyacylcarnitines was predictive of further cardiovascular events in those patients and most significant differences persisted after adjustment for cad risk factors . metabolic changes in human atrial fibrillation ( af ) have been investigated by nmr , performing a quantitative analysis of 24 previously selected metabolites . significant differences were found for beta - hydroxybutyrate , ketogenic amino acids and glycine , all of which augmented in af patients when compared to control subjects , suggesting a pathological role for ketone bodies . metabolic profiles enable more than 80% of patients at risk of af at the time of coronary artery bypass grafting to be classified , as a discordant regulation of energy metabolites was found to precede post - operative af . the effect of drug treatment on apoe mice was investigated by nmr analysis of metabolites in urine , showing allantoin to act as a marker for drug treatment , and xanthine and ascorbate as possible markers of plaque formation ( both were elevated in untreated mice ) . the application of metabolic analysis to cardiovascular diseases is an emerging field , and at this incipient stage it is not possible to clearly define a metabolic picture which is responsible for cvd prediction and progression . further metabolomic investigation promises to improve researchers and clinicians knowledge of these diseases in three critical ways . firstly , a complete description of the metabolites altered in a disease will better define the pathophysiology of the disease . secondly , metabolic profiling will enhance the feasibility of high - throughput patient screening to diagnose the disease state or risk evaluation . indeed , the identification of clinically relevant changes in circulating metabolites that may be considered as potential new biomarkers will also help with the evaluation of prognosis and will contribute to the development of new therapeutic strategies . thirdly , metabolite profiling will enable the effects of pharmacological treatments to be monitored , in particular , assessing the individual 's response to a particular drug . in contrast to genomics , metabolomics defines dynamic states that reflect the actual status of an organism , which requires the control of many variables ( from an individual 's status to metabolite degradation following sample collection ) . failure to do so may lead to the production of erroneous results and misleading conclusions . minimal protocol - specific differences can produce inconsistent findings , which must be clearly overcome prior to proposing the use of a biomarker to the scientific community . similarly , the results must be confirmed in a validation cohort composed by a different set of samples than that used in the discovery phase . adequate follow - up studies must corroborate earlier predictions , and adjustment for conventional risk factors to assess significant contribution of a discovered metabolite to current knowledge should be included . to date , there have been considerable efforts in improving instrumentation ( e.g. , mass spectrometry ) and the analytical methods suitable to complement these approaches ( e.g. , based on nmr ) , resulting in an expansion of the metabolites with potential roles in the development of atherosclerosis that can be quantified . however , further research is still needed prior to proposing an ideal platform for metabolite analysis that can replace conventional cvd diagnosis in clinical practice . with the growth of public metabolite databases , further improvements in the sensitivity and selectivity of analytical techniques and the development and routine use of novel platforms of demonstrated potential ,
metabolomics involves the identification and quantification of metabolites present in a biological system . three different approaches can be used : metabolomic fingerprinting , metabolic profiling , and metabolic footprinting , in order to evaluate the clinical course of a disease , patient recovery , changes in response to surgical intervention or pharmacological treatment , as well as other associated features . characteristic patterns of metabolites can be revealed that broaden our understanding of a particular disorder . in the present paper , common strategies and analytical techniques used in metabolomic studies are reviewed , particularly with reference to the cardiovascular field .
1. Introduction 2. Metabolomic Strategies, Analytical Approaches and Variability 3. Metabolic Fingerprinting in CVD 4. Metabolic Profiling in CVD 5. Conclusion
the term metabolome , derived from the word genome , refers to the complete set of metabolites in an organism and its organelles [ 7 , 8 ] or the total complement of metabolites in a cell . in this way , metabolomics and metabonomics refer to the use of analytical methods to identify and quantify all metabolites in a biological system , as well as the monitoring of changes in the metabolome of a biofluid , cell culture or tissue sample following perturbation [ 8 , 10 ] . in parallel with genomics , transcriptomics and proteomics , application of metabolomic technologies to the study of cvd will increase our understanding of the pathophysiological processes involved and this should help us to identify potential biomarkers to develop new therapeutic strategies [ 11 , 12 ] . indeed , the identification and quantification of these low molecular weight molecules ( e.g. in terms of personalized medicine , pharmacometabonomic approaches can serve to predict the action of specific drugs in a particular individual based on the predose urinary metabolite profile . there are several analytical strategies that can be used to analyse the metabolome , such as nuclear magnetic resonance ( nmr ) , fourier transformation infrared spectroscopy ( ft - ir ) [ 17 , 18 ] , and mass spectrometry ( ms ) coupled to separation techniques such as high performance liquid chromatography ( hplc ) , gas chromatography ( gc ) , or capillary electrophoresis ( ce ) . in some cases , a chemical derivatization step is required prior to the chromatographic separation in order to render polar metabolites volatile . in order to obtain a full overview of the detectable molecules , electrospray ionization ( esi ) irrespective of the analytical approach used in metabolomics , particular care has to be taken in preparing the sample . in this context , biological variation would be expected to be higher than the analytical variability and thus , it is essential to pay particular attention to : ( a ) the precise definition of a clinical phenotype ( in this sense , network - based analysis on associations among genes , proteins , metabolites , and environmental factors would be encouraged to increase sensitivity and selectivity of the diagnosis ) and ( b ) group matching in terms of sex , age , lifestyle , diet , or pharmacological treatment , otherwise attempts may fail in terms of disease prediction . if all these considerations are kept in mind , metabolomic research can set out to identify characteristic patterns that can be used for diagnostic purposes and risk prediction , substituting traditional , more expensive clinical approaches ( e.g. for ce analysis three complementary approaches are used for metabolic research ( see figure 3 ) : metabolic fingerprinting , metabolic profiling , and metabolic footprinting . in the first case , and like proteomics strategies , an unbiased analysis is performed that is oriented towards defining clinically relevant differences rather than identifying all the molecules present in a sample . alternatively , metabolic profiling involves a preselection of a set of metabolites , or a specific class of compounds , that might participate in a targeted pathway . metabolic fingerprinting does not aim to identify the entire set of metabolites but rather to compare patterns or fingerprints of metabolites that change in response to a disease state , pharmacological therapies or environmental alterations , for example . a wide variety of biological matrices can be used for metabolic fingerprinting , such as urine , plasma / serum , tissues / cells and saliva . this approach can be used as a diagnostic tool to evaluate the disease state by comparing healthy controls and disease subjects , or to assay the success of a particular treatment ( prognosis / recovery ) . once a differential pattern is discovered , which provides information that can be considered as the pathological phenotype , further steps to identify the participating compounds ( qualitative ) and to determine the absolute amounts of metabolites that participate in the processes studied ( quantitative ) must be followed . the decreased in 4oh - pro was especially interesting because circulating 4oh - pro is thought to prevent the binding of ldl to lipoprotein previously deposited in the vascular wall , as well as releasing already deposited ldl from the atherosclerotic lesions . serial blood from 36 patients were obtained before and at various intervals after pmi , and the changes in circulating levels of metabolites were identified by mass spectrometry - based metabolite profiling . most alterations produced by pmi were observed in the tricarboxylic acid cycle , in purine and pyrimidine catabolism , and in the pentose phosphate pathway . the effect of drug treatment on apoe mice was investigated by nmr analysis of metabolites in urine , showing allantoin to act as a marker for drug treatment , and xanthine and ascorbate as possible markers of plaque formation ( both were elevated in untreated mice ) . indeed , the identification of clinically relevant changes in circulating metabolites that may be considered as potential new biomarkers will also help with the evaluation of prognosis and will contribute to the development of new therapeutic strategies . thirdly , metabolite profiling will enable the effects of pharmacological treatments to be monitored , in particular , assessing the individual 's response to a particular drug . similarly , the results must be confirmed in a validation cohort composed by a different set of samples than that used in the discovery phase .
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a great variety of animal viruses encode for proteases that accomplish crucial functions during the biological cycle of the virus . usually , the main function of these proteases is to proteolyze viral polypeptide precursors to render mature viral proteins that form part of viral capsids or participate in virus vegetative processes . although both dna and rna viruses can encode proteases , the proteolytic tailoring of polypeptide precursors is most common among viruses with positive single - stranded rna genomes , such as picornaviruses , flaviviruses , caliciviruses , and retroviruses [ 37 ] . this mechanism of gene expression by proteolytic processing serves to compress the genetic information of viruses in the limited space provided by the genome . in this manner , viruses reduce the genetic space occupied by 5 and 3 untranslated regions ( utrs ) , the signals devoted for mrna transcription and to initiate translation are minimal , such that , for instance , in the case of picornaviruses or flaviviruses , only one 5 and 3 utr is necessary for viral replication , transcription , translation , and morphogenesis , despite the fact that several viral proteins are synthesized by the infected cells . in addition , a number of polypeptide precursors may exhibit functions that differ from those present in their mature products . in the case of poliovirus ( pv ) , eleven mature proteins are produced from a single translation initiation event , and at least two precursors , 2bc and 3cd , accomplish functions which are not present in their mature proteins . taking together all these considerations , the proteolytic strategy provides the small rna viruses with an advantageous and efficient mechanism for distribution of the genome to accomplish all the viral biological functions with the smaller genetic space . apart from generation of active viral proteins that participate in capsid morphogenesis and genome replication , viral proteases may also target a number of cellular proteins . proteolysis of these cellular substrates can very much affect a variety of cellular processes and play an important role in virus - induced cytopathogenesis [ 8 , 9 ] . in this regard , productive poliovirus infection induces rapid morphological alterations in host - cell . among them , the most prevalent is the accumulation of numerous membranous vesicles in the cytoplasm , derived from endoplasmic reticulum where the viral proteins 2c and 2bc play a central role . in addition , cellular shape is modified upon viral replication giving rise to cell rounding , which is most probably induced by disorders in the cytoskeletal network . finally , chromatin condensates at late times postinfection , associated with the nuclear envelope except for sites where nuclear pores are placed . interestingly , individual expression of the viral proteases 2a and 3c leads to the induction of most of these cytopathic effects , supporting the idea that these proteases actively contribute to the viral - induced morphological changes . indeed , long - term expression of either 2a or 3c triggers the activation of caspases and , thus , cell death by apoptosis [ 11 , 12 ] , reflecting the strong cytotoxicity of both proteases . in addition to the cytopathic effects induced by 2a and 3c , hydrolysis of host proteins may impact on other cellular functions such as the antiviral responses to virus infection . activation of innate immunity pathways , as well as the establishment of an antiviral response , is absolutely dependent on signals traversing the nuclear membrane through the nuclear pore complex . therefore , many viruses block cellular gene expression at different levels , that is , translation , transcription or protein and rna trafficking between nucleus and cytoplasm . the blockade of active trafficking can inhibit the nuclear import of antiviral signals or prevent the export of cellular mrnas detrimental to virus processes . the precise number of cellular proteins degraded by a viral protease , which is known as the degradome , still remains unknown for a given viral protease . perhaps , one of the best - studied proteases in this respect is pv 2a . the discovery that pv 2a bisects the initiation factor of translation eif4 g leading to the regulation of translation in the infected cells has attracted much attention from many laboratories during the past three decades [ 13 , 14 ] . more recently , 2a has been involved in the alteration of rna and protein trafficking between the nucleus and the cytoplasm upon proteolysis of several nucleoporins [ 1517 ] . the present paper focuses on the multifaceted activities of 2a and its regulation of different viral and cellular processes . pv is a prototype member of the picornaviridae family that infects cells of human or simian origin cytolytically or persistently and is responsible for poliomyelitis in humans . the rna genome is housed in a naked capsid formed by 60 copies of each of the four structural proteins : vp1 , vp2 , vp3 , and vp4 . the infectious cycle commences by the attachment of a viral particle to cellular receptors present at the cell surface [ 19 , 20 ] . this interaction leads to virion internalization and destabilization of the capsid , which adopts a less compact structure . once the rna is released in the cytoplasm , it interacts with the translational machinery , directing the synthesis of viral proteins during the early phase of infection . the pv genome is composed of a single - stranded rna copy of positive polarity of about 7.4 kb [ 21 , 22 ] . this rna molecule is uncapped and contains a poly(a ) tail at its 3 end and a single open reading frame , which encodes for a polyprotein of about two thousands amino acid residues . this polyprotein is proteolytically processed giving rise to the mature viral proteins ( figure 1(a ) ) . three different cleavages can be distinguished on the viral polyprotein : ( i ) polysomal cleavages that are produced on the nascent polypeptide chain . the first of these cleavages is catalyzed by 2a at its amino terminus separating the p1 precursor that encodes for the structural proteins from the rest of nonstructural polypeptides ( figure 1(b ) ) . the second cleavage still on polysomes is performed by 3c , releasing the p2 precursor ( 2abc ) from p3 ( 3abcd ) ; ( ii ) cytoplasmic cleavages that are mostly exerted by 3c and ( iii ) hydrolysis of vp0 ( vp4vp2 ) , which is concomitant with the morphogenesis of virus particles [ 2 , 23 ] . all these hydrolytic events lead to the formation of eleven mature proteins and several precursors such as p1 , p2 , p3 , vp0 , vp3 , vp1 , 2bc , 3ab , and 3cd . this last precursor , 3cd , can be used as substrate by 2a or 3c . the alternative cleavage carried out by 2a renders the mature products 3c and 3d , whereas 3c generates the canonical proteins 3c and 3d . however , the biological significance of this alternative cleavage is obscure because pv mutated at 2a - cleavage site on 3cd does not exhibit defects in virus replication . the nonstructural proteins that are generated participate in the replication of viral genomes [ 25 , 26 ] . to this end , the positive rna genome is recognized at its 3 end by proteins of the replication complex to synthesize the complementary rna strand of negative polarity . in this process , 3b protein , also known as vpg , acts as a primer to initiate viral rna transcription . this leads to the formation of a double - stranded rna molecule , also known as the replicative form . the negative rna synthesized serves in turn as a template to direct the synthesis of several copies of positive rna , so this process leads to the production of several nascent rna molecules with a vpg molecule bound to their 5 ends on the negative rna molecule forming a replicative intermediate . the positive rna molecules synthesized may participate in three processes ( i ) to serve as templates for synthesizing more negative rna molecules ; ( ii ) as mrnas that will be engaged in translation , and ( iii ) as genomes that will be encapsidated in new viral particles . in picornaviruses , the only type of mrna molecule known is exactly the same as the genome . once the synthesis of several thousands of positive rna molecules is performed , the late phase of translation takes place , giving rise also during this period to a great amount of viral proteins , some of which will participate in virus morphogenesis . this late phase of infection is preceded by the abrogation of cellular mrna translation , such that only viral proteins are being synthesized late in the pv life cycle . in the case of picornaviruses thus , inhibition of viral mrna translation provokes the sudden blockade of viral rna synthesis . moreover , translation is coupled to transcription , such that viral rnas transfected into picornavirus - infected cells are not able to direct protein synthesis . therefore , these two processes of viral macromolecular biosynthesis are tightly coupled , making it difficult to determine exactly the function affected in some pv mutants . notably , continuous lipid and cellular membrane synthesis is also necessary for pv rna synthesis . the morphogenesis of progeny virions in pv - infected cells is observed concomitantly with viral rna translation and replication . the release of new viral particles takes place by cell lysis , due to membrane permeabilization that occurs at the late phase of infection . viroporin 2b and its precursor 2bc are responsible for this permeabilization upon the formation of pore channels in cellular membranes [ 32 , 33 ] . pv has represented a useful model to gain insight into diverse aspects of molecular biology and gene expression . a number of discoveries concerning animal viruses with rna genomes were initially made in pv . for example , the presence of uncapped mrna , the sequencing and development of an infectious cdna clone , the three - dimensional structure of a virus particle , the discovery of the ires elements , the synthesis of an infectious virus in a cell - free system , the chemical synthesis of a complete viral genomes , among others , were initially reported in pv [ 3438 ] . in addition , the first time that eif4 g was found proteolytically cleaved was in pv - infected cells . picornaviruses encode different proteases depending on the virus species although it is common to all of them to encode 3c and its precursor 3cd ( figure 2 ) . in pv , both these exhibit protease activity , and they execute most of the hydrolytic events on the viral polyprotein [ 2 , 23 , 40 ] . apart from these two proteases , 3c and 3cd , picornaviruses also contain a 2a gene , whose product in some species exhibits proteolytic activity , as is the case for pv ( figure 2 ) . 2a has a limited proteolytic effect on the polyprotein and its function is most probably one of altering cellular functions by the cleavage of a number of cellular proteins . in this regard , the best studied of these cleavages is the bisection of eif4 g ( figure 3 ) . the general organization of the picornavirus genomes is to encode for p1-p2-p3 precursors giving rise to 4 - 3 - 4 mature products . some picornavirus species , in addition , encode a leader protein ( l ) placed before p1 ( figure 2 ) . in the case of aphthoviruses , such as foot - and - mouth disease virus ( fmdv ) , the l protein has proteolytic activity and it is known as l [ 42 , 43 ] . during polyprotein synthesis l is the first protein synthesized and its autoproteolytic activity releases itself from the rest of the polypeptide chain . thus , the only known hydrolysis executed by l on the polyprotein is to hydrolyze between its carboxy terminus and the amino terminus of vp4 ( figure 2 ) . because l does not play a direct role in viral replication and its protease activity has a limited impact in the viral polyprotein , this protease may be involved in the interaction with the host - cell [ 4547 ] . indeed , l also exhibits proteolytic activity on eif4 g acting at a position close to that of pv 2a ( figure 3 ) [ 4850 ] . in the case of fmdv , the 2a protein is reduced to a small peptide of 18 residues that does not hydrolyze eif4 g ; instead it induces the release of 2a from its carboxy terminus by a ribosomal skip mechanism [ 51 , 52 ] . this model proposes that fmdv 2a modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2a)-trna(gly ) ester linkage at the c - terminus of 2a , thereby releasing the polypeptide from the translational complex . however , not all l or 2a proteins from picornaviruses exhibit protease activity , since in the case of emcv , which encodes both proteins , neither has been demonstrated to possess proteolytic activity . all known proteases have been classified in four classes and many subgroups according to three parameters : ( i ) their catalytic center , ( ii ) their substrate specificity , and ( iii ) their three - dimensional structure . the classification of the different picornavirus proteases initially relied upon the effect of protease inhibitors . compounds that blocked sulphydryl groups abrogated the proteolytic activity of 2a and 3c , suggesting that the nucleophilic aminoacid in the active site was cysteine [ 54 , 55 ] . however , another cysteine inhibitor such as e64 had no effect on these proteases , while l activity was inhibited not only by sulphydryl - active compounds but also by e64 [ 56 , 57 ] . these findings together with structural observations imply that l belongs to the class of papain - like cysteine proteinases [ 43 , 5860 ] . comparison of the structure of picornavirus proteases with prototypes of cellular ones revealed that both 2a and 3c are similar in structure to the chymotrypsin like group [ 6163 ] . picornavirus 3c reflects similarities to the staphylococcus aureus proteinase , whereas 2a is more akin to streptomyces griseus proteinase a. pv 2a is a protein composed of 149 amino acids that belongs to the cysteine protease group . pv 2a is autocatalytically processed at its amino terminus between the capsid protein vp1 and 2a ( see figure 1(b ) ) . the determinants of substrate specificity of picornaviral pv 2a have been investigated in detail by identification of cleavage sites by n - terminal edman degradation , mutational analysis and using synthetic peptides as substrates [ 34 , 6567 ] . pv 2a can recognize a wide variety of amino acid residues at the p1 position . the determinants of substrate specificity for pv 2a lie at positions p4 , p2 , p1 , and p2 , which are preferentially occupied with ile / leu , thr / ser , gly , and pro , respectively . moreover , the determinants of substrate specificity of hrv and coxsackievirus 2a are very similar to those found for pv 2a [ 6567 ] . the yeast two - hybrid system has been used to identify the substrate sequence interacting with pv 2a . all the sequences identified contain the leu - x - thr - z motif ( x for any amino acid ; z for a hydrophobic residue ) in positions from p4 to p1 suggesting the presence of a common interacting site on pv 2a substrates . several 2a variants have been generated in the entire pv genome or in the isolated 2a gene . generation of pv 2a mutants was initially used to identify the cys106 , his18 , and asp35 as the residues that form part of the catalytic triad of 2a [ 69 , 70 ] . the role of the conserved cys and his residues in the structure - function relationship has also been studied by mutagenesis . the residues cys55 , cys57 , cys115 , and his117 play a critical role in the cis and trans proteolytic activity by maintaining 2a structure . the structure of hrv2 2a shows that the zn ion is coordinated tetrahedrally by the side chains of these conserved cys and his residues . this zn ion is tightly bound near to the c - terminal domain and may be important for the stability of the 2a [ 71 , 73 , 74 ] . the yeast saccharomyces cerevisiae has been used as a system to obtain pv 2a variants . the fact that this protease is very toxic for yeast has been exploited to generate 2a variants devoid of this cytotoxicity . using this approach , the characterization of these mutants revealed a region in 2a involved in the interaction with substrates but none of the mutations were found in the catalytic triad . a parallelism has been observed between the ability of these pv 2a variants to block protein synthesis and to cleave eif4 g . pv mutants in 2a gene that lack trans but not cis proteolytic activity have been also identified . normal processing of the viral polyprotein is observed upon infection with these pv variants , whereas eif4 g remains intact in these cells . interestingly , rna replication of those mutant viruses is hampered , suggesting that there is a correlation between pv rna replication and the trans activity of 2a . although for many years it was thought that pv 2a plays a direct role in pv replication , more recent studies have shown that a full - length dicistronic pv construct lacking 2a is capable to give rise to progeny viruses . moreover , virus yields of pv variants lacking the p1 coding region is partially restored when p1 is expressed in trans , suggesting that cleavage of the viral polyprotein by pv 2a is not essential for viral replication . . however , it is known that 2a is important for inducing the cytophatic effect and for avoiding the inhibition of pv replication in interferon ( ifn ) treated cells . in agreement with the idea that 2a participates in viral rna replication , a fraction of this protease localizes in pv replicative foci although the majority of 2a is associated with the matrix structure in the cytoplasm of infected cells . however , the presence of 2a in the proximity of replication complexes does not demonstrate that it participates directly in the replication process . the process of translation can be divided in different steps : initiation , elongation , termination and ribosome recycling . the synthesis of cellular proteins is highly regulated , and in this sense , the most precisely controlled step is the initiation of translation ( for a recent review , see [ 81 , 82 ] ) . for most eukaryotic mrnas , the initiation of translation commences with the recognition of the cap structure ( mgpppn ) and the poly(a ) tail by the heterotrimeric complex eif4f and the poly(a)-binding protein ( pabp ) , respectively , followed by the recruitment of the 43s preinitiation complex containing the 40s ribosomal subunit , the ternary complex met - trnai - eif2-gtp , and the eukaryotic initiation factors ( eifs ) , 1 , 1a , 3 , and 5 . then , the preinitiation complex scans along the 5 untranslated region until an aug initiation codon is encountered in a favourable context . the perfect complementarity between the aug start codon and the anticodon of met - trnai leads to the arrest of scanning and the hydrolysis of gtp in the ternary complex . the release of eif2-gdp and other factors triggers the interaction of the preinitiation complex with the 60s ribosomal subunit to form the 80s initiation complex , proceeding to translation of the mrna coding region . a number of eukaryotic initiation factors participate in both mrna binding and scanning of the 5 utr of the mrna by the small ribosomal subunit . the cap - binding protein eif4e , together with the dead - box helicase eif4a and the translation initiation factor eif4 g , forms the protein complex eif4f . the heterotrimeric complex eif4f is required for recruiting the 43s preinitiation complex onto the cap structure located at the 5 end of the mrna . in this sense , eif4 g , the larger polypeptide of eif4f , functions as an adaptor molecule that bridges the mrnas to ribosomes via interactions with factors eif4e ( which binds the 5cap structure ) , pabp ( which binds the poly(a ) tail ) , and eif3 , which interacts with the 40s ribosome subunit ( figure 3 ) [ 81 , 8486 ] . in addition , eif4 g also contains binding sites for other polypeptides involved in translation , such as the rna helicase eif4a ( figure 3 ) , which is required to unwind the secondary structure within the mrna 5 leader sequence that would otherwise inhibit ribosome scanning . the simultaneous interaction of eif4 g with eif4e and pabp promotes circularization of the mrna in a closed loop that facilitates the initiation of new rounds of translation by the proximity of the 5 and 3 ends [ 88 , 89 ] . furthermore , eif4 g also interacts with the mitogen - activated protein kinase 1 ( mnk1 ) ( figure 3 ) , which phosphorylates eif4e , although the role of this phosphorylation in the initiation of translation in still unclear [ 9094 ] . two forms of eif4 g , known as eif4gi and eif4gii , have been identified in mammalian cells . both forms show only 46% amino acid sequence identity but they are thought to be functionally interchangeable due to the high homology in key domains that interact with other factors . evidence obtained by specific depletion of each eif4 g form or differential cleavage of each of them by specific proteases ( see below ) points to the idea that both factors should be lacking for complete abolition of protein synthesis [ 95 , 96 ] . eif4gi is the dominant form in hela cells , in which the ratio between eifgi and eif4gii is 9 : 1 . however , the specific role of each form of eif4 g in the initiation of translation remains unknown . it was proposed that both eif4 g forms are differentially regulated by different kinases , supporting the hypothesis that eif4gi and eif4gii could drive differentially translation initiation . eif4gi is phosphorylated in response to serum and in a rapamycin - dependent manner at ser 1148 , 1188 , and 1232 , although the role of these posttranslational modifications is still under investigation . in addition , eif4gi is phosphorylated by p21-activated protein kinase ( pak-2 ) that is induced under stress conditions . this phosphorylation takes place in the eif4e - binding site of eif4 g and avoids the interaction between these two factors , inhibiting cap - dependent initiation of translation . on the other hand , therefore , activity of eif4gi and eif4gii might be tightly and reversibly regulated by phosphorylation under different physiological conditions . nevertheless , this factor is also subjected to irreversible modifications such as caspase - mediated proteolysis , which is triggered during apoptosis and leads to shutoff of protein synthesis . cleaves directly eif4gi in positions 532 and 1175 removing pabp , mnk1 , and one eif4a - binding domain from the eif4gi core ( figure 3 ) [ 103 , 104 ] . in contrast , eif4gii is degraded during apoptosis with a delayed kinetics in relation to eif4gi proteolysis , correlating with the shutoff of the protein synthesis . furthermore , many eif4gi isoforms have been detected in hela cells and these are synthesized from several distinct mrnas via alternative promoter usage and alternative splicing . the largest is the eif4gi - a isoform , which contains 1,600 residues , while the eif4gi - b , -c , -d , and e are shorter variants . it has been described that the longer isoforms are more active in translation initiation , most probably because they contain the pabp - binding site . infection of cells with pv results in a rapid shutoff of host - cell protein synthesis , whereas viral mrna translation takes place efficiently . it was initially observed that the inhibition of host - cell translation in pv - infected cells correlated with the proteolysis of a component of the eif4f complex with a molecular mass of about 220 kda ( later identified as eif4 g ) [ 39 , 109 ] . this cleavage is exerted by 2a and can be prevented by both insertion of mutations that abolish the protease activity and addition of 2a inhibitors [ 76 , 110 , 111 ] . interestingly , this proteolysis is more effective when eif4e is interacting with eif4 g , suggesting that pv 2a preferentially acts on the eif4 g pool involved in translation . cleavage of eif4gi also occurs in cells infected with other picornaviruses such as hrv , coxsackievirus , and fmdv [ 48 , 113 , 114 ] . interestingly , 2a from pv , hrv , and coxsackieviruses cleave eif4gi at positions 681/682 ( figure 3 ) , suggesting the conservation of the specificity of enterovirus 2a proteases for the substrate determinants present in eif4gi [ 114 , 115 ] . cleavage at position 681/682 separates eif4e- and eif3-binding sites of eif4gi , contained in n - terminal and c - terminal fragments respectively , thus decoupling mrna and ribosome recruiting activities . the pv 2a cleaves eif4gi directly and does not require any additional proteins for this process to occur [ 116 , 117 ] . however , the fact that pv 2a is not copurified with eif4gi fragments from pv - infected cell extracts suggest that 2a induced the activation of a host protease , which in turn cleaves eif4 g during pv infection . in addition , it has been proposed that eif3 and an unknown host - cell protein could act as cofactors for eif4gi cleavage by pv 2a . in this sense , zamora and colleagues suggested that pv infection activates at least two host - cell proteases , which together with pv 2a , cleave eif4gi . nevertheless , no additional evidence has been put forward to support this hypothesis and the identity of these host proteases has not yet been determined . many reports have demonstrated that the kinetics of protein synthesis shutoff and eif4gi cleavage are not correlated in pv - infected cells [ 120122 ] . these data clearly indicate that additional translation factors may be cleaved to achieve an efficient inhibition of cellular mrna translation . in this regard , additional reports showed that eif4gii is also proteolyzed by 2a during pv and hrv infections and that this cleavage is exerted between amino acids 699/700 leading to a proteolytic pattern similar to eif4gi . interestingly , eif4gii is significantly more resistant to 2a - mediated cleavage than eif4gi and the kinetics of protein synthesis shutoff close correlates with eif4gii cleavage in pv- and rhv - infected cells [ 122 , 123 ] . in those studies , gradi and colleagues proposed that hydrolysis of both eif4gi and eif4gii is required for achieving pv- and hrv - mediated inhibition of host - cell mrna translation and that the cleavage of eif4gii is the rate - limiting step in the shutoff of host - cell translation after infection with those viruses [ 122 , 123 ] . a variety of approaches have been devised to express pv 2a in order to cleave eif4 g in culture cells or in cell - free systems . of these approaches , the most straightforward system has been the addition of the purified pv 2a , usually as a hybrid protein such as mbp-2a , to cell - free systems such as rabbit reticulocyte lysates ( rrls ) , hela and krebs-2 extracts [ 76 , 118 , 124127 ] . in this sense , the addition of about 1 to 5 g mbp-2a suffices to hydrolyze eif4 g in those cell - free systems [ 76 , 125127 ] . an alternative method to cleave eif4 g in an in vitro system is the translation of an mrna encoding pv 2a in translation competent extracts . this assay has the advantage of providing genuine and freshly made pv 2a , leading to total cleavage of eif4 g in the test tube after several minutes of translation . many different approaches have been explored in culture cells , the most popular being transfection of plasmids encoding pv 2a in different eukaryotic cell types [ 75 , 76 , 128134 ] . several plasmids have been utilized in this respect , and perhaps the most successful one is ptm1 - 2a , which is transfected in mammalian cells that transiently express t7 rna polymerase by infection with a recombinant vaccinia t7 virus [ 76 , 130 , 131 , 133 , 134 ] . the amount of protease synthesized in this system is similar to that found in pv - infected cells at late times of infection , but these amounts are reached 1 - 2 hours after transfection . similar results have been obtained in cells constitutively expressing t7 polymerase , which comprises a less pleiotropic system , because vaccinia virus proteins are not expressed ( unpublished data ) . since pv 2a targets a number of different cellular proteins , which affect several cellular functions depending on the amount of protease synthesized ( see below ) , in some instances , it is useful to express 2a at low levels . we have explored many alternative methods trying to get a system that allows us to control the levels of pv 2a into the cells . novoa and colleagues developed a protocol based on the addition of hybrid proteins bearing pv 2a . these recombinant proteins enter into the cytoplasm on cell membrane permeabilization by different methods such as addition of mbp-2a mixed with replicationally inactive chicken adenovirus particles . cleavage of eif4 g following these protocols takes place after incubation for 810 hours , suggesting that the amount of protease internalized is probably low but sufficient to hydrolyze eif4gi in virtually all culture cells . probably one of the most attractive systems is a stable cell line that inducibly express pv 2a , obtained in two different laboratories including ours [ 137 , 138 ] . in these cell lines , pv 2a is synthesized when tretracycline is removed from the culture medium , leading to low expression of pv 2a that induces efficient cleavage of eif4 g after 13 h post induction correlating with a potent inhibition of cellular translation . finally , long term expression of pv 2a in tet off cell lines triggers apoptosis [ 12 , 137 , 138 ] . the main drawback of this cell line is the low pv 2a escape under repression conditions that gives rise to a basal cytotoxicity . probably , the most efficient method is electroporation of an mrna encoding 2a under the control of emcv leader sequence ( ires-2a ) . the biggest advantage of this method is the capacity to regulate levels of 2a expression by controlling the amounts of ires-2a transfected . for example , electroporation of 9 g of ires-2a into ~1.5 10 hela cells leads to total cleavage of both eif4gi and eif4gii in only 2 h , resulting in an almost complete shutoff of cellular protein synthesis . in contrast , electroporation of low amounts of ires-2a ( 1 g ) into hela cells induces efficient cleavage of eif4gi , whereas eif4gii remains largely intact . therefore , 9-fold more ires-2a mrna is required to cleave eif4gii compared to eif4gi . based on the ires-2a mrna electroporation method we were able to induce the differential proteolysis of eif4gi and eif4gii in a time- and dose - dependent manner kinetics of protein synthesis shutoff and eif4gii cleavage is closely correlated in hela cells , resembling what was found in pv - infected cells . in agreement with what was observed with the addition of exogenous recombinant proteins , translation of de novo synthesized mrnas showed higher susceptibility to low doses of pv 2a than mrnas already engaged in translational machinery . these results suggested a possible specific role of eif4gi in the pioneer round of translation in agreement with a previous report . however , specific ablation of eif4gi using sirnas induced a moderate inhibition of luciferase synthesis from de novo synthesized and preexisting mrna ( about 40% in both cases ) . these findings reported by welnowska and colleagues indicated that the higher susceptibility of de novo synthesized mrna translation to low doses of ires-2a might be produced by an additional effect of pv 2a on another gene expression step . in this regard , further studies demonstrate that the stronger impact of 2a on de novo synthesized mrnas is due to the concomitant inhibition of rna nuclear export by nucleoporin 98 cleavage , which is also achieved under these conditions ( see below ) . interestingly , cellular mrnas are able to initiate translation after a polysome runoff with high salt treatment when eif4gi is totally cleaved by pv 2a , whereas it is completely abolished when both forms of eif4 g are proteolyzed . taken together these set of data from cell expressing 2a [ 95 , 136 ] as well as from pv - infected cells [ 120122 ] we can conclude that complete shutoff of the protein synthesis induced by pv 2a is achieved when both eif4gi and eif4gii are completely cleaved . therefore , when the levels of one of the two populations of eif4 g remain unaffected either because it is not cleaved by pv 2a or it is not depleted by sirnas , extensive host protein synthesis takes place . the infection of pv and coxsackievirus also leads to hydrolysis of pabp [ 140 , 141 ] . this cleavage is carried out by pv 3c and coxsackievirus 2a and 3c and it might actively contribute to the host translational shutoff induced by these viruses [ 142 , 143 ] . in conclusion , the proteolysis of different components of the translation initiation machinery by picornavirus proteases can account for the shutoff of host translation induced after infection although the specific contribution of hydrolysis of eif4gi , eif4gii , and pabp remains still unclear . infection of animal cells with fmdv also leads to proteolysis of eif4 g and to rapid inhibition of cellular translation . the proteolysis of eif4 g is carried out by the two virally encoded proteases l and 3c [ 144 , 145 ] . l cleaves both eif4gi and eif4gii extremely rapidly at positions 674 ( figure 3 ) and 700 , respectively , located seven and one amino acids upstream of the 2a cleavage sites on eif4gi and eif4gii [ 50 , 146 ] . the cleavage of eif4 g by fmdv l results in the rapid shutoff of host - cell protein synthesis . although the initial cleavage of eif4gi can be carried out by fmdv l in the absence of virus replication , a sequential cleavage of the c - terminal fragment of eif4gi by fmdv 3c also occurs in bhk cells at early stages of infection concomitant with the shutdown of viral translation . the 3c cleavage site on eif4gi has been located at position 712 , 38 amino acids downstream of the l cleavage site although the role of this sequential cleavage is still unclear . the amino acid segment of eif4 g located between the l and 3c cleavage sites binds rna and was suggested to be critical for mrna scanning by the preinitiation complex . interestingly , this secondary cleavage does not occur in human cell lines due to an amino acid substitution at the cleavage site on eif4gi . infection of cells with other picornaviruses such as cardioviruses ( emcv and mengovirus ) leads to a shutoff of host - cell protein synthesis . these findings indicate that apart from eif4 g cleavage , there are other mechanisms that may block host translation by picornaviruses . in addition to picornavirus l and 2a , proteases from other viruses can also cleave eif4 g . the protease of human immunodeficiency virus type-1 ( hiv-1 ) hydrolyzes eif4gi during infection of human cd4 + cells . the cleavage of eif4gi takes place at positions 718 , 721 , and 1125 , separating it in three domains ( figure 3 ) [ 149 , 150 ] . interestingly , hiv-1 protease efficiently cleaves eif4gi , but not eif4gii , both in cell - free systems and in mammalian cells . the differential sensitivity of eif4gi and eif4gii to hiv-1 protease is more selective than that observed with picornaviral 2a proteases [ 122 , 123 ] . hiv-1 protease also cleaves pabp at positions 237 and 477 separating the two first rna - recognition motifs from the c - terminal domain of pabp . cleavage of eif4gi and pabp by hiv-1 protease is sufficient to inhibit the translation of capped and polyadenylated mrnas in cell - free systems , as well as in transfected cells [ 127 , 151 ] . in contrast , ires - driven translation is unaffected or even enhanced by hiv-1 pr after cleavage of both eif4gi and pabp [ 127 , 151 ] . moreover , the translation of capped and polyadenylated hiv-1 genomic mrna remains unaffected in hela extracts under these conditions suggesting that viral protein synthesis might persist at late phases of hiv-1 infection where those factors are cleaved . in contrast , a previous report claimed that the hydrolysis of eif4gi impaired the translation of both capped and ires - driven mrnas in reticulocyte lysate assays . however , the different effect observed by these authors on ires - driven translation can be due to differences already reported between hela extract and rrl [ 143 , 153 ] . in addition , eif4 g cleavage is executed by proteases from other retrovirus species , such as hiv-2 , simian immunodeficiency virus ( siv ) , human t - cell leukemia virus ( htlv-1 ) , moloney murine leukemia virus ( momlv ) , and mouse mammary tumor virus ( mmtv ) . these proteases hydrolyze eif4gi and eif4gii with different cleavage patterns and kinetics . and indeed , several retroviruses , including hiv , siv , and momlv , promote the translation of their gag gene products by internal ribosome entry , indicating that eif4 g cleavage could be compatible with viral protein synthesis in infected cells [ 154156 ] . furthermore , cleavage of eif4gi and eif4gii also occurs in feline calicivirus - infected cells although the cleavages occur at different sites to those observed for picornavirus proteases . in addition , the 3c - like protease of two caliciviruses , like pv 3c , cleaves pabp perhaps as a complementary strategy to inhibit cellular translation . the fact that proteases from many picornaviruses , retroviruses and caliciviruses , target eif4 g , and , in some cases , pabp , strongly suggest that those viruses may share a common mechanism to regulate cellular and viral translation . however , further investigation is required to determine the specific contribution of eif4gi , eif4gii and pabp to the shutoff of host - cell translation and virus protein synthesis . the biological cycle of picornaviruses is confined to the cytoplasm of infected cells . however , some of the pv proteins are able to target nuclear proteins such as transcription and splicing factors and proteins involved in nuclear - cytoplasmic trafficking . one of the best studied cases in this regard is the cleavage of nucleoporins ( nups ) , components of the nuclear pore complex ( npc ) , by pv and hrv 2a , which directly impacts on nuclear - cytoplasmic trafficking of proteins and rnas [ 15 , 16 , 159 ] . complementarily , emcv l protein affects on the phosphorylation status of nup62 and induces similar effects to those described for pv 2a in the transport of macromolecules through npc . nevertheless , nups are not only targets for picornavirus proteins but also for matrix ( m ) protein from vesicular stomatitis virus ( vsv ) and nonstructural protein 1 ( ns1 ) from influenza virus , which also impair components of the nuclear export - import host machinery following analogous mechanisms . taking into account that proteins from different positive and negative strand rna viruses target nups , we highlight in this paper npc as a key target for viral proteins , although the possible role of npc in virus biological cycle is still under intensive research . nucleus and cytoplasm are physically separated by a semipermeable barrier known as the nuclear envelope . due to this compartmentalization , a large number of macromolecules traverse the nuclear envelope to reach their biological destination . for example , proteins are synthesized by ribosomes in the cytoplasm , but some of them such as polymerases , transcription factors , nucleosome components and splicing factors , have to traverse the npc to reach the nucleoplasm . conversely , all rna species are transcribed and processed in the nucleus and later , most of mature rnas are exported through the npc to the cytoplasm , where their biological roles take place . therefore , the regulation of rna and protein trafficking between nucleus and cytoplasm directly impacts on gene expression [ 163 , 164 ] . npc forms large structures ( ~125 mda ) embedded in the nuclear envelope with a polarized eightfold symmetrical core . it is sandwiched by a cytoplasmic and nuclear ring , which projects eight filaments of about 50 nm into the cytoplasm and a basket - like structure of about 100 nm into the nucleoplasm [ 165 , 166 ] . the npc is composed of multiple copies ( 8 , 16 or 32 ) of ~30 different proteins , called nups , that are grouped in three major classes : ( i ) the phenylalanine - glycine ( fg)-containing nucleoporins that actively work in the nuclear - cytoplasmic trafficking of macromolecules ; ( ii ) the structural components , which lack fg - rich domains and ( iii ) the membrane integral proteins , which anchor the npc to the nuclear envelope [ 163 , 167 ] . whereas the two last groups of nucleoporins play a role in the architecture and localization of the npc , the fg - nucleoporins directly regulate the transport of rnas and proteins through the npc , and they are the main nucleoporin class targeted by viruses ( figure 4 ) . movement of ions , metabolites and other small molecules between nucleus and cytoplasm takes place by passive diffusion ; however , transport cargos larger than 40 kda require the participation of specific receptors and carriers [ 168 , 169 ] . fg nucleoporins are placed on both cytoplasmic and nucleoplasmic sides of the npc and play a central role in the active transport of macromolecules . the fg domains of nucleoporins are unfolded regions that participate in energy - independent transient interactions with the cargo receptors during the docking and translocation processes . nevertheless , the delivery of the cargo and some of the directional steps require hydrolysis of gtp . trafficking of proteins through npc is mediated by a family of conserved transport receptors named karyopherins , which recognize short peptides known as nuclear localization signal ( nls ) and nuclear export signal ( nes ) [ 169 , 171 ] . in addition , karyopherins also recognize nucleotide sequences during the export of some classes of rnas . due to their key role , karyopherins involved in cargo import are known as importins and those involved in export are known as exportins . the rangtp cycle also plays a central role in karyopherin activity and , therefore , in trafficking of macromolecules through the npc . importins bind the cargo in the cytoplasm and release it on binding to rangtp in the nucleus . in contrast , exportins bind the cargo in the nucleus together with rangtp ; and then the ran - associated gtp is hydrolyzed in the cytoplasm by rangap and cargo is liberated [ 163 , 164 ] . proteins containing constitutively active nls are predominantly nuclear ; but in some cases , the accessibility of nls or the nls itself is modified to selectively regulate the localization of the protein . a similar regulatory mechanism is also exerted for control of nes activity . for example , the nls of the transcription factor nfb is masked by the interaction with the inhibitor ib. however , ib is degraded on proinflammatory stimuli , exposing the nls of nfb for importin recognition . therefore , under proinflammatory conditions , nfb is imported to the nucleus , where it triggers a specific gene response . in addition to masking strategies , phosphorylation , ubiquitination or methylation of nls or nes also influences ( negatively or positively ) their recognition by importins or exportins . therefore , trafficking of proteins between nucleus and cytoplasm could be finely regulated by posttranslational modifications in the nls and nes [ 164 , 174 ] . conceptually , export of most of nuclear rna follows a similar mechanism to that described above for protein trafficking . this process also involves cargo receptors , export factors , and nucleoporins to deliver mature rna to the cytoplasm , but in this case , structure and function of nuclear export signals are not well understood . aminoacylated trnas are necessary in the cytoplasm for protein synthesis , but trnas are transcribed in the nucleus . trna export to the cytoplasm is mediated by exportin - t , which belongs to the karyopherin family . exportin - t forms a complex together with rangtp in the nucleus , and once the exportin - t - rangtp - trna complex reaches the cytoplasm , rangap induces the hydrolysis of the ran - associated gtp and the release of the trna [ 175 , 176 ] . it has been proposed that exportin-5 could act as an auxiliary protein in this process . however , later reports in yeast have opened the possibility of alternative pathways for trna nuclear export . snrnas are transcribed by the rna polymerase ( pol ) ii ( with the exception of u6 snrna which is produced by poliii ) and then they are capped but not polyadenylated . cap - binding proteins ( cbp ) 80 and 20 interact with the cap of snrnas in the nucleus and recruit an export adaptor known as phax . this adaptor is phosphorylated and in this state , it is able to interact with crm1 , another member of karyopherin family . hydrolysis of rangtp and dephosphorylation of phax lead to the release of the snrna in the cytoplasm . ribosomal proteins are assembled together with the different rrnas in the nucleolus following a complex process of maturation to give rise to the ribosomal subunits 40s and 60s . although is known that preribosomal subunits are exported by separate routes that involve crm1 and rangtp , nowadays the exact mechanism followed by 40s preribosomal subunits to leave the nucleus remains unclear . however , 60s preribosomal subunit relies on nmd3 adaptor , which mediates the interaction with crm1 [ 179 , 180 ] . the release of the 60s preribosomal subunit requires two gtpase steps : ( i ) the hydrolysis of the ran - associated gtp induces the liberation of crm1 ; and ( ii ) the hydrolysis of gtp mediated by the cytoplasmic gtpase lsg1 , which induces the release of nmd3 . finally , mrnas compose the most heterogeneous group of rnas , varying in length and structure . mrnas are transcribed by polii , and , concomitantly with this process , a number of rna - binding proteins assemble with them . these rna - binding proteins exert different modifications in immature mrnas such as polyadenylation , splicing and capping . in addition , export factors and adaptor proteins are also recruited to nascent pre - mrnas , playing a further function in nuclear export . trex ( transcription - coupled export ) complex is recruited to the 5 end of nascent pre - mrnas in a splicing - dependent manner by means of the interaction of one of its components , namely aly / ref , with the subunit of the nuclear cap - binding complex cbp80 . the trex complex also recruits the conserved rna - helicase uap56 that is important for mrnp biogenesis . tap - p15 , ( also known as nxf1-nxt1 ) directly binds the mrna immediately after splicing and actively participates in mrna export . aly / ref , tap - p15 and uap56 associate with exon junction complexes ( ejc ) , which are deposited in a splicing - dependent manner at 2024 nt of every exon - exon junction [ 184 , 185 ] . this interaction network makes spliced mrnas more susceptible to export and couple splicing and mrna - trafficking . in fact , unspliced mrnas are exported by an alternative and less efficient pathway that involves crm1 and rangtp . this alternative route is also followed by mrnas encoding some protoncogenes and cytokines [ 186 , 187 ] . it is important to mention that although mrna can follow different nuclear export pathways , in all cases the interaction of export receptors with nucleoporins plays an essential role in the transport of the mrnps throughout the npc . pv infection strongly impacts on host - cell protein localization , giving rise to an unusual cytoplasmic distribution of nuclear proteins . this particular effect has been characterized by different laboratories for a number of nuclear factors involved in several cellular processes and containing different types of nlss . nevertheless , not all nuclear proteins are re - localized after pv infection , evidencing the presence of a viral - specific mechanism affecting protein subcellular distribution . gustin and colleagues proposed the inhibition of nuclear protein import machinery as the cause of the cytoplasmic accumulation of nuclear proteins in pv - infected cells ( figure 4 ) . they demonstrated that pv impairs protein trafficking across the npc by expressing gfp proteins encoding classical or transporting nlss in mock and infected cultured cells [ 15 , 16 , 159 ] . these recombinant proteins accumulate in the cytoplasm after pv infection , being almost completely depleted from the nucleus . nevertheless , pv does not affect gfp distribution when the nls is mutated or deleted , since the small size of gfp allows its inefficient efflux throughout the npc . interestingly , cell - free nuclear import assays demonstrated that nls - containing gfp is unable to traverse the npc when cells are previously infected with pv . in agreement with these findings , shuttling endogenous proteins , such as heterogeneous nuclear ribonucleoprotein ( hnrnp ) a1 and hnrnp k , are detected in the cytoplasm of infected cells from 3 hpi but are undetectable in the nucleus at 4.5 hpi . however , cytoplasmic accumulation of nuclear resident proteins such as hnrnp c requires longer times of infection . these findings support the idea that the distribution of a protein that shuttles between nucleus and cytoplasm may be strongly altered by the disruption of protein trafficking pathways , as compared to nuclear resident proteins . however , not all the nuclear factors are redistributed to the cytoplasm of pv - infected cells . this is the case for sc35 ( a serine / arginine - rich splicing factor ) , fibrillarin or tata - binding protein ( tbp ) , which remain in the nucleus for the duration of infection . the different behaviour of these groups of proteins could arise as a consequence of different turnover ; thus , the distribution of highly stable nuclear proteins might be less affected by the inhibition of protein nuclear import than those proteins with low stability . alternatively , pv might not impair all protein import pathways , and some might remain operative , thereby allowing the nuclear import of several families of nuclear proteins . interestingly , some nuclear factors such as la antigen , ptb , sam68 , or nucleolin have been shown to interact with pv rna or viral proteins . these proteins accumulate in the cytoplasm of pv - infected cells and , consequently , their availability for viral replication is increased [ 188 , 193195 ] . cytoplasmic accumulation of nuclear factors might be produced mostly by the blockade of nuclear - cytoplasmic trafficking . however , loss of the nls after the cleavage of ptb and la proteins by pv 3c may also contribute to the subcellular relocalization of those proteins in pv - infected cells [ 193 , 195 ] . redistribution of nuclear proteins as well as impairment of cellular import machinery was also observed in cells infected with other picornaviruses such as hrv or emcv . these findings indicate that most picornaviruses might share similar strategies to impair nuclear - cytoplasmic trafficking machinery . an alternative hypothesis has been proposed as the cause of the redistribution of nuclear factors . belov and colleagues reported that the nuclear envelope is permeabilized on pv infection , allowing nuclear proteins to diffuse across the nuclear membrane . however , this hypothesis did not clarify why other proteins resident in the nucleus do not diffuse to the cytoplasm after pv infection . both protein nuclear import and permeabilization of nuclear envelope protein import blockade is detected early after infection , correlating with the first modifications in the npc ( see below ) . however , prolonged expression of viral proteins might induce nuclear membrane leakiness , reflected by stronger alterations in npc architecture . nevertheless , what might the biological relevance of these events be for a cytoplasmic virus ? the most evident answer , which was extensively commented above , is that inhibition of nuclear protein import and further nuclear envelope leakiness might increase the presence of nuclear proteins in the cytoplasm of infected cells , which has been proposed in some cases to play a relevant role in pv replication . in addition , many transcription factors are arrested in the cytoplasm of uninfected cells ( e.g. , nf-b , irf7 , and irf3 ) , but they are immediately activated and imported to the nucleus after proinflammatory extracellular signals or on the activation of intracellular sensors as a consequence of the viral replication . once in the nucleus , these factors trigger the transcription of a set of genes involved in the antiviral response . inhibiting the import of these transcription factors , pv might prevent or , at least attenuate , the establishment of a hostile intracellular environment . interestingly , nup98 , nup153 and nup62 , components of the npc belonging to fg nup family , were found to be degraded in pv- as well as hrv - infected cells ( figure 4 ) [ 15 , 16 , 159 ] . these proteins are essential factors of the nuclear - cytoplasmic trafficking machinery since their n - terminal fg - rich domains serve as docking sites for soluble transport factors [ 163 , 198 ] . nup98 , nup153 and nup62 are proteolyzed in pv - infected cells following different kinetics . thus , nup98 is the cleaved early after infection ( from 1 hpi ) , whereas nup153 and nup62 are targeted at late times after infection ( from 4 hpi ) [ 15 , 16 ] . in agreement with these findings , cleavage of nup98 is induced even in presence of inhibitors of pv replication , suggesting that small amounts of viral proteins are sufficient for this proteolysis to occur . however , cleavage of nup153 , and nup62 are efficiently prevented on arrest of viral replication , probably because they are only efficiently achieved when large amounts of viral proteins are produced . therefore , nup98 , nup153 , and nup62 exhibit different susceptibilities to pv replication , thus pv might have a gradual impact on the nuclear - cytoplasmic trafficking machinery . nup153 is also proteolyzed by caspase-3 and caspase-9 during apoptosis induction ; however , the involvement of these cellular proteases in pv - induced nup cleavage has been ruled out by different laboratories . first , nup153 cleavage products generated upon caspase activation differ to those found in pv - infected cells . in addition , nup62 is cleaved in pv - infected cells , but it remains intact despite caspase activation . most importantly , pv - induced npc structural damage takes place in cells lacking caspase 3 and 9 , and nup153 and nup62 are efficiently cleaved in pv - infected cells even in presence of the caspase inhibitor z - vad . all together , these data support the idea that one or more viral proteins play a direct role in the cleavage of those nups . in agreement with this hypothesis , pv - induced npc damage is prevented by pv 2a inhibitors such as elastatinal , elastase , and mpcmk , suggesting an involvement of this viral protease in the alteration of npc . indeed , individual expression of pv 2a in hela cells as well as addition of this protease to cell - free systems gives rise to nup98 , nup62 , and nup153 cleavage [ 16 , 17 , 200 ] . in agreement with the data obtained from pv - infected cells , on pv 2a expression in hela cells , nup98 is cleaved faster than nup62 and nup153 , which suggests the presence of optimal cleavage sites in this protein . proteolysis of nup98 in pv - infected cells as well as in cell - free systems generates two different cleavage products of around 5065 kda and 35 kda . there are two optimal cleavage sites in nup98 for pv 2a located between aminoacids 373 - 374 and 551 - 552 , containing gly at p1 , thr at p2 , and leu at p4 . hydrolysis at both sites results in n- and c - terminal products with predicted molecular masses of 37 and 53 or 55 and 35 kda , in good agreement with the size of the peptides detected experimentally in pv - infected cells and 2a - treated hela extracts [ 16 , 17 ] . an explanation for the delayed kinetics of nup62 and nup153 with respect to nup98 is that optimal pv 2a cleavage sites were not found in these nups ( unpublished data ) . recently , park and colleagues have reported that pv 2a directly cleaves nup62 at six different positions rendering multiple proteolytic products . these cleavage sites are located between aminoacids 103 and 298 , thus releasing the fg - rich region from the protein core . functionally , loss of the fg - rich region might make nup62 inactive for interaction with cargo receptors . this hypothetical mechanism of nup62 functional decoupling could be extrapolated to nup98 and nup153 ( figure 4 ) . however , it remains unknown whether pv 2a is able to directly cleave nup98 and nup153 or where cleavage might occur . nup98 , nup153 and nup62 are also involved in rna export from the nucleus and therefore , cleavage by pv 2a might also impact on this process ( figure 4 ) . however , oligo d(t ) hybridization studies showed that pv infection does not affect distribution of the polyadenylated mrna bulk after 3 hpi . a more detailed analysis revealed that expression of pv 2a in hela cells induces a number of disorders in rna location . nuclear export of cellular mrnas is inhibited in 2a - expressing cells in a dose dependent manner concomitantly with nup98 , nup62 and nup153 cleavage . interestingly , mrna export of constitutively expressed mrnas such as -actin is less affected than that of newly synthesized mrnas . for example , tetracycline - induced luciferase mrna was almost totally retained in the nucleus when these nups are cleaved by pv 2a . this effect was also observed for endogenous mrnas such as il-6 , c - myc or p53 mrnas which are induced on pv 2a expression . therefore , pv 2a could counteract the induction of proapoptotic ( c - myc and p53 ) and proinflammatory ( il-6 ) responses by accumulating the c - myc , p53 and il-6 mrnas in the nucleus . this export blockage may prevent the establishment of a host - cell response against pv infection . these findings could explain why pv 2a is essential for replication of pv in cells pre - treated with ifn- . furthermore , impairment of mrna export strongly alters the localization of mrnas with high turnover as compared to constitutively expressed and highly stable mrnas such as -actin . as observed in pv - infected cells , oligo d(t ) hybridization revealed that pv 2a expression hardly affects the distribution of the polyadenylated mrna pool after short times of expression ( 8 h ) . however , nuclear accumulation of polyadenylated mrna bulk is detected when cells are exposed to pv 2a for longer times ( 16 and 24 h ) . in this regard , the progression of the alterations of polyadenylated mrna localization in 2a - expressing cells takes place as follows : ( i ) disruption of nuclear mrna - containing foci ( ii ) appearance of mrna - containing granules in the cytoplasm ( most probably stress granules ) and ( iii ) depletion of cytoplasmic mrnas . these events were more clearly observed when high amounts of pv 2a are synthesized , reflecting that nuclear accumulation of mrnas is a time- and dose - dependent process . nevertheless , pv 2a is not only able to block mrna export , but also rrna and snrna transport . both 18s rrna and u2 snrna accumulate in the nucleus of 2a - expressing cells in a dose - dependent manner . most probably , cleavage of nup98 , nup62 and nup153 is involved in these effects , since rrna , snrna , and mrna are exported using different cargo receptors and auxiliary proteins ( see above ) but all of them relay in nup activity to traverse npc . importantly , trnas ( val - trna ) are exported normally despite pv 2a expression , indicating that some rna nuclear export pathways are not affected by this viral protease . in fact , nup98 , nup62 , and nup153 are not directly involved in trna export , reinforcing the idea that nucleoporin cleavage plays a central role in the impairment of protein and rna trafficking by pv ( figure 4 ) . notably , ifn- induced a specific increase of nup98 levels in hela cells that counteracts the inhibition of mrna export by pv 2a [ 17 , 201 ] . collectively , these findings reflect the central role of nup98 in pv infection and in antiviral response , since its overexpression by itself prevents , at least in part , blockade of nuclear rna export . therefore , secretion of ifn- by immune cells might allow the induction of antiviral response by neighbouring cells by increasing the levels of nup98 in order to protect nuclear - cytoplasmic protein and rna trafficking pathways . the physiological relevance of the crosstalk between nup98-pv 2a in pv ( and other viruses ) infection might be studied in the future with cellular and animal systems . as mentioned above , hrv also induces cleavage of nup62 , nup153 and most probably nup98 , leading to the impairment of nuclear protein import . nevertheless , pv and hrv 2a exhibit high homology and both proteases are therefore expected to share common targets . in contrast , the 2a gene of cardioviruses encodes a short peptide with autoproteolytic activity but lacks trans - protease activity as occurs in pv - infected cells , these cardioviruses induce both protein nuclear import inhibition and late membrane leakiness but they do not induce the cleavage of nups [ 160 , 202 , 203 ] . cardioviruses encode an additional protein known as l protein , which is highly cytopathic although it lacks protease activity ( in contrast to aphthovirus l ) . l protein contains a zinc finger domain , and an acidic region , which is proposed to be phosphorylated in infected cells . individual expression of this protein in cultured cells or in cell - free systems induces several cellular disorders including the inhibition of protein nuclear import that resembles that observed in emcv - infected cells [ 160 , 203 ] . several studies reported emcv l protein mutations that resulted in defective virus growth phenotypes in cell culture [ 202 , 204 ] . in particular , mutations in the zinc finger domain ( cys19ala and cys22ala ) or in the acidic region ( thr47ala ) partially avoid blockade of protein trafficking between the nucleus and the cytoplasm . taken together , these data support the involvement of cardiovirus l protein in npc damaging and in the inhibition of protein nuclear import , inducing nups phosphorylation rather than their cleavage . indeed , nup62 is quickly and strongly phosphorylated after emcv infection ( 2 hpi ) , and it was clearly detected by conventional western blotting . nevertheless , analysis with pro - q diamond phosphoprotein stain revealed that nup153 and nup214 are also phosphorylated to a certain extent upon emcv infection . notably , nups phosphorylation was avoided when cys19ala mutation was inserted in the l protein sequence , suggesting that the zinc finger domain is essential for this posttranslational modification to occur . however , the exact role of nups phosphorylation in nuclear - cytoplasmic trafficking is still unknown . the idea that nups phosphorylation could regulate protein import and rna export as a switch that turns the different pathways on / off should be pursued in more detail . as mentioned above , ran is essential for the regulation of most of the nuclear export and import pathways , because it acts as a cofactor modulating the affinity of importins and exportins for the cargo . it has been described that emcv l directly interacts with ran , and this interaction is abrogated by the insertion of c19a mutation in l . however , the potential role of l / ran interaction in the modulation of rangtp cycle and its impact in nuclear import and export pathways have not yet been studied . alteration of nuclear - cytoplasmic trafficking is not only restricted to picornaviruses but has also been observed with negative strand viruses such as vesicular stomatitis virus ( vsv ) and influenza virus . her and collaborators reported that rna export and protein import are strongly inhibited by vsv matrix ( m ) protein , by microinjection of oocytes with radiolabeled rnas and proteins . radiolabeled trnas , mrnas , u snrnas , and rrnas were injected directly into the nucleus of xenopus laevis oocytes , and the subcellular localization of those rnas was monitored by autoradiography . the conclusion of this work is that mrna , u snrna , and rrna but not trna trafficking is blocked by vsv m protein , in agreement with our findings on 2a - expressing hela cells . in addition , protein nuclear import was monitored by microinjection of radiolabeled proteins containing nls in the oocytes cytoplasm . this assay revealed that vsv m protein abrogates protein nuclear import to the same extent as treatment with specific inhibitors of this pathway such as wga . these interesting findings support the idea that pv 2a and vsv m protein could target similar host proteins to impair macromolecule trafficking between nucleus and cytoplasm . in agreement with this possibility , it was found that the vsv m protein interacts with nup98 , which is one of the primary targets of pv 2a . the n - terminal domain of vsv m is sufficient to block rna nuclear export and aa 5254 may play an essential role in this blockade , because their mutations to ala completely abrogate this inhibitory effect . indeed , the n - terminal domain of m protein is involved in the interaction with nup98 and , in particular , aa 5254 , because their mutation to ala blocks the binding of vsv m to nup98 . in addition , binding of m to nup98 requires active mrna export pathways since , treatment with inhibitors such as wga hampers this interaction . vsv m is also able to induce the accumulation of endogenous polyadenylated mrnas in the nucleus of hela cells , and this effect is prevented again by mutations in aa 5254 of m . furthermore , vsv m interacts with rae1 , which plays an essential role in mrna nuclear export by its interaction with nup98 and mrnps . overexpression of either nup98 or rae-1 prevents the nuclear accumulation of polyadenylated mrnas , suggesting that both factors may play a role in the blockade of mrna nuclear export by vsv m protein . interestingly , ifn- specifically increases the level of both nup98 and rae-1 and indicates a potential antiviral effect of these proteins . indeed , overexpression of both proteins by ifn- treatment counteracts the inhibitory effects of vsv m protein on mrna nuclear export , highlighting the possibility of a crosstalk between m and ifn- that might control the fate of the viral replication in infected animals [ 201 , 206 ] . influenza virus replication also impacts on nuclear - cytoplasmic trafficking and leads to the nuclear accumulation of host mrnas [ 162 , 207 ] . influenza virus ns1 protein is a major virulence factor that is essential for pathogenesis , because it impairs innate and adaptive immunity by inhibiting host signal transduction and gene expression [ 208 , 209 ] . ns1 forms a complex with nxf1/tap , p15/nxt , rae1 , and e1b - ap5 , which are components of the mrna nuclear export machinery ( see above ) . individual expression of ns1 in 293 t cells induces the accumulation of polyadenylated mrnas in the nucleus , suggesting that the interaction of ns1 with these export factors yields an inactive complex for mrna export . influenza virus also induces a strong reduction of nup98 steady - state levels although the viral mechanisms involved in this process are still unknown . expression of reporter luciferase mrna synthesized from a nuclear plasmid , this inhibition is overcome by overexpression of nxf1 , p15 , rae-1 or nup98 , evidencing the role of ns1 interaction with these factors in the impairment of mrna nuclear export . furthermore , mouse cells expressing low levels of nup98 or / and rae-1 show greater susceptibility to influenza infection , resulting in a significant increase in cell death and virus production . in addition , mrnas encoding antiviral factors or immunomodulators such as irf-1 , mhc i and icam1 accumulated more in the nucleus of those cells than in cells expressing normal levels of nup98 or rae-1 . all these data support the physiological role of ns1 interaction with rna export factors as well as the reduction of nup98 levels in influenza pathogenicity . interestingly , vsv m , influenza ns1 and pv 2a expression gives rise to similar effects on mrna trafficking . all these viral proteins target nup98 and other components of the cellular machinery involved in nuclear - cytoplasmic trafficking . survival of motor neurons ( smn ) complex is composed by smn and a class of proteins called gemins , which localize in both cytoplasm and nucleoplasm [ 210 , 211 ] . gemin7 and gemin8 constitute the core of the complex where the other gemins associate by means of numerous protein - protein interactions from the periphery . the smn complex is involved in the biogenesis of uridine - rich small nuclear ribonucleoprotein ( u snrnp ) in the cytoplasm and then the u snrnp carries out the splicing of pre - mrnas in the nucleus [ 212214 ] . the snrnps are composed of the major u snrnas u1 , u2 , u4 , u5 , and u6 as well as a group of seven proteins known as sm ribonucleoproteins that collectively make up the extremely stable sm core of the snrnp . gemins ( except gemin-2 ) associate with sm proteins to form a heptameric ring structure in the presence of u snrnas . after sm core assembly , the u snrnps are imported to the nucleus , localizing in foci known as cajal bodies , where further maturation processes take place . gemin-3 , one of the main components of smn complex , is cleaved in pv - infected hela cells leading to a 50 kda cleavage product . scission of gemin-3 negatively impacts on the kinetics of sm core assembly , which is prevented in presence of inhibitors of pv replication . these results indicated that high levels of pv proteins are required for this process to occur , as is the case of nup153 , nup62 , and eif4gii , [ 16 , 122 ] . pv 2a is able to hydrolyze purified gemin-3 in vitro , rendering a cleavage product similar to that found in pv - infected hela cells . only one potential 2a - cleavage site , between the amino acids tyr462 and gly463 ( vhtyg ) , was found in this smn complex component . proteolysis of gemin-3 at this position would render two cleavage products of about 5030 kda , in agreement with the polypeptide of about 50 kda found in pv - infected and 2a - expressing cells . in addition , g463e mutation avoids direct hydrolysis of gemin-3 exerted by pv 2ain vivo and in vitro . taken together , these findings support the notion that vhtyg is the cleavage site for pv 2a in gemin-3 . although hydrolysis of gemin-3 is exerted in cells transfected with plasmid encoding pv 2a , it does not take place when this protease is expressed from exogenous mrnas ; contrary to that found with eif4gi , eif4gii , nup98 , nup153 and nup62 ( and unpublished data ) . a probable explanation for this difference is that pv 2a is expressed at lower levels from transfected mrnas than from plasmids ( castello et al . , 2006 ) . thus , gemin-3 cleavage may be a very late event in pv - infected cells because it requires expression of high amounts of pv 2a . gemin-3 hydrolysis may directly impact on pre - mrna splicing since this event reduces the availability of smn complexes , which is involved in u snrnps biogenesis . nevertheless , alstead and colleagues could not detect any apparent effect of gemin-3 proteolysis in splicing of cellular pre - mrnas . in addition to eifs , nups and proteins from smn complex , pv 2a is able to cleave proteins involved in other cellular processes , such as transcription . tbp is cleaved by pv 2a between amino acids tyr34 and gly35 in vitro , although this cleavage only removes the first 34 aa located at the n - terminus and does not inhibit transcription carried out by rna polymerase ii [ 217 , 218 ] . these findings are in agreement with the fact that host mrna transcription takes place in 2a - expressing cells when both translation and rna nuclear export are inhibited , upon cleavage of eif4 g and nups . one attractive hypothesis is that pv 2a could cleave specific initiation factors affecting specific rather than general mrna transcription in order to modulate host - cell response to viral infection . further studies in this direction can be carried out using microarray platforms to detect precise alterations in cellular transcriptome after pv - infection or 2a expression . these studies could be complemented by screening for new host factors cleaved by pv 2a using different in silico and experimental approaches . the study of viral proteases is crucial to understand the mechanism used by animal viruses to replicate their genomes and to translate viral mrnas at the molecular level . in addition , we wish to draw attention to the concept that viral proteases can be used as tools to reveal the exact functioning of their target cellular proteins . in this regard , pv 2a has been very useful for examining the requirements for eif4 g to translate different cellular or viral mrnas . in addition to this , in this paper , we have highlighted the role of pv 2a not only in the processing of the poliovirus polyprotein but also in the interaction with the host - cell . interestingly , a single viral protein is able to modulate many steps of gene expression in order to generate an optimal intracellular environment for the viral biological cycle . in particular , pv 2a cleaves cell proteins involved in transcription , pre - mrna splicing , nucleus / cytoplasm transport and translation , in order to hijack those host functions and to concentrate the cellular resources on the production of the viral progeny . for example , pv 2a inactivates host translational machinery for capped cellular mrnas by cleaving eif4 g , whereas viral protein synthesis takes place under those conditions by ires - driven translation . because of the decrease of cellular mrna translatability in pv - infected cells , host ribosomes are available for viral protein synthesis . probably , the inhibition of host protein synthesis may prevent the production of antiviral proteins . similarly , the blockade of nucleus / cytoplasm transport of macromolecules might isolate nuclear processes from cytoplasmic cellular ones , hampering the arrival of specific proinflammatory transcription factors to the nucleus and of mrnas encoding proinflammatory , antiviral or proapoptotic proteins to the cytoplasm of infected cells . finally , transcription and pre - mrna splicing could be also modulated by pv 2a and might reduce the availability of mature mrnas . nevertheless , very little is known about the potential role of pv 2a in transcription and pre - mrna splicing , and further studies of these steps of gene expression in pv - infected and 2a - expressing cells should be carried out . making use of the omics technologies it would be possible to identify changes in the host transcriptome in those cells , allowing us to understand the readjustment of host gene expression to viral infection and to the cytotoxic effect of pv 2a . gene ontology tools can be used to cluster the pathways ( kegg ) , molecular activities and biological functions of the genes which are transcriptionally up- or downregulated or not affected after these unfavourable stimuli . in silico analysis could be carried out in order to identify whether these gene clusters belong to particular networks controlled by specific transcription factors . complementarily , deep sequence ( rnaseq ) , specific microarrays types and conventional rt - pcr could be used to screen for nonspliced or abnormal spliced mrna variants in pv - infected and 2a - expressing cells . finally , microarrays can be employed to identify the cytoplasmic and nuclear transcriptome in those cells to determine whether mrna nuclear export inhibition induced by pv 2a impacts on the distribution of the entire host mrna bulk or in specific mrna pools . taken together , all this information may provide us with a general and deep vision of the modification induced by pv 2a on the different steps of mrna metabolism . additionally , the physiological role of nups and gemin-3 cleavage might be studied using different models . one possible and interesting approach is to engineer stable cell lines expressing noncleavable versions of nup98 , nup62 , nup153 and/or gemin-3 and then to analyze the fitness of pv in the different cell types , especially in presence of extracellular antiviral stimuli such as ifns or interleukins , which will activate different epigenetic programs . these studies will provide essential information to help us understand the specific role that those host factors play in pv infection . the total number of cellular proteins targeted by pv 2a ( degradome ) remains unknown , but it can be anticipated that with the expanding use of proteomic methodologies , this analysis will be known soon not only for pv 2a , but also for other viral proteases of interest . furthermore , the analysis of the pv 2a - induced degradome in human cells will be of general interest for many researchers , including virologists and cellular biologists . this goal could be achieved combining in silico prediction of 2a cleavage sites and experimental tools such as proteomics . in the first case , blom and collaborators developed a bioinformatics tool using neural network algorithms to predict cellular targets for picornavirus proteases . this approach has been successfully used to predict the cleavage of dystrophin by coxsackievirus 2a although most of the predicted human targets for rhinovirus and enterovirus 2a have not been proved yet . in addition , the algorithm did not predict the cleavage of cellular targets that have been later demonstrated to be proteolyzed by picornaviral 2a such as nup98 and cytokeratin 8 [ 16 , 17 , 221 ] . thus , it would be necessary to develop an improved algorithm able to find optimal cleavage sequences in the host proteome by implementing the proteolytic sites known for newly described 2a targets . many parameters have to be taken into account , including the protein localization ( cytoplasmic and nuclear protein will be considered , but not proteins resident in the lumen of other organelles such as re or peroxisomes ) , the exposure of the cleavage site to the solvent ( the sequence must be accessible to the protease ) , and the secondary structure in which the proteolytic site is included ( optimally , unstructured regions ) . potential targets could be ordered by their degree of homology with optimal cleavage sequences , as well as with the degree in which they fulfill the above prerequisites . on the other hand , novel pv 2a targets can be identified by proteomic tools such as two - dimensional differential gel electrophoresis ( dige ) or quantitative proteomics such as stable isotope labeling by amino acids in cell culture ( silac ) coupled to monodimensional electrophoresis . following these two methods , it will be feasible to identify proteins with reduced levels on 2a expression and , in addition , to detect the cleavage products that will appear as lower size peptides . by the uncovering of novel 2a targets we will be able to map the cellular networks impacted by pv 2a and to integrate them in the context of pv infection . in fact , the role of 2a hijacking host processes could be potentially expanded to other cellular pathways with direct impact on control of viral infection . such knowledge will provide more insight into our understanding of the cytopathogenicity of viral proteases at the molecular level .
after entry into animal cells , most viruses hijack essential components involved in gene expression . this is the case of poliovirus , which abrogates cellular translation soon after virus internalization . abrogation is achieved by cleavage of both eif4gi and eif4gii by the viral protease 2a . apart from the interference of poliovirus with cellular protein synthesis , other gene expression steps such as rna and protein trafficking between nucleus and cytoplasm are also altered . poliovirus 2apro is capable of hydrolyzing components of the nuclear pore , thus preventing an efficient antiviral response by the host cell . here , we compare in detail poliovirus 2apro with other viral proteins ( from picornaviruses and unrelated families ) as regard to their activity on key host factors that control gene expression . it is possible that future analyses to determine the cellular proteins targeted by 2apro will uncover other cellular functions ablated by poliovirus infection . further understanding of the cellular proteins hydrolyzed by 2apro will add further insight into the molecular mechanism by which poliovirus and other viruses interact with the host cell .
1. Introduction 2. The Poliovirus Life Cycle: An Overview 3. Picornavirus Proteases: L, 2A and 3C 4. The PV 2A. Structure-Function Relationship 5. PV 2A Hijack Host Protein Synthesis Machinery by Cleaving eIF4G 6. The Nuclear Pore and the Inhibition of Nucleus-Cytoplasm Trafficking by PV 2A 7. Other Cellular Proteins Hydrolyzed by PV 2A 8. Conclusions and Future Prospects
in this manner , viruses reduce the genetic space occupied by 5 and 3 untranslated regions ( utrs ) , the signals devoted for mrna transcription and to initiate translation are minimal , such that , for instance , in the case of picornaviruses or flaviviruses , only one 5 and 3 utr is necessary for viral replication , transcription , translation , and morphogenesis , despite the fact that several viral proteins are synthesized by the infected cells . therefore , many viruses block cellular gene expression at different levels , that is , translation , transcription or protein and rna trafficking between nucleus and cytoplasm . more recently , 2a has been involved in the alteration of rna and protein trafficking between the nucleus and the cytoplasm upon proteolysis of several nucleoporins [ 1517 ] . in the case of aphthoviruses , such as foot - and - mouth disease virus ( fmdv ) , the l protein has proteolytic activity and it is known as l [ 42 , 43 ] . in those studies , gradi and colleagues proposed that hydrolysis of both eif4gi and eif4gii is required for achieving pv- and hrv - mediated inhibition of host - cell mrna translation and that the cleavage of eif4gii is the rate - limiting step in the shutoff of host - cell translation after infection with those viruses [ 122 , 123 ] . since pv 2a targets a number of different cellular proteins , which affect several cellular functions depending on the amount of protease synthesized ( see below ) , in some instances , it is useful to express 2a at low levels . for example , electroporation of 9 g of ires-2a into ~1.5 10 hela cells leads to total cleavage of both eif4gi and eif4gii in only 2 h , resulting in an almost complete shutoff of cellular protein synthesis . taken together these set of data from cell expressing 2a [ 95 , 136 ] as well as from pv - infected cells [ 120122 ] we can conclude that complete shutoff of the protein synthesis induced by pv 2a is achieved when both eif4gi and eif4gii are completely cleaved . in contrast , ires - driven translation is unaffected or even enhanced by hiv-1 pr after cleavage of both eif4gi and pabp [ 127 , 151 ] . one of the best studied cases in this regard is the cleavage of nucleoporins ( nups ) , components of the nuclear pore complex ( npc ) , by pv and hrv 2a , which directly impacts on nuclear - cytoplasmic trafficking of proteins and rnas [ 15 , 16 , 159 ] . nevertheless , nups are not only targets for picornavirus proteins but also for matrix ( m ) protein from vesicular stomatitis virus ( vsv ) and nonstructural protein 1 ( ns1 ) from influenza virus , which also impair components of the nuclear export - import host machinery following analogous mechanisms . therefore , the regulation of rna and protein trafficking between nucleus and cytoplasm directly impacts on gene expression [ 163 , 164 ] . these findings support the idea that the distribution of a protein that shuttles between nucleus and cytoplasm may be strongly altered by the disruption of protein trafficking pathways , as compared to nuclear resident proteins . the physiological relevance of the crosstalk between nup98-pv 2a in pv ( and other viruses ) infection might be studied in the future with cellular and animal systems . all these viral proteins target nup98 and other components of the cellular machinery involved in nuclear - cytoplasmic trafficking . these results indicated that high levels of pv proteins are required for this process to occur , as is the case of nup153 , nup62 , and eif4gii , [ 16 , 122 ] . in addition to this , in this paper , we have highlighted the role of pv 2a not only in the processing of the poliovirus polyprotein but also in the interaction with the host - cell . in particular , pv 2a cleaves cell proteins involved in transcription , pre - mrna splicing , nucleus / cytoplasm transport and translation , in order to hijack those host functions and to concentrate the cellular resources on the production of the viral progeny . making use of the omics technologies it would be possible to identify changes in the host transcriptome in those cells , allowing us to understand the readjustment of host gene expression to viral infection and to the cytotoxic effect of pv 2a . the total number of cellular proteins targeted by pv 2a ( degradome ) remains unknown , but it can be anticipated that with the expanding use of proteomic methodologies , this analysis will be known soon not only for pv 2a , but also for other viral proteases of interest .
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image registration is used to find the spatial correspondence between two images and plays an important role in pulmonary image analysis . in a sequence of pulmonary scans , the computed correspondences describe the motion of the lung between a pair of images at the voxel level . registration of lung volumes across time or across modalities has been utilized to establish lung atlases , estimate regional ventilation and local lung tissue expansion [ 25 ] , assess lobar slippage during respiration [ 6 , 7 ] , and measure pulmonary function change following radiation therapy . lung registration methods are typically intensity - based [ 2 , 5 , 913 ] or feature - based [ 1418 ] . intensity - based methods consider intensity patterns of the whole lung regions to define similarity measures . they take advantage of the strong contrast between the lung parenchyma and the chest wall , and between the parenchyma , the blood vessels and larger airways . commonly used intensity - based methods include minimizing intensity difference [ 3 , 10 ] , maximizing mutual information or normalized cross correlation [ 5 , 9 ] , and preserving tissue volume or lung mass [ 12 , 13 ] . since intensity - based methods do not use anatomical knowledge , these methods can get stuck in local minima resulting in mismatches of important anatomical structures such as bifurcations of smaller airway and vessel branches . on the other hand , they usually utilize corresponding landmarks and local intensity patterns [ 14 , 16 , 17 ] and surfaces correspondences [ 14 , 15 , 18 ] . however , due to the sparsity of features , good alignment of features can not guarantee satisfactory matching accuracy for all lung regions . since registration methods using either intensity - only or feature - only registration have their limitations , it is desirable to design lung registration methods that utilize both intensity and feature information [ 1923 ] . it has been shown that hybrid registration methods that combine intensity and feature information can help improve matching accuracy . most of these methods incorporate distributed landmark pairs selected at airway or vessel branch points , that were identified manually or semiautomatically , centerline of the airway and vessel tree structures , and lung surface information . therefore , fast feature extractions and effective methods to utilize feature information are needed to improve registration accuracy . in this paper , we couple intensity and feature information together to match 3d lung ct images acquired during breath hold at two different levels of inflation . we propose a feature - based similarity criterion utilizing the information of vessel locations and shapes in the registration process . this vesselness preserving cost function is added to four existing intensity - based similarity costs , and comparison experiments show that this criterion helps improve the registration accuracy . higher matching accuracy makes the postanalysis of regional tissue mechanical properties more plausible and reliable . our preliminary work on the effectiveness of using a vesselness matching similarity term was described in [ 2527 ] . the work presented in this paper provides a complete description of our vesselness matching approach . this paper extends our previous work by describing how to choose weighting factors for the different cost terms , describes a multiresolution optimization scheme , and provides more validation . this paper studies the effect of the vessel matching when used with various intensity similarity metrics such as the sum of squared intensity difference , sum of squared tissue volume difference , mutual information , and normalized cross correlation . this paper also examines the effect of using a linear - elastic constraint to regularize the displacement fields . in this study , six pairs of volumetric ct data sets from six human subjects in the supine orientation were collected on a siemens sensation 64 multidetector ct scanner . each image pair was acquired during breath - holds near functional residual capacity ( frc ) and total lung capacity ( tlc ) in the same scanning session . for subject 1 , data were acquired at functional residual capacity ( frc ) with 21.8% of the vital capacity ( vc ) and total lung capacity ( tlc ) with 95.6% of the vc . for subject 2 , data were acquired at frc with 30.5% of the vc and tlc with 89.6% of the vc . for subject 3 , data were acquired at frc with 26.3% of the vc and tlc with 95.7% of the vc . for subject 4 , data were acquired at frc with 11.0% of the vc and tlc with 68.9% of the vc . for subject 5 , data were acquired at frc with 25.8% of the vc and tlc with 92.9% of the vc . for subject 6 , data were acquired at frc with 26.5% of the vc and tlc with 102.0% of the vc . each volumetric data set was acquired at a section spacing of 0.5~0.6 mm and a reconstruction matrix of 512 512 . in - plane the parenchyma regions in the frc and tlc data sets were segmented using the method described in . figure 1 gives an illustration of pulmonary ct images with renderings of the lung segmentations . the goal of image registration is to estimate a transformation h that defines the pointwise correspondences between two images i1 and i2 . more formally , let d r define the domain of the images i1 : d r and i2 : d r. in this work , the transformation h : d d is assumed to be a diffeomorphism . let denote the set of all diffeomorphisms from d to d. the optimal transformation h is estimated by minimizing a cost function that consists of a similarity cost function and a regularizing term , that is , ( 1)h^=argminhcsim(i1,i2,h)+creg(h ) . the similarity cost function describes the characteristics of two images that should agree for corresponding image points . the regularization cost function is used to enforce desired properties of the transformation such as minimum distortion . the constant is used to balance the influence of the regularization cost with respect to the similarity cost . in general , the similarity and the regularization costs can be decomposed into linear combinations of more specific cost functions . for intensity - based image registration , it is usually assumed that intensities of corresponding voxels are related to each other in some way . many criteria to construct the intensity relationship between corresponding points have been suggested as the cost function for aligning two images . examples of intensity - based cost functions are the mean square difference ( msd ) , correlation coefficient , mutual information , pattern intensity , and gradient correlation [ 29 , 30 ] . in this work , we investigated three commonly used intensity - based cost functions and one intensity - based cost function designed for matching lung ct images . sum of squared difference ( ssd)minimizing the intensity difference at corresponding points between two images is an intuitive method to register grayscale images . a simple and common cost function is the sum of squared difference ( ssd ) defined by ( 2)cssd=x[i2(x)i1(h(x))]2 , where i1 and i2 are the template and target image intensity functions , respectively . r denotes the union of lung regions in target image and deformed template image . the underlying assumption of ssd is that the image intensity at corresponding points between two images should be similar . however , considering the change in ct intensity as air inspired and expired during the respiratory cycle , the grayscale range is different within the lung region in two ct images acquired at different inflation levels . to balance this grayscale range difference , for example , a histogram matching procedure can be used before ssd registration to modify the histogram of template image so that it is similar to that of target image . minimizing the intensity difference at corresponding points between two images is an intuitive method to register grayscale images . a simple and common cost function is the sum of squared difference ( ssd ) defined by ( 2)cssd=x[i2(x)i1(h(x))]2 , where i1 and i2 are the template and target image intensity functions , respectively . r denotes the union of lung regions in target image and deformed template image . the underlying assumption of ssd is that the image intensity at corresponding points between two images should be similar . however , considering the change in ct intensity as air inspired and expired during the respiratory cycle , the grayscale range is different within the lung region in two ct images acquired at different inflation levels . to balance this grayscale range difference , for example , a histogram matching procedure can be used before ssd registration to modify the histogram of template image so that it is similar to that of target image . mutual information ( mi)mutual information ( mi ) similarity cost function can accommodate intensity difference between two images and is therefore well - suited to accommodate the ct intensity change during inflation and deflation of the lung . mutual information expresses the amount of information that one image contains about the other one . analogous to the kullback - leibler measure , the negative mutual information cost of two images is defined as [ 9 , 32 ] ( 3)cmi=ijp(i , j)logp(i , j)pi1h(i)pi2(j ) , where p(i , j ) is the joint intensity distribution of transformed template image i1h and target image i2 ; pi1h(i ) and pi2(j ) are their marginal distributions , respectively . the histogram bins of i1h and i2 are indexed by i and j. the experiments of mi - driven registration use 50 50 histogram bins to estimate joint distribution . mutual information ( mi ) similarity cost function can accommodate intensity difference between two images and is therefore well - suited to accommodate the ct intensity change during inflation and deflation of the lung . mutual information expresses the amount of information that one image contains about the other one . analogous to the kullback - leibler measure , the negative mutual information cost of two images is defined as [ 9 , 32 ] ( 3)cmi=ijp(i , j)logp(i , j)pi1h(i)pi2(j ) , where p(i , j ) is the joint intensity distribution of transformed template image i1h and target image i2 ; pi1h(i ) and pi2(j ) are their marginal distributions , respectively . the histogram bins of i1h and i2 are indexed by i and j. the experiments of mi - driven registration use 50 50 histogram bins to estimate joint distribution . normalized cross correlation ( ncc)normalized cross correlation can be used for multimodality registration problems since it is insensitive to a constant multiplicative factor between the images . this cost function measures the pixel - wise cross - correlation between image intensities normalized by the square root of the autocorrelation of each image . mathematically , the negative normalized cross correlation measure is given by ( 4)cncc=xi2(x)i1(h(x))xi2(x)2xi1(h(x))2 , where the negative sign was added so that the optimal transformation h is found by minimization . when the factor of the intensity patterns from two images is a constant , the measure equals 1 . misalignments between the images will result in decrease of the normalized cross correlation , and thus , increase of the similarity cost cncc . normalized cross correlation can be used for multimodality registration problems since it is insensitive to a constant multiplicative factor between the images . this cost function measures the pixel - wise cross - correlation between image intensities normalized by the square root of the autocorrelation of each image . mathematically , the negative normalized cross correlation measure is given by ( 4)cncc=xi2(x)i1(h(x))xi2(x)2xi1(h(x))2 , where the negative sign was added so that the optimal transformation h is found by minimization . when the factor of the intensity patterns from two images is a constant , the measure equals 1 . misalignments between the images will result in decrease of the normalized cross correlation , and thus , increase of the similarity cost cncc . sum of squared tissue volume difference ( sstvd)the sum of squared tissue volume difference ( sstvd ) cost function accounts for the variation of intensity in the lung ct images during respiration . assume that lung is a mixture of two materials : air and tissue / blood ( nonair ) . then the hounsfield units ( hu ) in lung ct images is a function of tissue and air content . from the hu of ct lung images , the regional tissue volume and air volume can be estimated following the air - tissue mixture model by hoffman and ritman . let v(x ) be the volume element at location x. then the tissue volume v(x ) within the volume element can be estimated as v(x ) = v(x)((hu(x ) huair)/(hutissue huair ) ) , where we assume that huair = 1000 and hutissue = 0 . the intensity similarity cost function sstvd is defined as ( 5)csstvd=[v2(x)v1(h(x))]2dx = [v2(x)i2(x)+10001055 v1(h(x))i1(h(x))+10001055]2dx . the jacobian of a transformation j(h ) estimates the regional volume changes resulted from mapping corresponding regions . thus , the tissue volumes in image i1 and i2 are related by v1(h(x ) ) = v2(x ) j(h(x ) ) . the sum of squared tissue volume difference ( sstvd ) cost function accounts for the variation of intensity in the lung ct images during respiration . assume that lung is a mixture of two materials : air and tissue / blood ( nonair ) . then the hounsfield units ( hu ) in lung ct images is a function of tissue and air content . from the hu of ct lung images , the regional tissue volume and air volume can be estimated following the air - tissue mixture model by hoffman and ritman . let v(x ) be the volume element at location x. then the tissue volume v(x ) within the volume element can be estimated as v(x ) = v(x)((hu(x ) huair)/(hutissue huair ) ) , where we assume that huair = 1000 and hutissue = 0 . the intensity similarity cost function sstvd is defined as ( 5)csstvd=[v2(x)v1(h(x))]2dx = [v2(x)i2(x)+10001055 v1(h(x))i1(h(x))+10001055]2dx . the jacobian of a transformation j(h ) estimates the regional volume changes resulted from mapping corresponding regions . thus , the tissue volumes in image i1 and i2 are related by v1(h(x ) ) = v2(x ) j(h(x ) ) . feature information extracted from the grayscale image is important to help guide the registration process . during the respiration cycle , therefore , the shape and spatial information of vessels can be utilized to help improve the registration accuracy . in ct images , this intensity difference between parenchyma and blood vessels can effectively help intensity - based registration . therefore , grayscale information of the small vessels give almost no contribution to the intensity - based registration . in order to better utilize the information of blood vessel locations , we utilize the vesselness measure ( vm ) computed from intensity images rather than using their grayscales directly . sum of squared vesselness measure difference ( ssvmd)the vesselness measure is based on the analysis of eigenvalues of the hessian matrix of image intensity . the eigenvalues , which are geometrically interpreted as principal curvatures , can be used to indicate the shape of underlying object . in 3d lung ct images , isotropic structures such as parenchyma tissues ( dark ) are associated with three similar nonzero positive eigenvalues . tubular structures such as blood vessels ( bright ) are associated with one negligible eigenvalue and two similar nonzero negative eigenvalues . ordering the eigenvalues of a hessian matrix by magnitude |1 | |2 | |3| , the frangi 's vesselness function is defined as ( 6)f()={(1era2/22)erb2/22(1es2/22 ) if 2<0 and 3<0,0 otherwise , with ra=|2|/|3|,rb=|1|/|23|,s=12+22+32 , where ra distinguishes between plate - like and tubular structures , rb accounts for the deviation from a blob - like structure , and s differentiates between tubular structures and noise . , , and control the sensitivity of the vesselness measure . the experiments in this paper use = 0.5 , = 0.5 , and = 5 . the vesselness measure is based on the analysis of eigenvalues of the hessian matrix of image intensity . the eigenvalues , which are geometrically interpreted as principal curvatures , can be used to indicate the shape of underlying object . in 3d lung ct images , isotropic structures such as parenchyma tissues ( dark ) are associated with three similar nonzero positive eigenvalues . tubular structures such as blood vessels ( bright ) are associated with one negligible eigenvalue and two similar nonzero negative eigenvalues . ordering the eigenvalues of a hessian matrix by magnitude |1 | |2 | |3| , the frangi 's vesselness function is defined as ( 6)f()={(1era2/22)erb2/22(1es2/22 ) if 2<0 and 3<0,0 otherwise , with ra=|2|/|3|,rb=|1|/|23|,s=12+22+32 , where ra distinguishes between plate - like and tubular structures , rb accounts for the deviation from a blob - like structure , and s differentiates between tubular structures and noise . , , and control the sensitivity of the vesselness measure . the experiments in this paper use = 0.5 , = 0.5 , and = 5 . the hessian matrix is computed by convolving the intensity image with second and cross derivatives of the gaussian function . in a multiscale analysis , the response of the vesselness filter will achieve the maximum at a scale , which approximately matches the size of vessels to detect . therefore , the vesselness measure is estimated by computing ( 6 ) for a range of scales and selecting the maximum response : f = maxminmaxf( ) , where is the standard deviation of the gaussian function . the vesselness image is rescaled to [ 0 , 1 ] and can be considered as a probability - like estimate of vesselness features . larger vesselness value indicates that the underlying object is more likely to be a vessel structure , as shown in figure 2 . let f1(x ) and f2(x ) represent the vesselness measures of images i1 and i2 at location x , respectively . in order to match similar vesselness patterns between two images , the sum of squared vesselness measure difference ( ssvmd ) is proposed as ( 7)cssvmd=x[f2(x)f1(h(x))]2 . continuum mechanical models such as linear elasticity [ 11 , 37 , 38 ] and viscous fluid [ 37 , 39 ] can be used to regularize the transformation . a common way to constraint the deformation is applying a differential operator on the transformation and formulating an additive cost term in the objective cost function [ 11 , 22 , 4046 ] . in our registration algorithms , a linear - elastic constraint was used to regularize the displacement fields u , where u = h(x ) x. this regularization term is formed as ( 8)creg(u)=||lu(x)||2dx , where l can be any nonsingular linear differential operator . here the linear elasticity operator l is formed as l u(x ) = u(x ) (u(x ) ) + u(x ) , where = [ /x1 , /x2 , /x3 ] and = = [ /x1 + /x2 + /x3 ] . using the linear elasticity differential operator can help smooth the transformation and help eliminate abrupt changes in the displacement fields . the linear elasticity operator is used in this work to help avoid the transformation from folding onto itself . however , it can not prevent the jacobian of the transformation from going negative , that is , destroying the image topology under the transformation . finally , the total cost is defined as a linear combination of the intensity - based costs , vesselness measure preserving cost , and regularization constraint ( 9)ctotal(h)=cintensity(i1,i2,h)+cssvmd(i1,i2,h)+creg(h).cintensity can be one of the four intensity - based similarity cost functions : cssd , cmi , cncc , or csstvd . constants and are weights to adjust the significance of the three terms . the transform defines how points from the template image i1 are mapping to their corresponding points in the target image i2 . in three dimensional space , let x = ( x1 , x2 , x3 ) define a voxel coordinate in the image domain of the target image i2 . the transformation h is a ( 3 1 ) vector - valued function defined on the voxel lattice of target image , and h(x ) gives the corresponding location in template image to the point x. the lung is composed of nonhomogenous soft tissue . since lung expansion is nonuniform , nonrigid transformations are required to model the lung motion across different inflation levels . to represent the locally varying geometric distortions , the transformation can be represented by various forms of basis function , such as fourier transform , thin - plate splines , and b - splines . b - splines are well suited for image registration and are able to capture the local nonrigid deformation between two images [ 40 , 45 ] . considering the computational efficiency and accuracy requirement , let i = [ x(xi ) , y(xi ) , z(xi ) ] be the coefficients of the ith control point xi on the spline lattice g along each direction . the transformation is represented as ( 10)h(x)=x+igi(xxi ) , where (x , y , z ) = (x)(y)(z ) is a separable convolution kernel . (x ) is the uniform cubic b - spline basis function defined as ( 11)(x)={(46x2 + 3|x|3)6 , 0|x|<1,(2|x|)36 , 1|x|<2,0 , |x|2 . there are several existing methods in numerical analysis such as the partial differential equation ( pde ) solvers used to solve for elastic and fluid transformations , steepest gradient descent method , conjugate gradient method , and so forth . similar to [ 9 , 13 ] , our similarity cost functions were optimized using a limited memory quasi - newton minimization method with bounds ( l - bfgs - b ) algorithm . the bound constraints are applied on b - spline coefficients to guarantee the local injectivity ( one - to - one property ) of the transformation , that is , the transformation maintains the topology of two images . according to their analysis , the displacement fields are locally injective over the domain if the b - spline coefficients satisfy the conditions that x x / k , y y / k , and z z / k , where x , y , and z are the b - spline grid sizes along each direction , and k is a constant approximately equal to 2.479772335 . validation and evaluation of image registration accuracy is an important task to quantify the performance of registration algorithms . due to the absence of a gold standard to judge a registration algorithm , intrasubject ct images of the lung contain identifiable landmarks such as airway - tree and vascular - tree branch points . for each pair of frc and tlc data , 100150 distinctive landmark pairs were selected as branch points of the vascular tree using a semiautomatic method . the euclidean distance between the registration - predicted landmark position and its true position is defined as landmark error . let pk and qk be the location of landmark k on template image i1 and target image i2 , respectively . vessels in the lung keep their tubular shape and tree structures during the respiratory process . therefore , evaluating the alignment on vessel trees is an important approach to validate the matching accuracy at the lung feature level . the registration accuracy on the vessel tree was evaluated by vessel matching distance , which is calculated as the distance between a point on the target vessel tree and its closet point on warped template vessel tree . mathematically , this distance can be stated as the vessel positioning error ( vpe ) ( 12)vpe(x)=minyv2 d(x , h(y ) ) for a given point x in v1 , where v1 and v2 , respectively , are the set of all points in the vessel trees extracted from image i1 and i2 , respectively , and d( ) defines the euclidean distance . examples of the vessel tree extractions are shown as red curves in figure 3 . lobar fissures are defined as the division between adjacent lung lobes and represent important physical boundaries within the lungs . fissure locations are extracted from the images by segmenting the lobes using and then identifying voxels adjacent to two lobe segmentations . the fissure positioning error ( fpe ) is defined as the minimum distance between a point on the deformed fissure and the closest point on the corresponding target fissure ( 13)fpe(x)=minyf2 d(x , h(y ) ) for a given point x in f1 , where f1 and f2 , respectively , are the set of all points in the fissure in image i1 and i2 , respectively . examples of the lobe segmentations are shown in different colors in figure 3 . the lung tissue deformation pattern can be used as an index to assess lung function . in this work , the jacobian determinant of the transformation field derived by image registration is used to estimate the local tissue deformation . the jacobian determinant ( often simply called the jacobian ) [ 11 , 48 , 54 ] is a measurement to estimate the pointwise expansion and contraction during the deformation . the jacobian of the transformation j(h(x ) ) is defined as ( 14)j(h(x))=|h1(x)x1h1(x)x2h1(x)x3h2(x)x1h2(x)x2h2(x)x3h3(x)x1h3(x)x2h3(x)x3| . using a lagrangian reference frame local tissue expansion corresponds to a jacobian greater than one , and local tissue contraction corresponds to a jacobian less than one . preprocessing starts by identifying the lung regions in all images using the method described in . images and masks are downsampled by a factor of 2 in each dimension to reduce computation time . for each pair of data sets , frc images are used as the target image , and tlc images are used as the template image . in order to evaluate how the vesselness cost function affects the registration algorithm results , we performed registration experiments using different similarity costs on parenchyma region in each pair of data sets for comparison . there were four registration methods driven by intensity - only similarity cost functions described in section 2.2.1 , and the same four registration methods that included the feature - based similarity cost ssvmd . after registration , the results from each method were evaluated and compared through matching accuracy on landmarks , vessels , fissures , and underlying transformation properties . we designed experiments to discover good parameter settings on intensity - based cost , feature - based cost cssvmd , and regularization term creg in ( 9 ) . here parameter settings for registration algorithms using other intensity - based cost functions , for example , cssd , cmi , and cncc can be tuned in the same way . table 1 and figure 4 show the results for 20 ct - to - ct registration experiments , as the weighting values ( ved ) and ( smooth ) are varied . the values of and ranged from 0 to 2 and 0 to 0.5 , respectively . experiment ct01 corresponds to unconstrained estimation , in which the transformation was estimated only according to the intensity similarity cost . this experiment produced relatively the worse registration results as evident by the large values of csstvd , cssvmd , and creg in the respective tables . experiments ct02 , ct03 , ct04 , and ct05 demonstrate the effect of estimating the transformations without minimizing the vesselness similarity cost while varying the weight of the linear elastic cost . minimizing the linear elastic cost is good for optimizing the other two similarity costs csstvd and cssvmd , as we can see from figures 4(a ) and 4(b ) . values larger than 0.2 are not recommended since they may cause the csstvd to increase dramatically . experiments ct06 , ct11 , ct16 , and ct21 demonstrate the effect of using vesselness similarity cost function without enforcing the linear elasticity constraint . the cssvmd values for these experiments are much lower than the previous cases since they are being minimized . the intensity similarity costs csstvd also decreased using registration with vesselness constraint , especially when is in the range of [ 0.5 , 1 ] . the remaining experiments show the effect of jointly estimating the transformations , while varying the weights on both the vesselness similarity cost and the linear elasticity constraint . these experiments show that it is possible to find a set of parameters that produce better results using both constraints than only using one or none . the optimal set of parameters should be chosen to provide a good intensity match and vesselness match , while producing less spatial distortion as measured by an acceptable level of linear elastic cost . from the experiments , we observe that = 0.5 ~ 1 and = 0.05 ~ 0.1 are good for minimizing the three costs at the same time . in this work , weighting parameters were set to = 1 and = 0.1 when using sstvd as intensity cost function . parameters in registration using other three intensity cost functions are set in the same way . a spatial multiresolution optimization procedure from coarse to fine was used to improve speed , accuracy , and robustness of the registration . in the experiments , the spatial multiresolution strategy proceeds from low to high resolution , starts at one - eighth of the spatial resolution , and increases by a factor of two until the full resolution is reached . meanwhile , a hierarchy of b - spline grid spacings from large to small was also used . the finest b - spline grid space used in an example multiresolution scheme design for minimizing the total cost function is listed in table 2 . the image spatial resolution and b - spline grid spacing figure 5 lists the cost values at each iteration of one registration . at 1/8 and 1/4 the optimization is stopped before reaching the maximum iterations if the total cost change is nominal between consecutive iterations . registration at full resolution further adjusts the local structures matching . during the registration procedure , the original average landmark error is 27.40 14.37 mm with a maximum landmark error of 72.79 mm . table 3 shows the mean and standard deviation of landmark errors through all six subjects after using different registration methods . table 4 shows the vessel positioning errors for six subjects after using different registration methods . the average errors and standard deviations were all decreased after adding the vesselness constraint . figure 7 shows the distance map on frc vessel tree from one subject after using eight different registration methods . these results show that large errors between the deformed source and target vessel trees are reduced after adding the ssvmd constraint . for each pair of frc and tlc images , the parenchyma regions were segmented into five lobes , and the three fissures were identified , where the lobe segmentations touched each other . the mean and standard deviation of fissure positioning error over all three fissures after using eight registration methods are shown in table 5 . the average fissure positioning error across the six subjects was 9.20 mm and decreased to around 1.50 mm and 1.00 mm without and with ssvmd cost , respectively . the distance map on fissure planes from one subject after using eight different registration methods is shown in figure 8 . notice that adding ssvmd helped improve registration accuracy in the lung regions near the thoracic cage . registration methods producing similar matching accuracy on the feature locations may result in different underlying parenchymal tissue functions . in order to reveal the lung tissue deformation pattern , the jacobian determinant j at a given point gives important information about the behavior of transformation h near that point . arrows denote regions that show different deformation patterns using intensity - only registration methods , but they are more similar after adding vesselness cost function . the experiments presented in this paper were designed to evaluate the performance of the vesselness constraint when it was added to intensity - based registration algorithms . the experimental results of tuning weighting parameters in the total cost function suggest that using both vesselness and smoothing constraint can help minimize the similarity cost , as shown in figure 4 . figure 5 shows that the multiresolution scheme is important and useful for solving complex registration problem efficiently . this is because the registration is first performed at a coarse scale , where the images have much fewer voxels , which are fast and can help eliminate local minima . table 3 and figure 6 demonstrate that adding the ssvmd cost function reduced the mean landmark errors of the four basic registration methods . landmarks with large errors , shown as outliers in the box plot , are aligned much better when ssvmd is used . figure 7 reflects the fact that the ssvmd constraint helps improve matching accuracy over all four basic methods on small vessels , around lung boundaries and in the region near diaphragm . the reason for this is that blood vessels in those regions are usually small and have low intensity contrast , and thus they contribute little to conventional intensity similarity criteria . the vesselness measurement enhances blood vessel information and strengthens contribution of small vessels to registration process when using the ssvmd similarity cost . figure 8 indicates that the ssvmd not only helps match vessel structures , but also helps improve registration accuracy in other regions , such as positions on the fissure planes near the thoracic cage . good matching accuracy on the feature locations does not guarantee that the parenchymal tissue is correctly aligned . rather than evaluates the alignment accuracy , it reveals how well the transformation preserves topology and measures the differential lung volume change . in figure 9 , the left column shows that the jacobian maps generated by the four registration methods without ssvmd have a similar ventral to dorsal gradient as expected since the subjects were imaged in the supine orientation . however , the local tissue deformation patterns derived from these methods are different even in the methods pair ssd and sstvd , which have similar landmark errors as shown in table 3 . this is consistent with the findings that while the intermethod variability on the landmark error is small , there may be discriminating difference in the jacobian maps . the right column shows that adding the ssvmd constraint produces jacobian images that are much more similar across different registration methods and reveal more detailed deformation patterns especially near vessel locations . the four jacobian maps produced using registration methods with ssvmd are similar , which may imply that the derived local deformation patterns are more reliable . comparing the four intensity - only registration methods , registration driven by sstvd achieved lower landmark error and lower vessel and fissure positioning errors than other three methods driven by ssd , mi , and ncc . figures 7 and 8 reflect that sstvd - driven registration resulted in more accurate matching within the region near the thoracic cage . the reason may be that sstvd cost function contains a local jacobian factor , which can constrain incorrect large displacement and help prevent distortion near the thoracic cage . after adding ssvmd on the four intensity - based cost functions ssvmd helps improve matching accuracy in regions near the thoracic cage and near the diaphragm . in our experiments , registration method using sstvd + ssvmd resulted in the smallest matching errors . therefore , we may consider that sstvd + ssvmd is the best similarity cost function to register lung ct images according to our evaluation . the effectiveness of the vesselness preserving metric was tested on a variety of lung ct data sets as part of the grand challenge more than 20 individual registration algorithms from different groups were applied to 30 pairs of lung ct scans in the empire10 challenge . besides our tissue volume and vesselness preserving method , one other method called the robust treereg also combined intensity and feature information . the robust treereg algorithm performed a robust tree registration ( rtr ) and added correspondences between bifurcations of the vessel tree to the voxel - based mutual information driven registration . for this registration challenge , our tissue volume and vesselness preserving method had better performance than the robust treereg method . this result may imply that the vesselness measure provides more feature information than the bifurcation landmarks of the vessel tree . in addition , our tissue volume ( or mass ) and vesselness preserving method was shown to improve matching results compared to methods that only incorporated mass preservation . this paper presented nonrigid registration algorithms driven by commonly used intensity - based criteria for lung registration , a feature - based vesselness constraint , and a linear elastic smoothing constraint . results were presented to show that adding the ssvmd constraint to existing similarity cost functions such as ssd , mi , ncc , and sstvd reduces landmark error and improves overlap on vascular tree and fissure planes . the purpose of adding the vesselness cost in registration process is that it can help correct the mismatches of small vessels and their surrounding lung tissues . using the ssvmd constraint was shown to produce a more detailed expansion pattern for local tissue , especially near vessel locations . this demonstrates that using the ssvmd constraint not only helps match on feature structures , but also helps align corresponding parenchymal tissues providing a more reliable pattern of local lung tissue deformation . in this paper , registration method preserving both tissue volume and vesselness measurement performed best on matching 3d lung ct data according to our evaluation .
accurate pulmonary image registration is a challenging problem when the lungs have a deformation with large distance . in this work , we present a nonrigid volumetric registration algorithm to track lung motion between a pair of intrasubject ct images acquired at different inflation levels and introduce a new vesselness similarity cost that improves intensity - only registration . volumetric ct datasets from six human subjects were used in this study . the performance of four intensity - only registration algorithms was compared with and without adding the vesselness similarity cost function . matching accuracy was evaluated using landmarks , vessel tree , and fissure planes . the jacobian determinant of the transformation was used to reveal the deformation pattern of local parenchymal tissue . the average matching error for intensity - only registration methods was on the order of 1 mm at landmarks and 1.5 mm on fissure planes . after adding the vesselness preserving cost function , the landmark and fissure positioning errors decreased approximately by 25% and 30% , respectively . the vesselness cost function effectively helped improve the registration accuracy in regions near thoracic cage and near the diaphragm for all the intensity - only registration algorithms tested and also helped produce more consistent and more reliable patterns of regional tissue deformation .
1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusions
in a sequence of pulmonary scans , the computed correspondences describe the motion of the lung between a pair of images at the voxel level . this vesselness preserving cost function is added to four existing intensity - based similarity costs , and comparison experiments show that this criterion helps improve the registration accuracy . in this study , six pairs of volumetric ct data sets from six human subjects in the supine orientation were collected on a siemens sensation 64 multidetector ct scanner . more formally , let d r define the domain of the images i1 : d r and i2 : d r. in this work , the transformation h : d d is assumed to be a diffeomorphism . the regularization cost function is used to enforce desired properties of the transformation such as minimum distortion . in this work , we investigated three commonly used intensity - based cost functions and one intensity - based cost function designed for matching lung ct images . however , considering the change in ct intensity as air inspired and expired during the respiratory cycle , the grayscale range is different within the lung region in two ct images acquired at different inflation levels . however , considering the change in ct intensity as air inspired and expired during the respiratory cycle , the grayscale range is different within the lung region in two ct images acquired at different inflation levels . when the factor of the intensity patterns from two images is a constant , the measure equals 1 . when the factor of the intensity patterns from two images is a constant , the measure equals 1 . finally , the total cost is defined as a linear combination of the intensity - based costs , vesselness measure preserving cost , and regularization constraint ( 9)ctotal(h)=cintensity(i1,i2,h)+cssvmd(i1,i2,h)+creg(h).cintensity can be one of the four intensity - based similarity cost functions : cssd , cmi , cncc , or csstvd . validation and evaluation of image registration accuracy is an important task to quantify the performance of registration algorithms . the registration accuracy on the vessel tree was evaluated by vessel matching distance , which is calculated as the distance between a point on the target vessel tree and its closet point on warped template vessel tree . in this work , the jacobian determinant of the transformation field derived by image registration is used to estimate the local tissue deformation . the jacobian determinant ( often simply called the jacobian ) [ 11 , 48 , 54 ] is a measurement to estimate the pointwise expansion and contraction during the deformation . in order to evaluate how the vesselness cost function affects the registration algorithm results , we performed registration experiments using different similarity costs on parenchyma region in each pair of data sets for comparison . there were four registration methods driven by intensity - only similarity cost functions described in section 2.2.1 , and the same four registration methods that included the feature - based similarity cost ssvmd . experiments ct02 , ct03 , ct04 , and ct05 demonstrate the effect of estimating the transformations without minimizing the vesselness similarity cost while varying the weight of the linear elastic cost . a spatial multiresolution optimization procedure from coarse to fine was used to improve speed , accuracy , and robustness of the registration . notice that adding ssvmd helped improve registration accuracy in the lung regions near the thoracic cage . in order to reveal the lung tissue deformation pattern , the jacobian determinant j at a given point gives important information about the behavior of transformation h near that point . arrows denote regions that show different deformation patterns using intensity - only registration methods , but they are more similar after adding vesselness cost function . the experiments presented in this paper were designed to evaluate the performance of the vesselness constraint when it was added to intensity - based registration algorithms . figure 8 indicates that the ssvmd not only helps match vessel structures , but also helps improve registration accuracy in other regions , such as positions on the fissure planes near the thoracic cage . in figure 9 , the left column shows that the jacobian maps generated by the four registration methods without ssvmd have a similar ventral to dorsal gradient as expected since the subjects were imaged in the supine orientation . comparing the four intensity - only registration methods , registration driven by sstvd achieved lower landmark error and lower vessel and fissure positioning errors than other three methods driven by ssd , mi , and ncc . after adding ssvmd on the four intensity - based cost functions ssvmd helps improve matching accuracy in regions near the thoracic cage and near the diaphragm . results were presented to show that adding the ssvmd constraint to existing similarity cost functions such as ssd , mi , ncc , and sstvd reduces landmark error and improves overlap on vascular tree and fissure planes .
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leishmaniasis is a parasitic disease that can manifest in three forms : 1 ) mucosal , 2 ) cutaneous , and 3 ) visceral ( who 2010 , maroli et al . 2013 ) . visceral leishmaniasis ( vl ) is the most severe form caused by the protozoan flagellate , leishmania donovani ( east africa and indian subcontinent ) and l. infantum ( also known as l. chagasi , found in europe , north africa , and latin america ) . as this review focuses on elimination efforts in india , we will only refer to l. donovani in this article . globally , the annual incidence rate is approximately 200,000400,000 cases , the majority of cases are present in bangladesh , nepal , and india . furthermore , two - thirds of those cases occur in india where vl ( also known as kala - azar ; black fever ) is endemic in the states of uttar pradesh , jharkhand , west bengal , and bihar ( alvar et al . reports have noted the annual incidence in india as 146,700282,800 cases with a mortality rate of at least 2.4% . however , other studies involving active searches at the village level have discovered mortality rates from 1020% , partly due to a delay in diagnosis ( alvar et al . vl carries a mortality rate over 90% when left untreated ( desjeux 1996 , jeronimo et al . transmission of l. donovani occurs via the bite of a female phlebotomus argentipes sand fly . of the nearly 50 species of sand flies present in india , p. argentipes is the only one known to transmit vl in this country ( kumar et al . once the parasite is in the human body it rapidly invades macrophages and eventually moves in this way to the liver , spleen , and lymph nodes ( chappuis et al . symptoms include : fever lasting weeks to months , splenomegaly , hepatomegaly , and anemia ( desjeux 1996 , guerin et al . go on to develop a rash known as post kala - azar dermal leishmaniasis ( pkdl ) , this can occur anywhere from several months to several years following treatment ( ramesh and mukharjee 1995 , rahman et al . 2010 ) . pkdl patients do not experience other symptoms outside of the rash , however , they are thought to serve as a reservoir for l. donovani where feeding sand flies can acquire the parasite ( addy and nandy 1992 , rahman et al . are the only known reservoir for l. donovani in india , pkdl patients are another concern for vl elimination efforts . visceral leishmaniasis is a poverty - associated disease linked to poor housing and sanitary conditions and malnutrition , these factors have led to a number of difficulties regarding treatment and elimination ( cerf et al . easy access to medical care in the rural vl - endemic regions of india is still limited , meaning patients are not routinely identified or wait until the disease has progressed to more severe symptoms before seeking treatment . moreover , until recently treatment regimens required medication injections over the course of 2028 days leading to poor compliance rates ( clem 2010 , moore and lockwood 2010 , stockdale and newton 2013 ) . in addition to hurdles with medical treatment , increased sand fly density has been associated with certain types of housing , owning livestock , and nearby vegetation ( ranjan et al 2005 , singh et al . 2010 , poch et al . 2011 , poch et al . 2012 , perry et al . the risk of exposure to p. argentipes is even higher for some , as many people in these poor regions of india live in close proximity to their cattle , keeping them inside their dwellings ( singh et al . 2014 ) . as a by - product of ddt spraying for malaria elimination programs in the 1950s and 1970s , however , when ddt spraying ended , sand fly numbers and cases of vl rose again , leading to several major epidemics ( kishore et al . in 2005 , the governments of bangladesh , nepal , and india began a concerted vl elimination effort , with a target goal of 2015 for elimination ( 1 case in 10,000 people ) . indoor residual spraying ( irs ) and insecticide - treated bed nets ( itn ) have been the main forms of control regimens tried in india , while environmental modification ( evm ) and feed - through insecticides ( fti ) have been less studied . despite significant efforts to control sand flies and treat infected persons as such , we sought to review the current practices regarding vector / vl control programs in india . india in combination with the following keywords : phlebotomus argentipes , sand fly , kala - azar , and/or visceral leishmaniasis . the following paper is a summation of the articles collected in hopes of highlighting what has worked , what has not , and what can be learned as we move forward towards elimination goals . irs has been the main line of defense against vector - borne diseases in india . in the 1950s and again in the 1970s there were aggressive irs initiatives to eliminate malaria ( kishore et al . , households in malaria - endemic regions , many of which were the same as vl - endemic areas , were sprayed with ddt to cull mosquitoes . sand fly populations declined in kind , and with them , cases of vl . however , at the end of those campaigns , with no systematic irs programs , sand fly populations and cases of vl quickly rebounded ( kishore et al . since then , irs using ddt has been employed to fight vl transmission in india with mixed results . currently , the national vector borne disease control programme of india dictates that irs should be performed on all homes and cattle sheds in endemic regions . they recommend two annual applications , the first to occur in february / march and the second in may / june ( nvbdcp 2014 ) . applied properly , irs has been shown to dramatically impact p. argentipes density ( joshi et al . 2009 ) . in that carefully controlled study involving all homes in six clusters in each of four villages , there was a 72.4% decline in sand fly numbers . other studies that have assessed the efficacy of irs in india have instead highlighted a number of issues limiting its success in controlling sand flies and vl . huda et al . monitored local irs programs for control of vl in bangladesh , nepal , and india ( 2011 ) . in india , ( though it was not unique for some of these issues ) they found functional pumps and spare parts were lacking and 23.5% of the pumps were leaking . spraying staff were not adequately trained and as such , proper mixing of ddt was done only 29.4% of the times observed . additionally , proper distance and swath coverage during spraying were maintained only 49% and 58.8% of the time , respectively ( huda et al . 2011 ) . these results are echoed in a similar report that found the same issues with equipment , training , mixing , and distance from surface , in addition to storage and quality issues for the ddt ( chowdhury et al . this group also noted that the ddt residue levels on the walls varied at the village level from 66%90% of the intended concentration while at the household level , the concentration varied as much as 9.1% to 330% ( chowdhury et al . furthermore , spraying must be applied to > 80% of the homes in an area for mass effect ( cdc 2012 ) . according to huda another point of concern with irs in india is resistance of p. argentipes to ddt . resistance to ddt was documented for the district of samastipur in bihar , india as early as 1990 ( mukhopadhyay et al . 1992 ) . in general , p. argentipes remains largely susceptible to ddt with the majority of flies succumbing to the insecticide ( singh et al . however , select districts are showing signs of this trend shifting ( kishore et al . documented only a 54% mortality rate after 24 h for sand flies exposed to walls sprayed with ddt while another group found up to 70% were killed when exposed to surfaces that had been sprayed 2 weeks prior ( chowdhury et al . it is possible that the results obtained by huda and chowdhury et al . may have been partly due to improper storage , mixing , spraying , and/or active ingredient concentration which were all problems documented by those studies ( chowdhury 2011a , huda 2011a ) . a controlled study involving irs in three states in india found mortality rates for p. argentipes ranging from 3189% , indicating moderate to significant levels of resistance to ddt , even with proper use ( singh et al . 2012 ) . despite these many problems recorded by groups studying irs , in all of the aforementioned studies , post - treatment sand fly abundance was significantly reduced in the short term ( chowdhury et al . in fact , joshi et al . demonstrated a negative effect on sand fly numbers up to five months post - treatment ( joshi et al . both nepal and bangladesh employ pyrethroids for their irs regimens and have shown them to be effective ( joshi et al . some work has been conducted using different insecticides in india . in one report they found sand flies were resistant to ddt but not deltamethrin ( dhiman et al . a more recent study documented that deltamethrin was effective in nearly 100% of the locations tested in india ( singh et al . the government of india is currently testing the efficacy of synthetic pyrethroids on sand fly control in bihar ( nvbdp 2014 ) . a comprehensive table of susceptibility studies can be found in the review by ostyn et al . p. argentipes are peridomestic , found in cattle enclosures as well as vegetation ( poch et al . another matter that is infrequently addressed in studies regarding irs is the health effects to humans , animals , and the environment as a result of frequent and long - term irs . the stockholm convention on persistent organic pollutants has banned ddt ( van den berg 2009 ) . however , there is yet no consensus on whether ddt exposure leads to deleterious health effects among humans ( sharpe and stewart 2004 , beard 2006 ) . regardless , there are well documented studies regarding its toxicity in birds and a variety of aquatic species ( blus 2003 , sparling 2010 , beckvar and lotufo 2011 ) . while further work is needed to verify them , these data indicate that alternative irs compounds may be more efficacious in controlling p. argentipes abundance . research looking at effects on human health due to long - term exposure would still need to be addressed . ultimately , these data suggest that with proper execution , irs could be an even more effective tool against vl in india but is not sufficient as a standalone given that p. argentipes is found outdoors in significant numbers . bed nets , in particular , itns and long - lasting insecticide nets ( llins ) , have been suggested as alternatives and/or complements to irs for the control of sand fly populations and vl . itn / llins have been effective against other vector - borne diseases , including cutaneous leishmaniasis ( lengeler 2004 , kulkarni et al . 2007 , wilson et al . 2014 ) but there are mixed results when it comes to sand flies in india . a trial using untreated nets found that the number of female , blood - fed p. argentipes declined by 85% following the introduction of the nets ( picado et al . however , the authors of that study note that they lacked concurrent controls and thus , blood - feeding rates could potentially be attributed to changes in environmental factors ( temperature , humidity , precipitation ) or changes in host availability ( i.e. an increase in domestic animals ) . even so , other groups in bangladesh and nepal have found similar results ( bern et al . the report by joshi et al . investigated the usefulness of irs , llins , and evm to mitigate sand fly density in india , nepal , and bangladesh . when llins were in place , there was a village - wide reduction in p. argentipes numbers by 43.7% in india when measured 5 months post - intervention ( joshi et al . a subsequent village - wide study in india noted a 25% decline in sand fly density ( picado et al . in that study , 16 clusters were enrolled , of those , 10 were used for sand fly capture studies . the reduction appeared to be at the community level and not displacement as p. argentipes abundance did not increase in cattle enclosures ( picado et al . however , this observation does not exclude other environmental refuges , such as vegetation ( poch 2011 , poch 2012 ) . as a part of the same study by picado et al . they also investigated rates of vl in these same villages and found no protective effect of llins on the rate of seroconversion ( picado et al . over the course of 24 months , incidence of l. donovani infection was found to be 5.4% in the intervention group and 5.5% in the control group while clinical vl rates were 0.38% and 0.40% in the intervention and control groups , respectively . one explanation for this result was that llins do not prevent outdoor transmission ( picado et al . an earlier study of 48 homes and mixed dwellings ( homes shared with livestock ) in bihar compared two types of llins to two types of untreated nets and looked at sand fly densities . at all time - points , up to 9 weeks post - treatment , there were no differences in p. argentipes abundance between the groups ( dinesh et al . it should be noted , that during the course of the aforementioned studies , unplanned irs took place , and in both cases there was no noticeable effect on the number of sand flies captured in either study ( dinesh et al . furthermore , when the statistical models used to analyze the vl seroconversion data were adjusted for irs , no changes were found ( picado et al . while the number of studies has been limited , the efficacy of bed nets in india to combat vl have not shown the same promise as they have in bangladesh ( chowdhury et al . the potential reasons for this are many , including : study design , environmental factors ( temperature , humidity , flooding etc . ) , and susceptibility to insecticides - p. argentipes in bangladesh may be more susceptible , given their history of irs campaigns is more recent compared to india ( bern et al . as breeding sites are yet unknown , and sand flies have been associated with vegetation in india , these remain confounding factors when accounting for differences in intervention strategies ( poch et al . non - compliance and net quality are another concern . in the studies by picado and joshi , even so , compliance with nets can be difficult as sand flies are much smaller than mosquitoes requiring finer mesh that people find stifling to sleep under in the heat of summer ( ostyn et al . while in theory , the impregnated insecticide should provide repellent activities , thus allowing for a larger mesh , efficacy in the field has not been shown ( picado et al . moreover , 93% of 1,217 people surveyed in 20092011 in vl - endemic regions reported sleeping outside at some point during the hottest months of the year ( perry et al . 2013 ) . assuming this trend holds true throughout other vl - afflicted areas , this is a serious hurdle to be faced in regards to the efficacy of irs , itns , and evm as none mitigate transmission that occurs outside of a person s dwelling . typical dwellings in the regions of india afflicted by vl are often made of mud and thatch , or brick / plaster . 2014 ) . in all cases , cracks and crevices where sand flies can rest and hide are prevalent . all home types are susceptible to habitation by p. argentipes , but thatched homes in particular cater to high densities ( malaviya et al . evm is a method little studied when compared to irs and itns as a means to control vl in india . evm has generally meant alterations to the home or surrounding environment by means of covering or filling in cracks and crevices in walls and floors . a pilot study involving 15 homes saw a reduction in sand fly numbers using a mud and lime plaster mix to seal cracks in homes and cattle enclosures ( kumar et al . the same well - controlled study comparing the efficacy of irs and itns in india , nepal , and bangladesh found a 42% decline in sand fly abundance five months after the walls of homes and cattle enclosures were plastered with a mud and lime mixture . the negative effect on sand fly populations may be due to the lime ph or limiting available moisture , thus , inhibiting breeding of p. argentipes , though breeding sites for these flies have not been confirmed ( kumar et al . indeed , a study in bangladesh saw no effect on sand fly populations when crevices were filled with mud ( chowdhury et al . importantly , another study in india found mud - plastered walls themselves to be risk factor for vl ( ranjan et al . however , while mud / lime mixes are effective , this method requires continual maintenance and is costly compared to irs and itns ( das et al . 2008 ) . but like irs and itn , it is only effective against the sand flies that are inside homes ; populations that reside in outdoor enclosures and vegetation would remain a source of vl . a newer , relatively untested , yet promising addition to the vector - control arsenal is the use of ftis . several compounds including ivermectin , fipronil , and imidacloprid have been tested in rodents to cull p. papatasi , a vector for cutaneous leishmaniasis ( mascari et al . , the agents have been effective at controlling adult and larval sand flies when fed on blood or feces from treated animals , respectively . in regards to p. argentipes , ivermectin and fipronil as well as diflubenzuron and eprinomectin were tested as ftis in rats ( ingenloff et al . fipronil at 150 ppm was shown to result in the quickest mortality and its effects had greater longevity than the other three compounds , even when they were used at greater concentrations . cattle and other livestock act as a major source of blood meals for female sand flies . blood - meal analysis in one study found that 39.3% of the flies tested had fed on some form of livestock ( garlapati et al . so as to target p. argentipes that are feeding off of cattle and therefore , possibly residing outdoors , a controlled study involving cattle dosed with 0.5 , 1.0 , 2.0 , and 4.0 mg / kg body weight of fipronil was conducted ( poch et al . 2013 ) . that investigation demonstrated that fipronil as an fti is effective , even at the lowest dose , at killing both adult and larval sand flies . the majority of adult sand flies had a 100% mortality rate within four days when fed on cattle up to 21 days post - treatment for all but the lowest dose ( which had a 22% mortality rate at 21 days post - treatment ) . nearly all of the flies that were fed on days 1 , 3 , and 5 post - treatment succumbed on the same day . for larval p. argentipes , the mean - time to death when fed on feces from cattle 1 or 3 days post - treatment was 4.5 days , larva fed on feces with the lowest dose ( 0.5 mg / kg ) on day 1 post - treatment had 100% mortality within 5.5 days while the highest dose ( 4.0 mg / kg ) was 4.0 days . additionally , for all doses tested , 100% mortality of larval sand flies was achieved by day 15.5 when fed on dung collected 21 days post - treatment ( poch et al . 2013 ) . given that this method is untested under field conditions , conclusions regarding its effect on p. argentipes abundance or vl transmission can not be drawn at this time . while no effects to cattle health were noted in the aforementioned study , future work will need to be done to assess the health of cattle treated long - term . moreover , at the lowest dose , fipronil can remain in the animal for up to 35 days but it is at concentration below international allowable limits ( poch et al . studies regarding the efficacy of fipronil as an fti are ongoing and if they prove positive , this method would be a valued addition to irs and itns as it would better address exophilic p. argentipes , making control measures more comprehensive . irs has been the main line of defense against vector - borne diseases in india . in the 1950s and again in the 1970s there were aggressive irs initiatives to eliminate malaria ( kishore et al . , households in malaria - endemic regions , many of which were the same as vl - endemic areas , were sprayed with ddt to cull mosquitoes . sand fly populations declined in kind , and with them , cases of vl . however , at the end of those campaigns , with no systematic irs programs , sand fly populations and cases of vl quickly rebounded ( kishore et al . since then , irs using ddt has been employed to fight vl transmission in india with mixed results . currently , the national vector borne disease control programme of india dictates that irs should be performed on all homes and cattle sheds in endemic regions . they recommend two annual applications , the first to occur in february / march and the second in may / june ( nvbdcp 2014 ) . applied properly , irs has been shown to dramatically impact p. argentipes density ( joshi et al . 2009 ) . in that carefully controlled study involving all homes in six clusters in each of four villages , there was a 72.4% decline in sand fly numbers . other studies that have assessed the efficacy of irs in india have instead highlighted a number of issues limiting its success in controlling sand flies and vl . huda et al . monitored local irs programs for control of vl in bangladesh , nepal , and india ( 2011 ) . in india , ( though it was not unique for some of these issues ) they found functional pumps and spare parts were lacking and 23.5% of the pumps were leaking . spraying staff were not adequately trained and as such , proper mixing of ddt was done only 29.4% of the times observed . additionally , proper distance and swath coverage during spraying were maintained only 49% and 58.8% of the time , respectively ( huda et al . 2011 ) . these results are echoed in a similar report that found the same issues with equipment , training , mixing , and distance from surface , in addition to storage and quality issues for the ddt ( chowdhury et al . this group also noted that the ddt residue levels on the walls varied at the village level from 66%90% of the intended concentration while at the household level , the concentration varied as much as 9.1% to 330% ( chowdhury et al . furthermore , spraying must be applied to > 80% of the homes in an area for mass effect ( cdc 2012 ) . according to huda another point of concern with irs in india is resistance of p. argentipes to ddt . resistance to ddt was documented for the district of samastipur in bihar , india as early as 1990 ( mukhopadhyay et al . 1992 ) . in general , p. argentipes remains largely susceptible to ddt with the majority of flies succumbing to the insecticide ( singh et al . however , select districts are showing signs of this trend shifting ( kishore et al . documented only a 54% mortality rate after 24 h for sand flies exposed to walls sprayed with ddt while another group found up to 70% were killed when exposed to surfaces that had been sprayed 2 weeks prior ( chowdhury et al . it is possible that the results obtained by huda and chowdhury et al . may have been partly due to improper storage , mixing , spraying , and/or active ingredient concentration which were all problems documented by those studies ( chowdhury 2011a , huda 2011a ) . a controlled study involving irs in three states in india found mortality rates for p. argentipes ranging from 3189% , indicating moderate to significant levels of resistance to ddt , even with proper use ( singh et al . 2012 ) . despite these many problems recorded by groups studying irs , in all of the aforementioned studies , post - treatment sand fly abundance was significantly reduced in the short term ( chowdhury et al . in fact , joshi et al . demonstrated a negative effect on sand fly numbers up to five months post - treatment ( joshi et al . both nepal and bangladesh employ pyrethroids for their irs regimens and have shown them to be effective ( joshi et al . some work has been conducted using different insecticides in india . in one report they found sand flies were resistant to ddt but not deltamethrin ( dhiman et al . a more recent study documented that deltamethrin was effective in nearly 100% of the locations tested in india ( singh et al . the government of india is currently testing the efficacy of synthetic pyrethroids on sand fly control in bihar ( nvbdp 2014 ) . a comprehensive table of susceptibility studies can be found in the review by ostyn et al . p. argentipes are peridomestic , found in cattle enclosures as well as vegetation ( poch et al . another matter that is infrequently addressed in studies regarding irs is the health effects to humans , animals , and the environment as a result of frequent and long - term irs . the stockholm convention on persistent organic pollutants has banned ddt ( van den berg 2009 ) . however , there is yet no consensus on whether ddt exposure leads to deleterious health effects among humans ( sharpe and stewart 2004 , beard 2006 ) . regardless , there are well documented studies regarding its toxicity in birds and a variety of aquatic species ( blus 2003 , sparling 2010 , beckvar and lotufo 2011 ) . while further work is needed to verify them , these data indicate that alternative irs compounds may be more efficacious in controlling p. argentipes abundance . research looking at effects on human health due to long - term exposure would still need to be addressed . ultimately , these data suggest that with proper execution , irs could be an even more effective tool against vl in india but is not sufficient as a standalone given that p. argentipes is found outdoors in significant numbers . bed nets , in particular , itns and long - lasting insecticide nets ( llins ) , have been suggested as alternatives and/or complements to irs for the control of sand fly populations and vl . itn / llins have been effective against other vector - borne diseases , including cutaneous leishmaniasis ( lengeler 2004 , kulkarni et al . 2007 , wilson et al . 2014 ) but there are mixed results when it comes to sand flies in india . a trial using untreated nets found that the number of female , blood - fed p. argentipes declined by 85% following the introduction of the nets ( picado et al . however , the authors of that study note that they lacked concurrent controls and thus , blood - feeding rates could potentially be attributed to changes in environmental factors ( temperature , humidity , precipitation ) or changes in host availability ( i.e. an increase in domestic animals ) . even so , other groups in bangladesh and nepal have found similar results ( bern et al . the report by joshi et al . investigated the usefulness of irs , llins , and evm to mitigate sand fly density in india , nepal , and bangladesh . when llins were in place , there was a village - wide reduction in p. argentipes numbers by 43.7% in india when measured 5 months post - intervention ( joshi et al . a subsequent village - wide study in india noted a 25% decline in sand fly density ( picado et al . , 16 clusters were enrolled , of those , 10 were used for sand fly capture studies . the reduction appeared to be at the community level and not displacement as p. argentipes abundance did not increase in cattle enclosures ( picado et al . however , this observation does not exclude other environmental refuges , such as vegetation ( poch 2011 , poch 2012 ) . as a part of the same study by picado et al . they also investigated rates of vl in these same villages and found no protective effect of llins on the rate of seroconversion ( picado et al . nearly 20,000 people were enrolled in the seroconversion study . over the course of 24 months , incidence of l. donovani infection was found to be 5.4% in the intervention group and 5.5% in the control group while clinical vl rates were 0.38% and 0.40% in the intervention and control groups , respectively . one explanation for this result was that llins do not prevent outdoor transmission ( picado et al . . an earlier study of 48 homes and mixed dwellings ( homes shared with livestock ) in bihar compared two types of llins to two types of untreated nets and looked at sand fly densities . at all time - points , up to 9 weeks post - treatment , there were no differences in p. argentipes abundance between the groups ( dinesh et al . it should be noted , that during the course of the aforementioned studies , unplanned irs took place , and in both cases there was no noticeable effect on the number of sand flies captured in either study ( dinesh et al . furthermore , when the statistical models used to analyze the vl seroconversion data were adjusted for irs , no changes were found ( picado et al . while the number of studies has been limited , the efficacy of bed nets in india to combat vl have not shown the same promise as they have in bangladesh ( chowdhury et al . the potential reasons for this are many , including : study design , environmental factors ( temperature , humidity , flooding etc . ) , and susceptibility to insecticides - p. argentipes in bangladesh may be more susceptible , given their history of irs campaigns is more recent compared to india ( bern et al . as breeding sites are yet unknown , and sand flies have been associated with vegetation in india , these remain confounding factors when accounting for differences in intervention strategies ( poch et al . non - compliance and net quality are another concern . in the studies by picado and joshi , even so , compliance with nets can be difficult as sand flies are much smaller than mosquitoes requiring finer mesh that people find stifling to sleep under in the heat of summer ( ostyn et al . while in theory , the impregnated insecticide should provide repellent activities , thus allowing for a larger mesh , efficacy in the field has not been shown ( picado et al . moreover , 93% of 1,217 people surveyed in 20092011 in vl - endemic regions reported sleeping outside at some point during the hottest months of the year ( perry et al . 2013 ) . assuming this trend holds true throughout other vl - afflicted areas , this is a serious hurdle to be faced in regards to the efficacy of irs , itns , and evm as none mitigate transmission that occurs outside of a person s dwelling typical dwellings in the regions of india afflicted by vl are often made of mud and thatch , or brick / plaster . 2014 ) . in all cases , cracks and crevices where sand flies can rest and hide are prevalent . all home types are susceptible to habitation by p. argentipes , but thatched homes in particular cater to high densities ( malaviya et al . evm is a method little studied when compared to irs and itns as a means to control vl in india . evm has generally meant alterations to the home or surrounding environment by means of covering or filling in cracks and crevices in walls and floors . a pilot study involving 15 homes saw a reduction in sand fly numbers using a mud and lime plaster mix to seal cracks in homes and cattle enclosures ( kumar et al . the same well - controlled study comparing the efficacy of irs and itns in india , nepal , and bangladesh found a 42% decline in sand fly abundance five months after the walls of homes and cattle enclosures were plastered with a mud and lime mixture . the negative effect on sand fly populations may be due to the lime ph or limiting available moisture , thus , inhibiting breeding of p. argentipes , though breeding sites for these flies have not been confirmed ( kumar et al . indeed , a study in bangladesh saw no effect on sand fly populations when crevices were filled with mud ( chowdhury et al . importantly , another study in india found mud - plastered walls themselves to be risk factor for vl ( ranjan et al . however , while mud / lime mixes are effective , this method requires continual maintenance and is costly compared to irs and itns ( das et al . 2008 ) . but like irs and itn , it is only effective against the sand flies that are inside homes ; populations that reside in outdoor enclosures and vegetation would remain a source of vl . a newer , relatively untested , yet promising addition to the vector - control arsenal is the use of ftis . several compounds including ivermectin , fipronil , and imidacloprid have been tested in rodents to cull p. papatasi , a vector for cutaneous leishmaniasis ( mascari et al . 2013 , derbali et al . 2014 ) . in these reports , the agents have been effective at controlling adult and larval sand flies when fed on blood or feces from treated animals , respectively . in regards to p. argentipes , ivermectin and fipronil as well as diflubenzuron and eprinomectin were tested as ftis in rats ( ingenloff et al . fipronil at 150 ppm was shown to result in the quickest mortality and its effects had greater longevity than the other three compounds , even when they were used at greater concentrations . cattle and other livestock act as a major source of blood meals for female sand flies . blood - meal analysis in one study found that 39.3% of the flies tested had fed on some form of livestock ( garlapati et al . so as to target p. argentipes that are feeding off of cattle and therefore , possibly residing outdoors , a controlled study involving cattle dosed with 0.5 , 1.0 , 2.0 , and 4.0 mg / kg body weight of fipronil was conducted ( poch et al . that investigation demonstrated that fipronil as an fti is effective , even at the lowest dose , at killing both adult and larval sand flies . the majority of adult sand flies had a 100% mortality rate within four days when fed on cattle up to 21 days post - treatment for all but the lowest dose ( which had a 22% mortality rate at 21 days post - treatment ) . nearly all of the flies that were fed on days 1 , 3 , and 5 post - treatment succumbed on the same day . for larval p. argentipes , the mean - time to death when fed on feces from cattle 1 or 3 days post - treatment was 4.5 days , larva fed on feces with the lowest dose ( 0.5 mg / kg ) on day 1 post - treatment had 100% mortality within 5.5 days while the highest dose ( 4.0 mg / kg ) was 4.0 days . additionally , for all doses tested , 100% mortality of larval sand flies was achieved by day 15.5 when fed on dung collected 21 days post - treatment ( poch et al . 2013 ) . given that this method is untested under field conditions , conclusions regarding its effect on p. argentipes abundance or vl transmission can not be drawn at this time . while no effects to cattle health were noted in the aforementioned study , future work will need to be done to assess the health of cattle treated long - term . moreover , at the lowest dose , fipronil can remain in the animal for up to 35 days but it is at concentration below international allowable limits ( poch et al . studies regarding the efficacy of fipronil as an fti are ongoing and if they prove positive , this method would be a valued addition to irs and itns as it would better address exophilic p. argentipes , making control measures more comprehensive . with humans as the only known reservoir for vl in india , rapid diagnosis and treatment would go a long way in controlling epidemics . however , if pkdl patients serve as a source of l. donovani , vector - control programs will also be needed to reach the goal of elimination . in this way , surveillance and reporting programs may need to be reevaluated as under - reporting of vl remains an issue within india ( mubayi et al . taking into account the diverse feeding and living habits of p. argentipes , it is likely that a combination of control measures will be necessary to eliminate vl in india . switching insecticides may be beneficial with mounting evidence of increased resistance of p. argentipes to ddt coupled with potential health effects on humans , animals , and the environment ( blus 2003 , van den berg 2009 , sparling 2010 , huda et al . 2015 ) . in the end , the efficacy of irs is dependent on organized government programs that coordinate the spraying schedule and train the technicians , which can be either a benefit or hindrance depending on the resources available to these groups ( joshi et al . 2011a , huda et al . 2011 ) . even still , irs works well to curb adult p. argentipes abundance . efforts should also be placed on identifying breeding grounds . to this end , irs could be focused and effective at eliminating both adult and larval flies . insecticide treated nets have shown limited success in india despite reports of their usefulness in other countries in combating sand fly densities ( dinesh et al . 2008 , picado et al . while there is some level of personal protection from sand flies afforded to people who use bed nets , more work would need to be done to confirm that the cost of these initiatives is validated . itns can often be more effective control measures against disease - transmitting vectors as proper use is in the hands of the affected persons instead of an outside program . that being said , non - compliance in india could be a problem as many people report sleeping outdoors during the hot summer months ( perry et al . long - lasting insecticide nets can have repellent and insecticidal properties for years , making them a relatively cheap supplement to irs , however , given the impoverished state of most of the afflicted regions , nets may still need to be provided by government or non - profit groups . the most expensive of the three most studied intervention strategies is evm ( das et al . a mud and lime mixture to seal cracks and crevices in walls and floors has shown some negative effects on sand fly abundance , yet these studies have been limited ( kumar et al . given the cost and need for continual maintenance , evm is a strategy that may be best left for use on a case - by - case basis rather than a district - wide , vector - control measure . the state of dwellings in vl - endemic villages is a by - product of the greater issue of region - wide poverty , if that issue were better addressed , evm would be a moot point . this relatively new addition to vl vector - control has been effective at killing both adult and larval p. argentipes under controlled settings ( inglenoff et al . there is no known resistance by sand flies to fipronil and the tactic of dosing cattle begins to address outdoor transmission of vl . however , ftis would still rely on proper usage by individuals and like itns , would carry similar benefits and risks of proper use . similar to irs , ftis will result in the potential for some human , animal , and environmental exposure . although , reported residue levels of fipronil in milk is below international standards ( poch et al . work would need to be done to monitor for any adverse effects to humans , animals , and the environment , following long - term use of ftis to treat cattle . should ftis prove effective under field conditions , they would begin to fill the gap in available treatments that target sand flies outside of homes and cattle enclosures . similar to fti and irs is the use of natural botanicals as insecticides in place of synthetic chemicals . one study demonstrated that a 2% concentration of neem oil mixed with either coconut or mustard oil was effective at repelling sand flies from human subjects in india ( sharma and dhiman 1993 ) . while no work has been done regarding the use of potential biochemicals as insecticides in india , studies conducted in other countries on different sand fly species showed that various plant - derived compounds were effective at killing adult and larval stages of the insects ( dinesh et al . although many of the tested biochemicals only had a 50% mortality rate , this area of research is still relatively unexplored and may be a useful alternative to synthetic insecticides . with millions of homes that would require intervention , vector - control programs need to balance rapid efficacy with long - term cost to ensure that if a treatment measure is terminated the country does not experience the rapid resurgence as occurred at the end of ddt irs in the 1970s . one year of irs costs on average , $ 5.90 per household which is more than llins ( $ 4.50/house / year ) but less than evm ( $ 8.70/house / year ) , and those costs have likely only gone up since that report was conducted ( das et al . there is a real need for thorough cost - effectiveness studies using combinations of control measures and disease prevalence / sand fly population scenarios . research documenting the direct and indirect costs , estimated expenditures for effective training and supervision of staff , as well as equipment maintenance would help to make informed decisions on the best use of resources for this endeavor . moreover , modelling has been done to estimate what percent of the sand fly population would need to be culled in order for vl to be eliminated . in that report , if sand fly life expectancy was reduced ( eg via irs , itns , or ftis ) , there would need to be a 67% decline in abundance in order for vl to be eliminated ( stauch et al . only the fti report and some of the irs studies meet that threshold ( chowdhury et al . 2013 ) . further focusing of efforts and resources could be done by implementing remote sensing and gis data . preliminary work done in brazil and india modeling both climate and land data has been used to try and identify vector habitat as well as regions that may be the focus of vl outbreaks due to various weather and geo - environmental factors ( bhunia et al . while standardization of analysis methods and data acquisition are still needed , gis and remote sensing could greatly help to target high - risk areas before an outbreak occurs . vector - control and vl - transmission studies should begin to focus on combination intervention strategies as well as enhanced public education programs . if the public are unaware of the risk , the symptoms , and treatments for vl , control programs will struggle ( singh et al . 2010 , malaviya et al 2013 ) . from what is known at this point , irs is efficacious in quickly curbing adult sand fly populations , itns may have some benefit for personal protection and might also be useful if irs is not performed correctly 100% of the time . lastly , fti is deleterious to both adult and larval p. argentipes and has the potential to disrupt populations of exophilic sand flies . what has been lacking outside of the one study by picado et al . ( 2010b ) , are studies that attempt to link a decline in sand fly abundance with a subsequent drop in cases of vl . a broader review of 84 studies in 22 countries involving all forms of human leishmaniasis found that only 35% measured leishmania infection as an outcome ( stockdale and newton 2013 ) . this is an issue afflicting many vector / disease - control studies ( wilson et al . future studies should concentrate their efforts on making this connection . while a decline in sand fly numbers should in theory correlate to a decline in vl infections , this link has yet to be demonstrated clearly by the research performed to date . future work investigating the connection between reduced sand fly abundance and vl infection rates as well as shifts in parasite availability and sand fly feeding behaviors should be a priority . in combination , these tactics may work to bring sand fly numbers down quickly in the short - term , allowing for vl - patient identification and treatment . currently , the state of bihar is beginning tests for use of synthetic pyrethroids in place of ddt for irs . the national roadmap laid out by the government of india emphasizes case detection and treatment as well as surveillance for pkdl . while they state the need for integrated vector management , irs remains the mainstay for vector control ( nvbdp 2014 ) . ongoing work with sand fly breeding site identification , and implementation of more novel technologies like gis and remote sensing the global initiative to eliminate vl in all endemic regions has been strong , however a coordinated effort between groups employing the various control tactics will be vital to see the elimination goal met . once caseloads have been brought to 1:10,000 , the use of irs , itns , ftis and all of the aforementioned technologies and trainings should be evaluated to determine which would be best for continued control measures .
visceral leishmaniasis is a deadly parasitic disease that is transmitted via the bite of a female sand fly , phlebotomus argentipes . the highest burden of this disease is in northern india . in 2005 , india embarked on an initiative with nepal , bangladesh , and the world health organization to eliminate visceral leishmaniasis by 2015 . with the goal of 1 case in 10,000 people still unmet , it is prudent to evaluate the tools that have been used thus far to reduce vector numbers and cases of the disease . herein , we present a review of studies conducted on vector - control strategies in india to combat visceral leishmaniasis including indoor residual spraying , insecticide - treated bed nets , environmental modification , and feed - through insecticides . this review suggests that the quality of indoor residual spraying may enhance control measures while a combination of spraying , nets , and feed - through insecticides would best confront the diverse habitats of p. argentipes .
Introduction Control Strategies Indoor Residual Spraying Bed Nets Environmental Modification Feed-Through Insecticides Conclusion
leishmaniasis is a parasitic disease that can manifest in three forms : 1 ) mucosal , 2 ) cutaneous , and 3 ) visceral ( who 2010 , maroli et al . as this review focuses on elimination efforts in india , we will only refer to l. donovani in this article . globally , the annual incidence rate is approximately 200,000400,000 cases , the majority of cases are present in bangladesh , nepal , and india . transmission of l. donovani occurs via the bite of a female phlebotomus argentipes sand fly . visceral leishmaniasis is a poverty - associated disease linked to poor housing and sanitary conditions and malnutrition , these factors have led to a number of difficulties regarding treatment and elimination ( cerf et al . as a by - product of ddt spraying for malaria elimination programs in the 1950s and 1970s , however , when ddt spraying ended , sand fly numbers and cases of vl rose again , leading to several major epidemics ( kishore et al . in 2005 , the governments of bangladesh , nepal , and india began a concerted vl elimination effort , with a target goal of 2015 for elimination ( 1 case in 10,000 people ) . indoor residual spraying ( irs ) and insecticide - treated bed nets ( itn ) have been the main forms of control regimens tried in india , while environmental modification ( evm ) and feed - through insecticides ( fti ) have been less studied . india in combination with the following keywords : phlebotomus argentipes , sand fly , kala - azar , and/or visceral leishmaniasis . sand fly populations declined in kind , and with them , cases of vl . another matter that is infrequently addressed in studies regarding irs is the health effects to humans , animals , and the environment as a result of frequent and long - term irs . bed nets , in particular , itns and long - lasting insecticide nets ( llins ) , have been suggested as alternatives and/or complements to irs for the control of sand fly populations and vl . investigated the usefulness of irs , llins , and evm to mitigate sand fly density in india , nepal , and bangladesh . while the number of studies has been limited , the efficacy of bed nets in india to combat vl have not shown the same promise as they have in bangladesh ( chowdhury et al . the same well - controlled study comparing the efficacy of irs and itns in india , nepal , and bangladesh found a 42% decline in sand fly abundance five months after the walls of homes and cattle enclosures were plastered with a mud and lime mixture . sand fly populations declined in kind , and with them , cases of vl . bed nets , in particular , itns and long - lasting insecticide nets ( llins ) , have been suggested as alternatives and/or complements to irs for the control of sand fly populations and vl . investigated the usefulness of irs , llins , and evm to mitigate sand fly density in india , nepal , and bangladesh . while the number of studies has been limited , the efficacy of bed nets in india to combat vl have not shown the same promise as they have in bangladesh ( chowdhury et al . the same well - controlled study comparing the efficacy of irs and itns in india , nepal , and bangladesh found a 42% decline in sand fly abundance five months after the walls of homes and cattle enclosures were plastered with a mud and lime mixture . for larval p. argentipes , the mean - time to death when fed on feces from cattle 1 or 3 days post - treatment was 4.5 days , larva fed on feces with the lowest dose ( 0.5 mg / kg ) on day 1 post - treatment had 100% mortality within 5.5 days while the highest dose ( 4.0 mg / kg ) was 4.0 days . however , if pkdl patients serve as a source of l. donovani , vector - control programs will also be needed to reach the goal of elimination . taking into account the diverse feeding and living habits of p. argentipes , it is likely that a combination of control measures will be necessary to eliminate vl in india . switching insecticides may be beneficial with mounting evidence of increased resistance of p. argentipes to ddt coupled with potential health effects on humans , animals , and the environment ( blus 2003 , van den berg 2009 , sparling 2010 , huda et al . while no work has been done regarding the use of potential biochemicals as insecticides in india , studies conducted in other countries on different sand fly species showed that various plant - derived compounds were effective at killing adult and larval stages of the insects ( dinesh et al .
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the hunt for highly potent new drug molecules , often targeting receptors with lipophilic molecular requirements , has resulted in a general trend of increasing number of highly lipophilic , poorly water - soluble drugs . between 75 and 90% of all new drug molecules have a solubility which is too low to allow complete dissolution of the dose in the intestinal fluid after oral administration . therefore , the bioavailability of the drug is compromised due to low and erratic absorption . in the gastrointestinal tract lipophilic drug molecules are solubilized in mixed lipid aggregates in the intestinal fluids . in the fasted state , these are composed of bile salts and phospholipids . for lipophilic drug molecules , the interaction of the drug with the bile salts and phospholipids can have a significant effect on the partitioning into these structures and , hence , impact the solubilization and , consequently , the bioavailability . the composition of the intestinal fluid shows high interindividual variability in the fasted state , and the large variation in bile secretion and composition adds another unpredictable element to drug delivery . unpredictable bioavailability can be critical when the drug has a limited therapeutic window ; i.e. , a low dose may be ineffective while a high dose results in toxicity . the two primary components in simulated human intestinal fluid are the bile salt taurocholate ( tch ) and the phospholipid phosphatidylcholine 1,2-dilinoleoyl - sn - glycero-3-phosphocholine ( dlipc ) . phospholipids have polar headgroups and long ( nonpolar ) acyl chains ( figure 1 ) . they therefore tend to self - assemble into ordered and extended bilayered membranes and micelles in water to minimize the exposure of their nonpolar groups to water . bile salts are amphiphilic surfactants that easily self - assemble into small micelles in water solutions at millimolar concentrations , with the capacity to solubilize both polar and nonpolar compounds . bile salts originate from cholesterol in the liver and contain a hydrophilic side chain attached to a large , rigid , and boat - shaped tetracyclic ring system . the ring system is characterized by a hydrophilic and a hydrophobic face , where the overall amphiphilic properties stem from the hydrophilic hydroxyl and hydrophobic methyl groups located on different sides of the ring system ( see figure 1 ) . united - atom molecular dynamics snapshots and lewis structures illustrating the deprotonated bile salt taurocholate ( tch , top ) and the phospholipid 1,2-dilinoleoyl - sn - glycero-3-phosphocholine ( dlipc , bottom ) . note the stereochemistry of the tch hydroxyl groups . experimental characterization and drug dissolution studies show that mixtures of dlipc and tch produce different mixed lipid aggregates that may increase the solubility of certain drugs by several tenfold . the conditions of formation of these mixed lipid aggregates and their colloidal structure ( micellar , uni - multilamellar vesicles ) are hence of fundamental importance for the solubilization of drug molecules in the intestine . the aggregate size and the type of the structures formed in the three - component systems of water / bile salt / phospholipid differ significantly compared to two - component systems ( water / bile or water / phospholipid ) that typically form micelles or vesicles . in this study , we used md simulations to study drug partitioning into colloidal structures typically found in fasted intestinal fluid . as the first step , we investigated spontaneous aggregation of mixed tch and dlipc systems and simulated a range of systems with different water / tch / dlipc ratios on the united - atom ( ua ) level , starting from either random or ordered bilayer structures . then , to quantify and further investigate the mixed lipid aggregates , we performed simulations of embedded tch clusters in dlipc bilayers using free energy calculations via umbrella sampling ( us ) simulations . finally , we investigated the free energy barriers of the interaction of selected model drug compounds with representative dlipc bilayer membranes , either without or in the presence of embedded tch at a tch : dlipc ratio of 1:2 , using us simulations on the all - atom level . initially , several different attempts to probe the tendency for tch and dlipc molecules to spontaneously aggregate into ordered bilayers were performed on the united - atom level using the berger / gromos54a7 force fields by simulating systems having random initial configurations and different tch and dlipc compositions and sizes . these initial simulations were performed in view of the findings by marrink et al . , who showed that several phospholipids ( 1,2-dipalmitoyl - sn - glycero-3-phosphocholine ( dppc ) , 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) , 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc ) , and 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine ( dope ) ) spontaneously aggregate into an equilibrated bilayer phase in water within a few tens of nanoseconds , using random starting configurations . in contrast to the reported systems and simulations , only a handful of the simulated dlipc and mixed dlipc / tch systems performed in this study from random starting configurations yielded well - defined lipid aggregates after more than 100 ns of simulations . as for dlipc , this can be explained by the polyunsaturated acyl chains yielding a low gel to liquid phase transition temperature ( tm ) of 57 c . for comparison , this is approximately 100 c lower than for the saturated 16- and 18-carbon analogues , dppc and dspc ( tm = 41 and 55 c , respectively ) . with this in mind , it appeared that the full equilibration of the self - assembly between mixed dlipc / tch systems may require much longer time scales than those used in this study and that by marrink et al . in line with previous studies , however , the obtained lipid structures were found to be highly dependent on the properties of the periodic boundary conditions ( pbc ) and pressure control . for instance , 64 dlipc molecules ( with or without 16 tch molecules saturated with 55 water molecules per lipid ) did form a bilayered phase when semi - isotropic pressure control was used . however , cylindrical micelles were formed with isotropic pressure control . it is interesting to note that both types of structures can be found experimentally and are known to coexist under certain conditions . given that fully equilibrated self - assemblies of the dlipc / tch lipid systems typically appeared to be outside the time scale of our simulations and likely subjected to pbc artifacts , the stability of preformed bilayer structures of dlipc with tch molecules were also investigated for six systems having 128 dlipc molecules and tch : dlipc ratios of 0:1 , 1:4 , 1:2 , 1:1 , 2:1 , and 4:1 , respectively , containing 55 water molecules per lipid ( i.e. , fully water saturated conditions ) . the resulting bilayer structures displayed remarkably high stability on the time scale of the simulations . interestingly , even at a 1:1 ratio , the dlipc sustained a seemingly normal bilayer structure with the polar headgroups extending toward the aqueous phase , well beyond the tch polar side chain . this was further confirmed by the similar positions of the dlipc main functional groups ( choline , phosphate , glycerol ) in the system with a 1:2 tch : dlipc ratio ( figure 2 , center ) as compared with the pure dlipc system ( not shown ) . as for tch ( figure 2 , bottom ) , most of the sterol groups are buried deep into the dlipc acyl chains in the bilayer interior , and the polar side chains face the aqueous phase , although not beyond the dlipc polar headgroups . interestingly , with increasing tch concentration in the simulated systems , increasing amounts of water was also found within the bilayer interior , increasing from 0 wt % to 0.2 , 1 , 1.7 , 3.3 , and 12 wt % with increasing tch : dlipc ratio . top : density profiles of mixed tch / dlipc united - atom systems containing 0512 tch ( green ) and 128 dlipc ( black ) molecules at ratios of 0:1 , 1:4 ; 1:2 , 1:1 , 2:1 , and 4:1 tch to dlipc hydrated with 55 water molecules per lipid . dashed lines show the pure dlipc system . middle and bottom : density profiles of the 32:128 tch : dlipc system , displaying the relative positions of the molecular moieties of dlipc ( middle ) and tch ( bottom ) . for clarity , add the following at the end : ( abbreviations in the middle panel refer to the nitrogen group ( n grp ) , the phosphate group ( p grp ) and the glycerol linker ( glyc grp ) . the apparent stability of the mixed tch : dlipc bilayers under the given conditions might be due to the ability of the tch molecules to organize into transmembrane clusters in the normal direction within the dlipc bilayers ( figure 3 ) . at low tch : dlipc ratios , most tch molecules were found slightly below the dlipc headgroups ; the nonpolar groups faced the bilayer interior , and the polar groups interacted with the dlipc headgroups and water molecules . however , with increasing tch concentrations within the bilayer , the number of transmembrane clusters increased , extending throughout the bilayer in the normal direction . this behavior can be explained by the amphiphilic nature of tch , which maximizes the hydrophilic and hydrophobic interactions of the polar and nonpolar groups simultaneously . left : snapshot of a mixed tch : dlipc united - atom bilayer system . the equilibrated structure from a bilayered system contains 32 deprotonated tch and 128 dlipc molecules . water molecules are omitted for clarity ; charge - balancing na ions are shown in blue . right : an isolated 3/4 tch cluster having three and four tch molecules in the top and bottom bilayer leaflet , respectively . the molecules are stabilized by hydrogen bonds between the tch molecules within the same bilayer leaflet and between tch molecules in the opposing leaflet . note the single water molecule in the upper tch cluster ; it penetrates deep into the bilayer interior due to the interaction with the buried hydrogen donors and acceptors of the tch . pdb files with these structures are available as supporting information . to study the stability of the transmembrane tch clusters as a function of number of tch molecules per cluster , a set of simulations were performed with systems of 64 dlipc lipids containing a single isolated tch cluster . one to four tch molecules were kept in the upper leaflet and four in the lower one . the tch transmembrane clusters in systems with 1 and 2 tch in the upper leaflet were not stable during the timespan of the simulation , and the cluster disaggregated . these systems had a lower probability of transmembrane hydrogen bonding , in contrast to the systems with 3 or 4 tch upper leaflet tch molecules ; hence , the tch clusters were not stabilized . with only 1 or 2 tch molecules in the upper leaflet , the initial hydrogen bonds with the tch in the opposing bilayer leaflet were disrupted , and the tch molecules positioned themselves adjacent to and below the dlipc headgroups after a few tens of nanoseconds . however , the systems with 3 or 4 tch in the upper leaflet remained virtually unchanged even after 200 ns , indicating superior stability over the smaller clusters . hydrogen bond analysis ( with the maximum bond length and angle between the hydrogen bond donor and acceptor being 0.35 nm and 30 ) further revealed a slightly larger number of h - bonds per tch molecule between both tch / tch and tch / dlipc for clusters with 3 tch compared to 4 tch molecules , indicating greater stability of the former type ( figure 4 ) . hydrogen bond participation per united - atom tch in transmembrane clusters containing 3 ( blue ) and 4 tch ( red ) molecules per bilayer leaflet . the number of inter - tch hydrogen bonds ( approximately 3.5 ) is highest in clusters with 3 tch molecules ; in general , this number of tch molecules also forms more hydrogen bonds with its surroundings . the tch clusters with 3 tch molecules in the same bilayer leaflet were often slightly tilted relative the normal direction of the bilayer , with the polar oh groups in the sterol moiety facing each other . the clusters with 4 tch in the same bilayer leaflet typically had one peripheral and partly excluded tch , or one to two tchs slightly twisted around their principal axis , exposing oh groups toward the dlipc acyl chains . analysis of the external surface area of the entire tch transmembrane clusters with a 0.14 nm probe ( i.e. , the equivalent of the water molecule radii ) was also performed on the hydrophobic and hydrophilic parts of those clusters with atom charges less or greater than 0.2 . this analysis revealed that the 3 tch clusters had an overall 15% larger hydrophobic area and 4% larger hydrophilic area per molecule than the clusters with 4 tch . however , analysis of the external surface area of the sterol moieties only ( without the polar side chain ) resulted in a 12% increase and 10% decrease in the hydrophobic and hydrophilic areas , respectively . this shows that the sterol groups of the 3 tch cluster display a more optimal packing geometry when embedded in the dlipc bilayer than the corresponding 4 tch cluster , by exposing more hydrophobic and less hydrophilic atoms toward the lipid bilayer interior . spontaneous formation of the transmembrane tch clusters was also investigated , i.e. , tch cluster formation independent of the possible artifacts introduced by using ordered starting configurations . for this , we performed simulations using a pre - equilibrated micelle composed of 8 tch embedded in the center of 64 dlipc bilayer . upon initial equilibration , predominately pairs of tch molecules interacting with their hydrophilic groups formed within 12 ns , with some orienting the headgroup toward the center of the bilayer . however , during the production run ( 500 ns ) , a 3 + 3 transmembrane tch cluster formed within a few nanoseconds with the two residual tch molecules positioned adjacent to the dlipc headgroups on each side of the bilayer . this result further indicated that tch transmembrane clusters could form naturally in bilayers of dlipc . to further investigate the tch and dlipc interactions in mixed lipid bilayers , the potential of mean force ( pmf ) for the tch molecule perpendicular the bilayer ( z - direction ) was determined using us simulations followed by the weighted histogram analysis . the overall pmf profile also reveals the free energy barriers along the chosen reaction coordinate . figure 5 shows the pmf profiles for tch molecules in different transmembrane clusters over half the bilayer . for a tch molecule not interacting with any other tch molecules ( denoted 1/0 in figure 5 ) , the pmf profile is even positive ( + 8 kj / mol ) in the center of the bilayer . this clearly shows that the interior of the bilayer is an unfavorable environment for the tch alone . however , with 4 tch molecules in the opposing bilayer leaflet ( denoted as 1/4 in the figure ) , the corresponding value decreased to 68 kj / mol , indicating a gain in stability by formation of a transmembrane cluster . it is also notable that the minimum of the pmf profile ( the optimal position of the molecule com ) of the 1/4 system was more shifted toward the bilayer center and away from the dlipc headgroups than the 1/0 system . with additional tch molecules present in the same bilayer leaflet ( i.e. , systems with 2/4 and 3/4 tch molecules in the upper and lower bilayer leaflets , respectively ) , there were even deeper pmf profiles , demonstrating a greater overall stability of the tch clusters . for clusters with up to 3 tch molecules in the upper bilayer leaflet , the minimum of each pmf curve shifted away from the bilayer center due to the increasing interactions with adjacent tch . for the same systems , this trend was accompanied by an increasing free energy penetration barrier , gpen ( defined analogously to eq 1 ) , as indicated in figure 5 . however , the same trend was broken upon additional tch , suggesting that clusters with > 3 tch are less rigid due to the small energy gain achieved by adding an additional tch to the otherwise stable and rigid 3 tch - molecule clusters . top : simulation snapshot showing the system setup for the us simulations with black arrows indicating the approximate position of the on average 40 umbrella windows used . middle : density profiles of dlipc and the tch cluster , with five separate pmf curves for tch along the z - direction , normal to the dlipc bilayer . note the shift in the position of the pmf minimum with a changing number of tch molecules . bottom : density of dlipc and tch and the pmf profiles for a 3/4 tch cluster without and with additional ( xtch ) of surface tch . to better represent physiological conditions where free tch molecules are also present , an additional system was created by adding 64 tch to the aqueous bulk phase . after equilibration , the aqueous phase tch mostly accumulated at the dlipc / water interface , which drastically changed the pmf profile of the probe tch molecule ( figure 5 , bottom ) . nevertheless , the minimum value and position of the pmf profile strongly resembled systems without bulk phase tch . hence , tch was energetically favored to interact with the transmembrane tch clusters in the lipid bilayer rather than interacting with the tch - rich water phase in contact with the dlipc bilayer . bile salts and phospholipids such as tch and dlipc solubilize drugs and alter lipid membrane properties . therefore , additional us simulations were performed to investigate the interactions of drug molecules with the mixed lipid bilayers of dlipc and tch ( figure 6 ) . the simulations were performed at the all - atom level using the slipids and gaff force fields , as recommended by a recent benchmarking study , and to better account for specific small molecule lipid interactions . because of the computational expense of all - atom us simulations for three - component systems , the simulations were limited to five different molecules : water , ethanol , carbamazepine , felodipine , and danazol ( figure 7 ) . these molecules were chosen to cover a wide range of lipophilicity , and the resulting log p values , as obtained from eq 1 , are summarized in table 1 . density profiles of a mixed lipid bilayer containing 48 dlipc and 24 tch molecules . experimental log poct . left : molecular structures of ethanol ( top left ) , carbamazepine ( top right ) , felodipine ( middle ) , and danazol . right : symmetrized pmf profiles of water ( red ) , ethanol ( green ) , carbamazepine ( blue , denoted car ) , felodipine ( purple , denoted fel ) , and danazol ( black , denoted dan ) over half an all - atom bilayer ( centered at z = 0 ) containing only dlipc or ( dotted line ) mixed tch / dlipc ( solid line ) . for water , there was a clear decrease in the otherwise positive pmf profile in the mixed tch / dlipc system compared to the pure dlipc bilayer when investigating deep partitioning into the membrane ( > 1 nm ) . inspection of the trajectories revealed in line with the results in figure 4 and the united - atom simulations occasional penetration of water molecules into the transmembrane tch clusters , facilitated by hydrogen bonding with the tch sterol hydroxyl groups . the overall free energy difference ( glipid / water ) of water in the mixed tch / dlipc and the dlipc reference was 18.6 2.3 and 27.6 0.6 kj / mol , respectively , well in line with previous studies . this demonstrates that the water partitioning of a dlipc bilayer increases by a factor of 30 when tch is present ( calculated from glipid / water and eq 1 ) . the pmf profiles over the mixed tch / dlipc and pure dlipc bilayer revealed only small differences in partitioning of the drug molecules and ethanol ( table 1 ) . however , intricate details governing the molecular interactions between the different compounds and tch / dlipc were found by interaction energy analysis ( separating out electrostatic and van der waals interactions ) . for the lipophilic compound danazol ( log poct of 4.53 ) , the corresponding log plipid / water value was higher in the mixed tch / dlipc and the pure dlipc systems , respectively , than that observed for octanol . the increased partitioning of danazol in the mixed tch / dlipc bilayer and the shift of the minimum value in the pmf profile toward the center of the bilayer are explainable by the van der waals interactions with the embedded tch molecules ; these were much stronger than the electrostatic interactions . this was further indicated by the higher total density of atoms in the mixed bilayer than in the pure bilayer ( figure 6 ) . despite these findings , the simulated trajectories did not point to greater extent of binding between danazol and tch as compared to danazol and dlipc . for felodipine ( log poct of 5.58 ) , the corresponding log plipid / water values were lower in the mixed tch / dlipc than the pure dlipc system . the decrease in partitioning of felodipine in the mixed tch / dlipc bilayer resulted from both weaker van der waals and electrostatic interactions with dlipc . unlike in the danazol system , these were not compensated for by the interactions with the embedded tch . figure s1 of the supporting information shows the overlap of the umbrella sampling windows , as well as the effect of the symmetrization procedure on the resulting pmf . the differences in the pmf profiles for the tch / dlipc and pure dlipc bilayers were smaller for the more hydrophilic carbamazepine and ethanol ( log poct values of 2.32 and 0.30 , respectively ) . an inspection of the respective trajectories identified that these molecules interacted with the tch oh groups in the center region , which was highly accessible due to low particle density in the bilayer center . although the differences in the pmf profiles were small , the embedded tch may accelerate drug diffusion over the lipid bilayer by lowering the gpen . the resulting log plipid / water for carbamazepine was slightly lower in the mixed tch / dlipc compared to the pure dlipc system , which was equivalent to the reported log poct value . the corresponding log plipid / water values for ethanol were , on the other hand , slightly higher in the mixed tch / dlipc compared to the pure dlipc system ; i.e. , there was a slightly increased partitioning of ethanol when tch was embedded . the md simulations strongly suggest that in mixtures of tch and dlipc , reflecting the composition of fasted state simulated intestinal fluid , embedded transmembrane tch clusters constitute an integral component in phospholipid bilayers composed of dlipc . phospholipid micelle and bilayer systems by revealing the preferred number and configuration of bile salt molecules embedded in the phospholipid bilayers . both unconstrained and us free energy united - atom simulations showed that the transmembrane tch clusters were ( i ) stabilized by mutual hydrogen bonds ( oh3oh3 ) across the bilayer center and ( ii ) preferably consisted of 3 or 4 ( 3 + 1 ) adjacent and clustered tch molecules per bilayer leaflet . in the latter type , the additional tch constituted a more peripheral member to the overall cluster . from simulations on the all - atom level , the effect of embedded tch in dlipc bilayers on the partitioning of five model substances ( water , ethanol , carbamazepine , felodipine , and danazol ) showed that only water displayed a positive free energy profile over the entire bilayer regardless of embedded tch . for water this positive free energy barrier was however reduced in the presence of tch due to penetration into the transmembrane clusters . for the other hydrophilic solutes ethanol and carbamazepine the overall lipid / water partitioning into the lipid bilayer was similar to and without embedded tch . the effect of embedded tch on the overall lipid / water partitioning ( i.e. , from glipid / water ) was most significant for danazol , demonstrating that the embedded tch may facilitate partitioning into , and hence , solubilization of lipophilic solutes in the mixed bilayer . however , this was not observed for the other highly lipophilic drug felodipine , which displayed a slightly reduced log plipid / water in the presence of embedded tch . the gromacs suite of programs was used for all simulations . unless otherwise stated , all lipid simulations were conducted at a physiological nacl concentration of approximately 0.15 m. for the united - atom simulations ( spc / gromos54a7/berger ) and the all - atom simulations ( tip3p / gaff / slipids ) we used a universal short - range cutoff at 1.2 nm for both electrostatic and lennard - jones interactions . long - range electrostatic interactions were treated by particle mesh ewald ( pme ) . all covalent bond lengths were constrained using lincs , and a time step of 2 fs was used in all production simulations . simulation systems of dlipc with and without tch were constructed to have either random starting configurations or ordered bilayers . the latter type was obtained by initially distributing the lipid monomers on a square grid and pre - equilibrating bilayered systems for > 50 ns while weakly restraining the lipid movements in the direction normal to the bilayer . to model the mixed lipid systems at the united - atom level , berger force field parameters for dlipc were adapted from a dopc topology , by enforcing a cis - conformation of the double bonds in the acyl chains . for tch , the topology and all interaction parameters were obtained from the automated topology builder server using the gromos54a7 force field . however , the partial charges were calculated from restricted electrostatic potential fitting using the pyred server as opls compatible charges ( resp - o1 ) , i.e. , by gaussian09 calculations at the hf/6 - 31 g * level followed by resp . similarly , a slipids compatible topology for dlipc was adapted from a dopc topology . this was done by removal of two hydrogen atoms and by enforcing a cis - conformation of the two neighboring double bonds and by increasing the partial charges of the ch2 atoms in the adjacent methylene group by + 0.03 ( to + 0.06 ) to maintain charge neutrality . for the all - atom tch and drug molecules , the general amber force field ( gaff ) was used , having molecular topologies constructed by antechamber software through the acpype.py script ( available online : http://code.google.com/p/acpype/ ) applied to molecular coordinates obtained from the zinc database . all regular and unconstrained md productions runs were typically preceded by ( i ) energy minimization using the steepest descent algorithm with a tolerance of 1000 kj mol nm , followed by ( ii ) a 50 ps simulation in the canonical ( nvt ) ensemble using position restraints on all solute particles , followed by ( iii ) a 5 ns volume equilibration simulation in the isothermal the latter simulations used the berendsen barostat for semi - isotropic pressure control , whereas all production runs used the parrinello the modified berendsen and velocity - rescaled thermostats were used for equilibration and production runs , coupling the non - water and water molecules to separate thermostats . by analogy , removal of center of mass ( com ) translation was applied to the lipids and water molecules independently ; for bilayer simulations , the com of the upper and lower bilayer leaflets were controlled separately . pure dlipc and mixed tch : dlipc us systems with either a random or an ordered bilayered initial configuration were simulated for a minimum of 100 ns , with either 64 , 128 , or 256 dlipc molecules and with a tch : dlipc ratio from 0 to 4:1 . to ensure full water saturation , the systems contained a minimum of 30 water molecules per lipid , in contrast to 21.5 , the reported experimental value for dlipc . in the following studies we focused on free energy calculations making use of the bilayers since it is well - known that unilamellar vesicles are naturally present in fasted intestinal fluids . free energy profiles for tch molecules over the bilayers , i.e. , the potential of mean force ( pmf ) , were computed from us simulations . to generate the starting configurations for the sampling , a single tch molecule was pulled to the aqueous bulk phase from the center of a representative tch cluster embedded in a bilayer consisting of 64 dlipc molecules . the systems contained 04 adjacent tch molecules within the same bilayer leaflet and 4 tch in the opposing bilayer leaflet . the total number of lipids in each leaflet was set constant by removal of dlipc molecules from the opposing bilayer leaflet . each starting structure was equilibrated with 90 water molecules per lipid , after which a tch molecule was pulled by a harmonic force at a rate of 0.00025 nm / ps with the gromacs pull code . for the us simulations , more than 40 configurations were chosen from an initial set of 2500 . in these 40 configurations , the probed tch molecule was displaced approximately 0.15 nm along the reaction coordinate , i.e. , in the normal direction ( z - axis ) to the bilayer . each configuration was then simulated for 5 ns with a harmonic force constant of 800 kj mol nm . to ensure proper overlap of the sampled umbrella windows , each umbrella simulation was performed twice with different starting configurations . from the resulting potential of mean force ( pmf ) profiles an equivalent bilayer / water partitioning ( log plipid / water ) was calculated as1where glipid / water is the free energy of transfer from the aqueous phase to the lipid bilayer , expressed as the difference in the free energy of solubilization , gwater glipid , i.e. , analogous to calculations of the traditional octanol and water partition coefficient log poct / water . the free energy profiles over the bilayers for the small molecules ( danazol , felodipine , carbamazepine , water , and ethanol ) were similarly computed from us simulations , this time using all - atom systems . to evaluate the effect of tch in the mixed lipid bilayers , umbrella simulations the mixed lipid bilayer was constructed with 24 tch and 48 dlipc molecules , whereas 64 dlipc molecules were used in the systems without tch . similarly to the tch - only us simulations , representative configurations were chosen with a spacing of 0.15 nm along the pull vector . each chosen configuration was simulated for 20 ns using a harmonic force constant of 700 kj mol nm . unlike the tch us simulations , however , 2 ( or 4 for ethanol ) equally spaced probed molecules were pulled simultaneously through the bilayer , i.e. , sampling both sides of the bilayer leaflet at the same time . this was possible because of the small size and neutral charge of these compounds . to avoid overlap of the inserted molecules , a slow growth protocol over 50 consecutive simulation steps was applied , after which the complete drug / solvent / tch / dlipc structure was energy minimized and equilibrated . because of this , a lower pull rate of 0.000 05 nm / ps was used to avoid structural artifacts in the bilayer caused by the pulled drug molecules . to obtain the final pmf along the reaction coordinate , i.e. , the free energy profiles , all us simulations were analyzed with the weighted histogram analysis method as implemented in the gromacs g_wham utility . standard deviations were computed from bootstrapping over 200 runs using a tolerance of 1 10 . in the mixed tch / dlipc system , the pmf with calculated standard deviations for water was obtained from the averaged boltzmann - weighted density profiles of water in the drug molecule simulations , i.e. , from unconstrained conditions and a total simulation time of approximately 2 s .
we performed molecular dynamics ( md ) simulations to obtain insights into the structure and molecular interactions of colloidal structures present in fasted state intestinal fluid . drug partitioning and interaction were studied with a mixed system of the bile salt taurocholate ( tch ) and 1,2-dilinoleoyl - sn - glycero-3-phosphocholine ( dlipc ) . spontaneous aggregation of tch and dlipc from unconstrained md simulations at the united - atom level using the berger / gromos54a7 force fields demonstrated that intermolecular hydrogen bonding between tch molecules was an important factor in determining the overall tch and dlipc configuration . in bilayered systems , these intermolecular hydrogen bonds resulted in embedded transmembrane tch clusters . free energy simulations using the umbrella sampling technique revealed that the stability of these transmembrane tch clusters was superior when they consisted of 3 or 4 tch per bilayer leaflet . all - atom simulations using the slipids / gaff force fields showed that the tch embedded in the bilayer decreased the energy barrier to penetrate the bilayer ( gpen ) for water , ethanol , and carbamazepine , but not for the more lipophilic felodipine and danazol . this suggests that diffusion of hydrophilic to moderately lipophilic molecules through the bilayer is facilitated by the embedded tch molecules . however , the effect of embedded tch on the overall lipid / water partitioning was significant for danazol , indicating that the incorporation of tch plays a crucial role for the partitioning of lipophilic solutes into e.g. lipidic vesicles existing in fasted state intestinal fluids . to conclude , the md simulations revealed important intermolecular interactions in lipidic bilayers , both between the bile components themselves and with the drug molecules .
Introduction Results and Discussion Conclusions Methods
the two primary components in simulated human intestinal fluid are the bile salt taurocholate ( tch ) and the phospholipid phosphatidylcholine 1,2-dilinoleoyl - sn - glycero-3-phosphocholine ( dlipc ) . united - atom molecular dynamics snapshots and lewis structures illustrating the deprotonated bile salt taurocholate ( tch , top ) and the phospholipid 1,2-dilinoleoyl - sn - glycero-3-phosphocholine ( dlipc , bottom ) . as the first step , we investigated spontaneous aggregation of mixed tch and dlipc systems and simulated a range of systems with different water / tch / dlipc ratios on the united - atom ( ua ) level , starting from either random or ordered bilayer structures . finally , we investigated the free energy barriers of the interaction of selected model drug compounds with representative dlipc bilayer membranes , either without or in the presence of embedded tch at a tch : dlipc ratio of 1:2 , using us simulations on the all - atom level . initially , several different attempts to probe the tendency for tch and dlipc molecules to spontaneously aggregate into ordered bilayers were performed on the united - atom level using the berger / gromos54a7 force fields by simulating systems having random initial configurations and different tch and dlipc compositions and sizes . to further investigate the tch and dlipc interactions in mixed lipid bilayers , the potential of mean force ( pmf ) for the tch molecule perpendicular the bilayer ( z - direction ) was determined using us simulations followed by the weighted histogram analysis . the simulations were performed at the all - atom level using the slipids and gaff force fields , as recommended by a recent benchmarking study , and to better account for specific small molecule lipid interactions . because of the computational expense of all - atom us simulations for three - component systems , the simulations were limited to five different molecules : water , ethanol , carbamazepine , felodipine , and danazol ( figure 7 ) . inspection of the trajectories revealed in line with the results in figure 4 and the united - atom simulations occasional penetration of water molecules into the transmembrane tch clusters , facilitated by hydrogen bonding with the tch sterol hydroxyl groups . the increased partitioning of danazol in the mixed tch / dlipc bilayer and the shift of the minimum value in the pmf profile toward the center of the bilayer are explainable by the van der waals interactions with the embedded tch molecules ; these were much stronger than the electrostatic interactions . the md simulations strongly suggest that in mixtures of tch and dlipc , reflecting the composition of fasted state simulated intestinal fluid , embedded transmembrane tch clusters constitute an integral component in phospholipid bilayers composed of dlipc . both unconstrained and us free energy united - atom simulations showed that the transmembrane tch clusters were ( i ) stabilized by mutual hydrogen bonds ( oh3oh3 ) across the bilayer center and ( ii ) preferably consisted of 3 or 4 ( 3 + 1 ) adjacent and clustered tch molecules per bilayer leaflet . from simulations on the all - atom level , the effect of embedded tch in dlipc bilayers on the partitioning of five model substances ( water , ethanol , carbamazepine , felodipine , and danazol ) showed that only water displayed a positive free energy profile over the entire bilayer regardless of embedded tch . for the other hydrophilic solutes ethanol and carbamazepine the overall lipid / water partitioning into the lipid bilayer was similar to and without embedded tch . the effect of embedded tch on the overall lipid / water partitioning ( i.e. , from glipid / water ) was most significant for danazol , demonstrating that the embedded tch may facilitate partitioning into , and hence , solubilization of lipophilic solutes in the mixed bilayer . the free energy profiles over the bilayers for the small molecules ( danazol , felodipine , carbamazepine , water , and ethanol ) were similarly computed from us simulations , this time using all - atom systems .
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graphene electrochemistry has gained paramount interest owing to its unique role in energy conversion and storage devices such as fuel cells , water splitting cells , and supercapacitors . one of the most extensively studied electrocatalytic applications of graphene is the oxygen reduction reaction ( orr ) occurring at the cathode of fuel cells and metal - air batteries , in which advanced metal - free graphene - based electrocatalysts are considered as promising alternatives to the state - of - the - art precious pt catalysts due to their low cost , fuel tolerance , and long - term durability . tremendous efforts have been undertaken , both on the experimental and theoretical level , to improve the orr performance of graphene - based materials by tuning their electronic properties through doping nonmetallic heteroatoms into a graphene matrix . however , the key orr activity properties ( e.g. , exchange current density , on - set potential , 4e pathway selectivity , and kinetic current density ) of metal - free materials are still incomparable with those of pt - based catalysts , which stimulates the ongoing debate on whether graphene is indeed an efficient catalyst for orr . this issue is unresolved , largely due to the lack of knowledge on the origin of graphene activity toward orr and the effect of the doping of nonmetallic heteroatoms on the graphene s ( electro)chemical properties . in other words , understanding the nature of the orr process on the surface of doped graphene and recognizing the origin of its electrocatalytic activity open a new era in the design of graphene - based materials with superior electrocatalytic activity toward orr . in general , monitoring the reaction intermediates formed on the surface of catalysts represents a possible platform for understanding the pathway and mechanism of orr electrocatalysis ; however , it is difficult to observe in situ these adsorbed species due to their extremely short lifetimes . importantly , the development of computational quantum chemistry provides a feasible methodology to predict the possible intermediates formed during the orr process and to evaluate their stabilities on the surface of catalysts in terms of the adsorption free energies . for example , a theoretical methodology has been developed for studying orr on a wide variety of metal catalyst surfaces , and the free energy diagram for this reaction was constructed together with a volcano - shaped plot that correlates the apparent electrocatalytic orr activity with inherent oxygen adsorption strength . consequently , various pt and nonprecious metal alloys with superior experimentally measured orr activities comparable to that of a pure pt catalyst were designed and developed guided by the prediction of this theoretical methodology . however , such advanced methodology has not been applied to metal - free catalysts such as the most popular graphene - based catalysts ; whether these metal - free counterparts can possess similar catalytic behavior or be even more active than metallic electrocatalysts is unknown from both theoretical and experimental viewpoints . here , we extend for the first time the aforementioned methodology used for metal catalysts to metal - free systems by exploring the relationship between the experimentally measured electrocatalytic orr performance and the theoretically predicted free energy of reaction intermediates for a series of graphenes doped with nonmetal elements . the starting point of this methodology is the construction of the orr free energy diagrams for various doped graphene models by density functional theory ( dft ) calculations . next , the orr exchange current density , the on - set potential , and the reaction pathway selectivity are theoretically predicted on the basis of the free energy diagrams for each model catalyst . also , all these descriptors of the orr activity are obtained on the basis of the electrochemical voltammograms measured for the synthesized catalysts that correspond to the models studied . the origin of the oxygen reduction activity of the graphene - based catalysts studied is explained in - depth in terms of the molecular orbital theory . by combining experimental and theoretical data , it was possible to predict for the first time the electrocatalytic orr performance of an ideal graphene - based catalyst ( x - graphene ) which possesses a 2.1 10 a / cm orr exchange current density , a 0.33 v on - set potential ( vs normal hydrogen electrode , nhe ) , and a nearly 100% 4e pathway selectivity ; these values are comparable with or even better than those of the state - of - the - art pt catalyst . this combined computational and experimental study reveals the nature of orr electrocatalysis for graphene - based materials and paves the way to the molecular design of more highly efficient metal - free electrocatalysts for applications beyond orr . to study the effect of different dopants on the orr activity of graphene , we designed and synthesized nitrogen ( n)- , boron ( b)- , oxygen ( o)- , sulfur ( s)- , and phosphorus ( p)-doped graphenes , respectively , as well as undoped graphene . all graphene - based samples were synthesized from chemically exfoliated graphene oxide ( go ) prepared by a slightly modified hummer s method from graphite . after dialysis for one week , the graphite oxide dispersion was diluted , exfoliated , and centrifuged with the resulting go concentration of 0.5 mg / ml . graphite powder was considered as a representative of pure graphene ( g ) without any heteroatom doping . oxygen - doped graphene ( o - graphene ) , which can be considered as reduced go , was synthesized by thermally reducing initial go powder at 900 c in flowing ar for 3 h without any additional precursor . due to the strong oxidation process preceding chemical exfoliation of graphite oxide , the latter possesses many oxygen - containing functional groups . during high - temperature thermal reduction ( 900 c in ar atmosphere ) , most of the oxygen species are removed ( oxygen concentration decreases from 50 atomic % in pristine go to 5 atomic % in o - graphene ) ; only four relatively stable species are retained . nitrogen- , boron- , sulfur- , phosphorus - doped graphenes ( n - graphene , b - graphene , s - graphene , p - graphene ) were produced by annealing go powder with various heteroatom - containing precursors ( the mass ratio of the initial go to the respective precursor = 1:10 ) at 900 c in flowing ar for 3 h. melamine ( c3h6n6 ) , boron oxide ( b2o3 ) , benzyl disulfide ( c6h5ch2ssch2c6h5 ) , and triphenylphosphine ( ( c6h5)3p ) were used as the sources of n , b , s and p elements , respectively . the x - ray photoelectron spectra ( xps ) were measured on a kratos axis ultra x - ray photoelectron spectrometer equipped with a 165 mm hemispherical electron energy analyzer . the incident radiation was monochromatic al k x - rays ( 1486.6 ev ) at 225 w ( 15 kv , 15 ma ) . the survey scans were taken at the analyzer pass energy of 160 ev , and the multiple high - resolution scans were recorded at 20 ev . transmission electron microscope ( tem ) imaging was conducted on a fei tecnai g2 spirit tem at a voltage of 120 kv . nitrogen - sorption isotherms were collected on a tristar ii , micromeritics nitrogen sorption analyzer at 77 k. prior to each measurement , the samples were degassed at 150 c for at least 10 h. the brunauer teller ( bet ) specific surface area was calculated using adsorption data at a relative pressure range of p / p0 = 0.050.25 ( figure s1 , supporting information [ si ] ) . raman spectra were recorded on a horiba labram with 514.3 nm ar laser ( figure s2 , si ) . all electrochemical measurements were performed using the same mass of catalyst ( 0.2 mg / cm ) . linear sweep voltammograms ( lsv ) were recorded using a glassy carbon rotating disk electrode ( rde , 0.196 cm , pine research instrumentation , u.s.a . ) with a scan rate of 5 mv / s or a rotating ring - disk electrode ( rrde , 0.283 cm , pine research instrumentation , u.s.a . ) with a scan rate of 2 mv / s . the data were recorded using a chi 760 d potentiostat ( ch instruments , inc . the reference electrode was an ag / agcl in 4 m kcl solution ( all potentials were referenced to nhe by adding a value of 0.205 v ) and the counterelectrode was platinum wire . the kinetic current for orr occurring on the electrode can be calculated from the intercept of koutecky levich plot using the following equation ( figures s3 and s4 , si):1where jk is the kinetic current density at a constant potential , jd is the measured current density on rde , is the electrode rotating speed in rpm , and b , the reciprocal of the slope , could be determined from the slope of koutecky n is the number of electrons transferred per oxygen molecule , f is the faraday constant , do2 is the diffusion coefficient of o2 in 0.1 m koh , v is the kinetic viscosity , and co2 is the bulk concentration of o2 . the constant 0.2 is adopted when the rotating speed is expressed in rpm . the overall electron transfer numbers per oxygen molecule involved in a typical orr process were determined on the basis of rrde voltammograms recorded by using a rrde configuration with a 320 m gap pt ring electrode . the disk electrode was scanned cathodically at a rate of 2 mv / s , and the ring potential was constant at + 0.5 v for oxidizing any ooh intermediate . the electron transfer number ( n ) and ooh intermediate production percentage ( % ooh , which serves as 2e pathway selectivity ) were determined as follows ( figure s5 , si):34where i d is the disk current , ir is the ring current , and n is the current collection efficiency of the pt ring , which was determined to be 0.37 . the tafel analysis of the orr polarization was evaluated by using the relation between the kinetic current density ( jk ) and overpotential ( = unhe 0.455 ) as:5where a ( v ) is the tafel constant related to the exchange current density ( j0 ) and b ( v / dec ) is the tafel slope . a more complete version of this equation can be derived by simplifying the butler volmer equation as:6where r is the ideal gas constant , is the transfer coefficient , n is the number of electrons transferred , and j0 is the exchange current density . combination of eq 6 with eq 5 gives the following expression for calculation of the experimental j0 ( table s1 , si):7 the electrocatalytic active sites and the pathways of orr on the surface of various electrocatalysts were examined on the basis of hybrid dft calculations performed by using gaussian 09 program . all the calculations were carried out using ub3lyp/6 - 31g(d , p ) level of theory with all atoms fully relaxed . the solvent ( water ) effect was considered by the polarizable continuum model ( pcm ) . only intermediate states in the orr process , as well as the reactant and product states , were proposed and evaluated ; extra energy barriers might exist but were not considered due to the unbalanced electron numbers borne by different states . the calculation of the free energy diagrams was performed by setting up the reference level as that of the reaction product ( table s2 , si ) , and the reference electrode was set up to be the nhe ; at ph = 0 and 0 v vs nhe , reaction ( h + e ) 1/2 h2 is at the equilibrium under standard conditions . the free energies of reactants and each intermediate state at an applied electrode potential u were calculated as follows : g(u ) = g neu , where n is the electron number of such state and g is the free energy obtained by frequency calculations at room temperature ( 298.15 k ) after geometry optimization . hence , the equilibrium potential u for orr ( eq 8) at ph = 13 was determined to be 0.455 v vs nhe where the reactant and product are at the same energy level . the free energy of h2o(l ) was derived as gh2o(l ) = gh2o(g ) + rt ln(p / p0 ) since only gh2o(g ) can be directly obtained by dft calculations , where r is the ideal gas constant , t = 298.15k , p = 0.035 bar , and p0 = 1 bar . the free energy of o2(g ) was derived as go2(g ) = 2gh2o(l ) 2gh2 4.92 ev since the high - spin ground state of oxygen molecule is notoriously poorly described in dft calculations . the free energy of oh was derived as goh = gh2o(l ) gh+ , where gh+ = 1/2gh2 kbtln 10 ph ( kb is boltzmann s constant ) . the overall reaction scheme of o2 reduction to oh in alkaline environment is:8with three possible reaction pathways ( one dissociative and two associative ) as demonstrated in scheme s1 , si . specifically , since the surface of a doped graphene features the relatively high energy barrier ( > 1.2 ev ) in the dissociative pathway ( figure s7 , si ) , the following associative mechanism is dominant and considered in our calculations:9a9b9c9dwhere * refers to a given atom in the specific graphene model ( i.e. , possible active site ) . calculation of the current density at low overpotentials includes the following assumptions : ( 1 ) the amount of active sites on different graphene surfaces is the same ; ( 2 ) the rate constant represents the upper bound of the overall reaction rate ; if there are additional barriers to ooh * formation / oh * desorption , the overall reaction rate should be lower . according to reference ( 36 ) , the exchange current density for a certain electrocatalytic process can be theoretically calculated as follows:10where n is the electron transfer number , f is faraday s constant , k is the standard rate constant , ctotal is the total number of active sites , is the transfer coefficient ( a measure of the symmetry of the potential energy surface , ranging from 0 to 1 ) , and is a quantity related to the highest free energy change of the whole reaction ( see si eqs s59 , figure s8 for a detailed derivation of this equation and parameter fitting ) . we selected five nonmetallic elements ( b , n , p , o , s ) with various electron negativities to dope graphenes to obtain single - doped graphene materials . all the doped graphene electrocatalysts were chemically prepared from go by using appropriate doping procedures ( see the methods section ) . in all five resultant samples ( b - graphene , n - graphene , p - graphene , o - graphene , s - graphene ) the nanosheet morphology of the pristine go was well preserved without noticeable residues of the solid precursors , as shown in the transmission electron microscopy ( tem ) images ( figure 1 ) . note that the incorporation of different heteroatoms could not change the physicochemical properties of doped graphene samples such as morphology , surface area , and defects . nitrogen - adsorption studies indicate that all samples show similar surface areas in a range between 100 and 150 m / g , which also assures similar concentrations of the orr active sites present on the samples studied ( considering that the concentrations of all doped heteroatoms are also similar , between 3 to 5 atomic % based on the xps survey results ; not shown here ) . the raman results indicate that all samples have similar concentrations of defects with the id / ig values in a range of 1.01.2 , which are closely related to the electrical conductivity of these samples . therefore , it is presumed that the differences in the electrocatalytic orr activity of various doped graphene samples do not originate from their physicochemical properties but only from the nature of the dopant affecting their orr activities . the nature of doping was investigated by analyzing high - resolution xps spectra shown in figure 2 . peak deconvolutions were conducted as in the case of the reported respective single - doped graphenes . the five heteroatoms chemically substituted edge or central carbon atoms in the graphene matrix , yielding 13 different species in- or out - of the graphene basal plane . specifically , natural graphite was considered as a defect - free ( nondoped ) graphene with an intensive typical sp - hybridized carbon ( figure 2a ) . b - graphene has two boron containing species as central b-3c and edge b-2c o species ( excluding b2o3 precursor residue ) ( figure 2b ) . n - graphene has three nitrogen species as central graphitic nitrogen and edge pyridinic and pyrrolic n in the graphene plane ( figure 2c ) . o - graphene has two kinds of oxygen species as in - plane central pyran - type oxygen , edge carbonyl and hydroxyl oxygens , and out - of - plane epoxy oxygen ( figure 2d ) . p - graphene and s - graphene both have one heteroatom doping configuration as p3c(o)-type phosphorus ( excluding ph3p precursor residue ) ( figure 2e ) and edge c s c sulfur ( figure 2f ) , respectively . the inset values represent the id / ig ratios and the specific surface areas obtained from raman spectra and nitrogen adsorption isotherms , respectively for each sample ( see figures s1 and s2 , si for details ) . high - resolution xps spectra of different heteroatoms in doped graphenes : ( a ) graphite ; ( b ) b - graphene ; ( c ) n - graphene ; ( d ) o - graphene ; ( e ) p - graphene ; ( f ) s - graphene . we constructed the cluster models ( figure s6 , si ) for the aforementioned doped graphenes according to each heteroatom s chemical environments obtained from the xps spectra discussed above . h bonds , which have been previously adopted for investigation of orr on graphene doped with nitrogen or boron . five heteroatoms could induce 13 different doping configurations in graphene clusters with very different electronic properties ; concomitantly , 32 possible orr active sites , either heteroatoms themselves or adjacent carbon atoms , were theoretically studied to build the orr free energy diagram for each catalyst by obtaining the free energy for each reaction step . according to eq 9 , the associative 4e reaction pathways for each doped graphene at the equilibrium potential u = 0.455 v vs nhe can be illustrated on a free energy diagram as presented in figure 3a . from such diagrams , nitrogen- and boron - doped graphene models ( gn - g and gb - g ) exhibit the lowest overall reaction free energy change at u , suggesting their orr performance is the best from the theoretical viewpoint , which has been confirmed by previous reports and the experiments reported in this work ( figure s3 , si ) . taking the gn - g cluster model as an example ( black line ) , the diagram indicates that the first electron transfer step to form ooh * ( eq 9a ) is an endothermic reaction with a free energy change geq 9a(u ) = 0.70 ev , while the second electron transfer step ( eq 9b ) to form chemisorbed o * and the third electron transfer step ( eq 9c ) to form oh * are all exothermic with geq 9b(u ) = 0.25 ev and geq 9c(u ) = 0.54 ev , respectively . the last electron transfer step reflecting oh * desorption ( eq 9d ) possesses an easily surmountable free energy difference of geq 9d(u ) = 0.09 ev . therefore , geq 9a(u ) is the largest one among the free energy changes of all four reaction steps , which indicates that this step is the most sluggish one and represents the highest resistance for the whole orr . such trend is true for all other heteroatom - doped graphene models , indicating that the orr rate - determining step for these catalysts is the same . note that each graphene doped by one heteroatom can result in several different cluster configurations , which possess their own reaction pathways . the lines in figure 3a only represent the model with best performance ( i.e. , the lowest overall reaction free energy change ) among all the investigated cluster models for each dopant . the free energy diagrams for orr on all possible active sites of each model are presented in figures s9s14 , si . ( a ) free energy diagram of different heteroatom - doped graphenes at the equilibrium potential u. ( b ) the adsorption free energies of intermediates ooh * ( gooh * ) and oh * ( goh * ) on the investigated sites of different doped graphene models . blue points were not considered in the linear fitting because normal ooh * chemisorption did not occur on the corresponding graphene clusters . the specific values of gooh * and goh * for different models are provided in table s3 , si . ( c ) calculated free energy diagram of the predicted x - graphene at the equilibrium potential ; data for gn - g model are also included for the purpose of comparison . reaction intermediates for each step are shown as inset animation . according to the developed theories for metal surfaces , the orr electrocatalytic activity descriptors ( specifically , the exchange current density , on - set potential , 4e pathway selectivity , and kinetic current density ) of a given catalyst are governed by the adsorption free energies of orr intermediates including ooh * , o * , and oh * , which are represented by gooh * , go * , and goh * , respectively ( equation s1 , si ) . as shown in figure 3b , gooh * for a wide variety of the doped graphene surfaces studied scales roughly with goh * , similarly as in the case of metal surfaces . additionally , the o * chemisorption is more complicated than that of oh * and ooh * , since it could either form single bonding with the adsorption center on graphene or form epoxy - type bonding ; within each bonding type , a linear relationship is also observed ( figure s15 , si ) . on the basis of these linear relationships ( gooh * vs goh * or go * ) , we can predict the orr free energy diagram of an optimal x - graphene , which is the basis to obtain its corresponding electrocatalytic properties at the macroscopic level . the free energy diagram of x - graphene can be obtained on the basis of the sabatier principle that an ideal catalyst should bind the reaction intermediates not too strongly nor too weakly ; therefore , at equilibrium potential u , the optimal overall reaction pathway on an ideal x - graphene should follow the relationship of geq9a(u ) = geq9d(u ) . at the same time , geq9a(u ) and geq9d(u ) are associated to each other via the linear relationship of gooh * and goh * as shown in figure 3b ; therefore by obeying these requirements , geq9a(u ) for x - graphene is determined to be 0.35 ev , representing the largest free energy change on the overall pathway as shown in figure 3c ( red line , see eq s4 , si for free energy changes of other steps ) . this predicted free energy difference is lower than those for all other investigated doped graphene models ( for example , gn - g denoted by black line ) and is close to that calculated for pt by dft . ( a ) experimentally determined tafel plots for different catalysts from orr polarization curves shown in figure s3 , si , data were collected at rde = 1600 rpm . ( b ) volcano plot between j0 and gooh * with charge - transfer coefficient = 0.5 ( red dashed line ) . blue hollow squares are j0 obtained from tafel plots and dft - derived gooh * for each doped graphene catalyst . gooh * for x - graphene ( blue solid square ) was obtained from eq s4e , si and its j0 was obtained from eq 10 . the j0 value for pt was also shown by the blue dashed line as a reference . for a given catalyst j0 is defined as the current density at the equilibrium potential in one direction for a given reaction , which reflects the intrinsic catalytic activity of the catalyst . the measured exchange current densities , j0 , for each synthesized graphene surface can be obtained from the respective tafel plot as shown in figure 4a ( specific values could be found in table s1 , si ) . simultaneously , knowing the free energy diagram for each graphene model , the theoretical exchange current density , j0 , can be calculated by using a microkinetic model with one prefactor fitted from j0 by eq 10 . the predicted j0 values for various graphene models form a volcano - shaped plot versus gooh * ( figure 4b red line ) , while j0 for each synthesized sample perfectly follows the trend of this plot ( blue squares ) , similarly as in the case of metal surfaces . additionally , due to the weak binding of ooh * on graphene - based surfaces , the calculated points are all on the right branch of the volcano plot , while an optimal catalyst should possess a higher j0 induced by a gooh * closer to the volcano center . following such trends , the j0 value for an ideal x - graphene should be located at the summit of the volcano , with a calculated value of 2.12 10 a / cm , which is even 5 times higher than that of pt / c catalyst at same testing conditions ( figure s4c , si ) . due to the existence of a high free energy difference for the first electron transfer step geq ( 9a)(u ) as shown in figure 3a , the initialization of oxygen reduction requires a potential bias = u u to reduce the free energy difference to geq9a(u ) that could be overcame at room temperature . taking chemically synthesized n - graphene as an example , the measured on - set potential un = 0.029 v vs nhe ( from the polarization curve shown in figure 5a ) . from the theoretical viewpoint ( gn - g model ) , under this potential un , the free energies of the reactant and intermediates states ( figure 3c black line ) shift upward as compared to those under the equilibrium potential ( figure 5b red line ) ; as a result , geq9a(un ) reduces to an easily surmountable value of 0.26 ev . in the zone of u > un , geq9a(u ) keeps decreasing with increasing u , which indicates the first electron transfer step is no longer the rate - limiting step for the overall reaction , i.e. the whole orr process is activated. a similar trend was also observed for other graphene models as demonstrated in figures s9s14 , si . ( a ) enlarged lsvs plots at the orr initial region for different catalysts on rde at 1600 rpm in an o2-saturated 0.1 m solution of koh . inset illustrates the first electron transfer step that is o2 to adsorbed ooh*. ( b ) potential corrected free energy diagram for gn - g at experimentally observed on - set potential un ( red ) and theoretically predicted un and un which meet geq9a(un ) = 0.43 ev and geq9a(un ) = 0.22 ev , respectively ( blue ) . ( c ) experimentally derived on - set potentials of doped graphenes ( red squares ) , and the predicted values ( blue bars ) . theoretically , the on - set potential for a given graphene model can be thermodynamically predicted from its free energy diagram at equilibrium potential by eqs s23 , si . first , a range for geq9a is defined as [ 0.22 and 0.43 ] ev on the basis of classical reaction thermodynamics ( table s4 , si ) . it is assumed that , if geq9a at a given electrode potential possesses a value within this range , the free energy difference can be overcome . the obtained theoretical on - set potentials that satisfy 0.22 ev geq9a 0.43 ev as well as the experimentally determined ones for all chemically synthesized graphenes are shown in figure 5c . most of the experimentally obtained values ( red squares ) are in the theoretically predicted range ( blue bars ) , except for s - graphene and pure graphene samples ( slightly above the theoretical range ) , which could be attributed to the influence of nafion and glassy carbon working electrodes on the overall current density . furthermore , based on the same criterion , the predicted on - set potential for x - graphene that corresponds to geq9a = 0.43 ev was 0.33 v , which is closer to the orr equilibrium potential than the experimental value obtained for pt catalyst . theoretically , as demonstrated in scheme 1 in si , orr can proceed either by two sequential two - electron reactions ( 2e ) with formation of ooh intermediate or a more efficient direct four - electron reaction ( 4e ) . therefore understanding the nature of the 2e pathway is essential to design more appropriate catalysts for orr that possesses high 4e pathway selectivity to enhance the electrocatalytic efficiency . in alkaline solution , the mechanism of 2e pathway is:11a11b according to previous studies , 2e and 4e pathway selectivity is associated with the desorption of ooh * and oh * on a metal surface , respectively ; therefore , at a given potential u , the probability of 2e reaction pathway depends on the value of geq 11b(u ) in comparison to geq 9d(u ) . taking gn - g model as an example ( figure 6a ) , at equilibrium electrode potential for 2e pathway u2e ( 0.08 v vs nhe at ph = 13 ) , the desorption of ooh * ( eq 11b ) is exothermal with an energy difference of geq 11b(u2e ) = 0.16 ev , which means 2e pathway is unavoidable to compensate the 4e pathway on gn - g model . experimentally , we used the rotating ring - disk electrode ( rrde ) technique to verify this theoretically derived pathway selectivity prediction by monitoring the formation of intermediate peroxide species ( e.g. , ooh in the alkaline solution ) under different electrode potential as shown in figure 6b . the measured electron - transfer number at u2e was 3.35 for chemically synthesized n - graphene ; it corresponds to a mixed 33% 2e pathway selectivity and 67% 4e pathway selectivity as shown in figure 6c . also note that at more negative electrode potentials , 2e pathway is always unavoidable due to the negative geq 11b , which is consistent with the rrde observation ( figure 6c ) . additionally , the same property ( mixed 2e and 4e pathways for orr ) has been observed for all other chemically synthesized graphene catalysts . to fundamentally eliminate the 2e pathway , a catalyst surface that binds ooh * strongly enough to induce an endothermic ooh * desorption process this criterion is met by the ideal x - graphene model , as shown by its free energy diagram at u2e in figure 6a ( red line ) : at the equilibrium electrode potential for 2e pathway u2e , ooh * desorption on x - graphene is endothermic while oh * desorption is exothermic , which therefore avoids the 2e reduction pathway to present a nearly 100% 4e pathway selectivity , similar as in the case of pt catalyst . jk is the cathodic oxygen reduction current under the kinetic limitation zone when the reactant mass transfer is efficient enough to keep the concentration of o2 at the electrode surface equal to the bulk value , which represents the orr kinetic electrochemical property at a given electrode potential ( always more negative than orr on - set potential ) . experimentally , for n - graphene ( figures 7a , b ) and other chemically synthesized graphenes ( figure s3 , si ) , jk was measured by rotating disk electrode ( rde ) voltammogram with different rotating speeds followed by performing koutecky levich plot as presented in eqs 1 and 2 ( see the methods section ) . ( a ) free energy diagrams of 2e orr pathway for gn - g model ( black ) and x - graphene ( red ) at equilibrium electrode potential for 2e pathway u2e = 0.08 v vs nhe . under this potential , the rate - limiting step of orr on gn - g is the activation of o2 to ooh * , the atomic configuration of which is shown in figure 5a inset , whereas that for x - graphene is the reduction of ooh * to ooh , which atomic configuration is shown in the inset of this figure . ( b ) experimental ring and disk currents for the synthesized n - graphene catalyst on rrde at 1600 rpm in an o2-saturated 0.1 m solution of koh . ( c ) rrde measured electron transfer numbers ( black ) and corresponding 2e pathway selectivity ( blue ) for n - graphene catalyst according to eqs 3 and 4 ( a ) electrochemically measured lsv of n - graphene catalyst at different rotating speeds in an o2-saturated 0.1 m solution of koh . inset illustrates the last electron transfer step : oh * desorption to generate oh . ( b ) koutecky levich plots for n - graphene at 0.3 v , 0.2 v , and 0.1 v vs nhe , data were collected from panel a. ( c ) potential corrected free energy diagram for gn - g and x - graphene models at u = 0 v ( u = 0.77 v ) . ( d ) the relationship between goh * and jk for various synthesized graphene catalysts under different potentials ( open symbols ) , data were collected from figure s3 , si . the values in parentheses of the legend are jk values for x - graphene , which are predicted by extending the fitted lines to goh * = 0.10 ev , shown as closed symbols . from the theoretical perspective , the above electrochemical measured jk values can be related to the free energy change of the last electron transfer step ( goh * ) by a linear relationship shown in figure 7d . the microkinetic scenario of this relationship might be due to the fact that the last step in the reaction pathway , oh * desorption as shown in the inset of figure 7a , now becomes the rate - determining step of the whole orr as shown in figure 7c . on the basis of this trend , the predicted jk for x - graphene can be obtained from the value of the extended fitted lines at goh * = 0.10 ev ( obtained by eq s4 , si ) , as shown in figure 7d closed symbols , which is 6.01 ma / cm at 0.3 v , comparable to pt catalyst s value ( 7.35 ma / cm at 0.3 v , calculated from pt s lsv plots in figure s4c , si ) . it should be noted that these predicted jk values for x - graphene were obtained on the basis of a mixture of 2e and 4e orr pathways ( since the experimentally observed jk for line fitting all contain 2e pathway as shown in figure 6c and figure s5 , si ) , which could be further enhanced if only 4e pathway exists like in the case of pt catalyst . on the basis of the former theoretical and experimental observations of j0 , u , 4e pathway selectivity , and jk for various graphene - based catalysts , we found that all these electrochemical quantities relate well to the binding strength of intrinsic oxygen - containing intermediates ( adsorbed species ) on the catalyst surface . inspired by the success of the d - band center theory that the energy level of a metal atom s d - band center serves as the activity descriptor for metal surfaces , we searched for a simple activity descriptor suitable for metal - free catalysts that could accomplish a similar correlation between the binding strength and each orr active atom s molecular valence orbital levels . we first investigated the origin of the binding strength for different graphene cluster models via natural bond order ( nbo ) analysis to explore the orbital information of each active site . since the valence orbital of each active center participates in the bond formation with an oxygen - containing intermediate on the graphene surface ( e.g. , oh * ) , the valence orbital level should greatly influence its adsorption energy goh*. therefore , we introduced a descriptor ediff which is defined as the difference between lowest valence orbital energy of the active center and the highest valence orbital energy of the entire graphene cluster ( fermi energy level in the form of natural atomic orbitals ) to quantitively represent the valence orbital level . as shown in figure 8a , goh * data plotted against ediff formed a linear relationship for a wide variety of graphene active sites . the principle that underlies this linear relationship is that the valence band ( v ) of the active sites hybridizes with the bonding ( ) orbital of the adsorbed species to form bonding ( v- ) and antibonding ( v- ) * states , as illustrated in figure 8b . for the investigated graphene models , the ( v- ) state is full , while the filling of ( v- ) * state depends on the valence orbital levels of the active atom on the graphene surface . an increased filling of the antibonding ( v- ) * state , induced by a lower valence band , could lead to destabilization of the graphene adsorbate interaction and hence diminish the binding between them ; on the other hand , a decreased filling of ( v- ) * state corresponds to an enhanced binding between orr intermediates and the graphene surface . as a result , a better graphene - based orr catalyst such as x - graphene should possess higher valence orbital energies of the orr active atom to induce a smaller ediff and lead to stronger adsorption of ooh * and oh * intermediates , as marked in figure 8a , resulting in better orr activity . such an x - graphene could in principle be realized by doping with multiple elements , introducing structural defects , or any possible combination . ( a ) the relationship between goh * and ediff ; data were collected for the most active site of various doped graphene models and labeled according to the corresponding molecular configurations shown in figure s6 , si . ( b ) scheme of orbital hybridization of valence band from active sites and adsorbates bonding orbital . in summary , by combining experimental data and dft calculations , we systematically investigated the nature and origin of orr activity of a series of heteroatom - doped graphene catalysts . although the stability of each model , the electric double layer effect , and hydrogen bonding were not considered in the quantum chemistry study , our models are in good accordance with the experimental observations , from the viewpoint of the orr exchange current density , on - set potential , pathway selectivity , and the kinetic current density . this agreement also validates the predictive capability of the powerful dft model employed beyond traditional metallic catalysts . our study shows further that graphene - based metal - free catalysts possess the potential to surpass the orr performance of the state - of - the - art pt catalyst . using orr as a probe reaction , the explored methodology should be applicable to other energy - related electrocatalysis processes such as oxygen evolution and hydrogen evolution reactions .
the mutually corroborated electrochemical measurements and density functional theory ( dft ) calculations were used to uncover the origin of electrocatalytic activity of graphene - based electrocatalysts for oxygen reduction reaction ( orr ) . a series of graphenes doped with nonmetal elements was designed and synthesized , and their orr performance was evaluated in terms of four electrochemical descriptors : exchange current density , on - set potential , reaction pathway selectivity and kinetic current density . it is shown that these descriptors are in good agreement with dft calculations , allowing derivation of a volcano plot between the orr activity and the adsorption free energy of intermediates on metal - free materials , similarly as in the case of metallic catalysts . the molecular orbital concept was used to justify this volcano plot , and to theoretically predict the orr performance of an ideal graphene - based catalyst , the orr activity of which is comparable to the state - of - the - art pt catalyst . moreover , this study may stimulate the development of metal - free electrocatalysts for other key energy conversion processes including hydrogen evolution and oxygen evolution reactions and largely expand the spectrum of catalysts for energy - related electrocatalysis reactions .
Introduction Methods Results and Discussion Conclusions
one of the most extensively studied electrocatalytic applications of graphene is the oxygen reduction reaction ( orr ) occurring at the cathode of fuel cells and metal - air batteries , in which advanced metal - free graphene - based electrocatalysts are considered as promising alternatives to the state - of - the - art precious pt catalysts due to their low cost , fuel tolerance , and long - term durability . , exchange current density , on - set potential , 4e pathway selectivity , and kinetic current density ) of metal - free materials are still incomparable with those of pt - based catalysts , which stimulates the ongoing debate on whether graphene is indeed an efficient catalyst for orr . in other words , understanding the nature of the orr process on the surface of doped graphene and recognizing the origin of its electrocatalytic activity open a new era in the design of graphene - based materials with superior electrocatalytic activity toward orr . importantly , the development of computational quantum chemistry provides a feasible methodology to predict the possible intermediates formed during the orr process and to evaluate their stabilities on the surface of catalysts in terms of the adsorption free energies . here , we extend for the first time the aforementioned methodology used for metal catalysts to metal - free systems by exploring the relationship between the experimentally measured electrocatalytic orr performance and the theoretically predicted free energy of reaction intermediates for a series of graphenes doped with nonmetal elements . the starting point of this methodology is the construction of the orr free energy diagrams for various doped graphene models by density functional theory ( dft ) calculations . next , the orr exchange current density , the on - set potential , and the reaction pathway selectivity are theoretically predicted on the basis of the free energy diagrams for each model catalyst . the origin of the oxygen reduction activity of the graphene - based catalysts studied is explained in - depth in terms of the molecular orbital theory . by combining experimental and theoretical data , it was possible to predict for the first time the electrocatalytic orr performance of an ideal graphene - based catalyst ( x - graphene ) which possesses a 2.1 10 a / cm orr exchange current density , a 0.33 v on - set potential ( vs normal hydrogen electrode , nhe ) , and a nearly 100% 4e pathway selectivity ; these values are comparable with or even better than those of the state - of - the - art pt catalyst . this combined computational and experimental study reveals the nature of orr electrocatalysis for graphene - based materials and paves the way to the molecular design of more highly efficient metal - free electrocatalysts for applications beyond orr . according to reference ( 36 ) , the exchange current density for a certain electrocatalytic process can be theoretically calculated as follows:10where n is the electron transfer number , f is faraday s constant , k is the standard rate constant , ctotal is the total number of active sites , is the transfer coefficient ( a measure of the symmetry of the potential energy surface , ranging from 0 to 1 ) , and is a quantity related to the highest free energy change of the whole reaction ( see si eqs s59 , figure s8 for a detailed derivation of this equation and parameter fitting ) . according to the developed theories for metal surfaces , the orr electrocatalytic activity descriptors ( specifically , the exchange current density , on - set potential , 4e pathway selectivity , and kinetic current density ) of a given catalyst are governed by the adsorption free energies of orr intermediates including ooh * , o * , and oh * , which are represented by gooh * , go * , and goh * , respectively ( equation s1 , si ) . furthermore , based on the same criterion , the predicted on - set potential for x - graphene that corresponds to geq9a = 0.43 ev was 0.33 v , which is closer to the orr equilibrium potential than the experimental value obtained for pt catalyst . although the stability of each model , the electric double layer effect , and hydrogen bonding were not considered in the quantum chemistry study , our models are in good accordance with the experimental observations , from the viewpoint of the orr exchange current density , on - set potential , pathway selectivity , and the kinetic current density . our study shows further that graphene - based metal - free catalysts possess the potential to surpass the orr performance of the state - of - the - art pt catalyst .
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epicardial fat directly surrounding the major epicardial coronary arteries is a rich source of free fatty acids and numerous bioactive adipocytokines that play important roles in the development of atherosclerosis . recently , due to advances in temporal and spatial resolution , multidetector computed tomography ( mdct ) has emerged as a noninvasive diagnostic modality that allows for simultaneous assessment of coronary artery calcium ( cac ) , coronary artery stenosis and coronary plaque , and epicardial fat volume ( efv ) without increased radiation exposure or cost . several studies using mdct have shown a relationship between efv and cac , the presence of coronary plaque , plaque composition , and plaque vulnerability . however , it is unknown whether elevated efv is associated with a greater likelihood of developing atherosclerosis over time , and whether increases in efv are accompanied by a parallel increase in coronary plaque volume . the relationship between serial changes in efv and coronary plaque volume has not yet been investigated ; therefore , the aim of the present study was to determine whether baseline indexed efv ( efvi ) and serial changes in efvi are related to increase in coronary plaque volume as assessed by mdct . we reviewed the cardiac computed tomography ( ct ) patient data set from april 2007 to december 2014 , retrospectively , and there were 237 patients who underwent mdct examination more than once . after excluding patients who underwent coronary artery bypass graft ( n = 48 ) and percutaneous coronary angioplasty ( n = 5 ) , we reviewed the mdct data of the remaining 184 patients . we also excluded patients ( n = 63 ) who had no coronary artery plaques detected by ct and those ( n = 34 ) who underwent cardiac ct using a different tube voltage . as a result , this study ultimately examined data of 87 patients . this study was approved by the ajou institutional review board , in which there was no need to take informed consent due to the retrospective design . the pretest probability of coronary artery disease ( cad ) was estimated for each patient based on age , sex , and symptoms . medical records were obtained by reviewing the electronic medical database , and cardiovascular ( cv ) risk factors were analyzed at the time of the cardiac ct examination . body mass index ( bmi ) was calculated in each patient , and a bmi value of 25 kg / m was deemed to be obese . systolic blood pressure of 140 mm hg , diastolic blood pressure of 90 mm hg , or current use of an antihypertensive agent was considered hypertension . a confirmed diagnosis of diabetes mellitus or current use of any antidiabetic medication was evaluated . finally , the 10-year risk of coronary heart disease ( chd ) was calculated using the framingham risk score . cardiac ct examinations were performed using a brilliance 64-slice ct scanner ( philips medical systems , eindhoven , the netherlands ) from january 2009 to february 2011 or a somatom definition flash dual - source 128-slice ct scanner ( siemens healthcare , forchheim , germany ) from march 2011 to april 2013 . using the 64-slice ct scanner , nonenhanced imaging was performed using 120 kv and 55 ma s with prospective electrocardiogram ( ecg ) triggering to calculate calcium score and volume . coronary computed tomography angiography ( ccta ) was performed with retrospective ecg gating using the following parameters : detector collimation of 64 0.625 mm , gantry rotation of 400 milliseconds , pitch of 0.2 , tube voltage of 120 kv , and an effective tube current of 600 to 900 ma with ecg modulation . contrast medium ( 80 ml , iomeron 400 ; bracco spa , milan , italy ) was administered intravenously , at a rate of 4.5 ml / s . using the dual - source 128-slice ct scanner , nonenhanced imaging was performed using 120 kv and 80 ma s with tube current modulation . a prospective ecg tube current modulation technique , based on the patient 's bmi , and the mindose protocol ( siemens healthcare ) were used . contrast medium was injected using a split - bolus technique : first , 60 to 80 ml of pure iodine - containing contrast material ( iomeron 400 ; bracco spa ) was administered intravenously at 4.5 ml / s , based on the patient 's body weight . then , 40 ml of a 60%-to-40% mixture of contrast medium and saline was administered . cardiac ct images were assessed by a single observer with 15 years of experience in cardiac ct . for the cac score , agatston calcium scores were calculated using semiautomated software ( ebw ; philips medical systems , best , the netherlands ) , which identified areas of at least 0.5 mm and a density 130 hu as calcification . the software also measured the corresponding calcified plaque volume . to evaluate the coronary arteries , images at 75% r if there were severe motion artifacts , additional images at other cardiac cycles were used to obtain a better - quality image . maximum intensity projection , volume rendering , multiplanar reformation , and curved multiplanar reformation were routinely constructed using a commercial workstation ( ebw , philips medical systems ) . according to the american heart association classification , a 15-segment model was used . if the ramus intermedius was present , segment 16 was also used . however , if there were motion artifacts that lowered the image quality , nonevaluable segments were excluded from the analysis . if an abnormal segment was identified , that coronary artery was evaluated using an aquarius workstation ( terarecon , san mateo , ca ) and the volume of noncalcified plaque was measured . referring to previous studies , noncalcified plaques were divided into 2 categories based on the ct value ( hu ) : low ( 049 hu ; deemed a lipid - rich plaque ) and intermediate attenuation compositions ( 50129 hu ; deemed a fibrous plaque ) . the plaques were color coded and the volume of each component was measured ( fig . the color - coded area was adjusted manually , including the full thickness of the vessel wall , and surrounding tissues were excluded . the 2 curved multiplanar reconstruction images of the baseline and follow - up ct were displayed in parallel and then identical segments were compared side by side using the aquarius workstation . the lesions were matched on baseline and follow - up images using adjacent anatomical landmarks . the ct values of the proximal segments of the right coronary artery , left anterior descending artery , and left circumflex artery were measured and the mean intracoronary lumen density ( hu ) was calculated . a 54-year - old male patient with low pretest probability underwent calcium score ct ( a ) and coronary ct angiography ( b ) . ( a ) the semiautomated software was used to identify an area of at least 0.5 mm with a density 130 hu as calcified plaque , and then to measure the coronary calcium score and volume . noncalcified plaque was color - coded according to ct value ( hu ) and classified into low attenuation plaque ( 049 hu ; designated as lipid - rich plaque ) and intermediate attenuation plaque ( 50129 hu ; designated as fibrous plaque ) . lipid - rich and fibrous plaque volumes of the patient were 13.13 and 31.59 mm , respectively . the observers were blinded to the patients clinical histories , cac scores , and ccta results . epicardial fat was defined as the adipose tissue between the surface of the myocardium and the visceral layer of the pericardium . the superior boundary of epicardial fat was set at the center of the right pulmonary artery , and the inferior extent was indicated by the end of the pericardial sac . efv was quantified by calculating the total volume of the tissue in which the ct density ranged from 190 to 30 hu within the epicardium . efv was reported in cubic centimeters and indexed ( efvi ) to body surface area . a 50-year - old male patient with low pretest probability . epicardial fat was defined as the adipose tissue between the surface of the myocardium and the visceral layer of the pericardium . ( a ) the border of the epicardium ( yellow line ) was traced semiautomatically . ( b ) efv was quantified by calculating the total volume of the tissue ( green color ) showing a ct density of 190 to 30 hu within the epicardium . ( c ) the computer software constructed a 3-dimensional image of the epicardial fat automatically , with the data reported in cubic centimeters . the efv of the patient was 119 cm , and that indexed to body surface area ( efvi ; 1.91 m ) was 62.3 m / m . ct = computed tomography , efv = epicardial fat volume , efvi = indexed epicardial fat volume . the excel 2010 software ( microsoft corp , redmond , wa ) was used for data collection . comparisons of data between baseline and follow - up were performed using the test for categorical data and the paired t test for continuous variables . relationships between clinical variables , efvi , and plaque volume were explored by regression analysis . annual changes in plaque volume and efvi were calculated for each plaque by subtracting the values measured at baseline ct from the values measured at follow - up . then , the difference value was divided by the time elapsed between the 2 ct scans . annual change values in the highest tertile for each plaque volume were considered to indicate rapid increases in plaque volume . logistic regression analysis was used to determine whether baseline clinical variables and efvi were predictors of rapid increase in plaque volume . variables that achieved significance in the univariate analysis were included in a stepwise logistic regression analysis . statistical analyses were performed using medcalc ( version 13.1.2.0 ; medcalc software , mariakerke , belgium ) . a p value < 0.05 was considered to indicate statistical significance . we reviewed the cardiac computed tomography ( ct ) patient data set from april 2007 to december 2014 , retrospectively , and there were 237 patients who underwent mdct examination more than once . after excluding patients who underwent coronary artery bypass graft ( n = 48 ) and percutaneous coronary angioplasty ( n = 5 ) , we reviewed the mdct data of the remaining 184 patients . we also excluded patients ( n = 63 ) who had no coronary artery plaques detected by ct and those ( n = 34 ) who underwent cardiac ct using a different tube voltage . as a result , this study ultimately examined data of 87 patients . this study was approved by the ajou institutional review board , in which there was no need to take informed consent due to the retrospective design . the pretest probability of coronary artery disease ( cad ) was estimated for each patient based on age , sex , and symptoms . medical records were obtained by reviewing the electronic medical database , and cardiovascular ( cv ) risk factors were analyzed at the time of the cardiac ct examination . body mass index ( bmi ) was calculated in each patient , and a bmi value of 25 kg / m was deemed to be obese . systolic blood pressure of 140 mm hg , diastolic blood pressure of 90 mm hg , or current use of an antihypertensive agent was considered hypertension . a confirmed diagnosis of diabetes mellitus or current use of any antidiabetic medication was evaluated . finally , the 10-year risk of coronary heart disease ( chd ) was calculated using the framingham risk score . cardiac ct examinations were performed using a brilliance 64-slice ct scanner ( philips medical systems , eindhoven , the netherlands ) from january 2009 to february 2011 or a somatom definition flash dual - source 128-slice ct scanner ( siemens healthcare , forchheim , germany ) from march 2011 to april 2013 . using the 64-slice ct scanner , nonenhanced imaging was performed using 120 kv and 55 ma s with prospective electrocardiogram ( ecg ) triggering to calculate calcium score and volume . coronary computed tomography angiography ( ccta ) was performed with retrospective ecg gating using the following parameters : detector collimation of 64 0.625 mm , gantry rotation of 400 milliseconds , pitch of 0.2 , tube voltage of 120 kv , and an effective tube current of 600 to 900 ma with ecg modulation . contrast medium ( 80 ml , iomeron 400 ; bracco spa , milan , italy ) was administered intravenously , at a rate of 4.5 ml / s . using the dual - source 128-slice ct scanner , nonenhanced imaging was performed using 120 kv and 80 ma s with tube current modulation . a prospective ecg tube current modulation technique , based on the patient 's bmi , and the mindose protocol ( siemens healthcare ) were used . contrast medium was injected using a split - bolus technique : first , 60 to 80 ml of pure iodine - containing contrast material ( iomeron 400 ; bracco spa ) was administered intravenously at 4.5 ml / s , based on the patient 's body weight . then , 40 ml of a 60%-to-40% mixture of contrast medium and saline was administered . cardiac ct images were assessed by a single observer with 15 years of experience in cardiac ct . for the cac score , agatston calcium scores were calculated using semiautomated software ( ebw ; philips medical systems , best , the netherlands ) , which identified areas of at least 0.5 mm and a density 130 hu as calcification . the software also measured the corresponding calcified plaque volume . to evaluate the coronary arteries , images at 75% r if there were severe motion artifacts , additional images at other cardiac cycles were used to obtain a better - quality image . maximum intensity projection , volume rendering , multiplanar reformation , and curved multiplanar reformation were routinely constructed using a commercial workstation ( ebw , philips medical systems ) . according to the american heart association classification , a 15-segment model was used . if the ramus intermedius was present , segment 16 coronary segments with a diameter > 2.0 mm were analyzed . however , if there were motion artifacts that lowered the image quality , nonevaluable segments were excluded from the analysis . if an abnormal segment was identified , that coronary artery was evaluated using an aquarius workstation ( terarecon , san mateo , ca ) and the volume of noncalcified plaque was measured . referring to previous studies , noncalcified plaques were divided into 2 categories based on the ct value ( hu ) : low ( 049 hu ; deemed a lipid - rich plaque ) and intermediate attenuation compositions ( 50129 hu ; deemed a fibrous plaque ) . the plaques were color coded and the volume of each component was measured ( fig . the color - coded area was adjusted manually , including the full thickness of the vessel wall , and surrounding tissues were excluded . the 2 curved multiplanar reconstruction images of the baseline and follow - up ct were displayed in parallel and then identical segments were compared side by side using the aquarius workstation . the lesions were matched on baseline and follow - up images using adjacent anatomical landmarks . the ct values of the proximal segments of the right coronary artery , left anterior descending artery , and left circumflex artery were measured and the mean intracoronary lumen density ( hu ) was calculated . a 54-year - old male patient with low pretest probability underwent calcium score ct ( a ) and coronary ct angiography ( b ) . ( a ) the semiautomated software was used to identify an area of at least 0.5 mm with a density 130 hu as calcified plaque , and then to measure the coronary calcium score and volume . noncalcified plaque was color - coded according to ct value ( hu ) and classified into low attenuation plaque ( 049 hu ; designated as lipid - rich plaque ) and intermediate attenuation plaque ( 50129 hu ; designated as fibrous plaque ) . lipid - rich and fibrous plaque volumes of the patient were 13.13 and 31.59 mm , respectively . the observers were blinded to the patients clinical histories , cac scores , and ccta results . epicardial fat was defined as the adipose tissue between the surface of the myocardium and the visceral layer of the pericardium . the superior boundary of epicardial fat was set at the center of the right pulmonary artery , and the inferior extent was indicated by the end of the pericardial sac . efv was quantified by calculating the total volume of the tissue in which the ct density ranged from 190 to 30 hu within the epicardium . efv was reported in cubic centimeters and indexed ( efvi ) to body surface area . a 50-year - old male patient with low pretest probability . epicardial fat was defined as the adipose tissue between the surface of the myocardium and the visceral layer of the pericardium . ( a ) the border of the epicardium ( yellow line ) was traced semiautomatically . ( b ) efv was quantified by calculating the total volume of the tissue ( green color ) showing a ct density of 190 to 30 hu within the epicardium . ( c ) the computer software constructed a 3-dimensional image of the epicardial fat automatically , with the data reported in cubic centimeters . the efv of the patient was 119 cm , and that indexed to body surface area ( efvi ; 1.91 m ) was 62.3 m / m . ct = computed tomography , efv = epicardial fat volume , efvi = indexed epicardial fat volume . the excel 2010 software ( microsoft corp , redmond , wa ) was used for data collection . comparisons of data between baseline and follow - up were performed using the test for categorical data and the paired t test for continuous variables . relationships between clinical variables , efvi , and plaque volume were explored by regression analysis . annual changes in plaque volume and efvi were calculated for each plaque by subtracting the values measured at baseline ct from the values measured at follow - up . then , the difference value was divided by the time elapsed between the 2 ct scans . annual change values in the highest tertile for each plaque volume were considered to indicate rapid increases in plaque volume . logistic regression analysis was used to determine whether baseline clinical variables and efvi were predictors of rapid increase in plaque volume . variables that achieved significance in the univariate analysis were included in a stepwise logistic regression analysis . statistical analyses were performed using medcalc ( version 13.1.2.0 ; medcalc software , mariakerke , belgium ) . a p value < 0.05 was considered to indicate statistical significance . the baseline patient characteristics are listed in table 1 . the mean age of the study population was 54.8 7.9 years at the baseline examination and 56.5 7.9 years at the follow - up examination . the mean interval between the baseline and the follow - up ct was 25.5 15.7 months . the icc for interobserver agreement was 0.975 ( 95% confidence interval : 0.962 , 0.984 ) for baseline efv and 0.970 ( 95% confidence interval : 0.954 , 0.980 ) for follow - up efv . both baseline and follow - up efvi were positively correlated with age and bmi ( table 2 ) . with the exception of lipid - rich plaque volume on follow - up ct , comparisons between the baseline and follow - up examination results are provided in table 3 . cac score and coronary plaque volumes increased significantly ( p = 0.010 to < 0.001 ) on follow - up ct . the mean annual change values were 4.1 26.8 mm / y for lipid - rich plaque , 5.9 26.8 mm / y for fibrous plaque , and 15.1 27.0 mm / y for calcified plaque volume . however , efv ( 116.0 37.5 vs 116.6 37.4 cm , p = 0.604 ) and efvi ( 65.7 21.8 vs 66.0 21.8 cm / m , p = 0.620 ) change values between baseline and follow - up ct were not significant . the mean intracoronary lumen density was not significantly different ( 424.5 58.1 vs 430.3 78.8 hu , p = 0.811 ) between baseline and follow - up ct examinations . the mean annual changes in efv and efvi were 0.8 8.0 cm / y and 0.5 4.8 cm / m / y , respectively . the annual change in efvi was not accompanied by a parallel change in coronary plaque volume ( p = 0.286 for lipid - rich plaque , 0.500 for fibrous plaque , and 0.096 for calcified plaque ) ( fig . the mean annual change values in plaque volume in the highest tertile were 32.4 17.9 mm / y for lipid - rich plaque , 32.6 18.5 mm / y for fibrous plaque , and 39.5 35.3 mm / y for calcified plaque ( table 4 ) . a 43-year - old male patient with an intermediate pretest probability underwent baseline cardiac ct ( a d ) . the baseline efv was 54.0 cm ( a , b ) , calcified plaque volume was 380.1 mm ( c ) , and noncalcified plaque volume ( d ) was 289.4 mm ( lipid - rich plaque volume , 103.9 mm ; fibrous plaque volume , 185.5 mm ) . after 31 months , follow - up ct images ( e h ) showed no rapid annual change in efv ( 1.9 cm / y ) or efvi ( 1.0 cm / m / y ) . although the calcified plaque volume ( 62.9 mm / y ) increased rapidly , the noncalcified plaque volume ( lipid - rich plaque volume , 9.4 mm / y ; fibrous plaque volume , 1.2 mm / y ) did not . ct = computed tomography , efv = epicardial fat volume , efvi = indexed epicardial fat volume . plaque and epicardial fat volume according to tertile group . on univariate analysis , predictors of rapid increases in lipid - rich and fibrous plaque volumes were smoking , hypercholesterolemia , 10-year chd risk , obesity , and baseline efvi ( table 5 ) . diabetes mellitus was the only significant predictor of a rapid increase in calcified plaque volume . on multivariate analysis , 10-year chd risk was an independent predictor of rapid increases in lipid - rich ( odds ratio [ or ] = 1.184 , p = 0.002 ) and fibrous ( or = 1.413 , p < 0.001 ) plaque volume . baseline efvi ( or = 1.029 , p = 0.016 ) was an independent predictor of a rapid increase in lipid - rich plaque volume , but not fibrous plaque volume ( table 6 ) . or for prediction of rapid plaque increases ( univariate analysis ) . or for prediction of rapid plaque increases ( multivariate analysis ) . djaberi et al reported that efv is a significant predictor of coronary atherosclerosis after adjusting for cv risk factors . sarin et al and iwasaki et al showed that higher efv ( > 100 ml ) was associated with the presence and severity of cad . bastarrika et al showed that patients with significant coronary artery stenosis had significantly greater efv than those without significant cad . however , there have been no previous studies on the relationship between efv and coronary plaque volume . to the best of our knowledge , first , we investigated the relationship between efvi and coronary plaque volume and found that efvi was correlated significantly and positively with age and bmi , which accords with previous reports of a strong association between efv and obesity and bmi . however , with the exception of lipid - rich plaque volume on follow - up ct , no index of plaque volume was correlated with efvi ( table 2 ) . second , we investigated serial changes in efvi and coronary plaque volume , which could be helpful to determine the relationship between efvi change and coronary plaque progression . the efvi did not change significantly from baseline to the time of follow - up ct , although plaque volumes increased significantly on follow - up ct ( table 3 ) . therefore , the annual change in efvi was not accompanied ( p = 0.0960.500 ) by a parallel change in any index of coronary plaque volume . third , we investigated whether baseline characteristics , including efvi , were predictors of rapid increase in plaque volume . in addition to smoking , hypercholesterolemia , 10-year chd risk ( framingham risk score ) , and obesity , baseline efvi was a predictor of rapid increases in lipid - rich and fibrous plaque volumes on univariate analysis ( table 5 ) . after controlling for cv risk factors , baseline efvi remained a significant independent predictor of a rapid increase in lipid - rich plaque volume ( table 6 ) . however , baseline efvi was not an independent predictor of a rapid increase in fibrous plaque volume . cac score measured by mdct may reflect the overall burden of coronary atherosclerosis and may predict the risk of future cv events better than the framingham risk score . an association between a high cac score and large efv has been reported by several studies . gorter et al and bettencourt et al observed that efv was positively related to the cac score . ahmadi et al observed that efv was higher in both males and females with higher cac scores . however , in our study , efv was not significantly related to cac score or calcified plaque volume . nakanishi et al reported that an increase in efv was associated with a greater progression of cac . at follow - up , efv , efvi , and the degree of change in efvi ( % ) were higher in the high progression group than in the low progression group . however , our result does not accord with this previous report . in our study , the increase in calcified plaque volume was not accompanied by an increase in efvi in serial studies ( fig . 3 ) . yerramasu et al reported that the median efv was significantly higher in patients who progressed compared with in those who did not progress ( 93.1 vs 68.8 cm , respectively , p < 0.001 ) . however , our results showed that baseline efvi did not predict a rapid increase in calcified plaque volume . our data agree with the results of otaki et al who reported that neither baseline efv nor the change in efv over time was associated with accumulation of cac after a median follow - up of 4 years in low - risk patients . despite extensive research , the mechanistic understanding of atherosclerotic calcification in humans remains limited . in the progression of atherosclerotic lesions , recurrent plaque rupture and hemorrhage with subsequent healing in addition , cac growth under treatment with statins represents plaque repair rather than continuing plaque expansion . lipid - rich plaque , however , occurs relatively early in the process of atherosclerosis , which might be more affected by efv compared with calcified plaque . in a report by greif et al , efv was elevated in patients with exclusively noncalcified plaque , compared to in those with mixed and calcified plaque . it is therefore likely that efv is associated with plaque formation per se , rather than plaque calcification . . however , plaque can also mix with various components , the volume of which we measured in our study . therefore , the predictive ability of efvi in our study is relevant to the plaque composition , but not the type of plaque . in previous reports , the association between epicardial however , assessment of epicardial fat by echocardiography is not optimal , because the method is highly acoustic and time - window - dependent , is associated with difficulties in differentiating between epicardial and pericardial fat , and has low reproducibility . mdct is a more accurate and highly reproducible method of quantifying efv due to its higher spatial resolution . however , volumetric quantification of epicardial fat using ct is time - consuming , requires an advanced cardiac imaging workstation , and should be performed only by an experienced observer . manual tracing of the pericardium is also somewhat difficult at the bifurcation level of the pulmonary artery , and there may be a partial volume averaging effect near the diaphragm . nevertheless , in our study , the icc for interobserver agreement was very high ( 0.975 for baseline efv and 0.970 for follow - up efv ) . first , the study was a retrospective observational trial with a limited number of heterogeneous patients . therefore , potential biases can not be ruled out despite use of multivariate regression analysis , especially because the model has not been evaluated . second , the study population was derived from a single center , and was composed exclusively of ethnic koreans who were referred for ccta . third , information regarding lifestyle ( e.g. , dietary changes and exercise ) and treatment that may have affected efv was not available . however , the efvi was indexed by body surface area to correct for weight fluctuations between ct examinations . fourth , it is somewhat doubtful that the accuracy of ccta is sufficient for detecting small noncalcified atherosclerotic plaques . in addition , the ct density of the plaque can be affected by the contrast media used within the coronary artery , and serial mdct for obtaining efv is subject to interscan variability . regarding this limitation , we found that the intracoronary lumen density was not significantly different ( p = 0.811 ) between baseline and follow - up ct examinations . finally , we did not evaluate stenosis severity or plaque instability . in conclusion , in addition to the 10-year chd risk based on the framingham risk score , efvi was shown to be an independent predictor of a rapid increase in lipid - rich plaque volume . however , changes in efvi were not associated with parallel changes in coronary plaque volume .
abstractwe explored whether baseline indexed epicardial fat volume ( efvi ) and serial changes in efvi were associated with increase in coronary plaque volume as assessed by multidetector computed tomography.we retrospectively reviewed 87 patients with coronary artery plaque , identified during either baseline or follow - up cardiac computed tomography ( ct ) examinations . each plaque volume was measured in volumetric units using a semiautomatic software tool . efvi was quantified by calculating the total volume of epicardial tissue of ct density 190 to 30 hu , indexed to the body surface area . clinical cardiovascular risk factors were extracted by medical record review at the time of the cardiac ct examinations . the relationship between efvi and coronary plaque volume was explored by regression analysis.although the efvi did not change significantly from baseline to the time of the follow - up ct ( 65.7 21.8 vs 66.0 21.8 cm3/m3 , p = 0.620 ) , the plaque volumes were increased significantly on the follow - up ct scans . the annual change in efvi was not accompanied by a parallel change in coronary plaque volume ( p = 0.0960.500 ) . on univariate analysis , smoking , hypercholesterolemia , 10-year coronary heart disease risk , obesity , and baseline efvi predicted rapid increases in lipid - rich and fibrous plaque volumes . on multivariate analysis , baseline efvi ( odds ratio = 1.029 , p = 0.016 ) was an independent predictor of a rapid increase in lipid - rich plaque volume.efvi was shown to be an independent predictor of a rapid increase in lipid - rich plaque volume . however , changes in efvi were not associated with parallel changes in coronary plaque volume .
Introduction Methods Patients and clinical risk factors Cardiac CT examination and reconstruction Cardiac CT image analysis Statistical analysis Results Discussion
recently , due to advances in temporal and spatial resolution , multidetector computed tomography ( mdct ) has emerged as a noninvasive diagnostic modality that allows for simultaneous assessment of coronary artery calcium ( cac ) , coronary artery stenosis and coronary plaque , and epicardial fat volume ( efv ) without increased radiation exposure or cost . however , it is unknown whether elevated efv is associated with a greater likelihood of developing atherosclerosis over time , and whether increases in efv are accompanied by a parallel increase in coronary plaque volume . the relationship between serial changes in efv and coronary plaque volume has not yet been investigated ; therefore , the aim of the present study was to determine whether baseline indexed efv ( efvi ) and serial changes in efvi are related to increase in coronary plaque volume as assessed by mdct . efv was quantified by calculating the total volume of the tissue in which the ct density ranged from 190 to 30 hu within the epicardium . ( b ) efv was quantified by calculating the total volume of the tissue ( green color ) showing a ct density of 190 to 30 hu within the epicardium . efv was quantified by calculating the total volume of the tissue in which the ct density ranged from 190 to 30 hu within the epicardium . ( b ) efv was quantified by calculating the total volume of the tissue ( green color ) showing a ct density of 190 to 30 hu within the epicardium . cac score and coronary plaque volumes increased significantly ( p = 0.010 to < 0.001 ) on follow - up ct . however , efv ( 116.0 37.5 vs 116.6 37.4 cm , p = 0.604 ) and efvi ( 65.7 21.8 vs 66.0 21.8 cm / m , p = 0.620 ) change values between baseline and follow - up ct were not significant . the annual change in efvi was not accompanied by a parallel change in coronary plaque volume ( p = 0.286 for lipid - rich plaque , 0.500 for fibrous plaque , and 0.096 for calcified plaque ) ( fig . the baseline efv was 54.0 cm ( a , b ) , calcified plaque volume was 380.1 mm ( c ) , and noncalcified plaque volume ( d ) was 289.4 mm ( lipid - rich plaque volume , 103.9 mm ; fibrous plaque volume , 185.5 mm ) . on univariate analysis , predictors of rapid increases in lipid - rich and fibrous plaque volumes were smoking , hypercholesterolemia , 10-year chd risk , obesity , and baseline efvi ( table 5 ) . on multivariate analysis , 10-year chd risk was an independent predictor of rapid increases in lipid - rich ( odds ratio [ or ] = 1.184 , p = 0.002 ) and fibrous ( or = 1.413 , p < 0.001 ) plaque volume . baseline efvi ( or = 1.029 , p = 0.016 ) was an independent predictor of a rapid increase in lipid - rich plaque volume , but not fibrous plaque volume ( table 6 ) . to the best of our knowledge , first , we investigated the relationship between efvi and coronary plaque volume and found that efvi was correlated significantly and positively with age and bmi , which accords with previous reports of a strong association between efv and obesity and bmi . however , with the exception of lipid - rich plaque volume on follow - up ct , no index of plaque volume was correlated with efvi ( table 2 ) . second , we investigated serial changes in efvi and coronary plaque volume , which could be helpful to determine the relationship between efvi change and coronary plaque progression . the efvi did not change significantly from baseline to the time of follow - up ct , although plaque volumes increased significantly on follow - up ct ( table 3 ) . therefore , the annual change in efvi was not accompanied ( p = 0.0960.500 ) by a parallel change in any index of coronary plaque volume . in addition to smoking , hypercholesterolemia , 10-year chd risk ( framingham risk score ) , and obesity , baseline efvi was a predictor of rapid increases in lipid - rich and fibrous plaque volumes on univariate analysis ( table 5 ) . after controlling for cv risk factors , baseline efvi remained a significant independent predictor of a rapid increase in lipid - rich plaque volume ( table 6 ) . however , baseline efvi was not an independent predictor of a rapid increase in fibrous plaque volume . in conclusion , in addition to the 10-year chd risk based on the framingham risk score , efvi was shown to be an independent predictor of a rapid increase in lipid - rich plaque volume . however , changes in efvi were not associated with parallel changes in coronary plaque volume .
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unconjugated hyperbilirubinemia ( uhb ) is common and is associated with a variety of physiologic and pathologic conditions . this occurs as a result of excessive bilirubin formation and because the neonatal liver can not clear bilirubin rapidly enough from the blood . greater awareness is needed among all health workers about the description , causes , risk factors , effective treatment , and sequelae of neonatal jaundice . the american academy of pediatrics ( aap ) has published guidelines outlining the management of healthy newborns with hyperbilirubinemia , which includes maternal abo and rh typing , direct coombs test , blood and rh ( d ) typing of infants cord blood , and a total serum bilirubin level . laboratory tests recommended in case of readmission of the newborn for phototherapy or exchange transfusion in addition to the above mentioned are complete blood count with differential and smear , reticulocyte count , etco ( if available ) , g6pd if suggested by ethnic group or geographic origin or if poor response to phototherapy , urine for reducing substances , sepsis workup if suggested by clinical picture . indirect hyperbilirubinemia can be associated with severe illnesses such as hemolytic disease , metabolic and endocrine disorders , enzymatic deficiencies of the liver , and infections . urinary tract infections ( uti ) are attributed as the main reason for prolonged jaundice , and it is well known that uti can occur without apparent signs , and jaundice is an important and sometimes the presenting feature of uti . the first objective of this study is to determine the incidence of urinary tract infections in asymptomatic , jaundiced newborns admitted to makassed general hospital ( mgh ) under 4 weeks of age , and the second objective is to find a relationship between prolonged neonatal jaundice and uti . charts of jaundiced newborns below 4 weeks of age who were admitted to icn or nursery at mgh from january 1 , 2004 till april 1 2009 were reviewed . the demographic features including name , sex , date of admission , age at presentation , mode of delivery , mode of anesthesia , use of oxytocin or cytotec , type of assistance used , presence of cephalohematoma , bruising or caput succedaneum , weight at birth and at admission , age at onset of jaundice , baby 's and mother 's blood group , direct and indirect coomb 's test , tsh and g6pd levels , mode of collection of the urine culture , type of organism revealed , result of urine analysis , bilirubin level at presentation and maximum level reached during hospitalization , crp , wbc , blood culture , type of feeding , use and duration of antibiotics , duration of phototherapy , intensive phototherapy and exchange transfusion , final diagnosis , duration of hospital stay , discharge status , vcug and ultrasound of the kidneys and urinary tract . urine was collected in a sterile way using a urine bag in males and catheterization in females . culture was considered positive when a single pathogen with more than 10,000 colony forming units / ml were discovered by catheterization or more than 10,000 colony forming units / ml if urine collected by bag . urine culture was repeated if more than one pathogen was discovered or if the number of colonies did not match the above criteria . excluded charts were those of newborns more than four weeks of age , cases with direct hyperbilirubinemia , those having uti not coinciding with the time of onset of jaundice , cases with co - morbidities causing indirect hyperbilirubinemia and septic condition or any sign of infection in the newborns during jaundice . data were analyzed using statistical package of social sciences ( spss ) version 16.0 program using whole numbers , frequencies , means ( sd ) , median ( minimum - maximum ) . data were analyzed using statistical package of social sciences ( spss ) version 16.0 program using whole numbers , frequencies , means ( sd ) , median ( minimum - maximum ) . data were analyzed using statistical package of social sciences ( spss ) version 16.0 program using whole numbers , frequencies , means ( sd ) , median ( minimum - maximum ) . all charts of newborns admitted to makassed general hospital intensive care unit from january 2004 till april 2009 was reviewed all of the newborns with positive urine culture result were full term whereas 114 ( 95% ) newborns of the negative urine culture group were full term and only 6 ( 5% ) were preterms . 19 ( 59.4% ) of the positive urine culture group and 57 ( 47.5% ) of the negative urine culture group were males , while 13 ( 40.6% ) of the first group and 63 ( 52.5% ) of the second group were females , male to female ratio was 1:1.46 . 6 ( 19.4% ) of the first group and 27 ( 23.1% ) of the second group started to get jaundiced before 24 hours of life , the majority of newborns in both groups ( 24 and 83 in the first and second groups respectively ) started to become jaundiced in the interval between 24 and 168 hours ( 8 days ) . only 1 ( 3.2% ) newborn from the first group and 7 ( 6% ) of the second group started to become jaundiced after the 8 day of life . concerning the mode of delivery 24 ( 75% ) newborns of the first group and 86 ( 53.44% ) of the second group were delivered vaginally and 8 ( 25% ) from the first group and 33 ( 27.7% ) of the second group were delivered by cesarean section . exclusive breast milk was given to 15 ( 48.4% ) and 55 ( 46.6% ) of newborns from the first and second group respectively , nearly the same number of the newborns received mixed formula and breast milk feeding in both groups ( 48.4% and 51.7% respectively ) . only a very small number received formula feeding ( 3.2% and 1.7% respectively ) . the demographic characteristics of patients with positive and negative urine culture are presented in table 1 . no statistically significant differences were found between the two groups with regard to gestational age , gender , age at onset of jaundice , mode of delivery and type of feeding . demographic characteristics of infants with positive and negative urine cultures of 152 cases enrolled , positive urine cultures were found in 32 neonates ( 21.1% ) . bacterial pathogens isolated from urine culture were : klebsiella 15 cases ( 46.87% ) , e. coli 12 cases ( 37.5% ) , pseudomonas 2 cases ( 6.25% ) , enterobacter 1 case ( 3.12% ) , gbs 1 case ( 3.12% ) , and staph aureus 1 case ( 3.12% ) ( table 2 ) . pathogens isolated from urine culture in hyperbilirubinemic neonates before the age of 8 days , jaundice was seen in 27 ( 84.3% ) newborns of the uti group and 100 ( 83.4% ) of the non uti group . while after the age of 8 days jaundice was seen in 5 ( 15.6% ) of the newborns with uti and 20 ( 16.6% ) of newborns without uti . 31 ( 96.7% ) of newborns in the uti group , and 110 ( 91.7% ) of newborns in the non - uti group started to have yellowish discoloration of skin before the age of 8 days while only one ( 3.3% ) newborn with uti and 10 ( 8.3% ) of newborns without uti started to have jaundice after the age of eight days . the maximum duration of treatment in the uti group was 4 days with the majority of newborns ( 14 cases ) receiving 2 days of phototherapy . in the non - uti group the maximum duration of phototherapy was 7 days with the majority of newborns ( 45 ) receiving 2 days of phototherapy . intensive phototherapy was used in 6 ( 18.7% ) in the uti group and 35 ( 29.16% ) of the non - uti group ( table 3 ) . none of the newborns in the uti group underwent exchange transfusion therapy versus the non - uti group where 4 ( 3.3% ) of the newborns underwent exchange ( figure 1 ) . intensive phototherapy duration of phototherapy in uti and non - uti groups although not part of the study design , 32 of 32 newborns diagnosed with uti had renal ultrasounds performed . urinary tract abnormalities were found in 7 ( 21.87% ) patients , which included pelvi - calyceal system dilatation . no abnormalities were found in voiding cystourethrogram obtained in 12 out of the 32 newborns in the uti group . all charts of newborns admitted to makassed general hospital intensive care unit from january 2004 till april 2009 was reviewed all of the newborns with positive urine culture result were full term whereas 114 ( 95% ) newborns of the negative urine culture group were full term and only 6 ( 5% ) were preterms . 19 ( 59.4% ) of the positive urine culture group and 57 ( 47.5% ) of the negative urine culture group were males , while 13 ( 40.6% ) of the first group and 63 ( 52.5% ) of the second group were females , male to female ratio was 1:1.46 . 6 ( 19.4% ) of the first group and 27 ( 23.1% ) of the second group started to get jaundiced before 24 hours of life , the majority of newborns in both groups ( 24 and 83 in the first and second groups respectively ) started to become jaundiced in the interval between 24 and 168 hours ( 8 days ) . only 1 ( 3.2% ) newborn from the first group and 7 ( 6% ) of the second group started to become jaundiced after the 8 day of life . concerning the mode of delivery 24 ( 75% ) newborns of the first group and 86 ( 53.44% ) of the second group were delivered vaginally and 8 ( 25% ) from the first group and 33 ( 27.7% ) of the second group were delivered by cesarean section . exclusive breast milk was given to 15 ( 48.4% ) and 55 ( 46.6% ) of newborns from the first and second group respectively , nearly the same number of the newborns received mixed formula and breast milk feeding in both groups ( 48.4% and 51.7% respectively ) . only a very small number received formula feeding ( 3.2% and 1.7% respectively ) . the demographic characteristics of patients with positive and negative urine culture are presented in table 1 . no statistically significant differences were found between the two groups with regard to gestational age , gender , age at onset of jaundice , mode of delivery and type of feeding . demographic characteristics of infants with positive and negative urine cultures of 152 cases enrolled , positive urine cultures were found in 32 neonates ( 21.1% ) . bacterial pathogens isolated from urine culture were : klebsiella 15 cases ( 46.87% ) , e. coli 12 cases ( 37.5% ) , pseudomonas 2 cases ( 6.25% ) , enterobacter 1 case ( 3.12% ) , gbs 1 case ( 3.12% ) , and staph aureus 1 case ( 3.12% ) ( table 2 ) . pathogens isolated from urine culture in hyperbilirubinemic neonates before the age of 8 days , jaundice was seen in 27 ( 84.3% ) newborns of the uti group and 100 ( 83.4% ) of the non uti group . while after the age of 8 days jaundice was seen in 5 ( 15.6% ) of the newborns with uti and 20 ( 16.6% ) of newborns without uti . 31 ( 96.7% ) of newborns in the uti group , and 110 ( 91.7% ) of newborns in the non - uti group started to have yellowish discoloration of skin before the age of 8 days while only one ( 3.3% ) newborn with uti and 10 ( 8.3% ) of newborns without uti started to have jaundice after the age of eight days . the maximum duration of treatment in the uti group was 4 days with the majority of newborns ( 14 cases ) receiving 2 days of phototherapy . in the non - uti group the maximum duration of phototherapy was 7 days with the majority of newborns ( 45 ) receiving 2 days of phototherapy . intensive phototherapy was used in 6 ( 18.7% ) in the uti group and 35 ( 29.16% ) of the non - uti group ( table 3 ) . none of the newborns in the uti group underwent exchange transfusion therapy versus the non - uti group where 4 ( 3.3% ) of the newborns underwent exchange ( figure 1 ) . intensive phototherapy duration of phototherapy in uti and non - uti groups although not part of the study design , 32 of 32 newborns diagnosed with uti had renal ultrasounds performed . urinary tract abnormalities were found in 7 ( 21.87% ) patients , which included pelvi - calyceal system dilatation . no abnormalities were found in voiding cystourethrogram obtained in 12 out of the 32 newborns in the uti group . all charts of newborns admitted to makassed general hospital intensive care unit from january 2004 till april 2009 was reviewed all of the newborns with positive urine culture result were full term whereas 114 ( 95% ) newborns of the negative urine culture group were full term and only 6 ( 5% ) were preterms . 19 ( 59.4% ) of the positive urine culture group and 57 ( 47.5% ) of the negative urine culture group were males , while 13 ( 40.6% ) of the first group and 63 ( 52.5% ) of the second group were females , male to female ratio was 1:1.46 . 6 ( 19.4% ) of the first group and 27 ( 23.1% ) of the second group started to get jaundiced before 24 hours of life , the majority of newborns in both groups ( 24 and 83 in the first and second groups respectively ) started to become jaundiced in the interval between 24 and 168 hours ( 8 days ) . only 1 ( 3.2% ) newborn from the first group and 7 ( 6% ) of the second group started to become jaundiced after the 8 day of life . concerning the mode of delivery 24 ( 75% ) newborns of the first group and 86 ( 53.44% ) of the second group were delivered vaginally and 8 ( 25% ) from the first group and 33 ( 27.7% ) of the second group were delivered by cesarean section . exclusive breast milk was given to 15 ( 48.4% ) and 55 ( 46.6% ) of newborns from the first and second group respectively , nearly the same number of the newborns received mixed formula and breast milk feeding in both groups ( 48.4% and 51.7% respectively ) . only a very small number received formula feeding ( 3.2% and 1.7% respectively ) . the demographic characteristics of patients with positive and negative urine culture are presented in table 1 . no statistically significant differences were found between the two groups with regard to gestational age , gender , age at onset of jaundice , mode of delivery and type of feeding . of 152 cases enrolled , positive urine cultures were found in 32 neonates ( 21.1% ) . bacterial pathogens isolated from urine culture were : klebsiella 15 cases ( 46.87% ) , e. coli 12 cases ( 37.5% ) , pseudomonas 2 cases ( 6.25% ) , enterobacter 1 case ( 3.12% ) , gbs 1 case ( 3.12% ) , and staph aureus 1 case ( 3.12% ) ( table 2 ) . before the age of 8 days , jaundice was seen in 27 ( 84.3% ) newborns of the uti group and 100 ( 83.4% ) of the non uti group . while after the age of 8 days jaundice was seen in 5 ( 15.6% ) of the newborns with uti and 20 ( 16.6% ) of newborns without uti . 31 ( 96.7% ) of newborns in the uti group , and 110 ( 91.7% ) of newborns in the non - uti group started to have yellowish discoloration of skin before the age of 8 days while only one ( 3.3% ) newborn with uti and 10 ( 8.3% ) of newborns without uti started to have jaundice after the age of eight days . all newborns in both groups were treated with phototherapy . the maximum duration of treatment in the uti group was 4 days with the majority of newborns ( 14 cases ) receiving 2 days of phototherapy . in the non - uti group the maximum duration of phototherapy was 7 days with the majority of newborns ( 45 ) receiving 2 days of phototherapy . intensive phototherapy was used in 6 ( 18.7% ) in the uti group and 35 ( 29.16% ) of the non - uti group ( table 3 ) . none of the newborns in the uti group underwent exchange transfusion therapy versus the non - uti group where 4 ( 3.3% ) of the newborns underwent exchange ( figure 1 ) . intensive phototherapy duration of phototherapy in uti and non - uti groups although not part of the study design , 32 of 32 newborns diagnosed with uti had renal ultrasounds performed . urinary tract abnormalities were found in 7 ( 21.87% ) patients , which included pelvi - calyceal system dilatation . no abnormalities were found in voiding cystourethrogram obtained in 12 out of the 32 newborns in the uti group . older children and adults , newborns have a high rate of hemoglobin catabolism and bilirubin production because of their elevated hematocrit and red blood cell volume per body weight , and their shorter life span of red blood cells ( 70 to 90 days ) . although bilirubin production is elevated in newborns , conjugation and clearance of bilirubin intake can cause delayed clearance of bilirubin . in most jaundiced neonates , only unconjugated bilirubin is found in the blood , and the accumulated bilirubin is distributed by the circulation throughout the body and produces clinical jaundice . it generally is assumed that to cross intact cell membrane barriers the bilirubin must be free , or dissociated , from its albumin binding . uti in newborn infants affects 1 in 3 babies with proven bacterial infection , and the incidence is higher in low birth weight and preterm infants as well as febrile or hyperbilirubinemic patients . bilgen in a series of 102 patients with asymptomatic unexplained indirect hyperbilirubinemia , uti was diagnosed in ( 8% ) of cases . garcia and nager in a series of 160 asymptomatic jaundiced infants found positive urine culture in 7.5% of the infants younger than 8 weeks . our study showed similar results but with greater prevalence of uti where in a series of 152 asymptomatic jaundiced newborns 32 ( 21% ) had urinary tract infection all documented by urine cultures ( table 2 ) . e. coli is the most common etiologic agent of urinary tract infection , as found in older patients . gram - positive bacteria , with the exception of enterococci , are rare causes of urinary tract infections . the most common organism discovered in our study was klebsiella with 15 ( 46.87% ) cases , the second most common was the e. coli with 12 ( 37.5% ) cases . a study conducted by jafarzadeh and mohammadzadeh in neonatal research center in iran had similar results of our study with klebsiella the most common organism encountered . garcia et al . reported that infants with the onset of jaundice after eight days were more likely to have uti , our study showed that almost all of the newborns with asymptomatic jaundice who were diagnosed to have uti presented before the age of 8 days . this data was supported by a study done by hlya bilgen et al . in 2003 where they found that the onset of jaundice was < 7 days in most of their cases . although garcia et al reported that patients with an elevated conjugated bilirubin fraction were more likely to have uti , none of our patients had a high direct bilirubin level . concerning the duration of phototherapy our data showed that patients from the uti group received phototherapy for a maximum of 4 days with the majority of patients receiving only 4 days , while patients in the non - uti group received a maximum of 7 days of phototherapy with a similar majority receiving 2 days of phototherapy . the fact that our patients needed phototherapy for only a short period of time and that intensive phototherapy was used in only 18% of the cases compared to 81% in the second group may be indicative of the fact that our practice of early detection of the uti in these newborns may lead to a lesser need of phototherapy intensity and duration as well . older children and adults , newborns have a high rate of hemoglobin catabolism and bilirubin production because of their elevated hematocrit and red blood cell volume per body weight , and their shorter life span of red blood cells ( 70 to 90 days ) . although bilirubin production is elevated in newborns , conjugation and clearance of bilirubin intake can cause delayed clearance of bilirubin . in most jaundiced neonates , only unconjugated bilirubin is found in the blood , and the accumulated bilirubin is distributed by the circulation throughout the body and produces clinical jaundice . it generally is assumed that to cross intact cell membrane barriers the bilirubin must be free , or dissociated , from its albumin binding . uti in newborn infants affects 1 in 3 babies with proven bacterial infection , and the incidence is higher in low birth weight and preterm infants as well as febrile or hyperbilirubinemic patients . bilgen in a series of 102 patients with asymptomatic unexplained indirect hyperbilirubinemia , uti was diagnosed in ( 8% ) of cases . garcia and nager in a series of 160 asymptomatic jaundiced infants found positive urine culture in 7.5% of the infants younger than 8 weeks . our study showed similar results but with greater prevalence of uti where in a series of 152 asymptomatic jaundiced newborns 32 ( 21% ) had urinary tract infection all documented by urine cultures ( table 2 ) . e. coli is the most common etiologic agent of urinary tract infection , as found in older patients . gram - positive bacteria , with the exception of enterococci , are rare causes of urinary tract infections . the most common organism discovered in our study was klebsiella with 15 ( 46.87% ) cases , the second most common was the e. coli with 12 ( 37.5% ) cases . a study conducted by jafarzadeh and mohammadzadeh in neonatal research center in iran had similar results of our study with klebsiella the most common organism encountered . garcia et al . reported that infants with the onset of jaundice after eight days were more likely to have uti , our study showed that almost all of the newborns with asymptomatic jaundice who were diagnosed to have uti presented before the age of 8 days . this data was supported by a study done by hlya bilgen et al . in 2003 where they found that the onset of jaundice was < 7 days in most of their cases . although garcia et al reported that patients with an elevated conjugated bilirubin fraction were more likely to have uti , none of our patients had a high direct bilirubin level . concerning the duration of phototherapy our data showed that patients from the uti group received phototherapy for a maximum of 4 days with the majority of patients receiving only 4 days , while patients in the non - uti group received a maximum of 7 days of phototherapy with a similar majority receiving 2 days of phototherapy . the fact that our patients needed phototherapy for only a short period of time and that intensive phototherapy was used in only 18% of the cases compared to 81% in the second group may be indicative of the fact that our practice of early detection of the uti in these newborns may lead to a lesser need of phototherapy intensity and duration as well . older children and adults , newborns have a high rate of hemoglobin catabolism and bilirubin production because of their elevated hematocrit and red blood cell volume per body weight , and their shorter life span of red blood cells ( 70 to 90 days ) . although bilirubin production is elevated in newborns , conjugation and clearance of bilirubin intake can cause delayed clearance of bilirubin . in most jaundiced neonates , only unconjugated bilirubin is found in the blood , and the accumulated bilirubin is distributed by the circulation throughout the body and produces clinical jaundice . it generally is assumed that to cross intact cell membrane barriers the bilirubin must be free , or dissociated , from its albumin binding . uti in newborn infants affects 1 in 3 babies with proven bacterial infection , and the incidence is higher in low birth weight and preterm infants as well as febrile or hyperbilirubinemic patients . bilgen in a series of 102 patients with asymptomatic unexplained indirect hyperbilirubinemia , uti was diagnosed in ( 8% ) of cases . garcia and nager in a series of 160 asymptomatic jaundiced infants found positive urine culture in 7.5% of the infants younger than 8 weeks . our study showed similar results but with greater prevalence of uti where in a series of 152 asymptomatic jaundiced newborns 32 ( 21% ) had urinary tract infection all documented by urine cultures ( table 2 ) . e. coli is the most common etiologic agent of urinary tract infection , as found in older patients . gram - positive bacteria , with the exception of enterococci , are rare causes of urinary tract infections . the most common organism discovered in our study was klebsiella with 15 ( 46.87% ) cases , the second most common was the e. coli with 12 ( 37.5% ) cases . a study conducted by jafarzadeh and mohammadzadeh in neonatal research center in iran had similar results of our study with klebsiella the most common organism encountered . garcia et al . reported that infants with the onset of jaundice after eight days were more likely to have uti , our study showed that almost all of the newborns with asymptomatic jaundice who were diagnosed to have uti presented before the age of 8 days . this data was supported by a study done by hlya bilgen et al . in 2003 where they found that the onset of jaundice was < 7 days in most of their cases . although garcia et al reported that patients with an elevated conjugated bilirubin fraction were more likely to have uti , none of our patients had a high direct bilirubin level . concerning the duration of phototherapy our data showed that patients from the uti group received phototherapy for a maximum of 4 days with the majority of patients receiving only 4 days , while patients in the non - uti group received a maximum of 7 days of phototherapy with a similar majority receiving 2 days of phototherapy . the fact that our patients needed phototherapy for only a short period of time and that intensive phototherapy was used in only 18% of the cases compared to 81% in the second group may be indicative of the fact that our practice of early detection of the uti in these newborns may lead to a lesser need of phototherapy intensity and duration as well . thus , jaundice may be the first sign of uti in these babies before other signs become evident . we recommend that testing for a uti be part of the diagnostic evaluation of asymptomatic jaundiced newborns especially when no other obvious reason of jaundice is found . this may lead to early detection and treatment of these newborns leading to lesser long term complications of the urogenital tract especially the kidneys . we could also achieve shorter duration of phototherapy and consequently hospital stay which decreases the risk of nosocomial infection and the cost of hospital stay .
background : jaundice is a common problem during the neonatal period . about 60% of the full term and 80% of premature infants develop jaundice . it can be associated with serious illnesses such as urinary tract infections.aims:the aim of this study is to evaluate the incidence and prevalence of urinary tract infection in newborns with indirect hyperbilirubinemia and to find a relationship with prolonged jaundice.patients and methods : we retrospectively evaluated asymptomatic , jaundiced neonates for evidence of a urinary tract infection . data reviewed including demographic and historical data were included with data of blood studies , radiological evaluation and treatment.results:32 neonates of 152 cases had urinary tract infection . most commonly isolated organisms were klebsiella and escherishia coli . maximum duration of phototherapy was 4 days in the urinary tract infection group versus 7 in the non - urinary tract infection group . intensive phototherapy was used in 18.7% in the urinary tract infection group versus 29.16% in the non - urinary tract infection group . none of the newborns in the urinary tract infection group underwent exchange transfusion therapy.conclusion:urinary tract infection can occur in asymptomatic , jaundiced newborns . thus , it may be the first in these babies before other signs become evident .
Introduction Patients and Methods None Statistical analysis Results None Study population and demographics Incidence of UTI Age at presentation Age at onset of jaundice Outcomes Discussion None Bilirubin Metabolism Conclusion
indirect hyperbilirubinemia can be associated with severe illnesses such as hemolytic disease , metabolic and endocrine disorders , enzymatic deficiencies of the liver , and infections . the first objective of this study is to determine the incidence of urinary tract infections in asymptomatic , jaundiced newborns admitted to makassed general hospital ( mgh ) under 4 weeks of age , and the second objective is to find a relationship between prolonged neonatal jaundice and uti . the demographic features including name , sex , date of admission , age at presentation , mode of delivery , mode of anesthesia , use of oxytocin or cytotec , type of assistance used , presence of cephalohematoma , bruising or caput succedaneum , weight at birth and at admission , age at onset of jaundice , baby 's and mother 's blood group , direct and indirect coomb 's test , tsh and g6pd levels , mode of collection of the urine culture , type of organism revealed , result of urine analysis , bilirubin level at presentation and maximum level reached during hospitalization , crp , wbc , blood culture , type of feeding , use and duration of antibiotics , duration of phototherapy , intensive phototherapy and exchange transfusion , final diagnosis , duration of hospital stay , discharge status , vcug and ultrasound of the kidneys and urinary tract . in the non - uti group the maximum duration of phototherapy was 7 days with the majority of newborns ( 45 ) receiving 2 days of phototherapy . intensive phototherapy was used in 6 ( 18.7% ) in the uti group and 35 ( 29.16% ) of the non - uti group ( table 3 ) . none of the newborns in the uti group underwent exchange transfusion therapy versus the non - uti group where 4 ( 3.3% ) of the newborns underwent exchange ( figure 1 ) . the maximum duration of treatment in the uti group was 4 days with the majority of newborns ( 14 cases ) receiving 2 days of phototherapy . in the non - uti group the maximum duration of phototherapy was 7 days with the majority of newborns ( 45 ) receiving 2 days of phototherapy . intensive phototherapy was used in 6 ( 18.7% ) in the uti group and 35 ( 29.16% ) of the non - uti group ( table 3 ) . none of the newborns in the uti group underwent exchange transfusion therapy versus the non - uti group where 4 ( 3.3% ) of the newborns underwent exchange ( figure 1 ) . in the non - uti group the maximum duration of phototherapy was 7 days with the majority of newborns ( 45 ) receiving 2 days of phototherapy . intensive phototherapy was used in 6 ( 18.7% ) in the uti group and 35 ( 29.16% ) of the non - uti group ( table 3 ) . none of the newborns in the uti group underwent exchange transfusion therapy versus the non - uti group where 4 ( 3.3% ) of the newborns underwent exchange ( figure 1 ) . concerning the duration of phototherapy our data showed that patients from the uti group received phototherapy for a maximum of 4 days with the majority of patients receiving only 4 days , while patients in the non - uti group received a maximum of 7 days of phototherapy with a similar majority receiving 2 days of phototherapy . the fact that our patients needed phototherapy for only a short period of time and that intensive phototherapy was used in only 18% of the cases compared to 81% in the second group may be indicative of the fact that our practice of early detection of the uti in these newborns may lead to a lesser need of phototherapy intensity and duration as well . our study showed similar results but with greater prevalence of uti where in a series of 152 asymptomatic jaundiced newborns 32 ( 21% ) had urinary tract infection all documented by urine cultures ( table 2 ) . concerning the duration of phototherapy our data showed that patients from the uti group received phototherapy for a maximum of 4 days with the majority of patients receiving only 4 days , while patients in the non - uti group received a maximum of 7 days of phototherapy with a similar majority receiving 2 days of phototherapy . the fact that our patients needed phototherapy for only a short period of time and that intensive phototherapy was used in only 18% of the cases compared to 81% in the second group may be indicative of the fact that our practice of early detection of the uti in these newborns may lead to a lesser need of phototherapy intensity and duration as well . our study showed similar results but with greater prevalence of uti where in a series of 152 asymptomatic jaundiced newborns 32 ( 21% ) had urinary tract infection all documented by urine cultures ( table 2 ) . the fact that our patients needed phototherapy for only a short period of time and that intensive phototherapy was used in only 18% of the cases compared to 81% in the second group may be indicative of the fact that our practice of early detection of the uti in these newborns may lead to a lesser need of phototherapy intensity and duration as well . thus , jaundice may be the first sign of uti in these babies before other signs become evident .
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gypsies and travellers are recognized as one of the most disadvantaged minority groups in the united kingdom and globally . research into the health needs of this group is an emerging field , and gypsies and travellers could justly be described as an invisible minority1 in being rarely captured in health statistics , unassertive of their health needs and with few champions.2 , 3 in the united kingdom , gypsies and travellers are known to have poorer health status and a higher risk of mortality than socioeconomically matched comparison groups4 and to experience health inequalities which are greater than could be expected simply from socioeconomic disadvantage or from belonging to a minority ethnic group.5 gypsies and travellers access to health services is also known to be poor.6 , 7 in several european countries , roma people have been shown to have poorer health than other ethnic groups and poorer access to health services,8 , 9 , 10 and a recent literature review identified a higher prevalence of both communicable and noncommunicable disease in the roma community with significantly shorter life expectancies than national averages.11 gypsies and travellers share a history of persecution and rejection by mainstream society , which continues today.12 , 13 , 14 , 15 the umbrella term gypsies and travellers covers a diversity of people , including continental roma , english and welsh gypsies and irish and scottish travellers.3 in the 2001 , uk census gypsy or irish traveller was included as an ethnic category for the first time . gypsies have a shared common history , language and oral literature4 and share with travellers beliefs about the centrality of the family as a social structure , nomadism and ideals of purity which influence hygiene in daily life and behaviour towards the opposite sex.13 , 16 a component of the gypsy traveller identity is a strong awareness of the differences between gypsy culture and that of the majority population,17 , 18 which has been described as contributing to gypsies and travellers ability to maintain a unique identity as internaloutsiders within often hostile host populations.16 some community values such as taking a pride in resilience and resourcefulness , particularly in children , and setting a higher value on lived experience above learning from books diverge from those of the settled community.13 the common use of the word gorgio to describe nongypsies emphasizes the status of gypsy travellers as a race apart who share defined cultural characteristics which define and confirm their unique identity.18 given the poor health outcomes experienced by gypsies and travellers , it is important to explore their views on the services they receive from health providers and to establish their primary health needs . it has been noted that roma women are often overlooked in health research due to dual discrimination , both ethnic and gender , within and outside the community,19 and there is currently little qualitative research exploring women 's views on maternal and child health and the health services provided . the uk healthy child progamme20 stipulates that mothers should be visited antenatally to support infant feeding and that postnatal visits should promote breastfeeding , with introduction of solids foods at around 6 months , as recommended by the world health organization.21 encouraging engagement with children 's preventive health services has been recognized as the starting place for reducing health inequalities among gypsies and travellers , particularly in the area of nutrition.11 how a baby is fed in the first year of life has an impact upon health in the long and short term.21 , 22 , 23 breastfed babies are less likely to experience morbidity in the first year of life24 and to have a reduced risk of diabetes , hypertension and obesity in later life.25 health benefits for mothers who breastfeed include a reduced risk of breast and ovarian cancer and type 2 diabetes.24 in the united kingdom , infant feeding behaviour is strongly related to socioeconomic class and ethnicity , with highly educated professional women most likely to breastfeed among the white community , but mothers from nonwhite ethnic minority groups more likely to breastfeed than the general population.22 despite exclusive breastfeeding for 6 months being advised , only 34% of babies in the united kingdom are breastfed at all at 6 months of age and only 1% exclusively.22 a small quantitative study of gypsies and travellers feeding practices in one primary care trust in england , suggested very low breastfeeding rates , with only 3% estimated to have initiated breastfeeding and none continuing to 68 weeks ( data based on health visitors reports ) ; however , a survey of 20 gypsy traveller women suggested a rate of 15% who had ever breastfed.26 breastfeeding rates were found to be slightly above average among roma mothers in serbia27 , but no difference was identified between the duration of breastfeeding for czech and roma children ( a median duration of 3 months breastfeeding in both groups).28 no studies have looked at weaning practices among gypsies and travellers , although these are recognized as being important in relation to continuation of breastfeeding,22 and to later eating habits which have an impact upon risk of obesity and cardiac disease.29 , 30 the aim of this study was to explore the views of gypsy traveller mothers and grandmothers on infant feeding and health service provision . semistructured interviews were conducted with a purposive sample of mothers and grandmothers from english gypsy , irish traveller and romanian roma communities between november 2011 and february 2012 . grandmothers were included as they are considered to have an influence upon mothers infant feeding choices.31 the sample size was designed to facilitate in depth exploration of the subject and allowed the comparison of the views of roma mothers and grandmothers with their english gypsy and irish traveller counterparts . twentytwo mothers and grandmothers were recruited , half from the roma community , and half from english gypsy and irish traveller backgrounds ( see table 1 for demographic details ) . inclusion criteria were as follows : demographic details of participants nvq , national vocational qualification ( a uk occupational qualification ) . mothers of roma , english gypsy or irish traveller ethnicity with a child aged 3 years or undergrandmothers of roma , english gypsy or irish traveller ethnicity with one or more grandchildren mothers of roma , english gypsy or irish traveller ethnicity with a child aged 3 years or under grandmothers of roma , english gypsy or irish traveller ethnicity with one or more grandchildren recruitment was carried out by a researcher ( lc ) , following introductions by local authority and voluntary workers who were either members of , or wellknown to , the community . these workers initially identified potential participants , and the researcher checked inclusion criteria at interview , including the participant 's own selfdefinition of ethnicity . one interviewee was excluded at interview as she did not agree that her ethnicity was irish traveller , despite the link worker having identified her as such . participants were interviewed in their homes or in a community setting , including a church . travelling participants were interviewed in their homes on both privately owned and council caravan sites . the sample size was achieved with consideration to ritchie et al . 's view that further data collection would lead to diminishing returns in terms of new insights and ideas gained.32 ethical consent was granted by a university ethics committee ( application number hsc/11/09/89 ) . all were given directly to study participants by a link worker who could explain the content ; the researcher visited a few days later in the company of a link worker to offer an interview . prior to interview , the researcher read the information sheet with each participant and completed a consent form , thus ensuring that participants were able to opt out if they wished at this stage . all written materials were translated into romanian for roma participants and also read through at interview by an interpreter . consent was requested for audiotaping and use of anonymized quotations , in addition to participation . to preserve anonymity , questions were based upon a topic guide developed in collaboration with the steering committee , which included local experts in infant feeding and nutrition in addition to two members of gypsy traveller ethnicity . the topic guide was focused on personal experience of milk and solid feeding , as well as perceived family and community views of infant feeding . only one roma participant spoke english , so interviews with the roma were conducted in romanian , with professional interpreters providing concurrent translation . for all roma participants , romanian was a second language ; no romanyspeaking interpreters existed in bristol . liamputtong33 suggests that in crosscultural research , interpreters are best considered as joint researchers , as in facilitating communication through translation , they play a part in shaping the data collected . both interpreters who took part in the project were wellknown to the community and often acted as interpreters in health and educational settings . permission was requested to audiotape the interview . where participants agreed to audiotaping , interviews were subsequently transcribed . for the remainder , the interviewer ( lc ) documented responses contemporaneously by hand . no participants objected to notes being taken , but some interviewees felt that no traveller would allow their voice to be recorded . a sample of audiotaped interviews with roma participants was checked for validity of translation by an independent romanian interpreter ; all were confirmed to be accurate . data were coded using nvivo 9 as part of data analysis using a framework approach by which data are classified and organized according to key themes and concepts.34 members of the steering group contributed to the preliminary identification of codes from iterative reading of transcripts and contemporaneous notes and reached agreement on emergent categories . following coding of all transcripts , lc and ds identified dominant themes and developed a conceptual framework from an interpretation of the data set as a whole . the framework approach allowed movement from raw data to abstraction in the analytical process , without losing the voice of participants.35 semistructured interviews were conducted with a purposive sample of mothers and grandmothers from english gypsy , irish traveller and romanian roma communities between november 2011 and february 2012 . grandmothers were included as they are considered to have an influence upon mothers infant feeding choices.31 the sample size was designed to facilitate in depth exploration of the subject and allowed the comparison of the views of roma mothers and grandmothers with their english gypsy and irish traveller counterparts . twentytwo mothers and grandmothers were recruited , half from the roma community , and half from english gypsy and irish traveller backgrounds ( see table 1 for demographic details ) . inclusion criteria were as follows : demographic details of participants nvq , national vocational qualification ( a uk occupational qualification ) . mothers of roma , english gypsy or irish traveller ethnicity with a child aged 3 years or undergrandmothers of roma , english gypsy or irish traveller ethnicity with one or more grandchildren mothers of roma , english gypsy or irish traveller ethnicity with a child aged 3 years or under grandmothers of roma , english gypsy or irish traveller ethnicity with one or more grandchildren recruitment was carried out by a researcher ( lc ) , following introductions by local authority and voluntary workers who were either members of , or wellknown to , the community . these workers initially identified potential participants , and the researcher checked inclusion criteria at interview , including the participant 's own selfdefinition of ethnicity . one interviewee was excluded at interview as she did not agree that her ethnicity was irish traveller , despite the link worker having identified her as such . participants were interviewed in their homes or in a community setting , including a church . travelling participants were interviewed in their homes on both privately owned and council caravan sites . the sample size was achieved with consideration to ritchie et al . 's view that further data collection would lead to diminishing returns in terms of new insights and ideas gained.32 all were given directly to study participants by a link worker who could explain the content ; the researcher visited a few days later in the company of a link worker to offer an interview . prior to interview , the researcher read the information sheet with each participant and completed a consent form , thus ensuring that participants were able to opt out if they wished at this stage . all written materials were translated into romanian for roma participants and also read through at interview by an interpreter . consent was requested for audiotaping and use of anonymized quotations , in addition to participation . to preserve anonymity , questions were based upon a topic guide developed in collaboration with the steering committee , which included local experts in infant feeding and nutrition in addition to two members of gypsy traveller ethnicity . the topic guide was focused on personal experience of milk and solid feeding , as well as perceived family and community views of infant feeding . only one roma participant spoke english , so interviews with the roma were conducted in romanian , with professional interpreters providing concurrent translation . for all roma participants , romanian was a second language ; no romanyspeaking interpreters existed in bristol . liamputtong33 suggests that in crosscultural research , interpreters are best considered as joint researchers , as in facilitating communication through translation , they play a part in shaping the data collected . both interpreters who took part in the project were wellknown to the community and often acted as interpreters in health and educational settings . permission was requested to audiotape the interview . where participants agreed to audiotaping , interviews were subsequently transcribed . for the remainder , the interviewer ( lc ) documented responses contemporaneously by hand . no participants objected to notes being taken , but some interviewees felt that no traveller would allow their voice to be recorded . a sample of audiotaped interviews with roma participants was checked for validity of translation by an independent romanian interpreter ; all were confirmed to be accurate . data were coded using nvivo 9 as part of data analysis using a framework approach by which data are classified and organized according to key themes and concepts.34 members of the steering group contributed to the preliminary identification of codes from iterative reading of transcripts and contemporaneous notes and reached agreement on emergent categories . following coding of all transcripts , lc and ds identified dominant themes and developed a conceptual framework from an interpretation of the data set as a whole . the framework approach allowed movement from raw data to abstraction in the analytical process , without losing the voice of participants.35 three dominant themes were identified as influencing infant feeding behaviour , which were common to all participants and related to the culture of their community . these themes were as follows : the centrality of the family , beliefs and traditions related to culture , and a travelling lifestyle . a fourth crosscutting theme was identified as interaction with health professionals ; this was interwoven with the themes relating to gypsy and traveller culture throughout . it was apparent that at many points , interactions with health professionals led to some conflict with beliefs and attitudes prevalent in the community , and these conflicts are brought out in the account below . large families are commonplace among gypsies and travellers , and there is an expectation that older children help care for younger siblings . as a result , women considered that they had preexisting knowledge and skills when they became parents . few participants described themselves as requiring professional support in caring for children irrespective of age or number of children : the travelling community are reared up with children . i was married at 13 when you are small you are with your mum , and she shows you how to do things . roma 11 , mother i 'll tell you again , my mum had 10 kids and i helped look after them , i did n't need to learn from scratch , i already knew . some people do n't know , they have to learn from scratch and they need people telling them what to do and what not to do . i was married at 13 when you are small you are with your mum , and she shows you how to do things . i 'll tell you again , my mum had 10 kids and i helped look after them , i did n't need to learn from scratch , i already knew . some people do n't know , they have to learn from scratch and they need people telling them what to do and what not to do . english gypsy 4 , mother young motherhood increased participants view of themselves as being highly experienced in all aspects of child care . compared with the experiential knowledge that was easily available within the family , the advice of health professionals was often set at a low value by mothers and grandmothers.i do n't think they need any advice in feeding their children because it 's passed on from their own mums and grandmothers . irish traveller 1 , grandmother i 've no idea when health visitors say to introduce baby dinners . i think the health visitor advises 3 months but i would do what i thought , from my experience . lots of health visitors haven't had a baby ; they do n't know anything about it , just what they have read . irish traveller 4 , mother i do n't think they need any advice in feeding their children because it 's passed on from their own mums and grandmothers . irish traveller 1 , grandmother i 've no idea when health visitors say to introduce baby dinners . i think the health visitor advises 3 months but i would do what i thought , from my experience . lots of health visitors haven't had a baby ; they do n't know anything about it , just what they have read . irish traveller 4 , mother contact with health services was described as less common in the past ( early on they did n't want anything to do with travellers , now it 's more accepted. irish traveller 5 , grandmother ) , but english gypsies and irish travellers now expected contact with midwifery and health visiting services . roma women were sometimes unclear about difference in roles , referring to professionals generically as being from the doctor's. all described a minimal service with little routine contact beyond the immediate postnatal period . mothers took a pride in being capable mothers and did not see themselves as requiring extensive contact with health professionals.the health visitor came once or twice and saw i could get along , now she does n't come . translated roma 3 , mother the health visitor came once or twice and saw i could get along , now she does n't come . translated health professionals were described by all groups as relying on written materials to give health promotion information , despite low literacy levels . although none of the roma interviewees read english , most mentioned having been given leaflets . several nonroma participants also could not read health promotion materials : we received leaflets in english , about how to breast feed and what to expect when you 're a mum , but we do n't actually know how to read in english . translated roma 8 , mother she gave me some [ leaflets ] , but i ca n't read , not unless i ask someone to read it , then i find out what 's in it . i just looked at the pictures , but i did n't often look in the book . irish traveller 4 , mother we received leaflets in english , about how to breast feed and what to expect when you 're a mum , but we do n't actually know how to read in english . translated she gave me some [ leaflets ] , but i ca n't read , not unless i ask someone to read it , then i find out what 's in it i just looked at the pictures , but i did n't often look in the book . irish traveller 4 , mother these contacts often served to highlight the differences between travelling people and the settled community . many participants had little contact with people outside their community and were aware only of their own infant feeding norms . english gypsy and irish traveller participants frequently expressed pride in their culture , and where there was opposition between family traditions and health professional advice , a common response was to reaffirm the positives about the community way. one mother commented to the researcher : me meself , i like me own way of life , i 've been brought up as a romany gypsy and that 's the way i want me boys to be brought up to be truthful . i 'm not racialist against outsiders , but what we call like gaujes or house people like yourself you likes your way , we got our own way . english gypsy 5 , mother me meself , i like me own way of life , i 've been brought up as a romany gypsy and that 's the way i want me boys to be brought up to be truthful . i 'm not racialist against outsiders , but what we call like gaujes or house people like yourself english gypsy 5 , mother few demands were made on health services by gypsy and traveller mothers at the time of pregnancy and birth , and most health professionals were described as offering little beyond the minimum routine service . roma mothers described breastfeeding as the usual method of feeding , which babies much enjoyed . while the overriding reason given for preferring breastfeeding was its participants placed more emphasis on ease and the fact it was common practice : it is better to breastfeed because it is healthier . . we do n't give the bottle , you just put the breast in his mouth and that 's it . translated roma 6 , mother well it 's easier because you do n't have to spend the money on powder milk and bottles and all that stuff . it 's not just about the money ; it 's just that i find it better . translated roma 8 , mother we raise them with the breast . translated roma 10 , grandmother it is better to breastfeed because it is healthier . we do n't give the bottle , you just put the breast in his mouth and that 's it . translated well it 's easier because you do n't have to spend the money on powder milk and bottles and all that stuff . and it 's not just about the money ; it 's just that i find it better . . translated roma 10 , grandmother when a mother had a baby in the roma community , help with breastfeeding was available from within the extended family . mothers could learn by watching others breastfeed , and some described being explicitly taught how to breastfeed by their own mother.my mum showed him my breasts and she was showing this is how you do it then you take it out and you.pat him and when you feed him you put him like this . translated roma 4 , mother my mum showed him my breasts and she was showing this is how you do it then you take it out and you.pat him and when you feed him you put him like this . translated by contrast , english gypsies and irish travellers described a predominantly bottlefeeding culture . as with the roma , feeding behaviours prevalent within the community were passed on to the next generation , but in this case , community knowledge was of how to prepare and store bottles of formula milk , and which brand to choose . some harmful practices continued among english gypsies and irish travellers including adding foods such as rusks in the bottle at an early age which was justified as being a customary practice and as having been recommended by health professionals in the past . some of the younger mothers were aware that adding solid foods to bottles was no longer advised , citing the immaturity of the baby 's digestive system as a reason to delay the introduction of solid foods . both irish traveller and english gypsy mothers continued with pured foods beyond the recommended date ( even up to 18 months ) , due to a fear that the baby would choke if offered finger foods . to avoid this , two mothers offered food which they had prechewed , a practice which was described as being a traditional custom among gypsies . some irish traveller and english gypsy mothers wished to try breastfeeding , primarily because they were aware of the health benefits.they said it was better for the baby , and so because they said that , i thought , go on then , i 'll try it . english gypsy 2 , mother there are more natural things in your own breast milk than are ever in formula i saw some traveller girls who would dare to breastfeed and i wanted to try . irish traveller 2 , mother they said it was better for the baby , and so because they said that , i thought , go on then , i 'll try it . english gypsy 2 , mother there are more natural things in your own breast milk than are ever in formula i saw some traveller girls who would dare to breastfeed and i wanted to try . irish traveller 2 , mother although most interviewees knew of a relative or friend who had breastfed , choosing to initiate breastfeeding carried a sense of breaking a taboo . breastfeeding was frequently described as not being part of the gypsy or traveller tradition , instead being something that gauje women would do . breastfeeding conflicted with ideals of female behaviour , such as covering the body at all times , particularly in the presence of the opposite sex . many mothers considered that breastfeeding could only be carried out alone in a private place , not even in the presence of other women.breastfeeding is something that i would not do . i 'd never do it , no one in my family would you ca n't pull out a boob in front of a man , it would a bit embarrassing like some travelling women do it , in the trailer . i 'd never do it , no one in my family would you ca n't pull out a boob in front of a man , it would a bit embarrassing like some travelling women do it , in the trailer . irish traveller 6 , mother one mother stated that because men did not understand the health benefits of breast milk , they preferred women to bottlefeed.most traveller men are old fashioned in their ways and strict about how women behave . these men think that giving the bottle is exactly the same without a woman exposing herself . irish traveller 2 , mother most traveller men are old fashioned in their ways and strict about how women behave . these men think that giving the bottle is exactly the same without a woman exposing herself . irish traveller 2 , mother the one instance where irish traveller and english gypsy interviewees described themselves as actively seeking help from midwives and health visitors was in initiating and continuing breastfeeding . advice was then sought from professionals because no skilled help was available from within their communities . while messages about the health benefits of breastfeeding were successfully assimilated , postnatal support for breastfeeding was described as insufficient , with professionals failing to grasp the extent of cultural taboos which made breastfeeding as a dangerously immodest act . a mother , who had bottle fed in hospital due to fear of being observed by visitors , received only telephone advice ( just carry on trying ) from her midwife when she subsequently asked for help in attempting to breastfeed her baby at home . this parsimonious support offered by health professionals contrasted unfavourably with the support which was always available from within the family and community . a warm , trusting relationship which had developed over time appeared a prerequisite for successfully supporting a mother to breastfeed.i do n't know what decided me to breastfeed , perhaps it was the midwife . with the last one i had the best service from the midwife . i do n't know what decided me to breastfeed , perhaps it was the midwife . with the last one i had the best service from the midwife . irish traveller 5 , grandmother names of helpful and respected health professionals were known throughout the community even if they worked in another city . participants considered that being informed about gypsy traveller culture was an important characteristic of the respected health professional . grandmothers described a tradition in romania of breastfeeding until around 2 years , with solid foods , such as polenta , pork and nettle soup , being introduced at around 56 months . however , once in the united kingdom , some mothers started giving formula feeds , even though this had led to some problems with milk supply , babies refusing the breast and subsequent constipation . commercially manufactured baby foods were given in place of solid family foods , sometimes as early as 4 weeks of age . more civilized and thinking it is better to give the bottle ( roma 10 , grandmother ) . several mothers stated that in the united kingdom , they could afford to bottlefeed and give commercially made baby foods , and therefore did so . there was no evidence of awareness that a very high standard of infant nutrition was being replaced with a less healthy diet.in romania i did n't have the possibility to buy all the things i needed to bottle feed , so that 's why i breast fed . translated roma 7 , grandmother in romania i did not have enough money for baby food ; i had to feed the same food as everyone else . roma 11 , mother in romania i did n't have the possibility to buy all the things i needed to bottle feed , so that 's why i breast fed . translated roma 7 , grandmother in romania i did not have enough money for baby food ; i had to feed the same food as everyone else . when asked about any potential longterm health benefits of breastfeeding for babies or mothers , no participant was aware of any impact on lifelong health . two roma women responded to this question by quoting a gypsy proverb , how will a man live if he does not eat? which places emphasis upon the importance of food to avoid starvation rather than seeking additional health benefits . for roma women , economic opportunities played a part in dictating infant feeding patterns . some introduced formula feeds in order to be able to do housework , shop or work outside the home , as other family members could bottlefeed . others clung to the traditional maternal role of which breastfeeding was seen as a part , in order to have freedom from paid labour.some [ women ] bottle feed their babies and they have other occupations like selling newspapers or doing something to get money . i just stay at home and take care of the children and my husband goes and works translated roma 1 , mother some [ women ] bottle feed their babies and they have other occupations like selling newspapers or doing something to get money . i just stay at home and take care of the children and my husband goes and works i 'd rather stay at home , rather than going and selling newspapers . translated for english gypsy and irish traveller , interviewees travelling could lead to disrupted contacts with health professionals due to moving between sites . for mothers who wanted to breastfeed , breastfeeding while living in a caravan placed a responsibility on the mother to avoid the risk of offending others . the sociability of travelling life contributed to the difficulties of finding an acceptable place to breastfeed , and for some shortened the duration of breastfeeding.i did try with the last one and i did it for a couple of months . if was difficult because i had no privacy i did n't have a problem with breastfeeding but it was very hard living in a caravan with people in and out i did n't mind giving up.irish traveller 5 , grandmother i did try with the last one and i did it for a couple of months . if was difficult because i had no privacy i did n't have a problem with breastfeeding but it was very hard living in a caravan with people in and out large families are commonplace among gypsies and travellers , and there is an expectation that older children help care for younger siblings . as a result few participants described themselves as requiring professional support in caring for children irrespective of age or number of children : the travelling community are reared up with children . i was married at 13 when you are small you are with your mum , and she shows you how to do things . roma 11 , mother i 'll tell you again , my mum had 10 kids and i helped look after them , i did n't need to learn from scratch , i already knew . some people do n't know , they have to learn from scratch and they need people telling them what to do and what not to do . i was married at 13 when you are small you are with your mum , and she shows you how to do things . i 'll tell you again , my mum had 10 kids and i helped look after them , i did n't need to learn from scratch , i already knew . some people do n't know , they have to learn from scratch and they need people telling them what to do and what not to do . english gypsy 4 , mother young motherhood increased participants view of themselves as being highly experienced in all aspects of child care . compared with the experiential knowledge that was easily available within the family , the advice of health professionals was often set at a low value by mothers and grandmothers.i do n't think they need any advice in feeding their children because it 's passed on from their own mums and grandmothers . irish traveller 1 , grandmother i 've no idea when health visitors say to introduce baby dinners . i think the health visitor advises 3 months but i would do what i thought , from my experience . lots of health visitors haven't had a baby ; they do n't know anything about it , just what they have read . irish traveller 4 , mother i do n't think they need any advice in feeding their children because it 's passed on from their own mums and grandmothers . irish traveller 1 , grandmother i 've no idea when health visitors say to introduce baby dinners . i think the health visitor advises 3 months but i would do what i thought , from my experience . lots of health visitors haven't had a baby ; they do n't know anything about it , just what they have read . irish traveller 4 , mother contact with health services was described as less common in the past ( early on they did n't want anything to do with travellers , now it 's more accepted. irish traveller 5 , grandmother ) , but english gypsies and irish travellers now expected contact with midwifery and health visiting services . roma women were sometimes unclear about difference in roles , referring to professionals generically as being from the doctor's. all described a minimal service with little routine contact beyond the immediate postnatal period . mothers took a pride in being capable mothers and did not see themselves as requiring extensive contact with health professionals.the health visitor came once or twice and saw i could get along , now she does n't come . translated roma 3 , mother the health visitor came once or twice and saw i could get along , now she does n't come . translated health professionals were described by all groups as relying on written materials to give health promotion information , despite low literacy levels . although none of the roma interviewees read english , most mentioned having been given leaflets . several nonroma participants also could not read health promotion materials : we received leaflets in english , about how to breast feed and what to expect when you 're a mum , but we do n't actually know how to read in english . translated roma 8 , mother she gave me some [ leaflets ] , but i ca n't read , not unless i ask someone to read it , then i find out what 's in it . i just looked at the pictures , but i did n't often look in the book . irish traveller 4 , mother we received leaflets in english , about how to breast feed and what to expect when you 're a mum , but we do n't actually know how to read in english . translated she gave me some [ leaflets ] , but i ca n't read , not unless i ask someone to read it , then i find out what 's in it . i just looked at the pictures , but i did n't often look in the book . irish traveller 4 , mother these contacts often served to highlight the differences between travelling people and the settled community . many participants had little contact with people outside their community and were aware only of their own infant feeding norms . english gypsy and irish traveller participants frequently expressed pride in their culture , and where there was opposition between family traditions and health professional advice , a common response was to reaffirm the positives about the community way. one mother commented to the researcher : me meself , i like me own way of life , i 've been brought up as a romany gypsy and that 's the way i want me boys to be brought up to be truthful . gaujes or house people like yourself you likes your way , we got our own way . english gypsy 5 , mother me meself , i like me own way of life , i 've been brought up as a romany gypsy and that 's the way i want me boys to be brought up to be truthful . i 'm not racialist against outsiders , but what we call like gaujes or house people like yourself you likes your way , we got our own way . english gypsy 5 , mother few demands were made on health services by gypsy and traveller mothers at the time of pregnancy and birth , and most health professionals were described as offering little beyond the minimum routine service . roma mothers described breastfeeding as the usual method of feeding , which babies much enjoyed . while the overriding reason given for preferring breastfeeding was its participants placed more emphasis on ease and the fact it was common practice : it is better to breastfeed because it is healthier . we do n't give the bottle , you just put the breast in his mouth and that 's it . translated roma 6 , mother well it 's easier because you do n't have to spend the money on powder milk and bottles and all that stuff . it 's not just about the money ; it 's just that i find it better . translated roma 8 , mother we raise them with the breast . translated roma 10 , grandmother it is better to breastfeed because it is healthier . we do n't give the bottle , you just put the breast in his mouth and that 's it . translated well it 's easier because you do n't have to spend the money on powder milk and bottles and all that stuff it 's not just about the money ; it 's just that i find it better . translated we raise them with the breast . translated roma 10 , grandmother when a mother had a baby in the roma community , help with breastfeeding was available from within the extended family . mothers could learn by watching others breastfeed , and some described being explicitly taught how to breastfeed by their own mother.my mum showed him my breasts and she was showing this is how you do it then you take it out and you.pat him and when you feed him you put him like this . translated roma 4 , mother my mum showed him my breasts and she was showing this is how you do it then you take it out and you.pat him and when you feed him you put him like this . translated by contrast , english gypsies and irish travellers described a predominantly bottlefeeding culture . as with the roma , feeding behaviours prevalent within the community were passed on to the next generation , but in this case , community knowledge was of how to prepare and store bottles of formula milk , and which brand to choose . some harmful practices continued among english gypsies and irish travellers including adding foods such as rusks in the bottle at an early age which was justified as being a customary practice and as having been recommended by health professionals in the past . some of the younger mothers were aware that adding solid foods to bottles was no longer advised , citing the immaturity of the baby 's digestive system as a reason to delay the introduction of solid foods . both irish traveller and english gypsy mothers continued with pured foods beyond the recommended date ( even up to 18 months ) , due to a fear that the baby would choke if offered finger foods . to avoid this , two mothers offered food which they had prechewed , a practice which was described as being a traditional custom among gypsies . some irish traveller and english gypsy mothers wished to try breastfeeding , primarily because they were aware of the health benefits.they said it was better for the baby , and so because they said that , i thought , go on then , i 'll try it . english gypsy 2 , mother there are more natural things in your own breast milk than are ever in formula breast milk has more vitamins and stuff that they ca n't put in sma i saw some traveller girls who would dare to breastfeed and i wanted to try . irish traveller 2 , mother they said it was better for the baby , and so because they said that , i thought , go on then , i 'll try it . english gypsy 2 , mother there are more natural things in your own breast milk than are ever in formula i saw some traveller girls who would dare to breastfeed and i wanted to try . irish traveller 2 , mother although most interviewees knew of a relative or friend who had breastfed , choosing to initiate breastfeeding carried a sense of breaking a taboo . breastfeeding was frequently described as not being part of the gypsy or traveller tradition , instead being something that gauje women would do . breastfeeding conflicted with ideals of female behaviour , such as covering the body at all times , particularly in the presence of the opposite sex . many mothers considered that breastfeeding could only be carried out alone in a private place , not even in the presence of other women.breastfeeding is something that i would not do . i 'd never do it , no one in my family would you ca n't pull out a boob in front of a man , it would a bit embarrassing like some travelling women do it , in the trailer . i 'd never do it , no one in my family would you ca n't pull out a boob in front of a man , it would a bit embarrassing like some travelling women do it , in the trailer . irish traveller 6 , mother one mother stated that because men did not understand the health benefits of breast milk , they preferred women to bottlefeed.most traveller men are old fashioned in their ways and strict about how women behave . these men think that giving the bottle is exactly the same without a woman exposing herself . irish traveller 2 , mother most traveller men are old fashioned in their ways and strict about how women behave . these men think that giving the bottle is exactly the same without a woman exposing herself . irish traveller 2 , mother the one instance where irish traveller and english gypsy interviewees described themselves as actively seeking help from midwives and health visitors was in initiating and continuing breastfeeding . advice was then sought from professionals because no skilled help was available from within their communities . while messages about the health benefits of breastfeeding were successfully assimilated , postnatal support for breastfeeding was described as insufficient , with professionals failing to grasp the extent of cultural taboos which made breastfeeding as a dangerously immodest act . a mother , who had bottle fed in hospital due to fear of being observed by visitors , received only telephone advice ( just carry on trying ) from her midwife when she subsequently asked for help in attempting to breastfeed her baby at home . this parsimonious support offered by health professionals contrasted unfavourably with the support which was always available from within the family and community . a warm , trusting relationship which had developed over time appeared a prerequisite for successfully supporting a mother to breastfeed.i do n't know what decided me to breastfeed , perhaps it was the midwife . with the last one i had the best service from the midwife . i do n't know what decided me to breastfeed , perhaps it was the midwife . with the last one i had the best service from the midwife . irish traveller 5 , grandmother names of helpful and respected health professionals were known throughout the community even if they worked in another city . participants considered that being informed about gypsy traveller culture was an important characteristic of the respected health professional . grandmothers described a tradition in romania of breastfeeding until around 2 years , with solid foods , such as polenta , pork and nettle soup , being introduced at around 56 months . however , once in the united kingdom , some mothers started giving formula feeds , even though this had led to some problems with milk supply , babies refusing the breast and subsequent constipation . commercially manufactured baby foods were given in place of solid family foods , sometimes as early as 4 weeks of age . more civilized and thinking it is better to give the bottle ( roma 10 , grandmother ) . several mothers stated that in the united kingdom , they could afford to bottlefeed and give commercially made baby foods , and therefore did so . there was no evidence of awareness that a very high standard of infant nutrition was being replaced with a less healthy diet.in romania i did n't have the possibility to buy all the things i needed to bottle feed , so that 's why i breast fed . translated roma 7 , grandmother in romania i did not have enough money for baby food ; i had to feed the same food as everyone else . roma 11 , mother in romania i did n't have the possibility to buy all the things i needed to bottle feed , so that 's why i breast fed . translated roma 7 , grandmother in romania i did not have enough money for baby food ; i had to feed the same food as everyone else . when asked about any potential longterm health benefits of breastfeeding for babies or mothers , no participant was aware of any impact on lifelong health . two roma women responded to this question by quoting a gypsy proverb , how will a man live if he does not eat? which places emphasis upon the importance of food to avoid starvation rather than seeking additional health benefits . for roma women , some introduced formula feeds in order to be able to do housework , shop or work outside the home , as other family members could bottlefeed . others clung to the traditional maternal role of which breastfeeding was seen as a part , in order to have freedom from paid labour.some [ women ] bottle feed their babies and they have other occupations like selling newspapers or doing something to get money . i just stay at home and take care of the children and my husband goes and works i 'd rather stay at home , rather than going and selling newspapers . translated roma 1 , mother some [ women ] bottle feed their babies and they have other occupations like selling newspapers or doing something to get money . i just stay at home and take care of the children and my husband goes and works i 'd rather stay at home , rather than going and selling newspapers . translated for english gypsy and irish traveller , interviewees travelling could lead to disrupted contacts with health professionals due to moving between sites . for mothers who wanted to breastfeed , breastfeeding while living in a caravan placed a responsibility on the mother to avoid the risk of offending others . the sociability of travelling life contributed to the difficulties of finding an acceptable place to breastfeed , and for some shortened the duration of breastfeeding.i did try with the last one and i did it for a couple of months . if was difficult because i had no privacy i did n't have a problem with breastfeeding but it was very hard living in a caravan with people in and out i did n't mind giving up.irish traveller 5 , grandmother i did try with the last one and i did it for a couple of months . if was difficult because i had no privacy i did n't have a problem with breastfeeding but it was very hard living in a caravan with people in and out this qualitative study presents the views of gypsy and traveller mothers and grandmothers in an under explored area . its strength lies in giving voice to women from a marginalized group to comment on the health services provided for them and to indicate in which aspects of infant feeding culturally sensitive health promotion could be developed . there are indications from this study that cultural beliefs about infant feeding between uk gypsy travellers are not identical , and a wider range of views may have emerged from a larger sample . limitations of the study include the use of romanian , rather than roma interpreters , which could reduce the ability of participants to express themselves , and the use of interpreters who had ongoing contact with families in all aspects of their lives . however , this study adds to research which suggests that infant feeding in the united kingdom is influenced by ethnicity and culture36 , 37 , 38 and indicates that a common gypsy traveller identity influences infant feeding practices . shared aspects of gypsy life , such as observing defined gender roles , having large families and living within a close community13 , 16 , influence how mothers feed their babies . this study has highlighted that common aspects of the gypsy traveller identity affect feeding differently in different groups , a finding which has important implications for policy and practice in health promotion . in the roma community , studies of other ethnic groups have shown that migrant mothers who interact least with the host community are most likely to retain traditional feeding habits.38 , 39 romanian roma mothers exhibited a strong sense of community and could describe traditional feeding behaviours but took a pragmatic approach to changing these , particularly if infant feeding needed to fit in with the demands of paid and unpaid work . the economic necessity to return to work soon after delivery has been previously identified as a factor reducing migrant women 's ability to breastfeed.40 roma women described limited involvement with health professionals and seemed unaware of the potential cost to infant and maternal health in switching to formula feeding and early weaning . ceasing to practice traditional infant feeding behaviours , which are superior to the usual feeding practices of the uk settled population , poses an increased risk to child and maternal health . as roma children are known to have a higher prevalence of health risk factors,41 it is important to recognize where new risks are posed to child and maternal health . in this study , english gypsies and irish travellers exhibited a strong awareness of their political and cultural identity and commonly described their behaviour as characteristic of their community and unlike that of the gauje or nongypsies . shared infant feeding practices may play a part in maintaining family traditions and values , hence contributing to the community cohesion and resilience which can combat the effects of social disadvantage and racism.42 gypsies and travellers have been noted to define their identity as much by what is rejected as by what is chosen,18 and in this study , the identification of practices , such as breastfeeding , as something that a gypsy would not do demonstrates this . as demonstrated in other studies , developing a relationship with a trusted health professional appeared to be a key factor in facilitating breastfeeding , particularly for mothers who face exceptional barriers.43 , 44 in a community , where family support is ubiquitously present , it is vital that health professionals offer a responsive and accessible service . to this end , guidelines have previously been developed to assist health professionals in working effectively with gypsies and travellers.45 despite these , this study has highlighted again the futility of promoting the health benefits of breastfeeding in the antenatal period while failing to provide an adequate service postnatally.46 the disadvantage experienced by gypsies and travellers is exemplified by the demographic details of participants ( table 1 ) , which showed that participants had larger than average families , younger age of maternity and low educational achievement in comparison with the majority uk population ; all these factors increase disadvantage and reduce life chances.47 given the emphasis on providing health promotion and support to the most disadvantaged in the healthy child programme , it is remarkable that mothers in this study described receiving very little targeted support from health professionals . the frequency with which health professionals were reported as giving health education leaflets to women who did not read english suggests that cultural competency is undeveloped and not prioritized by practitioners.48 mcfadden et al . suggest that current provision of community breastfeeding support may be inappropriate and inaccessible to some women from minority ethnic backgrounds,49 and this study lends corroboration to this view . the effects of social disadvantage , exclusion and racism compound the difficulties faced by gypsy it is important to explore the views of gypsies and travellers in order to gain insight into their health needs and to address the extreme health inequalities they experience . this study has implications for policy and the practice of health professionals , both in indicating the customary feeding behaviours of some gypsy and traveller groups , and in suggesting how culturally sensitive support could facilitate optimal infant feeding practices . in a culture which prizes children , improving health outcomes by ensuring the best infant feeding is a potentially powerful health promotion message . this study was internally funded by the university of the west of england as part of the spur early career researcher funding stream .
abstractbackgroundgypsies and travellers are known to have poor health status and access to health services , even in comparison with other ethnic minority groups . people from this stigmatized ethnic group are rarely consulted about their health needs or health service provision . optimal infant feeding in the first year of life has the potential to improve lifelong health.objectivethe aim of this study was to explore mothers and grandmothers views on feeding in the first year of life , including the support provided by health professionals.methodssemistructured interviews were conducted with a purposively selected sample of 22 mothers and grandmothers of english gypsy , irish traveller and romanian roma ethnicity between november 2011 and february 2012 in a city in southwest england.resultsfew women perceived themselves as requiring help from health professionals in infant feeding , as acceptable and accessible support was available from within their own communities . roma mothers described a tradition of breastfeeding and appropriately timed weaning , while english gypsies and irish travellers customarily practised less healthy infant feeding . when mothers requested support , health service provision was often found inadequate.conclusionexploring the views of gypsies and travellers is important to gain insight into the provision of health services for this marginalized ethnic group . this study has implications for policy and the practice of health professionals , in indicating the customary feeding behaviours of some gypsy and travellers , and highlighting areas meriting culturally sensitive health promotion .
Introduction Methods Research design Participants Ethics Data collection Data analysis Results Centrality of the family Beliefs and traditions Travelling Discussion Conclusion Funding Conflict of interest
research into the health needs of this group is an emerging field , and gypsies and travellers could justly be described as an invisible minority1 in being rarely captured in health statistics , unassertive of their health needs and with few champions.2 , 3 in the united kingdom , gypsies and travellers are known to have poorer health status and a higher risk of mortality than socioeconomically matched comparison groups4 and to experience health inequalities which are greater than could be expected simply from socioeconomic disadvantage or from belonging to a minority ethnic group.5 gypsies and travellers access to health services is also known to be poor.6 , 7 in several european countries , roma people have been shown to have poorer health than other ethnic groups and poorer access to health services,8 , 9 , 10 and a recent literature review identified a higher prevalence of both communicable and noncommunicable disease in the roma community with significantly shorter life expectancies than national averages.11 gypsies and travellers share a history of persecution and rejection by mainstream society , which continues today.12 , 13 , 14 , 15 the umbrella term gypsies and travellers covers a diversity of people , including continental roma , english and welsh gypsies and irish and scottish travellers.3 in the 2001 , uk census gypsy or irish traveller was included as an ethnic category for the first time . the uk healthy child progamme20 stipulates that mothers should be visited antenatally to support infant feeding and that postnatal visits should promote breastfeeding , with introduction of solids foods at around 6 months , as recommended by the world health organization.21 encouraging engagement with children 's preventive health services has been recognized as the starting place for reducing health inequalities among gypsies and travellers , particularly in the area of nutrition.11 how a baby is fed in the first year of life has an impact upon health in the long and short term.21 , 22 , 23 breastfed babies are less likely to experience morbidity in the first year of life24 and to have a reduced risk of diabetes , hypertension and obesity in later life.25 health benefits for mothers who breastfeed include a reduced risk of breast and ovarian cancer and type 2 diabetes.24 in the united kingdom , infant feeding behaviour is strongly related to socioeconomic class and ethnicity , with highly educated professional women most likely to breastfeed among the white community , but mothers from nonwhite ethnic minority groups more likely to breastfeed than the general population.22 despite exclusive breastfeeding for 6 months being advised , only 34% of babies in the united kingdom are breastfed at all at 6 months of age and only 1% exclusively.22 a small quantitative study of gypsies and travellers feeding practices in one primary care trust in england , suggested very low breastfeeding rates , with only 3% estimated to have initiated breastfeeding and none continuing to 68 weeks ( data based on health visitors reports ) ; however , a survey of 20 gypsy traveller women suggested a rate of 15% who had ever breastfed.26 breastfeeding rates were found to be slightly above average among roma mothers in serbia27 , but no difference was identified between the duration of breastfeeding for czech and roma children ( a median duration of 3 months breastfeeding in both groups).28 no studies have looked at weaning practices among gypsies and travellers , although these are recognized as being important in relation to continuation of breastfeeding,22 and to later eating habits which have an impact upon risk of obesity and cardiac disease.29 , 30 the aim of this study was to explore the views of gypsy traveller mothers and grandmothers on infant feeding and health service provision . semistructured interviews were conducted with a purposive sample of mothers and grandmothers from english gypsy , irish traveller and romanian roma communities between november 2011 and february 2012 . the framework approach allowed movement from raw data to abstraction in the analytical process , without losing the voice of participants.35 semistructured interviews were conducted with a purposive sample of mothers and grandmothers from english gypsy , irish traveller and romanian roma communities between november 2011 and february 2012 . to this end , guidelines have previously been developed to assist health professionals in working effectively with gypsies and travellers.45 despite these , this study has highlighted again the futility of promoting the health benefits of breastfeeding in the antenatal period while failing to provide an adequate service postnatally.46 the disadvantage experienced by gypsies and travellers is exemplified by the demographic details of participants ( table 1 ) , which showed that participants had larger than average families , younger age of maternity and low educational achievement in comparison with the majority uk population ; all these factors increase disadvantage and reduce life chances.47 given the emphasis on providing health promotion and support to the most disadvantaged in the healthy child programme , it is remarkable that mothers in this study described receiving very little targeted support from health professionals . this study has implications for policy and the practice of health professionals , both in indicating the customary feeding behaviours of some gypsy and traveller groups , and in suggesting how culturally sensitive support could facilitate optimal infant feeding practices .
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parkinson 's disease ( pd ) is an age - related neurodegenerative disease with progressive loss of dopaminergic ( da ) neurons in the substantia nigra pars compacta ( snpc ) . in patients , this depletion of neurons presents clinically with severe motor symptoms including uncontrollable resting tremor , bradikinesia , rigidity and postural imbalance ( lang and lozano , 1998a ; 1998b ; lotharius and brundin , 2002 ) . these symptoms , which affect 1% of individuals over the age of 65 , start to manifest when 70~80% of da neurons in the snpc are lost ( mandel et al . the exact etiology of pd remains to be fully elucidated , but the key theories propose either an environmental ( e.g. insecticides ( calne et al . , 1987 ; schoenberg , 1987 ; paolini et al . , 2004 ) ) or a genetic ( e.g. parkin ( kitada et al . , 1998 ) the market value for pd and ad therapies exceeded us$6.5 billion , ( schapira et al . , 2005 ) with projections that these will surpass cancer as the second most common cause of death of the elderly ( lang and lozano , 1998a ) . therefore , there is a real sense of urgency to discover novel therapies for the treatment or preferably , prevention of these diseases . currently the only therapies approved for the treatment of pd and ad are agents that attenuate the symptoms ( symptomatic ) of the disease without disease modifying activity except the anti parkinson drug rasagiline ( azilect ) ( olanow et al . , 2009 ) , which we developed ( youdim et al . , 2005 ) the mainstay for pd treatment focuses on the replacement of lost da with l - dopa , dopamine ( da ) agonists , monoamine oxidase b inhibitors and catechol - o - methyl tranferase inhibitors , thereby normalizing the patient symptomatically ( schapira et al . , 2005 ) . while for ad there are the cholinesterase inhibitors and the glutamate antagonist memantine . tragically , but important in view of the seriousness of disease progression , is the fact that the course of the disease is not affected by the utilization of these drugs , and the loss of neurons continues unabated even as symptoms may be controlled , at least following initial treatment . currently , no drugs with claimed neuroprotective activity have been approved by the fda for the treatment of pd or ad ( table 1 ) ( mandel et al . , 2003 ; stocchi and olanow , 2003 ) . significantly though , recent research has suggested that some drugs used for symptomatic relief in pd , such as azilect , pramipexole ( hall et al . , 1996 ; dooley and markham , 1998 ; piercey , 1998 ) and memantine ( rogawski and wenk , 2003 ; plosker and lyseng - williamson , 2005 ) may also possess neuroprotective activities , rasagiline is currently only drug that may have a disease modifying activity ( olanow et al . , 2009 ) . recent literature show that there has been a paradigm shift in the way researchers are considering the development and design of drugs to treat diseases with complex etiological pathways ( i.e. diseases with multiple drug targets ) ( morphy et al . , 2004 ; gal et al . , 2005 ; morphy and rankovic , 2005 ; youdim and buccafusco , 2005a ; 2005b ; van der schyf et al . , 2006a ; 2006b ; zimmermann et al . , 2007 ) a drug with a single - target mechanism of action can not always compensate or correct a complex pathway , which suggests that a complex pathway disease should be treated 1 ) with a multitude of molecules , each acting on different pathways in the disease ( polypharmacy ) , or 2 ) with one molecule that possesses promiscuous activity acting on different pathways ( multiple mechanism drugs ) . 1 ) therefore , is the clinical practice of combining two or more medications in a patient 's medication profile , with a view to treat one specific disease . for example , the combination use of salmeterol ( a 2-adrenergic agonist ) and fluticasone ( a glucocorticoid steroid ) in asthma , has led to the combination of these two medications in one ( advair ) . also , the combination ( in vytorin ) of simvastatin ( an hmg - coa reductase inhibitor ) and ezetimibe ( an inhibitor of dietary cholesterol uptake ) is used to treat hyperlipidemia ( zimmermann et al . , 2007 ) . the major dilemma encountered in a polypharmaceutical approach , is a significant chance increase in side effects , which may be reduced statistically with the use of only one compound . the recent appearance on the market of drugs that display two mechanisms to treat a particular disease has been a clear move in the direction of the latter paradigm . one example , duloxetine ( cymbalta ) , used in the treatment of depression , inhibits both serotonin and norepinepherine uptake in the central nervous system ( cns ) ( wong et al . , 1988 ; kihara and ikeda , 1995 ; the introduction of drugs such as duloxetine indicates the clinical feasibility of designing multi - functional ligands to treat cns disorders with complex disease pathways . in this review , we will consider examples of compounds with multi - functional neuroprotective - neurorescue ( table 1 for definitions ) properties that may have promise in the treatment of pd and similar approaches have been made for multimodal drugs for ad ( youdim et al . , 2006 ; zheng et al . , 2009 ) , but for the present discussion we shall focus on pd only . some of the compounds discussed were discovered through serendipity while others were the products of active drug design projects . rasagiline ( n - propargyl-1r - aminoindan ) is an anti - pd drug with selective mao - b inhibitory activity ( youdim et al . , 2005a ) . its s isomer , tv1022 ( n - propargyl-1s - aminoindan ) , is more than a 1,000 times less potent as an mao inhibitor than rasagiline , but still retains neuroprotective activity , which suggests that the propargylamine moiety ( even when ostensibly not involved in michael chemistry at the fad within the mao catalytic site as the processing group in suicide inhibition ) is responsible for the neuroprotective activity seen in both these compounds ( weinreb et al . the selectivity of rasagiline as an mao - b inhibitor compared with tvp-1022 , is thought to be associated with the ability of rasagiline to enter the catalytic site gorge of mao - b . on the other hand , the configuration of the s - isomer imparts a highly restrictive conformation on the enzyme - ligand complex , which prevents the molecule from entering the catalytic site , precluding it from acting as a mechanism - based inhibitor . interestingly , the neuroprotective activity associated with these compounds has now been shown to be associated with the ability of propargylamine ( weinreb et al . , 2004a ; bar - am et al . , 2005 ) to protect mitochondrial viability by activation of bcl-2 and protein kinase c ( pkc) and , and by down regulating proapoptotic fas and bax , and pkc and - ( youdim et al . , 2005a ) additionally , these drugs induce the release of the soluble neuroprotective - neurotrophic form of the amyloid precursor protein ( sapp ) through a pck - map mediated activation of -secretase ( youdim and buccafusco , 2005b ) . the identification of the propargylamine moiety as a key element that confers neuroprotective activity and , in cases such as rasagiline and selegiline , also mao inhibitory activity , led to the development of ache inhibitors such as ladostigil ( tv3326 , now in phase ii clinical studies ) , another anti - alzheimer ( ad)/anti - pd / antidepressant drug ( sterling et al . , 2002 ; weinstock et al . , 2002 ; youdim et al . , 2005a ; youdim and buccafusco , 2005b ) . 3 ) is a dual acetylcholine - butyrylcholine - esterase , and brain - selective mao - a / b inhibitor in vivo , designed by combining the carbamate cholinesterase inhibitory moiety found in the rivastigmine molecule , with the pharmacophore of rasagiline and tvp1022 both of which possess the propargylamine moiety . ladostigil has been shown to have antidepressant activity due to its ability to inhibit mao - a in the raphe nucleus , striatum , hippocampus , and hypothalamus , and to raise brain levels of da , norepinephrine , and serotonin ( weinstock et al . , 2002 ) . its ability to also inhibit mao - b attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) toxicity in mice , a rodent model of parkinsonism ( sagi et al . although a poor mao - b inhibitor , the s isomer of ladostigil , tv3279 , has shown similar neuroprotective activity to rasagiline and ladostigil in vitro and in laboratory animals ( youdim and buccafusco , 2005b ) , with molecular mechanisms apparently identical to that of rasagiline . degenerating nigrostriatal da neurons are the main pathological feature in the snpc of pd sufferers . in addition , many pd patients also experience dementia and depression that likely result from sporadic neurodegeneration in cholinergic , noradrenergic , and serotonergic pathways . in pd , accumulation of iron is found inside some melanin - containing da - ergic neurons and inside amyloid plaques and neurofibrillary tangles associated with pd dementia ( zecca et al . , it has been suggested that iron accumulation may contribute to the oxidative stress - induced apoptosis reported in both pd and pd dementia ( zecca et al . , 2004 ; youdim et al . , 2005b ) . such oxidative stress may result from increased glial monoamine oxidase ( mao ) activity leading to exacerbated hydrogen peroxide production that can generate reactive hydroxyl radical through fenton chemistry with intracellular ferrous iron . iron chelators such as desferoxamine , clioquinol and vk-28 have been shown to have neuroprotective activity in animal models of ad and pd ( zecca et al . , 2004 ) . ( 2005b ) developed neuroprotective compounds with dual iron chelating and mao - b inhibitory activity . these authors combined the antioxidant chelator moiety present in an 8-hydroxyquinoline derivative of the neuroprotective brain - permeable iron chelator vk-28 , with the propargylamine moiety ( found in compounds such as rasagiline and selegiline , as stated earlier ) . hla20 was identified as a potential lead compound for further studies having selectivity for mao - b with an ic50 value in the region of 110 m ( fig . 4 ; > 200 m for mao - a ) , as well as acting as a free radical scavenger . however , a related compound designated m30 , unlike hla20 was found in vitro , to be a highly potent mao - a and b inhibitor with brain selectivity for these enzymes in vivo , in addition to possessing iron chelating properties similar to desferoxamine ( gal et al . , 2005 ; zheng et al . , 2005a ; 2005b ) . m30 behaves similarly to other propargylamine mao inhibitors by acting as a suicide- or mechanism - based inhibitor after being identified and processed as a substrate by the enzyme and imparts similar neuroprotective properties as those found in rasagiline and ladostigil ( fig . m30 protects against mptp and kainate neurotoxicity in mice by virtue of both its mao inhibitory and iron chelating / radical scavenging properties in these two animal models of neurodegeneration . it has recently been shown to have dopaminergic neurorestorative activity in post treatment with mptp ( gal et al . , 2009 ) and lactacystin ( zhu et al . , 2007 ) models of pd . the neurogenic activity of m30 and hla-20 has been attributed to the inhibition of iron dependent prolyl-4-hydroxylase , via chelation of iron and activation of hif ( hypoxia inducing factor ) that regulates transcription of a series of neurotropins such as bdnf , gdnf , erythropoietin and vegf . the consequence of hif activation is inhibition of cell cycle g / g , that results in inhibition of cyclin d1 that causes cell arrest differentiation into neurons as seen in the neurorestorative activity of m30 in the two models of pd ( zhu et al . we have introduced a carbamate cholinesterase inhibitor ( chei ) moieties into m30 , such as m30c - n and into hla-20 to give hla-20a and have even added the glutamate antagonist , memantine , which is presently in the clinical use . these compounds 7 ) have been shown to have potent che i and mao - a and b inhibitory activity and possess similar neuroprotective activity to those of their parent compounds , hla-20 and m30 ( zheng et al . , 2009 ) . the accumulation of iron at sites where neurons degenerate in pd , is thought to be a major event that is linked to the neurodegenerative process ( zecca et al . , 2004 ) . the novel non - toxic lipophilic ( and therefore brain - permeable ) iron chelator vk-28 , and its multi - functional derivative , m30 ( both of which possess the mao inhibitory and neuroprotective propargyl moiety of rasagiline ) , offer potential therapeutic benefits for pd . m30 attenuates apoptotic events in sh - sy5y neuroblastoma cells in a serum deprivation model via multiple protection mechanisms , including 1 ) reduction of the pro - apoptotic proteins , bad and bax ; 2 ) reduction of apoptosis - associated ser139-phosphorylated h2a.x ; 3 ) induction of the anti - apoptotic protein , bcl-2 and 4 ) inhibition of the cleavage and activation of caspase-3 . m30 also promotes morphological changes , resulting in axonal growth - associated protein-43 ( gap-43 ) , which is implicated in neuronal differentiation . the compound markedly reduces the levels of cellular holo - app , the -c - terminal fragment ( -ctf ) , and levels of amyloidogenic a peptide in the medium of sh - sy5y and cho cells stably transfected with the app " swedish " mutation . in addition , levels of the nonamyloidogenic sapp in cell medium , as well as levels of -ctf in cell lysate were found to be elevated . these results are consistent with the presence of an iron - responsive element ( ire ) in the 5'-untranslated region ( 5'utr ) of app and demonstrate the effectiveness of m30 in limiting holo - app expression and a peptide secretion . therefore , the multifunctional properties of m30 suggest that it may offer extraordinary potential as a drug for the treatment of pd , especially pd dementia ( avramovich - tirosh et al . , 2006 ) and ad ( mandel et al . , 2007 ; 2008 ) and more recently in transgenic g93a sod model of als ( amyotrophic lateral sclerosis ) where it extends the life span of these animals and has neurogenic activity in ncs-34 rat motor neurons ( kupershmidt et al . , 2009 ) . in pd , a dual mechanism that includes inhibition of mao - b , as well as adenosine a2a receptor blockade as detailed earlier , mao - b plays a role in the catabolism of neurotransmitters such as da , serotonin and norepinephrine , leading to hydrogen peroxide formation which contributes to oxidative stress and neuronal cell death ( riederer et al . , 2004 ) . levels of mao - b are found to be increased in older patients ( saura et al . , 1994 ; 1997 ; mahy et al . , 2000 ) which has led to the rationale for the use of drugs such as selegiline ( deprenyl ) and lazabemide , ( sramek and cutler , 1999 ) and the design of drugs such as ladostigil , ( youdim and buccafusco , 2005b ) as described before . caffeine , a non - selective adenosine receptor antagonist , is under some scrutiny as a potential drug to counteract age - related cognitive decline ( fig . work in this regard is supported by evidence that critical changes in adenosine - related neurotransmission occur with aging and may be counteracted by adenosine receptor antagonists ( maiade and mendonca , 2002 ; dall'igna et al . , 2003 ; prediger et al . , 2005 ) . caffeine , in fact , has been suggested to protect against -amyloid neurotoxicity , ( dall'igna et al . , 2003 ) while acute treatment with caffeine and the a2a receptor antagonist zm241385 was recently found to reverse age - related olfactory deficits and memory decline in rats ( prediger et al . , 2005 ) clearly suggesting involvement of a2a , but not a1 receptors , in cognitive decline and possibly , neurodegenerative processes . evidence such as the preceding , and other evidence for neuroprotection also in parkinsonian models , led petzer et al . ( petzer et al . , 2003 ) to evaluate ( e)-8-styrylxanthinyl derived adenosine a2a receptor antagonists for inhibition also of brain mao - b . included in these studies were kw-6002 , a potent a2a receptor antagonist ( ki of 2.2 nm ) which is undergoing clinical trials for pd , and ( e)-8-(3-chlorostyryl)-caffeine ( csc ; fig . 8) , which has been shown to be neuroprotective in the mptp parkinsonian mouse model ( chen et al . , 2002 ) . ( petzer et al . , 2003 ) showed mao - b inhibition in the low micromolar to high nanomolar range , with the ki of kw-6002 at 21 m , and that of csc at 0.1 m . these results clearly suggest that the neuroprotective properties of kw-6002 and csc may in part be due to mao - b inhibition , in synergism with the a2a antagonism ( castagnoli et al . , 2003 ) . the divalent calcium cation plays an important role in neuronal cell death ( lipton , 1999 ; horn and limburg , 2000 ; kemp and mckernan , 2002 ; ovbiagele et al . , 2003 ) . one of the receptors activated by glutamate ( together with its co - agonist glycine ) , the nmda receptor , is a major conduit for the influx of calcium ions into cells under excitotoxic conditions . the prevention of such excessive influx of calcium ( known as excitotoxicity ) therefore remains a major drug target in the design of neuroprotective agents . excess accumulation of calcium in neuronal cells rapidly leads to cell death through a variety of mechanisms including activation of proteases , nucleases , phospholipases , nitric oxide synthase ( nos ) , and other degradative enzymes that not only lead to activation of death cascades , but also to free radical formation ( lipton , 1999 ) . nmda receptor antagonists such as dizocilpine ( mk-801 ) and memantine may possess a dual mechanism by which neuronal cells are protected , both by direct blockade of the nmda receptor and by attenuating tnf-induced potentiation of glutamate toxicity ( zou and crews , 2005 ) . brain injury after ischemic stroke also triggers a release of glutamate - associated excitotoxic events , and the incidence of cognitive impairment and dementia have both been reported to be elevated after cerebral stroke , especially in the elderly ( kalaria and ballard , 2001 ) . up to 25% of stroke patients exhibit symptoms of dementia , including symptoms reminiscent of pd dementia ( van kooten and koudstaal , 1998 ) . stroke is the third leading cause of death in the united states ( ovbiagele et al . , 2003 ) and there is a definitive need to develop drugs that can protect or save neurons after an ischemic incident since , to date , no effective treatment has been developed to prevent neuronal cells from dying during stroke conditions ( horn and limburg , 2000 ) . several studies have shown that nmda receptor antagonists , such as dizocilpine ( mk-801 ) and the polycyclic cage amine memantine , display neuroprotective effects in experiments using ischemia paradigms in neurons ( gorgulu et al . , 2000 ; horn and limburg , 2000 ; gerriets et al . an alternative pathway for calcium to enter into neuronal cells is through voltage - gated ion channels , such as l - type calcium channels . animal experiments with nimodipine have suggested that calcium channel antagonists may be neuroprotective in ischemia by antagonizing the influx of calcium into neuronal cells ( horn and limburg , 2000 ) . the importance of calcium overload during cell death , suggests that a dual calcium channel and nmda receptor antagonist might be useful as a neuroprotective drug in stroke and other neurodegenerative disease such as idiopathic pd , where it has been suggested that brain - permeable l - type calcium channel blockers may have a salutary effect on the disease . ngp1 - 01 ( 8-benzylamino-8,11-oxapentacycloundecane ) is a polycyclic cage amine derived from the reductive amination of benzylamine and cookson 's " bird cage " diketone of which the biology was first described by van der schyf ( van der schyf et al . , 1986 ) ( fig . the l - type calcium channel blocking activity of ngp1 - 01 was investigated utilizing electrophysiological experiments in isolated guineapig papillary muscle and sheep purkinje fibers ( van der schyf et al . , 1986 ) . the structural similarity of ngp1 - 01 to another polycyclic cage amine and nmda receptor antagonist , memantine , led to the evaluation of ngp1 - 01 for potential nmda receptor antagonism . memantine is an uncompetitive nmda receptor antagonist which is used clinically to treat ad , but has also been used for pd in germany ( parsons et al . , 1999 ; rogawski and wenk , 2003 ; plosker and lyseng - williamson , 2005 ) . its favorable fast on - off binding kinetics gives this compound an improved side effect profile compared with other nmda antagonists such as mk-801 ( parsons et al . ngp1 - 01 was shown to also be an uncompetitive nmda antagonist in murine whole brain synaptoneurosomes and blocked nmda - mediated ca uptake with an ic50 of 2.98 m ( geldenhuys et al . , 2007 ) . ( 2006 ) showed that ngp1 - 01 ( at 1 m ) inhibited depolarizationinduced calcium influx by 78% in cortical neurons preloaded with fura-2 am , with a potency similar to that of nimodipine , while simultaneously inhibiting nmda - induced ( 1 mm ) calcium influx by 52% , only slightly less potent than memantine . using in vivo - microdialysis , intraperitoneal injection of ngp1 - 01 ( 40 mg / kg ) reduced nmda - induced membrane breakdown by 31% ( p<0.01 ) while memantine ( 10 mg / kg ) reduced choline release by 40% . these results demonstrate that ngp1 - 01 simultaneously blocks both major neuronal calcium channels and is brain - permeable after peripheral administration . this dual mechanism of modulating calcium entry into neuronal cells might suggest that ngp1 - 01 may have utility as a neuroprotective agent in pd , stroke and other neurodegenerative diseases , especially in patients with comorbidity among these diseases . this promise of neuroprotection has recently been partly confirmed in in vivo studies using the middle cerebral artery occlusion ( mcao ) mouse model of stroke , wherein it was shown that ngp1 - 01 , administered 30 minutes before mcao , afforded substantial protection against cerebral ischemia - induced brain lesioning , as well as brain swelling measured 24 hours after mcao ( mdzinarishvili et al . , 2005 ) . another role assigned to cage amines such as ngp1 - 01 in pd therapy is the ability of these compounds to inhibit da re - uptake into nerve terminals ( fig . compounds that are able to block the da transporter ( dat ) have been suggested to be more useful in treating the motor symptoms in pd , as opposed to norepinephrine and serotonin re - uptake inhibitors ( hansard et al . , 2002 ) . additionally , compounds with the ability to block dat , may also have neuroprotective activity ( kirby et al . , 2002 ) . ngp1 - 01 was recently shown to block da re - uptake in murine synaptosomes with an ic50 of 57 m . one of ngp1 - 01 's derivatives , a phenylethylamine derivative , was even more potent with an ic50 of 23 m ( geldenhuys et al . , 2004 ) . the latter compound was also found to be neuroprotective in the mptp - parkinsonian mouse model , affording protection against a single 35 mg / kg ( ip ) dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) ( geldenhuys et al . , 2003 ) . polyphenols are natural products present in beverages such as red wine and tea ( weinreb et al . , 2004b ) . one of the classes of polyphenols which are pharmaceutically interesting is the flavenoids ( fig . these compounds are characterized by an aromatic ring which is condensed to a heterocyclic ring and attached to a second aromatic ring . an innovative therapeutic approach could be the use of natural plant polyphenol flavonoids , reported to have access to the brain and to possess multifunctional activities as iron chelators , radical scavengers , anti - inflammatory agents and neuroprotectants ( morel et al . , 1993 ; guo et al . , 1996 ; hider et al . , 2001 ; joseph et al . , 2005 ) . these compounds and their actions have been extensively reviewed ( mandel et al . , 2005 ) . in particular , the major constituent of green tea catechin extract ( -)-epigallocatechin-3-gallate ( egcg ; fig . 11 ) plays a major role in the prevention of neurodegeneration in a variety of cellular and animal models of neurodegenerative diseases ( mandel et al . , 2006 ) . this effect appears to be mediated through multiple pathways , including the participation of the pro - survival pkc and extracellular mitogen - activated protein kinase ( mapk ) signaling and the promotion of neurite outgrowth ( reznichenko et al . , 2005 ) . structurally important features defining their chelating potential are the 3',4'-dihydroxyl group in the b ring ( hider et al . , 2001 ) , as well as the gallate group ( kumamoto et al . , 2001 ) which may neutralize ferric iron to form redox - inactive iron , thereby protecting cells against oxidative damage ( grinberg et al . , 1997 ) . recent studies have shown that prolonged administration of egcg to mice induced a significant reduction in membrane - associated app levels in hippocampus ( levites et al . , 2003 ) and in cerebral a levels concomitant with reduced -amyloid plaques ( rezai - zadeh et al . , 2005 ) . this effect may be accounted for , in part , by the chelation of the intracellular free - iron labile pool , modulating app mrna translation via its ire - type ii ( reznichenko et al . , 2006 ) , as has recently been described for other metal chelators , such as desferoxamine , clioquinol and dimercaptopropanol ( payton et al . , 2003 ; rogers and lahiri , 2004 ) . rasagiline ( n - propargyl-1r - aminoindan ) is an anti - pd drug with selective mao - b inhibitory activity ( youdim et al . , 2005a ) . its s isomer , tv1022 ( n - propargyl-1s - aminoindan ) , is more than a 1,000 times less potent as an mao inhibitor than rasagiline , but still retains neuroprotective activity , which suggests that the propargylamine moiety ( even when ostensibly not involved in michael chemistry at the fad within the mao catalytic site as the processing group in suicide inhibition ) is responsible for the neuroprotective activity seen in both these compounds ( weinreb et al . the selectivity of rasagiline as an mao - b inhibitor compared with tvp-1022 , is thought to be associated with the ability of rasagiline to enter the catalytic site gorge of mao - b . on the other hand , the configuration of the s - isomer imparts a highly restrictive conformation on the enzyme - ligand complex , which prevents the molecule from entering the catalytic site , precluding it from acting as a mechanism - based inhibitor . interestingly , the neuroprotective activity associated with these compounds has now been shown to be associated with the ability of propargylamine ( weinreb et al . to protect mitochondrial viability by activation of bcl-2 and protein kinase c ( pkc) and , and by down regulating proapoptotic fas and bax , and pkc and - ( youdim et al . , 2005a ) . additionally , these drugs induce the release of the soluble neuroprotective - neurotrophic form of the amyloid precursor protein ( sapp ) through a pck - map mediated activation of -secretase ( youdim and buccafusco , 2005b ) . the identification of the propargylamine moiety as a key element that confers neuroprotective activity and , in cases such as rasagiline and selegiline , also mao inhibitory activity , led to the development of ache inhibitors such as ladostigil ( tv3326 , now in phase ii clinical studies ) , another anti - alzheimer ( ad)/anti - pd / antidepressant drug ( sterling et al . 3 ) is a dual acetylcholine - butyrylcholine - esterase , and brain - selective mao - a / b inhibitor in vivo , designed by combining the carbamate cholinesterase inhibitory moiety found in the rivastigmine molecule , with the pharmacophore of rasagiline and tvp1022 both of which possess the propargylamine moiety . ladostigil has been shown to have antidepressant activity due to its ability to inhibit mao - a in the raphe nucleus , striatum , hippocampus , and hypothalamus , and to raise brain levels of da , norepinephrine , and serotonin ( weinstock et al . , 2002 ) . its ability to also inhibit mao - b attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) toxicity in mice , a rodent model of parkinsonism ( sagi et al . , 2003 ) . although a poor mao - b inhibitor , the s isomer of ladostigil , tv3279 , has shown similar neuroprotective activity to rasagiline and ladostigil in vitro and in laboratory animals ( youdim and buccafusco , 2005b ) , with molecular mechanisms apparently identical to that of rasagiline . degenerating nigrostriatal da neurons are the main pathological feature in the snpc of pd sufferers . in addition , many pd patients also experience dementia and depression that likely result from sporadic neurodegeneration in cholinergic , noradrenergic , and serotonergic pathways . in pd , accumulation of iron is found inside some melanin - containing da - ergic neurons and inside amyloid plaques and neurofibrillary tangles associated with pd dementia ( zecca et al . , 2004 ) . it has been suggested that iron accumulation may contribute to the oxidative stress - induced apoptosis reported in both pd and pd dementia ( zecca et al . , 2004 ; . such oxidative stress may result from increased glial monoamine oxidase ( mao ) activity leading to exacerbated hydrogen peroxide production that can generate reactive hydroxyl radical through fenton chemistry with intracellular ferrous iron . iron chelators such as desferoxamine , clioquinol and vk-28 have been shown to have neuroprotective activity in animal models of ad and pd ( zecca et al . , 2004 ) . ( 2005b ) developed neuroprotective compounds with dual iron chelating and mao - b inhibitory activity . these authors combined the antioxidant chelator moiety present in an 8-hydroxyquinoline derivative of the neuroprotective brain - permeable iron chelator vk-28 , with the propargylamine moiety ( found in compounds such as rasagiline and selegiline , as stated earlier ) . hla20 was identified as a potential lead compound for further studies having selectivity for mao - b with an ic50 value in the region of 110 m ( fig . 4 ; > 200 m for mao - a ) , as well as acting as a free radical scavenger . however , a related compound designated m30 , unlike hla20 was found in vitro , to be a highly potent mao - a and b inhibitor with brain selectivity for these enzymes in vivo , in addition to possessing iron chelating properties similar to desferoxamine ( gal et al . , 2005 ; zheng et al . , 2005a ; 2005b ) . m30 behaves similarly to other propargylamine mao inhibitors by acting as a suicide- or mechanism - based inhibitor after being identified and processed as a substrate by the enzyme and imparts similar neuroprotective properties as those found in rasagiline and ladostigil ( fig . m30 protects against mptp and kainate neurotoxicity in mice by virtue of both its mao inhibitory and iron chelating / radical scavenging properties in these two animal models of neurodegeneration . it has recently been shown to have dopaminergic neurorestorative activity in post treatment with mptp ( gal et al . , 2009 ) and lactacystin ( zhu et al . , 2007 ) models of pd . the neurogenic activity of m30 and hla-20 has been attributed to the inhibition of iron dependent prolyl-4-hydroxylase , via chelation of iron and activation of hif ( hypoxia inducing factor ) that regulates transcription of a series of neurotropins such as bdnf , gdnf , erythropoietin and vegf . the consequence of hif activation is inhibition of cell cycle g / g , that results in inhibition of cyclin d1 that causes cell arrest differentiation into neurons as seen in the neurorestorative activity of m30 in the two models of pd ( zhu et al . we have introduced a carbamate cholinesterase inhibitor ( chei ) moieties into m30 , such as m30c - n and into hla-20 to give hla-20a and have even added the glutamate antagonist , memantine , which is presently in the clinical use . these compounds 7 ) have been shown to have potent che i and mao - a and b inhibitory activity and possess similar neuroprotective activity to those of their parent compounds , hla-20 and m30 ( zheng et al . , 2009 ) . the accumulation of iron at sites where neurons degenerate in pd , is thought to be a major event that is linked to the neurodegenerative process ( zecca et al . , 2004 ) . the novel non - toxic lipophilic ( and therefore brain - permeable ) iron chelator vk-28 , and its multi - functional derivative , m30 ( both of which possess the mao inhibitory and neuroprotective propargyl moiety of rasagiline ) , offer potential therapeutic benefits for pd . m30 attenuates apoptotic events in sh - sy5y neuroblastoma cells in a serum deprivation model via multiple protection mechanisms , including 1 ) reduction of the pro - apoptotic proteins , bad and bax ; 2 ) reduction of apoptosis - associated ser139-phosphorylated h2a.x ; 3 ) induction of the anti - apoptotic protein , bcl-2 and 4 ) inhibition of the cleavage and activation of caspase-3 . m30 also promotes morphological changes , resulting in axonal growth - associated protein-43 ( gap-43 ) , which is implicated in neuronal differentiation . the compound markedly reduces the levels of cellular holo - app , the -c - terminal fragment ( -ctf ) , and levels of amyloidogenic a peptide in the medium of sh - sy5y and cho cells stably transfected with the app " swedish " mutation . in addition , levels of the nonamyloidogenic sapp in cell medium , as well as levels of -ctf in cell lysate were found to be elevated . these results are consistent with the presence of an iron - responsive element ( ire ) in the 5'-untranslated region ( 5'utr ) of app and demonstrate the effectiveness of m30 in limiting holo - app expression and a peptide secretion . therefore , the multifunctional properties of m30 suggest that it may offer extraordinary potential as a drug for the treatment of pd , especially pd dementia ( avramovich - tirosh et al . , 2006 ) and ad ( mandel et al . , 2007 ; 2008 ) and more recently in transgenic g93a sod model of als ( amyotrophic lateral sclerosis ) where it extends the life span of these animals and has neurogenic activity in ncs-34 rat motor neurons ( kupershmidt et al . , 2009 ) . in pd , a dual mechanism that includes inhibition of mao - b , as well as adenosine a2a receptor blockade offer a novel therapeutic approach to prevent neuronal cell death ( fig . as detailed earlier , mao - b plays a role in the catabolism of neurotransmitters such as da , serotonin and norepinephrine , leading to hydrogen peroxide formation which contributes to oxidative stress and neuronal cell death ( riederer et al . , 2004 ) . levels of mao - b are found to be increased in older patients ( saura et al . , 1994 ; 1997 ; mahy et al . , 2000 ) which has led to the rationale for the use of drugs such as selegiline ( deprenyl ) and lazabemide , ( sramek and cutler , 1999 ) and the design of drugs such as ladostigil , ( youdim and buccafusco , 2005b ) as described before . caffeine , a non - selective adenosine receptor antagonist , is under some scrutiny as a potential drug to counteract age - related cognitive decline ( fig . work in this regard is supported by evidence that critical changes in adenosine - related neurotransmission occur with aging and may be counteracted by adenosine receptor antagonists ( maiade and mendonca , 2002 ; dall'igna et al . , 2003 ; prediger et al . , 2005 ) . caffeine , in fact , has been suggested to protect against -amyloid neurotoxicity , ( dall'igna et al . , 2003 ) while acute treatment with caffeine and the a2a receptor antagonist zm241385 was recently found to reverse age - related olfactory deficits and memory decline in rats ( prediger et al . , 2005 ) clearly suggesting involvement of a2a , but not a1 receptors , in cognitive decline and possibly , neurodegenerative processes . evidence such as the preceding , and other evidence for neuroprotection also in parkinsonian models , led petzer et al . ( petzer et al . , 2003 ) to evaluate ( e)-8-styrylxanthinyl derived adenosine a2a receptor antagonists for inhibition also of brain mao - b . included in these studies were kw-6002 , a potent a2a receptor antagonist ( ki of 2.2 nm ) which is undergoing clinical trials for pd , and ( e)-8-(3-chlorostyryl)-caffeine ( csc ; fig . 8) , which has been shown to be neuroprotective in the mptp parkinsonian mouse model ( chen et al . , 2002 ) . ( petzer et al . , 2003 ) showed mao - b inhibition in the low micromolar to high nanomolar range , with the ki of kw-6002 at 21 m , and that of csc at 0.1 m . these results clearly suggest that the neuroprotective properties of kw-6002 and csc may in part be due to mao - b inhibition , in synergism with the a2a antagonism ( castagnoli et al . , 2003 ) . the divalent calcium cation plays an important role in neuronal cell death ( lipton , 1999 ; horn and limburg , 2000 ; kemp and mckernan , 2002 ; ovbiagele et al . , 2003 ) . one of the receptors activated by glutamate ( together with its co - agonist glycine ) , the nmda receptor , is a major conduit for the influx of calcium ions into cells under excitotoxic conditions . the prevention of such excessive influx of calcium ( known as excitotoxicity ) therefore remains a major drug target in the design of neuroprotective agents . excess accumulation of calcium in neuronal cells rapidly leads to cell death through a variety of mechanisms including activation of proteases , nucleases , phospholipases , nitric oxide synthase ( nos ) , and other degradative enzymes that not only lead to activation of death cascades , but also to free radical formation ( lipton , 1999 ) . nmda receptor antagonists such as dizocilpine ( mk-801 ) and memantine may possess a dual mechanism by which neuronal cells are protected , both by direct blockade of the nmda receptor and by attenuating tnf-induced potentiation of glutamate toxicity ( zou and crews , 2005 ) . brain injury after ischemic stroke also triggers a release of glutamate - associated excitotoxic events , and the incidence of cognitive impairment and dementia have both been reported to be elevated after cerebral stroke , especially in the elderly ( kalaria and ballard , 2001 ) . up to 25% of stroke patients exhibit symptoms of dementia , including symptoms reminiscent of pd dementia ( van kooten and koudstaal , 1998 ) . stroke is the third leading cause of death in the united states ( ovbiagele et al . , 2003 ) and there is a definitive need to develop drugs that can protect or save neurons after an ischemic incident since , to date , no effective treatment has been developed to prevent neuronal cells from dying during stroke conditions ( horn and limburg , 2000 ) . several studies have shown that nmda receptor antagonists , such as dizocilpine ( mk-801 ) and the polycyclic cage amine memantine , display neuroprotective effects in experiments using ischemia paradigms in neurons ( gorgulu et al . , 2000 ; horn and limburg , 2000 ; gerriets et al . , 2003 ; richard green et al . , 2003 ) . an alternative pathway for calcium to enter into neuronal cells is through voltage - gated ion channels , such as l - type calcium channels . animal experiments with nimodipine have suggested that calcium channel antagonists may be neuroprotective in ischemia by antagonizing the influx of calcium into neuronal cells ( horn and limburg , 2000 ) . the importance of calcium overload during cell death , suggests that a dual calcium channel and nmda receptor antagonist might be useful as a neuroprotective drug in stroke and other neurodegenerative disease such as idiopathic pd , where it has been suggested that brain - permeable l - type calcium channel blockers may have a salutary effect on the disease . ngp1 - 01 ( 8-benzylamino-8,11-oxapentacycloundecane ) is a polycyclic cage amine derived from the reductive amination of benzylamine and cookson 's " bird cage " diketone of which the biology was first described by van der schyf ( van der schyf et al . , 1986 ) the l - type calcium channel blocking activity of ngp1 - 01 was investigated utilizing electrophysiological experiments in isolated guineapig papillary muscle and sheep purkinje fibers ( van der schyf et al . , 1986 ) . the structural similarity of ngp1 - 01 to another polycyclic cage amine and nmda receptor antagonist , memantine , led to the evaluation of ngp1 - 01 for potential nmda receptor antagonism . memantine is an uncompetitive nmda receptor antagonist which is used clinically to treat ad , but has also been used for pd in germany ( parsons et al . , 1999 ; rogawski and wenk , 2003 ; plosker and lyseng - williamson , 2005 ) . its favorable fast on - off binding kinetics gives this compound an improved side effect profile compared with other nmda antagonists such as mk-801 ( parsons et al . ngp1 - 01 was shown to also be an uncompetitive nmda antagonist in murine whole brain synaptoneurosomes and blocked nmda - mediated ca uptake with an ic50 of 2.98 m ( geldenhuys et al . , 2007 ) . in a recent paper kiewert et al . ( 2006 ) showed that ngp1 - 01 ( at 1 m ) inhibited depolarizationinduced calcium influx by 78% in cortical neurons preloaded with fura-2 am , with a potency similar to that of nimodipine , while simultaneously inhibiting nmda - induced ( 1 mm ) calcium influx by 52% , only slightly less potent than memantine . using in vivo - microdialysis , intraperitoneal injection of ngp1 - 01 ( 40 mg / kg ) reduced nmda - induced membrane breakdown by 31% ( p<0.01 ) while memantine ( 10 mg / kg ) reduced choline release by 40% . these results demonstrate that ngp1 - 01 simultaneously blocks both major neuronal calcium channels and is brain - permeable after peripheral administration . this dual mechanism of modulating calcium entry into neuronal cells might suggest that ngp1 - 01 may have utility as a neuroprotective agent in pd , stroke and other neurodegenerative diseases , especially in patients with comorbidity among these diseases . this promise of neuroprotection has recently been partly confirmed in in vivo studies using the middle cerebral artery occlusion ( mcao ) mouse model of stroke , wherein it was shown that ngp1 - 01 , administered 30 minutes before mcao , afforded substantial protection against cerebral ischemia - induced brain lesioning , as well as brain swelling measured 24 hours after mcao ( mdzinarishvili et al . , 2005 ) . another role assigned to cage amines such as ngp1 - 01 in pd therapy is the ability of these compounds to inhibit da re - uptake into nerve terminals ( fig . compounds that are able to block the da transporter ( dat ) have been suggested to be more useful in treating the motor symptoms in pd , as opposed to norepinephrine and serotonin re - uptake inhibitors ( hansard et al . , 2002 ) . additionally , compounds with the ability to block dat , may also have neuroprotective activity ( kirby et al . , 2002 ) . ngp1 - 01 was recently shown to block da re - uptake in murine synaptosomes with an ic50 of 57 m . one of ngp1 - 01 's derivatives , a phenylethylamine derivative , was even more potent with an ic50 of 23 m ( geldenhuys et al . the latter compound was also found to be neuroprotective in the mptp - parkinsonian mouse model , affording protection against a single 35 mg / kg ( ip ) dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) ( geldenhuys et al . , 2003 ) . polyphenols are natural products present in beverages such as red wine and tea ( weinreb et al . , 2004b ) . one of the classes of polyphenols which are pharmaceutically interesting is the flavenoids ( fig . these compounds are characterized by an aromatic ring which is condensed to a heterocyclic ring and attached to a second aromatic ring . an innovative therapeutic approach could be the use of natural plant polyphenol flavonoids , reported to have access to the brain and to possess multifunctional activities as iron chelators , radical scavengers , anti - inflammatory agents and neuroprotectants ( morel et al . , 1993 ; guo et al . , 1996 ; hider et al . , 2001 ; joseph et al . , these compounds and their actions have been extensively reviewed ( mandel et al . , 2005 ) . in particular , the major constituent of green tea catechin extract ( -)-epigallocatechin-3-gallate ( egcg ; fig . 11 ) plays a major role in the prevention of neurodegeneration in a variety of cellular and animal models of neurodegenerative diseases ( mandel et al . , 2006 ) . this effect appears to be mediated through multiple pathways , including the participation of the pro - survival pkc and extracellular mitogen - activated protein kinase ( mapk ) signaling and the promotion of neurite outgrowth ( reznichenko et al . , 2005 ) . structurally important features defining their chelating potential are the 3',4'-dihydroxyl group in the b ring ( hider et al . , 2001 ) , as well as the gallate group ( kumamoto et al . , 2001 ) which may neutralize ferric iron to form redox - inactive iron , thereby protecting cells against oxidative damage ( grinberg et al . , 1997 ) . recent studies have shown that prolonged administration of egcg to mice induced a significant reduction in membrane - associated app levels in hippocampus ( levites et al . , 2003 ) and in cerebral a levels concomitant with reduced -amyloid plaques ( rezai - zadeh et al . , 2005 ) . this effect may be accounted for , in part , by the chelation of the intracellular free - iron labile pool , modulating app mrna translation via its ire - type ii ( reznichenko et al . , 2006 ) , as has recently been described for other metal chelators , such as desferoxamine , clioquinol and dimercaptopropanol ( payton et al . , 2003 ; rogers and lahiri , 2004 ) . pd and ad are complex diseases with multiple pathways which contribute to its etiology and finally cell death of da - ergic , cholinergic and other neurons . to address this multiplicity , compounds that target more than one drug target in the cell death cascades the feasibility of moving these drugs to market has been shown through the success of rasagiline , which has been shown to have neuroprotective activity and has made it to the market as a pd therapeutic ( olanow et al . , 2009 ) . the development of multimodal drugs is not limited to neurodegenerative disease , but rather that similar approaches are under way with other complex disease such as cancer , aids , depressive illness , schizophrenia and possibly cardiovascular disorders ( youdim and van der schyf , 2009 ) .
parkinson 's disease ( pd ) and alzheimer 's disease ( ad ) are severe neurodegenerative disorders , with no drugs that are currently approved to prevent the neuronal cell loss characteristic in brains of patients suffering from pd and ad and all drug treatment are synptomactic . due to the complex pathophysiology , including a cascade of neurotoxic molecular events that results in neuronal death and predisposition to depression and eventual dementia and etiology of these disorders , an innovative approach towards neuroprotection or neurorestoration ( neurorescue ) may be the development and use of multifunctional pharmaceuticals . such drugs target an array of pathological pathways , each of which is believed to contribute to the cascades that ultimately lead to neuronal cell death . in this short review , we discuss examples of novel multifunctional ligands that may have potential as neuroprotective - neurorestorative therapeutics in pd and ad . the compounds discussed originate from synthetic chemistry as well as from natural sources .
INTRODUCTION Rasagiline Iron chelators with radical scavenging and brain-selective monoamine oxidase inhibitory activity Monoamine oxidase inhibition by A NMDA antagonism by calcium channel blockers Green tea polyphenols DISCUSSION
parkinson 's disease ( pd ) is an age - related neurodegenerative disease with progressive loss of dopaminergic ( da ) neurons in the substantia nigra pars compacta ( snpc ) . , 1996 ; dooley and markham , 1998 ; piercey , 1998 ) and memantine ( rogawski and wenk , 2003 ; plosker and lyseng - williamson , 2005 ) may also possess neuroprotective activities , rasagiline is currently only drug that may have a disease modifying activity ( olanow et al . for example , the combination use of salmeterol ( a 2-adrenergic agonist ) and fluticasone ( a glucocorticoid steroid ) in asthma , has led to the combination of these two medications in one ( advair ) . in this review , we will consider examples of compounds with multi - functional neuroprotective - neurorescue ( table 1 for definitions ) properties that may have promise in the treatment of pd and similar approaches have been made for multimodal drugs for ad ( youdim et al . , it has been suggested that iron accumulation may contribute to the oxidative stress - induced apoptosis reported in both pd and pd dementia ( zecca et al . in pd , a dual mechanism that includes inhibition of mao - b , as well as adenosine a2a receptor blockade as detailed earlier , mao - b plays a role in the catabolism of neurotransmitters such as da , serotonin and norepinephrine , leading to hydrogen peroxide formation which contributes to oxidative stress and neuronal cell death ( riederer et al . , 2000 ) which has led to the rationale for the use of drugs such as selegiline ( deprenyl ) and lazabemide , ( sramek and cutler , 1999 ) and the design of drugs such as ladostigil , ( youdim and buccafusco , 2005b ) as described before . the divalent calcium cation plays an important role in neuronal cell death ( lipton , 1999 ; horn and limburg , 2000 ; kemp and mckernan , 2002 ; ovbiagele et al . excess accumulation of calcium in neuronal cells rapidly leads to cell death through a variety of mechanisms including activation of proteases , nucleases , phospholipases , nitric oxide synthase ( nos ) , and other degradative enzymes that not only lead to activation of death cascades , but also to free radical formation ( lipton , 1999 ) . , 2003 ) and there is a definitive need to develop drugs that can protect or save neurons after an ischemic incident since , to date , no effective treatment has been developed to prevent neuronal cells from dying during stroke conditions ( horn and limburg , 2000 ) . an innovative therapeutic approach could be the use of natural plant polyphenol flavonoids , reported to have access to the brain and to possess multifunctional activities as iron chelators , radical scavengers , anti - inflammatory agents and neuroprotectants ( morel et al . it has been suggested that iron accumulation may contribute to the oxidative stress - induced apoptosis reported in both pd and pd dementia ( zecca et al . in pd , a dual mechanism that includes inhibition of mao - b , as well as adenosine a2a receptor blockade offer a novel therapeutic approach to prevent neuronal cell death ( fig . , 2000 ) which has led to the rationale for the use of drugs such as selegiline ( deprenyl ) and lazabemide , ( sramek and cutler , 1999 ) and the design of drugs such as ladostigil , ( youdim and buccafusco , 2005b ) as described before . the divalent calcium cation plays an important role in neuronal cell death ( lipton , 1999 ; horn and limburg , 2000 ; kemp and mckernan , 2002 ; ovbiagele et al . excess accumulation of calcium in neuronal cells rapidly leads to cell death through a variety of mechanisms including activation of proteases , nucleases , phospholipases , nitric oxide synthase ( nos ) , and other degradative enzymes that not only lead to activation of death cascades , but also to free radical formation ( lipton , 1999 ) . , 2003 ) and there is a definitive need to develop drugs that can protect or save neurons after an ischemic incident since , to date , no effective treatment has been developed to prevent neuronal cells from dying during stroke conditions ( horn and limburg , 2000 ) . an innovative therapeutic approach could be the use of natural plant polyphenol flavonoids , reported to have access to the brain and to possess multifunctional activities as iron chelators , radical scavengers , anti - inflammatory agents and neuroprotectants ( morel et al . pd and ad are complex diseases with multiple pathways which contribute to its etiology and finally cell death of da - ergic , cholinergic and other neurons .
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\usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~g}$$\end{document}g\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 bolometer surrounded by a 3 m vm2002 reflecting foil , monitoring a crystal face with a germanium bolometer acting as light detector . in coincidence with the heat released in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 we were able to detect the light emitted by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta /\gamma $ $ \end{document}/ particles , which amounted to 173\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~ev}$$\end{document}ev at 2,528\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~kev}$$\end{document}kev . the crystal was doped with natural samarium , which contains \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{147}$$\end{document}147sm , an \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document}-unstable isotope with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q=2310\mathrm { ~kev}$$\end{document}q=2310kev . the light detected from these decays was compatible with zero , confirming that at the 0\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \beta \beta $ $ \end{document} energy scale no light is emitted by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document}s . finally , room temperature tests confirmed that the light emitted by particles interacting in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 can be ascribed to the sole cherenkov emission , excluding a contribution from the scintillation . in this paper we present the results of a test conducted on a cuore bolometer , i.e. a 750\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~g}$$\end{document}g crystal , 6 times larger than that used in our previous work and without samarium doping . the results confirm that the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} discrimination in cuore is possible , but the light signal is small and requires light detectors with higher sensitivity than that provided by bolometers . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 crystal comes from samples of the cuore batches used to check the radiopurity and the bolometric performances during the production , and therefore is identical to the crystals that are currently being mounted in cuore . the crystal is a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$5\times 5\times 5\mathrm { ~cm^3}$$\end{document}555cm3 cube with translucent faces , two opposite of which have a better polishing quality , close to optical polishing grade . all faces are surrounded by the vm2002 light reflector except for an optical one that is monitored by a 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~cm}$$\end{document}cm in diameter , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$300\mathrm { ~\upmu m}$$\end{document}300m thick germanium light detector ( ld ) ( fig . 1 ) . both the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 crystal and the germanium are operated as bolometers , using a neutron transmutation doped germanium ( ntd - ge ) thermistor as temperature sensor . the detectors are held in a copper structure by means of teflon ( ptfe ) supports , anchored to the mixing chamber of a dilution refrigerator . the setup is operated in the cuore / lucifer r&d cryostat , in hall c of lngs .fig . 1the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 crystal in the copper holder , surrounded by a 3 m vm2002 light reflector and monitored by the germanium bolometric light detector the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 crystal in the copper holder , surrounded by a 3 m vm2002 light reflector and monitored by the germanium bolometric light detector as in ref . , the read - out of the thermistor is performed using the cuoricino electronics . the analog signals are filtered by 6-pole active bessel filters and then fed into an 18-bit national instrument pxi analog - to - digital converter ( adc ) , the same system being used in cuore-0 . the filter cutoff and the adc sampling frequency are set to 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~hz}$$\end{document}hz and 125\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~hz}$$\end{document}hz for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 , respectively , and to 120\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~hz}$$\end{document}hz and 2,000\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~hz}$$\end{document}hz for the ld , respectively . when it fires , waveforms 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~s}$$\end{document}s long on the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 and 250\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~ms}$$\end{document}ms long on the ld are saved on disk . additionally , when the trigger fires on the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 , the waveform on the ld is acquired irrespective of its own trigger . to maximize the signal to noise ratio , the waveforms are processed offline with the optimum filter algorithm [ 17 , 18 ] . on the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 the pulse is identified with a peak finder algorithm , and the amplitude is evaluated as the maximum of the peak . on the ld , to eliminate noise artifacts at the threshold , the pulse amplitude is evaluated at the characteristic time delay of the ld response with respect to the pulse on the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 , which is estimated in calibration runs using events generated by particles interacting in both detectors ( for more details see ref . ) . the light detector is exposed to a permanent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{55}$$\end{document}55fe source , providing 5.9 and 6.5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~kev}$$\end{document}kev calibration x - rays . the typical rise and decay times of the pulses are 2.6 and 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~ms}$$\end{document}ms , respectively , while the energy resolution at the iron peaks and at the baseline is 135 and 72\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~ev~rms}$$\end{document}evrms , respectively . to calibrate the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 and to generate events in the 0\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \beta \beta $ $ \end{document} region , the setup is illuminated by a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{232}\mathrm { th}$$\end{document}232th source placed outside the cryostat . the rise and decay times of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 pulses are 40 and 532\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~ms}$$\end{document}ms , respectively , values that are similar to the cuore-0 ones . the energy resolution at the 2615 kev \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{208}$$\end{document}208tl peak from the thorium source is 11.5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~kev~fwhm}$$\end{document}kevfwhm , worse than the 5.7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~kev}$$\end{document}kev fwhm obtained averaging all the cuore-0 bolometers . this might be due to the different working temperature , which was chosen higher than in cuore-0 ( 20 mk instead of 10 mk ) in order to improve the energy resolution of the light detector ( see ref . for details ) . the worse energy resolution of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 bolometer does not affect our results , since attention is focused on the light signal . the energy spectrum acquired from the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 bolometer in 6.86 days of data taking is shown in fig . 2 . the peak around 5400\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~kev}$$\end{document}kev is due to the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document}-decay of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{210}$$\end{document}210po , a natural contamination of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 crystal observed also in the 117\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~g}$$\end{document}g detector and in cuore-0 . the remaining peaks are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document}s from the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{232}\mathrm { th}$$\end{document}232th source , except for the peak at 1,461\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~kev}$$\end{document}kev , which is a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document} from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{40}$$\end{document}40k contamination of the cryostat . both the single escape ( se ) and the double escape ( de ) peaks of the 2615 kev \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document} from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{208}\mathrm { tl}$$\end{document}208tl are visible . the presence of the de peak is of particular interest because it is a single site production of a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e^-$$\end{document}e- and of a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e^+$$\end{document}e+ , a process similar to the 0\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \beta \beta $ $ \end{document}.fig . 2energy spectrum acquired by the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 crystal . all the labeled peaks are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document}s , except for the single and double escape peaks of the 2615\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~kev}$$\end{document}kev \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document} from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{208}\mathrm { tl}$$\end{document}208tl , which are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e^- + e^+ + \gamma $ $ \end{document}e-+e++ and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e^- + e^+$$\end{document}e-+e+ events , respectively , and for the events around 5.4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~mev}$$\end{document}mev , which are generated by the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document}-decay of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{210}$$\end{document}210po in the crystal energy spectrum acquired by the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 crystal . all the labeled peaks are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document}s , except for the single and double escape peaks of the 2615\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~kev}$$\end{document}kev \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document} from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{208}\mathrm { tl}$$\end{document}208tl , which are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e^- + e^+ + \gamma $ $ \end{document}e-+e++ and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e^- + e^+$$\end{document}e-+e+ events , respectively , and for the events around 5.4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~mev}$$\end{document}mev , which are generated by the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document}-decay of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{210}$$\end{document}210po in the crystal the light detected versus calibrated heat in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 crystal is shown in fig . 3 . the distribution of the light corresponding to each peak in fig . 2 ( blue dots in the figure ) is fitted with a gaussian , the mean of which is overlaid onto the figure . the mean light from the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document}-decay of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{210}$$\end{document}210po is found to be \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle l_\alpha \rangle = -3.9\pm 14.5\mathrm { ~ev}$$\end{document}l=-3.914.5ev , i.e. compatible with zero . the mean light from the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document} peaks is fitted with a line \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle l_{\beta /\gamma } \rangle = \mathrm{ly}\times ( \mathrm{energy}-e_\mathrm{th})$$\end{document}l/=ly(energy - eth ) , with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_\mathrm{th } } = 280\pm 60\mathrm { ~kev}$$\end{document}eth=28060kev and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm{ly } = 45\pm 2\mathrm { ~ev / mev}$$\end{document}ly=452ev / mev . the standard deviations of the light distributions are found compatible with the baseline noise of the ld , which therefore appears as the dominant source of fluctuation , hiding any possible dependence on the position of the interaction in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 crystal or statistical fluctuations of the number of photons . as in our previous work , the light from the de peak is compatible with the light from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document}s , indicating that the fitted line can be used to predict the amount of light detectable from 0\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \beta \beta $ $ \end{document} events . we compute \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$101.4\pm 3.4$$\end{document}101.43.4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~ev}$$\end{document}ev of light for a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta /\gamma $ $ \end{document}/ event with 0\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \beta \beta $ $ \end{document} energy , 72\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~ev}$$\end{document}ev less than the light detected at the same energy in the 117\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~g}$$\end{document}g detector.fig . 3(color online ) detected light versus calibrated heat in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 bolometer for all the acquired events ( gray ) and for the events belonging to the peaks labeled in fig 2 ( blue ) . the mean light is clearly energy dependent for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document} peaks ( red circles below 3 mev ) and compatible with zero for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document}-decay of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{210}$$\end{document}210po ( pink circle at 5.4 mev ) ( color online ) detected light versus calibrated heat in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 bolometer for all the acquired events ( gray ) and for the events belonging to the peaks labeled in fig 2 ( blue ) . the mean light is clearly energy dependent for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document} peaks ( red circles below 3 mev ) and compatible with zero for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document}-decay of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{210}$$\end{document}210po ( pink circle at 5.4 mev ) the detected light at the 0\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \beta \beta $ $ \end{document} is small , at the same level of the ld noise , and does not allow one to perform an event by event rejection of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} background . as indicated in ref . , the emitted cherenkov light amounts to several hundreds of ev , a much higher value than what we detect . to increase the light collection efficiency , we applied various modifications to the setup : we changed the vm 2002 light reflector to aluminum foils . aluminum is expected to have higher reflectivity in the uv band , the region where the cherenkov emission is more intense . nevertheless , the amount of light detected is 25 % less than in the case of vm 2002.we removed the vm 2002 , which is a specular light reflector , and wrapped the crystal with teflon tape , which is a light diffusor . the amount of light detected is compatible with the vm 2002 measurement.we changed the ld to an identical one , but we coated the side faced to the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 with 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~nm}$$\end{document}nm of sio\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_2$$\end{document}2 . it has been demonstrated , in fact , that in the red / infrared band this layer enhances the light absorption by up to 20 % [ 20 , 21 ] . in our application , however , the amount of light detected does not change significantly.we added a second ld , monitoring opposite faces with two different light detectors . the amount of light detected from each ld is found to be the 50 % of the amount detected with a single ld . this causes an overall decrease of the signal to noise ratio , because each ld adds its own noise.we replaced the crystal with a cylindrical one , 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~cm}$$\end{document}cm in diameter and in height . again the amount of light detected does not change.summarizing , none of the above trials succeeded in providing a significant increase of the light collection efficiency , indicating that most of the light is absorbed by the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 crystal . this is due to the high refractive index of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 crystal ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n\sim 2.4$$\end{document}n2.4 ): many photons are reflected internally several times up to absorption . this effect is confirmed by the higher light yield obtained with the small ( 117 g ) crystal and by preliminary results from simulations of the light collection . aluminum is expected to have higher reflectivity in the uv band , the region where the cherenkov emission is more intense . nevertheless , the amount of light detected is 25 % less than in the case of vm 2002 . we removed the vm 2002 , which is a specular light reflector , and wrapped the crystal with teflon tape , which is a light diffusor . we changed the ld to an identical one , but we coated the side faced to the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 with 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~nm}$$\end{document}nm of sio\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_2$$\end{document}2 . it has been demonstrated , in fact , that in the red / infrared band this layer enhances the light absorption by up to 20 % [ 20 , 21 ] . in our application , however , the amount of light detected does not change significantly . the amount of light detected from each ld is found to be the 50 % of the amount detected with a single ld . this causes an overall decrease of the signal to noise ratio , because each ld adds its own noise . we replaced the crystal with a cylindrical one , 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~cm}$$\end{document}cm in diameter and in height . again the setup providing the highest light signal , around 100\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~ev}$$\end{document}ev at the 0\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \beta \beta $ $ \end{document} , consists in a single ld with the crystal surrounded by the vm 2002 reflector or wrapped with teflon tape . the recent cuore-0 result restricted the prediction of the amount of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} background in cuore from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_\alpha = 0.01 - 0.04$$\end{document}b=0.01 - 0.04 to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_\alpha = 0.01\mathrm { ~counts/(kev\,kg\,year)}$$\end{document}b=0.01counts/(kevkgyear ) , while the ultimate source of background , due to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta /\gamma $ $ \end{document}/ radioactivity from the setup , still amounts to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b_{\beta /\gamma } = 0.001\mathrm { ~counts/(kev\,kg\,year)}$$\end{document}b/=0.001counts/(kevkgyear ) . from these numbers and from the specs of cuore we perform toy monte carlo simulations to estimate the 90 % \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~c.l.}$$\end{document}c.l . the outcome of a toy experiment is fitted in energy with a flat probability density function ( pdf ) for the background and a gaussian pdf for the signal , and is simultaneously fitted in light with gaussians pdfs for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta /\gamma $ $ \end{document}/ light distributions . the posterior pdf of the signal events is obtained integrating over the nuisance parameters and assuming a flat prior . the sensitivity of a single experiment ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n_{90}$$\end{document}n90 ) is computed as the number of signal events corresponding to the 90 % of the posterior cumulative distribution . several ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sim 3{,}000$$\end{document}3,000 ) experiments are generated , and the median of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n_{90}$$\end{document}n90 is used as estimator of the sensitivity . the entire procedure is repeated while varying the signal to noise ratio in the light detector ( fig . 4 ) . from the figure one sees that the application of light detectors to cuore would increase its 90 % c.l . sensitivity to the half - life of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{130}\mathrm { te}$$\end{document}130te to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2.7\times 10^{26}\mathrm { ~years}$$\end{document}2.71026years , a factor 3 higher than cuore without light detectors . to achieve this goal one needs a signal to noise ratio in the light detector greater than 5 , a value that is far from that featured by the setup in this work , equal to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$101/72=1.4\mathrm { ~ev / ev}$$\end{document}101/72=1.4ev / ev . from the results presented the increase of the light signal is difficult , and therefore to upgrade cuore light detectors able to provide a noise level below \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$20\mathrm { ~ev~rms}$$\end{document}20evrms are needed . other than trying to improve the ntd technology , there are at least two possible alternatives . the use of phonon - mediated transition edge sensors ( tes ) , as in the cresst dark matter experiment , or the use of phonon - mediated kinetic inductance detectors ( kid ) , as recently proposed in ref . . the tes technology has already proved to reach very good noise levels , but the implementation of 988 light detectors implies a complicated read - out , mainly because of the cryogenic squid amplifiers that are employed . kids already proved to be a highly multiplexable technology in astrophysical applications ( up to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$400$$\end{document}400 channels on the same read - out line ) but the required energy resolution in our application still needs to be demonstrated.fig . sensitivity to the half - life of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{130}\mathrm { te}$$\end{document}130te as a function of the signal to noise ratio of the light detectors , under the reasonable hypothesis of an \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} background index in cuore of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.01\mathrm { ~counts/(kev\,kg\,year)}$$\end{document}0.01counts/(kevkgyear ) . the sensitivity of the experiment without light detectors corresponds to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm{s / n = 0}$$\end{document}s / n=0 . when \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm{s / n > 5}$$\end{document}s / n>5 the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} background is hidden by the irreducible background predicted from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document} interactions , amounting to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.001\mathrm { ~counts/(kev\,kg\,year)}$$\end{document}0.001counts/(kevkgyear ) , and the sensitivity is maximal . the performance of the light detectors used in this work , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm{s / n } = 1.4$$\end{document}s / n=1.4 , is clearly too low 90 % c.l . sensitivity to the half - life of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{130}\mathrm { te}$$\end{document}130te as a function of the signal to noise ratio of the light detectors , under the reasonable hypothesis of an \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} background index in cuore of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.01\mathrm { ~counts/(kev\,kg\,year)}$$\end{document}0.01counts/(kevkgyear ) . the sensitivity of the experiment without light detectors corresponds to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm{s / n = 0}$$\end{document}s / n=0 . when \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm{s / n > 5}$$\end{document}s / n>5 the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} background is hidden by the irreducible background predicted from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document} interactions , amounting to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.001\mathrm { ~counts/(kev\,kg\,year)}$$\end{document}0.001counts/(kevkgyear ) , and the sensitivity is maximal . the performance of the light detectors used in this work , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm{s / n } = 1.4$$\end{document}s / n=1.4 , is clearly too low we tested the possibility to discriminate the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} background in cuore by tagging the signal from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} particles through the detection of cherenkov light . the detected light at the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{130}\mathrm { te}$$\end{document}130te\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q$$\end{document}q - value is around 100\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~ev}$$\end{document}ev for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta /\gamma $ $ \end{document}/ particles and no light is detected from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} interactions , confirming the validity of this technology . however , the signal is small at the same level of the noise of the bolometric light detectors we are using , and does not allow us to perform an event by event discrimination of the background . we tested modifications of the setup , by using different light reflectors or multiple light detectors , but the light yield did not increase . we are working on simulations to estimate the fraction of emitted light that escapes the crystal and is eventually absorbed by the light detector . critical parameters are the index of refraction and the absorbance of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { teo}_2$$\end{document}teo2 , which unfortunately are not available in the literature for low temperatures . to this end given the results obtained so far , we conclude that , to remove completely the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} background in cuore , light detectors with a noise of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$20\mathrm { ~ev~rms}$$\end{document}20evrms are needed , a factor 34 times better than the bolometric light detectors we used in this work . changing the technology to tes or kid devices could be an alternative , provided that the present read - out and sensitivity limits are overcome . cuore without \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} background would reach a 90 % c.l . sensitivity to the 0\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \beta \beta $ $ \end{document} half - life of more than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$3\times 10^{26}\mathrm { ~years}$$\end{document}31026years , a factor 3 better than the upcoming experiment . combining the light read - out with an enrichment in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{130}\mathrm { te}$$\end{document}130te from the natural 34 to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sim 90\,\%$$\end{document}90% would push the half - life sensitivity by another factor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sim 3$$\end{document}3 . depending on the choice of the nuclear matrix elements , this corresponds to an effective neutrino mass sensitivity in the range 1435 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { ~mev}$$\end{document}mev , down into the inverted hierarchy of neutrino masses .
cuore , an array of 988 teo\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_2$$\end{document}2 bolometers , is about to be one of the most sensitive experiments searching for neutrinoless double - beta decay . its sensitivity could be further improved by removing the background from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} radioactivity . a few years ago it was pointed out that the signal from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document}s can be tagged by detecting the emitted cherenkov light , which is not produced by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document}s . in this paper we confirm this possibility . for the first time we measured the cherenkov light emitted by a cuore crystal , and found it to be 100 ev at the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q$$\end{document}q - value of the decay . to completely reject the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} background , we compute that one needs light detectors with baseline noise below 20 ev rms , a value which is 34 times smaller than the average noise of the bolometric light detectors we are using . we point out that an improved light detector technology must be developed to obtain teo\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_2$$\end{document}2 bolometric experiments able to probe the inverted hierarchy of neutrino masses .
Introduction Experimental setup Results Perspectives Conclusions
the experimental signature is very clear , a peak in the sum energy spectrum of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document}s at the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q$$\end{document}q - value of the decay . the performance of the light detectors used in this work , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm{s / n } = 1.4$$\end{document}s / n=1.4 , is clearly too low we tested the possibility to discriminate the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} background in cuore by tagging the signal from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} particles through the detection of cherenkov light . to this end given the results obtained so far , we conclude that , to remove completely the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha $ $ \end{document} background in cuore , light detectors with a noise of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$20\mathrm { ~ev~rms}$$\end{document}20evrms are needed , a factor 34 times better than the bolometric light detectors we used in this work .
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polo - like kinase 1 ( plk1 ) is the most well - studied member of the polo - like family of kinases and is most commonly known for its regulatory role during mitosis , where it has been shown to be a critical component of centrosome maturation , kinetochore microtubule attachment , bipolar spindle formation , and cytokinesis . the overexpression of plk1 is common in aberrant cells and has been identified in roughly 60% of reported cancers including melanoma , breast , ovarian , thyroid , colon , prostate , pancreatic , head and neck , nonsmall cell lung , and non - hodgkin s lymphomas . furthermore , the overexpression of plk1 has been linked to poor disease prognosis and a decreased survival rate . in vitro studies have shown that specific inhibition of plk1 can significantly reduce the proliferation and viability of multiple melanoma cell lines by inducing g2/m phase cell cycle arrest and mitotic catastrophe without detriment to normal adult or neonatal human epidermal melanocytes ( hems ) . these data have made plk1 an attractive target for small - molecule inhibition and provoked the development of over a dozen plk1 inhibitors , six of which have gone to clinical trials . however , despite plk1 inhibition having great preclinical success for cancer treatment , the clinical potential of plk1-targeted inhibition for human cancers has yet to be realized . this emphasizes the need for a more in - depth understanding of plk1 signaling in cancer . this study addresses that need by employing a large - scale comparative proteomics analysis to examine the downstream effects of plk1 inhibition using the small - molecule inhibitor bi 6727 . bi 6727 ( volasertib ) is a second - generation plk1 inhibitor and is currently the most specific plk1 atp - competitive inhibitor commercially available with an ic50 of 0.87 nm . plk1 inhibition by bi 6727 has been shown to have potent antitumorigenic activity in multiple preclinical studies , but the downstream mechanism by which bi 6727 confers its therapeutic effect remains largely undefined . in an effort to identify novel regulatory effects of plk1 inhibition in melanoma , we used a label - free , relative quantitation strategy to compare the proteomes of a375 melanoma cells cultured in the presence or absence of bi 6727 . the a375 melanoma cell line has been reported to express high levels of plk1 when compared with hems and is sensitive to the targeted depletion of plk1 . furthermore , a375 cells harbor the braf mutation , a mutation present in roughly 50% of melanoma cases , thus making it an ideal candidate for our proteomics analysis . our comparative proteomics strategy was conducted by employing data - dependent nano - lc ms / ms analysis on a q - exactive with the resultant data being searched against the human proteome using the sequest search engine and further analyzed with the sieve software package to reveal proteins with altered expression . this analysis resulted in the positive identification of 1819 proteins ( 1% false discovery rate ( fdr ) ) , 343 of which had significantly altered expression . using ingenuity pathway analysis ( ipa ) to filter the data under more stringent conditions , we identified a subset of 23 proteins that were significantly altered due to bi 6727 inhibition of plk1 , most of which have no previously reported associations with plk1 . importantly , we have identified proteins involved in cellular metabolism , proteasomal degradation , and p53 translation , thus providing novel insight into plk1 s role in cancer cell survival . a375 human melanoma cells ( atcc , va ) were cultured in hyclone dulbecco s modified eagle s medium ( dmem , thermo , ca ) supplemented with 10% fetal bovine serum ( fbs , thermo , ca ) . 1 10 a375 human melanoma cells ( atcc , va ) were seeded in 100 mm tissue culture plates ( tpp , mo ) containing 10 ml of supplemented dmem ( thermo , ca ) and were allowed to recover in untreated medium for 24 h under standard cell culture conditions . following recovery , medium was aspirated , and cells were treated with 25 nm bi 6727 ( chemietek , in ) or vehicle control ( dmso ) in supplemented dmem . after 24 h , cells were collected by trypsin digestion and centrifugation at 300 g for 3 min at 4 c . the treatments were performed using identical procedures on two varying a375 cell passages three times each for a total of six experimental replicates per treatment group . cell pellets were lysed mechanically with a needle in the absence of protease inhibitors or lysis buffer according to the following protocol . 0.3 ml of ice cold pbs was added to frozen cell pellets , and the resulting mixture was lysed by passing through a 23 gauge needle 15 times . the cytosolic protein fraction was isolated by centrifugation at 10 000 g for 10 min at 4 c to remove cellular debris . protein concentration of the extracts was measured by microbca assay ( thermo fisher scientific , il ) . a total of 20 g of protein from each of the six replicates ( control and treated ) was digested with 1 g of sequencing grade trypsin ( promega , fitchburg , wi ) . following an overnight digestion at 37 c samples were acidified with 10% formic acid and prepared for lc ms / ms by c18 zip - tip purification according to the manufacturers protocol ( millipore , billerica , ma ) . peptide samples were resuspended in water with 0.1% formic acid ( v / v ) and analyzed by nano - lc ms / ms . for label - free , relative , quantitative analysis , six replicates of each sample were analyzed by nano - lc ms / ms . for each run , 1 g of the digest was injected on a 100 m 100 mm , reverse - phase c18 beh column with 1.7 um particles and a 300 pore size ( waters , milford , ma ) using a waters nanoacquity system . chromatography solvents were water ( a ) and acetonitrile ( b ) , both with 0.1% formic acid . peptides were eluted from the column with the following gradient 3 to 35% b ( 130 min ) . at 140 min , the gradient increased to 95% b and was held there for 10 min . at 160 min , the gradient returned to 3% to re - equilibrate the column for the next injection . a short 50 min linear gradient blank was run between samples to prevent sample carryover . peptides eluting from the column were analyzed by data - dependent ms / ms on a q - exactive orbitrap mass spectrometer ( thermo fisher scientific , ma ) . a top-15 method was used to acquire data . in brief , the instrument settings were as follows : resolution was set to 70 000 for ms scans and 17 500 for the data - dependent ms / ms scans to increase speed . the ms agc target was set to 10 counts , while ms / ms agc target was set to 10 . ms scans were recorded in profile mode , while the ms / ms was recorded in centroid mode , to reduce data file size . dynamic exclusion was set to a repeat count of 1 with a 25 s duration . following lc ms / ms acquisition , the data were searched using sequest ht proteome discoverer 1.4 search engine ( thermo fisher scientific ) , against the uniprot human database ( 6/23/2013 , 20 209 sequences ) at a false discovery cut off 1% . following protein identification , the lc quantitation of peptides eluting between 38 and 145 min was performed on the processed data using sieve 2.1 ( thermo fisher scientific ) , which uses ms intensities from raw lc ms data to find statistical proteomic differences between two samples . proteins ratios calculated had to be significant with a p value lower than 0.05 , and the cv raw ms intensities of the six replicates had to be within 30% . identified proteins from the sieve processing were initially analyzed and filtered using ipa ( ingenuity systems , ca ) under a trial license . a data set containing proteins with only uniquely identified amino acid sequences ( peptides ) with a high level of confidence ( p < 0.05 ) was uploaded into ipa with number of peptides identified , triggered ms / ms fragmentation scans ( hits ) , and the corresponding ratio ( treated / untreated ) set as the observational parameters with swiss - prot accession numbers used for gene i d . a secondary data set was then generated using ipa filters set to proteins with more than two identified peptides on no fewer than four hits with a greater than two - fold change in expression . another secondary data set was generated using the ipa connect tool to identify any plk1-associated proteins with an expression change greater than two - fold . the two secondary data sets were combined , and their molecular function and biological processes were assessed using panther ( protein analysis through evolutionary relationships ) . 5 10 a375 cells were plated and grown in a 10 cm culture dish and treated with bi 6727 at 25 nm , 100 nm , or vehicle control , as previously described . following 24 h treatment , cells were trypsinized , washed with ice - cold pbs , and lysed with ripa buffer ( 50 mm tris , 150 mm nacl , 1% np-40 , 0.5% deoxycholic acid , 0.1% sds ) with phenylmethylsulfonyl fluoride ( pmsf ) and protease inhibitor cocktail ( pierce , il ) . for immunoblot analysis , 30 g of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) using mini - protean tgx precast gels and transferred onto nitrocellulose membrane . blots were blocked in 5% nonfat dry milk in tris - buffered saline + 0.1% tween-20 ( tbst ) , followed by probing with desired primary antibodies : anti - ldha , aurkb , p53 , -actin ( cell signaling nos . 2012 , 3094 , 9282 , 4970 ) , psmb1 , psmb2 , or psmb5 ( abcam nos . blots were then incubated with the appropriate hrp - conjugated antibodies followed by chemiluminescent detection ( pierce , il ) . 5 10 a375 cells were plated and grown in a 10 cm culture dish and treated with bi 6727 at 25 nm , 100 nm , or vehicle control , as previously described . following 24 h of treatment , cells were trypsinized and washed with ice - cold pbs , followed by rna isolation using the rneasy plus mini kit ( qiagen , ca ) and first strand cdna created with m - mlv reverse transcriptase ( promega , wi ) according to vendor s protocol . quantitative real - time rt - pcr was performed in triplicate in 20 l reactions with sybr premix ex taq perfect real time ( takara , wi ) with 100 ng first - strand cdna and 0.2 g of each desired primer pair . the sequences for gpi , ldhb , and p21 ( primerbankid 296080692c3 , 291575126c2 , 310832423c1 ) were acquired from the primerbank online database . samples were cycled once at 95 c for 10 min , then 35 cycles of 95 , 58 , and 72 c for 5 , 15 , and 20 s , respectively . relative mrna was calculated using the ct method with gapdh as an endogenous control . for lactate assay , 8 10 a375 cells were plated and grown in a 96-well plate and treated with bi 6727 at 25 nm , 100 nm , or vehicle control , as previously described . 24 h following treatments , 500 l of spent media was removed from each group and immediately cleared of ldh proteins by centrifugation in 10 kda molecular weight cutoff spin columns and stored at 80 c . all samples were diluted by adding 10 l of media to 90 l of lactate assay buffer and subsequently used in a serial dilution containing a final concentration of either 1 , 0.5 , 0.25 , or 0.125% media in lactate assay buffer with a final volume of 50 l in black 96-well half area plates ( corning , ma ) . lactate assay was performed per manufacturer s protocol , and fluorescence intensity was measured on the biotek synergy h1 microplate reader at ex = 535/em = 587 . 3 10 a375 cells were plated and grown in 96-well half - volume white - wall plates ( corning , ma ) and treated with bi 6727 , as previously described . following 24 h treatments , samples were processed using the nad(p)h - glo detection system ( promega , wi ) or nad / nadh - glo assay ( promega , wi ) as per manufacturer s protocol . luminescence was detected using the biotek synergy h1 microplate reader . for assessing 20s proteasome activity , a375 cells were treated with bi 6727 at 25 nm , 100 nm , or vehicle control , as previously described . following 24 h treatments , cells were lysed in 50 l of ice - cold ripa buffer for 10 min at room temperature while shaking . next , 25 l of lysate was added to a 96-well plate containing bca reagent ( pierce , rockford , il ) and incubated at 37 c for 30 min , while 10 l of lysate was added to the 20s proteasome activity assay ( millipore , ma ) prepared per manufacturer s protocol and incubated at 37 c for 2 h. relative levels of protein were quantified using the bca assay absorbance detected using the biotek synergy h1 microplate reader at 562 nm . 20s proteasome activity was quantified using fluorescence intensity on the synergy h1 microplate reader at ex = 380/em = 460 . proteasome activity was then normalized to relative protein concentration , and the mean of relative activity for each treatment ( n = 6 ) was represented graphically . for immunofluorescence staining , cells were plated and grown on bd falcon cultureslides ( bd biosciences , san jose , ca ) and treated with bi 6727 as previously described . the cells were fixed with a 4% paraformaldehyde in pbs ( ph 7.1 ) for 15 min at room temperature , then blocked with 5% normal goat serum in 0.3% triton x-100 for 1 h. after blocking , cells were incubated overnight in anti--tubulin primary antibody in blocking buffer ( 1:100 , cell signaling no . 2128 ) , followed by a secondary incubation with alexa fluor 594 antirabbit igg antibody in blocking buffer ( 5 g / ml , invitrogen no . the cells were then counter - stained with hoecsht 33342 ( invitrogen , grand island , ny ) for nuclear staining , and a prolong antifade kit was applied per vendor s protocol ( molecular probes , eugene , or ) . slides were examined under a nikon ti microscope using the respective manufacturer s suggested filter sets . 1 10 a375 cells were plated and grown in a six - well tissue culture dish ( tpp , che ) and treated with bi 6727 , as previously described . following treatment , cells were trypsinized for 5 min and transferred to 5 ml falcon tubes containing cultured media . samples were centrifuged at 1000 rpm for 2 min , and the supernatant was aspirated prior to the cells being resuspended in ice - cold 100% ethanol added dropwise while gently vortexing . samples were stored at 20 c overnight prior to centrifugation and resuspension in 500 l of propidium iodide ( pi ) buffer ( pbs , 50 g / ml pi , 0.1 mg / ml rnase a , 0.05% triton - x ) . samples were incubated at 37 c for 40 min in dark and stored at 4 c until processed by flow cytometry in the fl-2a channel . cell cycle analysis was done using modfit lt software ( verity software , topsham , me ) . in an effort to identify the downstream molecular mechanisms of plk1 in melanoma , we elucidated quantitative changes in the proteome of human melanoma cells following plk1 inhibition with bi 6727 . ms / ms on a q - exactive spectrometer were processed with sieve software to reveal up- or down - regulated proteins following plk1 inhibition . to determine the most effective concentration of bi 6727 , we assessed a375 cell viability following a defined range of bi 6727 treatments ( 0.11000 nm , data not shown ) . we determined 25 nm of bi 6727 as the lowest effective concentration to cause a statistically significant decrease in cell viability after 48 h. to assess the molecular functions that contribute to the reduction of a375 cell viability , we terminated treatments after 24 h to limit the necrotic and late apoptotic cell population . labeling methods , including chemical modification approaches ( i - traq , etc . ) and stable isotope labeling , can be expensive but generally require fewer lc label - free approaches are becoming more popular as improvements in instrumentation ( resolution and mass accuracy ) , and improvements in data analysis software facilitate analysis of large data sets . the capabilities of our q - exactive spectrometer made a label - free approach attractive . the high scanning speed coupled to the resolving power and mass accuracy of the q - exactive orbitrap make it particularly useful for accurate , label - free , quantitative protein analysis . this is primarily due to the higher resolution ( smaller diameter ) orbitrap cell of the q - exactive . high - resolution and mass accuracy facilitate area - under - the - curve analysis with the sieve software by allowing accurate tracking of peptides through their chromatographic elution . this improves the duty cycle and allows for increased peptide identification at the ms / ms level . the q exactive has been shown to identify a higher number of compounds with better confidence in multiple comparison studies . thus , there is greater proteome coverage , which enhances the ability to quantitate lower abundant proteins than is achieved with spectral - counting approaches . indeed , the power of this combination of instrumentation and software was born out in the ability of our analysis to positively identify over 1800 proteins , at a cutoff of 1% fdr , with 343 of these identified proteins showing significantly altered expression ( tables s1 and s2 in the supporting information ) . a key step in label - free quantitative proteomic analysis is the evaluation of run - to - run and sample - to - sample reproducibility / variability . prior to the full analysis , we performed a pilot study of bi 6727-treated cells and control replicates , using identical sample preparation and lc ms parameters as previously described to assess variability . two a375 human melanoma aliquots were seeded in 100 mm tissue culture plates as previously described and grown for 24 h under standard cell culture conditions . two replicate injections of 1 g of both control and treated samples were run . resulting data were subject to sequest searches against uniprot human at 2% fdr , using proteome discoverer . a higher fdr was used in assessing the run - to - run variability to delve deeper into the data . these four control runs had an average of 715 proteins identified , with an average of 1500 unique peptides and over 3100 total scans . percent overlap between the two control biological replicates was 67% at the protein level and 55% at the peptide level ( data not shown ) . not surprisingly , percent overlap of the duplicate injections was higher with 70% overlap at the protein level and 62% at the peptide level ( data not shown ) . percent overlap between both injection and biological replicates is consistent with a recent review article highlighting the strengths and limitations of label - free proteomic quantification . our control runs emphasize the need for more than three experimental replicates of each sample type to accurately determine protein ratios in label - free studies . to maximize the statistical power of our analysis , we opted to analyze six replicates of control versus plk1 treated a375 cells . the first step in any direct comparison of treated versus control samples ms peak our data showed average alignment scores of 0.825 , with the treatment groups clustering together ( figure 1a ) . because sieve , like many other label - free methods , allows for conserved peptides to be equally considered for all candidate proteins this avoids inaccurate fold - change calculations due to differential regulation of proteins sharing a conserved peptide . the q - exactive s speed , was key in being able to take this stringent approach to label - free proteomics . label - free quantitation approaches do not rely on internal standards , and thus care needs to be taken to accurately filter the data to reproducibly quantitate proteins . sieve allows for calculated peptides ratios between samples to be filtered not only on p value using fisher s combined probability but also based on variation in the ms peak intensities between the experimental replicates . after data were normalized to the total ion current ( tic ) , we filtered all protein ratios to have a cv between the six replicates ( 25 nm bi 6727 or vehicle control ) to be 30% . peptides had to show up in all six replicates of a treatment at the ms level to be quantitated ( figure 1b , c ) . one of the benefits sieve has over spectral counting is as long as a peptide has a consistent ms peak , it only has to be positively identified once to determine its ratio . finally , peptide fold - change ratios had to be significant at a p value of 0.05 to be considered in our analysis . ( a ) for label - free relative quantitation , six replicates of the tryptic digests were analyzed . an overlay of the base peak chromatograms of control ( blue ) and treated ( red ) samples shows good alignment and comparable loading in the region of peptide elution ( 38145 min rt ) . ( b ) an example of a peptide that is down - regulated in response to treatment . shown is a sieve - aligned , extracted - ion chromatograms for peptide 524.9481 m / z ( metastasis - associated protein ) . ( c ) example of a peptide that is up - regulated in response to treatment . shown is a sieve - aligned , extracted - ion chromatogram for peptide 373.3691 m / z ( ph - domain leucine - rich protein ) . a data set containing proteins with only uniquely identified peptides with a high level of confidence ( p < 0.05 ) was uploaded into ipa with the number of peptides identified ( figure 2a ) and the corresponding ratio ( treated / untreated ) ( figure 2b ) set as the observational parameters . a secondary data set was then generated using ipa filters set to proteins with more than two identified peptides , no fewer than four hits , and greater than a two - fold change in expression . another secondary data set was generated using less stringent criteria allowing for one uniquely identified peptide and integrated the ipa connect tool to identify any plk1-associated proteins with more than four hits and an expression change greater than two - fold . these two analyses resulted in a data set containing 23 proteins of interest ( table 1 ) . next , we employed panther ( protein annalysis through evolutionary relationships ) to assess the molecular function of the collective proteins and identified catalytic activity and binding as the primary protein function ( figure 2c ) . interestingly , using panther to identify the biological processes , cell cycle was scored at 8% , while metabolic process was scored at 57% ( figure 2d ) . furthermore , when scored by protein classes , the hydrolase , nucleic acid binding and protease classes were the three highest ranks , respectively . given the known biological function of plk1 , it would be difficult to predict that plk1 inhibition would have considerable association with cellular metabolism , nucleic acid binding , and proteolytic activity . therefore , we sought to further investigate these observations and substantiate our proteomics findings . ( a ) graphical breakdown representing the number of peptides recognized in all identified proteins . ( b ) graphical representation of the calculated protein ratios showing bi 6727-treated samples compared with the vehicle control . ( c ) proteins of interest molecular function reported by panther ( protein analysis through evolutionary relationships ) as characterized by gene ontology . ( d ) proteins of interest biological processes reported by panther as characterized by gene ontology . proteins identified as having greater than a two - fold change and triggering no less than four separate ms / ms fragmentation scans ( hits ) of at least two uniquely identified amino acid sequences ( peptides ) for proteins with no known association to plk1 or one unique peptide for proteins with a plk1 association , as identified by ingenuity pathway analysis ( ipa ) . also included is the number of lc peaks within a well - defined rectangular region in the m / z versus retention time plane ( frames ) , where each unique peptide was identified . the results of our proteomics analysis have revealed that the inhibition of plk1 activity results in the decreased expression of multiple metabolic proteins including malate dehydrogenase 1 ( mdh1 ) , glutamic - oxaloacetic transaminase 2 ( got2 ) , and transketolase ( tkt ) . additionally , we observed a significant reduction in lactate dehydrogenase a ( ldha ) , confirmed by western blot ( figure 3a ) as well as glucose-6-phosphate isomerase ( gpi ) and lactate dehydrogenase b ( ldhb ) , which were further assessed at the mrna level ( figure 3b ) . of interest , ldha , ldhb , and gpi each play an essential role in glycolysis , a metabolic pathway that allows tumor cells to thrive under hypoxic conditions . glycolysis is an evolutionarily conserved reaction that allows cells to generate atp from glucose under hypoxic conditions . however , cellular metabolism under normoxic conditions is primarily driven by a much more efficient reaction , mitochondrial oxidative phosphorylation ( oxphos ) , yet a hallmark of many cancer cells is their propensity to use the inefficient glycolytic reaction for the production of energy in the presence of oxygen , a phenomenon known as the warburg effect or aerobic glycolysis . it is interesting to speculate that plk1 overexpression may contribute to the cancer - related switch from oxphos to aerobic glycolysis . ( a ) lactate dehydrogenase a ( ldha ) protein expression was significantly reduced following plk1 inhibition in both proteomics ( top ) and western blot ( bottom ) analyses . ( b ) qpcr analysis suggests a dose - dependent decrease in polo - like kinase 1 ( plk1 ) , lactate dehydrogenase b ( ldhb ) , and glucose-6-phosphate isomerase ( gpi ) transcript levels following bi 6727 treatment ( 25 nm , 100 nm ) . ( c ) bi 6727 treatment significantly reduces extracellular lactate levels ( p < 0.001 ) . ( d ) plk1 inhibition significantly reduces reduced nicotinamide adenine dinucleotide ( nadh ) and nicotinamide adenine dinucleotide phosphate ( nadph ) levels ( p < 0.001 ) . ( e ) nad and nadh levels are significantly reduced in bi 6727-treated cells when compared with control ( p < 0.001 ) . ( f ) nad / nadh ratio decreases in a bi 6727 dose - dependent manner ( * * p < 0.01 , * * * p < 0.001 ) . to assess the effect of plk1 inhibition on aerobic glycolysis , we first measured extracellular levels of lactate , the major byproduct of glucose breakdown . following a 24 h incubation with bi 6727 under normoxic conditions , there was a significant decrease in lactate levels in both treatment groups ( 25 nm , 100 nm ) when compared with control ( figure 3c ) . because these data suggest a disruption in metabolic activity , we next assessed the levels of nicotinamide adenine dinucleotide ( nadh ) and nicotinamide adenine dinucleotide phosphate ( nadph ) , the enzymatic cofactors produced during glucose breakdown by glycolysis and the pentose phosphate pathway , respectively . the nadh and nadph levels were significantly reduced in bi-6727-treated cells , resulting in nearly a five - fold decrease ( figure 3d ) . to determine if these observations correlate to energy production , we independently measured levels of nad , an essential coenzyme in atp production . interestingly , in addition to the nad levels being significantly decreased in both the oxidized ( nad ) and reduced ( nadh ) forms ( figure 3e ) , plk1 inhibition also significantly altered the nad / nadh ratio ( figure 3f ) . the nad / nadh ratio is considered to be an indicator of the metabolic state and is important in regulating the intracellular redox state , while a shift in the nad / nadh ratio is closely linked to physiological and pathological states . our data suggest a greater decrease in nad following plk1 inhibition , and given that the pyruvate to lactate conversion is a major route of nad regeneration , it is reasonable to expect that the observed decrease in ldha is a primary factor in the ratio shift . although our data suggest that plk1 inhibition decreases the metabolic activity of melanoma cells , the question of how plk1 signaling relates to glycolysis remains to be answered . the initial warburg hypothesis attributed its effects to dysfunctional oxphos ; however , recent studies have indicated that oxphos remains functional in cancer cells and the shift toward aerobic glycolysis is driven by tumor suppressors such as p53 and pten or the oncogenes ras , c - myc , and hypoxia - inducible factor-1 ( hif-1 ) . plk1 has not only been shown to be involved in p53 , pten , and c - myc signaling pathways ; recent studies suggest that plk1 has the potential to act as a mediator between them . for example , the loss of pten expression is a frequent occurrence in human malignancies and results in elevated phosphoinositide 3-kinase ( pi3k ) signaling . recently , tan et al . have shown that hyperactivation of the pi3k downstream target , pi3k - dependent protein kinase-1 ( pdk1 ) , activates plk1 , which in turn stabilizes myc through direct phosphorylation at ser-62 . while this study has shown a direct connection between two key tumorigenic pathways through plk1 , further correlations can be made when considering the numerous interactions between plk1 and p53 ( discussed later ) . given these data , it is interesting to hypothesize that plk1 overexpression may contribute to the glycolytic shift described by the warburg effect ; however , further studies are needed to define the role of plk1 in metabolic signaling . the ubiquitin - proteasome system ( ups ) plays an integral role in cellular homeostasis through a systematic process that is responsible for 8090% of intracellular protein degradation . the 26s proteasome is the primary proteolytic complex of the system and is composed of two subcomplexes , the catalytic 20s core particle ( cp ) and either one or two terminal 19s regulatory particles ( rps ) . the rps are responsible for protein capturing by ubiquitin recognition , followed by protein unfolding and translocation to the proteolytic channel of the cp . the cp is made up of four stacked rings , each containing seven - ( 1 - 7 , psma1 - 7 ) or -subunits ( 1 - 7 , psmb1 - 7 ) in an configuration ( figure 4a ) . the outer -subunits act as a physical barrier to the active -subunits and create a docking site for the rp , while the inner -rings are responsible for protein cleavage through caspase - like , trypsin - like , and chymotrypsin - like activities by the catalytically active subunits 1 , 2 , and 5 , respectively . plk1 inhibition significantly alters 20s proteasome expression and activity . ( a ) basic structure of the 20s proteasome . ( b ) proteomics analysis identified four 20s proteasome subunits as being down - regulated . ( c ) western blot analysis of the catalytically active 20s proteasome subunits , proteasome subunits 2 , 5 , and 1 ( psmb2 , psmb5 , and psmb1 ) following plk1 inhibition . ( d ) bi 6727-treated cells have significantly decreased 20s proteasome activity ( p < 0.001 ) . in our comparative proteomics analysis , we found that bi 6727 treatment results in significant downregulation of multiple 20s subunits in human melanoma cells . following our ipa analysis , we initially identified subunits 3 and 2 as being 2.20- and 7.41-fold downregulated , respectively . broadening our ipa analysis to include plk1-associated proteins with only one unique peptide but still having a greater than a two - fold change and no fewer than four hits , we further identified the 20s subunits 7 and 6 as being downregulated by 2.46 and 5.56 fold , respectively ( figure 4b ) . because these data suggest that plk1 inhibition has an effect on a wide range of subunits , we performed a western blot analysis of the catalytically active subunits 1 , 2 , and 5 following inhibition of plk1 by bi 6727 ( figure 4c ) . we confirmed a decrease in 2 , as reported by our proteomics analysis , and additionally observed a modest decrease in 5 , while there was no apparent effect on 1 . to assess if the decreased subunit expression reflected on proteasome cleavage , we measured 20s proteasome activity using a fluorophore - labeled proteasome substrate . this assay demonstrated a significant reduction in 20s activity in bi 6727-treated cells when compared with control ( figure 4d ) . these data suggest that plk1 activity influences proteasome cleavage and is potentially upstream of multiple proteasome subunits in a signaling pathway that has yet to be defined . however , there is evidence of plk1 indirectly associating with the proteasome through the transcription factors forkhead box m1 ( foxm1 ) and p53 . similar to plk1 , foxm1 is overexpressed in numerous human carcinomas and is correlated to poor disease prognosis . plk1 is involved in a positive feedback loop with foxm1 , where plk1-dependent phosphorylation is required for efficient foxm1 activation , which in turn is required for expression of multiple mitotic regulators , including plk1 . interestingly , proteasome inhibitors have been shown to suppress foxm1 transcriptional activity , suggesting that proteasome - dependent degradation may confer a level of indirect regulation to plk1 overexpression in human carcinomas . in addition to foxm1 , the classical tumor suppressor p53 also intersects with both plk1 and the proteasome . studies have revealed that plk1 contributes to p53 repression by directly binding to p53 in addition to phosphorylating gtse1 and topors , negative regulators of p53 . furthermore , plk1 has also been shown to stabilize the oncoprotein mdm2 , an e3 ubiquitin ligase and the principal cellular antagonist of p53 . mdm2 mediates p53 protein turnover through constant monoubiquitination , a critical step in the polyubiquitnation of p53 and targeted degradation by the 26s proteasome . because p53 negatively regulates plk1 , a negative feedback loop exists in the plk1-p53 axis . considering the findings presented in this study and given the relationships among plk1 , foxm1 , and p53 , these data suggest that increased proteasome activity would be conducive to plk1 overexpression . the heterogeneous ribonucleoprotein c1/c2 ( hnrnpc ) is a ubiquitous rna - binding protein that is expressed in two main isoforms , hnrnpc1 and hnrnpc2 . studies have shown hnrnpc s biological functions to include mrna transcript packaging , splicing , nuclear retention , and mrna stability . in this study , we report a significant up - regulation of hnrnpc following plk1 inhibition , which was confirmed at the protein level by western blot analysis ( figure 5a ) . interestingly , a recent study has shown that hnrnpc overexpression induces micronucleation through the repression of the mitotic protein aurora kinase b ( aurkb ) in hepatocellular carcinoma ( hcc ) cells . using an immunofluorescent nuclear stain , we were able to observe a similar micronucleation phenotype in melanoma cells following plk1 inhibition ( figure 5b ) . next , we wanted to determine if there was also a repression of aurkb . despite a significant g2/m arrest ( figure 5c ) , we did not observe any significant changes to aurkb expression at the protein level ; however , we did observe a significant reduction in aurkb mrna ( figure 5d ) . with the understanding that an accumulation of cells in g2/m would result in increased mitotic proteins such as aurkb and given the consistent protein expression we observed at 24 h , it is possible that we are seeing early evidence of aurkb protein degradation and the reduced transcript levels may be a better indicator of aurkb repression . this evidence suggests that inhibition of plk1 activity may negatively affect aurkb expression and the mitotic catastrophe associated with plk1 inhibition may in part be mediated through hnrnpc and aurkb . ( a ) heterogeneous ribonucleoprotein c1/c2 ( hnrnpc ) protein expression was significantly increased following plk1 inhibition in both proteomics ( top ) and western blot ( bottom ) analyses . ( b ) immunofluorescence microscopy of -tubulin ( red ) and hoecsht dna ( blue ) staining , demonstrating micronucleation following plk1 inhibition . ( c ) cell cycle analysis of bi 6727-treated cells demonstrates a pronounced g2/m arrest . ( d ) western blot analysis of aurora kinase b ( aurkb ) does not indicate significantly altered protein expression following bi 6727 treatment ( top ) , but qpcr analysis reveals decreased mrna expression levels ( bottom ) . ( e ) plk1 inhibition causes a marked increase in p53 protein expression , visualized by western blot analysis ( top ) and p53 activity , demonstrated by increased p21 mrna expression ( bottom ) . although we have already discussed numerous connections between plk1 and p53 , an additional association exists through hnrnpc . hnrnpc has been shown to enhance p53 translation by directly binding to a cis - element in the 5 coding region of p53 mrna . in accordance with these findings , we did find a corresponding increase in p53 protein expression and activity as determined by western blot and transcriptional analysis of p21 , respectively ( figure 5e ) . these data provide compelling evidence of a novel pathway in plk1-mediated p53 expression through regulation of hnrnpc . however , the plk1p53 axis is a complex signaling network , and extensive studies will be required to determine the role of hnrnpc . inhibition of plk1 by small - molecule inhibitors has become an extremely active area of research based on the preclinical success of plk1 inhibition in a wide variety of cancers . while it has been shown in multiple studies that plk1 inhibition selectively targets cancerous cells over that of their normal counterparts , the molecular mechanisms that contribute to plk1 s selectivity remains poorly understood . our proteomics study of plk1 inhibition by bi 6727 in human melanoma a375 cells has revealed the altered expression of multiple proteins that have yet to be identified as part of the plk1 signaling network . our proteomics analysis provides evidence of a novel relationship between plk1 and hnrnpc and that plk1 inhibition has considerable effect on anaerobic glycolysis and proteasome activity , two pathways that are essential in cancer cell survival . further studies are needed to determine the significance of these protein interactions , but overall these data provide a solid foundation for novel plk1 signaling pathways and potential candidates for future targeted therapies .
polo - like kinase 1 ( plk1 ) is a serine / threonine kinase that plays a key role during the cell cycle by regulating mitotic entry , progression , and exit . plk1 is overexpressed in a variety of human cancers and is essential to sustained oncogenic proliferation , thus making plk1 an attractive therapeutic target . however , the clinical efficacy of plk1 inhibition has not emulated the preclinical success , stressing an urgent need for a better understanding of plk1 signaling . this study addresses that need by utilizing a quantitative proteomics strategy to compare the proteome of brafv600e mutant melanoma cells following treatment with the plk1-specific inhibitor bi 6727 . employing label - free nano - lc ms / ms technology on a q - exactive followed by sieve processing , we identified more than 20 proteins of interest , many of which have not been previously associated with plk1 signaling . here we report the down - regulation of multiple metabolic proteins with an associated decrease in cellular metabolism , as assessed by lactate and nad levels . furthermore , we have also identified the down - regulation of multiple proteasomal subunits , resulting in a significant decrease in 20s proteasome activity . additionally , we have identified a novel association between plk1 and p53 through heterogeneous ribonucleoprotein c1/c2 ( hnrnpc ) , thus providing valuable insight into plk1 s role in cancer cell survival .
Introduction Materials and Methods Results and Discussion Conclusions
polo - like kinase 1 ( plk1 ) is the most well - studied member of the polo - like family of kinases and is most commonly known for its regulatory role during mitosis , where it has been shown to be a critical component of centrosome maturation , kinetochore microtubule attachment , bipolar spindle formation , and cytokinesis . however , despite plk1 inhibition having great preclinical success for cancer treatment , the clinical potential of plk1-targeted inhibition for human cancers has yet to be realized . this emphasizes the need for a more in - depth understanding of plk1 signaling in cancer . this study addresses that need by employing a large - scale comparative proteomics analysis to examine the downstream effects of plk1 inhibition using the small - molecule inhibitor bi 6727 . in an effort to identify novel regulatory effects of plk1 inhibition in melanoma , we used a label - free , relative quantitation strategy to compare the proteomes of a375 melanoma cells cultured in the presence or absence of bi 6727 . our comparative proteomics strategy was conducted by employing data - dependent nano - lc ms / ms analysis on a q - exactive with the resultant data being searched against the human proteome using the sequest search engine and further analyzed with the sieve software package to reveal proteins with altered expression . using ingenuity pathway analysis ( ipa ) to filter the data under more stringent conditions , we identified a subset of 23 proteins that were significantly altered due to bi 6727 inhibition of plk1 , most of which have no previously reported associations with plk1 . importantly , we have identified proteins involved in cellular metabolism , proteasomal degradation , and p53 translation , thus providing novel insight into plk1 s role in cancer cell survival . for label - free , relative , quantitative analysis , six replicates of each sample were analyzed by nano - lc ms / ms . peptides eluting from the column were analyzed by data - dependent ms / ms on a q - exactive orbitrap mass spectrometer ( thermo fisher scientific , ma ) . in an effort to identify the downstream molecular mechanisms of plk1 in melanoma , we elucidated quantitative changes in the proteome of human melanoma cells following plk1 inhibition with bi 6727 . ms / ms on a q - exactive spectrometer were processed with sieve software to reveal up- or down - regulated proteins following plk1 inhibition . the results of our proteomics analysis have revealed that the inhibition of plk1 activity results in the decreased expression of multiple metabolic proteins including malate dehydrogenase 1 ( mdh1 ) , glutamic - oxaloacetic transaminase 2 ( got2 ) , and transketolase ( tkt ) . ( b ) qpcr analysis suggests a dose - dependent decrease in polo - like kinase 1 ( plk1 ) , lactate dehydrogenase b ( ldhb ) , and glucose-6-phosphate isomerase ( gpi ) transcript levels following bi 6727 treatment ( 25 nm , 100 nm ) . because these data suggest that plk1 inhibition has an effect on a wide range of subunits , we performed a western blot analysis of the catalytically active subunits 1 , 2 , and 5 following inhibition of plk1 by bi 6727 ( figure 4c ) . the heterogeneous ribonucleoprotein c1/c2 ( hnrnpc ) is a ubiquitous rna - binding protein that is expressed in two main isoforms , hnrnpc1 and hnrnpc2 . in this study , we report a significant up - regulation of hnrnpc following plk1 inhibition , which was confirmed at the protein level by western blot analysis ( figure 5a ) . ( a ) heterogeneous ribonucleoprotein c1/c2 ( hnrnpc ) protein expression was significantly increased following plk1 inhibition in both proteomics ( top ) and western blot ( bottom ) analyses . inhibition of plk1 by small - molecule inhibitors has become an extremely active area of research based on the preclinical success of plk1 inhibition in a wide variety of cancers . our proteomics analysis provides evidence of a novel relationship between plk1 and hnrnpc and that plk1 inhibition has considerable effect on anaerobic glycolysis and proteasome activity , two pathways that are essential in cancer cell survival .
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the discovery of the green fluorescent protein ( gfp ) and its ensuing applications as a genetically encoded fluorescent label have significantly advanced our understanding of the complex biochemical processes in living systems . mutations of wtgfp and similar fluorescent and chromoproteins gave rise to a palette of biomarkers covering the entire visible range from blue to far - red . red fluorescent proteins ( rfps ) are particularly important for in vivo imaging as they enable better penetration depth and signal separation from cellular autofluorescence . fps enable studies of protein protein interactions , gene expression , protein localization , and intracellular protein targeting . the unique properties of fps have also been exploited for development of ph , metal , redox and hydrogen peroxide sensors , and phototoxic agents . however , fps as fluorescent markers suffer from irreversible ( photobleaching ) and reversible ( blinking or flickering ) loss of fluorescence that limits their applications . despite being crucial for engineering of better fluorescent labels , the mechanistic understanding of these processes in fps is quite rudimentary , in stark contrast to similar phenomena in synthetic dyes . several photobleaching and blinking pathways have been determined using a combination of theoretical and experimental approaches . photoinduced rapture of the -conjugated system in irisfp resulting in a ( dark ) chromophore with sp - hybridized c was shown to be responsible for blinking . similar chromophore structures with partially destroyed -conjugation have been suggested to explain the x - ray structure of a bleached state of killerred . among other suggested blinking / flickering pathways are transitions to triplet states , reversible conformational changes , and changes of the chromophore h - bonding network and its protonation state . although photobleaching of organic dyes is often initiated by electron attachment to ( or detachment from ) electronically excited chromophores , it is unclear whether such photoreduction and photo - oxidation processes play a role in fps . it was suggested that formation of radical species precedes photobleaching of irisfp upon x - ray irradiation . photoinduced electron transfer in fps leads to the so - called oxidative redding , the photoconversion resulting in the green - to - red shift of the fluorescence . however , the mechanism of oxidative redding and even the chemical identity of the red form are still unknown . apart from their relevance to photostability , formation of long - living radical species may have implications for the phototoxicity of fps . indeed , an epr spectrum was reported for the killerred protein , the most phototoxic fp , although the structure of the long - lived radical is not known . killerred is strongly phototoxic when irradiated with light of 540580 nm ( 2.302.14 ev ) in the presence of oxygen ; the phototoxicity was attributed to the formation of reactive oxygen species ( ros ) . figure 1 illustrates major pathways of ros formation including superoxide radical and singlet oxygen . the key point is that electronic excitation increases both reducing and oxidizing abilities of the chromophore , because chro and chro can accept an extra electron into the highest occupied molecular orbital ( homo ) or donate an electron from the lowest unoccupied molecular orbital ( lumo ) , in contrast to the ground - state chromophore ( chro ) that accepts an electron into the lumo and donates one from the homo . the photo - oxidation / photoreduction leads to a formation of doublet radicals , chro and chro , respectively . the latter can then donate an electron to a nearby oxygen molecule forming o2. superoxide can also be formed by direct oxidation of chro by oxygen . contrary to superoxide formation , o2 production must proceed through the triplet state of the chromophore , thus requiring an intersystem crossing step . the chromophore in killerred is linked to the protein via glu68 ( r1 ) and ile64 ( r3 ) , and r2 is a side chain of gln forming the chromophore . ( b ) photoinduced processes that can lead to the formation of reactive oxygen species ( ros ) , o2 ( singlet oxygen ) , and o2 ( superoxide ) . relevant chromophore states are the ground and excited singlet states ( chro and chro ) , a triplet state ( chro ) , and electron - attached and electron - detached doublet states ( chro and chro ) . ( c ) electronic configurations of the ground - state singlet , lowest excited singlet and triplet states , and two doublet radicals derived by electron attachment and detachment from chro . solid arrows denote pathways supported by the previous and present studies , whereas dotted lines represent merely speculative ( at this moment ) pathways of ros formation . the lack of singlet oxygen emission suggests that killerred is a radical - based type i photosensitizer , although superoxide s relatively low toxicity is difficult to reconcile with the very strong phototoxicity of killerred . photoreduction / photo - oxidation of the chromophore does not necessarily involve external redox agents ; that is , nearby amino acids may also serve as electron donors / acceptors . for example , photoinduced electron transfer from neighboring glu to the chromophore is believed to be an initial step in the photoinduced decarboxylation of gfp and dsred . in sum , the bulk of knowledge of synthetic dyes properties , along with emerging data on fps , suggests that understanding the dynamics of photoinduced electron transfer events in rfps as well as lifetimes and chemical identity of the species involved in these processes is of central importance for understanding the photobleaching and phototoxicity of fps . aiming to elucidate photoinduced transformations of fps , we performed a time - resolved spectroscopic study of rfp dynamics in the micro- to millisecond range bridging the gap between two commonly studied regimes , femto- to microseconds and seconds to minutes . this time scale is typical for processes involving triplet states and radical species in related systems . here , we report the first broadband transient absorption ( ta ) in the microsecond to second time domain of three rfps , dsred , mrfp , and killerred . all three share an identical anionic chromophore ( figure 1a ) and have similar steady - state absorption / emission properties ; however , variations in the local environment and the barrel s structure lead to different photostability , brightness , and phototoxicity . dsred is a tetrameric rfp from which many other fps , including mrfp , have been derived . despite the common chromophore structure , these proteins differ dramatically in their phototoxicity in the order dsred < mrfp < killerred . interestingly , dsred shows better photostability than , for example , monomeric rfps from the mfruit series , which is attributed to the structural weakness of their -barrels facilitating the diffusion of small species in and out of the protein interior . to understand their photocycle section ii describes experimental and computational details ( additional information is provided in the supporting information ( si ) ) . time - resolved emission and absorption spectra were acquired using the instruments described below . fluorescence lifetimes were measured using an edinburgh instruments time - correlated single photon counting ( tcspc ) system . in these measurements , picosecond diode laser ( picoquant ) and subnanosecond led excitation pulses ( edinburgh instruments ) emitting at 467 and 590 nm , respectively , were used as excitation light sources . the detection system consisted of a high - speed microchannel plate photomultiplier tube ( mcp - pmt , hamamatsu r3809u-50 ) and tcspc electronics . the time resolution of the system was 30 ps after deconvolution with an irf signal . subpicosecond excited - state absorption spectra of rfps were measured using a commercially available pump probe spectroscopic system ( helios , ultrafast systems ) pumped by the femtosecond laser system consisting of a ti : sapphire regenerative amplifier ( solstice , spectra - physics , 800 nm , 1 khz ) and an optical parametric amplifier ( opa , topas - c , spectra - physics , 2902800 nm , 100 fs fwhm ) . transient absorption spectroscopy measurements in the microsecond - to - second time domain were performed using a custom - built kinetic setup based on andor and basler vision microarray imaging cameras , allowing the broadband ta measurements with 1 s time resolution after a single pulse excitation . this setup , previously used to study multiheme o2-reducing electron - transfer enzymes , is located at the university of helsinki ( helsinki , finland ) , where measurements were performed . unit a from the 3gb3 ( pdb : id ) x - ray structure was used as a model of the killerred protein . the only amino acids found in the nonstandard protonation states are glu68 and glu218 ; they were protonated in our model . the protonation states of the amino acids were consistent with the hydrogen - bonding pattern in the x - ray structure ( see the si for detailed discussion ) . the protein was placed in the 100 100 100 water box . eight cl and 15 na counterions were added to neutralize the protein s surface charges . molecular dynamics ( md ) simulations were performed to sample the ground - state structure of the protein . the system was first equilibrated during consecutive nvt ( t = 300 k ) and npt ( p = 1 atm , t = 300 k ) 100 ps simulations . the equilibrium parameters ( bond lengths and angles ) were taken from the optimized b97x - d/6 - 31+g(d , p ) ( see ref ( 44 ) ) killerred chromophore structure . nbo partial charges computed with b97x - d/6 - 31+g(d , p ) were used . force constants were taken from ref ( 46 ) and from the charmm27 parameters for the chemically similar moieties . the list of all force field parameters for the killerred chromophore is given in the si . all excitation / attachment / detachment energies were computed using the geometries taken from the md snapshots for killerred with the chromophore in the closed - shell anionic form . the qm part included the chromophore , as shown in figure s10 in the si . the rest of the system was represented by the mm force field point charges . in our previous studies of gfp and mstrawberry , we found that the effects of extending the qm part by including nearby residues are insignificant . in the present study , we revisited this question and investigated the effect of including arg94 in the qm part . as discussed in the si , the shifts in excitation energies caused by including the arg94 side chain into the qm part were negligible ( si table s1 ) . the resulting nonzero total charge on a truncated residue was redistributed equally between the rest of the residue sos - cis(d ) and tddft(b5050lyp ) were used in the qm calculations for the anion and dianion excitation energies , respectively . sos - cis(d ) was demonstrated to provide accurate results for the excitation energies of the gfp chromophore and its analogues . however , the benchmark calculations for the dianion radical showed that sos - cis(d ) based on the uhf reference suffers from spin contamination ( s 1.5 ) , whereas rohf calculations exhibit problematic convergence behavior . we found that for this open - shell dianionic system , the tddft description is more reliable ( s 0.80.9 ) ; thus , this method was used for excitation energy calculation . electron attachment and detachment energies were computed with b97x - d ( see ref ( 44 ) ) within the same qm / mm scheme . b97x - d was previously shown to provide reliable estimates of ionization energies of organic molecules , for example , dna bases . we note that the computed excitation energies are almost identical for tddft(b97x - d ) , tddft(b5050lyp ) , and sos - cis(d ) with a rohf - like reference ( table 1 ) . the quantitative agreement between the three different approaches validates the applicability of the chosen tddft scheme for calculation of excitation energies of the open - shell dianion . the 6 - 31g(d , p ) basis was used for more expensive excitation energies calculations ( 50 calculations for the md trajectory ) . the cc - pvdz basis was employed for attachment / detachment energy calculations . in addition , extrapolation to aug - cc - pvtz was performed using the following protocol . in calculations of fluctuation of the excitation and electron attachment / detachment energies along the md trajectory , the calculations were performed for the md trajectory snapshots extracted each 20 ps using the 6 - 31g(d , p ) and cc - pvdz bases for excitation and attachment / detachment energies , respectively . each 200 ps the same calculations were performed with the aug - cc - pvtz basis set . these data were used for extrapolation to the aug - cc - pvtz basis set . uhf reference converged to the solution close to the rohf one using maximum overlap method; s = 1.2 . all quantum - chemical calculations were performed using the q - chem package . table 2 summarizes spectral features and lifetimes of the ta components in microsecond - to - second and subpicosecond decays . all data are taken in pbs buffer , ph 7.5 at 25 c . owing to their structural similarity , all three rfps have similar steady - state spectra . killerred and mrfp have identical absorption and emission maxima at 585 nm ( 2.12 ev ) and 609 nm ( 2.04 ev ) . dsred has absorption and emission maxima at 561 nm ( 2.21 ev ) and 595 nm ( 2.08 ev ) , respectively , which were close to the previously reported values . a spectroscopic signature of blinking or bleaching behavior is delayed recovery of the ground - state absorption relative to the decay of the bright excited state . to test whether bleaching is significant at our conditions , we measured the transient absorption spectra with subpicosecond time resolution in the 03 ns time window , a common time scale for the fluorescent - state lifetime . the subpicosecond transient absorption ( ta ) spectra of the three rfps show that the recovery of the ground - state absorption occurs on a longer time scale than the decay of the bright excited state . all three rfps exhibit ta bands in the 8001400 nm ( 1.550.89 ev ) range and a band at 430 nm ( 2.88 ev ) . analysis of the transient decays measured at different wavelengths yields lifetimes of 1.5 , 1.8 , and 3.9 ns in killerred , mrfp , and dsred , respectively . all lifetimes were equal to or close to the fluorescence lifetime of the proteins measured using the time - correlated single photon counting technique ( see the si ) . therefore , we attribute these transients to the simple decay of the excited singlet - state population . the s1 lifetimes decrease in the order dsred > mrfp > killerred , revealing the increased quenching in this order . this effect can be attributed to the increased permeability of the -barrels by the external quenchers , variations of the local structure around the chromophore , and/or increase of the isc rate . altogether , the properties of the s0 s1 and s1 s0 transitions of the three rfps are similar . however , we note that the recovery of the ground - state bleach is slower than the decay of the s1 state , indicating other possible deactivation pathways for chro . therefore , we investigated these pathways using measurements at longer ( s - to - s ) time scales . the photophysics of rfps on this time scale was previously studied using fluorescence correlation spectroscopy and single - molecule spectroscopy techniques . complex flickering dynamics was observed ; it was interpreted in the framework of kinetic models involving several dark and bright states . figure 2 shows ta spectra of the three rfps in the micro- to millisecond range . the main features of the spectra are ground - state bleach and one major red - shifted ta feature separated by isosbestic points . the best exponential fit ( global ) of the ta data gave three components for all rfps studied ( see the si for the kinetic fit details ) . the kinetic fits with their residuals at selected wavelengths are shown in figure s3 in the si . the major component , 2 , for all three rfps has a maximum centered close to 740 nm ( table 2 ) and absorption that spans beyond 1040 nm , where we reach the limit of our detector . the minor features beyond 800 nm could be attributed to the vibronic structure of the same absorption band . note that ground - state bleach recovery in killerred and mrfp is slower than the decay of 730 nm transient , which suggests that the recovery may proceed through additional intermediates or that there are additional pathways for reversible and irreversible bleaching . the detailed understanding of the mechanism of interconversion between the bright state of killerred protein and the red - shifted intermediate is the subject of future work and is beyond the scope of the current study . here , we focus on the assignment of the chemical nature of the intermediate . micro- to millisecond transient absorption of killerred ( a ) , mrfp ( b ) , and dsred ( c ) at various time points . for experimental details , see the si . notably , all three proteins exhibit a major similar transient feature peaking at 731 , 722 , and 740 nm for killerred , mrfp , and dsred , respectively . to elucidate the structure of this red - shifted intermediate ( rsi ) ( 720740 nm ) , qm a relatively short lifetime of this intermediate ( s ) suggests that the spectral changes are likely due to changes in the electronic structure , conformation , or protonation state of the chromophore , rather than dramatic changes in the chemical nature ( processes such as chromophore maturation and oxidative redding occur on the minutes to hours time scale ) . figure 3 illustrates the energetics of the relevant electronic states of killerred protein : the ground state ( chro ) , the first excited state ( chro ) , the lowest and excited triplets ( chro and chro ) , electronic states formed by electron detachment ( chro and chro * ) from and electron attachment ( chro and chro ) to the anionic chromophore . the left panel shows the values averaged along molecular dynamics trajectory sampling ground - state geometries of the protein . ( a ) excitation energy of chro ( black ) , electron attachment energy ( blue ) , excitation energy of chro ( red ) , electron detachment energy ( orange ) , and excitation energy of chro ( violet ) . excitation energies were computed using a qm / mm protocol with the mm part represented by point charges . the values are averaged over 50 snapshots taken from the md trajectory of the killerred protein . ( b ) fluctuation of the excitation and electron attachment / detachment energies along the md trajectory extrapolated to the aug - cc - pvtz values . the lowest excited state of the neutral radical ( photooxidation product ) lies 0.5 ev above the rsi absorption maximum and is a dark state ( fl < 0.01 ) . the only bright transitions that are energetically close to the observed intermediate absorption ( 1.70 ev or 731 nm ) are chro chro ( 1.3 ev or 954 nm ) and chro chro ( 1.7 ev or 729 nm ) . because the upper bound for triplet lifetime in killerred is 40 s , which is 1 order of magnitude shorter than the transient centered at 731 nm , we rule out the triplet state as a possible candidate for the rsi . thus , the only viable candidate responsible for the transient intermediate with max = 731 nm is an electron - attached dianionic state of the chromophore ( chro ) . such a doubly charged state is unstable in the gas phase ; however , it is stabilized by the interactions with the nearby charged amino acid residues and is 1.7 ev below the ground anionic state of the protein - bound chromophore . we attribute this stabilization to interaction with the positively charged arg94 residue forming an h - bond with the chromophore . indeed , calculations with arg excluded from the mm system show no stabilization of the dianion electron attachment becomes energetically unfavorable ( + 1.0 ev ) . although electron attachment to the anionic chromophore in killerred is 1.7 ev exothermic ( computed value ) even for the ground - state chromophore , there should be a reducing agent providing an electron with matching or greater oxidation potential . however , amino acids have much higher ionization energies , and it is unlikely that the environmental effects can make electron transfer from nearby amino acids to the ground - state chromophore energetically favorable ( otherwise , chro would undergo spontaneous reduction ) . thus , we posit that the intermediate can be formed only via an excited state of the chromophore . the preliminary ta data taken with nanosecond resolution demonstrate that the rsi is formed within the 0.5 ns pulsewidth of the excitation laser . this indeed supports the direct formation of this intermediate from the chro singlet excited states . direct formation of the dianion from the lowest singlet excited state of the chromophore will be accompanied by a 4 ev energy gain . we note that the ionization energies of the amino acids are still higher ; for example , ionization energy of 7.3 0.2 ev was reported for tryptophan on the basis of vacuum ultraviolet single - photon ionization mass spectrometry experiments . however , the local environment may significantly alter the ionization / attachment energies as well as the resulting oxidation / reduction potentials of the amino acids , similarly to the observed effect of the nearby residues on the chromophore s energetics . to interrogate the identity of the main transient , we investigated the effect of external oxidative / reducing agents on the observed ta for all three rfps . first , the ta was measured for samples in ambient , aerobic ( oxygen - saturated ) , and fully anaerobic conditions . the lifetime of the main ta component decreased significantly in oxygen - saturated conditions for killerred and mrfp ( table 3 ) , whereas in dsred it remained mostly unchanged ( si figure s5 ) . the effects of several oxidant and reductants ( cytochrome c , -mercaptoethanol , nad , potassium ferricyanide , flavin mononucleotide ) were also tested on the kinetics of the rsi feature in killerred , mrfp , and dsred . in all three cases , these oxidants and reductants are likely too large or hydrophilic and , therefore , are unable to access the chromophore buried inside the protein barrels . thus , we tested a smaller molecule , h2o2 , that can act as either a relatively strong oxidant or a weak reductant . remarkably , h2o2 had a profound effect on the lifetime of the transient in killerred , decreasing the lifetime to 95 s in the presence of 5 m h2o2 . the effect was much weaker in mrfp and dsred , for which the lifetimes decreased to 147 and 810 s , respectively . we attribute this phenomenon to the unique structural feature of killerred s -barrel interior , a long water - filled channel . it has been hypothesized that this channel facilitates the escape of ros from the protein , thereby contributing to killerred s phototoxicity . in the control experiments , we observe that the same levels of h2o2 have little to no effect on the ground - state absorption spectrum in all three proteins ( si ) . thus , the transient is efficiently quenched by h2o2 , which is consistent with the proposed strongly reducing dianionic state . the dianionic radical chromophore ( chro ) can then donate an electron to o2 to generate superoxide , which is likely the main mechanism of phototoxicity in killerred . kinetics of transient absorption signal at 731 nm ( 1.70 ev ) for killerred ( top ) , at 722 nm ( 1.72 ev ) for mrfp ( middle ) , and at 745 nm ( 1.66 ev ) for dsred in the absence and presence h2o2 . for experimental details , see the si . among the three cytotoxic proteins studied , the phototoxicity and radical production increase in the order dsred < mrfp < killerred . thus , there is a direct correlation between phototoxicity and our quenching experiments in which the main ta component ( rsi ) is quenched with o2 and h2o2 . for example , the transient in the most phototoxic fluorescent protein , killerred , shows the greatest quenching with h2o2 and o2 , whereas the lifetime of the transient in dsred remains mostly unaffected . this observation suggests using weak reducing / oxidative pairs to control the phototoxicity and photostability of fps , as successfully done for organic dyes in solution . it is quite interesting that an agent as small as o2 is unable to penetrate the -barrel of dsred ; however , it is not that surprising because previous researchers had to use guanadinium hcl to add flexibility to a gfp mutant to detect singlet oxygen production . recent studies of rfps from the mfruit family attribute their poor photostability to the structural weakness of their -barrel that facilitates oxygen diffusion . we report an observation of a novel strongly red - shifted ( max 720740 nm ) photoinduced intermediate that is common for killerred , mrfp , and dsred and decays on the microsecond time scale . on the basis of the electronic structure calculations we interpret the signal as absorption by the dianionic open - shell state of the chromophore formed by photoreduction . the rsi is effectively quenched by oxygen and hydrogen peroxide in killerred , which features a large water channel facilitating access to the chromophore , whereas its lifetimes in dsred and mrfp are not strongly affected . the results raise the question of whether photoreduction is common for all fps and if the corresponding electron - attached states are common gateway states for photobleaching and blinking . in such a case , understanding the protein environment effects on the formation and lifetime of the radical species is crucial for development of more photostable fps , of phototoxic agents for photodynamic therapy , and of more stable fps with respect to photoinduced decarboxylation . further studies including time - resolved epr , ir , and raman spectroscopies are needed for a detailed characterization of the structure and , especially , of the formation mechanism of these species , which possibly play a crucial role in the photochemistry of photoactive proteins .
red fluorescent proteins ( rfps ) are indispensable tools for deep - tissue imaging , fluorescence resonance energy transfer applications , and super - resolution microscopy . using time - resolved optical spectroscopy this study investigated photoinduced dynamics of three rfps , killerred , mrfp , and dsred . in all three rfps , a new transient absorption intermediate was observed , which decays on a microsecond millisecond time scale . this intermediate is characterized by red - shifted absorption at 1.681.72 ev ( max = 720740 nm ) . on the basis of electronic structure calculations , experimental evidence , and published literature , the chemical nature of the intermediate is assigned to an unusual open - shell dianionic chromophore ( dianion - radical ) formed via photoreduction . a doubly charged state that is not stable in the isolated ( gas phase ) chromophore is stabilized by the electrostatic field of the protein . mechanistic implications for photobleaching , blinking , and phototoxicity are discussed .
Introduction Experimental Setup and Computational Details Results and Discussion Conclusions
red fluorescent proteins ( rfps ) are particularly important for in vivo imaging as they enable better penetration depth and signal separation from cellular autofluorescence . in sum , the bulk of knowledge of synthetic dyes properties , along with emerging data on fps , suggests that understanding the dynamics of photoinduced electron transfer events in rfps as well as lifetimes and chemical identity of the species involved in these processes is of central importance for understanding the photobleaching and phototoxicity of fps . here , we report the first broadband transient absorption ( ta ) in the microsecond to second time domain of three rfps , dsred , mrfp , and killerred . all three share an identical anionic chromophore ( figure 1a ) and have similar steady - state absorption / emission properties ; however , variations in the local environment and the barrel s structure lead to different photostability , brightness , and phototoxicity . we found that for this open - shell dianionic system , the tddft description is more reliable ( s 0.80.9 ) ; thus , this method was used for excitation energy calculation . the subpicosecond transient absorption ( ta ) spectra of the three rfps show that the recovery of the ground - state absorption occurs on a longer time scale than the decay of the bright excited state . analysis of the transient decays measured at different wavelengths yields lifetimes of 1.5 , 1.8 , and 3.9 ns in killerred , mrfp , and dsred , respectively . here , we focus on the assignment of the chemical nature of the intermediate . micro- to millisecond transient absorption of killerred ( a ) , mrfp ( b ) , and dsred ( c ) at various time points . notably , all three proteins exhibit a major similar transient feature peaking at 731 , 722 , and 740 nm for killerred , mrfp , and dsred , respectively . to elucidate the structure of this red - shifted intermediate ( rsi ) ( 720740 nm ) , qm a relatively short lifetime of this intermediate ( s ) suggests that the spectral changes are likely due to changes in the electronic structure , conformation , or protonation state of the chromophore , rather than dramatic changes in the chemical nature ( processes such as chromophore maturation and oxidative redding occur on the minutes to hours time scale ) . such a doubly charged state is unstable in the gas phase ; however , it is stabilized by the interactions with the nearby charged amino acid residues and is 1.7 ev below the ground anionic state of the protein - bound chromophore . the effects of several oxidant and reductants ( cytochrome c , -mercaptoethanol , nad , potassium ferricyanide , flavin mononucleotide ) were also tested on the kinetics of the rsi feature in killerred , mrfp , and dsred . remarkably , h2o2 had a profound effect on the lifetime of the transient in killerred , decreasing the lifetime to 95 s in the presence of 5 m h2o2 . in the control experiments , we observe that the same levels of h2o2 have little to no effect on the ground - state absorption spectrum in all three proteins ( si ) . kinetics of transient absorption signal at 731 nm ( 1.70 ev ) for killerred ( top ) , at 722 nm ( 1.72 ev ) for mrfp ( middle ) , and at 745 nm ( 1.66 ev ) for dsred in the absence and presence h2o2 . for example , the transient in the most phototoxic fluorescent protein , killerred , shows the greatest quenching with h2o2 and o2 , whereas the lifetime of the transient in dsred remains mostly unaffected . we report an observation of a novel strongly red - shifted ( max 720740 nm ) photoinduced intermediate that is common for killerred , mrfp , and dsred and decays on the microsecond time scale . on the basis of the electronic structure calculations we interpret the signal as absorption by the dianionic open - shell state of the chromophore formed by photoreduction . further studies including time - resolved epr , ir , and raman spectroscopies are needed for a detailed characterization of the structure and , especially , of the formation mechanism of these species , which possibly play a crucial role in the photochemistry of photoactive proteins .
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asthma is a chronic inflammatory lung disease , characterized by recurrent wheezing , and is the leading cause of morbidity and mortality in children worldwide . most infants who experience wheezing episodes also exhibit evidence of an ongoing viral respiratory infection . the viral agents respiratory syncytial virus ( rsv ) , influenza a ( flu a ) , and particularly human rhinovirus ( hrv ) appear to be the most prevalent and recurring threats . hrvs have been noted as pathogens of the common cold for over 50 years ; however , recent advances in viral molecular diagnostics have led to an appreciation of their role in more significant respiratory diseases . these include asymptomatic infections , frequent common colds , and severe lower respiratory infections , especially infantile bronchiolitis , childhood pneumonia , and acute exacerbations of chronic respiratory diseases , such as asthma . hrvs can be divided into three genetically distinct species : hrv - a , hrv - b , and the recently identified hrv - c . a proposed classification protocol based on the sequence of the hrv capsid genes revealed approximately 78 a types , 30 b types , and 51 c types . as with many other viruses , genetically , hrv - c is most closely related to hrv - a and hrv - b , but even small genetic variance can result in significant differences in clinical impact . it is well - known that hrvs are a major trigger for asthma exacerbations , and hrv - c is now under investigation for its potential role in the development of asthma . this is a preliminary study to assess the associations between different hrv species , particularly hrv - c , and asthma in young children in china . nasopharyngeal aspirates ( npas ) were obtained for respiratory virus screening from three groups of patients who visited the children 's hospital affiliated to the capital institute of pediatrics in beijing , china . these groups included : ( 1 ) asthma group : the inclusion criteria were pediatric patients younger than 16 years of age , diagnosed with asthma characterized by recurrent episodes ( 3 ) of wheezing , breathlessness , chest tightness , and coughing , particularly at night or in the early morning . the exclusion criteria were pediatric patients diagnosed with bronchiolitis or other diseases with symptoms of wheezing , breathlessness , chest tightness , and coughing . ( 2 ) nonasthma , acute respiratory infection ( ari ) group : the inclusion criteria were pediatric patients younger than 16 years of age , diagnosed with ari , including rhinitis , laryngitis , tonsillitis , tracheitis , bronchitis , bronchiolitis , and pneumonia . ( 3 ) control group : the inclusion criteria were pediatric patients with underlying surgical therapy . the exclusion criterion was pediatric patients confirmed with respiratory infections within the 10 days prior to collection of npa . the study was approved by the ethics committee of the capital institute of pediatrics . written informed consent was obtained from the parents or guardians of participants in the control group . the capital institute of pediatrics determined informed consent was not needed for the participants in the asthma and nonasthma ari groups for using the leftover npa for respiratory virus screening . supernatants were inoculated onto madin darby canine kidney cell cultures to isolate flu a and b , onto cultured hep-2 and vero cell lines to isolate rsv and human adenovirus ( hadv ) , and llc - mk2 cell cultures for parainfluenza virus ( piv ) types 13 . cell pellets from all npa samples were re - suspended and spotted onto an acetone - cleaned slide . then , individual monoclonal antibody reagents , labeled with fluorescein isothiocyanate ( fitc ) against rsv , hadv , flu a and b , and piv 13 , were used for specific virus identification by direct immunofluorescence assay ( dfa ) with a d ultra dfa respiratory virus screening & i d kit ( diagnostic hybrids inc . , athens , oh , usa ) . in addition , fitc - labeled monoclonal antibody against human metapneumovirus ( hmpv ) ( diagnostic hybrids inc . , athens , oh , usa ) was also used to detect hmpv . next , dna and rna were extracted from 150 l of each npa specimen using trizol reagent ( invitrogen inc . , carlsbad , ca , usa ) and solubilized in 30 l of 8 mmol / l naoh for dna or 30 l of diethylpyrocarbonate - treated water for rna , according to the manufacturer 's instructions . reverse transcription - polymerase chain reaction ( rt - pcr ) and pcr assays were performed for detection of piv types 14 , human bocavirus , and wu and ki polyomaviruses . both rt - pcr and dfa methods can detect piv types 13 from specimens ; however , rt - pcr can detect piv-4 ; therefore , the piv results in this study refer to those of rt - pcr . semi - nested pcr was performed for hrv screening using primers p1 ( 163181 ) : 5-c(a / g)a ( g / a)ca ctt ctg t(t / c)(t / a ) ccc c-3 , p2 ( 533551 ) : 5-c(a / g)a cta ctt tgg gtg tcc g-3 , and r2 ( 10821064 ) : 5-c(t / g / c / a)g g(t / c / a / g)a ( a / g)(t / c ) t tcc a(c / a)c acc a-3 , designed according to the conserved vp4/vp2 region of hrv ( genbank sequence l24917 ) , resulting in amplification of a 920-bp fragment in the first run with primers p1 and r2 and a 550-bp fragment in the second run with primers p2 and r2 . a previously published protocol was used , and all procedures , including specimen processing , rna and dna extraction , rt - pcr / pcr amplification , and gel electrophoresis , were performed in separate areas of the laboratory to avoid pcr contamination . amplified products from the second semi - nested pcr run were sequenced by invitrogen inc . , and phylogenetic analysis performed via an ncbi blast search ( http://blast.ncbi.nlm.nih.gov ) and the mega version 6.0 software package , to identify hrv species . phylogenetic trees were constructed with the neighbor - joining method and maximum composite likelihood model , using hrv sequences from our study and from genbank . a discrete gamma distribution used to model evolutionary rate differences among sites ( 1 category , + g ) was constructed with mega 6.0 . bootstrap resampling ( 1000 replications ) was used to assess the reliability of individual nodes in each phylogenetic tree . chi - square and analysis of variance were used to compare the prevalence of the three hrv species in each study group . to identify the correlation of different species of hrv infection with asthma , spearman 's rank correlation was used to compare the positive rates of hrv - a , b , or c between the asthma group and control group or nonasthma ari group . using odds ratio ( or ) as an indicator , regression analysis was used to determine the effect of different species of hrv infection on asthma . all tests were two - tailed , and p < 0.05 was considered statistically significant . all analyses were conducted in spss statistics version 22.0 ( ibm , ny , usa ) . nasopharyngeal aspirates ( npas ) were obtained for respiratory virus screening from three groups of patients who visited the children 's hospital affiliated to the capital institute of pediatrics in beijing , china . these groups included : ( 1 ) asthma group : the inclusion criteria were pediatric patients younger than 16 years of age , diagnosed with asthma characterized by recurrent episodes ( 3 ) of wheezing , breathlessness , chest tightness , and coughing , particularly at night or in the early morning . the exclusion criteria were pediatric patients diagnosed with bronchiolitis or other diseases with symptoms of wheezing , breathlessness , chest tightness , and coughing . ( 2 ) nonasthma , acute respiratory infection ( ari ) group : the inclusion criteria were pediatric patients younger than 16 years of age , diagnosed with ari , including rhinitis , laryngitis , tonsillitis , tracheitis , bronchitis , bronchiolitis , and pneumonia . ( 3 ) control group : the inclusion criteria were pediatric patients with underlying surgical therapy . the exclusion criterion was pediatric patients confirmed with respiratory infections within the 10 days prior to collection of npa . the study was approved by the ethics committee of the capital institute of pediatrics . written informed consent was obtained from the parents or guardians of participants in the control group . the capital institute of pediatrics determined informed consent was not needed for the participants in the asthma and nonasthma ari groups for using the leftover npa for respiratory virus screening . supernatants were inoculated onto madin darby canine kidney cell cultures to isolate flu a and b , onto cultured hep-2 and vero cell lines to isolate rsv and human adenovirus ( hadv ) , and llc - mk2 cell cultures for parainfluenza virus ( piv ) types 13 . cell pellets from all npa samples were re - suspended and spotted onto an acetone - cleaned slide . then , individual monoclonal antibody reagents , labeled with fluorescein isothiocyanate ( fitc ) against rsv , hadv , flu a and b , and piv 13 , were used for specific virus identification by direct immunofluorescence assay ( dfa ) with a d ultra dfa respiratory virus screening & i d kit ( diagnostic hybrids inc . , athens , oh , usa ) . in addition , fitc - labeled monoclonal antibody against human metapneumovirus ( hmpv ) ( diagnostic hybrids inc . , athens , oh , usa ) was also used to detect hmpv . next , dna and rna were extracted from 150 l of each npa specimen using trizol reagent ( invitrogen inc . , carlsbad , ca , usa ) and solubilized in 30 l of 8 mmol / l naoh for dna or 30 l of diethylpyrocarbonate - treated water for rna , according to the manufacturer 's instructions . reverse transcription - polymerase chain reaction ( rt - pcr ) and pcr assays were performed for detection of piv types 14 , human bocavirus , and wu and ki polyomaviruses . both rt - pcr and dfa methods can detect piv types 13 from specimens ; however , rt - pcr can detect piv-4 ; therefore , the piv results in this study refer to those of rt - pcr . semi - nested pcr was performed for hrv screening using primers p1 ( 163181 ) : 5-c(a / g)a ( g / a)ca ctt ctg t(t / c)(t / a ) ccc c-3 , p2 ( 533551 ) : 5-c(a / g)a cta ctt tgg gtg tcc g-3 , and r2 ( 10821064 ) : 5-c(t / g / c / a)g g(t / c / a / g)a ( a / g)(t / c ) t tcc a(c / a)c acc a-3 , designed according to the conserved vp4/vp2 region of hrv ( genbank sequence l24917 ) , resulting in amplification of a 920-bp fragment in the first run with primers p1 and r2 and a 550-bp fragment in the second run with primers p2 and r2 . a previously published protocol was used , and all procedures , including specimen processing , rna and dna extraction , rt - pcr / pcr amplification , and gel electrophoresis , were performed in separate areas of the laboratory to avoid pcr contamination . amplified products from the second semi - nested pcr run were sequenced by invitrogen inc . , and phylogenetic analysis performed via an ncbi blast search ( http://blast.ncbi.nlm.nih.gov ) and the mega version 6.0 software package , to identify hrv species . phylogenetic trees were constructed with the neighbor - joining method and maximum composite likelihood model , using hrv sequences from our study and from genbank . a discrete gamma distribution used to model evolutionary rate differences among sites ( 1 category , + g ) was constructed with mega 6.0 . bootstrap resampling ( 1000 replications ) was used to assess the reliability of individual nodes in each phylogenetic tree . chi - square and analysis of variance were used to compare the prevalence of the three hrv species in each study group . to identify the correlation of different species of hrv infection with asthma , spearman 's rank correlation was used to compare the positive rates of hrv - a , b , or c between the asthma group and control group or nonasthma ari group . using odds ratio ( or ) as an indicator , regression analysis was used to determine the effect of different species of hrv infection on asthma . all tests were two - tailed , and p < 0.05 was considered statistically significant . all analyses were conducted in spss statistics version 22.0 ( ibm , ny , usa ) . between october 2013 and november 2014 , 155 young children with a mean age 2.7 1.5 years , 116 ( 74.8% ) male , and 39 ( 25.2% ) female were enrolled in the asthma group , 461 children with a mean age 3.0 3.3 years , 288 ( 62.5% ) male , and 173 ( 37.5% ) female were enrolled in the nonasthma group . between august 2014 and march 2015 , 86 children with a mean age 3.0 2.3 years , 70 ( 81.4% ) male , and 16 ( 18.6% ) female were enrolled in the control group . the viral pathogen screening for all patients [ table 1 ] revealed that hrvs were the most common pathogens , with a prevalence of 15.4% ( 108/702 ) . similarly , in the asthma group , hrvs were the most common pathogens , with a prevalence of 25.8% ( 40/155 ) . in the nonasthma ari group , 11.1% ( 51/461 ) patients were positive for hrv . in the control group , 19.8% ( 17/86 ) of patients were hrv - positive . the prevalence of hrv was significantly different ( f = 10.680 , p < 0.001 ) among the three study groups ( 25.8% ; 11.1% ; 19.8% ) . while no significant difference was found between the asthma and control groups ( f = 1.113 , p = 0.293 ) , there was a significant difference between the asthma and nonasthma ari groups ( f = 20.633 , p = 0.000 ) . results of viral detection in different patient groups ari : acute respiratory infection ; rsv : respiratory syncytial virus ; hadv : human adenovirus ; flu a : influenza virus a ; flu b : influenza virus b ; hmpv : human metapneumovirus ; hrvs : human rhinoviruses ; hbov : human bocavirus ; wu : wu polyomavirus ; ki : ki polyomavirus ; piv1 , piv2 , piv3 , and piv4 : parainfluenza viruses 1 , 2 , 3 , and 4 ; dfa : direct immunofluorescence assay ; pcr : polymerase chain reaction . in the 108 samples positive for hrv by semi - nested pcr , there were 105 ( 97.2% ) specimens with sufficient pcr product for sequencing . by sequencing the 105 pcr products , partial vp4/vp2 nucleotide sequences were obtained , then blast - searched against available sequences of hrv - a , hrv - b , and hrv - c from genbank . figure 1 shows these 105 sequences from the three patient groups were divided into three hrv species groups ( a , b , and c ) . the hrv - a cluster was closer to hrv - b than hrv - c . the hrv - c cluster , with 56 of the 105 sequences , is the largest one of these three clusters , revealing hrv - c as the dominant species in the study population . the hrv - a cluster , with 41 sequences , is the second largest , and the hrv - b cluster , with eight sequences , is the smallest . the sequences belonging to different hrv species from the different patient groups were stratified into different subclusters , together with sequences of different hrv serotypes from genbank , and there are sequences from different patient groups clustered into one subcluster [ figure 1 ] . the 105 partial vp4/vp2 gene sequences from this study and hrv species a , b , and c sequences from genbank were used for tree construction . turquoise circles denote hrv sequences from the control group , blue triangles those from the asthma group , and purple diamonds the sequences from the nonasthma group . the sequence analysis results showed that 41 cases ( 5.8% , 41/702 ) were confirmed as hrv - a , including 22 from the nonasthma ari group ( 4.8% , 22/461 ) , 13 from the asthma group ( 8.4% , 13/155 ) , and six from the control group ( 7.0% , 6/86 ) , eight cases ( 1.4% , 8/702 ) were confirmed as hrv - b , including four from the nonasthma ari group ( 0.8% , 4/461 ) , one from the asthma group ( 0.6% , 1/155 ) , and three from the control group ( 3.5% , 3/86 ) , and 56 ( 8.0% , 56/702 ) were confirmed as hrv - c , including 22 from the nonasthma ari group ( 4.8% , 22/461 ) , 26 from the asthma group ( 16.8% , 26/155 ) , and 6 from the control group ( 9.3% , 6/86 ) . the overall proportion of hrv - a and hrv - c positive samples in the study were similar ( = 2.171 , p > 0.05 ) , and both were higher than that of hrv - b ( = 21.654 , p < 0.01 and = 36.164 , p < 0.01 , for hrv - a and hrv - c , respectively ) . in the nonasthma ari group , there were significant correlations between hrv - a and hrv - c and asthma , with r values of 0.08 ( p = 0.037 , p < 0.05 ) and 0.20 ( p < 0.001 ) , respectively [ table 2 ] . no significant correlations were identified for hrv - a , b , and c infections between the asthma group and control group ( p > 0.05 ) . correlation between different species hrv infection with asthma ari : acute respiratory infection ; hrvs : human rhinoviruses . sex and age were not evenly distributed , with more male cases and more young patients in all groups ; therefore , the distribution of sex and age in different groups was adjusted prior to regression analysis [ table 3 ] . the or between the asthma group and nonasthma ari group for hrv - a and hrv - c were 2.2 ( 95% confidence interval [ ci ] , 1.0654.409 ) and 4.2 ( 95% ci , 2.2627.906 ) , respectively , which confirmed patients with hrv - c infection were 4.2 times more likely to have asthma ( p < 0.001 ) , and patients with hrv - a infection were 2.2 times more likely to have asthma ( p < 0.05 ) . association between different species of hrv infection and asthma in a sample of 702 children in beijing * adjusted . hrv : human rhinoviruses ; ci : confidence interval ; or : odds ratio ; ari : acute respiratory infection . between october 2013 and november 2014 , 155 young children with a mean age 2.7 1.5 years , 116 ( 74.8% ) male , and 39 ( 25.2% ) female were enrolled in the asthma group , 461 children with a mean age 3.0 3.3 years , 288 ( 62.5% ) male , and 173 ( 37.5% ) female were enrolled in the nonasthma group . between august 2014 and march 2015 , 86 children with a mean age 3.0 2.3 years , 70 ( 81.4% ) male , and 16 ( 18.6% ) female were enrolled in the control group . the viral pathogen screening for all patients [ table 1 ] revealed that hrvs were the most common pathogens , with a prevalence of 15.4% ( 108/702 ) . similarly , in the asthma group , hrvs were the most common pathogens , with a prevalence of 25.8% ( 40/155 ) . in the nonasthma ari group , 11.1% ( 51/461 ) patients were positive for hrv . in the control group , 19.8% ( 17/86 ) of patients were hrv - positive . the prevalence of hrv was significantly different ( f = 10.680 , p < 0.001 ) among the three study groups ( 25.8% ; 11.1% ; 19.8% ) . while no significant difference was found between the asthma and control groups ( f = 1.113 , p = 0.293 ) , there was a significant difference between the asthma and nonasthma ari groups ( f = 20.633 , p = 0.000 ) . results of viral detection in different patient groups ari : acute respiratory infection ; rsv : respiratory syncytial virus ; hadv : human adenovirus ; flu a : influenza virus a ; flu b : influenza virus b ; hmpv : human metapneumovirus ; hrvs : human rhinoviruses ; hbov : human bocavirus ; wu : wu polyomavirus ; ki : ki polyomavirus ; piv1 , piv2 , piv3 , and piv4 : parainfluenza viruses 1 , 2 , 3 , and 4 ; dfa : direct immunofluorescence assay ; pcr : polymerase chain reaction . in the 108 samples positive for hrv by semi - nested pcr , there were 105 ( 97.2% ) specimens with sufficient pcr product for sequencing . by sequencing the 105 pcr products , partial vp4/vp2 nucleotide sequences were obtained , then blast - searched against available sequences of hrv - a , hrv - b , and hrv - c from genbank . figure 1 shows these 105 sequences from the three patient groups were divided into three hrv species groups ( a , b , and c ) . the hrv - a cluster was closer to hrv - b than hrv - c . the hrv - c cluster , with 56 of the 105 sequences , is the largest one of these three clusters , revealing hrv - c as the dominant species in the study population . the hrv - a cluster , with 41 sequences , is the second largest , and the hrv - b cluster , with eight sequences , is the smallest . the sequences belonging to different hrv species from the different patient groups were stratified into different subclusters , together with sequences of different hrv serotypes from genbank , and there are sequences from different patient groups clustered into one subcluster [ figure 1 ] . the 105 partial vp4/vp2 gene sequences from this study and hrv species a , b , and c sequences from genbank were used for tree construction . turquoise circles denote hrv sequences from the control group , blue triangles those from the asthma group , and purple diamonds the sequences from the nonasthma group . the sequence analysis results showed that 41 cases ( 5.8% , 41/702 ) were confirmed as hrv - a , including 22 from the nonasthma ari group ( 4.8% , 22/461 ) , 13 from the asthma group ( 8.4% , 13/155 ) , and six from the control group ( 7.0% , 6/86 ) , eight cases ( 1.4% , 8/702 ) were confirmed as hrv - b , including four from the nonasthma ari group ( 0.8% , 4/461 ) , one from the asthma group ( 0.6% , 1/155 ) , and three from the control group ( 3.5% , 3/86 ) , and 56 ( 8.0% , 56/702 ) were confirmed as hrv - c , including 22 from the nonasthma ari group ( 4.8% , 22/461 ) , 26 from the asthma group ( 16.8% , 26/155 ) , and 6 from the control group ( 9.3% , 6/86 ) . the overall proportion of hrv - a and hrv - c positive samples in the study were similar ( = 2.171 , p > 0.05 ) , and both were higher than that of hrv - b ( = 21.654 , p < 0.01 and = 36.164 , p < 0.01 , for hrv - a and hrv - c , respectively ) . in the nonasthma ari group , there were significant correlations between hrv - a and hrv - c and asthma , with r values of 0.08 ( p = 0.037 , p < 0.05 ) and 0.20 ( p < 0.001 ) , respectively [ table 2 ] . no significant correlations were identified for hrv - a , b , and c infections between the asthma group and control group ( p > 0.05 ) . correlation between different species hrv infection with asthma ari : acute respiratory infection ; hrvs : human rhinoviruses . sex and age were not evenly distributed , with more male cases and more young patients in all groups ; therefore , the distribution of sex and age in different groups was adjusted prior to regression analysis [ table 3 ] . the or between the asthma group and nonasthma ari group for hrv - a and hrv - c were 2.2 ( 95% confidence interval [ ci ] , 1.0654.409 ) and 4.2 ( 95% ci , 2.2627.906 ) , respectively , which confirmed patients with hrv - c infection were 4.2 times more likely to have asthma ( p < 0.001 ) , and patients with hrv - a infection were 2.2 times more likely to have asthma ( p < 0.05 ) . association between different species of hrv infection and asthma in a sample of 702 children in beijing * adjusted . hrv : human rhinoviruses ; ci : confidence interval ; or : odds ratio ; ari : acute respiratory infection . in this study , respiratory virus screening ( including hrvs ) by dfa , virus isolation , and pcr revealed that hrvs were the most common pathogens , with a prevalence of 15.4% ( 108/702 ) in all patients , and 25.8% ( 40/155 ) in the asthma group , 11.1% ( 51/461 ) in the nonasthma ari group , and 19.8% ( 17/86 ) in the control group . the prevalence of hrv was higher in the asthma group , compared with the nonasthma group , which may imply an association between hrvs and asthma . hrvs are the most common cause of respiratory illness in children and are commonly associated with asthma symptoms , requiring escalation of care and emergency room visits in many patients . a previous study in beijing reported 19.0% ( 14/73 ) of patients diagnosed with asthma were hrv - positive , and wheezing was a common symptom in children with ari . this suggests that hrv is an important pathogen for children with ari , particularly those with lower respiratory infections . however , the epidemiology and clinical significance of different species of hrvs , especially hrv - c , are poorly characterized . based on the phylogenetic analysis , these hrvs were divided into different species , hrv - a , b , and c. hrv - c cluster was the largest among the three clusters , followed by hrv - a , then hrv - b , identifying hrv - c as the dominant species in the study . hrv - c was also the dominant species in the asthma ( 16.8% ) and control groups ( 9.3% ) but not in the nonasthma ari group , where the prevalence of hrv - c and hrv - a was equal . in a study aiming to characterize the clinical and demographic features associated with different hrv species in northeast brazil , fawkner - corbett et al . believed that hrv - a was the dominant species , with a prevalence of 73% , compared with 27% in hrv - c and 0 hrv - b . in addition , hrv - c was detected more frequently than hrv - a in children with asthma or episodic viral wheeze , which supports the association of hrv - c with asthma , identified in the present study that the or between the asthma and nonasthma ari group for hrv - a and hrv - c confirmed patients with hrv - c infection were 4.2 times more likely to have asthma ( p < 0.001 ) , and patients with hrv - a infection were 2.2 times ( p < 0.05 ) . therefore , hrv - c had the strongest correlation with asthma in the study population . there was no significant difference in the prevalence of hrv - b among the three groups , which may be because there was no correlation between hrv - b and asthma . however , the proportion of hrv - b in the control group ( 3.5% , 3/86 ) is higher than that in the asthma group ( 0.6% , 1/155 ) and the nonasthma ari group ( 0.8% , 4/461 ) . in addition , there were few hrv - b positive specimens in this study , as in a previous study that nine infants had hrv - a , 13 had hrv - c , and one infant had hrv - b . therefore , more data are needed to evaluate the association between hrv - b infection and asthma . there was a high prevalence of hrvs in the control group ( 19.8% ) , which may suggest a high prevalence of asymptomatic hrv infection in children . however , this contradicts the association identified between hrv - a and hrv - c infection and asthma . chen et al . reported that the prevalence of hrv species differed with age , and hrv - c infection was more common in children compared with adults , with the majority of adults being infected with hrv - a . other research has described a high prevalence of hrv - c in children , regardless of clinical status . in the present study , compared with 155 patients in the asthma group , more data are needed to determine the clinical significance of a high prevalence of hrvs in the control group . the sequences of different clusters from the different patient groups in the phylogenetic analysis were blast - searched against hrv serotypes from genbank , and some patient group sequences were clustered into one subcluster . further studies are needed to understand the relationship of hrv serotype with the severity of illness or specific disease , especially with asthma . in conclusion , the results in this study suggest there is a high prevalence of hrvs in children in beijing , regardless of clinical status , and hrv - c may be a key factor in determining an association between hrvs and asthma . this work was supported by the grants from beijing municipal science and technology commission ( no . 2011 - 3 - 068 ) and national health and family planning commission special funds for public welfare projects this work was supported by the grants from beijing municipal science and technology commission ( no . 2011 - 3 - 068 ) and national health and family planning commission special funds for public welfare projects ( no .
background : human rhinoviruses ( hrvs ) are divided into three genetic species : hrv - a , hrv - b , and hrv - c . the association of different hrv species with asthma in children in china has not yet been evaluated . this preliminary study aimed to assess the associations between different hrv species , particularly hrv - c , and asthma in young children in china.methods:a total of 702 nasopharyngeal aspirates were obtained from 155 children with asthma ( asthma group ) , 461 children with acute respiratory infection ( ari ) without asthma ( nonasthma ari group ) , and 86 children from the control group . semi - nested polymerase chain reaction ( pcr ) was used to detect hrvs , and pcr products were sequenced for species identification . epidemiological characteristics of hrv - positive cases were analyzed.results:hrvs were the most common pathogen ( 15.4% ; 108/702 ) in the patients in this study . the prevalence of hrv was significantly different ( f = 20.633 , p = 0.000 ) between the asthma ( 25.8% ) and nonasthma ari groups ( 11.1% ) . phylogenetic analysis indicated that in the 108 cases positive for hrvs , 41 were identified as hrv - a , 8 as hrv - b , and 56 as hrv - c . comparing the asthma with the nonasthma ari group , spearman 's rank correlation analysis revealed an association between hrv - a ( p < 0.05 ) and c ( p < 0.01 ) and asthma , confirmed by regression analysis , with odds ratios of 2.2 ( hrv - a ) and 4.2 ( hrv - c).conclusions : our data revealed a high prevalence of hrvs in children in china , regardless of clinical status . hrv - c was the dominant species and may be one of the key factors in the association of hrvs with asthma .
I M Patients and specimens Screening for respiratory viruses Identification of human rhinoviruses Sequencing and analysis of second run polymerase chain reaction products Statistical analysis R Respiratory virus screening Sequencing and phylogenetic analysis of human rhinoviruses Correlation between human rhinovirus species and asthma D Financial support and sponsorship Conflicts of interest
this is a preliminary study to assess the associations between different hrv species , particularly hrv - c , and asthma in young children in china . to identify the correlation of different species of hrv infection with asthma , spearman 's rank correlation was used to compare the positive rates of hrv - a , b , or c between the asthma group and control group or nonasthma ari group . to identify the correlation of different species of hrv infection with asthma , spearman 's rank correlation was used to compare the positive rates of hrv - a , b , or c between the asthma group and control group or nonasthma ari group . in the nonasthma ari group , there were significant correlations between hrv - a and hrv - c and asthma , with r values of 0.08 ( p = 0.037 , p < 0.05 ) and 0.20 ( p < 0.001 ) , respectively [ table 2 ] . the or between the asthma group and nonasthma ari group for hrv - a and hrv - c were 2.2 ( 95% confidence interval [ ci ] , 1.0654.409 ) and 4.2 ( 95% ci , 2.2627.906 ) , respectively , which confirmed patients with hrv - c infection were 4.2 times more likely to have asthma ( p < 0.001 ) , and patients with hrv - a infection were 2.2 times more likely to have asthma ( p < 0.05 ) . the sequence analysis results showed that 41 cases ( 5.8% , 41/702 ) were confirmed as hrv - a , including 22 from the nonasthma ari group ( 4.8% , 22/461 ) , 13 from the asthma group ( 8.4% , 13/155 ) , and six from the control group ( 7.0% , 6/86 ) , eight cases ( 1.4% , 8/702 ) were confirmed as hrv - b , including four from the nonasthma ari group ( 0.8% , 4/461 ) , one from the asthma group ( 0.6% , 1/155 ) , and three from the control group ( 3.5% , 3/86 ) , and 56 ( 8.0% , 56/702 ) were confirmed as hrv - c , including 22 from the nonasthma ari group ( 4.8% , 22/461 ) , 26 from the asthma group ( 16.8% , 26/155 ) , and 6 from the control group ( 9.3% , 6/86 ) . in the nonasthma ari group , there were significant correlations between hrv - a and hrv - c and asthma , with r values of 0.08 ( p = 0.037 , p < 0.05 ) and 0.20 ( p < 0.001 ) , respectively [ table 2 ] . the or between the asthma group and nonasthma ari group for hrv - a and hrv - c were 2.2 ( 95% confidence interval [ ci ] , 1.0654.409 ) and 4.2 ( 95% ci , 2.2627.906 ) , respectively , which confirmed patients with hrv - c infection were 4.2 times more likely to have asthma ( p < 0.001 ) , and patients with hrv - a infection were 2.2 times more likely to have asthma ( p < 0.05 ) . in this study , respiratory virus screening ( including hrvs ) by dfa , virus isolation , and pcr revealed that hrvs were the most common pathogens , with a prevalence of 15.4% ( 108/702 ) in all patients , and 25.8% ( 40/155 ) in the asthma group , 11.1% ( 51/461 ) in the nonasthma ari group , and 19.8% ( 17/86 ) in the control group . hrv - c was also the dominant species in the asthma ( 16.8% ) and control groups ( 9.3% ) but not in the nonasthma ari group , where the prevalence of hrv - c and hrv - a was equal . in addition , hrv - c was detected more frequently than hrv - a in children with asthma or episodic viral wheeze , which supports the association of hrv - c with asthma , identified in the present study that the or between the asthma and nonasthma ari group for hrv - a and hrv - c confirmed patients with hrv - c infection were 4.2 times more likely to have asthma ( p < 0.001 ) , and patients with hrv - a infection were 2.2 times ( p < 0.05 ) . in conclusion , the results in this study suggest there is a high prevalence of hrvs in children in beijing , regardless of clinical status , and hrv - c may be a key factor in determining an association between hrvs and asthma .
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peripheral blood collection and cd14 cell isolation - peripheral blood was obtained from healthy volunteers after providing informed written consent according to the ethical research committee or israel institute of education and research ( 105/02 ) . mononuclear cells were purified from the peripheral blood using density gradient ficoll - paque plus ( ge healthcare , buckinghamshire , uk ) according the manufacturer 's instructions . the cd14 cell population was isolated from the mononuclear fraction by magnetic bead separation according to the manufacturer 's protocol ( miltenyi biotec , bergisch gladbach , germany ) . dc generation and treatment with vegf a - the cd14 cell population was dispensed into six - well plates containing x - vivo 15 medium ( cambrex , walkersville , md , usa ) supplemented with antibiotic - antimycotics ( gibco , north andover , ma , usa ) . to generate immature dcs , the cells were cultured in the presence of 20 ng / ml recombinant il-4 ( ril-4 ) ( r&d system ) and 50 ng / ml rgm - csf ( r&d system ) for six days ( d6 ) . mature dcs were obtained after 24 h ( d7 ) of stimulation of the immature dc culture with 10 ng / ml rtnf- ( r&d system ) plus 0.01 mmol / l pge2 ( sigma , st . louis , mo , usa ) , referred to in this paper as pg - tnf ; the concentrations used were previously described ( szabolcs et al . alternatively , the immature dc cultures were stimulated to maturity by adding 20 g bcg ( ataulpho de paiva foundation , rio de janeiro , brazil ) . the bcg suspension was previously titrated to obtain the lowest dose capable of stimulating efficient dc maturation ( data not shown ) . to study the effect of vegf on dcs , the cells were cultivated after the initial day of culture in the presence or absence of 20 ng / ml of human rvegf a , isoform165 ( calbiochem - millipore , billerica , ma , usa ) , which it the predominant form of vegf during angiogenesis . flow cytometry analysis - staining of the cells for flow cytometry analyses was performed using commercially available monoclonal antibodies ( mabs ) according to the manufacturer 's instructions . briefly , the cells were stained with the selected mabs and incubated in the dark for 30 min at room temperature . the cells were then washed and fixed with 1% paraformaldehyde , with the exception of the cells tested for apoptosis , which were resuspended in the annexin v binding buffer ( bd pharmingen , san diego , ca , usa ) provided with the reagent set . intracellular staining was performed in previously fixed and permeabilised surface - stained cells prior to adding the labelled intracellular mab . the mabs used to evaluate dc markers or maturation were as follows : cd11c - pe clone : b - ly6 , cd14-fluorescein isothiocyanate ( fitc ) clone : m5e2 , cd80-pe clone : l307.4 , cd83-pe clone : hb15e , cd86-pe clone:2331 , cd123-pe clone:9f5 and cd209-pe clone : dcn46 ( all purchased from bd pharmingen ) ; hla - dr - percp - cy5.5 clone : l243 and isotype controls ( purchased from bd biosciences , san jose , ca , usa ) . to assess lymphocyte proliferation , we used ki-67-fitc clone : b56 and cd3-percp cy5.5 clone : sk7 in addition to isotype controls ( bd biosciences ) . monoclonal abs against the vegf receptors , including anti - vegfr1-pure clone:49560 and vegfr2-pe clone:89106 ( r&d system ) and vegfr3-pure clone:9d9f9 ( chemicon , temecula , ca , usa ) and isotype controls were also used . annexin v - fitc , propidium iodide , anti - caspase-3 active form - pe and isotype controls were purchased from bd pharmingen . the secondary abs included goat anti - mouse - fitc , goat anti - rabbit - fitc and sheep anti - mouse - pe and were purchased from chemicon . the data were acquired using the facsaria flow cytometer ( bd biosciences ) and the analyses were performed using the facsdiva software ( bd biosciences ) and/or flowjo ( tree star , ashland , or , usa ) . ag - specific proliferation assay - mature dcs cultured in the presence or absence of vegf ( 10 cells/100 l ) were co - cultivated in 96-well tissue culture plates ( bd biosciences - discovery labware , san jose , ca , usa ) for four days with autologous lymphocytes ( 10 cells/100 l ) previously stained with 5 m / ml of carboxyfluorescein succinimidyl ester ( cfse ) ( invitrogen ) . ag - specific stimulation was performed by priming the dcs with lyophilised candida albicans ( 10 g / ml ) and non - specific stimulation of the cultures was performed by adding the mitogen phytohaemagglutinin ( pha ) 1 g / ml ) . the dcs were -irradiated at 1,500 rads ( gammacell 1000 cs source ) prior to co - cultivation . after four days in culture , lymphocyte proliferation was monitored according to the cfse fluorescence levels in the 530/30 channel . the lymphocyte populations were gated by size and granularity [ side light scatter ( ssc ) vs. forward light scatter ( fsc ) ] and the events selected in the lymphocyte gate were analysed in a second plot ( histogram ) . the results were expressed using the division index , which represents the average number of divisions that a cell has undergone . we also analysed non - specific ( pha ) lymphocyte proliferation in co - cultures arranged as described , in which a specific inhibitor of ido , 1-methyl - d - tryptophan ( 1-mt ) at 1 mm ( aldrich , st . louis , mo , usa ) , was added at the beginning of the culture together with the vegf - matured dcs . after four days in culture , lymphocyte proliferation was monitored for the cd3 population using ki-67 nuclear staining as a proliferation marker . the lymphocytes were gated based on ssc vs. fsc , followed by cd3 gating ; the selected events were analysed in a second plot for ki-67 expression . rna isolation and quantitative real - time polymerase chain reaction ( qpcr ) - monocytes , immature dcs and mature dcs were cultured in the presence or absence of vegf for subsequent rna isolation . total rna was isolated using the kit rneasy mini kit ( qiagen , germany ) . the extracted rna was then reverse transcribed into cdna using oligo , dntpmix and reverse transcriptase from the superscript ii reverse transcriptase kit ( invitrogen ) . the primers were based on the homo sapiens gene sequences for ido and glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) obtained in the genbank database ( ncbi.nlm.nih.gov ) . the primers were designed using primer - select ( dnastar inc , usa ) and were synthesised by invitrogen as ido ( forward , 5'-ggcaacccccagctatcaga-3 ' ; reverse , 5'-cagggagaccagagctttcaca-3 ' ) and gapdh ( forward , 5'-ggagaaggctggggctcat-3 ' ; reverse , 5'-gtccttccacgataccaaagtt-3 ' ) . the qpcr was performed using the quanti tect sybr green pcr kit according to the manufacturer 's instructions ( qiagen , germany ) . the qpcr data were analysed by normalising the cycle threshold ( ct ) values of the gene of interest to the ct values of the housekeeping gene gapdh ( livak & schmittgen 2001 ) . the results are displayed showing monocytes as the baseline for comparison . ultrastructural characterisation by transmission electron microscopy ( tem ) - the ultrastructures of monocytes and mature dcs grown in presence or absence of vegf was examined by tem . the pellets of these cells were fixed in 0.2 m cacodylate buffer with 1% glutaraldehyde for 2 h at 48c . post - fixation was performed with 1% osmium tetroxide for 1 h at 48c , followed by washing for 15 min in the same buffer . for contrast , the pellet was immersed in a solution of uranyl acetate for 30 min . after dehydration , the material was embedded in epon resin diluted in acetone ( 1:1 ) and incubated at 48c for 24 h with agitation . the pellet was then transferred to pure epon resin and incubated at 60c for 72 h until completely polymerised . the semithin sections were stained with azure ii ( 1% ) and methylene blue ( 1% ) . the ultrathin sections were placed on copper grids and stained with uranyl acetate and citrate . statistical analysis - statistical analyses were performed by employing a two - tailed student t test , with statistical significance set at a p - value < 0.05 . dc phenotypes after maturation induced pg - tnf or bcg - the differentiation of monocytes into dcs was assessed according to the expression of markers on d6 of culture in comparison to that of the original monocyte population at the start of the culture ( d0 ) . the expression of maturation markers was determined one day after the maturation stimuli were added to the six - day - old cultures ( d7 ) . the frequencies of cd14 populations on d0 , d6 and d7 are shown in fig . 1 . 1a ) ( d0 ) . after six days of culture in the presence of il-4 + gm - csf , the frequency of cd14 cells 1a ) ( d6 ) and was maintained at low levels in either the pg + tnf or bcg - matured dcs ( d7 ) ( fig . in contrast , cd209 , a marker exclusively expressed on dcs ( geijtenbeek et al . 2000 ) , was not observed on d0 , but was found in nearly 90% of the cells on d6 and its frequency was maintained in the matured dcs ( fig . no differences in the frequency of cells expressing cd11c or hla - dr were detected when comparing the cells at the d0 , d6 and d7 stages of culture ( fig . 1c , d ) ; however , the mean fluorescence index ( mfi ) showed increases on the order of two - three - fold . significant increases in the frequency of cd123 cells were also observed as the monocytes differentiated into dcs and subsequently matured ( fig . these results indicate that the phenotypic differentiation of monocytes into dcs occurred in vitro . 1a - hexpression of cell surface markers by human monocytes differentiated in vitro to dendritic cells ( dcs ) and cultured in the presence of prostaglandin e2 + tumour necrosis factor alpha ( pg - tnf ) or bacillus calmette - gurin ( bcg ) as maturation factors . blood monocytes were isolated from peripheral blood mononuclear cells obtained from normal donors and cultured , day zero ( d0 ) , in the presence of rgm - csf and recombinant interleukin four - six days to differentiate them to immature dc ( d6 ) and further cultured for 24 h in pg - tnf or bcg ( d7 ) as maturation factors . the cell suspensions were initially sorted by side light scatter ( ssc ) vs. forward light scatter ( fsc ) to exclude debris . the means and standard deviation ( of 5 independent experiments ) of the frequencies of positive cells for the different markers are shown in the graphs and significant ( p < 0.05 ) differences are indicated by an asterisk . the respective mean fluorescence intensities ( mfi ) for each fluorescent marker are shown as insets to each graph accompanied by its isotype - matched irrelevant fluorescent igg control . dc maturation was evidenced by increases in the frequency and expression of the co - stimulation molecule cd80 and the maturation marker cd83 from d0-d6 and d7 . in contrast , the mfi value of the co - stimulation molecule cd86 increased from d0-d6 and further increased as the cells matured ( d7 ) , although the cd86 cell frequencies were maintained from d0-d7 ( fig . pg + tnf was more effective at promoting the maturation of dcs than bcg , as the mfis and frequencies of cells expressing cd80 , cd83 and cd86 were lower after bcg treatment ( fig . we also examined the monocytes and dcs for the presence of vegfr and found that 90% of the monocytes expressed vegfr1 , vegfr2 or vegfr3 ( fig . 2a - c ) , however , among the differentiated dcs ( d6 ) , the frequency of cells positive for each receptor fell to approximately 30% , as did the vegfr1 and vegfr3 mfi ( but not for vegfr2 ) . at 24 h after the addition of maturation stimuli , the mean frequency of cells expressing vegfr1 increased to 50% , but this increase was only significant for pg + tnf and not for bcg . this result was most likely due to the greater intensity of the pg + tnf maturation stimulus and therefore its effect on the cells could be better observed and evaluated ( fig . 2expression of vascular endothelial growth factor ( vegf ) receptors during human dendritic cell differentiation and subsequent maturation in the presence of prostaglandin e2 + tumour necrosis factor alpha ( pg - tnf ) or bacillus calmette - gurin ( bcg ) . human peripheral blood monocytes were cultured and differentiated as described in the legend to fig . 1 . the mean frequencies and standard deviation ( n = 5 independent experiments ) of dendritic cell expressing vegfr1 ( a ) , vegfr2 ( b ) or vegfr3 ( c ) are shown in the graphs accompanied by the mean values of mean fluorescence intensities ( mfi ) . significant differences ( p < 0.05 ) are indicated by an asterisk . vegf stimulates ido expression by dcs treated with pg + tnf - pg has been broadly used as a potent dc maturation factor and has been shown to induce ido expression ( braun et al . ido mrna expression in monocytes is very low and the mean value was used as a baseline for the comparison of the values after differentiation and maturation . as the monocytes differentiated into immature dcs ( d6 ) , the ido relative mrna expression levels were not significantly altered ( fig . however , a 1,000-fold increase in ido mrna expression was observed in dcs that had been matured with pg + tnf for 24 h ( fig . 3a ) and the presence of vegf in the maturation cultures further increased ido mrna expression ( fig . indeed , we found that vegf stimulated 100% increases in ido mrna expression in mature dcs ( d7 v vs. d7 ) . when these cell populations were stained for ido and analysed by flow cytometry , the frequency of the cells matured with vegf was 30% higher than those cultivated without vegf ( fig . 3effect of vascular endothelial growth factor ( vegf ) during dendritic cell ( dc ) maturation on the relative expression of indoleamine 2,3-dioxygenase ( ido ) mrna and on intracellular ido content detected by flow cytometry . human peripheral blood monocytes were cultured and differentiated until day six ( d6 ) as described in the legend of fig . maturation was carried out with prostaglandin e2 + tumour necrosis factor alpha ( pg - tnf ) in the presence of vegf : d7 ( v ) or just pg - tnf ( d7 ) . a : ido mrna relative expression in logarithmic scale ; b : data in linear scale ( asterisk means significant difference at p < 0.05 ) . for comparison , very low levels of ido mrna of immature dc ( d6 ) compare to monocytes ( baseline ) and mature dc [ d7 : dc matured in the absence of vegf ; d7v : dc matured in the presence of vegf ( representative of 3 independent experiments ) ] are shown in a. the difference between d7v - d7 is better seem in b due to linear scale ; c : flow cytometry data of mature dc stained for intracellular ido accompanied by the mean fluorescence intensities ( mfi ) values ; igg1 : fluorescein isothiocyanate - labelled isotype control . vegf influence on the morphology of pg + tnf - treated dcs , as revealed by tem - under tem observation , the preparations of monocytes ( d0 ) showed a predominance of regular , round - shaped cells containing ovoid nuclei with loose chromatin and evident nucleoli ( fig . the mature dcs that had been treated with pg + tnf were irregular and often star - shaped , exhibiting characteristic dendritic - like prolongments ( fig . these cells also demonstrated ovoid - shaped nuclei with loose chromatin and nucleoli ; in addition , the mitochondria and rough endoplasmic reticulum were well developed and few lysosomes were observed ( fig . the morphology of the dcs treated with vegf ( in addition to pg + tnf ) was not much different than the above - described features , although some cells displayed intense cytoplasmic vacuolation ( fig . 4h ) ; these changes were compatible with the initial senescent cell population . 4transmission electron micrographs of monocytes and of dendritic cells ( dcs ) matured with prostaglandin e2 + tumour necrosis factor alpha ( pg - tnf ) in the presence or absence of vascular endothelial growth factor ( vegf ) . 3 . a : monocyte culture on day zero ; b - d : dcs matured in pg - tnf ( d7 ) ; e - h : dcs matured in pg - tnf + vegf ( d7v ) ; c : cytoplasm ; cp : cytoplasmic prolongations ; li : lysossome ; mi : mitochondria ; mt : microtubule ; n : nuclei ; nu : nucleoli ; rer : rough endoplasmic reticulum ; v : vacuolation . vegf effect on apoptosis in pg + tnf - treated dcs , as revealed by flow cytometry - the cell cultures matured with pg + tnf + vegf showed an increased frequency of annexin v+ cells ( a sign of early apoptosis ) by 24 h ( dc7v ) and 48 h ( dc8v ) in comparison to cells cultured in the absence of vegf ( fig . was also evidenced in the vegf - treated cultures after 48 h , as shown by higher frequencies of double staining for annexin and pi ( dc8v ) ( fig . these data were also confirmed for late apoptosis , as a higher frequency of cells were positive for active caspase-3 ( dc8v ) ( fig . these results suggest that vegf enhanced the apoptosis of dcs when present during the maturation process and were in agreement with the ultrastructural changes described for these cell cultures . 5effect of vascular endothelial growth factor ( vegf ) on the expression of early and late markers of apoptosis and/or necrosis by dendritic cell ( dc ) cultures . 3 with the maturation step carried out for 24 h with prostaglandin e2 + tumour necrosis factor alpha in the presence ( dc7v ) of vegf or in its absence ( dc7 ) . in additional cultures the maturation step was carried out for 48 h in similar conditions , identified respectively as dc8v and dc8 . the cells were double - stained for annexin v / propidium iodide ( apoptosis and/or necrosis ) or stained only for caspase-3 . a : cells that stained for annexin v only ( early apoptosis ) ; b : cells double stained for annexin v and propidium iodide only ( late apoptosis / necrosis ) ; c : frequencies of cells staining for annexin v / pi + annexin indicating the total number of cells undergoing apoptosis ( early and late ) and necrosis ; d : cells expressing caspase 3 active ( late apoptosis ) . dcs exposed to vegf during maturation are less capable of inducing ag - specific or mitogen - triggered lymphocyte proliferation - to determine whether the changes observed in vegf - exposed dcs ( e.g. , ido over - expression and accelerated apoptosis / senescence ) would also translate into functional alterations , we next assessed ag - specific and pha - triggered lymphocyte proliferation . to this end , we employed a 2 x 2 factorial design in which we examined the level of autologous lymphocyte proliferation in the presence of dcs treated or not with vegf . we used two independent stimuli , a specific stimulus dependent on presentation ( c. albicans ) and a non - specific mitogen , pha , for four days of culture . we also repeated the experiment with pha , evaluating lymphocyte proliferation by ki-67 expression . to assess the effect of ido on the maturation step , the dcs were matured in the presence of pg + tnf + vegf and the ido - inhibitor 1-mt was simultaneously added to the cultures ; the resulting dc population was then tested in proliferation assays . the vegf - treated dcs caused a reduction in lymphocyte proliferation in response to candida ( fig . 6d - f ) in comparison to the dcs matured in the absence of vegf . this result was further confirmed by a reduction in lymphocyte proliferation to pha according to ki-67 expression ( fig . 7d , e ) . of note , the addition of 1-mt to vegf - treated dcs generated dcs that were as affective as dcs matured without vegf ( fig . 7d , f ) , indicating that the impaired proliferation observed in the co - cultures of vegf - treated dcs and lymphocytes was related to the activation of the ido pathway during the maturation phase . 6 t cell proliferation is impaired in the presence of dendritic cells ( dcs ) matured in the presence of vascular endothelial growth factor ( vegf ) lymphocytes were co - cultivated with autologous dc that were treated with vegf in addition to prostaglandin e2 + tumour necrosis factor alpha ( pg - tnf ) or only with pg - tnf during their maturation phase . candida albicans was used to stimulate antigen - specific t lymphocyte proliferation ( a - c ) . histograms for lymphocytes labelled with side light scatter ( ssc ) vs. forward light scatter ( fsc ) are shown in b for co - cultures with pg - tnf - dc and in c for co - cultures with pg - tnf - v - dc . a : respective calculated indexes of cell division ; d : respective calculated indexes of cell division for phytohemagglutinin ( pha)-stimulated cultures ; d - f : pha was used as t - cell mitogen ; e , f : histograms of cfs - labelled lymphocytes co - cultured with dc matured without ( e ) or with vegf ( f ) . 7addition of the inhibitor 1-methyl - d - tryptophan ( 1-mt ) of indoleamine 2,3-dioxygenase ( ido ) prevents vascular endothelial growth factor ( vegf)-matured dendritic cell ( dc ) from suppressing lymphocyte proliferation . t cell proliferation stimulated by phytohemagglutinin ( pha ) was estimated by flow - cytometry of cd3 + lymphocytes expressing the antigen ki-67 ( a ) lymphocytes gated on side light scatter ( ssc ) vs. forward light scatter ( fsc ) ( b ) , histogram of gated cd3 + cells ( c ) , ki-67 expression on cd3 + lymphocytes without stimuli ( 1.56% of the cells express the marker ) ( d ) , ki-67 expression on pha - stimulated cd3 + lymphocytes co - cultured with autologous dc matured without vegf ( frequency of positive cells - 37.4% ) ( e ) , ki-67 expression on pha - stimulated cd3 + lymphocytes co - cultured with autologous dc matured with vegf ( frequency of positive cells - 17.6% ) and ki-67 expression on pha - stimulated cd3 + lymphocytes co - cultured with autologous dc matured with vegf in the presence of 1-mt as inhibitor of ido ( frequency of positive cells - 35% ) ( representative of 3 independent experiments ) ( f ) . although it is well known that vegf affects dc maturation , our results demonstrate for the first time that vegf increases the expression and activity of ido , which has a suppressive effect on ag - specific and mitogen - stimulated lymphocyte proliferation . bcg was originally used as a maturation factor because of its similarity to lps and because it was already in use as a human therapy . however , we observed that pg + tnf induced a more robust phenotype than bcg in dc maturation ; thus , we used only pg + tnf in these experiments . because we did not use bcg in the ensuing steps , these evaluations are valid for inflammatory environments , but are not necessarily relevant to infectious pathways . 1998 ) ; furthermore , tnf- in combination with pg induces ido activity during dc maturation ( trinchieri 1995 , braun et al . ido - expressing dcs are considered to be immunological tolerance inducers because they suppress lymphocyte activation and proliferation ( munn et al . although pge2 induces ido mrna expression through its receptor e - prostanoid 2 ( ep2 ) , enzyme activation is dependent on a second signal provided by tnfr or by tlr ligands ( braun et al . in contrast to these findings , it was reported that the induction of ido expression by pge2 occurs through its receptor ep4 instead of ep2 and that pge2-matured dcs were more capable of inducing both allogeneic and ag - specific t cell proliferation when compared to dcs matured in the absence of this molecule ( krause et al . 2007 ) . more recently ( lanzinger et al . 2012 ) , it was shown that the stimulatory effect of pge2-matured dcs on allogeneic t cells is variable and may be highly context dependent . when the ido activity in the microenvironment is low , dcs act as effective stimulators of immune responses ; however , once the enzymatic activity of ido predominates , these cells suppress t cell responsiveness and/or promote regulatory t cell responses . because of the importance of ido as a regulator of immune responses and the ubiquitous presence of vegf in tumoural or inflammatory microenvironments , it was of interest to investigate whether vegf would affect ido expression by dcs . indeed , our results show that ido expression by dcs was significantly increased in the presence of vegf compared to untreated dcs . in fact , both pg - tnf and vegf contributed to augment ido expression , suggesting a synergistic effect . corroborating these data , we also observed that the induction of dc maturation by pg + tnf significantly increased the expression of vegfr1 , thereby facilitating synergic signalling by vegf ( dikov et al . our data concerning the ultrastructure of dcs treated with vegf revealed morphological changes , predominantly vacuolation , which were compatible with processes that ultimately lead to cell death . this was further confirmed by annexin v / propidium iodide staining of vegf - treated dcs , which is indicative of apoptosis / necrosis ( henics & wheatley 1999 , orabona et al . the accelerated apoptosis of vegf - treated dcs was most likely caused by a combination of factors . first , as has been previously shown , pg can stimulate programmed cell death by inducing the pro - apoptotic protein bax ( lalier et al . 2011 ) ; second , vegf acting in synergy would most likely further enhance apoptosis . in addition , tryptophan is an amino acid that is essential to cell survival and ido activation reduces its availability to the cell ( broker et al . 2005 ) . one important question , however , was how dcs exposed to vegf would function as regulators of the immune response . we found that specific ( c. albicans ) and mitogen - induced ( pha ) lymphocyte proliferation were reduced in the presence of vegf - treated dcs . this effect was due to ido activity , as confirmed by experiments in which the addition of a specific ido inhibitor ( 1-mt ) during the maturation of vegf - dcs completely abolished their suppressive activity on lymphocyte proliferation . another mechanism by which dcs develop an immunosuppressive phenotype is the ingestion of apoptotic cells ( da costa et al . because the dcs added to the co - cultures were mature and also -irradiated , their phagocytic activity was much reduced ; thus , it is unlikely that such a mechanism was important to the observed suppression of lymphocyte proliferation in our assays . in addition , phagocytosis of apoptotic cells was not observed in our tem examination of sections of vegf - matured dcs . taken together , our results suggest that vegf plays a role in the complex process of immunological tolerance , as it can stimulate dcs to over - activate the ido pathway . the ensuing tryptophan depletion leads to the inhibition of t cell activation and expansion ( grohmann & bronte 2010 , kushwah & hu 2010 ) . in addition , dcs that express ido can stimulate the cellular general control non - depressible 2 kinase - dependent stress response in nave and mature t cells and in functionally quiescent t regulatory cells , leading to active bystander suppression ( fallarino et al . these mechanisms have broad implications in the study of immunological responses and tolerance under conditions as diverse as cancer , graft rejection and autoimmunity .
dendritic cells ( dcs ) are antigen ( ag)-presenting cells that activate and stimulate effective immune responses by t cells , but can also act as negative regulators of these responses and thus play important roles in immune regulation . pro - angiogenic vascular endothelial growth factor ( vegf ) has been shown to cause defective dc differentiation and maturation . previous studies have demonstrated that the addition of vegf to dc cultures renders these cells weak stimulators of ag - specific t cells due to the inhibitory effects mediated by vegf receptor 1 ( vegfr1 ) and/or vegfr2 signalling . as the enzyme indoleamine 2,3-dioxygenase ( ido ) is recognised as an important negative regulator of immune responses , this study aimed to investigate whether vegf affects the expression of ido by dcs and whether vegf - matured dcs acquire a suppressor phenotype . our results are the first to demonstrate that vegf increases the expression and activity of ido in dcs , which has a suppressive effect on ag - specific and mitogen - stimulated lymphocyte proliferation . these mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer , graft rejection and autoimmunity .
MATERIALS AND METHODS RESULTS DISCUSSION
1a - hexpression of cell surface markers by human monocytes differentiated in vitro to dendritic cells ( dcs ) and cultured in the presence of prostaglandin e2 + tumour necrosis factor alpha ( pg - tnf ) or bacillus calmette - gurin ( bcg ) as maturation factors . 2expression of vascular endothelial growth factor ( vegf ) receptors during human dendritic cell differentiation and subsequent maturation in the presence of prostaglandin e2 + tumour necrosis factor alpha ( pg - tnf ) or bacillus calmette - gurin ( bcg ) . 3effect of vascular endothelial growth factor ( vegf ) during dendritic cell ( dc ) maturation on the relative expression of indoleamine 2,3-dioxygenase ( ido ) mrna and on intracellular ido content detected by flow cytometry . 4transmission electron micrographs of monocytes and of dendritic cells ( dcs ) matured with prostaglandin e2 + tumour necrosis factor alpha ( pg - tnf ) in the presence or absence of vascular endothelial growth factor ( vegf ) . 5effect of vascular endothelial growth factor ( vegf ) on the expression of early and late markers of apoptosis and/or necrosis by dendritic cell ( dc ) cultures . dcs exposed to vegf during maturation are less capable of inducing ag - specific or mitogen - triggered lymphocyte proliferation - to determine whether the changes observed in vegf - exposed dcs ( e.g. , ido over - expression and accelerated apoptosis / senescence ) would also translate into functional alterations , we next assessed ag - specific and pha - triggered lymphocyte proliferation . 7d , f ) , indicating that the impaired proliferation observed in the co - cultures of vegf - treated dcs and lymphocytes was related to the activation of the ido pathway during the maturation phase . 6 t cell proliferation is impaired in the presence of dendritic cells ( dcs ) matured in the presence of vascular endothelial growth factor ( vegf ) lymphocytes were co - cultivated with autologous dc that were treated with vegf in addition to prostaglandin e2 + tumour necrosis factor alpha ( pg - tnf ) or only with pg - tnf during their maturation phase . 7addition of the inhibitor 1-methyl - d - tryptophan ( 1-mt ) of indoleamine 2,3-dioxygenase ( ido ) prevents vascular endothelial growth factor ( vegf)-matured dendritic cell ( dc ) from suppressing lymphocyte proliferation . although it is well known that vegf affects dc maturation , our results demonstrate for the first time that vegf increases the expression and activity of ido , which has a suppressive effect on ag - specific and mitogen - stimulated lymphocyte proliferation . in contrast to these findings , it was reported that the induction of ido expression by pge2 occurs through its receptor ep4 instead of ep2 and that pge2-matured dcs were more capable of inducing both allogeneic and ag - specific t cell proliferation when compared to dcs matured in the absence of this molecule ( krause et al . when the ido activity in the microenvironment is low , dcs act as effective stimulators of immune responses ; however , once the enzymatic activity of ido predominates , these cells suppress t cell responsiveness and/or promote regulatory t cell responses . because of the importance of ido as a regulator of immune responses and the ubiquitous presence of vegf in tumoural or inflammatory microenvironments , it was of interest to investigate whether vegf would affect ido expression by dcs . this was further confirmed by annexin v / propidium iodide staining of vegf - treated dcs , which is indicative of apoptosis / necrosis ( henics & wheatley 1999 , orabona et al . we found that specific ( c. albicans ) and mitogen - induced ( pha ) lymphocyte proliferation were reduced in the presence of vegf - treated dcs . this effect was due to ido activity , as confirmed by experiments in which the addition of a specific ido inhibitor ( 1-mt ) during the maturation of vegf - dcs completely abolished their suppressive activity on lymphocyte proliferation . in addition , phagocytosis of apoptotic cells was not observed in our tem examination of sections of vegf - matured dcs . these mechanisms have broad implications in the study of immunological responses and tolerance under conditions as diverse as cancer , graft rejection and autoimmunity .
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preconference workshop a : the nuts and bolts of antibody development : accelerating antibody drugs to the clinic . co - chairs : james larrick ( panorama research institute and velocity pharmaceutical development llc ) and mark alfenito ( engen bio , inc . ) the silver anniversary antibody engineering & therapeutics meeting will be kicked off with a dynamic , audience - participatory workshop on antibody drug development chaired by veterans jim larrick ( panorama research institute and velocity pharmaceutical development llc ) and mark alfenito ( engen bio , inc . ) . larrick 's introductory remarks regarding an investor 's perspective on therapeutic antibody drugs , max vasquez ( adimab ) will describe state - of - the - art bioinformatic and in silico methods to facilitate preclinical antibody development . akbar nayeem ( molecular discovery technologies ) will expand on this topic describing computational methods to optimize the structure of clinical candidate antibodies . next , nicola beaucamp ( roche innovation center penzberg ) will describe roche 's integrated approach to chinese hamster ovary ( cho ) cell line selection , upstream process , downstream process and analytics to deliver high - quality bispecific antibodies . the first , by dorina saro ( johnson & johnson ) , on the development of analytical and biophysical tools to select bispecific monoclonal antibody ( mab ) candidates will focus on product develop - ability , and the second , by thi - sau migone ( igenica biotherapeutics ) , will cover proteomic - based discovery of a novel hematologic cancer target and ind - enabling studies of a site - specific antibody - drug conjugate ( adc ) . preconference workshop b : advances in precision targeting chair : paul whi parren ( genmab ) precise targeting by biopharmaceuticals remains a major challenge in almost all therapeutic areas . this workshop brings together a number of experts who will present recent key knowledge - based advances to optimize target binding for increased specific activity . juergen schanzer ( roche innovation center penzberg ) discusses xgfr , a novel glycoengineered bispecific antibody scaffold targeting egfr and igf-1r that combines potent signaling inhibition and antibody - dependent cell - mediated cytotoxicity ( adcc ) , demonstrating improved targeting properties compared with tetravalent bispecific formats . christopher thanos ( halozyme therapeutics ) and sanjay khare ( immungene , inc . ) will discuss advances in adc targeting to improve therapeutic index . thanos engineered an egfr antibody for increased tumor specificity , leading to activity against kras / braf - mutated tumors in vivo . khare demonstrates selective targeting of an interferon ( ifn ) payload with reduced systemic toxicity . after the break , davide corti ( humabs biomed sa ) will turn our attention to infectious disease , in which escape from antibody targeting remains a critical topic . corti shows studies for a number of antibodies derived from human infection that employ various antiviral mechanisms to allow for extraordinary breadth in activity against a range of virus subtypes and subfamilies . rudolf kerschbaumer ( baxter innovations gmbh ) will discuss novel data on the well - known pleiotropic proinflammatory cytokine macrophage migration inhibitory factor ( mif ) . kerschbaumer isolated antibodies that target oxidized mif ( ox - mif ) , a previously unrecognized disease - related variant of the cytokine with interesting activity against cancer - associated inflammation and in vivo tumor growth inhibition . finally , yuki iwayanagi ( chugai pharmaceutical co , ltd ) will complete the session by discussing a novel approach to enhance the clearance of soluble antigens by making use of engineered antibodies that combine ph - dependent binding activity with enhanced selectivity for the igg fc receptor fcriib . overall , the session highlights a number of novel insights and important advances in the selection or engineering of antibodies that , through better precision , bear great promise for the development of improved immunotherapies . keynote presentations chair : james d. marks ( university of california , san francisco ; san francisco general hospital ) the meeting will open with keynote presentations by five luminaries in the field of molecular therapeutics development . douglas a. lauffenburger ( massachusetts institute of technology ) will discuss systems analysis of cell communication network dynamics for therapeutic biologics design . although the targets of antibodies and other protein drugs are identifiable and specifically addressable , understanding the effects of corresponding perturbations within complex tissue pathophysiology contexts requires multi - variate analysis of key components of the myriad of cellular and molecular actors potentially involved in execution and regulation of phenotypic behaviors in integrated manner . lauffenburger will inform meeting participants of how his group is pursuing development of in vivo systems biology approaches to meet this challenge . ira pastan ( national cancer institute ) will discuss his extensive experience with the development of recombinant immunotoxins ( rits ) composed of a cancer cell - targeted fv or fab fused to a portion of pseudomonas exotoxin a. rits targeting either cd22 on leukemias or mesothelin on mesotheliomas have produced complete and dramatic remissions in some patients , but their efficacy is limited by immunogenicity and capillary leak syndrome ( cls ) . to overcome these deficiencies , pastan 's group has identified and removed b- and t - cell epitopes and sequences responsible for cls to produce new immunotoxins with high efficacy , low immunogenicity and low side effects . james wells ( university of california , san francisco ) will give his views on engineered antibodies for challenging targets . antibody phage display technologies have proven useful for selecting high quality , reliable and renewable antibodies . many technological challenges remain , however , for selecting antibodies that can activate functions or bind specific ptms , and for developing high through - put technologies . dr . wells will present recent work to select conformationally selective antibodies as activators or inhibitors , new scaffolds for anti - peptide antibodies for ptms , and a new automation platform for large - scale antibody phage selections . ian tomlinson ( glaxosmithkline ) will discuss the past , present and future of antibody repertoires . over twenty years have passed since the first high - affinity antibody was selected from a naive library without the use of animals or any form of immunization . today , many large libraries and many different display technologies can be used to identify such antibodies , but only a few antibodies made this way have made it to the market . tomlinson will present his views on events of the last two decades , and address the questions : what were the barriers ? what were the key advances ? and what is state of the art today ? in commemoration of the 25 anniversary of the conference , anthony rees ( rees consulting ab and emeritus professor , university of bath ) will provide a historical perspective on the antibody molecule and the remarkable journey scientists have taken while trying to understand its functions . as prof . rees will recount , this journey has included controversy , excitement beyond belief and disappointment , and , if truth be told , where we are today is as much an accident as the result of a logical itinerary . track 1 : immunocytokine engineering co - chairs : james s. huston ( the antibody society : huston bioconsulting , llc ) and stephen d. gillies ( provenance biopharmaceuticals ) the potential merits of cytokine therapy , such as interleukin ( il)-2 administration in treating cancer , have long been recognized but it was blocked from routine use by adverse side effects that were generally not tolerated by patients at the dosages necessary for therapeutic effects . this barrier was eliminated with the observation in 1992 by stephen d. gillies and co - workers that antibody - targeted il-2 stimulates cytotoxic t - cell killing of tumor cells at doses far below the threshold for il-2 toxicity . they genetically fused il-2 to the c - termini of both h chains in the targeting antibody . since antibody - cytokine fusion proteins are localized in tumors by virtue of association of the antibody with its tumor target , the il-2 fusion partner can be concentrated exactly where it is needed , so it is locally effective at doses far below the systemic therapeutic window required for il-2 alone . this discovery was made when antibody engineering had emerged with a new generation of methods and molecules that provided for antibody - targeted delivery of human therapeutic fusion proteins . i.e. , a variety of fusion proteins , alternative cytokines and distinct targets , will discuss their work , along with two more recent converts to the development of immunocytokine ( ic ) therapeutics . the session marks a pivotal juncture for these singular immunotherapeutics , and will include presentations on the latest advances in ic engineering to the most recent clinical results . stephen d. gillies ( provenance biopharmaceuticals ) will open the session by orienting the audience with some background on ics and the overall field . he will then discuss his progress in the design and engineering of entirely new formats of ics . these are intended to be a new generation of ics that can integrate cytokine immunomodulatory effects with antibody effector functions to achieve significant improvements in ic capabilities . dario neri ( eth zrich ) will describe their vigorous activities on the engineering of advanced ics that utilize il-2 , il-4 , il-10 , and il-12 for the treatment of different diseases , three of which have already advanced to phase 2 clinical trials . his most recent preclinical advancement involves a trifunctional diabody immunotherapeutic that combines an il-2 ic with site - specific conjugation to the dm1 maytansinoid microtubular inhibitor . it displays exceptionally potent anti - tumor activity and thus opens the prospect for a new class of tripartite targeted anti - cancer agents . sherie l. morrison ( university of california los angeles ) has contributed many firsts to engineered antibody therapeutics . these include chimerized antibodies ( human c domains and murine v domains , as exemplified by rituximab ) and scfv fusions to the c - termini of the h chains in igg ( currently a favored design for tetravalent bispecific antibodies ) . she has contributed to the ic field from its early years , developing an interest in targeted ifn . she will discuss her group 's research , emphasizing her interest in anti - tumor antibody - ifn fusion proteins for cancer therapy . paul sondel ( university of wisconsin ) has maintained a major interest in ic therapy after beginning studies with anti - gd2 chimeric 14.18 ab-(il-2 ) fusion proteins . as an m.d. ph.d . scientist , he is particularly well - qualified to bridge the gap between preclinical studies and clinical investigations with patients . he will describe insights with patients that are sometimes distinct from mouse model - derived conclusions . he has combined his ic interests with a dedication to developing a cure for childhood neuroblastoma and continues to pursue this goal with important clinical trials . k. dane wittrup ( massachusetts institute of technology ) invented antibody - yeast display libraries and has leveraged that technology to propel his research into diverse areas of cancer biology and antibody engineering . he has worked on aspects of cellular immunology during much of the past decade , and recently became interested in the definition of boundary conditions and design objectives for ic engineering , which he will discuss in this session . ekkehard moessner ( roche innovation center zrich ) is the head of protein engineering for roche pharmaceutical research and early development . he will discuss their progress in development of a novel class of ics that include an optimized il-2 . they have tried to separate the cytokine contributions from adcc by making antibody fusions with or without the fc region . this will be a stimulating conclusion to a session that offers many examples of how scientific creativity is an ongoing process in the ic field . conceptual progress in understanding ic mechanisms of action provide the basis of more advanced molecular designs and improved protocols for ic administration . this will ultimately reinforce the addition of ic therapy to routine clinical use , in oncology and other areas of medicine . track 2 : the high - hanging fruit : targeting difficult antigens chair : andreas plckthun while the different selection methods ( phage display , ribosome display , yeast display ) have become rather mature over the years , the great majority of targets have been individual proteins , which can be expressed and purified , and thus handled in a well understood and rather similar manner . in this session chaired by andreas it will probably be emphasized by the speakers that an important part to success is the ability to produce the target in the first place . the first part of the session will be devoted to integral membrane proteins particularly those which do not have a significant extracellular domain , and where the problem can not simply be solved by expressing only the extracellular piece . the two key examples of such difficult proteins are g - protein - coupled receptors and ion channels . it goes without saying that these two protein classes have a huge potential as disease - relevant targets . markus enzelberger ( morphosys ) will talk on the challenges of selecting antibodies binding to gpcrs and how the use of stabilized gpcrs , obtained either by directed evolution or by alanine scanning , has helped in this endeavor . furthermore , advanced screening methods play a decisive role . a different approach will be presented by michael gallo ( innovative targeting solutions ) , who has generated antibodies against the glp-1 receptor and glucagon receptor , two class b gpcrs . here the antibodies were engineered to contain pieces of the natural ligand and libraries were generated on this principle . stefan zahn ( novo nordisk ) will present approaches to target t - cell specific ion channels such as crac , and the approaches which are necessary for achieving this . in the talk by andreas plckthun ( university of zurich ) a very different challenge will be addressed : is it possible to direct a targeting agent to the cytoplasm with high efficiency ? the challenge is to engineer a cell - specific uptake mechanism that is much more efficient and much less cell - toxic than the traditionally used cell - penetrating peptides or hypercharged proteins . yet another challenge is the generation of specific antibodies against non - proteins , e.g. , glycans . this can be of interest since some glycans are massively overexpressed in tumors , and might therefore constitute very generically useful targets . lindy durrant ( university of nottingham ) will present new results on antibodies targeting such specific glycans . much of the above technology development might be short - cut if it was possible to design an antibody binding site de novo to any structurally defined surface . sarel fleishman ( weizmann institute ) will give an update on what has been possible in using the rosetta software package , and the experimental validation of novel binders . morning track 1 : high quality research antibodies against the proteome chair : andrew bradbury ( los alamos national laboratory ) the current lack of standardized , renewable and low - cost protein affinity reagents impedes progress in biomedical research . this session will update participants on a number of initiatives intended to produce and distribute such reagents . in his extended chairman 's introductory remarks , andrew bradbury will provide his insights to the problems existing with research antibodies , as related to functionality , characterization and reproducibility , proposing possible solutions . he will be followed by andreas plckthun ( university of zrich ) , who in his inimitable manner will open the session by examining whether creating a repertoire of binding agents against all proteins is practical , as compared to an alternative approach in which technologies are developed , and implemented , to create affinity reagents rapidly and in high throughput on demand to the requirements of the intended experiment(s ) . david l. rimm ( yale university school of medicine ) will relate his group 's experience with the use of antibodies both in the research and clinical setting , and show how errors in assessment or validation can produce flawed data . he will also provide guidelines for antibody validation that can be used to avoid these problems . anthony kossiakoff ( university of chicago ) will update participants on the activities of the recombinant antibody network ( ran ) , which is an international consortium of 3 expert centers at the university of chicago , university of toronto and the university of california at san francisco . these centers are unified under a common set of goals , technologies and operational procedures , and each has a robotic technology platform capable of generating high - quality recombinant antibodies in a high throughput manner . ran is currently undertaking large proteomics - based projects to rapidly produce , validate and distribute high impact antibodies to the community . andrew bradbury ( los alamos national laboratory ) will describe the generation of renewable recombinant polyclonal antibodies using a combination of phage and yeast display , and the analysis of these polyclonals using next - generation sequencing . roberto d. polakiewicz ( cell signaling technology , inc . ) will discuss the current problem of inadequate validation of marketed research antibodies , and the potential for problems with the quality of the large quantities of antibodies that would be needed to cover the whole proteome . he will then present new strategies for effective discovery and validation of high quality mabs . a progress report on the national institutes of health 's protein capture reagents initiative will be given by seth blackshaw ( johns hopkins university school of medicine ) . the goal of the initiative is to generate and distribute standardized , renewable and low - cost protein affinity reagents to the scientific community . blackshaw will update participants on the initiative 's progress toward generation of monospecific mabs targeting a broad range of human transcription factors . chair : ian tomlinson ( glaxosmithkline ) the concept of hard - wiring 2 binding specificities into a single antibody molecule has been around for over 25 y and there are now over a hundred of these bispecifics in preclinical and clinical development . despite the availability of several validated platforms for making such bispecifics and the obvious application for combination targeting in cancer or immune - mediated disorders , one of the real challenges remains choosing the precise pair of targets to go after in such a format . haijun sun ( f - star biotechnology ) will open the session with a presentation on fcabs , which are fc fragments with mab - like antigen binding capability . fcabs can serve as a modular platform for testing bispecific target combinations through both rational design and empirical approaches . they can also be developed as drugs or replace the fc of a mab to create a bispecific molecule . simon chell ( glaxosmithkline ) will discuss effective target identification , stressing the importance of the manufacturability of new antibody architectures such as bispecific antibodies . martin steegmaier ( roche innovation center penzberg ) will describe how careful target selection and the development of the crossmab technology for creation of bispecific heterodimeric antibodies has led to the discovery of molecules that are efficiently transferred from the blood to the brain . in a preclinical model , the brain shuttle , which exploits receptor - mediated transcytosis and dual targeting , was shown to increase target engagement of investigational antibodies in the brain by over 50-fold compared to the parent antibody . janine schuurman ( genmab ) will draw on her experience with the duobody platform to provide general strategies and considerations for bispecific antibody discovery . the importance of using the final format for bispecific discovery approaches will be illustrated with surprising results found during the selection of a lead cmetxegfr bispecific antibody . michael t. stumpp ( molecular partners ) will update participants on the latest results with multispecific darpins , including inhibitors of soluble targets , receptors , and localized action darpins . these non - antibody small binding proteins have proven useful in understanding the biology of target combinations . in concluding the session , jijie gu ( abbvie biopharmaceuticals ) will discuss how to select bispecific target pairs and how to select the right molecules with desired pharmacological and development properties while taking the safety and efficacy of the molecules into consideration . afternoon track 1 : antibody - based therapeutics for diabesity chair : james larrick ( panorama research institute , velocity pharmaceutical development ) novel therapies to address the global pandemic of diabesity diabetes and obesity- are urgently needed . session chair james larrick ( panorama research institute , velocity pharmaceutical development ) will kick off this session by providing a perspective on the problem and the potential role of antibody - based therapeutics . next , gerd wallukat ( max delbrch center for molecular medicine ) will describe work on autoantibodies ( aabs ) against gpcrs found in sera of patients with cardiovascular diseases . these aabs are directed against epitopes localized on the first or second extracellular loop of the membrane - spanning hormone receptors and act like the corresponding agonists . the aabs directed against these epitopes induce their agonist - like effect by cross - linking and stabilization of the active receptor conformation . in contrast to the classical agonists , the functional aabs prevent the receptor desensitization normally seen if the receptors were stimulated for a longer time with the agonists . because these aabs act like the corresponding receptor agonists and prevent the receptor desensitization , such aabs may play a role in the pathogenesis of several cardiometabolic diseases . various glucagon - like peptide ( glp)-1 therapies ( e.g. , liraglutide , exenatide , dulaglutide ) exhibit efficacious glycemic control , but limited weight reduction in patients . bo yu ( larix bioscience llc ) will describe efforts to improve glp-1-fc fusions utilizing antibody membrane switch ( ams ) technology , which employs a switchable cell - surface display of the glp-1-fc fusion protein . sachdev sidhu ( university of toronto ) has developed a comprehensive set of synthetic antibodies that act as antagonists and agonists of the fgfr / klotho signaling pathway . antibodies displaying different effects in the modulation of signaling pathways related to diabesity will be described . after a break , maria groves ( medimmune ) will describe approaches employed by medimmune to generate potent antibody therapeutics to a selection of targets implicated in diabesity . lead isolation , affinity maturation strategies and preclinical data for the antibody therapeutics will be discussed . next , mingyue zhou ( amgen ) will describe development of novel lcat proteins for modulating hdl metabolism and reverse cholesterol transport ( rct ) . a modified lcat protein with fc fusion ( mlcat - fc ) exhibited enhanced enzyme activity , improved manufacturability , and desirable pharmacokinetic and pharmacodynamic properties in preclinical models , including cynomolgus monkeys . mlcat - fc administration to rabbits generated large hdl particles , promoted reverse cholesterol transport ( rct ) , and attenuated progression of atherosclerosis . a panel of novel agonistic human anti - lcat antibodies suitable for therapeutic development will also be discussed . there is enormous interest in the blockbuster potential of proprotein convertase subtilisin / kexin type 9 ( pcsk9 ) antibodies . the final presentation by jesper gromada ( regeneron pharmaceuticals ) will focus on antibody therapies to pcsk9 for the regulation of plasma ldl - c and angiopoietin - like protein 3 ( angptl3 ) to increase lipoprotein lipase activity and lower circulating triglyceride levels . we anticipate an exciting afternoon of first - rate science in this most important field . afternoon track 2 : preclinical and clinical case studies : emerging targets , new approaches chair : kerry a chester ( university college london ) in her opening remarks , chair kerry chester ( university college london ) will introduce a series of studies focused on emerging targets and approaches with potential to expand the scope of current immunotherapeutic and diagnostic imaging agents . mesothelin ( msln ) is becoming an increasingly attractive target for delivery of potent toxins , as it is highly expressed in a range of epithelial cancers but has very limited expression in healthy tissue , predicting low non - specific toxicity . klaus bosslet ( roche diagnostics gmbh ) will begin by giving an update on their latest work with rg7787 , a recombinant antibody - toxin fusion protein that targets msln with a de - immunized 24-kd fragment of pseudomonas exotoxin . rg7787 is effective against both quiescent and proliferating tumor cells and has shown potential for clinical development in triple - negative breast and gastric cancers . the development of therapeutic antibodies capable of targeting intracellular proteins will be addressed by david a. scheinberg ( memorial sloan kettering cancer center ) . the work constitutes a paradigm shift for pharmaceutical anti - cancer antibodies which , despite the abundance of intracellular tumor targets , have traditionally been directed to cell - surface or extracellular molecules . the approach exploits the fact that degraded small peptides from intracellular proteins are presented on the cell surface in the pocket of major histocompatibility complex ( mhc ) class i molecules . the presentation will focus on wilms tumor protein ( wt1 ) , an intracellular , oncogenic transcription factor that is overexpressed in a wide range of cancers , but has limited expression in normal adult tissues . scheinberg 's group has developed antibodies to rmfpnapyl ( rmf ) , a wilms ' tumor1-derived hla - a*02:01 epitope . the antibodies have been generated in multiple formats , including a glycoengineered human igg1 with enhanced adcc function ( eskm ) and bispecific forms . mark cobbold ( university of birmingham ) will present an intriguing approach to selectively harness the power of adaptive immunity using immunogenic viral epitopes covalently coupled to clinically relevant antibodies . this novel therapeutic entity , termed an antibody - peptide epitope conjugate ( apec ) , has been designed to release the viral antigens by proteolytic cleavage only in close proximity to the surface of the tumor . the technology renders tumors susceptible to immune attack by mimicking viral infection and engaging a potent t - cell response , with engineered control of which t cells engage with the target cell . in vitro and in vivo work will be used to demonstrate proof - of - concept . chimeric antigen receptors ( cars ) are fusion proteins that link an extracellular antigen binding domain , usually a single chain fv ( scfv ) , with intracellular t - cell signaling domains . in clinical studies , patient 's peripheral blood t - cells are engineered to express cars by transduction with integrating vectors , resulting in generation of antigen specific car t - cells that can target and kill cancer cells . car t - cells are highly potent cellular therapeutics which , despite the complexity of the treatment , are emerging as a realistic therapeutic option for the clinic . after the refreshment break , martin pule ( university college london ) will present his work on clinical development of cars and will discuss the challenges of finding tumor targets with acceptable on - target off - tumor toxicity . a new approach in directing cars against multiple myeloma using a high - affinity natural ligand rather than an scfv will be presented . in addition , the concept of using a combinatorial approach will be introduced , exploring the ability of engineered t cells to sample and trigger in response to patterns of antigen expression on target cells . ror1 is a type tyrosine - kinase - like onco - embryonic surface antigen that is expressed in early development and has an emerging role in cancer biology . ror1 is expressed by a variety of human cancers , including solid tumors of the colon , lung , and pancreas and chronic lymphocytic leukemia ( cll ) , the most common blood malignancy in adults . expression of ror1 enhances the growth , survival , and migration of cancer initiating cells in vitro and in vivo . thomas j. kipps ( moores ucsd cancer center ) will show the latest results from an evaluation of a first - in - class anti - cancer stem cell antibody targeting ror1 . the mab drug has recently entered a phase 1 trial to evaluate whether it is a safe and well - tolerated cancer stem cell - targeted agent in patients with cll . michel eisenblaetter ( king 's college london ) will conclude the session with a discussion of multi - modal molecular imaging of immune cell activity with antibodies that target the exosome - associated calcium binding proteins s100a8/a9 , which are key markers of activated monocytes . results from in vivo fluorescence and radionuclide imaging of s100a8/a9 expression indicate that s100a8/a9 has utility as new target for diagnostic imaging of immunosuppressive inflammation . the work is ground breaking because , although early detection , localization and monitoring of inflammation are crucial for tailoring individual therapies , reliable biomarkers to detect local inflammatory activities and predict disease outcome are not yet available . morning track 1 : antibody effector functions co - chairs : dennis r burton ( the scripps research institute ) and paul whi parren ( genmab ) on wednesday at 8 am , chairs dennis r burton and paul whi parren will be ready to kick - off an exciting session on antibody effector functions . so fill up on plenty of coffee , and join us for the first part of the session , which focuses on the interaction of antibody with igg fc receptors ( fcr ) . mark cragg ( southampton university ) will show data on a novel class of antagonistic anti - fcriib antibody for tumor therapy that overcomes previously experienced drawbacks of inhibitory signaling and internalization , and that bears promise for combination therapies with contemporary therapeutic mabs . mark hogarth ( burnett institute ) will discuss recent data identifying critical functional polymorphisms between human and macaque fcr with important implications for research , as well as non - clinical safety studies with human mab in non - human primates . david szymkowski ( xencor ) will present 2 case studies of antibodies with fc domains exhibiting enhanced fcriib binding . by making use of specific fcriib functions , an anti - cd19 antibody was derived that inhibits b cell function without inducing depletion , whereas an anti - ige antibody , among others , exploits fcriib 's role in antigen clearance as its mechanism of action . following the break , this is a novel platform to enhance effector function after antibody binds its cognate antigen on the cell surface . development of the platform was built on the novel insight provided by the observation that fc - mediated hexamerization on cell surfaces is required for optimal complement activation . sophia karagiannis ( king 's college london school of medicine ) will discuss ige as an exciting novel class of antibody for immunotherapy of cancer with unexpected and superior activity compared to the matching igg counterparts . richard blumberg ( brigham and women 's hospital ) will conclude the session by presenting his recent work on the critical , but previously unappreciated , role of the interaction between the neonatal fc receptor fcrn and antibody in modulating both innate and adaptive immune responses . in summary , you can look forward to an eye - opening session in which a number our best studied and favorite proteins are shown to convey novel functions , activity and therapeutic promise . morning track 2 : new targets and applications in immune checkpoint inhibitors chair : gregory p adams ( fox chase cancer center ) . antibodies targeted against inhibitory or activating receptors on immune effector cells are rapidly emerging as powerful agents for the treatment of cancer and other diseases . the approval of ipilimumab ( anti - ctla-4 ) and more recently pembrolizumab ( anti - pd-1 ) for the treatment of melanoma represent the first of what will likely be a large panel of clinically - approved antibodies capable of modulating the immune response to treat a variety of diseases . blocking inhibitory receptors to promote immune responses against cancer and blocking activating receptors to diminish autoimmune responses represent the most obvious applications , but other potential indications will likely include prevention of the rejection of organ transplants and stimulation of the immune system to fight infectious diseases . this session will focus on recent efforts in developing new agents for these and related applications . the session will be opened by sumit k. subudhi ( md anderson cancer center ) who will describe how the recent successes with anti - ctla-4 and anti - pd-1/pd - l1 antibodies can be improved on by targeting a variety of other immune checkpoints , including icos , b7-h3 , b7-h4 , lag-3 , 41bb , ox40 , cd40 , that have shown promise in preclinical studies . he will discuss the identification of promising targets and the design of agents with potential therapeutic properties . stephen willingham ( stanford university school of medicine ) will then discuss the role played by cd47 in the protection of cancer cells from phagocytosis by the innate immune system . he will describe a humanized monoclonal antibody ( hu5f9-g4 ) , which was developed by his group to block the interaction between cd47 and its ligand sirp- on phagocytic cells . willingham will share the results of in vivo preclinical studies demonstrating the ability of hu5f9-g4 to inhibit tumor growth and metastasis of hematologic malignancies and solid tumors . a novel approach to inhibit the suppressive activity of tregs using neutralizing antibodies directed against garp ( glycoprotein a repetitions predominant ) will be presented by michael saunders ( argen - x ) . antibodies targeting critical epitopes on garp expressed on the surface of tregs block its ability to bind tgf--1 , which plays a key role in treg - mediated immunosuppression . saunders will share evidence of the ability of one of their anti - garp antibodies , mhg-8 , to inhibit immune suppression mediated by tregs in a graft - versus - host disease model induced by transplantation of human pbmcs ( + /- autologous tregs ) into immunocompromised nod / scid / il2rg-/- mice , suggesting a potential role of anti - garp antibodies in the treatment of cancer or chronic infections . holbrook kohrt ( stanford university ) will discuss the role played by activated cd137 ( 41bb ) on nk cell - mediated adcc of cancer . he will present the results of studies showing that the addition of an agonistic anti - cd137 antibody enhanced the antitumor activity of rituximab , trastuzumab and cetuximab both in vitro and in vivo . the inhibition of t cell costimulation has emerged as an attractive alternative to the use of highly toxic immunosuppressive drugs in the prevention of organ transplant rejection . however , clinical trials with belatacept , a novel fusion receptor biologic that binds to cd80/86 with high affinity and inhibits costimulation signals to t cells did not achieve the success seen in preclinical studies . flavio vincenti ( university of california , san francisco ) will describe new approaches using combinations of belatacept with inhibition of the cd40/cd154 pathway , anti - il6 or possibly infusion of t regulatory cells to increase belatacept efficacy in the prevention of renal transplant rejection . while therapies that interfere with ctla-4 and pd-1 have been very promising in the clinic , many cancer patients fail to respond to these therapies . ana carrizosa anderson ( harvard medical school ) will describe her work studying how the tim family of molecules regulate t cell responses and the development and evaluation of agents that block signaling through tim-3 and other novel checkpoint targets . afternoon track 1 : engineering antibody developability : expression , solubility and polyreactivity chair : k dane wittrup ( massachusetts institute of technology ) . to advance in development as therapeutics , antibody leads must have appropriate biophysical properties , as well as suitable binding affinity , target specificity and functional activity . in this session , speakers will provide practical advice on the design and selection of antibodies that should have minimal downstream problems . yan wu ( genentech ) will open the session with a discussion of antibody lead selection . yingda xu ( adimab ) will describe high throughput assays that target detection of antibody self- and cross - interaction to predict the fate of an antibody during expression , purification , storage and serum clearance . bojana popovic ( medimmune ) will illustrate how an in silico spatial aggregation propensity tool was used in conjunction with antibody engineering to improve the stability and pharmacokinetic profile of medi-1912 , an anti - nerve growth factor mab , without compromising potency or affinity . silke hansen , ( roche innovation center penzberg ) will describe the production of high quality , complex format antibody molecules , including bispecific antibodies and antibody fusion proteins consisting of up to 4 different polypeptide chains , in a single cho cell line at . as dr . hansen will discuss , the application of diligent cell line selection strategies , supported by high throughput analytical methods , were critical to the successful identification of cell clones with well - characterized cell properties giving rise to a stable product profile and high product quality . ernest smith ( vaccinex , inc . ) will provide details of an antibody discovery platform that enables efficient mammalian cell - based expression of a library of human antibodies in full - length igg format on the surface of vaccinia virus . upon infection of mammalian cells , the antibody is incorporated into newly produced virus and displayed on the surface of the host cell . this approach combines the advantages of virus panning and cell sorting into one technology . to conclude the session , diana bowley ( abbvie bioresearch center ) will give an overview of an informatics platform that supports the design , cloning , expression , and purification of large panels of bispecific dual - variable domain immunoglobulin ( dvd - ig ) candidates bowley will describe how the platform is also used to assess properties of the molecules , including selectivity , cross reactivity , epitope binding , and stability . the system can thus enable the parallel engineering of 1,000s of dvd - ig molecules via combinatorial design of v - domains , linker positions and lengths , and fc chains . afternoon track 2 : emerging clinical data with therapeutic antibodies and adcs chair : louis m weiner ( georgetown university medical center ) therapeutic antibodies and adcs have important clinical applications in the treatment of cancer and other diseases . this session will focus primarily on emerging data from antibodies that target immune checkpoints to treat cancer . starting with antibodies targeting ctla4 , as presented by nils lonberg ( bristol - myers squibb ) , the field has rapidly expanded to include antibodies targeting the immune checkpoints pd-1 , as presented by omid hamid ( the angeles clinic and research institute ) and jonathan cheng ( merck ) , and pdl1 , as presented by edward cha ( genentech ) , and antibodies against additional immune checkpoints are being developed . moreover , combinations of immune modulating antibodies , such as ipilimumab plus anti - pd-1 demonstrate extremely promising activity in patients with advanced malignant melanoma . important emerging clinical indications include non - small cell lung cancer , as presented by hossein borghaei ( fox chase cancer center ) . finally , adcs are beginning to fulfill their clinical promise , as exemplified by the success of ado - trastuzumab emtansine , an anti - her2 : maytansinoid conjugate . as presented by kyle holen ( abbvie ) , another agent that is showing early promise , abt-414 , targets activated egfr and delivers a cytotoxic payload . the antibody society 's special session rounding out wednesday 's program , meeting participants will get a glimpse of the future at the antibody society 's special session on antibodies to watch in 2015 . the commercial pipeline of antibody therapeutics is highly dynamic , with a multitude of transitions occurring during the year as molecules advance through the clinical phases and onto the market . the president of the antibody society , janice m. reichert ( reichert biotechnology consulting llc ; editor - in - chief , mabs ) , will recap the important events of 2014 , including the first approvals of 5 antibody therapeutics ( vedolizumab , ramucirumab , siltuximab , nivolumab and pembrolizumab ) , and discuss expectations for 2015 . recent results suggest that activity in 2015 may be even greater than 2014 , which was a banner year for transitions into pivotal studies , as well as for first marketing approvals . relevant data for transitions to first phase 3 studies that occurred in 2014 and those projected for 2015 will be summarized . metrics for novel antibody therapeutics development , and the evolving field of biosimilar antibody development , will also be discussed . track 1 : antibody repertoires : next generation sequencing , data analysis , storage and sharing chair : jamie k scott ( simon fraser university ) the advent of next - generation sequencing ( ngs ) allows deep analysis of immune repertoires , a term collectively referring to antibody repertoires ( comprising the b - cell receptors ( bcrs ) and antibodies produced by b - cells and plasma cells , respectively ) , and to t - cell receptor ( tcr ) repertoires . productively rearranged vl and vh genes encode the variable domains of antibodies and b - cell receptors ; similarly , those of v and v genes encode the variable domains of / t - cell receptors . b- and t - cell mrna encoding antibodies and tcrs , or plasmid dna encoding phage libraries , constitute the material from which ngs immune repertoires are derived . v regions from tens of millions of cells or phage clones can be sequenced in bulk with high coverage ( i.e. , in the 100 s of millions ) to arrive at dna sequences reflecting a repertoire 's sequence diversity and relative copy numbers . from such data , the progress of phage library screenings can be precisely followed ; antibody and tcr repertoires can be assessed in depth ( e.g. , following residual disease after treatment for leukemia or lymphoma ) , and differences among b- and/or t - cell populations can be assessed between healthy vs. diseased states , and after vaccination . as well , somatic mutations among clonally related antibodies can be identified , and analyzed to reveal the the purpose of this session is to provide participants in this year 's antibody engineering and therapeutics conference a taste of current progress in immune - repertoire ngs data acquisition , analysis , storage and sharing . the first half of the session will focus on immune repertoire library technology and data analysis . brandon dekosky ( university of texas , austin ) will present new methods for high - throughput analysis of paired antibody / bcr heavy and light chain variable - region ( vh and vl ) sequences from b cells and plasma cells . ( los alamos national laboratory ) will discuss ngs data - analysis tools her group has used to identify selected antibody clones from a phage - displayed , single - chain ( scfv ) , nave antibody library . and finally , ramy arnaout ( beth israel deaconess medical center ) will present his analysis of ngs data from murine bone marrow and splenocytes , which identified vh genes from non - productive and nave follicular and marginal - zone b - cells subsets . his analysis reveals biased vh gene - segment usage among these b - cell subsets , and from this , differential pathways for follicular and marginal - zone b cell maturation . the second half of this session will focus on the storage and sharing of immune repertoire ngs data . felix breden ( simon fraser university ) will present ireceptor , a distributed data management system for sharing and comparing immune repertoires . in this scheme , users store primary data at their local sites , which they bring to the ireceptor system for analysis of their and other users data . david johnson ( gigagen , inc .. ) will present clonalysis , a cloud - based web portal for the storage and analysis of ngs immune repertoire data . he has developed tools to rapidly perform cdr3 analysis , v - gene assignment , and clustering of related sequences . finally , lindsay cowell ( ut southwestern medical center ) will present vdjserver , a web portal for storing , analyzing , sharing and archiving immune repertoire data . she is also developing standards for data sharing and interoperability , and analysis pipelines to support data reproducibility . track 2 : the best of both worlds : antibodies with enhanced or multiple functionalities chair : mark r alfenito ( engen bio , inc . ) as a class of drugs , antibodies are uniquely complex because they can come ready - armed with multiple functions , including a targeting function represented by the variable regions that can have blocking , agonizing or antagonizing functions , and effector functions , contained within the c domains , conferring adcc , opsonization and complement functions . this session will cover several strategies to take advantage , and enhance the flexible nature , of antibody functions . zhenping zhu ( kadmon china ) will discuss several formats of an anti - pd - l1 ( programmed death ligand ) antibody , including immunoconjugates , and show both in vitro and in vivo data of their efficacy . takehisa kitazawa ( chugai pharmaceutical co ) will show the use of a bispecific antibody to recreate a very novel function , that of binding coagulation factors ixa and x , for the treatment of hemophilia a. this is the first time a hemophilia treatment has been reported at this antibody conference . the molecular design of the molecule will be discussed , along with preclinical and early clinical data . bent jakobsen ( immunocore ltd ) will also speak about bispecific antibodies , the immtacs ( immune mobilizing monoclonal t cell receptors against cancer ) . this talk will cover engineering of immtacs , evidence for potent and specific killing of tumor cells and details of their mechanism of action . in addition , the latest clinical data on the most advanced immtac , imcgp100 , will be presented . kristian jensen ( dutalys ) will discuss dutamabs , a form of bispecific antibody with 2 binding sites in each fv region . the presentation will illustrate their benefits , using a case study of a novel bispecific angiogenesis blocker with best - in - class properties in the areas of affinity , potency , stability , solubility , manufacturability and tolerability . ronit mazor ( national cancer institute ) will speak on the engineering of antibody - toxin conjugates that minimize patient toxicity and immunogenicity by silencing human t cell epitopes contained within the molecule . mazor will present data for a redesigned immunotoxin with t cell epitope mutations that demonstrates high cytotoxicity to cells isolated from cancer patients , and that produces complete remissions in mice with human cancer xenografts . finally , david miao ( concortis biosystems ) will discuss the enhancement of an antibody 's efficacy by delivery of a toxic payload via their adc technology . antibody therapeutics for non - cancer indications chair : trudi veldman ( abbvie ) the last session of the meeting highlights the diversity of approaches to disease modification with antibody therapies . in addition to blocking inflammatory and disease promoting processes driven by pro - inflammatory cytokines , the focus has now been extended to blocking negative regulators such as myostatin and lingo-1 to achieve muscle growth and remyelination , respectively . clinical experience is now emerging with several of these antibodies , and these studies will inform us whether the preclinical promise of these approaches will translate to the clinical setting . tony de fougerolles ( ablynx ) will present the unique scientific opportunities for the use of single domain antibodies ( nanobodies ) and the experience in preclinical and clinical development through several case studies in the fields of inflammation and host defense . next , dimiter dimitrov ( national cancer institute ) will discuss the significant efforts to develop mabs against several emerging and biodefense - related viruses as candidate therapeutics and prophylactics . three exceptionally potent new mabs against mers - cov and their potential for therapy of humans will be described . lioudmila tchistiakova ( pfizer ) will present interesting results from studies of 2 antibodies to myostatin ( gdf-8 ) , a negative regulator of muscle growth , which were both evaluated in clinical trials . detailed studies of the structural interactions of the antibody rk35 with the target may provide insight into the superior clinical efficacy that was obtained with this antibody . dupilumab , an anti- il4r antibody that inhibits both il4 and il13 signaling , blocks th2 inflammation in patients with moderate - to - severe atopic dermatitis and eosinophilic asthma . neil graham ( regeneron pharmaceuticals ) will present data on the clinical efficacy and safety of dupilumab in these two conditions . then , john latham ( alder biopharmaceuticals ) will discuss a novel strategy to prevent migraine using a potent anti - cgrp antagonistic antibody . ald403 is a humanized , high - affinity mab that targets the neuropeptide cgrp , which has been shown to play an important role in migraine initiation and propagation . a summary of the properties of this antibody in preclinical studies and the efficacy in a human clinical trial will be presented . multiple sclerosis ( ms ) researchers are looking beyond anti - inflammatory drugs to halt disease progression and are interested in exploring neuronal repair mechanisms that have the potential to be groundbreaking therapies in the treatment of ms and other neurodegenerative diseases . of special interest are the ongoing studies with biib033 , an antagonist antibody to lingo-1 , which is a negative regulator of oligodendrocyte differentiation and myelination . werner meier ( biogen idec ) will discuss the discovery , engineering and development of biib033 for the treatment of ms .
the 25th anniversary of the antibody engineering & therapeutics conference , the annual meeting of the antibody society , will be held in huntington beach , ca , december 711 , 2014 . organized by ibc life sciences , the event will celebrate past successes , educate participants on current activities and offer a vision of future progress in the field . keynote addresses will be given by academic and industry experts douglas lauffenburger ( massachusetts institute of technology ) , ira pastan ( national cancer institute ) , james wells ( university of california , san francisco ) , ian tomlinson ( glaxosmithkline ) and anthony rees ( rees consulting ab and emeritus professor , university of bath ) . these speakers will provide updates of their work , placed in the context of the substantial growth of the industry over the past 25 years .
Sunday, December 7, 2014 Monday, December 8, 2014 Tuesday, December 9, 2014 Wednesday, December 10, 2014 Thursday, December 11, 2014
the silver anniversary antibody engineering & therapeutics meeting will be kicked off with a dynamic , audience - participatory workshop on antibody drug development chaired by veterans jim larrick ( panorama research institute and velocity pharmaceutical development llc ) and mark alfenito ( engen bio , inc . ) keynote presentations chair : james d. marks ( university of california , san francisco ; san francisco general hospital ) the meeting will open with keynote presentations by five luminaries in the field of molecular therapeutics development . douglas a. lauffenburger ( massachusetts institute of technology ) will discuss systems analysis of cell communication network dynamics for therapeutic biologics design . ira pastan ( national cancer institute ) will discuss his extensive experience with the development of recombinant immunotoxins ( rits ) composed of a cancer cell - targeted fv or fab fused to a portion of pseudomonas exotoxin a. rits targeting either cd22 on leukemias or mesothelin on mesotheliomas have produced complete and dramatic remissions in some patients , but their efficacy is limited by immunogenicity and capillary leak syndrome ( cls ) . james wells ( university of california , san francisco ) will give his views on engineered antibodies for challenging targets . ian tomlinson ( glaxosmithkline ) will discuss the past , present and future of antibody repertoires . in commemoration of the 25 anniversary of the conference , anthony rees ( rees consulting ab and emeritus professor , university of bath ) will provide a historical perspective on the antibody molecule and the remarkable journey scientists have taken while trying to understand its functions . k. dane wittrup ( massachusetts institute of technology ) invented antibody - yeast display libraries and has leveraged that technology to propel his research into diverse areas of cancer biology and antibody engineering . he has worked on aspects of cellular immunology during much of the past decade , and recently became interested in the definition of boundary conditions and design objectives for ic engineering , which he will discuss in this session . in the talk by andreas plckthun ( university of zurich ) a very different challenge will be addressed : is it possible to direct a targeting agent to the cytoplasm with high efficiency ? he will be followed by andreas plckthun ( university of zrich ) , who in his inimitable manner will open the session by examining whether creating a repertoire of binding agents against all proteins is practical , as compared to an alternative approach in which technologies are developed , and implemented , to create affinity reagents rapidly and in high throughput on demand to the requirements of the intended experiment(s ) . anthony kossiakoff ( university of chicago ) will update participants on the activities of the recombinant antibody network ( ran ) , which is an international consortium of 3 expert centers at the university of chicago , university of toronto and the university of california at san francisco . chair : ian tomlinson ( glaxosmithkline ) the concept of hard - wiring 2 binding specificities into a single antibody molecule has been around for over 25 y and there are now over a hundred of these bispecifics in preclinical and clinical development . flavio vincenti ( university of california , san francisco ) will describe new approaches using combinations of belatacept with inhibition of the cd40/cd154 pathway , anti - il6 or possibly infusion of t regulatory cells to increase belatacept efficacy in the prevention of renal transplant rejection . afternoon track 1 : engineering antibody developability : expression , solubility and polyreactivity chair : k dane wittrup ( massachusetts institute of technology ) . starting with antibodies targeting ctla4 , as presented by nils lonberg ( bristol - myers squibb ) , the field has rapidly expanded to include antibodies targeting the immune checkpoints pd-1 , as presented by omid hamid ( the angeles clinic and research institute ) and jonathan cheng ( merck ) , and pdl1 , as presented by edward cha ( genentech ) , and antibodies against additional immune checkpoints are being developed . the president of the antibody society , janice m. reichert ( reichert biotechnology consulting llc ; editor - in - chief , mabs ) , will recap the important events of 2014 , including the first approvals of 5 antibody therapeutics ( vedolizumab , ramucirumab , siltuximab , nivolumab and pembrolizumab ) , and discuss expectations for 2015 . ronit mazor ( national cancer institute ) will speak on the engineering of antibody - toxin conjugates that minimize patient toxicity and immunogenicity by silencing human t cell epitopes contained within the molecule . next , dimiter dimitrov ( national cancer institute ) will discuss the significant efforts to develop mabs against several emerging and biodefense - related viruses as candidate therapeutics and prophylactics .
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external fixation has gained wide acceptance in osteotomy , limb lengthening , and treatment of open fractures because it enables easy reduction of bone fragments , even under unfavorable soft tissue conditions , and secondary corrections and modifications can be made.1 despite these positive qualities , there is still a high incidence of pin site infection , which may cause mechanical deterioration of the bone - pin interface and lead to pin loosening and osteomyelitis.26 pin site infection is due to indigenous micro - organisms adhering to the pin surface and subsequently forming aggregates that may lead to formation of a biofilm and eventual spread of infection to the surrounding host tissue . reported rates of pin site infection vary widely in the literature , ranging from virtually zero to over 50%.3,5,6 generally , the cleaning and/or dressing of pin sites and systemic antibiotics are used for prevention and treatment of pin site infection.3,6 however these anti - infection measures have limitations . pins with deep infection should be removed and suppression of infection must be given priority , which means that continuation of treatment with external fixation for the primary disease becomes impossible . an additional problem arising from pin site infection is the risk of deep tissue infection , such as osteomyelitis , when converting external fixation to internal fixation , because of penetration of the skin barrier.7 therefore , a new strategy for prevention and treatment of pin site infection is required . modifications to biomaterial surfaces enable programming of cells to substratum events , thereby diminishing infection by inhibiting bacterial adhesion or by enhancing tissue compatibility or integration . recent studies by other groups have investigated the bactericidal effects of silver and antibiotics used as coatings for metal materials.813 however , mass et al aborted a study of the clinical application of silver - coated pins due to elevated blood levels of silver ions.9 furno et al concluded that the clinical failure of silver is mainly due to obliteration by proteinaceous materials in the human body.10 development of bacterial resistance to silver coating has also been reported.11 another group demonstrated the antibacterial effect of pins impregnated with tobramycin or gentamicin in an animal model . however , there are still concerns about the side effects of these agents and the emergence of resistant strains of bacteria.12,13 the bactericidal activity of photocatalytic titanium dioxide ( tio2 ) has recently been highlighted as an alternative strategy to assist in environmental purification of water and air because of its high catalytic activity , chemical stability , low energy requirements , and nontoxic properties.14,15 pure titanium and titanium alloys are widely used in modern orthopedic surgery because of their high resistance to corrosion and their biocompatibility . tio2 photocatalysts have strong oxidizing ability and can decompose various organic compounds when exposed to ultraviolet light by generating active oxygen species , such as free hydroxyl radicals , superoxide anion radicals , and hydrogen peroxide . this photodecomposition of organic compounds is also useful for killing bacteria , so self - sterilizing surfaces can be prepared . it has been confirmed that these active oxygen species can destroy the outer bacterial cell membrane , ultimately leading to nonselective cell death without fostering drug - resistant species , as well as decompose endotoxins and provide nourishment for bacteria.1620 in our previous in vitro studies , tio2 film had a strong photocatalytic bactericidal effect on staphylococcus aureus,21 which is one of the main bacteria causing pin site infection and can easily become antibiotic - resistant.9,2224 arciola et al found that s. aureus accounted for about 68% of bacteria isolated from infected incision sites postoperatively.22 tio2 can act as a potent biocidal agent because of its high oxidation potential and nonselective reactivity , so we hypothesized that a tio2 photocatalyst might reduce the risk of harmful pin site infection and then conducted an in vitro experiment . the purpose of the current in vivo study was to evaluate the efficacy of photocatalytic tio2 in inhibition of infection when using percutaneous external fixation pins in a rat model under low energy ultraviolet illumination . the substrates were prepared using 2 mm diameter pin screws ( atlas sports and medicine co , ltd , nagasaki , japan ) made of stainless steel ( sus316l ) which is the basic material in external fixation pins . the native oxides were removed by cleaning the samples with distilled water and acetone and then immersing them in a mixture of aqueous hf and hno3 acids . the plasma source ion implantation apparatus used in this study has been described elsewhere.21,25 this implantation method has several advantages compared with other methods , including a large area , multiple targets , and reasonable costs . tio2 films were deposited onto the stainless steel pins using titanium tetraisopropoxide plasma in the chamber . a pulsed high negative bias voltage of 18 kv was applied to the sample holder at a pulse duration of 10 sec with a pulse repetition rate of 1 khz . the pin was annealed at 923 k for one hour to obtain a crystalline anatase tio2 film . the pins were divided into two groups ( 30 control pins and 30 pins coated with tio2 ) . glancing - angle x - ray diffraction patterns for the tio2 pins showed peaks at positions corresponding mainly to an anatase - type structure , including a rutile one , and x - ray photoelectron spectroscopy revealed the chemical composition was almost all tio2 . energy - dispersive spectroscopy indicated that the homogeneous microstructure was composed of titanium , oxygen , and elements of stainless steel . cross - sectional analysis of the tio2 layers revealed an average thickness of 1.0 m , with no interfaces evident between the substrate and the dense layer . the surface roughness parameters for tio2-coated stainless steel ( ra = 808 nm , rz = 1674 ) were almost the same as for untreated stainless steel ( ra = 837 nm , rz = 1755 ) , as reported in our previous study.21 micrographs of the surfaces of the tio2-coated pins were obtained using a field emission scanning electron microscope ( jsm 6400f , jeol , tokyo , japan ) . this series of in vivo experiments was performed in accordance with the principles stated in the established guidelines for the treatment of animal subjects , and animal welfare assurance was strictly maintained . s. aureus ( seattle 1945 strain , atcc 25923 ) was incubated for 6 hours at 37c in 10 ml of trypticase soy broth . the bacterial cells were harvested by centrifugation at 3000 rpm for 10 minutes and then suspended in sterilized distilled water to a concentration of 1 10 cells / ml . sixty female , 7-week - old sprague - dawley ( specific pathogen - free ) rats were obtained from charles river laboratories japan inc , yokohama , japan . the animals were housed individually in a secure , climate - controlled facility for the duration of the experiment . the operative procedure was performed under general anesthesia by intraperitoneal administration of a sodium pentobarbital ( 50mg / kg of body weight ) . the insertion areas on the hind limb of each animal were shaved , and the area over the femur was aseptically prepared with 70% ethanol . a small linear skin incision ( about 12 mm ) was made and the pins were inserted through the incision into the femoral bones ( one pin per bone ) , followed by bacterial contamination with 100 l of an identifiable s. aureus strain on the insertion area , ie , the skin - pin interface . ultraviolet a light was emitted using a black light source set 30 cm above the rats for 30 minutes . in our previous in vitro study , the tio2-coated material significantly suppressed the viability of bacteria after 30 minutes of ultraviolet illumination,21 so the illumination time was set to 30 minutes . the intensity of the light was 2.0 mw / cm at a peak wavelength of 352 nm . the pin sites were examined daily and the dressings were carefully changed to avoid cross - contamination of the pin sites . additional treatment , including incision closure , blood coagulation , or antibiotic dressing was not needed . on day 14 , visible clinical findings at each pin site were classified according to pin site infection criteria as no infection , inflammation or serous drainage without frank purulence , or frank purulence.26,27 clinical determination of infection was defined as all three observers being in agreement . after evaluation by eye , the animals were sacrificed and the bone and soft tissue around the pin were retrieved . we used an automated microbiology system ( bd phoenix system , nippon becton dickinson co , tokyo , japan ) to identify the bacteria isolated ( panel type pmic / id-68 ) . the tissues obtained were fixed for one day in a 10% formalin solution buffered at ph 7.2 , dehydrated through a series of graded alcohol solutions , and embedded in paraffin . immediately after removal of the pins , 4 m thick longitudinal sections in the sagittal plane were cut using a microtome ( hm325 , thermo scientific mircom , walldorf , germany ) with a 40 degree stainless steel knife , taking care not to damage the bone - implant interface . slices in the same bone plane were assessed according to bone infection score as : 1 ) abscess formation ; 2 ) sequestrum formation ; 3 ) enlargement of corticalis ; 4 ) destruction of corticalis ; and 5 ) general appearance.12,28 parameters 1 to 4 were scored as 0 ( absent ) or 1 ( present ) . parameter 5 was scored as 0 ( absent ) , 1 ( mild ) , or 2 ( severe ) . we then computed the rate of contact between bone and screw , on the assumption that the part in which it is possible to confirm the shape of a screw thread in the tissue specimen is the part contacted , based on a modification of the method reported by giavaresi et al and moroni et al.29,30 we did the actual image processing of the hematoxylin and eosin staining sample automatically using an optical microscope ( bx51 - 33 , olympus optical co , tokyo , japan ) connected to public domain image analysis software ( image j ) and computed the data . in addition , the shape of the bone for the part corresponding to a screw thread was evaluated by gram staining . we first chose a part in which it was possible to confirm the two consecutive screw threads from the entrance part of a pin screw and then made a planimetric rate of occupation for bacterial colonies and intraosseous neutrophils in the shape of a screw thread using image j. the clinical and histomorphometric results for the control group ( n = 30 ) were analyzed and compared with those of the tio2-coated pin group ( n = 30 ) . all statistical analyses were performed using the statistical package for social sciences version 20 software ( spss inc , chicago , il ) . the mean and standard deviation of bone infection score , bone - implant contact ratio , and planimetric rate of occupation for bacterial colonies and intraosseous neutrophils were analyzed using the student s t - test . the substrates were prepared using 2 mm diameter pin screws ( atlas sports and medicine co , ltd , nagasaki , japan ) made of stainless steel ( sus316l ) which is the basic material in external fixation pins . the native oxides were removed by cleaning the samples with distilled water and acetone and then immersing them in a mixture of aqueous hf and hno3 acids . the plasma source ion implantation apparatus used in this study has been described elsewhere.21,25 this implantation method has several advantages compared with other methods , including a large area , multiple targets , and reasonable costs . tio2 films were deposited onto the stainless steel pins using titanium tetraisopropoxide plasma in the chamber . a pulsed high negative bias voltage of 18 kv was applied to the sample holder at a pulse duration of 10 sec with a pulse repetition rate of 1 khz . the pin was annealed at 923 k for one hour to obtain a crystalline anatase tio2 film . the pins were divided into two groups ( 30 control pins and 30 pins coated with tio2 ) . glancing - angle x - ray diffraction patterns for the tio2 pins showed peaks at positions corresponding mainly to an anatase - type structure , including a rutile one , and x - ray photoelectron spectroscopy revealed the chemical composition was almost all tio2 . energy - dispersive spectroscopy indicated that the homogeneous microstructure was composed of titanium , oxygen , and elements of stainless steel . cross - sectional analysis of the tio2 layers revealed an average thickness of 1.0 m , with no interfaces evident between the substrate and the dense layer . the surface roughness parameters for tio2-coated stainless steel ( ra = 808 nm , rz = 1674 ) were almost the same as for untreated stainless steel ( ra = 837 nm , rz = 1755 ) , as reported in our previous study.21 micrographs of the surfaces of the tio2-coated pins were obtained using a field emission scanning electron microscope ( jsm 6400f , jeol , tokyo , japan ) . this series of in vivo experiments was performed in accordance with the principles stated in the established guidelines for the treatment of animal subjects , and animal welfare assurance was strictly maintained . s. aureus ( seattle 1945 strain , atcc 25923 ) was incubated for 6 hours at 37c in 10 ml of trypticase soy broth . the bacterial cells were harvested by centrifugation at 3000 rpm for 10 minutes and then suspended in sterilized distilled water to a concentration of 1 10 cells / ml . sixty female , 7-week - old sprague - dawley ( specific pathogen - free ) rats were obtained from charles river laboratories japan inc , yokohama , japan . the animals were housed individually in a secure , climate - controlled facility for the duration of the experiment . the operative procedure was performed under general anesthesia by intraperitoneal administration of a sodium pentobarbital ( 50mg / kg of body weight ) . the insertion areas on the hind limb of each animal were shaved , and the area over the femur was aseptically prepared with 70% ethanol . a small linear skin incision ( about 12 mm ) was made and the pins were inserted through the incision into the femoral bones ( one pin per bone ) , followed by bacterial contamination with 100 l of an identifiable s. aureus strain on the insertion area , ie , the skin - pin interface . ultraviolet a light was emitted using a black light source set 30 cm above the rats for 30 minutes . in our previous in vitro study , the tio2-coated material significantly suppressed the viability of bacteria after 30 minutes of ultraviolet illumination,21 so the illumination time was set to 30 minutes . the intensity of the light was 2.0 mw / cm at a peak wavelength of 352 nm . the pin sites were examined daily and the dressings were carefully changed to avoid cross - contamination of the pin sites . additional treatment , including incision closure , blood coagulation , or antibiotic dressing was not needed . on day 14 , visible clinical findings at each pin site were classified according to pin site infection criteria as no infection , inflammation or serous drainage without frank purulence , or frank purulence.26,27 clinical determination of infection was defined as all three observers being in agreement . after evaluation by eye , the animals were sacrificed and the bone and soft tissue around the pin were retrieved . we used an automated microbiology system ( bd phoenix system , nippon becton dickinson co , tokyo , japan ) to identify the bacteria isolated ( panel type pmic / id-68 ) . the tissues obtained were fixed for one day in a 10% formalin solution buffered at ph 7.2 , dehydrated through a series of graded alcohol solutions , and embedded in paraffin . immediately after removal of the pins , 4 m thick longitudinal sections in the sagittal plane were cut using a microtome ( hm325 , thermo scientific mircom , walldorf , germany ) with a 40 degree stainless steel knife , taking care not to damage the bone - implant interface . slices in the same bone plane were assessed according to bone infection score as : 1 ) abscess formation ; 2 ) sequestrum formation ; 3 ) enlargement of corticalis ; 4 ) destruction of corticalis ; and 5 ) general appearance.12,28 parameters 1 to 4 were scored as 0 ( absent ) or 1 ( present ) . parameter 5 was scored as 0 ( absent ) , 1 ( mild ) , or 2 ( severe ) . we then computed the rate of contact between bone and screw , on the assumption that the part in which it is possible to confirm the shape of a screw thread in the tissue specimen is the part contacted , based on a modification of the method reported by giavaresi et al and moroni et al.29,30 we did the actual image processing of the hematoxylin and eosin staining sample automatically using an optical microscope ( bx51 - 33 , olympus optical co , tokyo , japan ) connected to public domain image analysis software ( image j ) and computed the data . in addition , the shape of the bone for the part corresponding to a screw thread was evaluated by gram staining . we first chose a part in which it was possible to confirm the two consecutive screw threads from the entrance part of a pin screw and then made a planimetric rate of occupation for bacterial colonies and intraosseous neutrophils in the shape of a screw thread using image j. the clinical and histomorphometric results for the control group ( n = 30 ) were analyzed and compared with those of the tio2-coated pin group ( n = 30 ) . all statistical analyses were performed using the statistical package for social sciences version 20 software ( spss inc , chicago , il ) . the mean and standard deviation of bone infection score , bone - implant contact ratio , and planimetric rate of occupation for bacterial colonies and intraosseous neutrophils were analyzed using the student s t - test . the tio2 surface had a number of global microstructures with diameters of 12 m and were well separated and homogeneously distributed over the sample . all of the animals tolerated surgery well and survived until the end of the experiment . clinical evaluation revealed purulence and/or serous drainage in 23 of the 30 control pins ( 76.7% ) , compared with only 11 of the 30 tio2 pins ( 36.7% , p < 0.01 ) , as shown in table 1 . the automated microbiology system identified the micro - organisms isolated from the purulence or drainage around the infected pin as being the same strain as that from the initial pin inoculation . severe bone resorption and destruction caused by infection were seen in the control pin group , whereas the tio2 pin was directly in contact with bone tissue , and new vessels and osteoblast cells were seen . the mean bone infection score for the tio2 pins was significantly lower than that for the control pins ( 3.1 1.6 points versus 4.9 1.0 points , p < 0.01 ) . the mean bone - implant contact ratio for the untreated stainless steel pin group was 58.2% 8.1% , whereas the tio2 pin group showed a mean ratio of 71.4% 5.4% . the rate of contact in the control pin group was significantly lower than in the tio2-coated pin group ( p < 0.01 ) . in the group of untreated pins , the planimetric rate of occupation for bacterial colonies and intraosseous neutrophils in the shape of a screw thread was 24.7% 10.3% for the control pins and 13.3% 6.4% for the tio2-coated pins , so the rate of occupation for the group of tio2 pins was significantly lower ( p < 0.01 ) . pin site infection with external fixation can easily occur by contiguous spreading , ie , commensal skin bacteria pass subcutaneously along the surface of the device towards the internal tissues . infected pins may lead to pin loosening , need for pin removal , and chronic osteomyelitis , which is considered to be a risk factor for deep infection in the conversion to internal fixation.24,7 in terms of treatment , much money and time are needed , in addition to the distress caused to patients . although various studies have been conducted to develop antibacterial external fixation pins , there remain concerns about the side effects of the agents used and emergence of resistant bacteria . hence we tested the photocatalytic anti - infective activity of tio2 in a rat model of external fixation . tio2 mainly consists of three polymorphs , ie , anatase , rutile , and brookite . using different methods and conditions , single or multiple polymorphs can be formed , with various morphologies . anatase is the most photocatalytic polymorph and generates many free radicals , which can decompose bacteria and other organic compounds via its strong oxidative effects.14,15 therefore , anatase tio2 was used in this study . several investigations have been done to elucidate the mechanism of photokilling of escherichia coli and other bacteria.1618 sunada et al proposed that decomposition of the outer membrane by photocatalytic reaction ultimately leads to cell death.31 maness et al reached the more specific conclusion that photocatalysis promotes peroxidation of phospholipids in the membrane.19 meanwhile , matsunaga et al32 and saito et al20 reported a loss of respiratory activity resulting from oxidization of coenzyme a. however , there are few published studies concerning the use of tio2 for microbial inactivation and focusing on prevention and treatment for pin site infection after external fixation.33s . aureus is one of the most common pathogens in percutaneous implant infections and readily mutates into multidrug - resistant forms that can be extremely difficult to treat , eg , staphylococcus epidermidis , e. coli , and pseudomonas aeruginosa.9,2224 in this study , we introduced a new method for inhibiting s. aureus infection at percutaneous pin sites based on the photocatalytic bactericidal reactions of tio2 in a rat model under ultraviolet illumination . in our animal study , the tio2-coated pin group showed fewer clinical signs of infection and quantitative histomorphometric findings than the untreated pin group . this result indicates that the amount of contaminated bacteria decreased due to the photocatalytic activity of tio2 . tio2 pins also had better compatibility with bone tissue and prevented bacterial migration and deep tissue infection in vivo . these findings suggest that photocatalytic tio2 has the potential to decrease the risk of pin site infection clinically and prevent loosening of the interface between bone and the pin , and that the external fixation pin performs its function effectively . it is known that ultraviolet a has deleterious effects on bacterial cells , depending on the type of bacteria , the dose of light administered , and degree of bacterial sensitivity.34 there was no sign of infection around seven of the 30 untreated pins ( 23.3% ) and 19 of the 30 tio2 pins ( 63.3% ) on clinical evaluation . in our earlier study , ultraviolet a from the same light source had deleterious effects on this type of bacterium.21 therefore , there is no doubt that ultraviolet a killed bacteria in the two groups . however , evidence of significantly fewer signs of infection on clinical and quantitative histomorphometric analysis in the tio2 group compared with the control group suggests the presence of a photocatalytic bactericidal action against s. aureus cells . another problem is the extended period of ultraviolet a illumination required to suppress pin site infection . our method of contamination , which was direct inoculation of a large number of bacteria with no additional treatment given , does not reflect how a percutaneous implant would be infected clinically . photocatalysts are not particularly useful for breaking down large volumes of bacteria , but they are capable of destroying bacteria as they accumulate . bactericidal efficacy would be more evident in a clinical situation where appropriate pin care is provided and bacterial exposure is much lower . in this study , ultraviolet a illumination was carried out as a single course only , but we can expect improvement in photocatalytic sterilization activity with multidirectional or multiple illuminations . we acknowledge that the negative effects of ultraviolet rays on the human body pose potential problems in clinical application . the intensity of the black light used in our animal model was 2.0 mw / cm , which is as low an intensity as that experienced outdoors . although ultraviolet a is less harmful than ultraviolet b or ultraviolet c , research is underway to resolve these problems using materials with photocatalytic actions triggered by visible light , so the need to use ultraviolet light will decrease in the near future.35,36 other groups have developed silver composite - type materials in order to maintain long - lasting bactericidal properties , even in dark conditions.37,38 however , use of silver in biomaterials or medical devices is still controversial.811 in this study , we tested and confirmed the efficacy of photocatalytic anatase tio2 in inhibition of infection associated with external fixation . tio2 can kill bacteria independent of bacterial strain or antibiotic susceptibility without generating drug - resistant species . moreover , it has been confirmed that tio2-coated implants do not prevent osteointegration in a rabbit.29,39 essentially , titanium together with its natural oxide film is known to be bio - inert and to enable bioactivity for osteointegration . for orthopedic surgeons , although such a photocatalytic surface offers new and favorable properties from a hygiene point of view , it is very important that there should be no disadvantages in regard to achieving osteointegration . zhu et al reported that the tio2 gradient improved corrosion and polarization resistance remarkably , as well as biocompatibility.40 therefore , it can be said that a photocatalytic surface is advantageous for biocompatibility and durability . the high bone - implant contact ratio in the tio2 pin group might have resulted from inhibition of the influence of infection due to good bone compatibility , as well as from the prevention of destruction or absorption of bone tissue due to bacterial infection . we used a rat model to investigate the ability of photocatalytic tio2 to inhibit infection at the insertion site of external fixation pins in vivo . infection was inhibited on the tio2-coated pins compared with the untreated stainless steel control pins clinically and histomorphometrically . tio2 has the potential to reduce the pin site infection rates associated with external fixation .
background : pin site infection is the most common and significant complication of external fixation . in this work , the efficacy of pins coated with titanium dioxide ( tio2 ) for inhibition of infection was compared with that of stainless steel control pins in an in vivo study.methods:pins contaminated with an identifiable staphylococcus aureus strain were inserted into femoral bone in a rat model and exposed to ultraviolet a light for 30 minutes . on day 14 , the animals were sacrificed and the bone and soft tissue around the pin were retrieved . the clinical findings and histological findings were evaluated in 60 samples.results:clinical signs of infection were present in 76.7% of untreated pins , but in only 36.7% of tio2-coated pins . the histological bone infection score and planimetric rate of occupation for bacterial colonies and neutrophils in the tio2-coated pin group were lower than those in the control group . the bone - implant contact ratio of the tio2-coated pin group was significantly higher ( 71.4% ) than in the control pin group ( 58.2% ) . the tio2 was successful in decreasing infection both clinically and histomorphometrically.conclusion:the photocatalytic bactericidal effect of tio2 is thought to be useful for inhibiting pin site infection after external fixation .
Introduction Materials and methods Preparation of implant materials Evaluation of bactericidal effect Statistical analyses Results Discussion Conclusion
external fixation has gained wide acceptance in osteotomy , limb lengthening , and treatment of open fractures because it enables easy reduction of bone fragments , even under unfavorable soft tissue conditions , and secondary corrections and modifications can be made.1 despite these positive qualities , there is still a high incidence of pin site infection , which may cause mechanical deterioration of the bone - pin interface and lead to pin loosening and osteomyelitis.26 pin site infection is due to indigenous micro - organisms adhering to the pin surface and subsequently forming aggregates that may lead to formation of a biofilm and eventual spread of infection to the surrounding host tissue . the purpose of the current in vivo study was to evaluate the efficacy of photocatalytic tio2 in inhibition of infection when using percutaneous external fixation pins in a rat model under low energy ultraviolet illumination . after evaluation by eye , the animals were sacrificed and the bone and soft tissue around the pin were retrieved . we first chose a part in which it was possible to confirm the two consecutive screw threads from the entrance part of a pin screw and then made a planimetric rate of occupation for bacterial colonies and intraosseous neutrophils in the shape of a screw thread using image j. the clinical and histomorphometric results for the control group ( n = 30 ) were analyzed and compared with those of the tio2-coated pin group ( n = 30 ) . the mean and standard deviation of bone infection score , bone - implant contact ratio , and planimetric rate of occupation for bacterial colonies and intraosseous neutrophils were analyzed using the student s t - test . on day 14 , visible clinical findings at each pin site were classified according to pin site infection criteria as no infection , inflammation or serous drainage without frank purulence , or frank purulence.26,27 clinical determination of infection was defined as all three observers being in agreement . after evaluation by eye , the animals were sacrificed and the bone and soft tissue around the pin were retrieved . we first chose a part in which it was possible to confirm the two consecutive screw threads from the entrance part of a pin screw and then made a planimetric rate of occupation for bacterial colonies and intraosseous neutrophils in the shape of a screw thread using image j. the clinical and histomorphometric results for the control group ( n = 30 ) were analyzed and compared with those of the tio2-coated pin group ( n = 30 ) . the mean and standard deviation of bone infection score , bone - implant contact ratio , and planimetric rate of occupation for bacterial colonies and intraosseous neutrophils were analyzed using the student s t - test . the mean bone - implant contact ratio for the untreated stainless steel pin group was 58.2% 8.1% , whereas the tio2 pin group showed a mean ratio of 71.4% 5.4% . the rate of contact in the control pin group was significantly lower than in the tio2-coated pin group ( p < 0.01 ) . in the group of untreated pins , the planimetric rate of occupation for bacterial colonies and intraosseous neutrophils in the shape of a screw thread was 24.7% 10.3% for the control pins and 13.3% 6.4% for the tio2-coated pins , so the rate of occupation for the group of tio2 pins was significantly lower ( p < 0.01 ) . several investigations have been done to elucidate the mechanism of photokilling of escherichia coli and other bacteria.1618 sunada et al proposed that decomposition of the outer membrane by photocatalytic reaction ultimately leads to cell death.31 maness et al reached the more specific conclusion that photocatalysis promotes peroxidation of phospholipids in the membrane.19 meanwhile , matsunaga et al32 and saito et al20 reported a loss of respiratory activity resulting from oxidization of coenzyme a. however , there are few published studies concerning the use of tio2 for microbial inactivation and focusing on prevention and treatment for pin site infection after external fixation.33s . aureus is one of the most common pathogens in percutaneous implant infections and readily mutates into multidrug - resistant forms that can be extremely difficult to treat , eg , staphylococcus epidermidis , e. coli , and pseudomonas aeruginosa.9,2224 in this study , we introduced a new method for inhibiting s. aureus infection at percutaneous pin sites based on the photocatalytic bactericidal reactions of tio2 in a rat model under ultraviolet illumination . the high bone - implant contact ratio in the tio2 pin group might have resulted from inhibition of the influence of infection due to good bone compatibility , as well as from the prevention of destruction or absorption of bone tissue due to bacterial infection . infection was inhibited on the tio2-coated pins compared with the untreated stainless steel control pins clinically and histomorphometrically .
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all abstracts at the krs conference and the abstracts presented by korean investigators at the rsna and ecr from 2001 to 2002 were identified , using a search of the conference program books containing the abstracts . in addition , a search of the official websites of the krs ( http://www.radiology.or.kr/event/annual.html ) , the rsna ( http://archive.rsna.org/index.cfm ) , and the ecr ( http://www.myesr.org/cms/website.php?id=/en/congress/ecr_2007/about_the_congress/past_meetings.htm ) was performed on the internet . we only included original research studies . abstracts that illustrated an imaging technique or reported case(s ) of educational value or special interests were excluded . when more than one country was listed on the abstract , we considered that the abstract originated from korea if more than 50% of authors were korean nationals ( with a typical korean name ) . a computer - based search for each abstract was performed to determine whether it was published in a peer - reviewed journal . the publication status was assessed by using the pubmed database ( http://www.ncbi.nlm.nih.gov/pubmed/ ) and the korean medical database ( http://kmbase.medric.or.kr/ ) from january 1 , 2000 to june 30 , 2007 ( the date of completion of the search ) . therefore , the follow - up periods ranged from 55 months ( for the rsna 2002 conference ) to 76 months ( for the ecr 2001 conference ) . the searches began with the first name initial(s ) and the full last name of the first author . if no corresponding publication was found , this search was followed by a search for subsequent authors or keywords from the title of the abstract . a published manuscript was considered as a full publication of a corresponding abstract when it satisfied the following two criteria : 1 ) at least one author on the abstract was listed as an author of the full publication , and 2 ) at least one conclusion from the presented abstract was included in the conclusions of the final publication . six investigators ( an investigator for each of the six meetings ) performed primary data curation . another investigator performed validation of the data from the abstracts and differences were resolved by consensus . the following variables were evaluated : 1 ) the overall publication rate ( the ratio of the number of subsequent publications to the total number of abstracts ) for each of the respective meetings ; 2 ) publication rates according to the classification of presentations ; 3 ) time to publication ; 4 ) journals in which the study was published ; 5 ) consistency between the abstract and the final publication . based on the outcome of our search , overall rates of publication were calculated for each of the respective meetings . these publication rates were compared with each other and with the rates reported for other radiological and other medical disciplines . for evaluation of publication rates according to the classification of presentation , we collected the following information from each abstract : radiological subspecialty , presentation type ( oral or poster ) , sample size ( 20 , 21 - 50 , or > 50 ) , study design ( prospective [ including experimental studies ] or retrospective [ including no available information ] ) , statistical analysis ( present [ authors stated the method of statistical analysis used or reported p values ] or absent ) , and study outcome ( positive [ the studied variables produced beneficial or statistically significant results ] or negative ) . for purposes of analysis , the abstracts were subdivided into the following 10 radiology subspecialties based on generally accepted categorizations often found in the literature as well as on the grouping of abstracts in the final program of each respective meeting . these subspecialties included breast , cardiac , chest , gastrointestinal , genitourinary , musculoskeletal , neuroradiology / head and neck , pediatric , vascular / interventional , and others ( including nuclear medicine , physics , basic science , and radiation oncology ) . the time to publication was defined in months for the duration between the month of publication and the month of abstract presentation . when a journal was published every two months if a publication had occurred in the same month as the meeting or any time before the abstract presentation , the time to publication was recorded as zero months . the journal impact factor of medline - indexed journals was retrieved from the science citation report on the thompson scientific isi web of knowledge server ( http://scientific.thompson.com/webofknowledge/ ) . this impact factor is calculated by dividing the number of citations in the current year to the number of articles published in the past two years . the mean impact factor for each journal between 2002 and 2006 we also evaluated consistency between the abstract and the final publication , including differences in the title , the number of authors , the position of the first author in the abstract , study objective / hypothesis , study design , sample size , statistical analysis , study results , and conclusions . when a full publication was confirmed , two investigators independently compared the content of the abstracts of the presentations and the summaries of the published studies ; discrepancies were resolved by discussion with another investigator . statistical analyses for overall publication rates and predictive factors for publication were determined using tests . a p value < 0.05 was considered as statistically significant . all statistical analyses were performed by using spss statistical software ( version 10.0 ; spss , chicago , il ) . all abstracts at the krs conference and the abstracts presented by korean investigators at the rsna and ecr from 2001 to 2002 were identified , using a search of the conference program books containing the abstracts . in addition , a search of the official websites of the krs ( http://www.radiology.or.kr/event/annual.html ) , the rsna ( http://archive.rsna.org/index.cfm ) , and the ecr ( http://www.myesr.org/cms/website.php?id=/en/congress/ecr_2007/about_the_congress/past_meetings.htm ) was performed on the internet . we only included original research studies . abstracts that illustrated an imaging technique or reported case(s ) of educational value or special interests were excluded . when more than one country was listed on the abstract , we considered that the abstract originated from korea if more than 50% of authors were korean nationals ( with a typical korean name ) . a computer - based search for each abstract was performed to determine whether it was published in a peer - reviewed journal . the publication status was assessed by using the pubmed database ( http://www.ncbi.nlm.nih.gov/pubmed/ ) and the korean medical database ( http://kmbase.medric.or.kr/ ) from january 1 , 2000 to june 30 , 2007 ( the date of completion of the search ) . therefore , the follow - up periods ranged from 55 months ( for the rsna 2002 conference ) to 76 months ( for the ecr 2001 conference ) . the searches began with the first name initial(s ) and the full last name of the first author . if no corresponding publication was found , this search was followed by a search for subsequent authors or keywords from the title of the abstract . a published manuscript was considered as a full publication of a corresponding abstract when it satisfied the following two criteria : 1 ) at least one author on the abstract was listed as an author of the full publication , and 2 ) at least one conclusion from the presented abstract was included in the conclusions of the final publication . six investigators ( an investigator for each of the six meetings ) performed primary data curation . another investigator performed validation of the data from the abstracts and differences were resolved by consensus . the following variables were evaluated : 1 ) the overall publication rate ( the ratio of the number of subsequent publications to the total number of abstracts ) for each of the respective meetings ; 2 ) publication rates according to the classification of presentations ; 3 ) time to publication ; 4 ) journals in which the study was published ; 5 ) consistency between the abstract and the final publication . based on the outcome of our search , overall rates of publication were calculated for each of the respective meetings . these publication rates were compared with each other and with the rates reported for other radiological and other medical disciplines . for evaluation of publication rates according to the classification of presentation , we collected the following information from each abstract : radiological subspecialty , presentation type ( oral or poster ) , sample size ( 20 , 21 - 50 , or > 50 ) , study design ( prospective [ including experimental studies ] or retrospective [ including no available information ] ) , statistical analysis ( present [ authors stated the method of statistical analysis used or reported p values ] or absent ) , and study outcome ( positive [ the studied variables produced beneficial or statistically significant results ] or negative ) . for purposes of analysis , the abstracts were subdivided into the following 10 radiology subspecialties based on generally accepted categorizations often found in the literature as well as on the grouping of abstracts in the final program of each respective meeting . these subspecialties included breast , cardiac , chest , gastrointestinal , genitourinary , musculoskeletal , neuroradiology / head and neck , pediatric , vascular / interventional , and others ( including nuclear medicine , physics , basic science , and radiation oncology ) . the time to publication was defined in months for the duration between the month of publication and the month of abstract presentation . when a journal was published every two months , we defined the time of publication as occurring halfway between the two months . if a publication had occurred in the same month as the meeting or any time before the abstract presentation , the time to publication was recorded as zero months . the journal impact factor of medline - indexed journals was retrieved from the science citation report on the thompson scientific isi web of knowledge server ( http://scientific.thompson.com/webofknowledge/ ) . this impact factor is calculated by dividing the number of citations in the current year to the number of articles published in the past two years . the mean impact factor for each journal between 2002 and 2006 we also evaluated consistency between the abstract and the final publication , including differences in the title , the number of authors , the position of the first author in the abstract , study objective / hypothesis , study design , sample size , statistical analysis , study results , and conclusions . when a full publication was confirmed , two investigators independently compared the content of the abstracts of the presentations and the summaries of the published studies ; discrepancies were resolved by discussion with another investigator . statistical analyses for overall publication rates and predictive factors for publication were determined using tests . a p value < 0.05 was considered as statistically significant . all statistical analyses were performed by using spss statistical software ( version 10.0 ; spss , chicago , il ) . a total of 1,230 abstracts ( 732 oral and 498 poster presentations ) presented at the six meetings were identified . one hundred thirty - three posters presented at the krs meeting were excluded from the study ( 108 educational exhibits , 17 technical exhibits , and 8 case reports ) . of these abstracts , 301 were subsequently published as full - text articles by june 30 2007 , as determined by the computerized search , giving an overall publication rate of 27% ( table 1 ) . the publication rate for studies presented at the ecr conference ( 50.5% ) was significantly higher than the rate for the rsna conference ( 35.4% ; p = 0.029 ) and krs conference ( 23.6% ; p < 0.001 ) . in addition , there is a statistically significant difference between the publication rate for the rsna and krs conferences ( p = 0.002 ) . however , there was no significant difference in publication rates between abstracts for oral ( 28.6% ) and poster presentations ( 25.2% ; p = 0.272 ) . " vascular / interventional radiology " had the highest publication rate ( 33.1% ) , whereas " musculoskeletal radiology " had the lowest publication rate ( 17.1% ) . studies in the field of " musculoskeletal radiology " were published significantly less often than the mean ( p = 0.03 ) . studies described in abstracts with a prospective design had higher publication rates than studies with a retrospective design ( 33.4% vs 25.1% , respectively , p = 0.007 ) . there was also a significant difference in publication rates based on statistical analysis and study outcome ( p < 0.0001 and p = 0.0001 , respectively ) . however , the sample size did not significantly influence the publication rate ( p = 0.136 ) . for published articles , the time course between abstract presentation and journal publication for each meeting is shown in table 4 . interestingly , 41 ( 3.7% of all presented abstracts and 14% of all published papers ) were published before the date of abstract presentation at a scientific meeting . the overall mean time to publication was 15.8 months ( standard deviation , 13.8 months ) , when studies published before the meeting were given a time to publication value of zero . most ( 81.7% ) of the studies presented in the abstracts were published within two years of presentation . only 10% of the studies presented in the abstracts were published more than three years after presentation . seventy - six studies ( 38.0% ) were published in journal of the korean radiological society ( jkrs ) . in contrast , 101 studies from the rsna and ecr meetings were published in a total of 27 journals including , in decreasing order of frequency , american journal of roentgenology ( n = 23 , 22.8% ) , radiology ( n = 21 , 20.8% ) , jkrs ( n = 15 , 14.9% ) , and korean journal of radiology ( n = 9 , 8.9% ) . we sought to identify any inconsistencies between the abstracts and the subsequent full - text publications . study information was consistent for the position of the first author , study objective / hypothesis , design , statistical analysis , results , and conclusions ( table 6 ) . in contrast , investigators altered presentations by changing the title for 41% of the studies , adding or deleting authors for 79% , of the studies and changing the sample size for 42% of the studies . a total of 1,230 abstracts ( 732 oral and 498 poster presentations ) presented at the six meetings were identified . one hundred thirty - three posters presented at the krs meeting were excluded from the study ( 108 educational exhibits , 17 technical exhibits , and 8 case reports ) . of these abstracts , 301 were subsequently published as full - text articles by june 30 2007 , as determined by the computerized search , giving an overall publication rate of 27% ( table 1 ) . the publication rate for studies presented at the ecr conference ( 50.5% ) was significantly higher than the rate for the rsna conference ( 35.4% ; p = 0.029 ) and krs conference ( 23.6% ; p < 0.001 ) . in addition , there is a statistically significant difference between the publication rate for the rsna and krs conferences ( p = 0.002 ) . however , there was no significant difference in publication rates between abstracts for oral ( 28.6% ) and poster presentations ( 25.2% ; p = 0.272 ) . table 2 shows publication rates according to subspecialty . " vascular / interventional radiology " had the highest publication rate ( 33.1% ) , whereas " musculoskeletal radiology " had the lowest publication rate ( 17.1% ) . studies in the field of " musculoskeletal radiology " were published significantly less often than the mean ( p = 0.03 ) . studies described in abstracts with a prospective design had higher publication rates than studies with a retrospective design ( 33.4% vs 25.1% , respectively , p = 0.007 ) . there was also a significant difference in publication rates based on statistical analysis and study outcome ( p < 0.0001 and p = 0.0001 , respectively ) . however , the sample size did not significantly influence the publication rate ( p = 0.136 ) . for published articles , the time course between abstract presentation and journal publication for each meeting is shown in table 4 . interestingly , 41 ( 3.7% of all presented abstracts and 14% of all published papers ) were published before the date of abstract presentation at a scientific meeting . the overall mean time to publication was 15.8 months ( standard deviation , 13.8 months ) , when studies published before the meeting were given a time to publication value of zero . most ( 81.7% ) of the studies presented in the abstracts were published within two years of presentation . only 10% of the studies presented in the abstracts were published more than three years after presentation . seventy - six studies ( 38.0% ) were published in journal of the korean radiological society ( jkrs ) . in contrast , 101 studies from the rsna and ecr meetings were published in a total of 27 journals including , in decreasing order of frequency , american journal of roentgenology ( n = 23 , 22.8% ) , radiology ( n = 21 , 20.8% ) , jkrs ( n = 15 , 14.9% ) , and korean journal of radiology ( n = 9 , 8.9% ) . we sought to identify any inconsistencies between the abstracts and the subsequent full - text publications . study information was consistent for the position of the first author , study objective / hypothesis , design , statistical analysis , results , and conclusions ( table 6 ) . in contrast , investigators altered presentations by changing the title for 41% of the studies , adding or deleting authors for 79% , of the studies and changing the sample size for 42% of the studies . the annual krs meeting is a national radiology meeting that provides an important forum for the dissemination of current research findings in korea . the rsna and ecr meetings are two internationally prominent meetings in the field of radiology . recently , many abstracts have been presented by korean investigators at both scientific meetings , and the number of presentations from korea has increased gradually . our survey of 1,097 abstracts from three radiology meetings revealed that 27% of abstracts presented at these meeting in 2001 and 2002 were subsequently published in peer - reviewed journals . in comparing the publication rates between the three meetings , the publication rates from two international meetings ( rsna and ecr ) were significantly higher than that from the krs meeting , perhaps because fewer but better quality studies were presented at international meetings . moreover , the publication rate for studies presented at the ecr conference was significantly higher than that for the rsna conference , although the exact reason for the difference is unclear . a few studies that were performed in the field of radiology revealed the publication rate for presentations at international meetings to be between 33% and 47% ( table 7 ) ( 5 - 8 , 11 ) , although all studies included only oral presentations . in our series , the publication rate in medline - indexed journals of abstracts presented by korean investigators at the rsna and ecr meetings was 33% ( 83/249 ) . although this rate is lower than the rates for the ecr 2000 and 2001 conferences ( 47% and 45% , respectively ) ( 7 , 8) , it is comparable to those observed at other usa - based national and international radiological meetings ( 33 - 40% ) . 10 ) reported that 25% of studies described in abstracts that were presented at the 1992 - 1996 krs meetings were subsequently published in jkrs . the publication rate in jkrs as determined in the present study from the krs meeting was 9% ( 76/848 ) . a possible explanation for this difference may include that many researchers published research in the medline - indexed journals , or korean researchers have less interest in publishing studies in jkrs . in contrast , the publication rate in medline - indexed journals for studies described in abstracts presented at the 2001 and 2002 krs meetings was 12% ( 102/848 ) . this publication rate is comparable to rates reported for a meeting in france ( 12 ) and turkey ( 13 ) , but markedly lower than the rates rate for australian and new zealand meetings ( 4 ) ( table 7 ) . failure to publish a study originally presented in an abstract is problematic for a variety of reasons . first , although some journals publish the abstracts of society meetings , in general the abstracts of meetings may be available only to meeting attendees . second , the abstracts presented at scientific meetings usually have not undergone rigorous peer review , and lack the necessary detail for readers to critically appraise a given study for its validity . third , nonpublication of negative results may lead to publication bias ( 14 , 15 ) . in the field of radiology last , failure to publish may be unethical and wasteful , leading to the potential replication of almost identical studies . in this study , we documented that only a relatively small percentage of studies presented at scientific meetings were finalized in the form of a published paper . although most investigators have identified " lack of time " as a reason for not publishing findings presented in abstracts , actual reasons are multifactorial . other reasons for failure of publication include low priority , ongoing preparation of results , lack of funds or other resources , lack of faith in the quality of research , rejection of a submitted paper , problematic relationships with co - authors , negative results , and the existence of other published reports with identical results ( 1 , 16 - 19 ) . in this study , we included six study variables ( presentation type , radiological subspecialty , sample size , study design , statistical analysis , and study outcome ) to identify factors predictive of publication . our findings suggest that a prospectively designed study , use of statistical testing , and positive outcome were significant predictors of publication . these results are similar to those found in some previous analyses ( 1 , 6 , 15 , 20 - 23 ) . in addition , studies in oral presentations are more likely to be published in peer - reviewed journals than studies in poster presentations , although this difference did not reach significance . the influence of an oral presentation on publication has been well documented in other specialties ( 1 , 19 , 20 , 24 - 26 ) . one might hypothesize that high - quality research abstracts would be selected for oral presentation by the program committee and authors of studies not selected for oral presentation are given the option of presenting a poster . the topic of the presentation may be related to the publication rate , but in our study the publication rate according to the 10 different subspecialties did not markedly differ . only the musculoskeletal radiology subspecialty had a markedly lower publication rate than the other subspecialties . although we did not specifically evaluate all variables that may influence the publication rate , a broad spectrum of predictor variables influencing publication among presentations at meetings has been identified . investigators of previous studies have reported that country of origin ( 7 - 8 ) , basic research ( 27 ) , originality of a study ( 19 ) , affiliation with a university ( 28 ) , a randomized study ( 28 ) , external financial support ( 22 , 29 ) , and international collaboration ( 9 ) appeared to influence the publication rate . we found a substantial change in sample size and authorship from the time of initial presentation to final publication . the sample size of a study changed in 43% of the published studies originally presented as abstracts ; this finding is similar to reported values of 39 - 46% found in other surveys ( 7 , 30 ) . in addition , in our study , 79% of published studies originally presented as abstracts had at least one different author than the original presented report , and this rate is higher than the rates observed at other meetings ( 59 - 73% ) ( 30 , 31 ) . however , the percentage of studies where the first author of the presentation had changed or disappeared in the derived article was lower in our study ( 18.3% ) than in surveys of other analyzed medical specialty meetings ( 22 - 36% ) ( 7 , 31 , 32 ) . an increase in sample size may be related to the continued enrollment of subjects after presentation of preliminary results , while a decrease in the sample size suggests possible tampering with the data to increase the quality of the paper . the possible reasons for changes in authorship are more complex and may include further analysis by another investigator , removal of authors if contribution is below standard required for authorship , limiting the number of authors in some meetings or journals , and so called " honorary " authorship . the fact that the first author of the presentation disappeared in 4% of publications is of interest . a possible reason for this result is that this author was chosen only for the presentation . first , we determined publication status based on a particular medical database ( pubmed and korean medical databases ) search ; therefore , it is possible that we missed some studies that were published in journals not indexed in the database . these errors may occur due to the misspelling of the name of an author or merely because of a faulty search . although we tried to minimize these errors by searching with two independent investigators , major changes of the author names or the key words from the title stand as possible causes of error during the search . second , we did not investigate the possibility of duplicate presentations ( presentations of the same study at multiple meetings ) or duplicate publications ( multiple publications resulting from a single abstract ) as one of our aims was to compare the publication rate in our study with that of other studies . only a few previous studies have addressed the situation of duplicate presentation or publication ( 19 , 25 ) . finally , in this study , no attempt was made to contact the authors of presentations ; further research needs to be conducted to determine the precise reasons why presentations were not published . in conclusion , the publication rate is significantly lower for the krs ( 23.6% ) as compared to the rsna ( 35.4% ) and ecr ( 50.5% ) meetings . prospective design , use of statistical testing , and a positive study outcome have a statistically significant effect on the publication rate .
objectiveto determine the publication rate of abstracts presented by korean investigators at national and international radiological meetings , and to identify predictive factors of publication.materials and methodsabstracts presented at the annual meetings of the korean radiological society ( krs ) , and abstracts presented by korean investigators at the annual meetings of the radiological society of north america ( rsna ) and european congress of radiology ( ecr ) from 2001 to 2002 were searched for subsequent publication , using pubmed and the korean medical database . the following variables were evaluated . 1 ) the overall publication rate ; 2 ) the publication rates according to the radiological subspecialty , presentation type ( oral or poster ) , sample size ( 20 , 21 - 50 , or > 50 ) , study design ( prospective or retrospective ) , statistical analysis ( present or absent ) , and study outcome ( positive or negative ) ; 3 ) the time to publication ; 4 ) the journal where the study was published ; 5 ) consistency between the abstract and the final publication.resultsof 1,097 abstracts , 301 ( 27.4% ) were subsequently published , at an average of 15.8 13.8 months after presentation in 48 journals . the publication rates for studies presented at the rsna ( 35.4% ) and ecr ( 50.5% ) conferences were significantly higher than that for the krs conference ( 23.6% , p < 0.05 ) . vascular / interventional radiology studies had the highest publication rate ( 33.1% ) , whereas musculoskeletal radiology studies had the lowest publication rate ( 17.1% ) . other factors associated with subsequent publication were prospective design , use of statistical testing , and a positive study outcome.conclusionthe publication rate is significantly lower for the krs ( 23.6% ) meeting abstracts as compared to those of the rsna ( 35.4% ) and ecr ( 50.5% ) . prospective design , use of statistical testing , and positive study outcome have a statistically significant effect on the publication rate .
MATERIALS AND METHODS Data Collection Data Analysis RESULTS Publication Rate Predictive Factors for Publication DISCUSSION
all abstracts at the krs conference and the abstracts presented by korean investigators at the rsna and ecr from 2001 to 2002 were identified , using a search of the conference program books containing the abstracts . the following variables were evaluated : 1 ) the overall publication rate ( the ratio of the number of subsequent publications to the total number of abstracts ) for each of the respective meetings ; 2 ) publication rates according to the classification of presentations ; 3 ) time to publication ; 4 ) journals in which the study was published ; 5 ) consistency between the abstract and the final publication . for evaluation of publication rates according to the classification of presentation , we collected the following information from each abstract : radiological subspecialty , presentation type ( oral or poster ) , sample size ( 20 , 21 - 50 , or > 50 ) , study design ( prospective [ including experimental studies ] or retrospective [ including no available information ] ) , statistical analysis ( present [ authors stated the method of statistical analysis used or reported p values ] or absent ) , and study outcome ( positive [ the studied variables produced beneficial or statistically significant results ] or negative ) . the mean impact factor for each journal between 2002 and 2006 we also evaluated consistency between the abstract and the final publication , including differences in the title , the number of authors , the position of the first author in the abstract , study objective / hypothesis , study design , sample size , statistical analysis , study results , and conclusions . all abstracts at the krs conference and the abstracts presented by korean investigators at the rsna and ecr from 2001 to 2002 were identified , using a search of the conference program books containing the abstracts . in addition , a search of the official websites of the krs ( http://www.radiology.or.kr/event/annual.html ) , the rsna ( http://archive.rsna.org/index.cfm ) , and the ecr ( http://www.myesr.org/cms/website.php?id=/en/congress/ecr_2007/about_the_congress/past_meetings.htm ) was performed on the internet . the following variables were evaluated : 1 ) the overall publication rate ( the ratio of the number of subsequent publications to the total number of abstracts ) for each of the respective meetings ; 2 ) publication rates according to the classification of presentations ; 3 ) time to publication ; 4 ) journals in which the study was published ; 5 ) consistency between the abstract and the final publication . for evaluation of publication rates according to the classification of presentation , we collected the following information from each abstract : radiological subspecialty , presentation type ( oral or poster ) , sample size ( 20 , 21 - 50 , or > 50 ) , study design ( prospective [ including experimental studies ] or retrospective [ including no available information ] ) , statistical analysis ( present [ authors stated the method of statistical analysis used or reported p values ] or absent ) , and study outcome ( positive [ the studied variables produced beneficial or statistically significant results ] or negative ) . the mean impact factor for each journal between 2002 and 2006 we also evaluated consistency between the abstract and the final publication , including differences in the title , the number of authors , the position of the first author in the abstract , study objective / hypothesis , study design , sample size , statistical analysis , study results , and conclusions . the publication rate for studies presented at the ecr conference ( 50.5% ) was significantly higher than the rate for the rsna conference ( 35.4% ; p = 0.029 ) and krs conference ( 23.6% ; p < 0.001 ) . vascular / interventional radiology " had the highest publication rate ( 33.1% ) , whereas " musculoskeletal radiology " had the lowest publication rate ( 17.1% ) . the publication rate for studies presented at the ecr conference ( 50.5% ) was significantly higher than the rate for the rsna conference ( 35.4% ; p = 0.029 ) and krs conference ( 23.6% ; p < 0.001 ) . vascular / interventional radiology " had the highest publication rate ( 33.1% ) , whereas " musculoskeletal radiology " had the lowest publication rate ( 17.1% ) . in comparing the publication rates between the three meetings , the publication rates from two international meetings ( rsna and ecr ) were significantly higher than that from the krs meeting , perhaps because fewer but better quality studies were presented at international meetings . moreover , the publication rate for studies presented at the ecr conference was significantly higher than that for the rsna conference , although the exact reason for the difference is unclear . in our series , the publication rate in medline - indexed journals of abstracts presented by korean investigators at the rsna and ecr meetings was 33% ( 83/249 ) . in this study , we included six study variables ( presentation type , radiological subspecialty , sample size , study design , statistical analysis , and study outcome ) to identify factors predictive of publication . in conclusion , the publication rate is significantly lower for the krs ( 23.6% ) as compared to the rsna ( 35.4% ) and ecr ( 50.5% ) meetings . prospective design , use of statistical testing , and a positive study outcome have a statistically significant effect on the publication rate .
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basic helix - loop - helix ( bhlh ) transcription factors belong to a large family of genes present in the shared ancestor of plants , animals , and fungi , and this family has undergone an expansion in the land plant lineage . often referred to as helix - loop - helix ( hlh ) proteins , a loosely defined basic domain is involved in dna binding and present in the great majority of characterized proteins in this family ; thus the term bhlh factors is used henceforth . bhlh transcription factors have been implicated in numerous biological processes in plants including responses to light , cold , and hormones , epidermal cell fate determination , developmental patterning in roots and flowers , and anthocyanin biosynthesis [ 314 ] . in many cases , the bhlh family is critically important for correct developmental and environmental responses , as demonstrated by a large number of mutants in arabidopsis with severe phenotypes as a result of a lesion in a bhlh - encoding gene . development and dehiscence of the seed and seed pod ( silique ) [ 13 , 15 , 16 ] and responses to light quality and photoperiod [ 9 , 1721 ] are particularly known to be under the control of bhlh factors , and these phenomena are important to soybean agronomic performance . characterization of the bhlh - encoding gene family can therefore be a useful step in the detailed functional characterization of the soybean genome . the bhlh transcription factors have been extensively characterized at the sequence and structural level . in animals , the best - known and most thoroughly characterized bhlhs are well - known regulators and proto - oncogenes such as c - myc , max , and e47 , where in many cases structural data on the proteins and their interaction with dna molecules is available . many animal bhlhs show a binding preference for the so - called e - box motif ( canntg ) and the residues within the protein that are required for sequence specific recognition are well defined ( reviewed in [ 2 , 22 , 23 ] ) . a number of plant bhlh proteins have been demonstrated to show a particular preference for binding the g - box ( cacgtg ) sequence ( a subset of e - box ) [ 3 , 19 , 2427 ] . homo- and heterodimer formation are also ubiquitous and required for dna binding within the bhlh family , a property that increases the combinatorial possibilities for regulation of transcription . the -helices in the bhlh domain and the other motifs outside the conserved bhlh domain are required for protein - protein interactions and function [ 2832 ] . classification of bhlh proteins is usually based on sequence homology within the conserved bhlh domain . in recent years , a number of proteins have been identified genetically that represent novel and atypical bhlh proteins that were not previously classified by homology - based approaches , in some cases because a basic domain was lacking [ 31 , 3336 ] . an intriguing feature of the bhlh family is that the proteins can often be very divergent outside of the highly conserved bhlh domain and contain a range of other motifs , not all of which have known functions [ 1 , 37 ] . for this reason , a sequence - homology - based search approach using the entire gene sequence ( such as blast of the canonical bhlhs from arabidopsis ) may not be the most appropriate tool to identify all the bhlh - encoding genes in a genome such as soybean . an alternative approach is to use data from the conserved hlh motif across the kingdoms of life to develop a hidden markov model ( hmm ) that allows the detection of the bhlh domain across highly divergent sequences without the need for extensive sequence identity . this type of approach has been successfully deployed for identifying conserved motifs in distantly related proteins and allows more sensitive and accurate discovery of a specific motif like the bhlh domain . the pfam project ( http://pfam.sanger.ac.uk/ ) uses a curated alignment approach to provide constantly updated hmms for most characterized protein families , including the hlhs . hmms for the subsidiary motifs of the different bhlh families can be generated using protein alignments . several recent genome - wide studies have examined and classified the bhlh protein family in arabidopsis thaliana , rice , poplar , and other plants with whole - genome sequences , but these studies have not examined the conservation and diversification of this family in the soybean [ 1 , 33 , 37 , 3942 ] . the bhlh transcription factor family is the second largest family of transcription factors in plants ( behind the myb family ) , and the complete whole - genome sequence of soybean revealed a number of genes predicted to encode bhlh proteins [ 4345 ] . a study of this family of genes in soybean and classification of the bhlhs into subfamilies orthologous with those present in other species is useful in order to provide a list of candidate genes that are likely to be upstream regulators of a number of processes . such lists of candidate genes enable association mapping approaches to proceed with knowledge of potential functions for regulatory genes likely involved in conferring seed and agronomic traits . they are also very helpful as a means to rapidly identify candidate genes within positional genomic intervals defined by conventional genetic fine mapping in mutant studies or as quantitative trait loci ( qtl ) in recombinant inbred populations of soybean . soybean , arabidopsis , and rice gene models were originally identified from annotation versions contained in the phytozome v7.0 package . the most recent version of the soybean assembly ( assembly v.2.0 and gene models for phytozome v.10 ) was compared with our data ; however since stringent new rules for inclusion of genes in the newer assembly and annotation resulted in the loss of 62 conserved bhlh genes including several with confirmed expression data , the legacy annotation and assembly were retained for this analysis ( http://www.phytozome.net/ ) [ 44 , 47 , 48 ] . to identify bhlh transcription factors in the soybean genome , a hidden markov model search ( hmmsearch v. 3.0 ; http://hmmer.janelia.org/ ) was applied to the predicted open reading frames ( orfs ) of soybean ( genome and assembly version gmax109 ; ) using the pfam hlh hidden markov model ( pf00010 ; http://pfam.sanger.ac.uk ) . no e - value cutoff was initially applied in order to maximize detection of poorly annotated and orphan bhlh sequences . a total of 329 hits to unique predicted proteins containing putative hlh domains were initially identified . these included 31 orfs not annotated as bhlhs by the soybean genome annotation and a small group of hlh proteins that lack the basic domain ( see section 3 ) . data on expression of soybean bhlh genes was obtained from http://www.soybase.org/ and is described in . sequences were manually curated to identify mispredictions of splice sites , which often led to omission of part of the bhlh domain in the genome annotation , reducing the hidden markov model score . sequences that included in - frame stop codons in the williams-82 genomic sequence or were missing part of the hlh domain were removed , and these soybean gene models are listed in additional file 2 ( see additional file 2 in supplementary material available online at http://dx.doi.org/10.1155/2015/603182 ) . we then examined the revised list for sequences that were atypical or were unlikely to be true bhlhs . several studies have used a stringent cutoff restricting the number of mismatches to the canonical bhlh motif found in mammals [ 22 , 39 , 40 , 50 ] . other studies have used a less stringent cutoff to identify additional , atypical bhlhs in characterized genomes . empirically , it was observed in the soybean bhlh domains that such a cutoff of 6 mismatches to the core 11 residues or 11 mismatches to the larger 19 defined residues would eliminate the soybean homologs of functionally characterized , bhlh - related proteins , and this also corresponded to the maximum number of mismatches observed in the soybean bhlh domains . therefore , no cutoffs were used , other than those previously described ( i.e. , the hmm search had to hit the sequence ( at any e - value ) and no sequences with incomplete bhlh domains or that contained stop codons were included ) . alignments were performed using mafft v.6.811b with the following parameters : alignments were visualized and edited using geneious pro v.5.4.6 for the macintosh ( biomatters ltd . , auckland , new zealand ) . the construction of the bootstrapped maximum likelihood phylogenetic tree was performed using the hpc - mpi version of raxml version 7.3.0 with geneious used to manage , curate , and reformat the mafft alignment files . the protcat option was used to select the appropriate model , and appropriate gamma model parameters were automatically selected by the software as was a maximum likelihood estimate of 25 per - site rate categories . 1052 bootstrap trees were generated , and the highest - scoring tree was visualized using the treeexplorer utility of mega 5.0 and used together with bootstrap confidence values ( as a percentage of trees ) to create the figures . assignment of subsidiary motifs in the bhlh or hlh proteins was performed using the alignment of motifs provided in supplemental material by pires and dolan . hidden markov models were generated from script - reformatted versions of these alignments using hmmbuild 2.3.2 ( the 3.0 version of hmmbuild lacks import filters for alignments in formats other than stockholm or selex ) . the presence of motifs was detected by running the full - length predicted proteins against each model using hmmsearch as described above , using a bash shell script for automation . for the apb domain , the sequences in the alignment described by khanna et al . were realigned ( using mafft --auto --reorder --clustalout ) and models generated and used for searching as described for the other motifs . using meme ( http://meme.sdsc.edu ) we searched for up to 10 new sites between 2 and 300 residues wide . using discriminative motif discovery a file was supplied containing the bhlh motif plus the sequences used to create the hidden markov models from the known bhlh secondary motifs . as above , the alignment supplied by meme for the new motif was used to create a hidden markov model , and this model was used to search the soybean bhlhs to determine which of them contained the motif . as before no e - value cutoff was applied , however the family x sequences all showed e < 10 while the two family ix sequences gmbhlh262 and 261 showed e - values of 0.0025 and 0.003 , respectively . in total , 319 gene models were identified as encoding bhlh transcription factors in soybean ( see section 2 ) . each soybean bhlh sequence in the alignment was assigned a number , as is the convention in other species . a table showing the correspondence of the gmbhlh numbers to the soybean gene models from glyma version 1.1 as well as version 2.0 and other bhlh classification information in tabular form can be found in additional file 1 . the bhlh domain consists of an n - terminal basic region of approximately 13 amino acids , followed by two alpha helices ( 14 - 15 residues in length ) separated by a loop that ranges from 5 to 14 amino acids ( figure 1 , additional file 3 ) . ( position numbers in figure 1 and additional file 3 follow the convention of , with the exception of the numbering of the second hlh domain which is numbered consistent with our soybean alignment . ) this pattern is conserved in multiple plant species as well as soybean [ 50 , 51 ] . within the two -helices , several hydrophobic residues are thought to be required to stabilize the secondary structure of the protein , and these residues are highly conserved in both plant and animal sequences [ 22 , 23 ] . at position 23 , over 99% of soybean bhlhs have a characteristic l residue . at position 55 , positions 45 and 52 are occupied by either i , l , or v , in 98.4% and 94% of soybean bhlh proteins , respectively ( figure 1 ) . additional file 3 contains a full text multiple sequence alignment for all of the soybean bhlh domains . the conservation of key residues within the basic region of the bhlh domain can predict both the potential to bind dna and the preferred recognition sequence . the e residue at position 9 within the basic domain has been shown in protein crystal structures to make contact with the major groove of the dna ( reviewed in ) . this position is conserved in 245 of 319 of the bhlhs identified in soybean ( 77% ) and has previously been shown to be present in 74% of bhlh sequences from other plants . it has been shown that e at position 9 and r at position 12 are required for the recognition of an e - box sequence 244 of the soybean bhlhs have this configuration and can be classed as e - box binding . another pattern has been described that includes h or k at position 5 , e at position 9 , and r at position 13 ( h5-e9-r13 ) ; in animals these bhlhs recognize the e - box subset cacgtg . this sequence is a commonly occurring promoter motif in plant genomes , where it is referred to as the g - box , and a number of plant bhlh proteins have demonstrated g - box binding activity [ 3 , 24 , 26 , 27 ] . 186 ( 58% ) of soybean bhlh proteins have a conserved h / k5-e9-r13 motif and thus could be candidates for binding the g - box sequence . the conservation of these residues that are potentially involved in protein - dna interaction within soybean is an indication that the dna - binding function and also the recognition sequence may be conserved . of the soybean bhlh proteins that lack the e9 residue ( 74 proteins ) only eight have five or more basic residues within the basic region . the number of basic residues has been previously used as a criterion to classify hlh proteins as having the potential to bind dna . since the e9 residue is thought to be required for e - box binding , it is possible that these proteins bind dna at a non - e - box dna sequence , although this has not yet been demonstrated for plant bhlhs . the remaining 66 hlh domains , which contain 4 or fewer basic residues within the basic region , are classified as hlh proteins and are unlikely to bind dna directly . many proteins identified through mutant studies in recent years in plants as atypical or nonbasic bhlhs have few or no basic residues in this region [ 7 , 31 , 34 , 35 , 57 ] . they do not bind dna but in some cases act in concert with the more typical bhlhs to regulate gene expression by negative interference , and similar non - dna binding hlhs exist in animal systems [ 22 , 31 , 58 , 59 ] . since these helix - loop - helixes are related to the bhlhs and are involved in many of the same pathways , they are included in our characterization of soybean bhlhs if they are recognized by the hlh hmm . most plant bhlhs have a conserved intron structure with three introns within the bhlh domain [ 33 , 39 , 40 ] . the intron / exon patterns were examined for soybean bhlh - encoding genes , and it was determined that 31% of soybean bhlhs contain the pattern of three conserved introns ( pattern a , figure 2 ) . 43% of the soybean bhlhs contain only one of these introns ( pattern d ) , and 4% of soybean bhlhs have another subset of these conserved introns ( patterns b , c , and e ) . only 8.7% of soybean bhlhs exhibit a different splicing pattern , which fall into other distinct classes ( figure 2 , patterns f , g , h , or other ) . the proportions of distinct intron patterns in soybean genes are consistent with the intron structure distribution in the arabidopsis and rice bhlh families [ 33 , 39 , 40 ] . as previously observed for the bhlh superfamily , intron distribution tends to be conserved within bhlh subfamilies and lends additional credence to these class distinctions . the bhlh superfamily in plants is composed of between 14 and 32 subfamilies based on phylogenetic analysis of the bhlh region [ 1 , 33 , 37 , 39 , 40 ] . supporting these classifications , it has been found that both the intron patterns , other domains of sequence homology outside the bhlh region , and dna binding potential are often conserved within these subfamilies . a phylogenetic reconstruction of the soybean bhlhs shown in figure 1 , together with at least one arabidopsis bhlh sequence representing each of the major subfamilies , was generated based on the alignment of the bhlh domain ( figure 1 and additional file 3 ) . the alignment used to generate the phylogenetic tree , which contains representative arabidopsis sequences and excludes all but one of any identical soybean sequences , is supplied as additional file 7 . a bootstrapped maximum likelihood tree ( 1,052 bootstraps ) the best scoring tree is displayed in figure 3 using a summary radiation diagram to show branch lengths and provide an overview of the similarities within 24 bhlh subfamilies found in soybean . the full phylogeny including bootstrap support values ( expressed as percentages ) is presented in additional file 4 . a number of intriguing aspects of the soybean bhlh proteins are apparent from this tree . family xiv has one functionally characterized member in arabidopsis , sac51/atbhlh142 , which is involved in spermidine synthase - mediated stem elongation . secondly , family viia / b has been repeatedly shown to be involved in light signalling [ 9 , 1721 ] . interestingly , pif3 ( atbhlh008 ) , which interacts directly with phytochromes and mediates light - responsive gene expression , has a number of highly conserved homologs in soybean family viia / b . the short branch lengths within this family may indicate higher levels of sequence or structural constraint . hfr1 ( atbhlh026 ) , also involved in light signalling and normally placed in family viia / b , has only very distant similarity to any soybean protein and , in our analysis , appears as an outlier of family xv ( figure 3 , additional file 4 ) . we did not observe any atypical bhlh proteins or families in soybean that clearly do not fit into one of the characterized arabidopsis families ; however several ambiguous sequences were observed in the phylogeny ( figure 3 , additional file 4 ) . we were able to assign families to all these sequences using branch length data , additional motif information , and blast searches ; however not all families formed monophyletic groups ( see additional file 4 ) . in addition to the bhlh domain analysis , any of the bhlh subfamilies can also be distinguished by the presence of one or more characteristic motifs outside the bhlh domain [ 1 , 33 , 37 , 55 ] . to identify these motifs in the predicted full - length sequences of the soybean bhlhs , hmms were created from alignments of the motifs from across the plant kingdom that were previously published . the soybean bhlhs and subfamilies that possess these defined motifs are highlighted in additional file 1 . all of the previously described motifs were detected with the exception of motif 28 , which is associated with family xiv , which was also found to be absent from soybean . the hmm created from the alignment of the active phytochrome binding ( apb ) domain described in matched precisely the same protein motifs as those identified using the hmm for motif 14 . a search was conducted using meme ( http://meme.sdsc.edu ) for new motifs , by excluding the known secondary motifs plus the bhlh domain . this sequence is strongly conserved in all the gmbhlh sequences 165184 ( family x ) . in the supplemental material this is named motif 40 , although it follows closely the distribution of motif 20 ; the ngadywap portion of this motif is similar to motif 20 and it likely represents an expanded version of this motif . much weaker similarity to motif 40 is also observed in gmbhlh261 and gmbhlh262 of family xi . this motif is also conserved in several arabidopsis bhlhs in family x. to compare the soybean bhlhs to arabidopsis and o. sativa homologs , blast searches of the predicted arabidopsis and rice proteomes were conducted with the predicted full - length coding sequences of the soybean bhlhs . a total of 141 soybean bhlhs hit arabidopsis proteins with > 50% amino acid sequence identity , and 288 had hits with an e - value of less than 10 ( additional file 5 ) . a total of 151 soybean bhlhs shared > 50% sequence identity with rice , 255 with an e - value of less than 10 . in order to increase confidence that true orthologs were detected , a reciprocal blast search of the soybean genome was performed using full - length arabidopsis and rice bhlh sequences , and the orthologs are presented in additional file 5 . of the arabidopsis bhlhs identified as matching soybean bhlhs , all but one of the arabidopsis sequences are known from previous classification of bhlh proteins in arabidopsis [ 33 , 37 , 40 ] . the one arabidopsis protein not previously classified as a bhlh is at2g40435 , a protein with homology to 5 soybean hlh proteins . the soybean proteins lack a complete basic domain , show a reasonably conserved hlh motif but have some divergence from previously described consensus sequences , and match the hlh hmm with relatively low scores . at2g40435 has strong similarity to these soybean proteins in this predicted hlh region , but hmmer does not find a match to the hlh hmm at all . five rice proteins ( corresponding to 16 soybean genes , all of which were identified as similar to known arabidopsis bhlhs ) were also not in previous classifications of rice bhlh proteins ( additional file 5 ) [ 33 , 39 ] . all but one of these proteins have a hlh domain predicted by the hmm search as used here for soybean ; the fifth has no hmm similarity to the hlh but does have similarity to the hlhmycn conserved domain . because of the recent duplication of the soybean genome , many soybean bhlh proteins have closely related homeologs ( recently duplicated paralogs arising as a result of polyploidy ) which may be as much as 98% identical at the dna sequence level . to determine which bhlhs were recent homeologs , we combined the data on recently duplicated genomic regions of soybean and validated potential homeologs by sequence identity ( tabulated data provided by dr . 135 are present as two copy loci , 21 are present in three copies , 52 are present in 4 copies , and 6 are present in 6 copies . information on the homeologous groups of soybean bhlhs is included in the table in additional file 1 . using a survey of the publicly available transcriptome data for soybean , it was investigated what fraction of the soybean bhlhs were actively expressed at some stage of plant development . it was determined that 281 of 319 ( 88% ) bhlhs showed some evidence of expression at the mrna level ( figure 4 ) . 47% of these are expressed across root and leaf , as well as developing seed tissues , while the remaining genes showed some evidence of tissue specific expression ( one or more tissue types ) . notably , this included 5 soybean bhlh mrnas that are expressed preferentially in nodules and are mostly absent from aerial tissues ( additional file 6 ) . the closest arabidopsis orthologs of these preferentially nodule - expressed bhlhs do not have known biological functions and do not cluster in any particular subfamily . are homologs of target of monopteros 5 and transparent testa 8 , both of which have known roles in arabidopsis embryo development ( additional file 6 ) [ 61 , 62 ] . the whole - genome duplication events in soybean that occurred 59 million years ago and 13 million years ago have clearly had a substantial impact on the size of the bhlh family in soybean when compared to other plant species [ 1 , 37 , 39 , 40 , 44 ] . most of the soybean bhlhs ( 213 ) were present in homeologous copies duplicated two or more times . expression evidence was identified for 281 of the bhlh genes , and 122 bhlhs were expressed in leaf , seed , and root tissue . these single - copy genes are likely the result of deletion of the homeolog after whole - genome duplication , perhaps because of negative selection due to deleterious effects from multigene dosage . speculatively , these genes may be involved in critical developmental or other processes in which these deleterious effects are seriously disadvantageous , and thus selection for loss of the homeologous copy has already taken place . several of the genes with strong evidence for expression and/or conservation were omitted from the current soybean genome annotation , suggesting that this version of the annotation omits a relatively large number of bhlh genes . based on sequence conservation within the basic domain , it can be predicted that the majority of soybean bhlhs ( 77% ) have the potential to bind dna , and 58% of all soybean bhlhs are likely to recognize the core g - box sequence , of which there are over 17,000 represented in the promoters of actively transcribed genes in the soybean genome ( meh , unpublished data ) . there is experimental evidence that different bhlh proteins have a preference for certain nucleotides flanking the core g - box sequence and that residues in the second -helix may also be involved in dna contact , increasing specificity of genomic sequence targets [ 24 , 56 , 63 ] . all but one of the bhlh subfamilies represented in other land plants are present in soybean , and the proportion of bhlhs in each subfamily was largely consistent between arabidopsis and soybean , although soybean contains many more bhlh - encoding genes , implying a broadly similar set of functions for each bhlh family may be conserved . all but one of the conserved motifs identified in bhlh subfamilies were found in soybean bhlh genes . a small number of these motifs are known to be involved interactions with other proteins ( such as the apb and leucine zipper motifs ) [ 32 , 55 ] . using meme a long , highly conserved motif was identified , which we term motif 40 . the strongly conserved ngadywap portion of this motif is similar to motif 20 , with which it shares very similar distribution . we do not believe motif 40 is related to motif 22 because flanking conserved residues of motif 22 are very different to those of motif 40 , and because the distribution of motif 40 is strongly correlated with and predictive of membership in family x. motif 20 also correlates with family x but is not found in all members with strong significance , perhaps due to greater discriminative power and sensitivity for the longer motif . recently the hmm method identified the bhlh domain in the closest soybean homologs of several of these genes including kidari , atbs1 , and pre1 , and these were used in our analysis . in the case of lonesome highway , a clear ortholog was present in soybean ( glyma12g31460 , at a blastp e - value of 10 ) but this gene was not predicted to contain a bhlh domain using our hmm search . in the case of par1 and par2 , by contrast , while par1 and par2 have blast hits in soybean , they are not clearly orthologs . the blastp e values of the best hits are in the range 10 to 10 , which represents strong similarity but is a lower level of confidence than for the orthologs of other genes such as kidari . this may indicate that the putative hlh domains of lonesome highway , par1 , and par2 are divergent to an extent where structural similarity to canonical bhlhs is limited , or it could indicate that the hmm we used is not sensitive to this type of hlh domain , since it did not include representatives of this type of atypical bhlh . however , five soybean predicted hlh genes were identified , highly similar to an arabidopsis gene ( with greater than 50% amino acid sequence identity and blast e - value of less than 2e 33 ) not within the set of classified bhlh proteins . interestingly , while all five soybean proteins are predicted to contain a hlh domain using the hmm approach , the same method does not predict a bhlh domain in the highly similar at2g40435 protein . the amino acid similarity between these proteins is very strong in the predicted hlh region of the soybean proteins , and the hmm score for the hlh prediction in the soybean proteins is not strong . the putative hlh domain is located at the extreme n - terminus of the soybean proteins , which fall into bhlh subfamily iii but are not predicted to bind dna . the intriguingly high level of sequence conservation in these genes across arabidopsis and soybean may point to an important , previously unknown biological function , possibly connected with the bhlh similarity . we have identified a large number of candidate genes from a family with likely regulatory roles in many processes critical for soybean growth and genetic improvement . these results establish a foundation for future functional genomics studies to target bhlh - controlled processes for modification and improvement in soybean .
the complete genome sequence of soybean allows an unprecedented opportunity for the discovery of the genes controlling important traits . in particular , the potential functions of regulatory genes are a priority for analysis . the basic helix - loop - helix ( bhlh ) family of transcription factors is known to be involved in controlling a wide range of systems critical for crop adaptation and quality , including photosynthesis , light signalling , pigment biosynthesis , and seed pod development . using a hidden markov model search algorithm , 319 genes with basic helix - loop - helix transcription factor domains were identified within the soybean genome sequence . these were classified with respect to their predicted dna binding potential , intron / exon structure , and the phylogeny of the bhlh domain . evidence is presented that the vast majority ( 281 ) of these 319 soybean bhlh genes are expressed at the mrna level . of these soybean bhlh genes , 67% were found to exist in two or more homeologous copies . this dataset provides a framework for future studies on bhlh gene function in soybean . the challenge for future research remains to define functions for the bhlh factors encoded in the soybean genome , which may allow greater flexibility for genetic selection of growth and environmental adaptation in this widely grown crop .
1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions
basic helix - loop - helix ( bhlh ) transcription factors belong to a large family of genes present in the shared ancestor of plants , animals , and fungi , and this family has undergone an expansion in the land plant lineage . often referred to as helix - loop - helix ( hlh ) proteins , a loosely defined basic domain is involved in dna binding and present in the great majority of characterized proteins in this family ; thus the term bhlh factors is used henceforth . development and dehiscence of the seed and seed pod ( silique ) [ 13 , 15 , 16 ] and responses to light quality and photoperiod [ 9 , 1721 ] are particularly known to be under the control of bhlh factors , and these phenomena are important to soybean agronomic performance . an alternative approach is to use data from the conserved hlh motif across the kingdoms of life to develop a hidden markov model ( hmm ) that allows the detection of the bhlh domain across highly divergent sequences without the need for extensive sequence identity . the bhlh transcription factor family is the second largest family of transcription factors in plants ( behind the myb family ) , and the complete whole - genome sequence of soybean revealed a number of genes predicted to encode bhlh proteins [ 4345 ] . the most recent version of the soybean assembly ( assembly v.2.0 and gene models for phytozome v.10 ) was compared with our data ; however since stringent new rules for inclusion of genes in the newer assembly and annotation resulted in the loss of 62 conserved bhlh genes including several with confirmed expression data , the legacy annotation and assembly were retained for this analysis ( http://www.phytozome.net/ ) [ 44 , 47 , 48 ] . to identify bhlh transcription factors in the soybean genome , a hidden markov model search ( hmmsearch v. 3.0 ; http://hmmer.janelia.org/ ) was applied to the predicted open reading frames ( orfs ) of soybean ( genome and assembly version gmax109 ; ) using the pfam hlh hidden markov model ( pf00010 ; http://pfam.sanger.ac.uk ) . sequences were manually curated to identify mispredictions of splice sites , which often led to omission of part of the bhlh domain in the genome annotation , reducing the hidden markov model score . as above , the alignment supplied by meme for the new motif was used to create a hidden markov model , and this model was used to search the soybean bhlhs to determine which of them contained the motif . the conservation of key residues within the basic region of the bhlh domain can predict both the potential to bind dna and the preferred recognition sequence . of the soybean bhlh proteins that lack the e9 residue ( 74 proteins ) only eight have five or more basic residues within the basic region . since these helix - loop - helixes are related to the bhlhs and are involved in many of the same pathways , they are included in our characterization of soybean bhlhs if they are recognized by the hlh hmm . the intron / exon patterns were examined for soybean bhlh - encoding genes , and it was determined that 31% of soybean bhlhs contain the pattern of three conserved introns ( pattern a , figure 2 ) . because of the recent duplication of the soybean genome , many soybean bhlh proteins have closely related homeologs ( recently duplicated paralogs arising as a result of polyploidy ) which may be as much as 98% identical at the dna sequence level . based on sequence conservation within the basic domain , it can be predicted that the majority of soybean bhlhs ( 77% ) have the potential to bind dna , and 58% of all soybean bhlhs are likely to recognize the core g - box sequence , of which there are over 17,000 represented in the promoters of actively transcribed genes in the soybean genome ( meh , unpublished data ) . all but one of the bhlh subfamilies represented in other land plants are present in soybean , and the proportion of bhlhs in each subfamily was largely consistent between arabidopsis and soybean , although soybean contains many more bhlh - encoding genes , implying a broadly similar set of functions for each bhlh family may be conserved . recently the hmm method identified the bhlh domain in the closest soybean homologs of several of these genes including kidari , atbs1 , and pre1 , and these were used in our analysis . the amino acid similarity between these proteins is very strong in the predicted hlh region of the soybean proteins , and the hmm score for the hlh prediction in the soybean proteins is not strong .
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in our clinical experience some parents with epilepsy express concern how the condition will affect their child . this is especially the case when the parent has therapy - resistant epilepsy and often suffers from seizures . on the other hand , there are also parents who are not at all concerned about the consequences of epilepsy for their children . the children of people with epilepsy ( pwe ) comprise a group that possibly can be affected by their parent s condition for different reasons . there is a potential risk that the child can be hurt in association with a seizure affecting their parent , but there are also possible psychosocial consequences associated with growing up with a parent with a chronic disease . epilepsy is associated with an increased prevalence of mental - health disorder compared with the general population1 and it is also associated with a higher risk of suicide . one may speculate that growing up with a parent under these conditions could be difficult for a child . our interest in this matter is explained by information from focus - group interviews that we undertook with young to middle - aged persons with epilepsy and memory problems . here , many informants were worried about how growing up with a parent with epilepsy affected the situation of their children,2 and gave a more detailed description of the psychosocial stress that pwe may suffer , in relation to a reduced capability to look after their children , that some of them report . there is not much knowledge about the situation of or risk for children of persons with a chronic disease such as epilepsy.3 one may suspect that such children could be at risk of developing psychosocial / psychological problems or even physical symptoms , but we have found no research on this subject . there are some reports on these issues from people with other conditions such as multiple sclerosis47 and cancer.8 a literature search revealed just a few reports on the situation for children of persons with epilepsy9,10 and none that focused on the risks for the children . studies indicate that epilepsy may cause psychosocial difficulties for family members , but have concentrated on the effect of children with epilepsy on adult family members.3 epilepsy also affects family members quality of life , but this has also been studied in adults exclusively.11 family members of adult pwe , especially of mothers , have increased levels of anxiety , depression , and somatic complaints.12 the literature search revealed no reports on the deeper meaning of being a parent with epilepsy and , to our knowledge , this area has not been investigated before . the aim of this paper is to describe aspects of what it means to be a parent with epilepsy , focusing the parent s perspective and their thoughts on having children . a qualitative method was used to obtain knowledge and understanding on this issue ; this method is often used to start research on a new area . this method does not produce any numbers , but can help to understand more complex human behavior . in the study reported here , we reused preexisting data and performed a secondary analysis.2 heaton describes secondary analyses as a way of reworking and analyzes preexisting qualitative data by asking new and expanded questions of the text.13 focus - group interviews of young adults , aged 1835 years old , with epilepsy , treated in our clinics , that aimed to investigate the impact of memory problems in epilepsy , actually turned out to reveal much information about epilepsy and parenthood . altogether , 18 young adults were invited to take part in the study , but four of them did not show up at the appointed time . most of the participants were interested in meeting other young people with the same condition as themselves , and therefore wanted to join the study . the interviews were conducted by two of the authors ( hg and aml ) and took place in four groups . each group contained three or four individuals , two groups with women and two groups with men . the participants have been described in a previous article2 and many of the participants had therapy - resistant epilepsy , but some of the participants were seizure free . the participants ( male [ m ] 52 years old and m 55 ) who were interviewed for the book leva med epilepsi [ to live with epilepsy]14 were interviewed in face - to - face interviews by one of the authors ( aml ) . for demographic information the participants in focus - group interviews were asked open questions on living with epilepsy and subjective memory problems . if there were feelings the participants did not want to share in the group , they were informed they could meet the doctor confidentially after the interviews . there were no questions about being a parent with epilepsy , but since the participants were free to discuss subjects of importance to them , this subject was raised spontaneously in the groups . such naturalistic data that have been collected with minimal interference by researchers , heaton13 describes as being very suitable for secondary analysis . interviews by the same method , but without concentrating on cognitive problems , were also performed with people of different ages with epilepsy when preparing the book leva med epilepsi.14 the focus - group interviews were recorded and verbatim transcribed , which means that every word , sound , and silent period during the interviews was written down as a long text . relevant areas and aspects covering the participants narratives and issues of being a parent with epilepsy were extracted from the original interview text . in addition , relevant aspects from interviews undertaken by aml , using open - ended questions , for the book leva med epilepsi were extracted . the two men taking part in these interviews with open - ended questions discussed issues of being a parent having epilepsy . these men were older than the other participants and they were included in the study to achieve a wider age range ( m 52 and m 55 ) . the text in the domain was analyzed in steps described by graneheim and lundman.15 first , all the authors read the transcripts several times , in order to get a sense of the whole and a good grasp of the content . in the next step , this analysis did not follow common grammatical or linguistic rules , but the text was divided when a shift in the sense of meaning could be found . in the next step , these meaning units were then concentrated without changing the inherent meaning and the implicit meaning in the text was interpreted . following this the interpretations were grouped together and abstracted to themes and are presented in the results . the regional ethical committee of linkping , linkping , sweden , approved this study ( dnr 2010/246 - 31 ) . the aim of this paper is to describe aspects of what it means to be a parent with epilepsy , focusing the parent s perspective and their thoughts on having children . a qualitative method was used to obtain knowledge and understanding on this issue ; this method is often used to start research on a new area . this method does not produce any numbers , but can help to understand more complex human behavior . in the study reported here , we reused preexisting data and performed a secondary analysis.2 heaton describes secondary analyses as a way of reworking and analyzes preexisting qualitative data by asking new and expanded questions of the text.13 focus - group interviews of young adults , aged 1835 years old , with epilepsy , treated in our clinics , that aimed to investigate the impact of memory problems in epilepsy , actually turned out to reveal much information about epilepsy and parenthood . altogether , 18 young adults were invited to take part in the study , but four of them did not show up at the appointed time . most of the participants were interested in meeting other young people with the same condition as themselves , and therefore wanted to join the study . the interviews were conducted by two of the authors ( hg and aml ) and took place in four groups . each group contained three or four individuals , two groups with women and two groups with men . the participants have been described in a previous article2 and many of the participants had therapy - resistant epilepsy , but some of the participants were seizure free . the participants ( male [ m ] 52 years old and m 55 ) who were interviewed for the book leva med epilepsi [ to live with epilepsy]14 were interviewed in face - to - face interviews by one of the authors ( aml ) . for demographic information see table 1 . the participants in focus - group interviews were asked open questions on living with epilepsy and subjective memory problems . if there were feelings the participants did not want to share in the group , they were informed they could meet the doctor confidentially after the interviews . there were no questions about being a parent with epilepsy , but since the participants were free to discuss subjects of importance to them , this subject was raised spontaneously in the groups . such naturalistic data that have been collected with minimal interference by researchers , heaton13 describes as being very suitable for secondary analysis . interviews by the same method , but without concentrating on cognitive problems , were also performed with people of different ages with epilepsy when preparing the book leva med epilepsi.14 the focus - group interviews were recorded and verbatim transcribed , which means that every word , sound , and silent period during the interviews was written down as a long text . relevant areas and aspects covering the participants narratives and issues of being a parent with epilepsy were extracted from the original interview text . in addition , relevant aspects from interviews undertaken by aml , using open - ended questions , for the book leva med epilepsi were extracted . the two men taking part in these interviews with open - ended questions discussed issues of being a parent having epilepsy . these men were older than the other participants and they were included in the study to achieve a wider age range ( m 52 and m 55 ) . the text in the domain was analyzed in steps described by graneheim and lundman.15 first , all the authors read the transcripts several times , in order to get a sense of the whole and a good grasp of the content . in the next step , this analysis did not follow common grammatical or linguistic rules , but the text was divided when a shift in the sense of meaning could be found . in the next step , these meaning units were then concentrated without changing the inherent meaning and the implicit meaning in the text was interpreted . following this the interpretations were grouped together and abstracted to themes and are presented in the results . the regional ethical committee of linkping , linkping , sweden , approved this study ( dnr 2010/246 - 31 ) . the discussions in the focus groups revealed the following key issues of being a parent with epilepsy : ( 1 ) a persistent feeling of insecurity , since a seizure may occur at any time and the child could be hurt ; ( 2 ) a feeling of inadequacy of not being able to take full responsibility for one s child ; ( 3 ) acknowledgment that one s children are forced to take more responsibility than other children do ; and ( 4 ) a feeling of guilt of not being able to fulfill one s expectations of being the parent one would like to be . the participants in the focus - group interviews talked about the difficulties for and dangers to the child of a parent with epilepsy . they described that they were always aware of the possibility of a seizure and tried to foresee what the consequences would be in each and every situation , so that the child would be safe if a seizure did occur . the participants took great care to prevent the child from being harmed and some of them explained that they were never left alone with their child . they tried to care for their child on the floor or on a couch , preventing the child from falling if a seizure did happen , and also avoided carrying their child . one woman described that she concentrated hard on the safety of her child in every situation . if i will carry her or if i am walking with the stroller , it is really frightening . if i am carrying her or do something with her i really shape up , i try to concentrate so that nothing will happen . i always take care of my baby on the floor or on the couch so she can not fall . ( female [ f ] 26)i was really scared when i took care of the child . if i would fall the child would fall too , so i was never alone with the boy until he was like 5 years old . ( m 32 ) i bite my tongue all the time i am with my baby . if i will carry her or if i am walking with the stroller , it is really frightening . if i am carrying her or do something with her i really shape up , i try to concentrate so that nothing will happen . i always take care of my baby on the floor or on the couch so she can not fall . ( female [ f ] 26 ) i was really scared when i took care of the child . if i would fall the child would fall too , so i was never alone with the boy until he was like 5 years old . ( m 32 ) one of the men ( m 24 ) , who was expecting his first child , was at first not concerned about the possible dangers for the child ( illustrated in the dialogue following ) . however after discussing with one of the other participants , he seemed to realize that this is a problem he must consider in the future : m 33 : how will it be when it is just you and your baby ? i think that is really difficult?m 24 : just me and the baby ; but that is not difficult?m 33 : yes , i mean when you have to bathe the baby or walk with the stroller.m 24 : i think i can take care of that ; i am good with kids.m 33 : yes , but i mean if you have a seizure.m 24 : but i do not have many seizures now ; i think it will be okay.m 33 : i think it is so hard ; i have just a few seizures , but am so afraid . a lot can happen if you have a seizure , even if you do not have them often [ ] .m 24 : i can understand that ; i feel the same now . m 33 : how will it be when it is just you and your baby ? m 33 : yes , i mean when you have to bathe the baby or walk with the stroller . m 24 : i think i can take care of that ; i am good with kids . m 24 : but i do not have many seizures now ; i think it will be okay . m 33 : i think it is so hard ; i have just a few seizures , but am so afraid . a lot can happen if you have a seizure , even if you do not have them often [ ] . one father was not worried for his daughter when she was a small child , but in retrospect , he understood that the situation must have been difficult for her : we were divorced but there was something i really wanted : my daughter . i had a few seizures ; but it is hard to know if my daughter was afraid . when she was older she got the telephone number for a nurse and could call if something happened . she was a clever girl and could make a phone call . at this time i used to have a couple of seizures every month . ( m 55 ) we were divorced but there was something i really wanted : my daughter . i had a few seizures ; but it is hard to know if my daughter was afraid . when she was older she got the telephone number for a nurse and could call if something happened . she was a clever girl and could make a phone call . at this time i used to have a couple of seizures every month . ( m 55 ) the participants described a feeling of inadequacy when discussing parenthood in the group . they wished to take the same responsibility as other parents , but understood it was important to accept help from other people . they expressed that it was difficult to accept that they could not be part of all aspects of parenthood . for some participants this meant that they felt they did not have the same value or importance as other parents : i feel like i am not really grown up , i can not even walk with the stroller . but it hurts , that i can not and should not , take the full responsibility . i did not have the energy to do all the things that i wanted to and my opinion was not always asked for . ( m 52 ) i feel like i am not really grown up , i can not even walk with the stroller . but it hurts , that i can not and should not , take the full responsibility . i did not have the energy to do all the things that i wanted to and my opinion was not always asked for . ( m 52 ) the parents were also worried that the children had to take on a big responsibility and grow up too fast because of epilepsy , giving the parents the feeling that their children were not allowed to be children . they often trusted in their children and got help from them , but the fact that they had to lean on their children made them sad . the children were always alert to recognizing if their parent might have a seizure and some children even observed their parent and did not let them out of sight . the children learned to take own initiative ; for example , by getting help from other people by calling them . in this way they had to take on a big responsibility even if they were very young : my daughters help me , with the seizures . they are children , but they are not allowed to be children , they are like adults actually . but i must try to let them be children now , as they are 8 and 9 years old . and i wake up after a seizure and i call on her to ask her to turn off the tv . she calls dad , but he does not answer and then she calls grandma instead . they are children , but they are not allowed to be children , they are like adults actually . but i must try to let them be children now , as they are 8 and 9 years old . and i wake up after a seizure and i call on her to ask her to turn off the tv . shall i call dad ? she calls dad , but he does not answer and then she calls grandma instead . ( f 29 ) the parents in the focus - group interviews often felt guilty because they could not be the parents they wanted to be . they also felt guilty because of all the difficulties their children had to experience when witnessing a seizure . they thought about what the seizure would look like and wondered if the child would be afraid when observing it . the parents were also worried about how these experiences in childhood would affect their children in the long run . they discussed whether the seizures would scare the children and how the children would react to them . the participants also experienced subjective memory decline and they believed that this affected the situation of their children . they explained that they often forgot to keep their promises and forgot to give information and they regarded this as being difficult for their children . they felt that they often let their children down and the children were often disappointed : i have been worried and i am so anxious . how will it be when she gets older and how will she cope with it when i have a seizure ? what will happen if i have a seizure , if it is just me and my children ? i am feeling bad when i have a seizure and since i have this seizure my children must take care of themselves , just because i am feeling bad . how will it be when she gets older and how will she cope with it when i have a seizure ? what will happen if i have a seizure , if it is just me and my children ? i am feeling bad when i have a seizure and since i have this seizure my children must take care of themselves , just because i am feeling bad . ( f 29 ) a friend of my daughter calls when my daughter is outside playing . the participants in the focus - group interviews talked about the difficulties for and dangers to the child of a parent with epilepsy . they described that they were always aware of the possibility of a seizure and tried to foresee what the consequences would be in each and every situation , so that the child would be safe if a seizure did occur . the participants took great care to prevent the child from being harmed and some of them explained that they were never left alone with their child . they tried to care for their child on the floor or on a couch , preventing the child from falling if a seizure did happen , and also avoided carrying their child . one woman described that she concentrated hard on the safety of her child in every situation . if i will carry her or if i am walking with the stroller , it is really frightening . if i am carrying her or do something with her i really shape up , i try to concentrate so that nothing will happen . i always take care of my baby on the floor or on the couch so she can not fall . ( female [ f ] 26)i was really scared when i took care of the child . if i would fall the child would fall too , so i was never alone with the boy until he was like 5 years old . ( m 32 ) i bite my tongue all the time i am with my baby . if i will carry her or if i am walking with the stroller , it is really frightening . if i am carrying her or do something with her i really shape up , i try to concentrate so that nothing will happen . i always take care of my baby on the floor or on the couch so she can not fall . ( female [ f ] 26 ) i was really scared when i took care of the child . if i would fall the child would fall too , so i was never alone with the boy until he was like 5 years old . ( m 32 ) one of the men ( m 24 ) , who was expecting his first child , was at first not concerned about the possible dangers for the child ( illustrated in the dialogue following ) . however after discussing with one of the other participants , he seemed to realize that this is a problem he must consider in the future : m 33 : how will it be when it is just you and your baby ? i think that is really difficult?m 24 : just me and the baby ; but that is not difficult?m 33 : yes , i mean when you have to bathe the baby or walk with the stroller.m 24 : i think i can take care of that ; i am good with kids.m 33 : yes , but i mean if you have a seizure.m 24 : but i do not have many seizures now ; i think it will be okay.m 33 : i think it is so hard ; i have just a few seizures , but am so afraid . a lot can happen if you have a seizure , even if you do not have them often [ ] .m 24 : i can understand that ; i feel the same now . m 33 : how will it be when it is just you and your baby ? m 33 : yes , i mean when you have to bathe the baby or walk with the stroller . m 24 : i think i can take care of that ; i am good with kids . m 24 : but i do not have many seizures now ; i think it will be okay . i think it is so hard ; i have just a few seizures , but am so afraid . a lot can happen if you have a seizure , even if you do not have them often [ ] . m 24 : i can understand that ; i feel the same now . being a single parent with epilepsy one father was not worried for his daughter when she was a small child , but in retrospect , he understood that the situation must have been difficult for her : we were divorced but there was something i really wanted : my daughter . i had a few seizures ; but it is hard to know if my daughter was afraid . when she was older she got the telephone number for a nurse and could call if something happened . she was a clever girl and could make a phone call . at this time i used to have a couple of seizures every month . ( m 55 ) we were divorced but there was something i really wanted : my daughter . i had a few seizures ; but it is hard to know if my daughter was afraid . when she was older she got the telephone number for a nurse and could call if something happened . she was a clever girl and could make a phone call . at this time i used to have a couple of seizures every month . they wished to take the same responsibility as other parents , but understood it was important to accept help from other people . they expressed that it was difficult to accept that they could not be part of all aspects of parenthood . for some participants this meant that they felt they did not have the same value or importance as other parents : i feel like i am not really grown up , i can not even walk with the stroller . but it hurts , that i can not and should not , take the full responsibility . i did not have the energy to do all the things that i wanted to and my opinion was not always asked for . ( m 52 ) i feel like i am not really grown up , i can not even walk with the stroller . but it hurts , that i can not and should not , take the full responsibility . i did not have the energy to do all the things that i wanted to and my opinion was not always asked for . the parents were also worried that the children had to take on a big responsibility and grow up too fast because of epilepsy , giving the parents the feeling that their children were not allowed to be children . they often trusted in their children and got help from them , but the fact that they had to lean on their children made them sad . the children were always alert to recognizing if their parent might have a seizure and some children even observed their parent and did not let them out of sight . the children learned to take own initiative ; for example , by getting help from other people by calling them . in this way they had to take on a big responsibility even if they were very young : my daughters help me , with the seizures . they are children , but they are not allowed to be children , they are like adults actually . but i must try to let them be children now , as they are 8 and 9 years old . and i wake up after a seizure and i call on her to ask her to turn off the tv . shall i call dad ? she calls dad , but he does not answer and then she calls grandma instead . they are children , but they are not allowed to be children , they are like adults actually . but i must try to let them be children now , as they are 8 and 9 years old . and i wake up after a seizure and i call on her to ask her to turn off the tv . she calls dad , but he does not answer and then she calls grandma instead . the parents in the focus - group interviews often felt guilty because they could not be the parents they wanted to be . they also felt guilty because of all the difficulties their children had to experience when witnessing a seizure . they thought about what the seizure would look like and wondered if the child would be afraid when observing it . the parents were also worried about how these experiences in childhood would affect their children in the long run . they discussed whether the seizures would scare the children and how the children would react to them . the participants also experienced subjective memory decline and they believed that this affected the situation of their children . they explained that they often forgot to keep their promises and forgot to give information and they regarded this as being difficult for their children . they felt that they often let their children down and the children were often disappointed : i have been worried and i am so anxious . how will it be when she gets older and how will she cope with it when i have a seizure ? what will happen if i have a seizure , if it is just me and my children ? i am feeling bad when i have a seizure and since i have this seizure my children must take care of themselves , just because i am feeling bad . how will it be when she gets older and how will she cope with it when i have a seizure ? what will happen if i have a seizure , if it is just me and my children ? i am feeling bad when i have a seizure and since i have this seizure my children must take care of themselves , just because i am feeling bad . ( f 29 ) a friend of my daughter calls when my daughter is outside playing . parents in the focus - group interviews expressed , without any direct questioning on this subject , that they were very worried about the consequences of epilepsy affecting their children . these parents took great care to prevent any accidents that could harm their children because of epilepsy . it was always in their minds to figure out the safest way to take care of the child in case they had a seizure . we have not found any study examining the risk of accidents for children of parents with epilepsy . in a study examining children drowning or near drowning in baths in england , four out of 44 cases were related to epilepsy but every case affected children with epilepsy and none was caused by a parent with epilepsy.16 the parents themselves often suffered from a feeling of being an inadequate parent , forgetting about schedules , and being forced to rest instead of taking care of their children . one may interpret this situation as putting more demand on the spouse , and also on the children themselves . these results can be involved in a future discussion on different forms of support for pwe with children , especially if they are single parents or the spouse can not support the identified needs . on the other hand , it is also important to strengthen the self - esteem in pwe , which obviously also is determined by their ability to care for their children . consequently , this is a matter of balance between the quality of life and self - esteem of pwe and the safety of their children . persons with epilepsy who have children seem to have a double trauma : first , the risk of getting seizures and the constant fear that this can create and , second , worries about generating psychological and physical problems for their children.2 the authors also suspect that there may be such risks for some children of pwe , that the pwe do not recognize themselves . however , parents who are concerned about the security of the child will , just like the parents in our study , try to foresee every possible risk associated with epilepsy . it is more dangerous if the parent does not acknowledge the possible risks associated with the seizure and ignores them . it is therefore important to scrutinize the attitude and knowledge of each parent with epilepsy . parents can also fear that the child will be taken into custody if they expose the possible risks to the social authorities . it is essential that parents with epilepsy can get support in their homes if necessary , so that they do not have to conceal their worries for the child s safety . interventions offered to pwe for supporting their children should be designed in a way that the person feels safe and the action taken should be beneficial to the whole family . parents with epilepsy are aware of the difficulties of being a parent who can be affected by seizures , but it is important to identify those parents that ignore the risks . cognitive profile is one factor that should be investigated in this situation ; low performance may be a risk factor , but further research is needed to know this for certain . support programs must be done in a way that respects the condition of the patient , knowing that the self - esteem of the patient is vulnerable in this disease . a previous study has shown that self - esteem among young adults with epilepsy decreases when they have passed adolescence and meet the different obstacles of adult life,17 such as getting a job and raising a child . interventions should address both pwe but also their family members and the safety of the child must be discussed with the whole family . it is essential that information about the difficulties for parents with epilepsy also reaches the pediatrician , general practitioner , and social authorities . one possible method is to let professional patients persons with epilepsy who have been educated about the condition spread information about the difficulties of being a parent with epilepsy . the young man in our study who at first could not see any risks for his future child because of epilepsy got new perspectives about this when discussing the subject with another parent with epilepsy . to talk with someone that has experience can be important for understanding the issue . even though this qualitative study is limited by the relatively small number of participants it shows that this is a field of importance to many pwe and further research could give more information . since a qualitative study generates no numbers we do not know how frequent these problems are . most participants in the interviews had therapy - resistant epilepsy and one could assume that patients who are seizure - free do not experience the same kinds of problems or find this issue as important . another option would be to analyze groups of parents with epilepsy before and after extra support interventions and providing them with information . material from the focus - group interviews suggests that parents with epilepsy are very concerned about the consequences of epilepsy affecting their children . the feeling of insecurity is persistent , as a seizure can happen at any time . the risk of seizures leads to a feeling of inadequacy and not being able to take full responsibility for the child . the parents take care to prevent accidents , but need support and guidance , especially when expecting their first child . fear of not being approved as a custodian can prevent parents from discussing the possible risks for their child . parents with epilepsy also feel the children must take on too much responsibility for their age and they feel guilty they can not be the ideal parent . we believe it is essential that supportive programs are made available to parents with epilepsy , since fear for the safety of the child increases the psychosocial burden of the condition , and that interventions should address both pwe and family members . to arrange study circles , in which pwe get the opportunity to meet each other , is one possible strategy for sharing information about the difficulties of being a parent with epilepsy . professional patients , who are persons with epilepsy who have been given education about the condition . other family members , such as spouses and siblings , could also be offered to attend a group of their own . associations for pwe could also recognize and work with this important aspect of living with epilepsy . this qualitative study on parents with epilepsy indicates that further research is of importance in this field .
objectiveparents with epilepsy can be concerned about the consequences of epilepsy affecting their children . the aim of this paper is to describe aspects of what it means being a parent having epilepsy , focusing the parents perspectives and their thoughts on having children.methodsfourteen adults aged 1835 years with epilepsy and subjective memory decline took part in focus - group interviews . the interviews were conducted according to a semi - structured guideline . material containing aspects of parenthood was extracted from the original interviews and a secondary analysis was done according to a content - analysis guideline . interviews with two parents for the swedish book leva med epilepsi [ to live with epilepsy ] by am landtblom ( stockholm : bilda ide ; 2009 ) were analyzed according to the same method.resultsfour themes emerged : ( 1 ) a persistent feeling of insecurity , since a seizure can occur at any time and the child could be hurt ; ( 2 ) a feeling of inadequacy of not being able to take full responsibility for one s child ; ( 3 ) acknowledgment that one s children are forced to take more responsibility than other children do ; and ( 4 ) a feeling of guilt of not being able to fulfill one s expectations of being the parent one would like to be.conclusionthe parents with epilepsy are deeply concerned about how epilepsy affects the lives of their children . these parents are always aware that a seizure may occur and reflect on how this can affect their child . they try to foresee possible dangerous situations and prevent them . these parents were sad that they could not always take full responsibility for their child and could not live up to their own expectations of parenthood . supportive programs may be of importance since fear for the safety of the child increases the psychosocial burden of epilepsy . there were also a few parents who did not acknowledge the safety issue of their child the authors believe that it is important to identify these parents and provide extra information and support to them .
Introduction Aim and methods Aim Participants and procedures Analysis Ethics Results A persistent feeling of insecurity, since a seizure can occur at any time and the child could be hurt A feeling of inadequacy of not being able to take full responsibility for ones child Acknowledgment that ones children are forced to take more responsibility than other children do A feeling of guilt of not being able to fulfill ones expectations of being the parent one would like to be Discussion Conclusion
on the other hand , there are also parents who are not at all concerned about the consequences of epilepsy for their children . here , many informants were worried about how growing up with a parent with epilepsy affected the situation of their children,2 and gave a more detailed description of the psychosocial stress that pwe may suffer , in relation to a reduced capability to look after their children , that some of them report . the aim of this paper is to describe aspects of what it means to be a parent with epilepsy , focusing the parent s perspective and their thoughts on having children . in the study reported here , we reused preexisting data and performed a secondary analysis.2 heaton describes secondary analyses as a way of reworking and analyzes preexisting qualitative data by asking new and expanded questions of the text.13 focus - group interviews of young adults , aged 1835 years old , with epilepsy , treated in our clinics , that aimed to investigate the impact of memory problems in epilepsy , actually turned out to reveal much information about epilepsy and parenthood . the participants ( male [ m ] 52 years old and m 55 ) who were interviewed for the book leva med epilepsi [ to live with epilepsy]14 were interviewed in face - to - face interviews by one of the authors ( aml ) . for demographic information the participants in focus - group interviews were asked open questions on living with epilepsy and subjective memory problems . interviews by the same method , but without concentrating on cognitive problems , were also performed with people of different ages with epilepsy when preparing the book leva med epilepsi.14 the focus - group interviews were recorded and verbatim transcribed , which means that every word , sound , and silent period during the interviews was written down as a long text . the aim of this paper is to describe aspects of what it means to be a parent with epilepsy , focusing the parent s perspective and their thoughts on having children . in the study reported here , we reused preexisting data and performed a secondary analysis.2 heaton describes secondary analyses as a way of reworking and analyzes preexisting qualitative data by asking new and expanded questions of the text.13 focus - group interviews of young adults , aged 1835 years old , with epilepsy , treated in our clinics , that aimed to investigate the impact of memory problems in epilepsy , actually turned out to reveal much information about epilepsy and parenthood . the participants ( male [ m ] 52 years old and m 55 ) who were interviewed for the book leva med epilepsi [ to live with epilepsy]14 were interviewed in face - to - face interviews by one of the authors ( aml ) . the participants in focus - group interviews were asked open questions on living with epilepsy and subjective memory problems . interviews by the same method , but without concentrating on cognitive problems , were also performed with people of different ages with epilepsy when preparing the book leva med epilepsi.14 the focus - group interviews were recorded and verbatim transcribed , which means that every word , sound , and silent period during the interviews was written down as a long text . the discussions in the focus groups revealed the following key issues of being a parent with epilepsy : ( 1 ) a persistent feeling of insecurity , since a seizure may occur at any time and the child could be hurt ; ( 2 ) a feeling of inadequacy of not being able to take full responsibility for one s child ; ( 3 ) acknowledgment that one s children are forced to take more responsibility than other children do ; and ( 4 ) a feeling of guilt of not being able to fulfill one s expectations of being the parent one would like to be . the participants in the focus - group interviews talked about the difficulties for and dangers to the child of a parent with epilepsy . the parents were also worried that the children had to take on a big responsibility and grow up too fast because of epilepsy , giving the parents the feeling that their children were not allowed to be children . parents in the focus - group interviews expressed , without any direct questioning on this subject , that they were very worried about the consequences of epilepsy affecting their children . in a study examining children drowning or near drowning in baths in england , four out of 44 cases were related to epilepsy but every case affected children with epilepsy and none was caused by a parent with epilepsy.16 the parents themselves often suffered from a feeling of being an inadequate parent , forgetting about schedules , and being forced to rest instead of taking care of their children . however , parents who are concerned about the security of the child will , just like the parents in our study , try to foresee every possible risk associated with epilepsy . parents with epilepsy are aware of the difficulties of being a parent who can be affected by seizures , but it is important to identify those parents that ignore the risks . material from the focus - group interviews suggests that parents with epilepsy are very concerned about the consequences of epilepsy affecting their children . the risk of seizures leads to a feeling of inadequacy and not being able to take full responsibility for the child . we believe it is essential that supportive programs are made available to parents with epilepsy , since fear for the safety of the child increases the psychosocial burden of the condition , and that interventions should address both pwe and family members .
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development of methods and the determination of inhaled volumes are important for the protection of wearers from airborne contaminants and assignment of minimal expected respirator protection factors . respirator protection factors are defined as contaminant concentration outside the facepiece divided by contaminant concentration inside the facepiece . these two concentrations are often measured by nondiscriminating particle counters that require a finite amount of time to reach a valid time - averaged measurement . although this is a relatively simple measurement to make , it can not be used accurately for rapidly changing particle counts . particle count is also dependent upon placement within the facepiece , and sharp spatial discontinuities in contaminant concentrations may exist that lead to erroneous conclusions regarding representative facepiece concentrations . concentration ratio is only valid as a measure of protection factor as long as there are no particle sources or sinks in the system . it is known that the respiratory system is a source for moisture particles , and these can be counted along with particles of the challenge substance . deposition of particles within the respiratory system can also occur as air is inhaled , leading to apparent protection factors higher than they ought to be . respirator protection factors may only be an approximate indication of inspiration of contaminants by the wearer . that is because contaminants penetrating the respirator facepiece may not reach the mouth to be inhaled . a more direct indicator of protection afforded by the respirator is wearer protection factor , which we have defined as the concentration of contaminant inhaled divided by ambient concentration . the difference between this and conventional protection factor use is that contaminants inside the respirator facepiece , but not inhaled , are measured conventionally but not for wearer protection factor . in addition , a tracer gas , such as co2 , can be used to determine the eventual outcome of air supplied by a papr blower . this determination we have called blower effectiveness . a blower effectiveness with a value of 1.0 means that all blower air supplied during the inhalation portion of the breathing cycle would contribute to wearer inhaled gas . some blower air may be lost to the environment directly through the exhalation valve ; in this case , blower effectiveness would be calculated as less than 1.0 . a blower effectiveness of less than 1.0 would indicate that some inspired air had to come from the ambient , bypassing the blower through respirator leakage . a blower effectiveness greater than 1.0 indicates that some blower air contributed during the exhalation phase would be inhaled in the subsequent inspiration . an apr , without a blower , should have an equivalent blower effectiveness close to 1.0 . in this study , we investigated measurement of respirator protection factors using a different method of a challenge gas and collection of exhaled breath . it was intended that results from this approach could be a better assessment of protection factors actually experienced by the wearer of the respirator . because this test used carbon dioxide as the tracer gas , it had to be conducted on a head form , but it did give quantitative assessments of wearer protection factors and blower effectivenesses . the co2 was traceable as originating in the ambient air surrounding the masked head form , and so could help to partition air supplied through the filter from air that bypassed the filter . respirator leakage was determined by operating the respirator inside a chamber containing co2 as a tracer gas . air supply to the respirator or papr blower came from the outside atmosphere containing a negligible concentration of the tracer gas . a breathing machine was used to simulate the effect of a human wearer , but exhaled air from the breathing machine was collected in a separate container . the presence of the tracer gas in the exhaled air was quantitative proof of papr inward leakage ( figure 1 ) . the chamber of dimensions 137 cm ( 54 in ) by 76 cm ( 30 in ) by 180 cm ( 71 in ) was constructed of plywood and lexan transparent plastic for visibility . the mouth of the head form was connected to a breathing machine ( krug life sciences , houston , tex , usa ) outside the chamber by means of a 3.8 cm ( 1.5 in ) flexible ventilator hose ( a - m systems spiral tubing , carlsborg , wash , usa ) . two one - way valves directed inhaled air from the respirator into the breathing machine and exhaled air into a separate container . the valves had been salvaged from u. s. army m17 air - purifying respirators the container to collect exhaled breath was constructed from several 2 l soft drink plastic bottles sealed with black electrical tape . the ability of the tracer gas to diffuse through the walls of the container was not investigated , but the short time between breaths , the type of plastic used for the containers , and the volume inside the container made significant concentration errors unlikely . the test chamber was filled with 6 - 7% co2 from a cylinder , and the gas concentration was monitored continuously with a mass spectrometer ( model 1100 , perkin - elmer , st . louis , mo ) . because co2 inside the test chamber was continuously being replaced with fresh air passing through the respirator , co2 was added continuously from the gas cylinder to maintain the target co2 concentration . the exhalation gas collection container began with a negligible co2 concentration , which proceeded to climb as exhaled air from more breaths displaced initial air . the air / co2 mixture was sampled continuously by the mass spectrometer and at 50/sec by the data acquisition system . when co2 concentration had reached its final steady - state value , this concentration was used as the value in exhaled air , and , by inference , inhaled air . there was no other place for co2 to go once it was inhaled than into the exhalation collection container . respirators tested were the racal airmate 3 ( racal , frederick , md , usa ) loose - fitting papr , breathe easy ( 3 m , st . paul , minn , usa ) tight - fitting papr , butyl head cover with cape , # 522 - 02 - 23 ( 3 m ) loose - fitting hood , centurion max ( martindale protection ; thetford , norfolk , uk ) multipurpose loose - fitting papr with scarfs in place , se 400 ( sea , meadowlands , pa , usa ) breath - responsive papr , and frm 40 ( 3 m ) air - purifying respirator . medgraphics ( st . paul , minn , usa ) # 5038773 pitot tube flowmeters were used to measure blower flow and breathing machine flow . these were carefully calibrated beforehand to ensure that they gave identical measurements for identical flow rates . flowmeters were adapted to blower inlets for the racal , centurion , and sea devices . flowmeters were placed in the connecting hose between blower and facepiece for the two 3 m powered devices . papr blowers were operated with fully charged batteries , and each test lasted approximately 2 min . hoses were attached to the inlets of each blower so that ambient air could be drawn from outside the chamber . hose inlets were located about 1 m above the chamber , and excess gas was exhausted from the chamber floor in order not to cycle co2 from the chamber back into the respirator inlets ( co2 is denser than air ) . the breathing machine was set to generate a minute volume of 112 l / min , tidal volume of 2.4 l , and a peak flow of 317 l / min . these settings have been used in this and previous experiments in order to induce respirator leakage if the respirator is going to leak at all . the breathing machine minute volume is nearly the same as most papr blower flow rates , but peak flows are much higher than blower flow rates . data were collected with an analog - to - digital data acquisition board ( national instruments , austin , tex , usa ) connected to the universal serial bus ( usb ) of a pc computer . custom software developed in labview 7 ( national instruments , austin , tex , usa ) recorded flow and concentration data , calculated flow differences , and exhaled volumes . the volume of inhaled co2 is the leakage volume times the concentration of co2 in the exhalation collection chamber atmosphere , which also equals the exhaled volume times the exhaled co2 concentration . thus , leakage volume can be obtained as the exhaled volume times the ratio of co2 concentrations in the exhaled breath and chamber atmosphere . the leftmost column includes values obtained from the readings ( v ) of flowmeter # 2 in figure 1 . where there is a range of values , the blower flow rate varied throughout the breathing cycle . inhaled volume ( vinh ) was obtained by integrating the flow signal ( vinh ) from flowmeter # 1 during the inhalation portion of the breathing cycle . exhaled volume ( vexh ) was obtained by integrating the flow signal ( vexh ) from flowmeter # 1 during the exhalation portion of the breathing cycle . normally , both inhaled volume and exhaled volume would have been expected to agree within the error rate of the measurement and integration process . there is a larger than expected difference of up to 10% between the two volumes , possibly attributed to some cooling of the air as it resided in the breathing machine or compression of the exhaled air as it left the breathing machine . because of the differences between to two volumes , the appropriate volume was chosen to calculate additional derived values . the co2 ratio in table 1 is the ratio of co2 concentration measured in the captured exhaled air ( cco2,exh ) divided by the co2 concentration in the ambient air in the chamber surrounding the respirator ( cco2,amb ) . the wearer protection factor was calculated as the inverse of the co2 ratio , ( cco2,amb / cco2,exh ) . leakage volume ( vleak ) represents the volume of air that leaked into the respirator facepiece and was subsequently inhaled by the breathing machine . this came from a co2 mass balance on the respirator : ( 1)amount of co2 inamount of co2 out+co2 generated = amount of co2 stored . the amount of co2 into the respirator was the net leakage volume times the co2 concentration in the ambient air in the chamber surrounding the respirator . any leakage of air and co2 out of the respirator was already included in the net leakage of air containing co2 into the respirator . there was no co2 generated in this apparatus . the amount of co2 stored was the amount of co2 collected in the container positioned to accumulate exhaled air and equaled the exhaled volume times the co2 concentration in the collected exhaled volume . thus , ( 1 ) became ( 2)vleak(cco2,amb)+0 + 0=vexh(cco2,exh),vleak = vexh(cco2,exhcco2,amb ) . the blower contribution to the inhaled volume was calculated from ( 3)vbl , inh = vinh[1cco2,inhcco2,amb ] . this equation indicates that the blower air volume contribution ( vbl , inh ) to total inhaled volume ( vinh ) is the proportion of air not identified as leakage ( 1 cco2,inh / cco2,amb ) . the total volume of filtered air passing through the blower during the time for inhalation , labeled total blower volume ( vbl , tot ) in table 1 , can be obtained from blower flow rate ( vbl ) measured by flowmeter # 2 in figure 1 , integrated over the total inhalation time ( tinh ) . blower effectiveness is the ratio of the blower contribution to inhaled air ( vbl , inh ) to total blower volume ( vbl , tot ) during the inhalation portion of the breathing cycle ( 4)eff = vbl , inhvbl , tot . in figure 2 are shown breathing machine flow rate , blower flow rate , and inhaled leakage volumes ( labeled inhaled contaminant volumes ) for the racal airmate 3 loose - fitting papr . this figure is intended to show that blower flow rate for this respirator is almost constant , and that inhaled contaminant volume ( integrated breathing machine flow rate times co2 concentration in the captured exhaled breath ) is slightly more than 2 l for a breathing machine tidal volume of 2.4 l. figure 3 is similar to figure 2 but shows that blower flow rate for the 3 m breathe easy tight - fitting papr varies throughout the inhalation phase of breathing . figure 4 illustrates responses by the se 400 breath - responsive tight - fitting papr . blower flow rate tracks breathing machine flow rate in an attempt to maintain positive pressure inside the facepiece . again , inhaled volume of contaminants is zero . it has been seen previously in our lab that papr blower flow rates vary during the breathing cycle , and this is also reflected in the entries for blower flow rates . exhaled tidal volumes were nearly constant at 2.32 to 2.42 l. the ratio of co2 concentrations in the exhaled breath and enclosing chamber is also shown . the inverses of these figures are the measured protection factors for each of the respirators as worn and used . in the column labeled leakage volume are found volumes of inhaled contaminant - laden air , obtained by multiplying the concentration ratios by exhaled volumes . these figures also represent nominal leakage volumes for the respirators . despite advances in filter technology , contaminant levels inside respirators can still become unacceptably high if the respirators can leak ambient air through alternate pathways . in fact , weak links in wearer protection are the facial fit and the exhalation valve . the figure of merit for respiratory protection has been the protection factor ( pf ) defined as the concentration of contaminant outside the respirator divided by the concentration inside the respirator . in this study , we used an alternative means to measure protection factor as it is related to respirator leakage . if the respirator was operated in an atmosphere containing a tracer gas , but the supply of air to the respirator through the filter circuit was free of tracer gas , then the only means for the gas to enter the facepiece and be inhaled was if the gas leaked inward from some path different from the filter circuit . if this test was conducted with a breathing machine , then the tracer gas would neither be deposited nor absorbed in the machine . the average concentration of the gas in the collected exhaled breath should then be equal to the average concentration as presented to the mouth inside the facepiece , thus averaging regions of high and low concentrations due to preferred contaminant flow pathways . the ratio of tracer gas concentration in the collected exhaled breath to the gas concentration in the surrounding atmosphere should then be the inverse of pf , at least from the wearer 's standpoint . it can be noted that , although the racal airmate 3 blower flow rate was higher than the centurion max blower flow rate , contaminant exposure with the racal is much higher and protection factor is much lower . with a protection factor nearly equal to 1.0 , the racal loose - fitting papr gives almost no protection against airborne contamination ; concentration of contaminant inside the respirator shield is nearly the same as outside the shield . the centurion max loose - fitting respirator gives somewhat better protection , but still not enough to be very effective . the se 400 breath - responsive respirator attempts to maintain positive pressure inside the facepiece . the se 400 blower flow can be seen to exceed the peak flow of 317 l / min produced by the breathing machine . when the blower was turned off , flow through the blower was much lower , and the respirator operated as an air - purifying respirator ( apr ) . the frm 40 apr has a similar flow rate through its filter , but a somewhat lower protection factor . these were the 3 m hood , 3 m papr , and se 400 papr . in very demanding environments , these respirators would afford the best protection . even if the power fails on the se 400 , the 3 m hood was , in effect , a loose - fitting respirator , yet had enough dead volume within its enclosure that contaminants did not reach the mouth . results from this study are not totally in agreement with some of the assigned protection factors published by osha . our results for the frm 40 give a protection factor of 17 , and for the unpowered se 400 a value of 20 . the osha value for full - face piece tight - fitting papr is 1000 ; our results for the 3 m papr and powered se 400 are extremely high , infinite in our tests . the osha value for the loose - fitting papr is 25 ; we obtained values of 1.1 for the racal , 4 for the centurion , and infinity for the 3 m hood . first is that contaminant concentration inside the respirator can be nonuniform , and , thus , the measurement can be dependent upon location . recent studies on flow visualization inside respirator facepieces have shown that flow pathways can twist and curl , with clear delineation between contaminant - filled air and clean air over very short distances . placing a contaminant - detection probe in a stagnant zone could yield measurements that probably underestimate contaminant concentration . placing the probe in the flow pathway , where contaminant concentration might be particularly high , could overestimate average concentration in the facepiece . present practice is to place the probe in front of the mouth ; this somewhat corrects the placement problem , because this is the place where inhaled air is to be drawn but still does not solve the problem of time variation in contaminant levels at the place where they are likely to be inhaled . the second difficulty with pf is that some contaminants can deposit or be absorbed in the respiratory system , thus making the respiratory airways into a contaminant filter . thus , it may not be surprising that results obtained in this study did not match those obtained with other methods . dead volume within a respirator facepiece is often thought to be detrimental because it accumulates exhaled co2 and recycles it into the next inhaled breath and limiting physical work performance . dead volume inside the facepiece , however , can be helpful if it acts as a buffer against leaked contaminants reaching the mouth . dead volume can be protective especially if air during the exhalation phase of breathing purges the enclosed air of contaminants leaked during inhalation . this can happen with papr blowers , for instance , when they supply filtered air even during exhalation . protective dead volumes of some of these respirators had previously been measured . for the centurion max , that value was about 1.4 l , and for the racal airmate , it was close to zero . dead volume of the frm 40 is about 1.0 l , and the other two tight - fitting facepieces were presumed to have about the same amount . the protective dead volume of the 3 m hood was measured on a mannequin by connecting the inlet port at the mannequin mouth to a vacuum hose and the inlet hose from the blower was closed off . mouth flow was recorded for the length of time for the fog to reach the mouth . as expected , fog entered the facepiece from below the ear and chin along the neck . the total volume of air inhaled before the fog reached the mouth was 2 l. how large should protective dead volume be ? it could be made large enough so that no contaminated air would reach the mouth even under extreme circumstances . inhaled tidal volumes during physical exertion are normally in the 1.5 l range , sometimes reach 2.0 l , and only rarely exceed 2.5 l. protective dead volumes greater than 2.53.0 l should then be at least as effective as continuous positive pressure in the face piece in providing wearer protection , as long as the blower can purge the dead volume during the exhalation phase of the breathing cycle . dead volumes larger than 2.53.0 l require larger blower flow rates and may be unnecessary and undesirable . comparing results from this and other recent fog flow visualization studies shows that protective respirator dead volume measurements are generally in agreement no matter what procedure is used . using flow visualization with a breathing machine gave an inhaled volume before fog reached the mouth of about 1.4 l for the centurion max papr . flow visualization with human subject breathing gave 1.1 l before fog reached the mouth for the same respirator . it is known that peak respiratory flows can often exceed blower flows [ 2 , 49 ] . there has also been concern expressed when the pressure inside a respirator facepiece intended to be positive pressure that becomes negative momentarily . as long as positive pressure is maintained , it is asserted , any leakage would flow from inside the facepiece to the outside , and contaminated air would not enter the facepiece . if papr blowers and batteries were made powerful enough to perform up to peak flow levels , bulk of the devices would probably increase greatly , and the extra weight could reduce work performance . an alternate strategy , one that has just recently been realized , is to ensure a large enough protective dead volume that any contaminants entering the facepiece do not reach the mouth . without accounting for exhalation air coming from the mouth , blower flow rate does not need to be any larger than that required to purge the dead volume of contaminants during the exhalation phase , so long as the distribution of blower air is wide enough to sweep the entire dead volume . taking exhaled air into account reduces required blower flow rate even further , at least as far as contaminants are concerned . the net result is that peak inhalation flow rates do not have to be met by the blower as long as stored clean air is available inside the facepiece . nevertheless , results in this and other recent experiments have shown that it is not necessary to maintain positive pressure inside the facepiece at all times just as long as contaminated ( leakage ) air never reaches the mouth . the blower must be able to remove contaminated air from the protective dead volume before the next negative pressure incident . many of the newer hood - type loose - fitting respirators use the protective dead volume principle , and so can be considered at least as protective as aprs and tight - fitting paprs . they have the advantage over aprs in that there is no high resistance to breathe through but have the disadvantage compared to tight - fitting paprs that , should the blower be inoperable , there is no contaminant protection afforded . the tracer gas used in this study was co2 , and we did not allow co2 to challenge the filter ; each filter inlet was supplied by clean air . hence , perfect filter efficiency was assumed , and we were mainly interested in respirator leakage . if a different gas was used , one that the filter should remove , then the inlet to the filter could be in contact with the same atmosphere that surrounds the respirator under test . clayton et al . calculated respirator protection factors for human wearers while they simulated asbestos removal operations . they used a method similar to that used in the present study , except that their subjects worked in a chamber containing a small concentration of sodium hexafluoride instead of carbon dioxide . they also continuously measured sf6 concentrations inside and outside the respirator , and thus , could measure protection factors as they varied throughout the breathing cycle . in the present study , we were more interested in knowing how much of the contaminated air was actually inhaled , so inhaled contamination ( co2 ) was collected when it was expelled from the breathing machine . obviously , co2 could not be used as a test gas with human test subjects ; sf6 or ch4 might be a better choice . however , collecting exhaled gas and determining contaminant levels there rather than monitoring contaminant levels inside the respirator facepiece give the actual protection factor experienced by the wearer as compared to the respirator protection factor ( insofar as there is no gas absorbed in the respiratory system ) . the average concentration of tracer gas in the exhaled breath would not be expected to equal the average concentration inside the facepiece . rather , the average exhaled - breath concentration reflects the concentration of contaminant actually inspired . this means that regions of high flow leading to the mouth are weighted substantially more than regions of nearly - stagnant flow . in this respect , measurement of exhaled - breath concentration is an honest measure of the exposure of the wearer . one reason for this , of course , is that some facial configurations result in large leaks , and thus , lower protection factors . however , results from this study indicate another possible cause , and that is flow pathway of contaminated air . different facial configurations could channel leakage flows differently in different people . depending on the exact position of the particle counter used in those studies [ 1315 ] , the counter could register higher average values or lower average values when contaminants were drawn into the respirator facepiece . although several of these studies were conducted with half facepiece or filtering facepiece respirators [ 1317 ] , there is no reason to suspect that preferential flow pathways discovered in studies with loose - fitting paprs or tight - fitting aprs are not also present in other types of respirators . facial configuration , especially nose protrusion , could easily affect leakage flow pathway to the mouth . calculation of net overbreathed volume as the integral of the difference between mouth flow and blower flow depends on the assumption that all the blower flow is captured within the facepiece . likewise , the statement made earlier that the blower needed to supply a flow rate no larger than the facepiece dead volume divided by the exhalation time is contingent upon no blower flow escaping the face piece before it sweeps the facepiece . it is likely that some blower flow escapes directly to the outside , either through leaks or through the exhalation valve . this represents inefficient use of blower capacity . at present , there are no known published measurements of ineffectual blower flow , but these measurements are able to be made with the same method as used in this experiment . if contaminated air can leak from outside the facepiece into the inhaled breath , then blower air can flow directly out of the facepiece without contributing to clean air in the protective dead volume . this indicates that most of the air propelled by the blower does not contribute to the volume of air inhaled and is consistent with other data relating to protection factor in this study and previous studies . the 3 m breathe easy tight - fitting papr has a blower effectiveness of about 1.0 , which indicates that nearly all of the blower flow contributes to inhalation . the same is true for the se 400 with blower turned off and frm 40 apr ; nearly all the air flowing through the blower or filter pathway contributes to inhaled air . when the se 400 blower was turned on , positive pressure is maintained in the facepiece at least most of the time , and some of the blower air leaks out , probably through the exhalation valve . blower effectivenesses for both the centurion max and 3 m hood were greater than 1.0 . in both of these cases , there apparently was enough protective dead volume that air supplied by the blowers during the exhalation phase of breathing contributed to inhaled volume . in these two cases , blowers conform to the recommendation we made that the blower need not supply all inhaled air volume but it does need to purge dead volume air during the exhalation interval . thus , the blower can contribute to inhaled air volume even during exhalation , although data in table 1 indicates that not all contaminant is evacuated . the flow situation inside the respirator face piece is complex , involving air leaking in as well as out simultaneously . breathing is periodic and not steady , and flow pathways inside the face piece most likely change between inhalation and exhalation , and probably over time within breathing phase as well . if significant physical movement accompanies work performance , then the respirator can shift on the face , or in the case of the hood , the volume of air inside the hood covering the torso can change greatly . under extremely difficult breathing conditions , the facepiece itself can deform , which changes the boundary of the flow domain . at this point , successful determination of flow dynamics within a respirator has not been accomplished , to our knowledge . these results demonstrated that there was a large range of wearer protection factors among different types of respirators . some of the respirators tested provided little to no protection to the wearer , whereas others provided extremely high amounts of protection . the results also showed that blowers have multiple contributions to papr performance , including providing inhalation air , cleaning contaminant from the respirator facepiece , and perhaps adding to papr leakage . the concept of blower effectiveness was introduced as a way to rate the ability of the blower to supply clean inhalation air to the wearer .
this experiment was conducted to determine how much contaminant could be expected to be inhaled when overbreathing several different types of respirators . these included several tight - fitting and loose - fitting powered air - purifying respirators ( paprs ) and one air - purifying respirator ( apr ) . co2 was used as a tracer gas in the ambient air , and several loose - and tight - fitting respirators were tested on the head form of a breathing machine . co2 concentration in the exhaled breath was monitored as well as co2 concentration in the ambient air . this concentration ratio was able to give a measurement of protection factor , not for the respirator necessarily , but for the wearer . flow rates in the filter / blower inlet and breathing machine outlet were also monitored , so blower effectiveness ( defined as the blower contribution to inhaled air ) could also be determined . wearer protection factors were found to range from 1.1 for the racal airmate loose - fitting papr to infinity for the 3 m hood , 3 m breath - easy papr , and se 400 breath - responsive papr . inhaled contaminant volumes depended on tidal volume but ranged from 2.02 l to 0 l for the same respirators , respectively . blower effectiveness was about 1.0 for tight - fitting aprs , 0.18 for the racal , and greater than 1.0 for two of the loose - fitting paprs . with blower effectiveness greater than 1.0 , some blower flow during the exhalation phase contributes to the subsequent inhalation . results from this experiment point to different ways to measure respirator efficacy .
1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions
a blower effectiveness greater than 1.0 indicates that some blower air contributed during the exhalation phase would be inhaled in the subsequent inspiration . it was intended that results from this approach could be a better assessment of protection factors actually experienced by the wearer of the respirator . because this test used carbon dioxide as the tracer gas , it had to be conducted on a head form , but it did give quantitative assessments of wearer protection factors and blower effectivenesses . when co2 concentration had reached its final steady - state value , this concentration was used as the value in exhaled air , and , by inference , inhaled air . respirators tested were the racal airmate 3 ( racal , frederick , md , usa ) loose - fitting papr , breathe easy ( 3 m , st . paul , minn , usa ) tight - fitting papr , butyl head cover with cape , # 522 - 02 - 23 ( 3 m ) loose - fitting hood , centurion max ( martindale protection ; thetford , norfolk , uk ) multipurpose loose - fitting papr with scarfs in place , se 400 ( sea , meadowlands , pa , usa ) breath - responsive papr , and frm 40 ( 3 m ) air - purifying respirator . the co2 ratio in table 1 is the ratio of co2 concentration measured in the captured exhaled air ( cco2,exh ) divided by the co2 concentration in the ambient air in the chamber surrounding the respirator ( cco2,amb ) . blower effectiveness is the ratio of the blower contribution to inhaled air ( vbl , inh ) to total blower volume ( vbl , tot ) during the inhalation portion of the breathing cycle ( 4)eff = vbl , inhvbl , tot . in figure 2 are shown breathing machine flow rate , blower flow rate , and inhaled leakage volumes ( labeled inhaled contaminant volumes ) for the racal airmate 3 loose - fitting papr . this figure is intended to show that blower flow rate for this respirator is almost constant , and that inhaled contaminant volume ( integrated breathing machine flow rate times co2 concentration in the captured exhaled breath ) is slightly more than 2 l for a breathing machine tidal volume of 2.4 l. figure 3 is similar to figure 2 but shows that blower flow rate for the 3 m breathe easy tight - fitting papr varies throughout the inhalation phase of breathing . if the respirator was operated in an atmosphere containing a tracer gas , but the supply of air to the respirator through the filter circuit was free of tracer gas , then the only means for the gas to enter the facepiece and be inhaled was if the gas leaked inward from some path different from the filter circuit . with a protection factor nearly equal to 1.0 , the racal loose - fitting papr gives almost no protection against airborne contamination ; concentration of contaminant inside the respirator shield is nearly the same as outside the shield . when the blower was turned off , flow through the blower was much lower , and the respirator operated as an air - purifying respirator ( apr ) . these were the 3 m hood , 3 m papr , and se 400 papr . even if the power fails on the se 400 , the 3 m hood was , in effect , a loose - fitting respirator , yet had enough dead volume within its enclosure that contaminants did not reach the mouth . the osha value for full - face piece tight - fitting papr is 1000 ; our results for the 3 m papr and powered se 400 are extremely high , infinite in our tests . the osha value for the loose - fitting papr is 25 ; we obtained values of 1.1 for the racal , 4 for the centurion , and infinity for the 3 m hood . dead volume of the frm 40 is about 1.0 l , and the other two tight - fitting facepieces were presumed to have about the same amount . inhaled tidal volumes during physical exertion are normally in the 1.5 l range , sometimes reach 2.0 l , and only rarely exceed 2.5 l. protective dead volumes greater than 2.53.0 l should then be at least as effective as continuous positive pressure in the face piece in providing wearer protection , as long as the blower can purge the dead volume during the exhalation phase of the breathing cycle . many of the newer hood - type loose - fitting respirators use the protective dead volume principle , and so can be considered at least as protective as aprs and tight - fitting paprs . the 3 m breathe easy tight - fitting papr has a blower effectiveness of about 1.0 , which indicates that nearly all of the blower flow contributes to inhalation . the same is true for the se 400 with blower turned off and frm 40 apr ; nearly all the air flowing through the blower or filter pathway contributes to inhaled air .
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detecting primary crc and crlm at an early stage results in better outcomes . at a molecular level , crc consists of a heterogeneous group of diseases with molecularly , as well as clinically , distinct tumors based on the primary site of origin ( eg , colon vs rectal , and right - sided vs left - sided ) . chromosomal instability , deficient mismatch repair ( dmmr ) with resultant microsatellite instability ( msi ) , aberrant dna methylation , as well as altered molecular signaling pathways all have been described in the transformation from normal mucosa to adenocarcinoma.12 , 13 , 14 , 15 , 16 the role of biologics in the adjuvant treatment of resected primary crc has been evaluated , including cetuximab for kirsten rat sarcoma viral oncogene ( kras ) wild - type cancers and the vascular endothelial growth factor inhibitor bevacizumab ; however , these targeted treatments have not shown the benefit seen in the metastatic or advanced setting.17 , 18 , 19 more recently , those altered pathways and mutations have been used for therapy modification and patient stratification in metastatic crc based on the sidedness of the primary tumor , supporting the use of different biologic agents for distinct primary biology underlying the disease.17 , 18 , 19 chromosomal anomalies with demonstrated importance in tumorigenesis , including dna gains or losses , result in changes in gene expressions that might lead to a differential response to chemotherapeutic agents . this recently was studied in an analysis of cell - free dna ( cfdna ) showing acquired resistance to anti epidermal growth factor ( egfr ) therapies , as well as recent investigations reporting a correlation between dna copy number losses and an association with response to fluorouracil ( 5-fu ) , irinotecan , and capecitabine . given the extensive molecular and clinical heterogeneity of crc , it is essential to individualize therapy on the basis of molecular profiling to avoid treatment - related toxicities without a realized survival benefit . some of the strongest data to support the need for identification of high - risk cohorts among patients with crlm come from adjuvant trials for primary crc . the 2004 adjuvant the multicenter international study of oxaliplatin/5-fluorouracil / leucovorin in the adjuvant treatment of colon cancer ( mosaic ) trial assessed the impact of an oxaliplatin - containing systemic regimen ( folinic acid , 5-fu , and oxaliplatin ) for patients with resected primary crc compared with 5-fu alone in patients with stage ii and iii disease . a significant survival benefit for patients with stage iii disease was found and has been maintained in recently updated 10-year results . however , these benefits come with significant morbidity impacting patient quality of life . for patients with stage iii crc treated with folinic acid , 5-fu , and oxaliplatin , instead of 5-fu and leucovorin ( lv ) , there is a consequent 4% decrease in mortality . however , to achieve this 4% reduction in mortality with oxaliplatin , 92% of those patients will suffer from treatment - associated peripheral neuropathy , with approximately 15% experiencing permanent neuropathy when followed up longitudinally for 2 years . it is clear that even among patients with stage iii disease there is an underappreciated disease heterogeneity that at present is being treated with an often - homogenous systemic approach . these data in the primary crc setting underscore the need for molecularly driven systemic treatment to avoid both the financial and quality - of - life costs to patients with liver - only metastatic crc . work is ongoing to identify molecular subsets of patients with crlm to personalize targeted treatments to maximize therapeutic interventions . in this review , we describe the role of liquid biopsies ( ie , analyses of tumor cells or tumor derived material that is circulating in the blood ) along with novel cancer and immunologic cell populations to both surveil and assess treatment response in patients with crlm . we also propose using this information to guide the design and development of therapeutic strategies for liver - directed treatments . for patients with liver - only metastatic crc , there is a pressing need for a more robust molecular characterization of the primary and metastatic lesions to direct perioperative management of patients at highest risk for disease recurrence . in the primary disease setting , the focus has been directed toward patients with high - risk stage ii crc those patients with negative lymph nodes but other high - risk features such as t4 lesions , obstruction or perforation , cancers with lymphovascular invasion , and poorly differentiated histology . one of the early investigations on the impact of adjuvant treatment on stage ii crc was the 2007 quick and simple and reliable ( quasar ) trial , in which patients with stage ii crc were randomized to treatment with adjuvant 5-fu / lv or observation after curative resection of their primary cancer . the results of this trial showed an approximate 3% improvement in outcome when 5-fu / lv was given in the adjuvant setting . in other words , 97% of patients were exposed to chemotherapy without any benefit . because standard stage ii patients do not benefit from adjuvant therapy as shown in the quasar , mosaic , and other trials , it is currently at the discretion of the treating clinician to decide if the high - risk features of the patient s primary crc support adjuvant treatment.22 , 23 , 26 according to the current national comprehensive cancer network guidelines the definition of high - risk stage ii colon cancer is clearly inadequate , because many patients with high - risk features do not have a recurrence whereas some patients deemed to be average - risk do . furthermore , no data point to features that are predictive of benefit from adjuvant chemotherapy , and no data correlate risk features and selection of chemotherapy in patients with high - risk stage ii disease . the challenges of selecting patients with high - risk stage ii crc are remarkably similar to and parallel the issues of directing perioperative treatment in patients with crlm . aside from the pathologic risk factors of stage ii crc , several investigators have sought a correlation between discrete gene signatures and higher - risk patient populations to stratify patients molecularly and to better direct adjuvant therapy . efforts to improve patient stratification have been ongoing through comprehensive molecular characterization of hypermutated genes , microsatellite instability , and hypermethylated genes to characterize colon cancer stages into subtypes to better predict outcomes as well as to further refine chemotherapy selection.28 , 29 , 30 rodriguez et al reported that loss of corticotropin - releasing hormone receptor-2 expression in crc specimens a well - characterized neuropeptide that participates in the regulation of intestinal inflammation based on clinicopathologic data they were able to show that a decreased expression of corticotropin - releasing hormone receptor-2 in patients with crc was associated with an increased risk of distant metastasis and a worse 5-year survival after initiation of treatment . in 2016 , dalerba et al reported on the expression of the caudal - type homeobox transcription factor 2 ( cdx2 ) , a critical regulator of intestinal development and oncogenesis , as a prognostic biomarker in patients with stage ii crc . their work combined insights from basic science discoveries in normal colon stem cells and cancer stem cells , the availability of public databases of sequenced tumors ( national center for biotechnology information gene expression omnibus and national cancer institute cancer diagnosis program ) , and the power of bioinformatics to query more than 2329 human samples . they identified 16 genes that were not present in colorectal epithelia that expressed high levels of the cancer stem cell marker , activated leukocyte cell adhesion molecule ( alcam or cd166 ) . of these genes , they focused on cdx2 , a protein already identified for pathologic assessments of resected crc specimens . in a discovery data set of patients with stage ii crc and a subsequent validation data set , the investigators went on to show that patients with cdx2-negative tumors had a worse 5-year disease - free survival ( dfs ) compared with patients with cdx2-positive cancers ( 49% among 15 patients with cdx2-negative tumors vs 87% among 191 patients with cdx2-positive tumors ; p = .003 ) . furthermore , using a discovery data set of stage iii crc tumors , investigators identified an association of cdx2 expression and the treatment of adjuvant therapy with survival . again , these findings were validated in a larger data set , in which the investigators found an increased 5-year dfs in patients treated with adjuvant chemotherapy in stage ii cdx2-negative tumors vs individuals who did not undergo adjuvant chemotherapy ( 91% vs 56% ; p = .006 ) . although the study population was small , these data show an identifiable profile in crc patients who may achieve a survival benefit from adjuvant treatment that outweighs the treatment - associated morbidity . this work was updated most recently in the metastatic crc population,33 , 34 in which patients with cdx2-negative metastatic crc were found to have a median os of 8 vs 39 months in individuals with cdx2-positive metastatic crc ( hazard ratio , 4.04 ; 95% confidence interval , 2.496.54 ; p < .0001 ) . cdx2-negative patients were more likely to have right - sided primary tumors , poorly differentiated cancers , distant lymphatic metastasis , and be women . although the prevalence of cdx2-negative disease is low , these insights continue to stratify a subgroup of patients with advanced crc who would derive a dfs benefit from adjuvant treatment after curative hepatic resection of their disease . the continued focus to elucidate the underlying biology driving disease recurrence in more diverse and larger subsets of patients will clarify the effective treatment for patients at all stages of disease . the majority of patients who have had an attempted curative hepatic resection of crlm will have recurrence of their disease . historically , several clinicopathologic factors ( nodal status of the primary cancer , preoperative carcinoembryonic antigen [ cea ] level , size of the largest liver lesion , and the number of hepatic metastases ) have been shown to be independent predictors of both poor outcomes and intrahepatic recurrence of disease in patients with resected crlm and collectively comprise the clinical risk score . similar to the tumor characteristics in patients with clinically high - risk stage ii crc , these factors unfortunately provide a limited description of the disease . in addition to the prediction models , oncologists now are using mutational data in the egfr pathways to select and treat patients who are most likely to respond to a given regimen ( kras mutation status predicting poor response to anti - growth factor receptor therapies35 , 36 ) and braf mutation status ( conferring resistance to anti - egfr therapy given beyond first - line treatment and associated with an increased risk of peritoneal disease).37 , 38 , 39 , 40 recent work has explored deriving cancer gene expression profiles as prognosticators of recurrence and survival for patients with crlm . balachandran et al reported a gene - expression classifier to correlate disease - specific survival as well as liver dfs in patients with resected crlm . by using gene expression microarray on resected crlm the investigators were able to identify and validate 20 genes that were associated with os . importantly , this so - called molecular risk score was shown to be an independent prognosticator of dfs , unlike the traditional clinical risk score . these findings suggest methods for identifying patients with high - risk primary crc and resected crlm who are at risk of recurrence and may benefit from directed and potentially prolonged adjuvant treatment . further identification of patients with molecular subsets of crlm that underlie discrete tumor biology , and subsequently predict treatment response and improve os , are essential to realize the benefit of perioperative treatment with both biologic and cytotoxic therapy . in patients with crlm who undergo a hepatic resection with curative intent , it is estimated that approximately 75% of all recurrences both intrahepatic and extrahepatic occur within the first 2 years after surgery . efforts over the past decades have sought to address the risk of recurrence , which is possibly the result of treatment - resistant micrometastatic disease . one avenue to obliterate micrometastatic disease in the liver focuses on maximizing locoregional therapy by exploiting basic tumor biology . cancer cells from gastrointestinal malignancies , especially crc , hematogenously spread via the portal circulation , often making the liver the first site of metastasis . once hepatic metastases grow to more than 2 mm in size , they derive their blood supply from the hepatic artery , while normal hepatocytes are perfused mostly from the portal circulation . understanding this biologic difference has led to treating select patients with crlm using hepatic arterial infusion ( hai ) therapy . this intense locoregional treatment is based on the extraction of chemotherapy from the hepatic arterial circulation , resulting in high local drug concentrations with the goal of minimizing systemic toxicity . the ideal agent should have a high dose - response curve , high extraction , and rapid total body clearance once the infusion is discontinued . of the various agents studied , hai - delivered floxuridine approximates this ideal with a short half - life ( < 10 min ) and more than 90% hepatic extraction , resulting in a 16-fold higher concentration in hepatic tumors compared with venous administration.42 , 43 by using floxuridine in combination with dexamethasone , patients with crlm can have their liver disease maximally treated with modest side effects compared with standard systemic treatment . several prospective trials45 , 46 , 47 , 48 have investigated using hai alone to circumvent the toxicity associated with systemic treatment of crlm , to maximize hepatic response in an effort to improve both os and progression - free survival , and potentially improve the patient s quality of life.49 , 50 the role of hepatic arterial infusion for patients with resected crlm initially was tested without concurrent systemic therapy , which at the time of the initial trials did not include modern systemic agents such as oxaliplatin and irinotecan . to date , there have been no prospective randomized controlled trials comparing adjuvant hai with modern systemic therapy vs modern systemic therapy alone in patients with resected crlm . in 2016 , kemeny et al reported on an analysis of 4 consecutive hai adjuvant trials for patients with resected crlm from 1991 to 2009 ( n = 287 ) . the patients were divided into 2 groups : those treated before and after 2003 , corresponding to the incorporation of modern systemic oxaliplatin or irinotecan - containing regimens . with a median follow - up period of 11 years , the investigators reported that patients treated after 2003 had a 5- and 10-year os of 78% and 61% , respectively , with the median survival not being reached . patients treated before 2003 had a 3- and 5-year dfs of 42% and 41% , respectively . taken together , these data support that properly selected patients with crlm can have hepatic resection of their disease followed by adjuvant systemic therapy plus hai and achieve a 5-year survival as high as 78% . however , similar to toxicity associated with systemic therapy , treatment with hai has risks , including biliary sclerosis in less than 5% of patients , that needs to be balanced with the anticipated benefit of treatment . for treatments such as hai that seek to maximally treat the liver , it is imperative to begin integrating preoperative prognostic indicators that are predictive of a patient s risk of intrahepatic recurrence after hepatic resection to select patients for intensive treatment regimens . ultimately , we must develop a noninvasive test to determine the risk for both local and distant recurrence of disease with monitoring the response to systemic therapy as well as an early signal for intrahepatic recurrence . in addition to discrete gene expression profiles that identify patients at high risk of recurrence , blood - based biomarkers for noninvasive monitoring of early detection of recurrent disease actively in development may serve as a more reliable marker to monitor response to treatment , and ultimately to monitor patients for recurrence of disease after curative treatment . although newly identified gene signatures can identify at - risk patient populations for defined treatment regimens , a second approach for improving patient survival is in developing biomarkers with enhanced specificity and sensitivity for early detection of primary and recurrent disease . the goal of this approach is to identify recurrent or persistent disease at a point when traditional clinical indicators , such as radiographic signs , still are negative , and to treat or alter treatment of disease at the earliest time point this almost certainly will improve overall disease control . genetic material sourced from blood - based material originating from primary and/or metastatic lesions can be used to inform noninvasive , blood - based biomarker discovery , and provide a nuanced view of the disease specifically the temporal evolution of disease over treatment to facilitate tailored therapy . the gold standard for a noninvasive early diagnosis test is the fecal occult blood test , but it has sensitivity limitations . detection of the plasma - based factor cea also widely is used to monitor disease status longitudinally in patients with treated crc , but it also has limitations . to improve specificity and sensitivity , tumor biologists are pursuing a new generation of blood - based biomarkers that have correlative or biologic value , as well as providing tumor genomic information on which to alter treatment . although still in development , these new factors , including new populations of circulating tumor cells ( ctcs ) , cfdna , micro - rna ( mirna ) , and exosomes have the potential for the further development of critical assays to surveil patients with crc and intervene at times that may improve disease control.53 , 54 conventionally isolated ctcs , defined by cell surface expression of epithelial cell adhesion molecule ( epcam ) or cytokeratin ( ck ) , and the absence of the pan - leukocyte marker , cd45 expression , have been shown to correlate with progression - free survival and os in patients with colorectal cancer , prostate cancer , and also with breast cancer . although these data are predictive of prognosis , ctcs are rare entities in the circulation , and , more importantly , they have failed to provide biologic insights that may guide informed therapeutic treatment . standard ctc detection methods rely on the expression of specific epithelial markers , ck and/or epcam , and the exclusion of leukocyte - specific markers , typically cd45 . ctcs also have been isolated based on size , density , charge , or various other properties that positively or negatively enrich a specific cell population . cellsearch ( janssen diagnostics , raritan , nj ) is the food and drug administration approved test to detect ctcs by magnetic separation of epcam cells followed by positive staining for ck and negative staining for cd45 . these existing approaches bias the subsets of ctcs captured , and may be excluding biologically relevant subpopulations . for example , zhang et al showed the high metastatic capability of an epcam ctc population isolated from patients with breast cancer in a mouse xenograft assay . this epcam ctc population may represent cancer cells that have undergone epithelial - to - mesenchymal transition , thereby losing expression of epcam , and therefore represent a more migratory and invasive cell . indeed , in crc , ctcs that have lost epcam expression or gained n - cadherin , vimentin , or fibronectin expression are hypothesized to have undergone epithelial it reasons that to gain their full metastatic potential , tumor cells must cross several cellular barriers to travel through the circulation and seed metastatic sites . such cells appear to have adapted by loss of epithelial differentiation and acquisition of advantageous phenotypes to modulate and balance differentiation , self - renewal , and homeostasis in the selected environment.53 , 54 an additional population of ctcs that have not been well studied are those expressing the leukocyte marker cd45 . peripheral blood cells from cancer patients , isolated by differential centrifugation and size exclusion , were found to harbor ctcs that expressed ck and cd45 , yet conferred robust growth in culture . in addition , cd45ck ctcs were identified in patients with metastatic pancreatic cancer from an epcam - enriched population , and in metastatic breast cancer patients , even with partial cd45 depletion with magnetic beads . interestingly , the breast cancer cd45 ctcs also expressed the macrophage marker cd68 , indicating that ctc populations may acquire proteins typically expressed by macrophages , possibly through a cell fusion mechanism . to fully appreciate these cd45 ctcs , direct visualization would rule cancer - immune cell clusters that could be construed as a cd45 ctc by flow cytometry . if these cells arise from leukocyte - cancer fusion , this novel tumor biology may provide important insights that may guide therapy more effectively . ongoing work is directed at investigating how these untapped and uninvestigated populations of ctcs potentially can contribute to our overall knowledge of disease and are being developed in parallel with the rapid advancements in other biomarker fields , such as that of cfdna . cfdna is hypothesized to arise from cells that die , whether by necrosis , cell lysis , or apoptosis , releasing naked dna into the circulation and creating a residual fingerprint . although cfdna was first detected in healthy individuals in the late 1940s , it was not until the 1970s1980s that neoplastic characteristics were identified and that cfdna was found to exist in higher concentrations in cancer patients relative to healthy controls.65 , 66 although quantification of cfdna was useful in some disease states when used alongside classic blood tests ( eg , cea ) , cfdna is being developed for the identification of gene mutations and microsatellite instability in early detection assays . current technologic advancements in amplifying dna and in sequencing supports the relevance of this biologic material . for example , de kok et al showed concordance of kras point mutations between primary tumors and serum cfdna amplified by polymerase chain reaction in 14 crc patients . in addition to point mutations , microsatellite abnormalities have been detected in patient blood from breast cancer , head and neck cancer , lung cancer , melanoma , and crc.69 , 70 , 71 , 72 isolated cfdna has many characteristics of tumor dna , including the presence of oncogenes and other global molecular classifiers such as msi , cpg island methylator phenotype , and chromosomal instability . el messaoudi et al conducted a multiparametric analysis correlating cfdna with os in metastatic crc patients ( n = 97 ) . higher cfdna levels were associated with a statistically significant decrease in os ( 18.07 vs 28.5 mo ; p = .0087 ) . furthermore , on multivariate analysis the investigators showed that a higher cfdna level is an independent prognostic factor ( p = .034 ) and that high levels of cfdna fragmentation were associated with decreased os in the mutant kras / braf population . newer technologies under development such as the plasmaselect assay ( personal genome diagnostics , baltimore , md ) allow for the identification of multiple mutations and genetic alterations resulting in a comprehensive genomic analysis of the tumor and the potential to track tumor evolution across treatment . analyses of cfdna along with evaluation of novel ctc populations have great potential to provide novel noninvasive approaches to diagnosis cancer , facilitate early detection of disease and recurrent disease , assessment of the evolving tumor biology , and provide a foundation for tailored treatment . aside from cfdna and ctcs , there are active investigations into tumor - derived or tumor microenvironment - derived exosomal stable mirnas that are released into the vasculature , glandular secretions , or waste excretions.77 , 78 , 79 , 80 mirnas are a small , recently discovered class of highly conserved noncoding rnas that play key roles in the regulation of gene expression . in their transcriptional regulation , mirnas possess differential short nucleotide length sequence complementarity to confer combinatorial diversity to affect hundreds of targets in similar gene networks . in this framework , a single mirna exists in reciprocal inhibition with an entire network of functionally related genes . thus , baseline activity or change in exosomal mirnas provides a molecular snapshot of intracellular activity from their tissue of origin . additional information can be derived because the presence of exosomal mirnas dictates potential paracrine and endocrine roles that have been observed in a number of malignancies including crc.81 , 82 , 83 properties of exosomal mirnas have been highlighted in recent clinical studies to ascertain their utility as novel minimally invasive biomarkers.84 , 85 , 86 , 87 in crc , ogata - kawata et al performed mirna microarray analyses on serum exosomes derived from 88 crc patients compared with healthy controls and identified a subset of 7 up - regulated diagnostic mirnas that outperformed conventional cea utility for surveillance of crc recurrence . in crlm , a similar study was performed in serum exosomes from both the liver metastases subgroup and the nonmetastatic group at different time points of treatment and resection . microarray analyses showed a distinct subset of 6 tumor - derived exosomal micro rnas ( mirna or mir ) , which were synchronized with liver metastasis development . among this subset , exosomal mir-19a was found to be up - regulated compared with normal healthy volunteers , and the level of exosomal mir-19a was found to correlate with more aggressive disease including nodal involvement , liver metastases , and higher tnm stage . taken together , these data provide a glimpse in the acquisition and analysis of exosomal mirna diagnostic and predictive biomarkers in primary and metastatic crc . over the past 5 years , there has been burgeoning interest and now demonstrated efficacy in exploiting the tumor immune microenvironment as a viable and effective treatment option for many cancers that previously had been recalcitrant to treatment . it has become increasingly clear that the tumor microenvironment plays a key role in tumor progression and response to therapies across many different cancer types . immune check - point inhibitors such as ipilimumab , pembrolizumab , and nivolumab are being widely studied in prospective trials in a variety of cancers , including in patients with liver - only metastatic crc . several recent studies have highlighted the role of non - neoplastic cells , particularly stromal cells and immune cells , as prognostic markers in human crc.91 , 92 , 93 , 94 immunologically , it has been shown that dmmr cancers harbor infiltrating tumor lymphocytes that actively are suppressed by immune - inhibitory signals such as the programmed death ( pd ) ligand-1 and pd-1 complexes . in 2015 , le et al reported the results of a phase ii trial of patients with treatment - refractory progressive metastatic cancer treated with the anti the results for 32 patients with metastatic crc were stratified by dmmr crc compared with patients with proficient mmr tumors . for patients with dmmr crc , the immune - related objective response and immune - related progression - free survival were 40% and 78% , respectively , as compared with 0% and 11% , respectively , for patients with proficient mmr crc . these findings currently are being evaluated in the keynote-177 trial in patients with msi - high or dmmr metastatic crc who have been randomized to treatment with pembrolizumab vs standard therapy . specific features of the tumor microenvironment such as an abundance of t - helper ( th ) 2 cytokines , proinflammatory molecules , pro - angiogenic molecules , and profibrotic molecules are immunosuppressive and considered protumorigenic . in contrast , an abundance of th1 cytokines , angiostatic factors , and immunostimulatory molecules , along with the mobilization and reinvigoration of the cd8 t cells , all are characteristic of a robust antitumorigenic microenvironment . therefore , understanding the recruitment and function of leukocytes in the cancer will enable the development of both targeted therapies and biomarkers that can predict emergence of treatment resistance and recurrence of cancer . interestingly , there are several nuances that dictate the function of even the same leukocyte subsets in different cancers.97 , 98 for example , protumorigenic macrophages are regulated by th2-cd4 t cells in mammary carcinomas and b cells in pancreatic adenocarcinomas and squamous cell carcinomas . similarly , the soluble mediators and signaling pathways that regulate this cellular cross - talk also are different , with il4/il13 and colony stimulating factor-1 playing key roles in driving macrophage function in mammary carcinomas whereas bruton tyrosine kinase and phosphoinositide 3-kinase regulating the macrophage function in pancreatic and squamous cell carcinoma . as such , it is possible that the immune checkpoint pathways also are regulated differently in different tissues , necessitating the development of tailored approaches to immunotherapy across different cancers . in this context , we propose that a multimodal biomarker - based approach would be optimal for immune - mediated cancer control and assessing treatment response . such an approach would rely on biomarkers that measure the protumorigenic and antitumorigenic factors elaborated earlier and provide multiple avenues to mobilize and reinvigorate the cytotoxic t - cell responses . this could include a combination of strategies such as neutralizing the th2 responses , cytotoxic and targeted agents , immune checkpoint blockade , vaccines , and chimeric antigen - receptor t cells . this multimodal approach also will sample the microenvironment whenever the cancers escape and recalibrate the specific reprogramming strategy , specific immune checkpoints that can be targeted , and specific pathways that drive the microenvironment so cancer regression and control can be re - established . in summary , an approach that dynamically engages the immune system by constantly sampling the microenvironment to detect recurrence and relapse is essential to incorporate into the management of patients with advanced crc and direct novel immunologic therapies . ultimately , the biology of the tumor both cell intrinsic and cell extrinsic underlies the clinical outcome for patients with metastatic crc . indeed , the concept of liver - only metastatic disease by definition implies a different biologic subtype . this difference is readily clinically apparent and our attempts to exploit this biology are the basis of all liver - directed therapy . future directions in treating patients with the crc liver - only subtype therefore must revolve around better molecular characterization and the development of improved therapeutic approaches . as liver - directed therapy continues to evolve with the development of other local treatment modalities including microwave ablation , irreversible electroporation , and transarterial radioembolization ( eg , y-90)our ability to identify and understand this biology becomes paramount . currently , clinicians use the biologic test of time to ascertain if a hepatic - only metastatic state can be maintained while first - line systemic agents are used and hence provide the rationale to attempt intensive liver - directed approaches , including hepatic resection and hai . banking tissue from these patients for molecular and clinicopathologic analyses , and correlating molecular and cellular correlates with high - quality clinical data , is essential and has become an integral aspect of modern prospective clinical trials . tissue samples collected and analyzed across the continuum of a patient s treatment are essential to facilitate discovery - based approaches to improve therapy . specimens collected at several time points ( eg , pretreatment , after each chemotherapeutic cycle , after completion of systemic therapy , and after hepatic resection ) along with tissue from the surrounding hepatic parenchyma can be analyzed for a number of genomic and cellular alterations , including mutational analysis , copy number variability , and circulating biomarkers that have the potential to shed insight into evolving tumor biology that may predict treatment response . data support that properly selected patients with crlm can have hepatic resection of their disease followed by adjuvant systemic therapy plus hai and achieve a 5-year os as high as 78% with a hepatic dfs of 62% at 5 years . the role of hai in the adjuvant treatment of patients with resected crlm additionally offers the unique opportunity to deliver novel agents in a liver - directed fashion . although floxuridine has been used for decades and represents a pharmacokinetically ideal agent , our rapidly expanding knowledge of crc tumor biology and microenvironment begs for the development and study of novel agents coupled with the rational design of clinical trials that can exploit this knowledge in a liver - targeted fashion . given the extensive molecular and clinical heterogeneity of the liver - only metastatic crc , it is of great importance to individualize targeted therapy on the basis of molecular profiling through logical implementation of biomarker assessment in liquid biopsies for early metastasis to monitor for intrahepatic recurrence . we are charged with moving beyond a blunt one - size - fits - all approach in treating patients with crlm . ultimately , we believe that molecularly driven treatments will lead to improved os , a reduction in intrahepatic recurrence , and will decrease the toxicity of perioperative therapies .
in patients with colorectal cancer ( crc ) that metastasizes to the liver , there are several key goals for improving outcomes including early detection , effective prognostic indicators of treatment response , and accurate identification of patients at high risk for recurrence . although new therapeutic regimens developed over the past decade have increased survival , there is substantial room for improvement in selecting targeted treatment regimens for the patients who will derive the most benefit . recently , there have been exciting developments in identifying high - risk patient cohorts , refinements in the understanding of systemic vs localized drug delivery to metastatic niches , liquid biomarker development , and dramatic advances in tumor immune therapy , all of which promise new and innovative approaches to tackling the problem of detecting and treating the metastatic spread of crc to the liver . our multidisciplinary group held a state - of - the - science symposium this past year to review advances in this rapidly evolving field . herein , we present a discussion around the issues facing treatment of patients with crc liver metastases , including the relationship of discrete gene signatures with prognosis . we also discuss the latest advances to maximize regional and systemic therapies aimed at decreasing intrahepatic recurrence , review recent insights into the tumor microenvironment , and summarize advances in noninvasive multimodal biomarkers for early detection of primary and recurrent disease . as we continue to advance clinically and technologically in the field of colorectal tumor biology , our goal should be continued refinement of predictive and prognostic studies to decrease recurrence after curative resection and minimize treatment toxicity to patients through a tailored multidisciplinary approach to cancer care .
Scope of the Clinical Problem for Patients With Colorectal Cancer Liver Metastasis Treatment Challenges for Patients With Liver-Only Metastases Maximizing Regional Treatment of Colorectal Cancer Liver Metastasis to Decrease Intrahepatic Recurrence Summary, Novel Molecules, and Future Clinical Trial Directions
chromosomal instability , deficient mismatch repair ( dmmr ) with resultant microsatellite instability ( msi ) , aberrant dna methylation , as well as altered molecular signaling pathways all have been described in the transformation from normal mucosa to adenocarcinoma.12 , 13 , 14 , 15 , 16 the role of biologics in the adjuvant treatment of resected primary crc has been evaluated , including cetuximab for kirsten rat sarcoma viral oncogene ( kras ) wild - type cancers and the vascular endothelial growth factor inhibitor bevacizumab ; however , these targeted treatments have not shown the benefit seen in the metastatic or advanced setting.17 , 18 , 19 more recently , those altered pathways and mutations have been used for therapy modification and patient stratification in metastatic crc based on the sidedness of the primary tumor , supporting the use of different biologic agents for distinct primary biology underlying the disease.17 , 18 , 19 chromosomal anomalies with demonstrated importance in tumorigenesis , including dna gains or losses , result in changes in gene expressions that might lead to a differential response to chemotherapeutic agents . the 2004 adjuvant the multicenter international study of oxaliplatin/5-fluorouracil / leucovorin in the adjuvant treatment of colon cancer ( mosaic ) trial assessed the impact of an oxaliplatin - containing systemic regimen ( folinic acid , 5-fu , and oxaliplatin ) for patients with resected primary crc compared with 5-fu alone in patients with stage ii and iii disease . in this review , we describe the role of liquid biopsies ( ie , analyses of tumor cells or tumor derived material that is circulating in the blood ) along with novel cancer and immunologic cell populations to both surveil and assess treatment response in patients with crlm . for patients with liver - only metastatic crc , there is a pressing need for a more robust molecular characterization of the primary and metastatic lesions to direct perioperative management of patients at highest risk for disease recurrence . efforts to improve patient stratification have been ongoing through comprehensive molecular characterization of hypermutated genes , microsatellite instability , and hypermethylated genes to characterize colon cancer stages into subtypes to better predict outcomes as well as to further refine chemotherapy selection.28 , 29 , 30 rodriguez et al reported that loss of corticotropin - releasing hormone receptor-2 expression in crc specimens a well - characterized neuropeptide that participates in the regulation of intestinal inflammation based on clinicopathologic data they were able to show that a decreased expression of corticotropin - releasing hormone receptor-2 in patients with crc was associated with an increased risk of distant metastasis and a worse 5-year survival after initiation of treatment . similar to the tumor characteristics in patients with clinically high - risk stage ii crc , these factors unfortunately provide a limited description of the disease . further identification of patients with molecular subsets of crlm that underlie discrete tumor biology , and subsequently predict treatment response and improve os , are essential to realize the benefit of perioperative treatment with both biologic and cytotoxic therapy . several prospective trials45 , 46 , 47 , 48 have investigated using hai alone to circumvent the toxicity associated with systemic treatment of crlm , to maximize hepatic response in an effort to improve both os and progression - free survival , and potentially improve the patient s quality of life.49 , 50 the role of hepatic arterial infusion for patients with resected crlm initially was tested without concurrent systemic therapy , which at the time of the initial trials did not include modern systemic agents such as oxaliplatin and irinotecan . for treatments such as hai that seek to maximally treat the liver , it is imperative to begin integrating preoperative prognostic indicators that are predictive of a patient s risk of intrahepatic recurrence after hepatic resection to select patients for intensive treatment regimens . in addition to discrete gene expression profiles that identify patients at high risk of recurrence , blood - based biomarkers for noninvasive monitoring of early detection of recurrent disease actively in development may serve as a more reliable marker to monitor response to treatment , and ultimately to monitor patients for recurrence of disease after curative treatment . although newly identified gene signatures can identify at - risk patient populations for defined treatment regimens , a second approach for improving patient survival is in developing biomarkers with enhanced specificity and sensitivity for early detection of primary and recurrent disease . although still in development , these new factors , including new populations of circulating tumor cells ( ctcs ) , cfdna , micro - rna ( mirna ) , and exosomes have the potential for the further development of critical assays to surveil patients with crc and intervene at times that may improve disease control.53 , 54 conventionally isolated ctcs , defined by cell surface expression of epithelial cell adhesion molecule ( epcam ) or cytokeratin ( ck ) , and the absence of the pan - leukocyte marker , cd45 expression , have been shown to correlate with progression - free survival and os in patients with colorectal cancer , prostate cancer , and also with breast cancer . analyses of cfdna along with evaluation of novel ctc populations have great potential to provide novel noninvasive approaches to diagnosis cancer , facilitate early detection of disease and recurrent disease , assessment of the evolving tumor biology , and provide a foundation for tailored treatment .
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s6 kinases are members of the agc serine / threonine kinases of the rsk family , exhibit high homology within their catalytic domain , and are activated by the phosphorylation of a critical residue within the activation loop by phosphoinositide dependent kinase 1 ( pdk1 ) . yeast contains one s6 kinase called sch9 , and humans contain two isoforms called s6k1 and s6k2 . s6 kinases act downstream of phosphatidylinositol ( 3,4,5)-triphosphate ( pip3 ) in the phosphatidylinositide 3-kinase ( pi3k ) pathway . phosphorylation of serine and threonine residues in the c - terminal regulatory domain leads to the phosphorylation of a s6k activation loop residue by pdk1 ( residue 252 on the longer splice variant of s6k1 ) . in addition to pdk1 , mtor is also involved in the activation of s6k1 and phosphorylates s6k1 at residue t412 . s6 kinases are associated with many cellular processes , including protein synthesis , mrna processing , cell growth , and cell survival . s6k1 and s6k2 phosphorylate and activate the 40s ribosomal protein s6 , which promotes protein synthesis through an increased rate of mrna transcription . s6k1 also regulates cell size and progression through the cell cycle , in addition to promoting cell survival by inactivating the proapoptotic protein bad . the aberrant activation of s6 kinases has been shown to play a role in many disease conditions , including diabetes , obesity , aging , and cancer . many melanoma cells harbor constitutive activation of the pi3k - akt pathway , which results in akt phosphorylation and leads to activation of the downstream targets mtor and s6k1 . treatment with rapamycin , an allosteric mtor inhibitor , leads to significant dephosphorylation of s6k1 and decreased cell growth . however , treatment with mtor inhibitors abrogates feedback inhibition of other pathways , which in part leads to side effects such as hyperglycemia , hypercholesterolemia , and hyperlipidemia . because of this , inhibition of s6k1 represents an alternative therapeutic strategy that may bypass the limitations of mtor inhibition . we have previously reported on the development of atp competitive organometallic kinase inhibitors with high potency and specificity . these inhibitors are structurally inspired by the class of indolocarbazole alkaloids , such as staurosporine , but use a transition metal ion that coordinates up to six ligands to replace the carbohydrate moiety of staurosporine . the scaffold design includes a bidentate ligand that is able to target the metal complexes to the atp - binding site . this mimics atp and conventional indolocarbazole inhibitors , while the increased size of the bulky transition metal complex allows for exploration of additional chemical space at the edges of the atp binding site specific to each kinase . despite being conventional atp - competitive inhibitors , the combination of unusual globular shape and rigid characteristic of these complexes it is worth noting that the coordinative bonds to the transition metal are considered to be kinetically stable and are expected to remain intact when exposed to the biological environment , thus avoiding metal - related cytotoxicities . however , druglikeness of such complexes , including metabolic stability , bioavailability , and pharmacokinetic properties , is not established yet and is subject to current studies . regardless , this strategy has led to the development of specific and potent kinase inhibitors for gsk3 , pim1 , pi3k , mst1 , and braf . here , we present data on the development of potent and specific organometallic s6k1 inhibitors , em5 and fl772 . we show that fl772 binds to s6k1 with an ic50 value in the single digit nanomolar range at 100 m atp and that the more potent fl772 compound has a greater than 100-fold specificity over s6k2 . crystal structures of the s6k1 domain bound to the pan - kinase inhibitor staurosporine , em5 , and fl772 reveal that the organometallic inhibitors bind in the atp binding pocket in a way that is distinct from staurosporine , likely explaining their more favorable potency and selectivity . cellular data demonstrate that fl772 is able to inhibit s6k phosphorylation in yeast cells . the data provide an important starting point for the development of s6k inhibitors for possible therapeutic applications . inhibitors for s6k1 were initially identified through millipore kinaseprofiler ( supporting table 9 ) . ten different staurosporine - inspired organometallic ruthenium complexes were screened against a diverse panel of 283 protein kinases . this screen led to the identification of em5 as a potential inhibitor of s6k1 , with 7% activity at a concentration of 100 nm in the presence of 10 m atp . em6 , a similar complex replacing the isothiocyanate functional group with an isocyanate ( figure 1a ) , inhibited significantly less , exhibiting 54% activity under the same conditions . in the kinase panel , the em5 inhibitor inhibited only 41 kinases ( 16% ) to less than 10% activity , including s6k1 and the related s6k family members rsk1 , rsk2 , rsk3 , and rsk4 . first generation organometallic ruthenium inhibitors exhibit potency and specificity for s6k1 . ( a ) em5 and em6 , two organometallic ruthenium compounds , use a similar structure to mimic the pan - kinase inhibitor , staurosporine . ( b ) a radioactive kinase assay was used to determine the activity of five different protein constructs of s6k1 . ( c ) staurosporine ( dotted line ) , em5 , and em6 were assayed against s6k1(1421 , t412e ) pdk1 activated in a radioactive kinase assay at 100 m atp . a radioactive kinase assay was used to determine the activity of s6k1 protein constructs prepared in baculovirus - infected insect cells in order to identify a construct that would be suitable for inhibitor testing . initial tests showed that the full - length i isoform of s6k1 ( s6k(1525 ) ) and the isolated kinase domain ( s6k(84384 ) ) had low kinase activity , although the full - length kinase showed more activity than the kinase domain ( figure 1b ) . we reasoned that the s6k1 protein constructs had low kinase activity because the full - length kinase contained the c - terminal autoinhibitory domain . to address this issue and express a more active kinase for further inhibitor studies , we prepared a s6k1(1421 ) construct including both the t252 and t412 phosphorylation sites , based on previous data from keshwani et al . indicating that the catalytic domain of the s6k1 aii isoform ( residues 1398 ) analogous to s6k1(1421 ) of the i isoform was highly expressed in insect cells . to further enhance the catalytic activity of s6k1(1421 ) , we prepared the t412e mutant to mimic phosphorylation at this position and coexpressed the protein with pdk1 to promote phosphorylation of t252 . preparation of the s6k1(1421 , t412e , pdk1 activated ) protein resulted in highly active kinase that was suitable for inhibition studies in vitro ( figure 1b ) . both em5 and em6 were assayed against s6k1(1421 , t412e , pdk1 activated ) in a radioactive kinase assay and determined to have ic50 values of 33.9 nm and 23.5 m , respectively , at 100 m atp ( figure 1c ) . for comparison , we also determined the ic50 of the nonspecific kinase inhibitor staurosporine , which had an ic50 value of 64.1 nm under the same conditions . given the apparent specificity and potency of em5 , it became the lead structure for the development of second - generation organometallic s6k1 inhibitors . our initial attempts to cocrystallize the s6k1 kinase domain ( s6k1kd , residues 84384 ) bound to em5 using several factorial screens were unsuccessful . however , we were able to reproduce the crystals reported by sunami et al . of the s6k1 kinase domain in complex with staurosporine . we then soaked these crystals with high concentrations of the em5 inhibitor in the hope of exchanging em5 for staurosporine in the crystals . the em5-soaked crystals diffracted to about 2.5 resolution and formed in space group p21 with two molecules per asymmetric unit . the structure was refined to rwork and rfree values of 19.15% and 22.21% , respectively , with excellent geometry ( table 1 ) . during the refinement process similar to the previously published structures of the s6k1 kinase domain , the kinase domain is bilobal , consisting of an n - lobe composed largely of -sheet and a c - lobe that is mostly -helical . the crystal structure revealed that one protein molecule in the asymmetric unit was bound to staurosporine , while the other molecule was bound to em5 in the same atp binding site . fc difference peak corresponding to the ruthenium atom in the atp binding sites of one of the molecules before the inhibitor models were built into the electron density map ( figure 2a ) . the staurosporine and em5-bound molecules in the asymmetric units are similar to each other with an overall rmsd of 0.68 for the shared atoms . ( a ) the electron density map of em5 ( contoured at 12 ) corresponds to the ruthenium atom of the inhibitor . hydrogen bonds are shown as a dashed line , while other interacting residues are labeled . ( d ) notable changes between the em5 and staurosporine bound structures are shown . this includes a change to the activation loop due to em5 interactions with g100 and v105 and a shortening of two turns to the d helix resulting from an interaction with e179 . ( e ) close - up around the c helix , which is more ordered in the em5-bound structure because of an interaction between f237 and l147 , and a hydrogen bond between k123 and e143 . although both staurosporine and em5 bind in the atp binding pocket , the more elaborate em5 compound makes more extensive interactions , correlating with its greater s6k1 potency than staurosporine . staurosporine forms hydrogen bonds to s6k1 through the backbone oxygen of glu-222 of the kinase with the nitrogen of the methylamine of staurosporine , and the backbone nitrogen of leu-175 and backbone oxygen of glu-173 of the kinase hinge region contact the pyrrolidone oxygen and nitrogen of staurosporine , respectively . the ring system of staurosporine also makes van der waals contacts to leu-97 , lys-99 , gly-98 , val-105 , ala-121 , tyr-174 , glu-179 , and met-225 ( figure 2b ) . the em5 compound retains two hydrogen bonds between the backbone atoms of the hinge residues ( glu-173 and leu-175 ) and the maleimide ring of em5 and all of the van der waals interactions with the em5 ring system ( figure 2c ) . however , in addition to these contacts , em5 makes additional protein interactions between the ruthenium coordination sphere and the protein . in particular , the additional isothiocyanate group of em5 makes van der waals interactions with gly-100 and val-105 of the kinase p - loop while the trithiacyclononane ligand makes van der waals contacts to gly-100 of the p - loop and glu-179 and glu-222 across from the p - loop where the protein substrate is likely to bind and thr-235 and asp-236 of the activation loop . notably , each of these residues ( glu-179 , glu-222 , thr-235 , and asp-236 ) undergoes a dramatic movement toward the em5 inhibitor relative to their positions in the staurosporine complex ( figure 2d ) . the binding of em5 to s6k1 also introduces significant structural changes in the kinase relative to the staurosporine complex , and these structural changes appear to be indirectly caused by the 1,4,7-trithiacyclononane ligand of the em5 inhibitor . the d helix of the staurosporine complex is about two turns longer at its n - terminus than the corresponding helix of the em5 complex where the corresponding segment takes on a -strand conformation . this structural difference appears to be driven by the interaction of the tridentate ligand of em5 with glu-179 . on the opposite side of the inhibitor , the staurosporine complex contains an activation loop that is folded toward the atp active site in an inactive conformation and does not have an ordered c helix , as previously reported . strikingly , the em5 complex contains a well - defined c helix of about two turns . the difference in disposition of the c helix in the two structures appears to be nucleated around the n - terminal region of the activation loop that undergoes about a 6 movement toward the em5 inhibitor relative to staurosporine . the movement of the activation segment toward the em5 inhibitor appears to be mediated by the van der waals interactions that are made between thr-235 and asp-236 with the trithiacyclononane ligand of em5 ( figure 2d ) . this in turn provides enough room for the c helix to form and to be stabilized by van der waals interactions between phe-237 of the activation loop and leu-147 of the c helix and a hydrogen bonding between lys-123 of the small domain and glu-143 of the c helix ( figure 2e ) . interestingly , these interactions are characteristic of the active conformations of kinases , even though the activation segment is in an inactive conformation . in contrast , the more out conformation of the activation loop of the staurosporine structure places phe-237 and asp-236 in positions that sterically occlude formation of the c helix ( figure 2d ) . taken together , while staurosporine binding to s6k1 places it in the inactive conformation , the s6k1/em5 complex has characteristics of both the inactive and active kinase conformations . the em5 inhibitor bound to s6k1 with an ic50 value in the mid - nanomolar range , and a cocrystal structure confirmed that the inhibitor was binding in the atp pocket of the kinase domain . as a result , em5 was a promising lead structure for the design of more selective and potent s6k1 inhibitors . previous data indicate that modifications to the pyridocarbazole moiety or the coordination sphere can have significant effects on binding affinities or kinase selectivity , and the structure of the s6k1/em5 complex indicated several positions where chemical elaboration could enhance specificity for the kinase . a series of 64 derivatives of em5 were designed with modifications at the pyridcarbazole heterocycle and the remaining ligand sphere and were tested for inhibition of s6k1 activity using both a radioactive kinase assay ( supporting information figure 3a ) and an adp - glo assay with 1 m compound . twenty - five of these inhibitors were further screened using 250 nm compound ( figure 3a ) . the eight compounds that inhibited s6k1 to less than 25% activity , the equivalent of em5 , were assayed to determine their ic50 values ( at 100 m atp ) . this analysis produced several compounds that inhibited s6k1 similarly or more potently than em5 including sek-222 ( ic50 = 18.9 nm ) , sek-243 ( ic50 = 15.9 nm ) , sek-214 ( ic50= 14.8 nm ) , sek-220 ( ic50= 7.7 nm ) , and fl772 ( ic50 = 7.3 nm ) ( supporting information figure 3b ) . compound fl772 ( figure 3b ) showed the most potent inhibition with an ic50 of 7.3 nm , ( 100 m atp and 2 nm of enzyme ) ( figure 3c ) . ( a ) the 25 top derivatives of em5 were assayed against s6k1 in a radioactive kinase assay , using 100 m atp and 250 nm s6k1 . ( b ) structure of the fl772 inhibitor , which showed the most potent inhibition of s6k1 . the 1,4,7-trithiacyclononane ligand of em5 is replaced by a 1,4,7-trithiacyclodecane bearing a methylated amino group in fl772 . ( c ) radioactive kinase assay of fl772 against s6k1 reveals an ic50 of 7.3 nm . ( d ) radioactive kinase assay of fl772 against s6k2 shows an ic50 of 975 nm . ( e ) radioactive kinase assay of pf-4708671 against s6k1 indicates an ic50 of 142.8 nm . panels c , d , and e employed 100 m atp and 2 nm of the respective kinase . testing the fl772 inhibitor at a range of concentrations from 1 m atp to 500 m atp resulted in an increase in the ic50 value concurrent with increasing atp concentrations from 3.91 nm at 1 m atp to 25.79 nm at 500 m atp , confirming that fl772 is an atp competitive inhibitor ( supporting information figure 4 ) . to assess the specificity of fl772 for the s6k1 isoform , we also assayed the fl772 compound against recombinant s6k2 ( figure 3d ) , which resulted in an ic50 value of 975.0 nm , more than 100-fold greater than the ic50 value for s6k1 . this confirms that the fl772 is indeed specific for the s6k1 isoform over the s6k2 isoform . for comparison , we tested the published s6k1 inhibitor pf-4708671 against s6k1 using the radioactive kinase assay in order to compare the potency of our compound with another specific s6k1 inhibitor ( figure 3e ) . the ic50 of pf-4708671 against s6k1 was determined to be 142.8 nm , nearly 20-fold higher than the ic50 of fl772 against s6k1 . to establish the kinase selectivity profile of fl772 , we submitted the compound at a concentration of 100 nm to the kinomescan profiling of lead hunter discovery services using 456 kinases ( supporting information table 10 ) . fl772 demonstrated a high degree of kinase selectivity , with only 10 of 456 ( 2.2% ) kinases showing less than 10% activity and only 26 of 456 ( 5.7% ) kinases showing less than 35% activity ( supporting information figure 2 ) . like em5 , fl772 showed binding to the cam , dap , flt , pim , and rsk family member kinases . unexpectedly , s6k1 itself exhibited 71% activity in the kinomescan set of lead hunter discovery services with 70 of 456 ( 15.3% ) showing a higher degree of binding than s6k1 . the potency of fl772 therefore appears to be greater against s6k1 prepared by us than s6k1 prepared by lead hunter discovery services . we hypothesize that the different s6k1 kinase preparation and/or phosphorylation state by lead hunter discovery services leads to the different fl772 potencies for s6k1 measured by us and lead hunter discovery services . nonetheless , taking together our analysis of fl772 against s6k1 and the kinase profiling results , we conclude that fl772 exhibits a high degree of kinase selectivity . fl772 is based on the em5 lead structure and differs by a methylated hydroxy group at the pyridocarbazole moiety and the thioether - containing tridentate ligand . the nine - membered ring of the symmetrical 1,4,7-trithiacyclononane ligand is replaced by a prochiral 1,4,7-trithiacyclodecane bearing a basic n - methylamino group in the 10-membered cyclic tridentate ligand . these structural changes in the tridentate ligand significantly increase the structural complexity of the inhibitor which is exemplified by the number of possible stereoisomers . to determine the molecular basis for the increased potency of fl772 over em5 , we determined the x - ray crystal structure of fl772 in complex with s6k1 to 2.7 resolution ( table 1 ) . the overall structure for the fl772-bound s6k1 is very similar to the em5-bound structure , with an rmsd of 0.54 for all atoms . in particular the p - loop and activation loop and d and c helices take on nearly identical conformations , although the c - helix is about one turn shorter at its n - terminal end ( figure 4a ) . in addition , the fl772 inhibitor retains all of the interactions made by em5 but makes some additional interactions including a hydrogen bond between the backbone carbonyl of lys-99 of the kinase p - loop with the amine ligand of the n - methyl-1,4,7-trithiacyclodecan-9-amine ligand while the methyl group makes a van der waals interaction with tyr-174 of the kinase hinge region ( figure 4b ) . these additional interactions of fl772 likely contribute to the greater potency of fl772 over em5 . the protrusion of the amine ligand into the region where protein substrate binds for phosphorylation probably also contributes to the greater inhibitor potency . ( a ) comparison of the s6k1 structures with em5 and fl772 . the c helix of s6k1 is one turn shorter at its n - terminal end of the fl772-bound structure compared to the em5-bound structure . hydrogen bonds are shown as a dashed line , while other interacting residues are labeled . fl772-specific interactions that are not made on the em5-bound structure are highlighted in darker shade . this includes interactions between y174 and the ether methyl group and the k99 backbone carbonyl with the amine group of the tridentate ligand . after establishing that fl772 functions as a potent atp competitive s6k inhibitor in the in vitro radioactive kinase assay , we carried out studies to characterize its activity in cells . we first tested fl772 for overall cell cytotoxicity and downregulation of phosphorylation of s6 in the 451lu ( braf mutant ) and 451lu - mr ( braf / mek - inhibitor resistant ) melanoma cell lines . cells were treated with vehicle or a dose of inhibitor ranging from 0.001 to 10 m for 22 h ( figure 5a ) . the 451lu or 451lu - mr cell lines exhibited neither a significant decrease in s6 phosphorylation nor a decrease in cell viability as indicated by the absence of cleaved parp . there was also no change in total s6 or peef2k levels , indicating that mtor was not a target of fl772 . fl772 does not inhibit s6 phosphorylation in human melanoma cells but does inhibit s6 phosphorylation in yeast cells . ( a ) 451lu or 451lu - mr cells were treated with increasing concentrations of fl772 or vehicle for 22 h. cells were lysed and blotted for ps6 and other downstream effectors of s6k1 . ( b ) 293 t cells were treated with fl772 , fl1324 ( a similar organometallic ruthenium compound ) , a previously reported s6k1 inhibitor , pf-671 ( 4708671 ) , or a previously reported dual mtorc1 and mtorc2 inhibitor , azd8055 , for 3 or 16 h. ( c ) western blot of yeast cells treated with fl772 . by4742 budding yeast cells were treated with fl772 for 4 h. cells were lysed and blotted for ps6 . quantitative western blot signals were detected by li - cor , and the relative ps6 levels were calculated by normalizing raw ps6 measurements to gapdh signals . ( ) p < 0.05 ( two - tailed student t test , n = 3 ) . we also investigated the effect of fl772 in 293 t cells at both 3 and 16 h of treatment ( figure 5b ) . as controls azd8055 is an atp - competitive dual mtorc1 and mtorc2 inhibitor that inhibits the phosphorylation of mtorc1 substrates s6k1 and 4ebp1 and the mtorc2 substrate akt . pf-4708671 is a reported s6k1 inhibitor that does not affect the phosphorylation of akt . the compound fl1324 is an fl772 analogue identified in our screen that inhibited s6k1 with an ic50 of 11 nm ( figure 3a ) . fl1324 replaces the fluorine of em5 with a hydroxyl group and is thus a less polar molecule . since previous studies using pf-4708671 demonstrated a significant reduction in s6 phosphorylation in 293 t cells in 30 min , we tried both a short ( 3 h ) and long ( 16 h ) time point for treatment . as expected , the azd8055 mtor inhibitor showed a significant decrease in ps6 levels at both the s235 and s240 sites , along with a decrease in pakt at t308 and s473 . the pf-4708671 compound showed a modest decrease in phosphorylation of s6 at the 3 h time point , but this phosphorylation returned to near basal levels by the 16 h time point . notably , neither the fl772 nor the fl1324 compound inhibited phosphorylation of s6 or akt . together , these results suggest either that the fl772 inhibitor has poor cell membrane permeability or that inhibition of s6k1 in cells does not significantly reduce s6 phosphorylation . the latter possibility is consistent with the fact that the structurally unrelated compound pf-4708671 also shows poor inhibition of s6 phosphorylation in cells and the observation that s6k2 also targets s6 for phosphorylation . to test if fl772 can inhibit s6 phosphorylation in a setting where s6k2 was not present , we investigated the ability of fl772 to inhibit s6 phosphorylation in budding yeast where a single kinase , sch9p , is orthologous to human s6k1 . we observed that treatment of wild - type budding yeast cells ( by4742 ) with fl772 significantly decreased the level of phosphorylated s6 in a dose - dependent manner ( figure 5c ) . at the highest dosage , these data suggest that fl772 functions as an inhibitor of s6 kinases in vivo in a yeast cellular system . in this study , we developed an organometallic ruthenium compound to inhibit s6k1 . using the millipore kinaseprofiler and radioactive kinase assays , we showed that the em5 lead compound was a potent and selective s6k1 inhibitor , with 100 nm compound inhibiting 93% of s6k1 activity and only inhibiting 16% of 283 kinases by less than 90% . we found that the em6 analogue in which an isocyanate group replaces an isothiocyanate is about 1000-fold less potent , implying that potency and specificity could be further optimized . a crystal structure of em5 bound to s6k1 provided important molecular insights into em5 inhibition of s6k1 and led to the development of fl772 , a compound containing a novel ligand scaffold with an ic50 in the single digit nanomolar range for s6k1 . a crystal structure of fl772 bound to s6k1 revealed the molecular basis for the compound s potent and selective inhibition of s6k1 . in order to investigate the efficacy of the fl772 inhibitor in cells , we evaluated the inhibitor in both human 293 t and braf mutant melanoma cells and in budding yeast . we found that fl772 was only able to inhibit s6 phosphorylation in yeast cells , suggesting that either the compound is unable to enter human cells , a significant shift in the ic50 of the compound occurs in the presence of physiological levels of atp , or the uninhibited activity of s6k2 in human cells was sufficient to maintain s6 phosphorylation . given that ruthenium compounds similar to fl772 have been used to successfully target mst1 , pak1 , and pi3k in cells , we do not believe that the ruthenium compounds are unable to penetrate cells . the radioactive kinase assay prohibits measurements at physiological levels of atp , but we were able to assay the activity of fl772 against s6k1 using an atp range from 1 to 500 m . this analysis revealed that the ic50 values increased with increasing atp concentrations , consistent with fl772 binding competitively with atp , as also confirmed with the crystal structure of the s6k1/fl772 complex . interestingly , the ic50 ranged from 3.91 nm at 1 m atp to only 25.79 nm ( a 6-fold increase ) at 500 m atp , suggesting that s6k1 binds atp relatively loosely and that fl772 is likely to displace atp even at the higher physiological concentration of atp . on the basis of these accumulated data , we propose that fl772 is unable to inhibit s6 phosphorylation in human cells because s6 is phosphorylated by the uninhibited s6k2 . s6k1 is closely related to s6k2 , sharing 83% sequence identity in the catalytic domain . a study involving s6k1/2 knockdown in mice suggests that both s6k1 and s6k2 are required for full phosphorylation of s6 but that s6k2 may be the more important of the two for phosphorylation of s6 . the mek inhibitor azd6244 showed additive effects on decreasing the phosphorylation of s6 in vitro when treated in combination with sirna inhibition of both s6k1 and s6k2 combined , indicating the importance of s6k2 in the phosphorylation of s6 . furthermore , while normal tissues often express low levels of s6k2 , overexpression of s6k2 is more common than overexpression of s6k1 in cancer cells . taking these data together suggests that targeting s6k2 either alone or in combination with s6k1 inhibition may be a more viable option for direct s6 inhibition in melanoma and potentially other cancers . despite the similarities in the catalytic domain , homology modeling between s6k1 and s6k2 indicates an important difference in residue tyr-174 that plays an important role in fl772 binding and is a cysteine in s6k2 . this residue is located in the hinge region of s6k1 and makes an important van der waals interaction with the methyl group of the secondary amine , which would not be made with a cysteine residue , suggesting that fl772 may not be a potent inhibitor for s6k2 . indeed , we confirmed in our study that fl772 inhibits s6k2 more than 100-fold more poorly than s6k1 . the lethality of s6k1/s6k2 knockout mice implies that s6k2 targeting may need to be selective for therapeutic value . to date , there are no commercially available s6k2-selective inhibitors , indicating a potential target for the next series of organometallic ruthenium inhibitors . taken together , the studies reported here provide a potent and selective s6k1 inhibitor that should be useful to probe s6k1 function and as a starting point for the development of efficacious s6k inhibitors for therapeutic use . protein kinase profiling of em5 was performed with the millipore kinaseprofiler in a panel of 263 human protein kinases . percentage of kinase activities were determined for em5 and em6 at 100 nm in the presence of 10 m atp . kinase profiling of fl772 was performed with the discoverx kinome screen using a panel of 456 kinases . active - site - directed competition binding was determined in the presence of 100 nm fl772 . em5 and em6 were synthesized in analogy to related compounds reported . a detailed synthesis and characterization of fl772 full length human s6k1 cdna ( 1525 ) was purchased from epitope ( catalogue number ihs1380 - 97652397 ) . s6k1 constructs ( 84384 , 1421 , 1421 t412e ) were subcloned into the pfastbac htb vector for protein expression . sf9 cells were transfected with the recombinant bacmid dna using cellfectin ( invitrogen ) . cells were harvested after being incubated for 48 h at 28 c and stored at 80 c . the 1421 t412e construct was coexpressed with pdk1 to phosphorylate the t412e residue ( cloned from cdna purchased from openbiosystems ) . frozen pellets of the s6k1 kinase domain , s6k1(84384 ) used for crystallography were resuspended in sonication buffer ( 50 mm kpi , ph 7.0 , 250 mm nacl , 5% glycerol , 1:1000 pmsf ) and sonicated at a power output of 5.5 for 120 s with 20 s intervals ( misonix sonicator 3000 ) . lysates were cleared by high - speed centrifugation at 18 000 rpm for 35 min at 4 c . equilibrated talon metal affinity resin ( clontech ) was added to cleared lysates and incubated at 4 c for 1 h with gentle shaking . the resin / lysate mixture was loaded into a gravity flow column , and the resin was extensively washed with wash buffer ( 50 mm kpi , ph 7.0 , 250 mm nacl , 5% glycerol ) . protein was then eluted with elution buffer ( 50 mm kpi , ph 7.0 , 250 mm nacl , 500 mm imidazole , and 5% glycerol ) in a single step . pooled talon eluent was diluted 3.5-fold in dilution buffer ( 50 mm kpi , ph 7.0 , 5% glycerol ) and loaded onto an sp anion exchange column pre - equilibrated with buffer a ( 50 mm kpi , ph 7.0 , 50 mm nacl , 5% glycerol ) . protein was eluted with buffer b ( 50 mm kpi , ph 7.0 , 500 mm nacl , 5% glycerol ) in a single step . elution after the q column was concentrated and loaded to a superdex s200 column equilibrated with 50 mm na citrate , ph 6.5 , 300 mm nacl , 1 mm dtt , 5% glycerol . the eluent was collected and concentrated to 3 mg / ml before protein was flash frozen in dry ice and stored at 80 c . purification of the 1421 t412e construct was completed as above , with gel filtration on the superdex s200 column using a buffer containing 25 mm tris , ph 7.5 , 200 mm nacl , 1 mm edta , and 5% glycerol . full - length human s6k2 cdna was purchased from ge healthcare dharmacon ( rps6kb2 , clone identification number 2959036 ) . the s6k2 1423 construct , equivalent to s6k1 1421 , was subcloned into the pfastbac htb vector for protein expression . each reaction mixture contained 5 l of 5 reaction buffer ( 100 mm mops , ph 7.0 , 150 mm mgcl2 ) , 2 l of inhibitor in 50% dmso , 3.6 l of s6k1 substrate peptide ( rrrlsslra ) , 1 l of bsa ( 20 mg / ml ) , 3.2 l of s6k1 ( concentration as described in results ) , and 5 l of atp / atp * mix ( concentration as described in results ) in a total reaction volume of 25 l . reaction mixtures were incubated for 1 h at room temperature before being transferred to whatman paper and washed with 0.75% phosphoric acid . ic50 values were determined using sigmoidal dose response with a variable curve in prism , version 5 . the s6k1 kinase domain crystals were obtained using room temperature hanging drop vapor diffusion by mixing equal volumes of protein ( 15 mg / ml ) preincubated with 1 mm staurosporine with 2025% ( w / v ) peg335 , 0.1 m bis - tris ( ph 5.55.7 ) , and 0.2 m liso4 . following the growth of crystals , crystal soaking was carried out by incubation with a final inhibitor concentration of 1 mm in cryoprotectant containing the well solution and 15% ( w / v ) glycerol for 4 h to overnight and flash frozen in liquid nitrogen . the structures were determined by molecular replacement using the reported s6k1/staurosporine complex ( pdb accession code 3a60 ) as a search model with the staurosporine removed from the coordinate file and refined with cns and coot . simulated annealing omit maps were employed to unambiguously confirm the modeled inhibitors . for the em5-soaked crystals , this revealed that one protein molecule in the asymmetric unit was bound to staurosporine while the other protein molecule was bound to em5 . for the fl772-soaked crystals , the asymmetric unit contained a single , domain - swapped monomer and only the fl772 inhibitor was modeled in the binding site . the structures were refined to convergence with a final rwork = 19.15% and rfree = 22.21% for the s6k1/em5 structure and a final rwork = 20.63% and rfree = 23.01% for the s6k1/fl722 structure with excellent geometry ( table 1 ) . human cell lines were cultured in rpmi ( 10 - 040-cm ; cellgro ) supplemented with 5% fetal bovine serum and harvested at 70% confluence . for immunoblotting , cells were treated for the specified times with the indicated drugs , washed with cold phosphate buffered saline ( pbs ) containing 100 mm na3vo4 , and lysed using tne buffer ( 150 mm nacl , 1% ( v / v ) np-40 , 2 mm edta , 50 mm tris - hcl , ph 8.0 ) supplemented with protease inhibitors ( 11697498001 ; roche ) . page and transferred to nitrocellulose membranes ( 9004700 ; biorad ) . after blocking for 1 h in 5% ( wt / vol ) dry milk / tris - buffered saline ( tbs)/0.1% ( v / v ) tween-20 , membranes were incubated overnight at 4 c with primary antibodies followed by incubation with alexa fluor - labeled secondary antibodies ( irdye 680lt goat - anti - mouse or irdye 800cw goat - anti - rabbit antibodies ( li - cor biosciences ) for 1 h. -actin ( a5441 ) and vinculin ( v9131 ) antibodies were obtained from sigma . p - akt ( 4056 , 4060 ) , s6 ( 2317 ) , p - s6 ( 4858 , 5364 ) , s6k1 ( 2708 ) , p - s6k1 ( 9234 ) , p - eef2k ( 3691 ) , peif4b ( 3591 ) , and cleaved parp ( 5625 ) were obtained from cell signaling technologies . overnight cultures of wild - type yeast cells ( by4742 ) were diluted in synthetic complete ( sc ) medium and regrown at 30 c to early log phase ( od600 of 0.2 ) . fl772 was added to an aliquot of culture to the final concentration of 1 , 10 , 100 , and 1000 nm . the treated cultures were further grown at 30 c for 4 h before harvesting . a culture of sch9 cells ( ks68 ) was grown and harvested in parallel as a control . yeast cell pellets were lysed by spinning down cultures at 3000 rpm for 3 min at 4 c , washing with ice - cold water , and broken in lysis buffer as previously described . whole cell extracts were separated on a 412% bolt gel with mops running buffer ( life technologies ) , followed by transfer to a pvdf membrane in a mini trans - blot cell ( bio - rad ) at 20 v overnight . the blot was blocked with 3% bsa at room temperature for 2 h and then at 4 c for 4 h , followed by incubation with primary antibodies , phospho - s6 ( cell signaling , catalog no . incubation with secondary antibodies ( anti - rabbit - dylight-680 and anti - mouse - dylight-800 , pierce , 1:10000 dilution ) was carried out at room temperature for 1 h before imaging with li - cor odyssey .
aberrant activation of s6 kinase 1 ( s6k1 ) is found in many diseases , including diabetes , aging , and cancer . we developed atp competitive organometallic kinase inhibitors , em5 and fl772 , which are inspired by the structure of the pan - kinase inhibitor staurosporine , to specifically inhibit s6k1 using a strategy previously used to target other kinases . biochemical data demonstrate that em5 and fl772 inhibit the kinase with ic50 value in the low nanomolar range at 100 m atp and that the more potent fl772 compound has a greater than 100-fold specificity over s6k2 . the crystal structures of s6k1 bound to staurosporine , em5 , and fl772 reveal that the em5 and fl772 inhibitors bind in the atp binding pocket and make s6k1-specific contacts , resulting in changes to the p - loop , c helix , and d helix when compared to the staurosporine - bound structure . cellular data reveal that fl772 is able to inhibit s6k phosphorylation in yeast cells . together , these studies demonstrate that potent , selective , and cell permeable s6k1 inhibitors can be prepared and provide a scaffold for future development of s6k inhibitors with possible therapeutic applications .
Introduction Results Discussion and Conclusions Experimental Section
the aberrant activation of s6 kinases has been shown to play a role in many disease conditions , including diabetes , obesity , aging , and cancer . we have previously reported on the development of atp competitive organometallic kinase inhibitors with high potency and specificity . the scaffold design includes a bidentate ligand that is able to target the metal complexes to the atp - binding site . here , we present data on the development of potent and specific organometallic s6k1 inhibitors , em5 and fl772 . we show that fl772 binds to s6k1 with an ic50 value in the single digit nanomolar range at 100 m atp and that the more potent fl772 compound has a greater than 100-fold specificity over s6k2 . crystal structures of the s6k1 domain bound to the pan - kinase inhibitor staurosporine , em5 , and fl772 reveal that the organometallic inhibitors bind in the atp binding pocket in a way that is distinct from staurosporine , likely explaining their more favorable potency and selectivity . cellular data demonstrate that fl772 is able to inhibit s6k phosphorylation in yeast cells . ( c ) staurosporine ( dotted line ) , em5 , and em6 were assayed against s6k1(1421 , t412e ) pdk1 activated in a radioactive kinase assay at 100 m atp . for comparison , we also determined the ic50 of the nonspecific kinase inhibitor staurosporine , which had an ic50 value of 64.1 nm under the same conditions . the crystal structure revealed that one protein molecule in the asymmetric unit was bound to staurosporine , while the other molecule was bound to em5 in the same atp binding site . ( e ) close - up around the c helix , which is more ordered in the em5-bound structure because of an interaction between f237 and l147 , and a hydrogen bond between k123 and e143 . although both staurosporine and em5 bind in the atp binding pocket , the more elaborate em5 compound makes more extensive interactions , correlating with its greater s6k1 potency than staurosporine . the binding of em5 to s6k1 also introduces significant structural changes in the kinase relative to the staurosporine complex , and these structural changes appear to be indirectly caused by the 1,4,7-trithiacyclononane ligand of the em5 inhibitor . the em5 inhibitor bound to s6k1 with an ic50 value in the mid - nanomolar range , and a cocrystal structure confirmed that the inhibitor was binding in the atp pocket of the kinase domain . previous data indicate that modifications to the pyridocarbazole moiety or the coordination sphere can have significant effects on binding affinities or kinase selectivity , and the structure of the s6k1/em5 complex indicated several positions where chemical elaboration could enhance specificity for the kinase . panels c , d , and e employed 100 m atp and 2 nm of the respective kinase . testing the fl772 inhibitor at a range of concentrations from 1 m atp to 500 m atp resulted in an increase in the ic50 value concurrent with increasing atp concentrations from 3.91 nm at 1 m atp to 25.79 nm at 500 m atp , confirming that fl772 is an atp competitive inhibitor ( supporting information figure 4 ) . a crystal structure of em5 bound to s6k1 provided important molecular insights into em5 inhibition of s6k1 and led to the development of fl772 , a compound containing a novel ligand scaffold with an ic50 in the single digit nanomolar range for s6k1 . we found that fl772 was only able to inhibit s6 phosphorylation in yeast cells , suggesting that either the compound is unable to enter human cells , a significant shift in the ic50 of the compound occurs in the presence of physiological levels of atp , or the uninhibited activity of s6k2 in human cells was sufficient to maintain s6 phosphorylation . this residue is located in the hinge region of s6k1 and makes an important van der waals interaction with the methyl group of the secondary amine , which would not be made with a cysteine residue , suggesting that fl772 may not be a potent inhibitor for s6k2 .
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it is well established that resistance training is good for adults health , regardless of age.13 increased strength and bone density , improved ability to complete activities of daily living , improvement in health - related quality of life , reduced signs and symptoms of chronic illness , and a reduction in sarcopenia are all benefits of regular participation in resistance ( strength ) training.4 the world health organization ( who ) and multiple national guidelines specifically include resistance training in their recommended physical activity guidelines , particularly the guidelines for those aged 60 years ( older people).5,6 nevertheless , the proportions of older people participating are low.79 older populations across the world are growing , and it is expected that by 2050 , there will be two billion people aged 60 years living worldwide , which is more than double the number in 2013.10 with this increase , it is important that older people stay as healthy as possible for as long as they can to avoid needing ongoing health and care services , hospitalization , or a move into residential aged care . physical activity plays an essential role in staying healthy , maintaining independence , reducing the risk of falling , and allowing older people to live their later life well.11,12 the physical activity of choice for older people , particularly in australia is walking , with the majority of the older population participating only in this mode of exercise.13 studies have shown that fewer than 15% of older people participate in resistance training twice a week ( the minimum guideline recommended frequency).14,15 a number of studies have explored motivators and barriers to older people participating ( or commencing participation ) in resistance training in an attempt to increase participation rates.1619 the main motivators specific to older people participating in resistance training were preventing deterioration or disability , building muscle , falls prevention , and feeling more alert or having better concentration.16 barriers are usually identified by asking participants who are not or have not participated in a given activity to provide their reasons for not taking part . for older people , barriers to participating in resistance training include the following : health issues , pain , tiredness or fatigue , lack of social support , and a lack of available exercise facilities.16 given the low uptake of resistance training by older people , it is important that strategies are implemented to support ongoing participation for those who commence such programs , so that they maintain long - term participation , which can assist to optimize health and well - being . evidence from two systematic reviews that examined the effects of resistance training for improving physical function4 and exercise for improving balance20 found withdrawals after 12 months participating ranged between 20% and 48% , respectively . this highlights the importance not only for research to guide improved uptake or commencement of resistance training programs but also for retaining those who commence programs for the longer term . this study differs from other studies described as investigating barriers or reasons for ceasing participation because participants in these previous studies were still engaged in a resistance training program or had never participated.19,21,22 however , this study explores reasons why older people stop participating in a structured resistance exercise program by asking people who have recently made this choice . exploring why this particular cohort ceases resistance training could benefit older people by assisting them to understand how to maintain their participation in resistance training . it is also important for gymnasium and fitness center owners , managers , and staff who provide such programs to better understand the reasons why older participants withdraw from attending resistance training programs after having commenced the program . therefore , the aim of this study was to identify the reasons why older people who had been participating in a resistance training program chose to discontinue participation . this was a cross - sectional descriptive study , in which participants were surveyed by mail . inclusion criteria were people aged 60 years who had been attending a structured , gymnasium - based , resistance training program specifically designed for older people ( ie , living longer living stronger)23 and who within the previous 15 months decided to no longer attend . during august and september 2015 , questionnaires were sent to participants who had ceased participation in a resistance training program between may 2014 and april 2015 . the managers from 15 gymnasiums advised the number of members who had ceased participation in their living longer living stronger resistance training programs ( n=293 ) for the research team , to ensure an adequate number of surveys were provided to them for distribution . due to confidentiality requirements , the managers could not provide actual contact details of these former participants to the research team . as a result , the researchers prepared 293 participant invitation letters , questionnaires , and reply paid envelopes into individual envelopes and posted them in bundles to each of the participating managers . the managers then added a name and address label to each individual envelope and posted them to past members meeting the study criteria . the participating gymnasiums were all delivering living longer living stronger resistance training programs specifically for seniors within their facility and agreed to be involved in the research . the questionnaire was developed by the researchers , research partners who came from a seniors advocacy organization , and a home care agency . two consumer representatives ( two older people , aged in their late 70s one who participated regularly in resistance training and one who did not ) also assisted to develop the wording and format of the questionnaire . this study was one section of a larger research project , and the consumer representatives were part of the project team for a period of 2 years . both consumer representatives had worked extensively in education ( professorial level ) and/or the sport industry in their previous working lives . their role in this project was to provide feedback on the appropriateness of all research documents , methods , language , and so on to older people in the community . the lack of research in this area prevented the use of validated scales in the data collection , but the questionnaire was based on eliciting responses , which would explain the reasons why participants no longer attended the resistance training program . the questionnaire included participant demographics ( eg , age , sex , area lived in , self - reported physical and mental health , and number of prescribed medications taken daily ) , physical activity levels , why they joined resistance training and for how long ( including sessions per week ) , type of program they had attended , why they withdrew , and whether they would consider participating again in the future and , if so , why . other questions related to how challenging they found the resistance training program , whether they noticed any physical or psychological changes due to participating , their perspectives on whether the program represented value for money , their confidence in completing sessions , support received , and motivation to participate . a combination of open and closed questions ( likert scales ) were utilized to avoid bias associated with checklists and to ensure opportunity for maximal responses . the physical activity level data in the questionnaire involved using the physical activity scale for the elderly ( pase ) , which is a valid and reliable tool for determining physical activity levels of older people.24,25 scores on the pase can range from 0 to 400 , where 0 being not active at all to 400 being very active . statistical analysis was performed using statistical package for the social sciences ( spss ) ( version 22 ) . the qualitative data derived from the open - ended responses were analyzed using inductive content analysis . content analysis is a research method for making replicable and valid inferences from data in a way which can generate new insights and can inform practical actions.26 inductive content analysis was used as it was felt that there was limited knowledge about barriers and enablers to resistance training in this specific cohort.27 these data were entered verbatim onto an excel spreadsheet ( microsoft corporation , washington , dc , usa ) , and color highlights were used to code . two researchers ( eb and a - mh ) coded the data independently , then compared and discussed the data until they reached a consensus . the results were then joined under higher order headings to reduce the number of categories through the collapse of like and unlike categories . the final data were presented using participant quotes to illustrate each category . to assist in clarifying connections among the categories , these data concept maps are graphical tools that are used for confirming relationships among concepts and validating ideas.28 the concept map was constructed with reference to the research question why do older people who had been participating in a resistance training program choose to discontinue ? to minimize bias , the analyses were verified at each stage by a second independent researcher ( a - mh ) . this was a cross - sectional descriptive study , in which participants were surveyed by mail . inclusion criteria were people aged 60 years who had been attending a structured , gymnasium - based , resistance training program specifically designed for older people ( ie , living longer living stronger)23 and who within the previous 15 months decided to no longer attend . during august and september 2015 , questionnaires were sent to participants who had ceased participation in a resistance training program between may 2014 and april 2015 . the managers from 15 gymnasiums advised the number of members who had ceased participation in their living longer living stronger resistance training programs ( n=293 ) for the research team , to ensure an adequate number of surveys were provided to them for distribution . due to confidentiality requirements , the managers could not provide actual contact details of these former participants to the research team . as a result , the researchers prepared 293 participant invitation letters , questionnaires , and reply paid envelopes into individual envelopes and posted them in bundles to each of the participating managers . the managers then added a name and address label to each individual envelope and posted them to past members meeting the study criteria . the participating gymnasiums were all delivering living longer living stronger resistance training programs specifically for seniors within their facility and agreed to be involved in the research . the questionnaire was developed by the researchers , research partners who came from a seniors advocacy organization , and a home care agency . two consumer representatives ( two older people , aged in their late 70s one who participated regularly in resistance training and one who did not ) also assisted to develop the wording and format of the questionnaire . this study was one section of a larger research project , and the consumer representatives were part of the project team for a period of 2 years . both consumer representatives had worked extensively in education ( professorial level ) and/or the sport industry in their previous working lives . their role in this project was to provide feedback on the appropriateness of all research documents , methods , language , and so on to older people in the community . the lack of research in this area prevented the use of validated scales in the data collection , but the questionnaire was based on eliciting responses , which would explain the reasons why participants no longer attended the resistance training program . the questionnaire included participant demographics ( eg , age , sex , area lived in , self - reported physical and mental health , and number of prescribed medications taken daily ) , physical activity levels , why they joined resistance training and for how long ( including sessions per week ) , type of program they had attended , why they withdrew , and whether they would consider participating again in the future and , if so , why . other questions related to how challenging they found the resistance training program , whether they noticed any physical or psychological changes due to participating , their perspectives on whether the program represented value for money , their confidence in completing sessions , support received , and motivation to participate . a combination of open and closed questions ( likert scales ) were utilized to avoid bias associated with checklists and to ensure opportunity for maximal responses . the physical activity level data in the questionnaire involved using the physical activity scale for the elderly ( pase ) , which is a valid and reliable tool for determining physical activity levels of older people.24,25 scores on the pase can range from 0 to 400 , where 0 being not active at all to 400 being very active . statistical analysis was performed using statistical package for the social sciences ( spss ) ( version 22 ) . the qualitative data derived from the open - ended responses were analyzed using inductive content analysis . content analysis is a research method for making replicable and valid inferences from data in a way which can generate new insights and can inform practical actions.26 inductive content analysis was used as it was felt that there was limited knowledge about barriers and enablers to resistance training in this specific cohort.27 these data were entered verbatim onto an excel spreadsheet ( microsoft corporation , washington , dc , usa ) , and color highlights were used to code . two researchers ( eb and a - mh ) coded the data independently , then compared and discussed the data until they reached a consensus . the results were then joined under higher order headings to reduce the number of categories through the collapse of like and unlike categories . the final data were presented using participant quotes to illustrate each category . to assist in clarifying connections among the categories , these data concept maps are graphical tools that are used for confirming relationships among concepts and validating ideas.28 the concept map was constructed with reference to the research question why do older people who had been participating in a resistance training program choose to discontinue ? to minimize bias , the analyses were verified at each stage by a second independent researcher ( a - mh ) . of the 293 questionnaires posted by the 15 gymnasiums , 56 were returned , a response rate of 19% . the mean age of respondents was 71.5 years ( sd : 9.0 ) , with over three quarters ( 79% , n=44 ) being female and 21% male ( n=12 ) . almost all ( 95% , n=53 ) respondents lived in the metropolitan area . over three quarters ( 79% , n=44 ) said that they had good ( 39% , n=22 ) , very good ( 30% , n=17 ) , or excellent ( 9% , n=5 ) physical health . the majority ( 87% , almost half were taking three or more prescribed medications ( 48% , n=27 ) , with only 16% taking none ( n=9 ) . the mean pase score for the group was 119.5 ( sd : 68.4 ) , males : 156.8 ( sd : 78.0 ) and females : 113.5 ( sd : 63.2 ) . given the pase norms for 7075 year olds are males : 102.4 ( sd:53.7 ) and females : 89.1 ( sd:55.5),29 these groups were more physically active than others of a similar age . over half ( 57.1% , n=32 ) of the respondents only 8.9% left in the first month ( n=5 ) , 12.5% during months 1 and 2 ( n=7 ) , 8.9% between months 2 and 3 ( n=5 ) , and 12.5% in months 3 and 4 ( n=7 ) . during the initial 4 months , most respondents attended class once ( 36.8% , n=21 ) or twice ( 47.4% , n=27 ) a week . only eight respondents ( 14% ) attended three classes a week and no one attended more than three . many respondents attended a group session ( 53.6% , n=30 ) or a combination of group and individual work ( 10.7% , n=6 ) , whereas 19.6% worked individually ( n=11 ) and 16.1% had a personalized assessment ( n=9 ) . ninety - three percent of respondents ( n=52 ) were confident or very confident that they could complete the exercises included in the sessions . only 7.2% were not confident ( n=2 ) or not confident at all ( n=2 ) in completing the exercises . twelve percent described themselves as neither motivated nor not motivated ( neutral ) and 8.9% ( n=5 ) as not or not at all motivated . the majority of respondents felt that they were given more than adequate ( 26.8% , n=15 ) or adequate ( 46.4% , however , 10% suggested that they were given inadequate ( 1.8% , n=1 ) or very inadequate ( 8.9% , n=5 ) support , and a further 16.1% ( n=9 ) were neutral on this subject . comments on where improvement could occur included : the instructor was more interested in their own exercise regime than considering the needs of their clients ( n=2 ) , the attention you received from the physio during the session depended on whether you were a patient at the practice or not and the instructors tended to respond to your questions rather than initiating any step - up in exercise . the instructor was more interested in their own exercise regime than considering the needs of their clients ( n=2 ) , the attention you received from the physio during the session depended on whether you were a patient at the practice or not and the instructors tended to respond to your questions rather than initiating any step - up in exercise . there were also a number of positive comments which included the following : staff being helpful and supportive ( n=10 ) , exercise individualized to my level and capabilities ( n=4 ) , and average group of 10 with an engaged and interested physio always present . respondents were asked to rate the adequacy of the information provided about safety while they were participating in the resistance training program . in total , 88% said that the information was either adequate ( n=25 ) or very adequate ( n=20 ) . twelve percent rated safety information as inadequate ( n=3 ) or very inadequate ( n=3 ) . these were coded into three subcategories , such as health , program / facility , and other . injury ( 32.1% , n=18 ) was the most common reason to emerge for the respondents ceasing participation in a resistance training program . table 1 shows the main categories , subcategories , and some example quotes from respondents . there were many reasons for discontinuing , and 22 of the 56 questionnaire respondents ( 39.3% ) reported two or more reasons for withdrawing . the three most commonly reported reasons within the subcategories for withdrawing included illness , holidays , and the program not being suitable . cost was only reported by two participants . over three quarters of the respondents thought that the resistance training program was good ( 40.4% , n=23 ) or very good ( 36.8% , n=21 ) value for money . ten percent found the cost barely acceptable ( n=6 ) , 1.8% poor ( n=1 ) , and 5.3% very poor ( n=3 ) . only 36 respondents ( 64.3% ) answered this question , the other 20 either left it blank ( n=10 ) or reported none ( n=10 ) . class time and places available within preferred classes were the most commonly identified negative aspects of the resistance training programs . waiting for machines and equipment issues were also highlighted as well as poor staff support and the program not satisfying the participants . table 2 shows the main categories , subcategories , and participant quotes of the most commonly reported negative aspects of participating in a resistance program . although the blank responses can not be interpreted , it is likely that those reporting the data were concept mapped to understand the flow of participation and withdrawal from the respondents ( figure 1 ) . this showed that there were several reasons why withdrawal occurred and there are opportunities to have older people participate again in the future . the map demonstrates the importance of the staff and facility in maintaining participation for this age group . when asked if they would like to return to the program in the future , 68.4% of the respondents ( n=39 ) said that they would , 19.3% reported being unsure ( n=11 ) , and 12.3% were not interested ( n=7 ) in participating again . the reasons given by those not interested in returning were time , not suitable , boring ( waiting for machine and no interaction ) , cost , attending gym elsewhere , and not interested . the most common reasons why past participants said that they would like to return to a resistance training program in the future were enjoyment ( 26.3% , n=10 ) , fitness ( 18.4% , n=7 ) , gaining health benefits and exercises ( 13.2% , n=5 each ) , and if the instructor improved ( 7.9% , n=3 ) . other reasons provided by individual respondents included the following : in the new year when my health fund will pay more , i would like to join more general classes , possibly when i m not working and i intend to . in the new year when my health fund will pay more , i would like to join more general classes , possibly when i m not working and i intend to . be able to return to the program at some point and around two thirds ( 67.3% , n=37 ) reported yes they would be able to , 21.8% were unsure ( n=12 ) , and 10.9% stated no ( n=6 ) . the reasons why respondents would return were similar to those described above , with additional reasons including : because i can or i am able to and if a class is available . reasons given for not being able to return were the following : do nt want to , i m active in other areas , not with this set up at the center , and time and cost factors . of the 293 questionnaires posted by the 15 gymnasiums , 56 were returned , a response rate of 19% . the mean age of respondents was 71.5 years ( sd : 9.0 ) , with over three quarters ( 79% , n=44 ) being female and 21% male ( n=12 ) . almost all ( 95% , n=53 ) respondents lived in the metropolitan area . over three quarters ( 79% , n=44 ) said that they had good ( 39% , n=22 ) , very good ( 30% , n=17 ) , or excellent ( 9% , n=5 ) physical health . the majority ( 87% , almost half were taking three or more prescribed medications ( 48% , n=27 ) , with only 16% taking none ( n=9 ) . the mean pase score for the group was 119.5 ( sd : 68.4 ) , males : 156.8 ( sd : 78.0 ) and females : 113.5 ( sd : 63.2 ) . given the pase norms for 7075 year olds are males : 102.4 ( sd:53.7 ) and females : 89.1 ( sd:55.5),29 these groups were more physically active than others of a similar age . over half ( 57.1% , n=32 ) of the respondents had attended the resistance program for > 4 months . only 8.9% left in the first month ( n=5 ) , 12.5% during months 1 and 2 ( n=7 ) , 8.9% between months 2 and 3 ( n=5 ) , and 12.5% in months 3 and 4 ( n=7 ) . during the initial 4 months , most respondents attended class once ( 36.8% , n=21 ) or twice ( 47.4% , n=27 ) a week . only eight respondents ( 14% ) attended three classes a week and no one attended more than three . many respondents attended a group session ( 53.6% , n=30 ) or a combination of group and individual work ( 10.7% , n=6 ) , whereas 19.6% worked individually ( n=11 ) and 16.1% had a personalized assessment ( n=9 ) . ninety - three percent of respondents ( n=52 ) were confident or very confident that they could complete the exercises included in the sessions . only 7.2% were not confident ( n=2 ) or not confident at all ( n=2 ) in completing the exercises . twelve percent described themselves as neither motivated nor not motivated ( neutral ) and 8.9% ( n=5 ) as not or not at all motivated . the majority of respondents felt that they were given more than adequate ( 26.8% , n=15 ) or adequate ( 46.4% , n=26 ) support during their sessions . however , 10% suggested that they were given inadequate ( 1.8% , n=1 ) or very inadequate ( 8.9% , n=5 ) support , and a further 16.1% ( n=9 ) were neutral on this subject . comments on where improvement could occur included : the instructor was more interested in their own exercise regime than considering the needs of their clients ( n=2 ) , the attention you received from the physio during the session depended on whether you were a patient at the practice or not and the instructors tended to respond to your questions rather than initiating any step - up in exercise . the instructor was more interested in their own exercise regime than considering the needs of their clients ( n=2 ) , the attention you received from the physio during the session depended on whether you were a patient at the practice or not and the instructors tended to respond to your questions rather than initiating any step - up in exercise . there were also a number of positive comments which included the following : staff being helpful and supportive ( n=10 ) , exercise individualized to my level and capabilities ( n=4 ) , and average group of 10 with an engaged and interested physio always present . respondents were asked to rate the adequacy of the information provided about safety while they were participating in the resistance training program . in total , 88% said that the information was either adequate ( n=25 ) or very adequate ( n=20 ) . twelve percent rated safety information as inadequate ( n=3 ) or very inadequate ( n=3 ) . these were coded into three subcategories , such as health , program / facility , and other . injury ( 32.1% , n=18 ) was the most common reason to emerge for the respondents ceasing participation in a resistance training program . table 1 shows the main categories , subcategories , and some example quotes from respondents . there were many reasons for discontinuing , and 22 of the 56 questionnaire respondents ( 39.3% ) reported two or more reasons for withdrawing . the three most commonly reported reasons within the subcategories for withdrawing included illness , holidays , and the program not being suitable . thought that the resistance training program was good ( 40.4% , n=23 ) or very good ( 36.8% , n=21 ) value for money . ten percent found the cost barely acceptable ( n=6 ) , 1.8% poor ( n=1 ) , and 5.3% very poor ( n=3 ) . only 36 respondents ( 64.3% ) answered this question , the other 20 either left it blank ( n=10 ) or reported none ( n=10 ) . class time and places available within preferred classes were the most commonly identified negative aspects of the resistance training programs . waiting for machines and equipment issues were also highlighted as well as poor staff support and the program not satisfying the participants . table 2 shows the main categories , subcategories , and participant quotes of the most commonly reported negative aspects of participating in a resistance program . although the blank responses can not be interpreted , it is likely that those reporting the data were concept mapped to understand the flow of participation and withdrawal from the respondents ( figure 1 ) . this showed that there were several reasons why withdrawal occurred and there are opportunities to have older people participate again in the future . the map demonstrates the importance of the staff and facility in maintaining participation for this age group . when asked if they would like to return to the program in the future , 68.4% of the respondents ( n=39 ) said that they would , 19.3% reported being unsure ( n=11 ) , and 12.3% were not interested ( n=7 ) in participating again . the reasons given by those not interested in returning were time , not suitable , boring ( waiting for machine and no interaction ) , cost , attending gym elsewhere , and not interested . the most common reasons why past participants said that they would like to return to a resistance training program in the future were enjoyment ( 26.3% , n=10 ) , fitness ( 18.4% , n=7 ) , gaining health benefits and exercises ( 13.2% , n=5 each ) , and if the instructor improved ( 7.9% , n=3 ) . other reasons provided by individual respondents included the following : in the new year when my health fund will pay more , i would like to join more general classes , possibly when i m not working and i intend to . in the new year when my health fund will pay more , i would like to join more general classes , possibly when i m not working and i intend to . be able to return to the program at some point and around two thirds ( 67.3% , n=37 ) reported yes they would be able to , 21.8% were unsure ( n=12 ) , and 10.9% stated no ( n=6 ) . the reasons why respondents would return were similar to those described above , with additional reasons including : because i can or i am able to and if a class is available . reasons given for not being able to return were the following : do nt want to , i m active in other areas , not with this set up at the center , and time and cost factors . the respondents in this study stopped attending resistance training programs for a number of reasons . most commonly reported reasons were the result of injury or illness , going away on holidays , and issues at the facility ( eg , class not available including type , age range , and times ; waiting for machines ; and poor staff support ) . previous research that explored the barriers preventing older people participating in resistance training and exercise , in general , also found injury and pain to be common reasons for nonparticipation , together with feeling too old and not being interested.16,17,30 it is unknown how many of the injuries occurred due to taking part in resistance training or for some other reason , such as falling downstairs on holiday . however , regardless of the reason and depending on the injury and also illness ( which was the second most common response ) , it may be possible for the older person to still attend ( either immediately following the injury or illness or after a period of time ) but be given a modified program to accommodate any new exercise constraints associated with their injury or recovery . illness for an older person can often make it difficult to bounce back and for some continuing with activities of daily living and living independently becomes challenging . fitness center and other health professionals should be aware that this may occur and provide regular advice , support , and referral to the appropriate health professional . it is also recommended that facilities provide screening for past injuries to reduce the likelihood of previous injuries reoccurring . regular physical activity and resistance training for improving strength , if appropriately moderated , can assist the older person to return to better health and fitness at a faster rate.4,31 indeed , national physical activity guidelines for older australians recommend older people who have stopped physical activity , or who are starting a new physical activity , should start at a level that is easily manageable and gradually build up the amount , type , and frequency of activity.32this study did not explore whether it is already common practice for fitness centers / others running resistance programs to follow - up those who cease participation and encourage them back with a program designed to help them regain fitness after injury or illness . this warrants further research , especially the costs and benefits for both the centers and participants . older people who have stopped physical activity , or who are starting a new physical activity , should start at a level that is easily manageable and gradually build up the amount , type , and frequency of activity.32 holidays were the third most commonly reported reason for withdrawing from the resistance training program . unlike people working full - time who receive 4 weeks annual leave a year ( in australia and less elsewhere ) , some older people have the option of holidaying for much longer periods of time . a proportion of older people in australia are well known for taking driving holidays to the northern parts of australia for up to 34 months each year to avoid the cold winters.33 older people in the usa do something similar in moving to the southern states of the usa,33 and many british seniors have spent prolonged periods of time in spain.34 it may be that the lengths of these long breaks make it difficult to recommence resistance training when settling back home ; especially , if it was necessary for the older person to withdraw rather than suspend their program membership when they were away . if this is the case , one strategy to help would be for gymnasiums and fitness centers to consider flexibility with their memberships , allow long suspensions , and make a personalized phone call to the older person on their return to encourage them back to the program . of note , over 65% of the people were keen to return to the program so a personalized phone call or written invitation may well be a means of easily getting these people to return . some of the participants also identified issues with the gymnasium or fitness center programs as being the reason(s ) they stopped participating . these issues included distance to travel to the program , class times and availability not suitable , dissatisfaction with the instructor , the facility being overcrowded , and the program provided not being suitable . older instructors are often more able to relate to this target group , and research has shown that using peer leaders ( older instructors ) can be beneficial.3538 using peer leaders could also provide a competitive point of difference for their business . there are obvious requirements for training and support for peer exercise trainers that would need to be considered . fitness facilities staff often put sessions for older people in off - peak times ( late morning , middle of the day , and early afternoon ) thinking older people can attend at any time . yet , many older people have multiple commitments and interests such as looking after grandchildren , doing volunteer or paid work , and attending classes , and as a result , prefer sessions to be held earlier in the mornings or later in the afternoon.21,39 with the projected increase in the proportion of people aged > 60 years in the forthcoming decades , businesses need to be flexible and consider the needs and preferences of this important and growing target group . this study had several limitations that need to be considered . the low - response rate to the questionnaire may limit the generalizability of the findings . because of privacy reasons , we were not able to contact the participants directly to follow - up on their completion of the survey , to interview them as a form of member checking , or to explore their responses in more depth . also , sections of the tool were nonvalidated because of this type of study not having been undertaken with this population previously ; therefore , it was not possible to use a validated tool . the authors also had no knowledge about participants health and injury status prior to them commencing participation resistance training . despite these limitations , this is the first study to actually look at a population of seniors who recently stopped attending a structured resistance training program of their own accord after having participated for a period , and we believe that it provides useful information for resistance training providers running programs for older people . the number of older people participating in the recommended minimum two resistance training sessions a week is currently fewer than one in six.79 this needs to increase so more older people can experience some of the many benefits that have been shown to be associated with resistance training . providing those working in this area with as much information as possible about older people s needs , preferences , and motivations is , therefore , essential if this needs to be achieved . this study identified reasons why older people who had been participating in resistance training for a period of time ceased taking part . injury , illness , holidays , and issues with the resistance training program , center , or staff were the most commonly reported reasons for stopping . to reduce the number of older people leaving resistance training programs , it is suggested that gymnasiums and fitness centers provide ongoing advice for prevention and return to training after injury and flexible programs and services ( membership types ) that accommodate older people s life choices in retirement .
purposethe proportion of the population , that is older , is growing at a faster rate than other age groups . physical activity is important for older people because it assists in living independently . participating in resistance training on a regular basis ( twice weekly ) is recommended for older people ; yet , fewer than 15% of people over 60 years achieve this level . the aim of this article was to investigate the factors contributing to older people s decisions to stop participation in a resistance training program.participants and methodsparticipants were older people who had chosen to participate in a structured resistance training program specifically designed for seniors and then after a period of time discontinued . this population received a questionnaire in the mail focused on factors contributing to their cessation of resistance training exercise . qualitative results were analyzed using inductive content analysis.resultsfifty-six survey responses were received ( average age 71.5 years , sd = 9.0 ; 79% females ) . injury , illness , and holidaying were the main reasons for ceasing participation . a small but important number of responses ( 11% ) reported that they considered they were not provided with sufficient support during the resistance training programs.conclusionsto attract and retain their senior clients , the results indicate that program organizers need to provide tailored support to return to resistance training after injury and offer flexible and individualized services that accommodate older people s life choices in retirement .
Introduction Participants and methods Study design and sample Questionnaire Statistical analysis and ethics Results Respondents Attendance Program support Reasons for withdrawing from the program Negative aspects of the resistance training program Returning to program in the future Discussion Limitations Conclusion
it is well established that resistance training is good for adults health , regardless of age.13 increased strength and bone density , improved ability to complete activities of daily living , improvement in health - related quality of life , reduced signs and symptoms of chronic illness , and a reduction in sarcopenia are all benefits of regular participation in resistance ( strength ) training.4 the world health organization ( who ) and multiple national guidelines specifically include resistance training in their recommended physical activity guidelines , particularly the guidelines for those aged 60 years ( older people).5,6 nevertheless , the proportions of older people participating are low.79 older populations across the world are growing , and it is expected that by 2050 , there will be two billion people aged 60 years living worldwide , which is more than double the number in 2013.10 with this increase , it is important that older people stay as healthy as possible for as long as they can to avoid needing ongoing health and care services , hospitalization , or a move into residential aged care . physical activity plays an essential role in staying healthy , maintaining independence , reducing the risk of falling , and allowing older people to live their later life well.11,12 the physical activity of choice for older people , particularly in australia is walking , with the majority of the older population participating only in this mode of exercise.13 studies have shown that fewer than 15% of older people participate in resistance training twice a week ( the minimum guideline recommended frequency).14,15 a number of studies have explored motivators and barriers to older people participating ( or commencing participation ) in resistance training in an attempt to increase participation rates.1619 the main motivators specific to older people participating in resistance training were preventing deterioration or disability , building muscle , falls prevention , and feeling more alert or having better concentration.16 barriers are usually identified by asking participants who are not or have not participated in a given activity to provide their reasons for not taking part . for older people , barriers to participating in resistance training include the following : health issues , pain , tiredness or fatigue , lack of social support , and a lack of available exercise facilities.16 given the low uptake of resistance training by older people , it is important that strategies are implemented to support ongoing participation for those who commence such programs , so that they maintain long - term participation , which can assist to optimize health and well - being . this study differs from other studies described as investigating barriers or reasons for ceasing participation because participants in these previous studies were still engaged in a resistance training program or had never participated.19,21,22 however , this study explores reasons why older people stop participating in a structured resistance exercise program by asking people who have recently made this choice . therefore , the aim of this study was to identify the reasons why older people who had been participating in a resistance training program chose to discontinue participation . inclusion criteria were people aged 60 years who had been attending a structured , gymnasium - based , resistance training program specifically designed for older people ( ie , living longer living stronger)23 and who within the previous 15 months decided to no longer attend . inclusion criteria were people aged 60 years who had been attending a structured , gymnasium - based , resistance training program specifically designed for older people ( ie , living longer living stronger)23 and who within the previous 15 months decided to no longer attend . regular physical activity and resistance training for improving strength , if appropriately moderated , can assist the older person to return to better health and fitness at a faster rate.4,31 indeed , national physical activity guidelines for older australians recommend older people who have stopped physical activity , or who are starting a new physical activity , should start at a level that is easily manageable and gradually build up the amount , type , and frequency of activity.32this study did not explore whether it is already common practice for fitness centers / others running resistance programs to follow - up those who cease participation and encourage them back with a program designed to help them regain fitness after injury or illness . older people who have stopped physical activity , or who are starting a new physical activity , should start at a level that is easily manageable and gradually build up the amount , type , and frequency of activity.32 holidays were the third most commonly reported reason for withdrawing from the resistance training program . yet , many older people have multiple commitments and interests such as looking after grandchildren , doing volunteer or paid work , and attending classes , and as a result , prefer sessions to be held earlier in the mornings or later in the afternoon.21,39 with the projected increase in the proportion of people aged > 60 years in the forthcoming decades , businesses need to be flexible and consider the needs and preferences of this important and growing target group . this study identified reasons why older people who had been participating in resistance training for a period of time ceased taking part . injury , illness , holidays , and issues with the resistance training program , center , or staff were the most commonly reported reasons for stopping . to reduce the number of older people leaving resistance training programs , it is suggested that gymnasiums and fitness centers provide ongoing advice for prevention and return to training after injury and flexible programs and services ( membership types ) that accommodate older people s life choices in retirement .
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ulcerations are characterized by defects in the epithelium , underlying connective tissue , or both . due to diversity of causative factors and presenting features , diagnosis of oral ulcerative lesions might be quite challenging [ 14 ] . this narrative review paper , however , focuses on the duration and the number of lesions in order to build a diagnostic decision tree . for the purpose of this article , if an ulcerative lesion lasts for two weeks or longer , it is considered chronic ; otherwise , it is regarded as an acute ulcer [ 1 , 2 ] . recurrent ulcers , on the other hand , present with a history of similar episodes with intermittent healing . the term solitary indicates the presence of a single ulcerative lesion whereas the term multiple describes the presence of several ulcerative lesions . in order to arrive at a definitive diagnosis , this is the cognitive process of integrating logic and knowledge into a series of stepwise decisions . all lesions that can not be excluded initially should be included in the differential diagnosis , followed by laboratory tests and additional investigations to narrow the diagnosis . according to the literature , many cases of oral malignant ulcerations were misdiagnosed as nonneoplastic lesions up to several months before the definite diagnosis was established [ 68 ] . valente et al . reported a case of squamous cell carcinoma misdiagnosed as a denture - related traumatic ulcer . meanwhile , de sant ' ana dos santos et al . reported misdiagnosis of lip scc as actinic cheilitis . a case of gingival scc masquerading as an aphthous ulcer was also reported by kumari et al . . this time elapse might jeopardize patients ' overall prognosis ; therefore , attempts should be done to come to timely diagnosis via more logical routes such as decision trees rather than test - and - error methods . a decision tree is a flowchart that organizes features of lesions so that the clinician can make a series of orderly decisions to reach a logical conclusion . to use the decision tree , the clinician begins from the left side of the tree , makes the first decision , and proceeds to the far right of the tree , where the names of entities are listed [ 9 , 10 ] . this narrative review article aims to introduce an updated decision tree for diagnosing oral ulcerative lesions on the basis of their diagnostic features . general search engines and specialized databases including pubmed , pubmed central , medline plus , ebsco , science direct , scopus , embase , and authenticated textbooks were used by the first author and the corresponding author to find relevant topics by means of mesh keywords such as oral ulcer , stomatitis , and mouth diseases . related english - language articles published since 1983 to 2015 in both medical and dental journals including reviews , meta - analyses , original papers ( randomized or nonrandomized clinical trials , prospective or retrospective cohort studies ) , case reports , and case series on oral ulcers , stomatitis , and oral disease were appraised . out of a total of 105 relative articles , 34 were excluded due to lack of full texts or being written in languages other than english . finally , 4 textbooks and 71 papers were selected including 32 reviews , 27 case reports or case series , and 12 original articles ( figure 1 ) . in this narrative review article , oral ulcerative lesions were categorized into three major groups : acute , chronic , and recurrent ulcers ( tables 13 ) and into five subgroups : solitary acute , multiple acute , solitary chronic , multiple chronic , and solitary / multiple recurrent , based on the number and duration of lesions . in total , 29 entities were organized in the form of a decision tree ( figure 2 ) in order to help clinicians establish a logical diagnosis by stepwise progression . the first decision to be made is whether the ulcerative lesion is of an acute , chronic , or recurrent nature ; thereafter , the lesion(s ) should be placed in one of the five subgroups . then , the clinician can consult the list of diagnoses in the relevant category . they are caused by mechanical damage ( contact with sharp foodstuff ; accidental biting during mastication , talking , or even sleeping ) and thermal , electrical , or chemical burns . traumatic ulcers are most common on the tongue , lips , and buccal mucosa . according to chen et al . , traumatic lesions of the oral cavity were mostly seen on the buccal mucosa ( 42% ) , followed by the tongue ( 25% ) and the lower lip ( 9% ) . noteworthy , traumatic ulcers are more common in men than women ( male : female ratio of 2.7 : 1 ) . these lesions may persist for a few days or even several weeks , especially in the case of tongue ulcers due to repeated insults to the tissues [ 5 , 33 ] . the borders of traumatic ulcers are usually slightly raised and reddish , with a yellowish - white necrotic pseudomembrane that can be readily wiped off ( figure 3 ) . traumatic ulcers normally become painless within three days after the injury had been eliminated and , in most cases , heal within 10 days . necrotizing sialometaplasia . necrotizing sialometaplasia ( ns ) is a self - limiting , benign , inflammatory disease of the salivary glands more frequently seen in middle - aged men . although the main etiology is not clear ; many authors believe that local infarction due to ischemia of the salivary tissue is the causative factor . meanwhile , a number of potential predisposing factors have been suggested such as sharp direct local trauma ( local anesthesia , intubation , and surgical procedures ) , use of ill - fitting dentures , violent or provoked vomiting ( in patients with bulimia ) , upper respiratory infection , and radiotherapy [ 11 , 34 ] . ns appears as a crater - like ulcer with indurated and well - delineated borders . more than 75% of all cases occur on the posterior part of the palate , followed by the lower lip , retromolar pad , sublingual region , tongue , and larynx [ 11 , 34 ] . the size of the lesion ranges from less than 1 cm to over 5 cm . however , complete healing is usually observed only after five to seven weeks [ 11 , 34 ] . primary herpetic gingivostomatitis . primary herpetic gingivostomatitis is the most common pattern of symptomatic herpes simplex virus ( hsv ) infection . over 90% of cases it can be asymptomatic or very mild in young patients but is associated with more severe general symptoms in the elderly . most cases occur between the ages of six months and 5 years , with peak prevalence between 2 and 3 years . oral manifestations consist of a generalized gingivitis followed , after 2 - 3 days , by pin - headed vesicles that readily rupture and give rise to painful ulcers covered by a yellowish pseudomembrane . can be affected , and the number of the lesions is quite variable . in many cases , punched - out erosions along the free gingival margin have been reported . submandibular lymphadenitis , halitosis , and difficulty in swallowing are noted in most cases [ 13 , 35 ] . in addition , involvement of the oral mucosa anterior to waldeyer 's ring is encountered in roughly 10% of patients . the ulcers usually heal spontaneously after 5 to 7 days , with no scarring , but may persist for two weeks in severe cases [ 13 , 35 , 36 ] . herpes zoster infection ( shingles ) . herpes zoster infection ( hzi ) is a less common viral infection brought about by the reactivation of varicella zoster virus ( vzv ) [ 14 , 37 , 38 ] , which may happen spontaneously or as a result of immune system deficiency . increased age , trauma ( from dental procedures ) , psychological stress , malignancy , radiotherapy , and immunocompromised conditions such as organ transplantation , immunosuppressive therapy , and hiv infection are contributory factors for vzv reactivation . the incidence of hzi is 1.53 cases per 1,000 persons , which increases to 10 per 1,000 in people over 75 years of age [ 15 , 37 ] . majority of hzi involve the thoracic and lumbar dermatomes , but nearly 13% of patients present with involvement of the trigeminal nerve branch , most commonly the ophthalmic branch . the condition is acutely painful and patients with involvement of the maxillary branch experience a prodromal phase of unilateral pain , burning , and tenderness , usually on the palate ( figure 4 ) . after several days , painful , clustered ulcers of 15 mm in diameter appear on the hard palate or buccal gingivae in a characteristic unilateral pattern . development of blisters and ulcers on the mandibular gingivae and tongue is indicative of mandibular branch involvement . this entity is self - limiting , and management of oral lesions is directed toward pain control , supportive care , and hydration . use of acyclovir , valacyclovir , or famciclovir is also effective in treating hzi when started within 72 hours of disease onset [ 3 , 13 ] . herpangina presents as multiple vesicular exanthema and ulcers of the oropharynx , soft palate , and tonsillar pillars [ 16 , 17 ] ( figure 5 ) . children under 10 years of age are usually affected , and outbreaks occur in epidemics in summer . coxsackie virus a genotypes 110 ( cav110 ) , a12 , a16 , and a22 and coxsackie virus b genotypes ( cbv25 ) and echo virus 18 and entero virus 71 identified as etiologic factors . it is a self - limiting disease and management directed toward control of oral pain and fever . hand - foot - and - mouth disease ( hfm ) is one of the common causes of morbidity among children below 10 years of age [ 16 , 39 ] . all of the patients have skin rash , especially on the hands and feet and 30% on the buttocks . oral ulcers are usually located on the tongue , hard and soft palate , and buccal mucosa . it is characterized by irregular red macules , papules , and vesicles that coalesce with each other to grow larger and make plaques on the skin called target lesions . oral lesions usually appear as erythematous macules on the lips and buccal mucosa , followed by bullae and ulcerations with irregular borders and inflammatory halo . bloody encrustations can be observed on the lips , which is a diagnostic feature [ 18 , 41 ] . em can be triggered by medications such as sulfonamide , penicillin , cephalosporins , quinolones , analgesics , and nonsteroidal anti - inflammatory drugs ( nsaids ) or several infections ( herpes simplex virus , epstein - barr virus , cytomegalovirus , varicella zoster virus , fungal agents , and parasites ) . em typically affects young adults ( 2040 years ) and teenagers , but the onset might be as late as 50 years of age or elder . there is a male predilection with male to female ratio of 3 : 2 . according to recent evidence , em has been categorized as minor , major , steven - johnson syndrome or toxic epidermal necrolysis . prodromal signs such as fever , lymphadenopathy , headache , malaise , cough , and sore throat may be noticed one week prior to onset of mucocutaneous erythema or blisters [ 18 , 40 , 41 ] . treatment mainly depends on the severity of clinical presentations . in the mild forms , healing takes place within 10 to 20 days ; therefore , patients only need local wound care , liquid diet , and topical analgesics or anesthetics for pain control [ 40 , 41 ] . necrotizing ulcerative gingivitis . it is characterized by punched - out ulcerations , and necrosis on the papillary and marginal gingivae ( figure 6 ) as well as severe gingival pain and bleeding [ 19 , 43 ] . there are some predisposing factors such as smoking , poor oral hygiene , preexisting gingivitis , malnutrition , psychological stress , and hiv infection . the above - mentioned factors usually lead to immunodysregulation including depressed polymorphonuclear leukocytes , antibody response , and lymphocyte mitogenesis . nug usually affects young adults ( 1820 years of age ) , and it is estimated to be seen in 0.5% to 11% of the population [ 19 , 43 ] . the diagnosis of nug is based on three essential symptoms : sore gums , bleeding gums , and , the most diagnostic criterion , ulceration and necrosis of the interdental papillae . treatment is based on mechanical removal of tartar with local ( chlorhexidine , 0.12% twice daily ) and systemic ( amoxicillin 250 mg and metronidazole 250 mg , three times a day for 7 days ) delivery of antimicrobial agents . healing is expected in a few days , whereas inadequate treatment can lead to deterioration of lesions in the form of necrotizing ulcerative periodontitis ( nup ) [ 19 , 43 ] . oral hypersensitivity reactions . oral hypersensitivity reactions ( ohrs ) have a variety of manifestations : acute onset of em ulcers , red and white reticular lesions such as lichenoid reactions , fixed drug eruption ( usually seen as ulcers on the lip vermilion after exposure to drugs with resolution on withdrawal and relapse on rechallenge ) , swelling of the lips , and oral allergy syndrome ( itching with or without swelling of oral structures and oropharynx ) [ 3 , 44 , 45 ] . plasma cell stomatitis ( pcs ) was first described in the late 1960s and early 1970s as a hypersensitivity reaction and likely a contact stomatitis to a component of chewing gum . this entity usually occurs few days after exposure and presents as erythematous macular areas of oral cavity . angular cheilitis with fissuring and dry atrophic lips have been found in patients with pcs . nevertheless , pain control and anti - inflammatory agents can help diminish the healing time [ 3 , 44 , 45 ] . chemotherapy - related ulcers . bone marrow suppression and immune response of oral mucosa , which leads to bacterial , fungal , or viral infections , happen during indirect effect of chemotherapeutic agents . other medications cause oral ulcerative lesions via direct impact on replication and growth of the oral epithelial cells [ 3 , 46 ] . kolbinson et al . demonstrated that early changes in the oral mucosa such as erythema and ulceration appear between 5 and 7 days after onset of chemotherapy . it is also noted that these lesions are considered as risk factors for systemic infections . after completion of chemotherapy , the lesions resolve spontaneously ; however , anti - inflammatory drugs may be useful in minimizing chemotherapy - related ulcers [ 3 , 46 ] . chronic injuries of oral mucosa may lead to solitary long standing ulcerative lesions ; therefore , traumatic ulcer can also be classified as a chronic solitary ulcer . this entity has been reported by pattison as a self - inflicted gingival lesion in patients who were seeking prescriptions for narcotic drugs . chronic traumatic ulcerations usually occur on the tongue , lips , and buccal mucosa as ulcerative areas surrounding a central removable , yellow fibrinopurulent membrane . in many cases , the lesion develops a raised , rolled border of hyperkeratosis immediately adjacent to the area of ulceration [ 11 , 51 , 52 ] . however , some lesions persist for several weeks because of continued traumatic insults , irritation by the oral liquids , or secondary infection . there are different treatment modalities , but coating the ulcerated surface with fluocinonide or triamcinolone acetonide in an emollient base after meals and before bed time usually relieves pain and decreases duration of healing . although this lesion usually occurs on the palate , it can be seen anywhere from oral mucosa , which contains salivary glands including the retromolar trigon and the lips . ns initially presents as a tender erythematous nodule , followed by a deep ulcer with a yellowish base . it resembles squamous cell carcinoma and ulcerated mucoepidermoid carcinoma to a large extent during its ulcerated phase . ns is mainly a self - limiting lesion , and healing time may be varied from 2 to 12 weeks according to the severity of the lesion [ 5 , 11 , 34 ] . therefore , ns can be classified as an acute or chronic solitary ulcer . eosinophilic ulcer . eosinophilic ulcer ( eu ) or traumatic ulcerative granuloma with stromal eosinophilia ( tugse ) is a chronic solitary ulcer of oral mucosa , which is most frequently seen in patients aged 4060 years , but occurs in young and elderly patients as well . male to female ratio is 1 : 1 , or slightly more prevalent in women . the most frequently affected site is the tongue ( about 60% of cases ) , followed by buccal mucosa , retromolar region , floor of the mouth , and lips [ 3 , 21 ] . eosinophilic ulcer manifests as a slow - healing ulcer with a rolled or elevated border mimicking a squamous cell carcinoma ( figure 7 ) . they ranged from 0.5 to several cm in size . in two - thirds of cases , the main etiology is not clear , but trauma has been elicited in 20% to 50% of cases . in addition , intralesional corticosteroids , oral corticosteroids , topical antibiotics , and cryotherapy have been also suggested . ulcerative squamous cell carcinoma . squamous cell carcinoma ( scc ) represents about 95% of all oral malignancies [ 22 , 54 ] . it presents as a red , white , red - white , exophytic , or ulcerative lesion . scc is a persistent ulcer in the oral cavity , which is of high importance especially on the lips . scc is often asymptomatic ; therefore , patients usually are not aware of it until it has become relatively progressive . the classic ulcerative scc is described as a craterlike lesion having a rolled , indurated border and a velvety base ( figure 8) . it may be covered with a crust when occurring on the vermilion [ 5 , 22 ] . the mostly affected sites in the oral cavity are lower lip , floor of the mouth , and ventral and lateral borders of the tongue . lesions are usually solitary , but in rare cases multifocal . according to wood and goaz , a lesion is most likely a scc if the patient is male , older than 40 years , smokes or drinks heavily , no evidence of trauma or systemic disease exists , serologic findings are negative , and the lesion is not located on the posterolateral region of the hard palate . ulcerative form of scc is locally destructive ; thereby timely and correct diagnosis of oral scc plays a key role in the improvement of patients prognosis and survival rate [ 3 , 55 ] . there is no single treatment for oral scc ; however , various therapeutic modalities from surgery , radiotherapy , and chemotherapy to combination different methods have been introduced . cytomegalovirus - related ulcers occur more commonly under conditions of significant immunodeficiency , especially severe hiv disease [ 3 , 55 ] . oral ulcers are usually single , painful , large , and necrotic , with minimally rolled border , which affects keratinized and nonkeratinized mucosa . on rare occasions , some granulomatous diseases such as tuberculosis and leprosy can cause ulcerative lesions in the oral cavity [ 23 , 56 , 57 ] . the world health organization ( who ) has estimated 9.4 million incident cases and 11.1 million prevalent cases of tb globally [ 23 , 57 ] . tuberculosis rarely affects oral mucosa , roughly 1.4% of all tb cases with a male to female ratio of 4 : 1 . the classic oral lesion presents as a solitary ulcer usually with an undermined edge most commonly on the tongue , followed by gingivae , floor of the mouth , palate , lips , and buccal mucosa . meanwhile , it may be ragged and indurated and is often painful [ 24 , 25 ] . the differential diagnosis of tuberculous ulcer includes traumatic ulcer , syphilitic ulcer , and oral scc . primary syphilitic ulceration usually occurs as a result of orogenital or oroanal contact with an infectious lesion . almost one to three weeks after acquisition , a chancre develops as a solitary ulcer usually on the lips or rarely on the tongue , pharynx , or tonsils . the upper lip is more commonly affected in males and the lower lip in females , probably due to the anatomy involved with fellatio and cunnilingus . the ulceration is usually deep , with a red purple or brown base ragged rolled border , and usually an accompanying cervical lymphadenopathy . detailed history of sexual and social life style helps approach the diagnosis of primary syphilis [ 26 , 58 , 59 ] . deep fungal ulceration ( histoplasmosis , blastomycosis , and mucormycosis ) . oral mucosal lesion in histoplasmosis is usually secondary to pulmonary involvement and occurs in a significant percentage of patients with disseminated histoplasmosis . the most frequent feature of oral blastomycosis is a nonspecific , painless , verrucous ulcer with indurated borders , often mistaken for oral scc . the most common oral sign of mucormycosis is ulceration of the palate , which results from necrosis due to invasion of a palatal vessel . lesion has also been observed on the gingivae , lips , and alveolar ridge . some vesiculobullous diseases such as pemphigus vulgaris ( pv ) , mucous membrane pemphigoid ( mmp ) , and bullous pemphigoid ( bp ) present as multiple and chronic oral ulcerative lesions . pemphigus vulgaris ( pv ) is a chronic vesiculobullous mucocutaneous autoimmune disease characterized by loss of cell adhesion ( acantholysis ) and blister formation . about 90% of patients with pv develop oral lesions , and in more than 50% of cases they are the first sign of disease . the edge of lesions continues to extend over a period of weeks until they involve large areas of oral cavity . the lesions usually start on the buccal mucosa ; however , palate and gingivae are other commonly affected sites . gingival involvement might be in the form of desquamative gingivitis , which is a characteristic feature for pv . there is a small group of pv patients whose disease remains confined to the oral cavity . early diagnosis is an important aspect of patient management when lower doses of medication can be used for shorter periods of time to control the disease . the mainstay of treatment remains high doses of systemic corticosteroids , usually given in dosages of 1 to 2 mg / kg / d [ 3 , 60 ] . mucous membrane pemphigoid ( mmp ) has been known by different names including benign mucous membrane pemphigoid , cicatricial ( scarring ) pemphigoid , and ocular cicatricial pemphigoid . mmp is a common immune - mediated subepithelial blistering disease mainly affecting oral mucosa ( over 90% ) ; however , skin lesions are also present in 20% to 30% of cases . the most affected site in the oral cavity is gingivae followed by buccal mucosa and palate . it occurs twice as frequently in females and is generally seen in patients more than 50 years old [ 3 , 5 , 62 , 63 ] . desquamative gingivitis is the most common presentation of the disease , which can be the only feature of mmp . blood blisters which result from bleeding into bullae are a diagnostic feature of mmp in the oral cavity . bullous pemphigoid ( bp ) is the most common subepithelial blistering disease , which occurs chiefly in patients over the age of 60 . in this entity , , oral mucosal lesions are found in only 16.6% of cases , which are similar to pv but are smaller and less painful . meanwhile , extensive labial involvement which is common in pv is not present in bp . desquamative gingivitis has been mentioned as the most frequent oral manifestation in bp and gingivae may be the only affected site . clinically , oral lesions are not distinguishable from pv or mmp , but early remission of bp is more common [ 64 , 65 ] . noteworthy , bp has been reported in conjunction with other diseases such as multiple sclerosis , malignancies , or medications particularly diuretics . bullous pemphigoid is self - limiting and may last from a few months to 5 years . topical clobetasol or betamethasone has been suggested in the management of localized oral lesions , whereas , in more extensive disease , use of systemic corticosteroids alone or in combination with immunosuppressive drugs is recommended [ 3 , 64 ] . lichen planus ( lp ) . lichen planus ( lp ) is a chronic , autoimmune , mucocutaneous disease . most lp patients are middle - aged , and it is rare in children . the disease occurs more commonly in women than in men with a female / male ratio of approximately 2 : 1 . the reported prevalence of lp is up to 5% , while the prevalence of oral lp ( olp ) is 0.1% to 2.2% [ 28 , 66 ] . there are different subtypes of olp with different clinical presentations : reticular ( lace - like keratotic mucosal configuration , wickham 's striae ) , atrophic ( keratotic changes combined with mucosal erythema ) , erosive / ulcerative ( pseudomembrane covered ulcerations combined with keratosis and erythema ) , bullous ( vesiculobullous presentation combined with reticular or erosive patterns ) , and popular / plaque like ( keratotic changes with elevation adjacent to the normal mucosa ) ( figure 9 ) . reticular , popular , and plaque - like lesions are generally asymptomatic whereas bullous , erosive and ulcerative forms are generally associated with pain the most frequently affected site in the oral cavity is buccal mucosa , followed by the tongue , gingivae , palate , and vermilion border . the risk of malignancy has been estimated to be 0.4%3.7% , which often develops after 10 years . topical or systemic corticosteroids are usually recommended in management of olp . linear iga disease . linear iga disease ( lad ) or linear iga dermatitis is an autoimmune subepithelial mucocutaneous disease . peak of incidence is between sixth and seventh decades of life in adult patients , with a twofold predilection for females . the pediatric variant is called chronic bullous dermatitis of childhood or juvenile dermatitis herpetiform . oral involvement in lad has been estimated to be between 5% and 70% in the form of vesicles , painful ulcerations or erosions , and erosive gingivitis / cheilitis . the most common affected site in the oral cavity is hard and soft palate , followed by tonsillar pillars , buccal mucosa , tongue , and gingivae . in a case report by chan et al . oral lesions are usually managed with the use of topical steroids , but dapsone therapy for more severe cases is recommended . recurrent aphthous stomatitis ( ras ) is the most common inflammatory disease of the oral mucosa with a global prevalence of 0.5% to 75% and female predilection . the lesions usually begin with prodromal burning sensation 2 to 48 hours before an ulcer appears . oral aphthous ulcers typically occur as painful , symmetrically round fibrin - covered mucosal defects with an erythematous border and most commonly on nonkeratinized mucosa in healthy patients ( figure 10 ) . however , it can be seen on the keratinized mucosa especially in patients with immune deficiency . three clinical types of ras have been identified : minor - type ( mikulicz ) is smaller than 1 cm in diameter ( usually 2 - 3 mm ) and heals spontaneously in two weeks . major - type ( sutton ulcer ) is usually 13 cm in size , and lasts for 10 days to 6 weeks or even longer . herpetiform aphthae appears as very small ( 1 - 2 mm ) , extremely painful , and numerous ulcers ( up to 100 lesions ) . laboratory evaluation is mandatory when ( a ) episodes of lesions become more severe , ( b ) lesions begin after the age of 25 , and ( c ) general symptoms are accompanied by lesions . ras is self - limiting , but in severe cases topical or systemic corticosteroids are recommended [ 70 , 71 ] . recurrent herpes stomatitis . herpes simplex virus can establish latency in the trigeminal ganglia and periodically reactivate to cause recurrent herpetic stomatitis ( rhs ) . there are two subgroups : recurrent herpes simplex labialis ( hsl ) ( figure 11 ) , which is more commonly seen in healthy subjects . it begins as vesicles that rupture soon , which leave superficial crusted ulcers and heal without scarring . however , rih in immune competent patients is limited to the keratinized mucosa , especially on the hard palate usually as clustered and unilateral vesicles . common triggers of rhs are physical / emotional stress , uv light , cold weather , hormonal changes , upper respiratory tract illness , and lip / mouth trauma [ 3 , 72 ] . although the lesions are self - limiting , symptomatic treatment by using ice or lanolin is recommended . applying acyclovir ointment 5% every 2 hours since the prodromal phase until the lesions subside has been suggested as well . elective dental treatments should be deferred in patients with active lesions to prevent aerosolization of the virus . erythema multiform ( em ) can be induced by several infectious agents , in particular hsv [ 73 , 74 ] . according to ng et al . , hsv dna has been detected in 50% of patients with recurrent idiopathic em typically affecting young adults ( 2040 years ) and is more common in men with the ratio of 3 : 2 . it can be found several days or weeks following an episode of hsv , and the lips are mostly affected . the diagnosis is based on clinical features and is more straightforward when target lesions with preceding or coexisting hsv infection present . the duration of disease ranges from 2 to 36 years ( mean of 10 years ) . acyclovir ( 200 mg , 5 times a day , for 5 days ) is recommended for management of lesions . cyclic neutropenia ( cn ) is an immunodeficiency syndrome , characterized by regular periodic oscillations in the circulating neutrophil count from normal to neutropenic levels every 21 days , and lasting for 36 days . patients with cyclic neutropenia are usually asymptomatic but during neutropenic episodes suffer from fever of unknown origin , gingivitis , stomatitis , aphthous - like ulceration , cellulitis , perirectal abscess , and severe systemic pyogenic infections . the severity and recurrence of oral ulcers in cn are similar to those of ulcers in major ras ; additionally , periodontal destruction in cn is as severe as what is seen in aggressive periodontitis . behet 's disease ( bd ) is a systemic immune - mediated vasculitis characterized by the presence of recurrent oral and genital ulcers , ocular inflammation , and skin lesions . bd can affect any age groups , but onset before puberty and after the sixth decade of life is relatively rare . the most common age of presentation is around the third decade of life , with a balanced male / female ratio . recurrent and painful oral ulcers are present in 90% to 100% of patients with bd . presence of recurrent oral aphthous - like ulcers ( minor , major , or herpetiform ulcers , which recur at least three times within a period of 12 months ) along with two of the following : genital ulcers , ocular lesions ( anterior uveitis , posterior uveitis , vitreous cellularity , or retinal vasculitis ) , and skin lesions ( erythema nodosum , pseudofolliculitis or papulopustular lesions , or acneiform papulae in postadolescent patients without any steroid treatment , and positive pathergy test ) establishes the diagnosis [ 3032 ] . glucocorticoids , colchicine , azathioprine , cyclosporine , tacrolimus , anti - tnf - alpha ( infliximab and etanercept ) , thalidomide , and rituximab are all recommended for treatment . oral ulcerative lesions are categorized into solitary acute , multiple acute , solitary chronic , multiple chronic , and recurrent lesions . acute oral ulcerations result from traumatic insults , viral or bacterial infections , allergy , or cancer chemotherapy . acute traumatic ulcers usually present as solitary lesions of nonspecific clinical shapes ( figure 3 ) [ 11 , 12 ] , whereas viral stomatitis appear as multiple small symmetrical ulcers , which sometimes coalesce to form larger lesions with scalloped borders ( figure 4 ) [ 11 , 13 ] . allergic stomatitis tend to involve any site intraorally with various clinical features usually with a history of synchronous exposure to contactants or systemic allergens [ 3 , 44 ] . although chemotherapy - induced stomatitis can be quite diffuse , its commencement is chronologically related to therapeutic agents , which differentiate it from other acute oral ulcerations [ 46 , 47 ] . some bacterial stomatitis present as either acute multiple punched- out necrotic ulcerations ( e.g. , nug and nup ) ( figure 6 ) in patients with malnutrition or immunocompromised state [ 19 , 43 ] , or single chronic ulceration of which tuberculous ulcer presents as an undermined lesion . when confronting a single chronic ulcer , it would appear to be prudent to preclude oral squamous cell carcinoma ( figure 8) first due to its poor prognosis and grave morbidity [ 5 , 22 ] . in addition , in the category of solitary chronic ulcers , history of lung disease or dealing with animals propose histoplasmosis or blastomycosis , while presence of debilitating disease such as diabetes mellitus suggest mucormycosis . on the other hand , multiple map - like or asymmetrical ulcerations suggest the presence of mucocutaneous vesiculobullous diseases ; among them , pemphigus is of high potency for extension , which might lead to death [ 3 , 27 ] . despite chronic lesions with ongoing progression or sustained clinical features a history of repeated resolution and relapse prompts the clinician to rank recurrent oral ulcerations higher in the differential diagnosis . recurrent aphthous stomatitis ( figure 10 ) and recurrent herpes are two common entities in this category with the former being more frequent in the nonkeratinized mucosa and the latter in the keratinized mucosa [ 3 , 27 ] . the newly updated decision tree includes 29 oral ulcerative lesions based on duration and number of lesions , which helps clinicians establish a stepwise method to rule out improbable conditions to arrive at a logical diagnosis .
diagnosis of oral ulcerative lesions might be quite challenging . this narrative review article aims to introduce an updated decision tree for diagnosing oral ulcerative lesions on the basis of their diagnostic features . various general search engines and specialized databases including pubmed , pubmed central , medline plus , ebsco , science direct , scopus , embase , and authenticated textbooks were used to find relevant topics by means of mesh keywords such as oral ulcer , stomatitis , and mouth diseases . thereafter , english - language articles published since 1983 to 2015 in both medical and dental journals including reviews , meta - analyses , original papers , and case reports were appraised . upon compilation of the relevant data , oral ulcerative lesions were categorized into three major groups : acute , chronic , and recurrent ulcers and into five subgroups : solitary acute , multiple acute , solitary chronic , multiple chronic , and solitary / multiple recurrent , based on the number and duration of lesions . in total , 29 entities were organized in the form of a decision tree in order to help clinicians establish a logical diagnosis by stepwise progression .
1. Introduction 2. Search Strategy 3. Results 4. Discussion 5. Conclusion
due to diversity of causative factors and presenting features , diagnosis of oral ulcerative lesions might be quite challenging [ 14 ] . this narrative review paper , however , focuses on the duration and the number of lesions in order to build a diagnostic decision tree . a decision tree is a flowchart that organizes features of lesions so that the clinician can make a series of orderly decisions to reach a logical conclusion . to use the decision tree , the clinician begins from the left side of the tree , makes the first decision , and proceeds to the far right of the tree , where the names of entities are listed [ 9 , 10 ] . this narrative review article aims to introduce an updated decision tree for diagnosing oral ulcerative lesions on the basis of their diagnostic features . general search engines and specialized databases including pubmed , pubmed central , medline plus , ebsco , science direct , scopus , embase , and authenticated textbooks were used by the first author and the corresponding author to find relevant topics by means of mesh keywords such as oral ulcer , stomatitis , and mouth diseases . related english - language articles published since 1983 to 2015 in both medical and dental journals including reviews , meta - analyses , original papers ( randomized or nonrandomized clinical trials , prospective or retrospective cohort studies ) , case reports , and case series on oral ulcers , stomatitis , and oral disease were appraised . in this narrative review article , oral ulcerative lesions were categorized into three major groups : acute , chronic , and recurrent ulcers ( tables 13 ) and into five subgroups : solitary acute , multiple acute , solitary chronic , multiple chronic , and solitary / multiple recurrent , based on the number and duration of lesions . in total , 29 entities were organized in the form of a decision tree ( figure 2 ) in order to help clinicians establish a logical diagnosis by stepwise progression . the first decision to be made is whether the ulcerative lesion is of an acute , chronic , or recurrent nature ; thereafter , the lesion(s ) should be placed in one of the five subgroups . more than 75% of all cases occur on the posterior part of the palate , followed by the lower lip , retromolar pad , sublingual region , tongue , and larynx [ 11 , 34 ] . can be affected , and the number of the lesions is quite variable . the diagnosis of nug is based on three essential symptoms : sore gums , bleeding gums , and , the most diagnostic criterion , ulceration and necrosis of the interdental papillae . healing is expected in a few days , whereas inadequate treatment can lead to deterioration of lesions in the form of necrotizing ulcerative periodontitis ( nup ) [ 19 , 43 ] . oral hypersensitivity reactions ( ohrs ) have a variety of manifestations : acute onset of em ulcers , red and white reticular lesions such as lichenoid reactions , fixed drug eruption ( usually seen as ulcers on the lip vermilion after exposure to drugs with resolution on withdrawal and relapse on rechallenge ) , swelling of the lips , and oral allergy syndrome ( itching with or without swelling of oral structures and oropharynx ) [ 3 , 44 , 45 ] . other medications cause oral ulcerative lesions via direct impact on replication and growth of the oral epithelial cells [ 3 , 46 ] . the mostly affected sites in the oral cavity are lower lip , floor of the mouth , and ventral and lateral borders of the tongue . according to wood and goaz , a lesion is most likely a scc if the patient is male , older than 40 years , smokes or drinks heavily , no evidence of trauma or systemic disease exists , serologic findings are negative , and the lesion is not located on the posterolateral region of the hard palate . ulcerative form of scc is locally destructive ; thereby timely and correct diagnosis of oral scc plays a key role in the improvement of patients prognosis and survival rate [ 3 , 55 ] . on rare occasions , some granulomatous diseases such as tuberculosis and leprosy can cause ulcerative lesions in the oral cavity [ 23 , 56 , 57 ] . some vesiculobullous diseases such as pemphigus vulgaris ( pv ) , mucous membrane pemphigoid ( mmp ) , and bullous pemphigoid ( bp ) present as multiple and chronic oral ulcerative lesions . gingival involvement might be in the form of desquamative gingivitis , which is a characteristic feature for pv . oral involvement in lad has been estimated to be between 5% and 70% in the form of vesicles , painful ulcerations or erosions , and erosive gingivitis / cheilitis . oral ulcerative lesions are categorized into solitary acute , multiple acute , solitary chronic , multiple chronic , and recurrent lesions . the newly updated decision tree includes 29 oral ulcerative lesions based on duration and number of lesions , which helps clinicians establish a stepwise method to rule out improbable conditions to arrive at a logical diagnosis .
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patients with unresectable or metastatic hepatocellular carcinoma ( hcc ) , who are not candidates for local / regional treatment , have a very limited number of therapeutic options ( 1 ) . to date , sorafenib is the only systemic therapy proven to prolong overall survival in patients with advanced hcc who are not candidates for surgical or local / regional therapies . however , the median survival achieved with sorafenib therapy was reported to be only 10.7 months compared with 7.9 months in the placebo arm ( hazard ratio [ hr ] , 0.69 ; 95% confidence interval [ ci ] , 0.55 to 0.87 ; p<0.001 ) , and was even shorter in asian patients ( 6.2 vs. 4.1 months ; hr , 0.67 ; 95% ci , 0.49 to 0.93 ; p=0.0155 ) ( 2,3 ) . for those patients who are not candidates for sorafenib treatment or for those who have had treatment failures after sorafenib , the only therapeutic option is systemic chemotherapy . however , there are no standard chemotherapy regimens that have been shown to improve overall survival . a large number of controlled and uncontrolled studies have been performed with most of the major classes of chemotherapy agents given as single or combination therapies . single or combination regimens of other drugs , including doxorubicin , 5-fluorouracil ( 5-fu ) , capecitabine , cisplatin , gemcitabine , and mitoxantrone have elicited 0% to 27% response rates , 2 to 6 months time to progression ( ttp ) or progression - free survival , and 3 to 12 months of overall survival ( os ) ( 4 - 9 ) . the most widely studied regimens in patients with advanced hcc are doxorubicin and cisplatin - based chemotherapies . doxorubicin was associated with a single agent response rate of 0 - 15% ( 4,10 ) . doxorubicin as a single agent , when compared to the combination of cisplatin , interferon , doxorubicin , and 5-fu ( piaf ) in a phase iii randomized trial , caused a lower response rate ( 10.5% vs. 20.9% ) but a similar os rate ( 6.83 vs. 8.67 months , p=0.83 ) . doxorubicin single agent used to be considered a standard treatment for patients with advanced hcc before the sorafenib era ( 11 ) . cisplatin - based combination regimens were reported in some studies to result in higher objective response rates than regimens excluding cisplatin . cisplatin administration had an objective response rate of 15% as single - agent chemotherapy , and a higher response rate when combined with doxorubicin , 5-fu and gemcitabine ( 5 - 9,11,12 ) . however , there are insufficient data to recommend any one regimen as the standard of care . systemic chemotherapy loses efficacy due to the frequently observed development of multidrug tumor resistance , which is related to the high rate of expression of the multidrug resistance gene ( mdr1 ) , and p53 tumor suppressor gene mutations ( 13,14 ) . poor hepatic function in patients with advanced hcc results in higher toxicity and precludes systemic chemotherapy in many patients . therefore , patient selection for those who would benefit from systemic chemotherapy is of vital importance before initiating systemic chemotherapy in patients with advanced hcc . the aim of this study was to determine the efficacy of cisplatin - based combination chemotherapy and identify a subgroup of patients with advanced hcc who would be candidates for cisplatin - based combination chemotherapy . the records of all consecutive patients with advanced hcc who received cisplatin - based combination chemotherapy at a single center , before the sorafenib era , were retrospectively analyzed by univariate and multivariate prognostic factor analyses . between january 2003 and october 2009 , consecutive patients with unresectable or metastatic hcc who received cisplatin - based combination as the first - line chemotherapy at seoul national university bundang hospital were retrospectively enrolled . patients had disease progression after a curative resection or other local treatment procedures , such as radiofrequency ablation ( rfa ) , cryoablation or transarterial chemoembolization ( tace ) , or were not amenable to local / regional treatment . the diagnosis of hcc was confirmed by histopathology or by computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and angiographic findings in addition to elevated values of alpha - fetoprotein ( afp ) using the guidelines proposed by the korea liver cancer study group ( 15 ) . using these criteria , a patient was diagnosed with hcc if one or more risk factors ( hepatitis b virus [ hbv ] or hepatitis c virus [ hcv ] infection , or cirrhosis ) were present , and one of the following was also present : a serum afp level>400 ng / ml and a positive result on at least one of the three typical liver imaging techniques ( spiral ct , contrast enhanced dynamic mri or hepatic angiography ) ; or a serum afp level<400 ng / ml and positive findings on at least two of the three imaging techniques . a positive finding for typical hcc with dynamic ct or mri is indicative of arterial enhancement followed by venous washout in the delayed portal / venous phase . tnm stages were used for tumor staging and clinical stages were classified according to the cancer of the liver italian program ( clip ) staging system ( 16,17 ) . the first - line chemotherapy regimens given to patients were cisplatin - based combination chemotherapy with doxorubicin ( ap ) , 5-fu ( fp ) , capecitabine ( xp ) and gemcitabine ( gp ) . the ap regimen consisted of doxorubicin 60 mg / m delivered as an intravenous infusion over 30 minutes on day 1 , followed by cisplatin 60 mg / m infused over 30 minutes on day 1 . the fp regimen consisted of 5-fu 1,200 mg / m administered continuously on days 1 to 4 , and cisplatin 60 mg / m infused over 30 minutes on day 1 . the xp regimen consisted of capecitabine 2,000 mg / m orally administered on days 1 to 14 , and cisplatin 60 mg / m infused over 30 minutes on day 1 . the gp regimen consisted of gemcitabine 1,200 mg / m infused on days 1 and 8 , and cisplatin 60 mg / m infused over 30 minutes on day 1 . response was assessed after every two cycles of chemotherapy by ct or mri scan using the response evaluation criteria in solid tumors ( recist ) criteria ( 18 ) . complete response ( cr ) was defined as the disappearance of all target and non - target lesions compared to baseline . partial response ( pr ) was defined as at least a 30% decrease in the sum of the longest diameters of all target lesions , taking as a reference the baseline sum of the diameters with no new lesions appearing . patients were considered to have progressive disease ( pd ) if any new lesion appeared , if the tumor size increased by at least 20% in the sum of the diameters of the target lesions , taking as reference the smallest sum on study , or if there was unequivocal progression of existing non - target lesions . a patient who failed to meet the definition of cr , pr or pd was classified as having stable disease ( sd ) . the percentage of patients who had the best responses ( other than pd ) according to the recist criteria , and had those responses maintained for at least 28 days after the first radiologic evaluation , was defined as the disease control rate . toxic effects of chemotherapy were evaluated according to the national cancer institute - common terminology criteria for adverse events version 3.0 . the ttp was calculated from the first day of chemotherapy to the day when progressive disease was documented . os ( in days ) was calculated from the first day of chemotherapy to death by any cause . all survival distributions were calculated using the kaplan - meier method and the log - rank test was used for univariate analysis . the cox proportional hazard model was used to assess the prognostic factors by multivariate analysis . the study was approved by an independent review board at the seoul national university bundang hospital ( irb approval number : b-1003 - 095 - 102 ) . between january 2003 and october 2009 , consecutive patients with unresectable or metastatic hcc who received cisplatin - based combination as the first - line chemotherapy at seoul national university bundang hospital were retrospectively enrolled . patients had disease progression after a curative resection or other local treatment procedures , such as radiofrequency ablation ( rfa ) , cryoablation or transarterial chemoembolization ( tace ) , or were not amenable to local / regional treatment . the diagnosis of hcc was confirmed by histopathology or by computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and angiographic findings in addition to elevated values of alpha - fetoprotein ( afp ) using the guidelines proposed by the korea liver cancer study group ( 15 ) . using these criteria , a patient was diagnosed with hcc if one or more risk factors ( hepatitis b virus [ hbv ] or hepatitis c virus [ hcv ] infection , or cirrhosis ) were present , and one of the following was also present : a serum afp level>400 ng / ml and a positive result on at least one of the three typical liver imaging techniques ( spiral ct , contrast enhanced dynamic mri or hepatic angiography ) ; or a serum afp level<400 ng / ml and positive findings on at least two of the three imaging techniques . a positive finding for typical hcc with dynamic ct or mri is indicative of arterial enhancement followed by venous washout in the delayed portal / venous phase . tnm stages were used for tumor staging and clinical stages were classified according to the cancer of the liver italian program ( clip ) staging system ( 16,17 ) . the first - line chemotherapy regimens given to patients were cisplatin - based combination chemotherapy with doxorubicin ( ap ) , 5-fu ( fp ) , capecitabine ( xp ) and gemcitabine ( gp ) . the ap regimen consisted of doxorubicin 60 mg / m delivered as an intravenous infusion over 30 minutes on day 1 , followed by cisplatin 60 mg / m infused over 30 minutes on day 1 . the fp regimen consisted of 5-fu 1,200 mg / m administered continuously on days 1 to 4 , and cisplatin 60 mg / m infused over 30 minutes on day 1 . the xp regimen consisted of capecitabine 2,000 mg / m orally administered on days 1 to 14 , and cisplatin 60 mg / m infused over 30 minutes on day 1 . the gp regimen consisted of gemcitabine 1,200 mg / m infused on days 1 and 8 , and cisplatin 60 mg / m infused over 30 minutes on day 1 . response was assessed after every two cycles of chemotherapy by ct or mri scan using the response evaluation criteria in solid tumors ( recist ) criteria ( 18 ) . complete response ( cr ) was defined as the disappearance of all target and non - target lesions compared to baseline . partial response ( pr ) was defined as at least a 30% decrease in the sum of the longest diameters of all target lesions , taking as a reference the baseline sum of the diameters with no new lesions appearing . patients were considered to have progressive disease ( pd ) if any new lesion appeared , if the tumor size increased by at least 20% in the sum of the diameters of the target lesions , taking as reference the smallest sum on study , or if there was unequivocal progression of existing non - target lesions . a patient who failed to meet the definition of cr , pr or pd was classified as having stable disease ( sd ) . the percentage of patients who had the best responses ( other than pd ) according to the recist criteria , and had those responses maintained for at least 28 days after the first radiologic evaluation , was defined as the disease control rate . toxic effects of chemotherapy were evaluated according to the national cancer institute - common terminology criteria for adverse events version 3.0 . the ttp was calculated from the first day of chemotherapy to the day when progressive disease was documented . os ( in days ) was calculated from the first day of chemotherapy to death by any cause . all survival distributions were calculated using the kaplan - meier method and the log - rank test was used for univariate analysis . the cox proportional hazard model was used to assess the prognostic factors by multivariate analysis . the study was approved by an independent review board at the seoul national university bundang hospital ( irb approval number : b-1003 - 095 - 102 ) . between january 2003 and october 2009 , 73 patients received systemic chemotherapy at seoul national university bundang hospital . among the 73 , we identified 46 who underwent cisplatin combination chemotherapy as the first - line chemotherapy . the diagnosis of hcc was confirmed by pathology in five patients ; for the remaining 41 patients , the diagnosis was established by ct , mri , and angiographic findings in addition to elevated values of afp using the guidelines proposed by the korea liver cancer study group ( 15 ) . a total of 46 patients with hcc were analyzed in this study , and their clinical characteristics are summarized in table 1 . the mean age of the patients was 53 years ( range , 21 to 73 years ) and the male to female ratio was 4.1 : 1 . hbv infection was documented in most of the patients ( 82.6% ) , hcv infection in a few ( 4.3% ) and both infections in even fewer ( 2.2% ) . the majority of patients had an eastern cooperative oncology group ( ecog ) performance ( ps ) score of 0 - 1 ( 84.8% ) with liver function classified as child - pugh classification a ( 69.6% ) . the sites of extrahepatic metastases included lung in 32 ( 69.6% ) , bone in 14 ( 30.4% ) and lymph nodes in 31 ( 67.4% ) patients . previous treatments included surgery in 13 patients ( 28.3% ) , tace in 31 ( 67.4% ) , and rfa in six patients ( 13.0% ) . an afp level higher than 400 ng / ml was recorded in 23 ( 50.0% ) patients , and 28 patients ( 60.9% ) had portal vein thrombosis . the hbeag was positive in 10 patients ( 25.6% among hbv - positive patients ) . eleven patients ( 23.9% ) had a clip score of 0 or 1 points , 23 ( 50.0% ) patients had 2 - 3 points , and 12 patients ( 26.1% ) had 4 - 6 points . patients received a mean of 2.3 cycles of cisplatin - based combination chemotherapy ( range , 1 to 6 cycles ) : 10 patients received doxorubicin combination ( ap ) ; 32 fluoropyrimidine combination ( fp and xp ) ; and 4 patients received gemcitabine combination ( gp ) chemotherapy . the best responses to first - line chemotherapy are shown in table 2 : no complete response was observed ; two patients ( 4.3% ) achieved partial remission ; 14 ( 30.4% ) had stable disease ; and 30 ( 65.2% ) had progressive disease . there were no significant differences in response rates among the different regimens ( p=0.28 ) . a mean of 2.8 cycles of chemotherapy ( range , 1 to 6 cycles ) was given . two patients received anthracycline - based chemotherapy , 4 fluoropyrimidine - based , 10 gemcitabine - based , and 3 patients received sorafenib . the response rate was 5.3% ( 1/19 ) and the disease control rate was 47.4% ( 9/19 ) . after a median follow - up duration of 5.5 months , the median time to progression for all patients was 1.8 months ( 95% ci , 1.1 to 2.5 ) . there was no statistically significant difference in the time to progression among the regimens : 2.3 ( 95% ci , 0.2 to 4.4 ) months for the ap regimen , 1.8 months ( 95% ci , 0.8 to 2.2 ) for the fluoropyrimidine combination ( fp , xp ) and 1.9 ( 95% ci , 0.1 to 2.5 ) months for the gp regimen . the median os from the start of chemotherapy was 7.2 ( 95% ci , 3.0 to 11.5 ) months . the overall survival was 8.5 ( 95% ci , 3.0 to 13.9 ) months for the ap regimen , 4.1 months ( 95% ci , 2.3 to 5.8 ) for the fluoropyrimidine combination and 4.7 months ( 95% ci , can not be calculated ) for the gp regimen ( table 2 ) . on univariate analysis , poor ecog ps , child classification b compared to a , high clip score , and presence of portal vein thrombosis were statistically significant factors that indicated a poor prognosis for both ttp and os ( table 3 ) ( fig . 1 ) . based on multivariate analyses using a model with factors entered for a significance level of p0.05 , poor ecog ps ( hr , 4.51 ; 95% ci , 1.61 to 12.6 ; p=0.004 ) and the presence of portal vein thrombosis ( hr , 2.12 ; 95% ci , 1.10 to 4.11 ; p=0.026 ) were independent poor prognosis factors for ttp . the presence of portal vein thrombosis ( hr , 2.77 ; 95% ci , 1.36 to 5.62 ; p=0.005 ) was the only significant poor prognosis factor for os ( table 4 ) . toxicities were mostly hematologic , with grade 3/4 leukopenia in six ( 13% ) , neutropenia in 14 ( 30.4% ) and thrombocytopenia in five ( 10.9% ) patients . a grade 3/4 elevation of the aminotransferases occurred in 11 ( 23.9% ) patients , and jaundice in eight ( 17.4% ) patients . there was no significant difference in hematologic and liver - related toxicities among the cisplatin - based combination ( table 5 ) treatment . however , grade 3/4 neutropenia was more frequent in the gp and ap regimens . between january 2003 and october 2009 , 73 patients received systemic chemotherapy at seoul national university bundang hospital . among the 73 , we identified 46 who underwent cisplatin combination chemotherapy as the first - line chemotherapy . the diagnosis of hcc was confirmed by pathology in five patients ; for the remaining 41 patients , the diagnosis was established by ct , mri , and angiographic findings in addition to elevated values of afp using the guidelines proposed by the korea liver cancer study group ( 15 ) . a total of 46 patients with hcc were analyzed in this study , and their clinical characteristics are summarized in table 1 . the mean age of the patients was 53 years ( range , 21 to 73 years ) and the male to female ratio was 4.1 : 1 . hbv infection was documented in most of the patients ( 82.6% ) , hcv infection in a few ( 4.3% ) and both infections in even fewer ( 2.2% ) . the majority of patients had an eastern cooperative oncology group ( ecog ) performance ( ps ) score of 0 - 1 ( 84.8% ) with liver function classified as child - pugh classification a ( 69.6% ) . the sites of extrahepatic metastases included lung in 32 ( 69.6% ) , bone in 14 ( 30.4% ) and lymph nodes in 31 ( 67.4% ) patients . previous treatments included surgery in 13 patients ( 28.3% ) , tace in 31 ( 67.4% ) , and rfa in six patients ( 13.0% ) . an afp level higher than 400 ng / ml was recorded in 23 ( 50.0% ) patients , and 28 patients ( 60.9% ) had portal vein thrombosis . the hbeag was positive in 10 patients ( 25.6% among hbv - positive patients ) . eleven patients ( 23.9% ) had a clip score of 0 or 1 points , 23 ( 50.0% ) patients had 2 - 3 points , and 12 patients ( 26.1% ) had 4 - 6 points . patients received a mean of 2.3 cycles of cisplatin - based combination chemotherapy ( range , 1 to 6 cycles ) : 10 patients received doxorubicin combination ( ap ) ; 32 fluoropyrimidine combination ( fp and xp ) ; and 4 patients received gemcitabine combination ( gp ) chemotherapy . the best responses to first - line chemotherapy are shown in table 2 : no complete response was observed ; two patients ( 4.3% ) achieved partial remission ; 14 ( 30.4% ) had stable disease ; and 30 ( 65.2% ) had progressive disease . there were no significant differences in response rates among the different regimens ( p=0.28 ) . a mean of 2.8 cycles of chemotherapy ( range , 1 to 6 cycles ) was given . two patients received anthracycline - based chemotherapy , 4 fluoropyrimidine - based , 10 gemcitabine - based , and 3 patients received sorafenib . the response rate was 5.3% ( 1/19 ) and the disease control rate was 47.4% ( 9/19 ) . after a median follow - up duration of 5.5 months , the median time to progression for all patients was 1.8 months ( 95% ci , 1.1 to 2.5 ) . there was no statistically significant difference in the time to progression among the regimens : 2.3 ( 95% ci , 0.2 to 4.4 ) months for the ap regimen , 1.8 months ( 95% ci , 0.8 to 2.2 ) for the fluoropyrimidine combination ( fp , xp ) and 1.9 ( 95% ci , 0.1 to 2.5 ) months for the gp regimen . the median os from the start of chemotherapy was 7.2 ( 95% ci , 3.0 to 11.5 ) months . the overall survival was 8.5 ( 95% ci , 3.0 to 13.9 ) months for the ap regimen , 4.1 months ( 95% ci , 2.3 to 5.8 ) for the fluoropyrimidine combination and 4.7 months ( 95% ci , can not be calculated ) for the gp regimen ( table 2 ) . on univariate analysis , poor ecog ps , child classification b compared to a , high clip score , and presence of portal vein thrombosis were statistically significant factors that indicated a poor prognosis for both ttp and os ( table 3 ) ( fig . 1 ) . based on multivariate analyses using a model with factors entered for a significance level of p0.05 , poor ecog ps ( hr , 4.51 ; 95% ci , 1.61 to 12.6 ; p=0.004 ) and the presence of portal vein thrombosis ( hr , 2.12 ; 95% ci , 1.10 to 4.11 ; p=0.026 ) were independent poor prognosis factors for ttp . the presence of portal vein thrombosis ( hr , 2.77 ; 95% ci , 1.36 to 5.62 ; p=0.005 ) was the only significant poor prognosis factor for os ( table 4 ) . toxicities were mostly hematologic , with grade 3/4 leukopenia in six ( 13% ) , neutropenia in 14 ( 30.4% ) and thrombocytopenia in five ( 10.9% ) patients . a grade 3/4 elevation of the aminotransferases occurred in 11 ( 23.9% ) patients , and jaundice in eight ( 17.4% ) patients . there was no significant difference in hematologic and liver - related toxicities among the cisplatin - based combination ( table 5 ) treatment . however , grade 3/4 neutropenia was more frequent in the gp and ap regimens . the results of this study confirmed the poor prognosis and poor response to cisplatin - based combination chemotherapy among patients with advanced hcc , who were not amenable to local / regional treatment outside of the clinical trial setting . in this study , cisplatin - based combination chemotherapy regimens had a response rate of 4.3% , a median ttp of 1.8 ( 95% ci , 1.1 to 2.5 ) months , and a median os of 7.2 ( 95% ci , 3.0 to 11.5 ) months . our results are consistent with earlier studies that investigated the efficacy of cisplatin - based combination chemotherapy , although the results of our study are modest compared to prior studies with regard to ttp . the results of our study might more accurately reflect real clinical practice , with unselected patients . a high proportion of patients with metastases ( 89.1% ) and portal vein thrombosis ( 60.9% ) in this population may also explain the poor response rates . the combination of gemcitabine with cisplatin was reported to have a response rate of approximately 20% ( 7 ) , a ttp of 18 weeks , and a median os of 21 weeks . the combination of capecitabine and cisplatin elicited a response rate of 6.3% , a ttp of 2 months , and a median os of 12.2 months ( 8) . although not statistically significant , the ap regimen had a longer ttp and os than the other regimens ( fp , xp , gp ) ( table 2 ) . there were no differences in clinical characteristics of patients who received ap compared to the other regimens in this study . ( 6 ) reported that the response rate and disease control rate associated with the ap regimen were 18.9% ( 7/37 ) and 35.1% ( 13/37 ) , respectively , and the median ttp and os were 6.6 and 7.3 months . ( 12 ) , found that the response rate and disease control rate of the ap regimen were , respectively , 58.6% ( 6/21 ) and 76.2% ( 46/21 ) , and the median ttp and os were , respectively , 5.4 and 10.7 months . the os achieved with the ap regimen showed consistency across trials , 7.3 - 10.7 months , and compared favorably to the effects of sorafenib reported in an asian trial ( 3 ) . further study comparing cisplatin - based combination chemotherapy , especially the ap regimen , with sorafenib or a combination of cytotoxic regimens with sorafenib is needed in patients with advanced hcc . based on the results of multivariate analysis , the only independent prognostic factors in this study population were ecog ps ( p=0.004 ) and portal vein thrombosis ( p=0.026 ) with regard to ttp , and portal vein thrombosis ( p=0.005 ) with regard to os ( table 4 ) . ecog ps , child - pugh classification , clip score and portal vein thrombosis were prognostic factors for ttp and os on univariate analysis . however , the child - pugh classification lost its statistical significance in the multivariate analysis , probably due to the small number of patients enrolled . the clip score was not included in the multivariate model , since the clip score represented a combination of other risk factors : child - pugh stage ; tumor morphology and extension ; serum afp levels ; and portal vein thrombosis . the overall survival of patients associated with clip scores of 0 - 1 , 2 - 3 and 4 - 6 were 13.3 ( 95% ci , 1.9 to 24.6 ) , 7.6 ( 95% ci , 2.7 to 12.4 ) and 2.2 ( 95% ci , 1.6 to 2.7 ) months , respectively ( table 3 ) . the median os of patients in this study was shorter compared to os reported in other studies ( 17,19 - 22 ) . all patients had disease progression after a curative resection or other local treatment procedures by enrollment criteria , and the os was assessed from day one of chemotherapy , not from the time of diagnosis , which may explain the short survival duration . however , the decrease in survival associated with high clip score remained consistent , validating the prognostic value of clip score in this advanced hcc population . the results of this study are consistent with the findings of prior studies regarding prognostic factors ( 23 - 25 ) . ( 23 ) reported that the prognosis for patients with unresectable hcc after systemic chemotherapy depended on pre - treatment liver function and the stage of disease . portal vein thrombosis causes stenosis or occlusion of the portal vein ; as a result , the blood supply to the liver parenchyma is decreased and further deterioration of liver function can occur . zhang et al . ( 24 ) reported that portal vein thrombosis was often associated with a poor prognosis , and suggested palliative 3-dimensional conformal radiotherapy , targeting the main portal vein thrombosis . ( 25 ) analyzed clinical prognostic factors in 397 untreated patients with hcc and found that poor performance status , presence of ascites , and high afp levels were statistically significant prognostic factors associated with decreased overall survival . if the prognostic factors associated with ttp identified in our study were used to select candidates for cisplatin - based combination chemotherapy ( ecog ps 0 - 1 vs. 2 , and presence of pvt ) , the patients who had no risk factors ( ecog ps 0 - 1 without pvt ) would have shown a statistically significant increase in ttp compared to patients with 1 or 2 poor prognostic factors ( 3.1 [ 95% ci , 1.8 to 4.3 ] vs. 1.3 [ 95% ci , 1.3 to 1.4 ] vs. 0.7 [ 95% ci , 0.5 to 0.9 ] months , p<0.0001 ) and also in os ( 14.5 [ 95% ci , 10.5 to 18.5 ] vs. 4.5 [ 95% ci , 0.0 to 9.8 ] vs. 2.2 ( 1.1 to 3.2 ) months , p<0.0001 ) . these results suggest that patient selection based on the prognostic factors described above could aid in selecting candidates who would benefit from cisplatin - based combination chemotherapy . fifteen patients ( 32.6% ) in the current study had ecog ps 0 - 1 and no pvt , and their median ttp and os were , respectively , 3.1 months ( 95% ci , 1.8 to 4.3 months ) and 14.5 months ( 95% ci , 10.5 to 18.5 months ) , which compares favorably with sorafenib data in an asian trial ( 3 ) . on the other hand , six patients ( 13% ) with ecog ps 2 and pvt in the current study had a very poor prognosis and could have been spared the toxicities of the cisplatin - based combination chemotherapy , if patient selection had been based on the above prognostic factors prior to commencing chemotherapy . in addition , the selection of the chemotherapy regimen was not randomly assigned , but rather decided based on physician and patient preference , which could have led to biased results . furthermore , the small sample size might have contributed to a lack of power in comparing the different chemotherapy regimens with regard to their toxicity , efficacy , and prognostic value . despite these limitations , our study confirmed the poor prognosis and efficacy of cisplatin - based combination chemotherapy in patients with advanced hcc in a real world setting . our data suggest that selection of candidates for cisplatin - based combination chemotherapy based on prognostic factors - ecog ps , and presence of pvt- would benefit select patients with advanced hcc , while sparing the others the unnecessary toxicities of combination chemotherapy . careful classification of patients according to these prognostic variables should be part of the study design of future investigations of hcc chemotherapies . cisplatin - based combination chemotherapy in patients with advanced hcc showed a short ttp and a low response rate regardless of the chemotherapy regimen used . patients with a good ecog performance status and absence of portal vein thrombosis had a longer ttp and os , and therefore can be considered as good candidates for cisplatin - based combination chemotherapy .
purposesystemic chemotherapy is the only option for patients with unresectable / metastatic hepatocellular carcinoma ( hcc ) who are not candidates for local / regional treatment . however , the response to such treatment and survival are poor , especially in hepatitis b virus ( hbv ) endemic areas . the aim of this study was to determine the efficacy of cisplatin - based combination chemotherapy and identify a subgroup of advanced hcc patients with favorable responses.materials and methodsthe medical records of all consecutive patients with unresectable / metastatic hcc who received cisplatin - based combination chemotherapy between january 2003 and october 2009 were reviewed . time to progression ( ttp ) and overall survival ( os ) were determined using kaplan - meier analysis . univariate and multivariate analyses were performed to identify prognostic factors for ttp and os.resultsdata for 46 patients were analyzed . first - line chemotherapies consisted of cisplatin - based combination treatment with doxorubicin , fluoropyrimidines and gemcitabine . the response rate for all patients was 4.3% . the median ttp and os were 1.8 ( 95%confidence interval [ ci ] , 1.1 to 2.5 ) and 7.2 ( 95% ci , 3.0 to 11.5 ) months , respectively . eastern cooperative oncology group ( ecog ) performance status ( ps ) , child classification , cancer of the liver italian program ( clip ) score and portal vein thrombosis ( pvt ) were identified by univariate analyses as prognostic factors for ttp and os . ecog ps ( hazard ratio [ hr ] , 4.51 ; 95% ci , 1.61 to 12.6 ; p=0.004 ) and pvt ( hr , 2.12 ; 95% ci , 1.10 to 4.11 ; p=0.026 ) were independent prognostic factors for ttp.conclusioncisplatin-based combination chemotherapy in patients with advanced hcc has a low response rate and short ttp regardless of the chemotherapy regimen used . patients with a good ecog ps and without pvt can be considered candidates for cisplatin - based combination chemotherapy .
Introduction Materials and Methods 1. Patients 2. Cisplatin-based combination chemotherapy 3. Response and toxicity evaluation 4. Statistical analysis Results 1. Patient characteristics 2. Efficacy 3. Survival analysis 4. Prognostic factors 5. Toxicity Discussion Conclusion
patients with unresectable or metastatic hepatocellular carcinoma ( hcc ) , who are not candidates for local / regional treatment , have a very limited number of therapeutic options ( 1 ) . however , the median survival achieved with sorafenib therapy was reported to be only 10.7 months compared with 7.9 months in the placebo arm ( hazard ratio [ hr ] , 0.69 ; 95% confidence interval [ ci ] , 0.55 to 0.87 ; p<0.001 ) , and was even shorter in asian patients ( 6.2 vs. 4.1 months ; hr , 0.67 ; 95% ci , 0.49 to 0.93 ; p=0.0155 ) ( 2,3 ) . the aim of this study was to determine the efficacy of cisplatin - based combination chemotherapy and identify a subgroup of patients with advanced hcc who would be candidates for cisplatin - based combination chemotherapy . the records of all consecutive patients with advanced hcc who received cisplatin - based combination chemotherapy at a single center , before the sorafenib era , were retrospectively analyzed by univariate and multivariate prognostic factor analyses . between january 2003 and october 2009 , consecutive patients with unresectable or metastatic hcc who received cisplatin - based combination as the first - line chemotherapy at seoul national university bundang hospital were retrospectively enrolled . between january 2003 and october 2009 , consecutive patients with unresectable or metastatic hcc who received cisplatin - based combination as the first - line chemotherapy at seoul national university bundang hospital were retrospectively enrolled . after a median follow - up duration of 5.5 months , the median time to progression for all patients was 1.8 months ( 95% ci , 1.1 to 2.5 ) . based on multivariate analyses using a model with factors entered for a significance level of p0.05 , poor ecog ps ( hr , 4.51 ; 95% ci , 1.61 to 12.6 ; p=0.004 ) and the presence of portal vein thrombosis ( hr , 2.12 ; 95% ci , 1.10 to 4.11 ; p=0.026 ) were independent poor prognosis factors for ttp . after a median follow - up duration of 5.5 months , the median time to progression for all patients was 1.8 months ( 95% ci , 1.1 to 2.5 ) . based on multivariate analyses using a model with factors entered for a significance level of p0.05 , poor ecog ps ( hr , 4.51 ; 95% ci , 1.61 to 12.6 ; p=0.004 ) and the presence of portal vein thrombosis ( hr , 2.12 ; 95% ci , 1.10 to 4.11 ; p=0.026 ) were independent poor prognosis factors for ttp . in this study , cisplatin - based combination chemotherapy regimens had a response rate of 4.3% , a median ttp of 1.8 ( 95% ci , 1.1 to 2.5 ) months , and a median os of 7.2 ( 95% ci , 3.0 to 11.5 ) months . based on the results of multivariate analysis , the only independent prognostic factors in this study population were ecog ps ( p=0.004 ) and portal vein thrombosis ( p=0.026 ) with regard to ttp , and portal vein thrombosis ( p=0.005 ) with regard to os ( table 4 ) . if the prognostic factors associated with ttp identified in our study were used to select candidates for cisplatin - based combination chemotherapy ( ecog ps 0 - 1 vs. 2 , and presence of pvt ) , the patients who had no risk factors ( ecog ps 0 - 1 without pvt ) would have shown a statistically significant increase in ttp compared to patients with 1 or 2 poor prognostic factors ( 3.1 [ 95% ci , 1.8 to 4.3 ] vs. 1.3 [ 95% ci , 1.3 to 1.4 ] vs. 0.7 [ 95% ci , 0.5 to 0.9 ] months , p<0.0001 ) and also in os ( 14.5 [ 95% ci , 10.5 to 18.5 ] vs. 4.5 [ 95% ci , 0.0 to 9.8 ] vs. 2.2 ( 1.1 to 3.2 ) months , p<0.0001 ) . cisplatin - based combination chemotherapy in patients with advanced hcc showed a short ttp and a low response rate regardless of the chemotherapy regimen used . patients with a good ecog performance status and absence of portal vein thrombosis had a longer ttp and os , and therefore can be considered as good candidates for cisplatin - based combination chemotherapy .
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the ability of the food and pharmacological substances to interactions with free radicals is their important property ( pawowska - gral et al . , 2013 ; rzepecka - stojko et al . , free radicals are responsible for a lot of negative effects in organism , and their inactivation is needed . free radicals have unpaired electrons , which cause major biochemical reactions and destroy the structures in cells . free radicals are dangerous during the diabetes , polyneuropathy , arteriosclerosis , and cancer ( eaton et al . , 1998 ; the substances used in medicine should not contain free radicals , and they should be antioxidants . pharmacological species as antioxidants react with free radicals , which loss their unpaired electrons and become diamagnetic . the activity of diamagnetic molecules is lower than paramagnetic free radicals , the risk of modification of chemical structures in tissues decreases , and their functions are not destroyed ( jaroszyk , 2008 ; bartosz , 2006 ) . the examination of contents of free radicals in food ( pawowska - gral et al . , 2013 ) , drugs ( ramos et al . , 2013 ) , herbs ( kurzeja et al . , 2013 ) , biopolymers ( chodurek et al . , 2012 ) , cells ( pawowska - gral and pilawa , 2011 ) , and tissues ( eaton et al . , 1998 ; bartosz , 2006 ) by electron paramagnetic resonance ( epr ) is known . tea ( wawer and zawadzka , 2004 ) , meat ( sin et al . , 2005 ) , dry fruits ( yordanov and pachowa , 2006 ) , and flour ( shimoyama et al . , free radicals may appear in drugs during sterilization processes , and such conditions accompanied by production of these paramagnetic dangerous molecules should be reject . the interacting factors killing the microorganisms during sterilization of drugs are radiation or high temperature ( skowroska et al . , 2012 ; wilczyski et al . , 2012 ) epr studies showed that gamma irradiation ( wilczyski et al . , 2012 ) or heating of drugs ( skowroska et al . , 2012 ; kocielniak - ziemniak and pilawa , 2012 ) or herbs ( pawowska - gral et al . , 2013 ; kurzeja et al . , epr spectroscopy was used to determine the optimal condition of radiative ( wilczyski et al . , 2012 ) and thermal sterilization of drugs ( skowroska et al . , 2012 ; kocielniak - ziemniak and pilawa , 2012 ) . thermal sterilization of herbs also forms free radicals in their molecular units ( pawowska - gral et al . , 2013 ; kurzeja et al . , 2012 ) and biradicals ( najder - kozdrowska et al . , 2010 ) were found by epr method in melanin biopolymers , model melanins , and their complexes with metal ions and drugs ( najder - kozdrowska et al . , 2010 ) . free radical concentration in melanins increased after adding diamagnetic metal ions , and it was lower after complex formation between melanin and paramagnetic metal ions ( najder - kozdrowska et al . , 2010 ) . epr spectra were measured for tumor cells ( pawowska - gral and pilawa , 2011 ) and tissues ( eaton et al . , 1998 ; laser irradiation of tumor cells with photosensitizer changed parameters of their epr spectra , and the changes depended on type of cells ( pilawa et al . , 2006 ) . this information was obtained by comparative analysis of epr spectra of free radicals in food , drugs , or biological samples ( pawowska - gral et al . , 2013 ; skowroska et al . , 2012 epr method is mainly used to study paramagnetic samples containing free radicals , but it is also possible to test antioxidant properties of diamagnetic samples by microwave absorption in this spectroscopy ( arshad et al . , 2013 ; rzepecka - stojko et al . , 2012 ; the antioxidative interactions of the samples reflect the quench of epr line of the paramagnetic reference after addition to its environment the tested molecules ( bartosz , 2006 ) . for example , it is known as epr measurement of antioxidative properties of bee pollen extracts ( rzepecka - stojko et al . , 2012 ) and the aim of this work was to show spectroscopic examination of the influence of uv irradiation on interactions of echinaceae purpureae with free radicals . the susceptibility of the antioxidative properties of tested drug on uv irradiation was checked to obtain practical knowledge about storage conditions for e.purpureae . echinaceae purpureae is the most popular herbal immune adjuvant ( ghedira et al . , 2008 ; e.purpureae preparations are consumed mainly in autumn and winter , when we need additional protection against bacteria and viruses . e. purpureae contains caffeic acid derivatives , flavonoids , polyacetylenes , polysaccharides , and small amounts of essential oil . e. purpureae also exhibits properties such as anti - inflammatory , antibacterial , antiviral , antifungal , antioxidant , diuretic , cholagogue , and antispasmodic , and stimulates the synthesis of collagen and elastin ( koevar et al . , the herb is used as a natural body tonic and shortens it the duration of colds . it has the prophylactic effect and helps in the treatment of respiratory infections , flu , and tonsillitis . it is also recommended by recurrent infections of the urinary tract and inflammation of the ascending cholangitis ( koevar et al . the herb is useful in healing wounds , ulcers , burns , frostbite , and pressure ulcers . it supports the treatment of acne , psoriasis , eczema and herpes , as well as inflammatory vagina and vulva ( koevar et al . , 2012 ; moraes et al . , 2011 ; ghedira et al . , 2008 ; schapowal , 2013 ) . the infusions used in the form of lotions relieve inflammation of the throat , mouth , and gums . in cosmetology , the herb is used as a moisturizer , regenerating , antioxidant , soothing irritation , and inflammation of the skin ( koevar et al . , 2012 ; schapowal , 2013 ) . we used the following times of irradiation : 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , and 110 min . the irradiation was performed by the use of medison 250 lamp with four radiators with power of 20 w. the uva wavelengths ( ) were in the range of 315400 nm . epr spectroscopy with microwaves of frequency of 9.3 ghz from an x - band was applied in the examination of e. purpureae interactions with free radicals . the paramagnetic reference dpph ( 2,2-diphenyl-1-picrylo - hydrazyl)was used as the model source of free radicals . epr spectra of free radicals of dpph in 10 % ethyl alcohol solution were measured . these spectra were compared with epr spectra of dpph in ethyl solution after adding of the tested nonirradiated and uv - irradiated e. purpureae samples . the antioxidative properties of the tested samples cause the decrease of amplitude of epr line of dpph . the quenching of the epr lines of dpph after addition of e. purpureae to the solution was observed . the measurements were done for the samples placed in the thin - walled glass tubes with the external diameter of 1 mm . the empty tubes did not contain paramagnetic impurities , and the epr signals were not observed for them . epr spectrometer with magnetic modulation of 100 khz produced by radiopan firm ( pozna , poland ) was used in this experiment . microwave frequency was measured by mcm101 recorder of eprad firm ( pozna , poland ) . epr spectra of dpph were numerically detected as the first derivatives by the the rapid scan unit of jagmar firm ( krakw , poland ) linked with the epr spectrometer . the short time of acquisition of the individual epr line was equal to 1 s. to avoid microwave saturation of the epr lines , the spectra were detected with low microwave power of 2.2 mw , which corresponds to 15 db of attenuation . the total microwave power produced by klystron of the epr spectrometer was 70 mw . the epr spectrum of the reference dpph in ethyl solution is presented in fig . 1 . the analyzed lineshape parameters of this spectrum a1 , a2 , b1 , and b2are shown in fig . 1 . differences between a1 and a2 , b1 and b2 , indicate on asymmetry of the epr spectrum . the values of a1/a2 , a1 a2 , b1/b2 , and b1 b2 , were calculated . amplitudes ( a ) of the epr spectra were obtained as a1 + a2 . amplitude of the epr line increases with increasing of the free radical contents in the sample ( wertz and bolton , 1986 ; weil and bolton , 2007 ) . linewidths ( bpp ) of the epr spectra were obtained as b1 + b2 . linewidths depend on magnetic interactions in the sample ( wertz and bolton , 1986 ; weil and bolton , 2007 ) . this value was used to obtain g - factor of free radicals existing in the source of free radicalsdpph.fig . the parameters of a 1 , a 2 , b 1 , and b 2 were used to analyze the asymmetry of epr spectra . the asymmetry parameters a 1/a 2 , a 1 a 2 , b 1/b 2 , and b 1 b 2were calculated . b is the magnetic induction of the field produced by electromagnet of the epr spectrometer . b r is the resonance magnetic induction epr spectrum of the reference dpph in ethyl alcohol solution . the parameters of a 1 , a 2 , b 1 , and b 2 were used to analyze the asymmetry of epr spectra . the asymmetry parameters a 1/a 2 , a 1 a 2 , b 1/b 2 , and b 1 b 2were calculated . b is the magnetic induction of the field produced by electromagnet of the epr spectrometer . b r is the resonance magnetic induction g - factors were calculated from the paramagnetic resonance condition as ( wertz and bolton , 1986 ) g = h/bbr , where h planck constant , microwave frequency , b bohr magneton , and br induction of resonance magnetic field . g - factor characterizes localization of unpaired electrons in the sample ( wertz and bolton , 1986 ) . the calculations were performed by the use of programs of jagmar firm ( krakw , poland ) and labview 8.5 of national instruments firm . echinaceae purpureae is the most popular herbal immune adjuvant ( ghedira et al . , 2008 ; e.purpureae preparations are consumed mainly in autumn and winter , when we need additional protection against bacteria and viruses . e. purpureae contains caffeic acid derivatives , flavonoids , polyacetylenes , polysaccharides , and small amounts of essential oil . e. purpureae also exhibits properties such as anti - inflammatory , antibacterial , antiviral , antifungal , antioxidant , diuretic , cholagogue , and antispasmodic , and stimulates the synthesis of collagen and elastin ( koevar et al . , the herb is used as a natural body tonic and shortens it the duration of colds . it has the prophylactic effect and helps in the treatment of respiratory infections , flu , and tonsillitis . it is also recommended by recurrent infections of the urinary tract and inflammation of the ascending cholangitis ( koevar et al . the herb is useful in healing wounds , ulcers , burns , frostbite , and pressure ulcers . it supports the treatment of acne , psoriasis , eczema and herpes , as well as inflammatory vagina and vulva ( koevar et al . , 2012 ; moraes et al . , 2011 ; ghedira et al . , 2008 ; schapowal , 2013 ) . the infusions used in the form of lotions relieve inflammation of the throat , mouth , and gums . in cosmetology , the herb is used as a moisturizer , regenerating , antioxidant , soothing irritation , and inflammation of the skin ( koevar et al . , 2012 ; schapowal , 2013 ) . we used the following times of irradiation : 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , and 110 min . the irradiation was performed by the use of medison 250 lamp with four radiators with power of 20 w. the uva wavelengths ( ) were in the range of 315400 nm . epr spectroscopy with microwaves of frequency of 9.3 ghz from an x - band was applied in the examination of e. purpureae interactions with free radicals . the paramagnetic reference dpph ( 2,2-diphenyl-1-picrylo - hydrazyl)was used as the model source of free radicals . epr spectra of free radicals of dpph in 10 % ethyl alcohol solution were measured . these spectra were compared with epr spectra of dpph in ethyl solution after adding of the tested nonirradiated and uv - irradiated e. purpureae samples . the antioxidative properties of the tested samples cause the decrease of amplitude of epr line of dpph . the quenching of the epr lines of dpph after addition of e. purpureae to the solution was observed . the measurements were done for the samples placed in the thin - walled glass tubes with the external diameter of 1 mm . the empty tubes did not contain paramagnetic impurities , and the epr signals were not observed for them . epr spectrometer with magnetic modulation of 100 khz produced by radiopan firm ( pozna , poland ) was used in this experiment . microwave frequency was measured by mcm101 recorder of eprad firm ( pozna , poland ) . epr spectra of dpph were numerically detected as the first derivatives by the the rapid scan unit of jagmar firm ( krakw , poland ) linked with the epr spectrometer . the short time of acquisition of the individual epr line was equal to 1 s. to avoid microwave saturation of the epr lines , the spectra were detected with low microwave power of 2.2 mw , which corresponds to 15 db of attenuation . the total microwave power produced by klystron of the epr spectrometer was 70 mw . the epr spectrum of the reference dpph in ethyl solution is presented in fig . 1 . the analyzed lineshape parameters of this spectrum a1 , a2 , b1 , and b2are shown in fig . 1 . differences between a1 and a2 , b1 and b2 , indicate on asymmetry of the epr spectrum . the values of a1/a2 , a1 a2 , b1/b2 , and b1 b2 , were calculated . amplitudes ( a ) of the epr spectra were obtained as a1 + a2 . amplitude of the epr line increases with increasing of the free radical contents in the sample ( wertz and bolton , 1986 ; weil and bolton , 2007 ) . linewidths ( bpp ) of the epr spectra were obtained as b1 + b2 . linewidths depend on magnetic interactions in the sample ( wertz and bolton , 1986 ; weil and bolton , 2007 ) . this value was used to obtain g - factor of free radicals existing in the source of free radicalsdpph.fig . the parameters of a 1 , a 2 , b 1 , and b 2 were used to analyze the asymmetry of epr spectra . the asymmetry parameters a 1/a 2 , a 1 a 2 , b 1/b 2 , and b 1 b 2were calculated . b is the magnetic induction of the field produced by electromagnet of the epr spectrometer . b r is the resonance magnetic induction epr spectrum of the reference dpph in ethyl alcohol solution . the parameters of a 1 , a 2 , b 1 , and b 2 were used to analyze the asymmetry of epr spectra . the asymmetry parameters a 1/a 2 , a 1 a 2 , b 1/b 2 , and b 1 b 2were calculated . b is the magnetic induction of the field produced by electromagnet of the epr spectrometer . b r is the resonance magnetic induction g - factors were calculated from the paramagnetic resonance condition as ( wertz and bolton , 1986 ) g = h/bbr , where h planck constant , microwave frequency , b bohr magneton , and br induction of resonance magnetic field . g - factor characterizes localization of unpaired electrons in the sample ( wertz and bolton , 1986 ) . the calculations were performed by the use of programs of jagmar firm ( krakw , poland ) and labview 8.5 of national instruments firm . the comparison of the epr spectra of dpph in ethyl solution and dpph in ethyl solution with e. purpureae indicates interactions between the tested herbs and free radicals . epr spectrum of dpph in ethyl solution with nonirradiated e. purpureae is shown in fig . amplitudes ( a ) and linewidth ( bpp ) of dpph line change upon interactions with e. purpureae ( figs . 1 , 2 ) . epr spectra of dpph in ethyl solution after adding of uv - irradiated e. purpureae for the herb exposed to electromagnetic waves during 10 and 110 min are presented in fig . the shape and parameters of the epr spectrum of dpph changed after the addition of e. purpureae to the solution . the parameters of the epr spectra of dpph as the reference , and dpph interacting with e. purpureae for the original nonirradiated herb and the herb uv irradiated are presented in table 1.fig . 2epr spectra of dpph in ethyl alcohol solution with e. purpureae nonirradiated ( a ) , and uv irradiated during 10 ( b ) , and 110 ( c ) minutes . b is the magnetic induction of the field produced by electromagnet of the epr spectrometertable 1the analyzed parameters of the epr spectra of the reference dpph interacting with nonirradiated and uv - irradiated e. purpureae sample a [ a.u . ] ( 0.1)b pp [ mt ] ( 0.02 ) a 1/a 2 ( 0.2 ) a 1 a 2 [ a.u . ] ( 0.2 ) b 1/b 2 ( 0.02 ) b 1 b 2 [ mt ] ( 0.04)dpph10.40.491.10.51.240.05nonirradiated echinaceae purpureae 0.80.481.20.10.620.11uv - irradiated echinaceae purpureae during time ( t):10 min0.90.480.90.10.900.0320 min1.20.611.10.11.230.0630 min1.40.531.30.21.000.0040 min1.20.641.20.11.080.0350 min1.10.520.90.11.360.0860 min1.10.541.10.10.610.1370 min1.50.440.80.10.860.0380 min1.40.701.10.10.640.1590 min1.20.401.30.21.250.04100 min1.30.561.10.01.060.02110 min1.50.591.00.10.860.04 a amplitude of the epr spectra ; bpp linewidth of the epr spectra ; lineshape parameters : a 1/a 2 , a 1 a 2 , b 1/b 2 , and b 1 b 2 . 1 . the times ( t ) of uv irradiation of the sample are in the range of 10110 min epr spectra of dpph in ethyl alcohol solution with e. purpureae nonirradiated ( a ) , and uv irradiated during 10 ( b ) , and 110 ( c ) minutes . b is the magnetic induction of the field produced by electromagnet of the epr spectrometer the analyzed parameters of the epr spectra of the reference dpph interacting with nonirradiated and uv - irradiated e. purpureae a amplitude of the epr spectra ; bpp linewidth of the epr spectra ; lineshape parameters : a 1/a 2 , a 1 a 2 , b 1/b 2 , and b 1 b 2 . 1 . the times ( t ) of uv irradiation of the sample are in the range of 10110 min g - factors of 2.0036 , typical for unpaired electrons localized on nitrogen atoms in dpph , were obtained . the amplitude ( a ) of epr lines of dpph in ethyl alcohol solution with nonirradiated e. purpureae was lower than the amplitude of epr signal of dpph in ethyl alcohol solution , before adding of the tested herb ( table 1 ) . similar amplitude ( a ) characterizes uv - irradiated e. purpureae during time 10 min relative to the sample nonirradiated ( table 1 ) . the higher amplitudes ( a ) of dpph lines in ethyl alcohol solution were obtained for e. purpureae irradiated by uv longer than 10 min 20110 min ( table 1 ) . 3a , it is clearly visible that all the relative amplitudes ( a / adpph ) of epr lines with the solution containing the tested herb are lower than one ( fig . 3b , the total amplitudes ( a ) of dpph interacting with nonirradiated and uv - irradiated e. purpureae are compared . the total amplitudes ( a ) are also lower for the uv - irradiated samples.fig . 3amplitudes of epr spectra of dpph in ethyl alcohol solution , and dpph interacting with nonirradiated and uv - irradiated e. purpureae in ethyl alcohol solution . a / adpph is the amplitude of epr line of dpph with the tested sample in alcohol solution divided by amplitude of epr line of the reference dpph in ethyl alcohol solution . the total amplitude a is the amplitude of epr line measured for dpph in ethyl alcohol solution . the times ( t ) of uv irradiation of the sample are in the range of 10110 min amplitudes of epr spectra of dpph in ethyl alcohol solution , and dpph interacting with nonirradiated and uv - irradiated e. purpureae in ethyl alcohol solution . the relative amplitudes a / adpph and the total amplitudes a are shown in fig . a / adpph is the amplitude of epr line of dpph with the tested sample in alcohol solution divided by amplitude of epr line of the reference dpph in ethyl alcohol solution . the total amplitude a is the amplitude of epr line measured for dpph in ethyl alcohol solution . the times ( t ) of uv irradiation of the sample are in the range of 10110 min the epr spectra of dpph in ethyl alcohol solution with e. purpureae were nonsymmetrical with the parameters of a1/a2 and b1/b2 which differ from 1 , and the parameters of a1 a2 and b1 b2 differ from 0 ( table 1 ) . the parameters of lineshape of epr spectrum of dpph ( a1/a2 , b1/b2 , a1 a2 , and b1 b2 ) changed with the time of uv irradiation of e. purpureae ( table 1 ) . the linewidths ( bpp ) of epr spectra of dpph in ethyl alcohol solution both for nonirradiated and uv - irradiated e. purpureae had the high values ( table 1 ; fig . 4 ) . 4linewidth ( bpp ) of epr spectra of dpph in ethyl alcohol solution , and dpph interacting with nonirradiated and uv - irradiated e. purpureae ethyl solution . a / adpph is the amplitude of epr line of dpph with the tested sample in alcohol solution divided by amplitude of epr line of the reference dpph in ethyl alcohol solution . the total amplitude a is the amplitude of epr line measured for dpph in ethyl alcohol solution . the times ( t ) of uv irradiation of the sample are in the range of 10110 min linewidth ( bpp ) of epr spectra of dpph in ethyl alcohol solution , and dpph interacting with nonirradiated and uv - irradiated e. purpureae ethyl solution . a / adpph is the amplitude of epr line of dpph with the tested sample in alcohol solution divided by amplitude of epr line of the reference dpph in ethyl alcohol solution . the total amplitude a is the amplitude of epr line measured for dpph in ethyl alcohol solution . the times ( t ) of uv irradiation of the sample are in the range of 10110 min application of epr spectroscopy at the x - band ( 9.3 ghz ) in food biophysics was confirmed . epr spectra of the paramagnetic reference were used to determine antioxidative properties of the popular herb as e. purpureae ( koevar et al . , 2012 ; moraes et al . , 2011 ; ghedira et al . , 2008 ; schapowal , 2013 ) with pharmacological interactions in human organism . the changes of shape and amplitudes of epr spectra of dpph in ethanol alcohol solution as the result of interactions of e. purpureae with free radicals of this reference were observed ( table 1 ; figs . 2 , 3 , 4 ) . the quenching of epr lines of the reference by the tested herb ( fig . the proposed method of examination of interactions of the herbs with free radicals has a lot of advantages . epr spectroscopy is a physical method , which uses the epr effect ( wertz and bolton , 1986 ; weil and bolton , 2007 ) . epr effect is caused by zeemann splitting of energy levels in magnetic field , and absorption of microwaves by electrons of the tested samples is studied . the energy of microwaves is fitted to the distances between the energy levels of electrons in magnetic fields . electrons after absorption of electromagnetic waves with the respective frequencies are excited , and after they relax via spin spin and spin lattice relaxation processes ( wertz and bolton , 1986 ; weil and bolton , 2007 ) . in practice , the magnetic field is produced by electromagnet of the epr spectrometer , and the tested samples are located in the resonance cavity . the type of free radicals and concentrations may be determined ( wertz and bolton , 1986 ; weil and bolton , 2007 ) . the economic costs of the epr measurements at x - band are very low , because only the cold water is used to decrease the temperature of electromagnet that is needed and the electrical current . the parameters of the epr spectra are analyzed numerically by the use of spectroscopic programs . application of epr in food biophysics ( pawowska - gral et al . , 2013 ; kurzeja et al . , 2013 ) , pharmacy ( skowroska et al . , 2012 ; wilczyski et al . , 2012 ) , medicine ( pawowska - gral and pilawa , 2011 ; pilawa et al . , 2006 ) , biology ( pawowska - gral et al . , 2013 ; kurzeja et al . , 2013 ) , free radicals ( chodurek et al . , 2012 ; najder - kozdrowska et al . , 2010 ) , techniques ( eaton et al . , 1998 ; wertz and bolton , 1986 ) , and biotechnology ( krzto et al . , 2009 ) is known . the obtained results broaden our knowledge about antioxidative properties of the famous herb e . the effect of uv irradiation on interactions of e. purpureae was not physically studied so far , and our proposition of epr analysis in this example has the innovatory character . the important result was obtained : the interactions of e. purpureae with free radicals decrease after uv irradiation ( table 1 ; fig . only the short time of uv irradiation ( 10 min ) does not negatively influence on antioxidative properties of e. purpureae , when the epr lines of dpph did not increase relatively to the nonirradiated herb ( table 1 ; fig . epr parameters of dpph changed with time of uv exposition ( table 1 ; figs . 3 , 4 ) the interactions of e. purpureae with free radicals had a complex character , and this fact was reflected by the changes of linewidths ( bpp ) ( fig . 4 ) and the asymmetry parameters ( a1/a2 , b1/b2 , a1 a2 , and b1 b2 ) of the dpph spectra with time of uv irradiation ( table 1 ) . the complex interactions are expected , because of the major transformations in e. purpureae under uv irradiation , when different chemical bonds may be broken and distances between unpaired electrons did not remain stable . the broadening of the epr lines of dpph interacting with e. purpureae is mainly caused by dipolar interactions between freer radicals . the obtained results proved the possibilities of epr studies of diamagnetic samples as e. purpureae by the use of paramagnetic probes dpph . the performed studies of e. purpureae by the use of an x - band ( 9.3 ghz ) epr spectroscopy proved thatnonirradiated and uv - irradiated e. purpureae reveal antioxidant properties ; it interacts with free radicals and as the result , it causes decrease of epr signal of the paramagnetic reference dpph in ethyl alcohol solution.uv irradiation changes interactions of e. purpureae with free radicals , and it decreases the antioxidative properties of this herb.the interactions of e. purpureae with free radicals depend on time of uv irradiation . the weaker interactions of e. purpureae with free radicals characterize the herb irradiated longer than 10 min ( irradiated 20110 min).taking to account of the antioxidative properties , e. purpureae should be stored without exposition on uv irradiations.usefulness of electron paramagnetic resonance spectroscopy with paramagnetic reference of dpph to determine interactions of diamagnetic herbs with free radicals was confirmed . nonirradiated and uv - irradiated e. purpureae reveal antioxidant properties ; it interacts with free radicals and as the result , it causes decrease of epr signal of the paramagnetic reference dpph in ethyl alcohol solution . uv irradiation changes interactions of e. purpureae with free radicals , and it decreases the antioxidative properties of this herb . the interactions of e. purpureae with free radicals depend on time of uv irradiation . the weaker interactions of e. purpureae with free radicals characterize the herb irradiated longer than 10 min ( irradiated 20110 min ) . taking to account of the antioxidative properties , e. purpureae should be stored without exposition on uv irradiations . usefulness of electron paramagnetic resonance spectroscopy with paramagnetic reference of dpph to determine interactions of diamagnetic herbs with free radicals was confirmed .
the effect of uva ( 315400 nm ) irradiation on echinaceae purpureae interactions with free radicals was examined by the use of electron paramagnetic resonance ( epr ) spectroscopy . the changes of antioxidant properties of e. purpureae with time of uv irradiation from 10 to 110 min ( 10 min steps ) were determined . dpph as the paramagnetic reference was used in this study . changes of epr signals of the reference after interactions with nonirradiated and uv - irradiated e. purpureae were detected . interactions of the tested e. purpureae samples caused decrease of the epr signal of dpph as the result of its antioxidant properties . the decrease of the amplitude of epr line of dpph was lower for interactions with uv - irradiated e. purpureae . epr examination confirmed antioxidant properties of e. purpureae . the weaker antioxidant properties of e. purpureae after uv irradiation were pointed out . e. purpureae should be storage in the dark . the tests bring to light usefulness of electron paramagnetic resonance with microwave frequency of 9.3 ghz ( an x - band ) in examination of storage conditions of pharmacological herbs .
Introduction Experimental method The studied samples EPR measurements Results Discussion Conclusions
, 2012 ) and the aim of this work was to show spectroscopic examination of the influence of uv irradiation on interactions of echinaceae purpureae with free radicals . epr spectroscopy with microwaves of frequency of 9.3 ghz from an x - band was applied in the examination of e. purpureae interactions with free radicals . these spectra were compared with epr spectra of dpph in ethyl solution after adding of the tested nonirradiated and uv - irradiated e. purpureae samples . epr spectroscopy with microwaves of frequency of 9.3 ghz from an x - band was applied in the examination of e. purpureae interactions with free radicals . these spectra were compared with epr spectra of dpph in ethyl solution after adding of the tested nonirradiated and uv - irradiated e. purpureae samples . the antioxidative properties of the tested samples cause the decrease of amplitude of epr line of dpph . b is the magnetic induction of the field produced by electromagnet of the epr spectrometer the analyzed parameters of the epr spectra of the reference dpph interacting with nonirradiated and uv - irradiated e. purpureae a amplitude of the epr spectra ; bpp linewidth of the epr spectra ; lineshape parameters : a 1/a 2 , a 1 a 2 , b 1/b 2 , and b 1 b 2 . the amplitude ( a ) of epr lines of dpph in ethyl alcohol solution with nonirradiated e. purpureae was lower than the amplitude of epr signal of dpph in ethyl alcohol solution , before adding of the tested herb ( table 1 ) . the times ( t ) of uv irradiation of the sample are in the range of 10110 min amplitudes of epr spectra of dpph in ethyl alcohol solution , and dpph interacting with nonirradiated and uv - irradiated e. purpureae in ethyl alcohol solution . the times ( t ) of uv irradiation of the sample are in the range of 10110 min linewidth ( bpp ) of epr spectra of dpph in ethyl alcohol solution , and dpph interacting with nonirradiated and uv - irradiated e. purpureae ethyl solution . the times ( t ) of uv irradiation of the sample are in the range of 10110 min application of epr spectroscopy at the x - band ( 9.3 ghz ) in food biophysics was confirmed . the changes of shape and amplitudes of epr spectra of dpph in ethanol alcohol solution as the result of interactions of e. purpureae with free radicals of this reference were observed ( table 1 ; figs . the effect of uv irradiation on interactions of e. purpureae was not physically studied so far , and our proposition of epr analysis in this example has the innovatory character . only the short time of uv irradiation ( 10 min ) does not negatively influence on antioxidative properties of e. purpureae , when the epr lines of dpph did not increase relatively to the nonirradiated herb ( table 1 ; fig . the performed studies of e. purpureae by the use of an x - band ( 9.3 ghz ) epr spectroscopy proved thatnonirradiated and uv - irradiated e. purpureae reveal antioxidant properties ; it interacts with free radicals and as the result , it causes decrease of epr signal of the paramagnetic reference dpph in ethyl alcohol solution.uv irradiation changes interactions of e. purpureae with free radicals , and it decreases the antioxidative properties of this herb.the interactions of e. purpureae with free radicals depend on time of uv irradiation . the weaker interactions of e. purpureae with free radicals characterize the herb irradiated longer than 10 min ( irradiated 20110 min).taking to account of the antioxidative properties , e. purpureae should be stored without exposition on uv irradiations.usefulness of electron paramagnetic resonance spectroscopy with paramagnetic reference of dpph to determine interactions of diamagnetic herbs with free radicals was confirmed . nonirradiated and uv - irradiated e. purpureae reveal antioxidant properties ; it interacts with free radicals and as the result , it causes decrease of epr signal of the paramagnetic reference dpph in ethyl alcohol solution . usefulness of electron paramagnetic resonance spectroscopy with paramagnetic reference of dpph to determine interactions of diamagnetic herbs with free radicals was confirmed .
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floating seaweeds are an important habitat for many accompanying or attached animals in offshore waters . among natural floating objects , several studies have highlighted the importance of rafting as a habitat for marine invertebrates and some fish species ( safran and omori 1990 ; inglfsson 1995 ; sano et al . for example , ascophyllum nodosum and fucus vesiculosus have been abundantly reported from the north atlantic coasts ( inglfsson 1995 ; vandendriessche et al . 2011 ) and macrocystis pyrifera along the californian and chilean coast ( hobday 2000 ; hinojosa et al . , sargassum species are the dominant floating seaweeds , especially around japan ( yoshida 1963 ) . in the sargasso sea , floating seaweeds pass their entire life in floating state through vegetative reproduction ( parr 1939 ) . on the other hand , sargassum species growing on rocky coasts form a luxuriant forest in spring and a scanty one in summer in the northwestern pacific ( komatsu et al . thus benthic sargassum forests have great influences on marine environments in spring such as water temperature ( komatsu et al . 1994 ) , ph ( komatsu and kawai 1986 ) , dissolved oxygen content of seawater ( komatsu 1989 ) , downward illumination ( komatsu 1989 ) , and water flow ( komatsu and murakami 1994 ) . during their maturation season from winter to spring when they become longer , waves and currents detach the seaweeds above or with holdfast on the bottom . this is because their drag force becomes greater than the attachment force due to higher buoyancy of large individuals , produced by their numerous gas vesicles ( yoshida 1963 ) . thus they contribute to the stocks of floating seaweeds in waters around japan ( yoshida 1963 ) , occasionally reaching similar extensions as observed in the sargasso sea ( parr 1939 ) . they serve as spawning substratum for flying fish and pacific saury ( ikehara and sano 1986 ) . around japan , one of the commercially most important pelagic fish , the yellowtail seriola quinqueradiata , also associates with seaweed rafts for reproductive purposes . juvenile s. quinqueradiata start to accompany floating seaweeds 1 month after hatching and leave them when their size attains 150 mm body length ( safran 1990 ; sakakura and tsukamoto 1997 ) . during the spring , fishermen catch wild juveniles accompanying floating seaweeds by scooping up these rafts together with the juvenile fishes , which are then used for aquaculture . this occurs especially in waters off kyushu island including the east china sea since1960s because the aquaculture of yellowtail depends on wild juveniles accompanying floating seaweeds . the dependence on wild seedstock is due to their cannibalism during its larvae and juvenile periods , which makes it difficult to produce artificial seedstock . yellowtails spawn in the region between the continental shelf and the oceanic front of the kuroshio current in the east china sea from late winter to spring ( yamamoto et al . 2007 ) . the earliest yellowtail larvae hatch in the end of january . yoshida ( 1963 ) reported that most floating seaweeds around japan are found in nearshore coastal waters , within 20 km from the shore , especially in areas where oceanic fronts come close to the coast . one important reason for the patchiness of floating items , such as macroalgae as well as any other floating objects , is their accumulation at fronts between two different water masses ( witherington 2002 ; acha et al . however , senta ( 1965 ) reported that massive amounts of floating seaweeds could not be found in the front between coastal waters and kuroshio current in the east china sea . recently , komatsu et al . ( 2007 ) surveyed seaweed rafts in the eastern east china sea in march and may and reported that they were distributed in waters on the continental shelf west of the kuroshio front ( komatsu et al . taking into account the yellowtail spawning season in the east china sea , the juvenile starts to accompany seaweed rafts in february . ( 1986 ) reported that survival rates of the juvenile reared with seaweed rafts or artificial seaweed rafts made by cellulose are higher than without rafts . thus , it is very important to know the distributions of seaweed rafts in the east china sea in february and early march . however , it was not well known whether abundant patches of floating seaweeds can be found in the east china sea in february and early march . this study thus aimed to elucidate the distribution of floating seaweeds in the east china sea in february and march by visual census and towed net samples . two research cruises using r / v tansei maru were organized to survey floating seaweeds in the east china sea in late winter to early spring . one was kt10 - 1 research cruise from 22 february to 6 march 2010 and the other was kt11 - 1 from 18 to 23 february 2011 . the observation course was planned to cover the waters west of kyushu islands and the area between the kuroshio current and the continental shelf in the eastern east china sea within the exclusive economic zone of japan . visual census of seaweed rafts were conducted from the deck of r / v tansei maru at a height of 11.5 m above the sea surface under good conditions ( i.e. , wave height less than 1 m ) , and from sunrise to sunset during the two cruises . the vessel navigated along designed transects . the following information was recorded at each occurrence of floating seaweeds : time , perpendicular distance from the vessel to a seaweed raft , and its diameter . seaweed rafts were sampled at 12 locations in february and march 2010 and 16 locations in february 2011 ( fig . 1 ) . some seaweed rafts were sampled randomly by using dip net or towing an ori ring net , cone - shaped plankton net , with a diameter of 1.6 m , and mesh size 1 mm . sampled floating seaweeds were divided into individual plants on the deck of the vessel to identify species composition . after species identification the wet weight of each individual plant was measured with the aid of a spring scale on the deck.fig . 1map showing sampling stations in the east china sea and pacific ocean south of shikoku island . triangles and circles indicate stations in 2010 and 2011 , respectively . filled and open marks are stations where floating seaweed rafts consists of only s. horneri and many seaweed species , respectively map showing sampling stations in the east china sea and pacific ocean south of shikoku island . triangles and circles indicate stations in 2010 and 2011 , respectively . filled and open marks are stations where floating seaweed rafts consists of only s. horneri and many seaweed species , respectively floating seaweed abundance was estimated by the line transect method using distance sampling ( buckland et al . this method uses the perpendicular distance from the transect line to the object and diameter of the object to correct for visibility bias , and allows estimating the probability of detecting objects and corrected densities . the probability of floating seaweed detection was estimated with models combining density function ( uniform , half - normal , and hazard - rate ) with adjustments ( cosine , simple , and hermite polynomials ) . ( 2002 ) , we estimated the parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{\mu } $ $ \end{document } called the effective strip half - width ( esw ) , and defined as the maximum limit for distinction.1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{d}=\frac{n}{2\widehat{\mu}l } $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{d } $ $ \end{document } , n , and l represent the abundance of seaweed rafts , the total number of rafts of floating seaweeds along a transect , and the length of the transect , respectively . in order to take into account the lack of homogeneity at sea and meteorological conditions between transects , the model was fitted to data separately for each transect . akaike s information criterion ( aic ) provides an objective , quantitative method for model selection . estimates from the model with the lowest aic [ defined by eq . 2 ] were selected ( buckland et al . 2001).2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mathrm{aic}=-2 { \log}_e\left(\varlambda \right)+2(q ) $ $ \end{document}where is the maximized likelihood and q the number of estimated parameters . this number of estimated parameters was the number of parameters based on combination key - function and adjustment - term provided by the program distance . rafts of floating seaweeds were classified into four classes depending on the perpendicular distances from the vessel to a raft , 010 , 1020 , 2030 , and 3040 m. rafts more than 40 m distant from the vessel were neglected due to the uncertainty of measuring distance . the number of rafts within the effective strip half - width was obtained from the best fitted model . the population mean for the wet weight along each transect in the surveys of 2010 and 2011 was estimated by using the relation between diameter and wet weight of seaweed rafts collected during both surveys of 2010 and 2011 . the diameter of floating seaweeds was converted to wet weight using the equation representing this relation . the mean wet weight ( m ) of all rafts along each transects was calculated . standing crop ( sc ) along a transect was calculated from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{d } $ $ \end{document } , the density of rafts , obtained by the program distance , and m , the mean wet weight of the rafts , estimated from the observation.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mathrm{sc}\left(\mathrm{kg}\;\mathrm{ww}\;{\mathrm{km}}^{-2}\right)=\widehat{d}\left(\mathrm{raft}\ { \mathrm{km}}^{-2}\right)\times m\left(\mathrm{kg}\kern0.3em \mathrm{ww}\;{\mathrm{raft}}^{-1}\right ) $ $ \end{document } the total biomass along a transect ( tb ) was calculated by multiplying the standing crop ( sc ) by the survey area ( a).\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mathrm{tb}\left(\mathrm{kg}\;\mathrm{ww}\right)=\mathrm{sc}\left(\mathrm{kg}\;\mathrm{ww}\;{\mathrm{km}}^{-2}\right)\times a\left({\mathrm{km}}^{-2}\right ) $ $ \end{document}where sc and a are standing crop of floating seaweeds along a transect and survey area obtained from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{\mu } $ $ \end{document } and l , respectively . two research cruises using r / v tansei maru were organized to survey floating seaweeds in the east china sea in late winter to early spring . one was kt10 - 1 research cruise from 22 february to 6 march 2010 and the other was kt11 - 1 from 18 to 23 february 2011 . the observation course was planned to cover the waters west of kyushu islands and the area between the kuroshio current and the continental shelf in the eastern east china sea within the exclusive economic zone of japan . visual census of seaweed rafts were conducted from the deck of r / v tansei maru at a height of 11.5 m above the sea surface under good conditions ( i.e. , wave height less than 1 m ) , and from sunrise to sunset during the two cruises . the vessel navigated along designed transects . the following information was recorded at each occurrence of floating seaweeds : time , perpendicular distance from the vessel to a seaweed raft , and its diameter . seaweed rafts were sampled at 12 locations in february and march 2010 and 16 locations in february 2011 ( fig . 1 ) . some seaweed rafts were sampled randomly by using dip net or towing an ori ring net , cone - shaped plankton net , with a diameter of 1.6 m , and mesh size 1 mm . sampled floating seaweeds were divided into individual plants on the deck of the vessel to identify species composition . after species identification the wet weight of each individual plant was measured with the aid of a spring scale on the deck.fig . 1map showing sampling stations in the east china sea and pacific ocean south of shikoku island . triangles and circles indicate stations in 2010 and 2011 , respectively . filled and open marks are stations where floating seaweed rafts consists of only s. horneri and many seaweed species , respectively map showing sampling stations in the east china sea and pacific ocean south of shikoku island . triangles and circles indicate stations in 2010 and 2011 , respectively . filled and open marks are stations where floating seaweed rafts consists of only s. horneri and many seaweed species , respectively floating seaweed abundance was estimated by the line transect method using distance sampling ( buckland et al . this method uses the perpendicular distance from the transect line to the object and diameter of the object to correct for visibility bias , and allows estimating the probability of detecting objects and corrected densities . the probability of floating seaweed detection was estimated with models combining density function ( uniform , half - normal , and hazard - rate ) with adjustments ( cosine , simple , and hermite polynomials ) . ( 2002 ) , we estimated the parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{\mu } $ $ \end{document } called the effective strip half - width ( esw ) , and defined as the maximum limit for distinction.1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{d}=\frac{n}{2\widehat{\mu}l } $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{d } $ $ \end{document } , n , and l represent the abundance of seaweed rafts , the total number of rafts of floating seaweeds along a transect , and the length of the transect , respectively . in order to take into account the lack of homogeneity at sea and meteorological conditions between transects , the model was fitted to data separately for each transect . akaike s information criterion ( aic ) provides an objective , quantitative method for model selection . estimates from the model with the lowest aic [ defined by eq . 2 ] were selected ( buckland et al . 2001).2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mathrm{aic}=-2 { \log}_e\left(\varlambda \right)+2(q ) $ $ \end{document}where is the maximized likelihood and q the number of estimated parameters . this number of estimated parameters was the number of parameters based on combination key - function and adjustment - term provided by the program distance . rafts of floating seaweeds were classified into four classes depending on the perpendicular distances from the vessel to a raft , 010 , 1020 , 2030 , and 3040 m. rafts more than 40 m distant from the vessel were neglected due to the uncertainty of measuring distance . the number of rafts within the effective strip half - width was obtained from the best fitted model . the population mean for the wet weight along each transect in the surveys of 2010 and 2011 was estimated by using the relation between diameter and wet weight of seaweed rafts collected during both surveys of 2010 and 2011 . the diameter of floating seaweeds was converted to wet weight using the equation representing this relation . the mean wet weight ( m ) of all rafts along each transects was calculated . standing crop ( sc ) along a transect was calculated from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{d } $ $ \end{document } , the density of rafts , obtained by the program distance , and m , the mean wet weight of the rafts , estimated from the observation.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mathrm{sc}\left(\mathrm{kg}\;\mathrm{ww}\;{\mathrm{km}}^{-2}\right)=\widehat{d}\left(\mathrm{raft}\ { \mathrm{km}}^{-2}\right)\times m\left(\mathrm{kg}\kern0.3em \mathrm{ww}\;{\mathrm{raft}}^{-1}\right ) $ $ \end{document } the total biomass along a transect ( tb ) was calculated by multiplying the standing crop ( sc ) by the survey area ( a).\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mathrm{tb}\left(\mathrm{kg}\;\mathrm{ww}\right)=\mathrm{sc}\left(\mathrm{kg}\;\mathrm{ww}\;{\mathrm{km}}^{-2}\right)\times a\left({\mathrm{km}}^{-2}\right ) $ $ \end{document}where sc and a are standing crop of floating seaweeds along a transect and survey area obtained from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{\mu } $ $ \end{document } and l , respectively . distribution of floating seaweeds in the east china sea , floating seaweeds were quite abundant in february and early march . most floating seaweeds observed in both years were distributed in waters on the continental shelf less than 200 m deep , which is the western edge of the kuroshio current in the east china sea except in the waters south of kyushu island ( fig . 2 ) . although floating objects were observed on all transects , no floating seaweeds occurred along the two transects located in waters east of kuroshio current ( fig . 2visual survey transects for floating seaweed rafts ( solid lines ) in the east china sea during r / v tansei maru cruises of kt10 - 1 ( a ) and kt11 - 1 ( b ) , and floating seaweed rafts ( green circle ) in intervals of 20 km distance of transect . black dash line represents 200 m isobaths visual survey transects for floating seaweed rafts ( solid lines ) in the east china sea during r / v tansei maru cruises of kt10 - 1 ( a ) and kt11 - 1 ( b ) , and floating seaweed rafts ( green circle ) in intervals of 20 km distance of transect . black dash line represents 200 m isobaths in 2010 , three species of sargassum , one rhodophyceae species and one ulvales species were observed at stn . . nine sargassum species , one colpomenia species , and one seagrass species were present at stn . more than one algal species was observed in waters close to the japanese coast ( shown as open triangle in fig . 1 ) . f1 , f3 , f4 , f5 , f6 , f8 , f9 , f10 , f11 , and all station on february 2011 ( table 1 and 2 ) . all these stations were included in the east china sea.table 1species of floating seaweed rafts and their number of individuals during kt10 - 1kt10 - 1species namestationf1f2f3f4f5f6f8f9f10f11f12f13 colpomenia sinuosa ( mertens ex roth ) derbes et solier1rhodophyceae sp1 sargassum filicinum harvey6 ( 4 ) s. fulvellum ( turner ) c. agardh s. fusiforme ( harvey ) setchell1 ( 1)1 s. horneri ( turner ) c. agardh2 25 10 13 5 21 60 136 51 111 36 ( 3 ) s. micracanthum endlicher1 s. muticum ( yendo ) fensholt1 s. patens c. agardh710 ( 1 ) s. siliquastrum ( turner ) c. agardh1 ( 1 ) s. fulvellum ( turner ) c. agardh15 ( 1 ) s. thunbergii ( mertens ex roth ) kuntze1 s. yamamotoi yoshida42 ( 34)138 ( 97 ) ulvales sp1 zostera marina l.9number in parenthesis shows number of individuals with holdfast dominant speciestable 2species of floating seaweed rafts and their stipe numbers during kt11 - 1kt11 - 1species namestationst1st2st3st4st5st6st7st8st9st10st11st12st13st14st15st16 s. horneri ( turner ) c. agardh2 4 2 1 5 2 10 10 15 6 9 7 16 2 14 2 no individuals with holdfast dominant species species of floating seaweed rafts and their number of individuals during kt10 - 1 number in parenthesis shows number of individuals with holdfast species of floating seaweed rafts and their stipe numbers during kt11 - 1 no individuals with holdfast some seaweeds at stns . f2 , f12 , and f13 still had their holdfasts ( table 1 ) . distances of these stations to the nearest land are about 48 , 4 , and 12 km , respectively ( fig . 1 ) . on the other hand , s. horneri collected at all stations on kt10 - 1 and kt11 - 1 had no holdfasts , except at stn . f13 near the coast . temperature of sampling stations ranged 16.8 to 22.8 c in 2010 and 15.3 to 18.4 c in 2011 . to obtain the relationship between the diameter of a raft and its weight , we applied various models to fit their distributions ( fig . 3 ) the exponential model was selected as a suitable model to explain the relationship between diameter ( di ) and wet weight ( wt ) of seaweed rafts : wt = 368.64di + 379.25di + 1,755.1di ( r = 0.796 ) . this relationship permits us to estimate the mean wet weight of seaweed rafts based on the estimated diameter.fig . 3regression curve ( r = 0.796 ) showing the relation between diameter ( di ) and wet weight ( wt ) of floating seaweed rafts collected during the r / v tansei maru cruises of kt10 - 1 ( closed squares ) and kt11 - 1 ( open triangles ) regression curve ( r = 0.796 ) showing the relation between diameter ( di ) and wet weight ( wt ) of floating seaweed rafts collected during the r / v tansei maru cruises of kt10 - 1 ( closed squares ) and kt11 - 1 ( open triangles ) in february and march 2010 , esw values for all transects ranged from 11.4 to 25.7 m ( table 3 ) . raft density of floating seaweed along the transect on 2 march was highest among transects in 2010 ( table 3 ) . the mean wet weight per raft was 3.86 kg ( table 3 ) , and the estimated biomass for this transect was 100.36 kg ww km ( fig . 4b).table 3results for models applied to the distribution of floating seaweed rafts along each transect during february and march 2010 by the program distance v6.0 r2datemodel ( key + adjustment)eswlength of transect ( km)number of raftsestimated mean wet weight ( kg ww ind ) of raftsdd lcld ucld cv22 febhalf + hermite11.486.8733.341.50.29.60.4523 febhalf + cos13.8179.74881.5817.714.122.30.1224 febunifo + cos25.7159.33141.861.71.12.70.2225 febhalf + hermite16.3117.64591.9015.212.418.60.102 marhalf + hermite16.0166.731373.8626.022.829.60.073 marhazard + cos15.9190.51951.4415.713.418.40.084 marunifo + simple17.2234.68162.491.91.52.60.13 aic akaike s information criteria , esw effective strip width , n total number of rafts within esw , d density of rafts , d lcl lower confidence limit of d , d ucl upper confidence limit of d , d cv coefficient of variation of d half , unifo , hermite , cos and hazard are half - normal , uniform , hermite , cosine and hazard - rate distributions , respectivelyfig . 4estimated biomass of floating seaweeds ( kg ww km ) along the observation transects ( solid lines ) during r / v tansei maru . cruises of kt10 - 1 in february 2010 ( a ) , march 2010 ( b ) , and kt11 - 1 in february 2011 ( c ) . ( source : hydrographic and oceanographic department of japan coast guard , 2010 and 2011 ) results for models applied to the distribution of floating seaweed rafts along each transect during february and march 2010 by the program distance v6.0 r2 aic akaike s information criteria , esw effective strip width , n total number of rafts within esw , d density of rafts , d lcl lower confidence limit of d , d ucl upper confidence limit of d , d cv coefficient of variation of d half , unifo , hermite , cos and hazard are half - normal , uniform , hermite , cosine and hazard - rate distributions , respectively estimated biomass of floating seaweeds ( kg ww km ) along the observation transects ( solid lines ) during r / v tansei maru . cruises of kt10 - 1 in february 2010 ( a ) , march 2010 ( b ) , and kt11 - 1 in february 2011 ( c ) . ( source : hydrographic and oceanographic department of japan coast guard , 2010 and 2011 ) in february 2011 , esw values for all transects ranged from 11.0 to 17.8 m. density of floating seaweeds along the 21 february located southernmost in survey area of 2011 was extremely high ( table 4 ) . total biomass of this transect was the highest , 504.12 kg ww km ( fig . 4c).table 4results for models applied to the distribution of floating seaweed rafts along each transect during february 2012 by the program distance v6.0 r2datemodel ( key + adjustment)eswlength of transect(km)number of raftsestimated mean wet weight ( kg ww ind ) of raftsdd lcld ucld cv18-febunifo/ cos14.779.9741.071.70.47.10.3419-febhazard / cos11.0134.16471.9816.06.340.60.4920-febhalf / cos17.8162.463512.6360.752.669.90.0721-febhalf / cos14.4138.468782.29220.2204.3237.40.0422-febhazard / cos17.1159.363971.6072.863.783.10.0723-febhalf / hermite17.4237.40201.692.41.73.50.18abbreviations are identical to the ones in table 3 results for models applied to the distribution of floating seaweed rafts along each transect during february 2012 by the program distance v6.0 r2 abbreviations are identical to the ones in table 3 in both surveys of 2010 and 2011 , the maximum biomass of floating seaweeds consisting of only s. horneri was distributed in waters on the continental shelf west of the kuroshio current around 25 n and 127 e. in february and march 2010 , esw values for all transects ranged from 11.4 to 25.7 m ( table 3 ) . raft density of floating seaweed along the transect on 2 march was highest among transects in 2010 ( table 3 ) . the mean wet weight per raft was 3.86 kg ( table 3 ) , and the estimated biomass for this transect was 100.36 kg ww km ( fig . 4b).table 3results for models applied to the distribution of floating seaweed rafts along each transect during february and march 2010 by the program distance v6.0 r2datemodel ( key + adjustment)eswlength of transect ( km)number of raftsestimated mean wet weight ( kg ww ind ) of raftsdd lcld ucld cv22 febhalf + hermite11.486.8733.341.50.29.60.4523 febhalf + cos13.8179.74881.5817.714.122.30.1224 febunifo + cos25.7159.33141.861.71.12.70.2225 febhalf + hermite16.3117.64591.9015.212.418.60.102 marhalf + hermite16.0166.731373.8626.022.829.60.073 marhazard + cos15.9190.51951.4415.713.418.40.084 marunifo + simple17.2234.68162.491.91.52.60.13 aic akaike s information criteria , esw effective strip width , n total number of rafts within esw , d density of rafts , d lcl lower confidence limit of d , d ucl upper confidence limit of d , d cv coefficient of variation of d half , unifo , hermite , cos and hazard are half - normal , uniform , hermite , cosine and hazard - rate distributions , respectivelyfig . 4estimated biomass of floating seaweeds ( kg ww km ) along the observation transects ( solid lines ) during r / v tansei maru . cruises of kt10 - 1 in february 2010 ( a ) , march 2010 ( b ) , and kt11 - 1 in february 2011 ( c ) . light blue stripe represents the path of kuroshio current . ( source : hydrographic and oceanographic department of japan coast guard , 2010 and 2011 ) results for models applied to the distribution of floating seaweed rafts along each transect during february and march 2010 by the program distance v6.0 r2 aic akaike s information criteria , esw effective strip width , n total number of rafts within esw , d density of rafts , d lcl lower confidence limit of d , d ucl upper confidence limit of d , d cv coefficient of variation of d half , unifo , hermite , cos and hazard are half - normal , uniform , hermite , cosine and hazard - rate distributions , respectively estimated biomass of floating seaweeds ( kg ww km ) along the observation transects ( solid lines ) during r / v tansei maru . cruises of kt10 - 1 in february 2010 ( a ) , march 2010 ( b ) , and kt11 - 1 in february 2011 ( c ) . ( source : hydrographic and oceanographic department of japan coast guard , 2010 and 2011 ) in february 2011 , esw values for all transects ranged from 11.0 to 17.8 m. density of floating seaweeds along the 21 february located southernmost in survey area of 2011 was extremely high ( table 4 ) . total biomass of this transect was the highest , 504.12 kg ww km ( fig . 4c).table 4results for models applied to the distribution of floating seaweed rafts along each transect during february 2012 by the program distance v6.0 r2datemodel ( key + adjustment)eswlength of transect(km)number of raftsestimated mean wet weight ( kg ww ind ) of raftsdd lcld ucld cv18-febunifo/ cos14.779.9741.071.70.47.10.3419-febhazard / cos11.0134.16471.9816.06.340.60.4920-febhalf / cos17.8162.463512.6360.752.669.90.0721-febhalf / cos14.4138.468782.29220.2204.3237.40.0422-febhazard / cos17.1159.363971.6072.863.783.10.0723-febhalf / hermite17.4237.40201.692.41.73.50.18abbreviations are identical to the ones in table 3 results for models applied to the distribution of floating seaweed rafts along each transect during february 2012 by the program distance v6.0 r2 abbreviations are identical to the ones in table 3 in both surveys of 2010 and 2011 , the maximum biomass of floating seaweeds consisting of only s. horneri was distributed in waters on the continental shelf west of the kuroshio current around 25 n and 127 e. floating seaweeds consist of several sargassum species in coastal waters of japan ( yoshida 1963 ) . ohno ( 1984 ) observed floating seaweeds south of kyusyu and shikoku island and reported that those in the coastal waters were composed of two to six species . 2001 ) investigated seasonal changes of the species composition of floating seaweeds in the coastal waters of izu peninsular , central japan . they stated that the number of species of floating plants in one patch ranged from one to 11 . hirosaki ( 1963 ) and gamo and matsuura ( 1975 ) also investigated seasonal changes in the species composition of floating seaweeds in coastal waters around japan , and found that floating seaweeds consisted of multiple seaweed species . on the other hand , komatsu et al . ( 2007 ) reported that floating seaweeds in offshore waters of the east china sea in mid march and may were only s. horneri . thus , the floating seaweeds in offshore waters of the east china sea in february and early march are also characterized by s. horneri , same as those in mid march and may . s. horneri growing in benthic habitats along the coasts east of the east china sea are not distributed from south of kyushu island to taiwan through ryukyu archipelago ( yoshida 1998 ) although the species composition of floating seaweeds in coastal waters around japan is affected by the flora of surrounding waters ( hirata et al . 2001 ) . considering the benthic distribution of s. horneri along the coasts around the east china sea ( tseng 1983 ) and the northeastward surface currents , such as the kuroshio current and taiwan warm current in offshore waters of the east china sea in spring ( zhu et al . 2004 ) , the possible origin of floating s. horneri in offshore waters of the east china sea in february and early march is suspected to be the chinese coast , similar as suggested by komatsu et al . it appears that the supply of floating seaweeds distributed in the east china sea depends on the benthic populations of s. horneri along the chinese coast . in previous surveys conducted in nearshore waters around japan , it is reported that floating seaweeds consisted of sargassum individuals with holdfasts ( ohno 1984 ; yatsuya 2005 ) . our study showed that no holdfasts were found in offshore waters in the east china sea . komatsu et al . ( 2007 ) deployed satellite tracking buoys attached to rafts of s. horneri from the chinese coast . it took about 1 to 2 months for the buoys to move from the chinese coast to offshore waters with currrents in the east china sea . this suggests that long - distance dispersal of floating sargassum is accompanied by damage and loss of gas vesicles . since the holdfasts have negative buoyancy , it is possible that floating seaweeds of s. horneri with a holdfast have sunk before reaching offshore waters because of their negative densities caused by the loss of gas vesicles . yatusya ( 2008 ) reported s. horneri had lower density and longer floating periods than other sargassum species . consequently , the floating individuals of s. horneri without holdfast can be transported far from their original habitats on rocky shores . studies on other floating macroalgae also show that at larger distances from potential source regions , these are more overgrown by epibionts and floating individuals are more disintegrated ( rothusler et al . no floating seaweeds were observed along the two transects located in the kuroshio current and east of the kuroshio current in february to march of 2010 . this is also true in surveys of floating seaweeds in mid march of 2004 and may of 2002 ( komatsu et al . 2008 ) . floating seaweeds apparently do not cross the kuroshio current from the continental shelf to outer waters east of the kuroshio current . thus , they are generally retained in the convergence region between warmer water of the kuroshio current and colder waters of the continental shelf . the highest biomass of floating seaweeds was located at the southernmost transect on the continental shelf in surveys of 2010 and 2011 . komatsu et al . ( 2008 ) reported that the biomass of floating seaweeds in the southernmost transect ( 20.35 kg ww km , around 30 n , 127 e ) was greater than in the transect located west of kyushu ( 2.18 kg ww km ) in mid march . in all of the surveyed month , there were floating seaweeds in offshore water in the east china sea much more than coastal water around kyusyu island in february and early march . in the east china sea , yellowtail starts to spawn in waters southeast of continental shelf edge in late january . yellowtail juveniles have been collected in surface water temperatures between 14 and 26 c ( yamamoto et al . optimal growth rates of yellowtail juveniles are at a temperature of around 22 c ( fujimoto et al . since yellowtail juveniles were also collected with floating seaweeds in this study , it is suggested that the juveniles retained on the spawning grounds by eddies start to accompany floating seaweeds in waters southeast of the continental shelf where sea surface temperatures are optimal for their growth . thus , yellowtail juveniles spawned earliest in the east china sea utilize floating seaweeds distributing on the continental shelf in the east china sea as their nursery in february and early march . floating s. horneri in the east china sea plays an indispensable role as nursery habitat for commercially important species in february and early march . it is important to identify their source populations along the chinese coast and to conserve s. horneri beds that support the unique ecosystem of floating seaweeds in the east china sea .
floating seaweeds play an important role as a habitat for many animals accompanying or attaching to them in offshore waters . it was in 2000 that the first report described abundant distributions of floating seaweeds in offshore waters in the east china sea in spring . young individuals of the yellowtail seriola quinqueradiata are captured for aquaculture purposes from floating seaweeds in the east china sea . therefore , a sound understanding of the distributions of floating seaweeds in the east china sea is needed . detailed information is especially important during the late winter to early spring , which corresponds to the juvenile period of the yellowtail . thus , field surveys using r / v tansei - maru were conducted in the japanese exclusive economic zone in the east china sea from late winter to early spring in 2010 and 2011 . we obtained positions of the vessel by gps and transversal distances from the vessel to a raft by visual observation . distance sampling method ( thomas et al . 2010 ) was applied to estimation of floating seaweed densities ( rafts km2 ) . seaweed rafts were also randomly sampled using nets during the research cruises . in the east china sea , seaweed rafts were distributed mainly on the continental shelf west of the kuroshio , especially in waters between 26 n and 30 n. collected rafts consisted of only one species , sargassum horneri ( turner ) c. agardh . taking into account surface currents and geographical distribution of s. horneri , it is estimated that these floating seaweeds originated from natural beds along the coast between mid and south china . considering the approximate travel times , it is suggested that floating patches are colonized by yellowtails early on during their trips , i.e. , close to the chinese coast .
Introduction Materials and methods Research cruises Results Abundance of floating seaweeds Discussion
( 2007 ) surveyed seaweed rafts in the eastern east china sea in march and may and reported that they were distributed in waters on the continental shelf west of the kuroshio front ( komatsu et al . two research cruises using r / v tansei maru were organized to survey floating seaweeds in the east china sea in late winter to early spring . two research cruises using r / v tansei maru were organized to survey floating seaweeds in the east china sea in late winter to early spring . 4c).table 4results for models applied to the distribution of floating seaweed rafts along each transect during february 2012 by the program distance v6.0 r2datemodel ( key + adjustment)eswlength of transect(km)number of raftsestimated mean wet weight ( kg ww ind ) of raftsdd lcld ucld cv18-febunifo/ cos14.779.9741.071.70.47.10.3419-febhazard / cos11.0134.16471.9816.06.340.60.4920-febhalf / cos17.8162.463512.6360.752.669.90.0721-febhalf / cos14.4138.468782.29220.2204.3237.40.0422-febhazard / cos17.1159.363971.6072.863.783.10.0723-febhalf / hermite17.4237.40201.692.41.73.50.18abbreviations are identical to the ones in table 3 results for models applied to the distribution of floating seaweed rafts along each transect during february 2012 by the program distance v6.0 r2 abbreviations are identical to the ones in table 3 in both surveys of 2010 and 2011 , the maximum biomass of floating seaweeds consisting of only s. horneri was distributed in waters on the continental shelf west of the kuroshio current around 25 n and 127 e. in february and march 2010 , esw values for all transects ranged from 11.4 to 25.7 m ( table 3 ) . ( source : hydrographic and oceanographic department of japan coast guard , 2010 and 2011 ) results for models applied to the distribution of floating seaweed rafts along each transect during february and march 2010 by the program distance v6.0 r2 aic akaike s information criteria , esw effective strip width , n total number of rafts within esw , d density of rafts , d lcl lower confidence limit of d , d ucl upper confidence limit of d , d cv coefficient of variation of d half , unifo , hermite , cos and hazard are half - normal , uniform , hermite , cosine and hazard - rate distributions , respectively estimated biomass of floating seaweeds ( kg ww km ) along the observation transects ( solid lines ) during r / v tansei maru . 4c).table 4results for models applied to the distribution of floating seaweed rafts along each transect during february 2012 by the program distance v6.0 r2datemodel ( key + adjustment)eswlength of transect(km)number of raftsestimated mean wet weight ( kg ww ind ) of raftsdd lcld ucld cv18-febunifo/ cos14.779.9741.071.70.47.10.3419-febhazard / cos11.0134.16471.9816.06.340.60.4920-febhalf / cos17.8162.463512.6360.752.669.90.0721-febhalf / cos14.4138.468782.29220.2204.3237.40.0422-febhazard / cos17.1159.363971.6072.863.783.10.0723-febhalf / hermite17.4237.40201.692.41.73.50.18abbreviations are identical to the ones in table 3 results for models applied to the distribution of floating seaweed rafts along each transect during february 2012 by the program distance v6.0 r2 abbreviations are identical to the ones in table 3 in both surveys of 2010 and 2011 , the maximum biomass of floating seaweeds consisting of only s. horneri was distributed in waters on the continental shelf west of the kuroshio current around 25 n and 127 e. floating seaweeds consist of several sargassum species in coastal waters of japan ( yoshida 1963 ) . considering the benthic distribution of s. horneri along the coasts around the east china sea ( tseng 1983 ) and the northeastward surface currents , such as the kuroshio current and taiwan warm current in offshore waters of the east china sea in spring ( zhu et al . since yellowtail juveniles were also collected with floating seaweeds in this study , it is suggested that the juveniles retained on the spawning grounds by eddies start to accompany floating seaweeds in waters southeast of the continental shelf where sea surface temperatures are optimal for their growth .
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attention - deficit / hyperactivity disorder ( adhd ) is a widespread neurodevelopmental disorder , the prevalence and significance of which is rapidly growing . increasing numbers of people the ever - increasing life s tempo , ever - growing stress , and last but not least , the onset of the ubiquitous electronic technology environment also tend to exacerbate adhd . the connection between adhd and time perception specifically using the methods laid out by zimbardo and sword and the zimbardo time perception inventory can bring a new and helpful tool in diagnosis and therapy of adhd.1,2 adhd is a disorder which is widely known and classically recognized in children but that is only recently getting studied in the adult population.3 simon et al report that prevalence of adult adhd is 2%3%.3 other sources state that adhd affects between 5%4 and 11%5 of the population aged 417 years in the us . fayyad et al report that the prevalence of adhd is around 5.3% in the pediatric population and 3.4% in the adult population.6 however , recent data suggest even more dramatic occurrence of adhd : almost 1 in 5 teenage boys and 11% of all school - aged children have been diagnosed with adhd.7 adhd is a neurodevelopmental disorder . however , rather than by its neurological substance and causes , it tends to be described by observable behavioral manifestations . the marked symptoms of adhd include general inattention , hyperactivity , impulsivity , and difficulty with self - control . adhd used to be classified as a childhood syndrome , but it has recently become a chronic , lifelong disorder with adhd , childhood adhd , and adult adhd defined as three different categories under diagnostic and statistical manual of mental disorders , fifth edition ( dsm - v).4 in the dsm system , the child or teenager can be labeled with the diagnosis of predominantly inattentive , predominantly hyperactive impulsive , or a mix of the two categories ( dsm ) ; the symptoms can later be carried on to adulthood . failure to observe a linear progression from childhood to adult adhd leads many to believe that with time , there is a catch up in development and the symptoms may remit into adulthood.8 the variety of causes and triggers is emphasized by the fact that the disorder can be managed to some extent by drug therapy and/or psychotherapy.9 the literature is abound with reports on the neurological and hormonal peculiarities of children with adhd.1012 furthermore , neuroimaging studies have shown developmental differences in children with adhd , especially disparity in the maturation of the cortex and the cerebellum.13,14 children and adults with adhd also seem to differ from non - adhd individuals in that various deficiencies in the striatum have been observed . this is specifically related to the dopaminergic system , the reward system and dopamine.15 dopamine is directly related to reward - seeking behavior , impulsivity , and addictions . however , to what degree dopamine serotonin interactions are at the source of adhd still remains a continuing debate.16,17 adhd was also found to have a genetic component.18,19 specific genetic differences have been reported which link adhd , impulsive behavior , delinquency , and also hedonism.20 it is also hypothesized that adhd may be connected with specific changes in physical growth.21,22 there is no single clinical picture of adhd . there are marked differences between the impulsive and inattentive types that have been isolated and described.23 although both the inattentive and hyperactive subtypes share the same diagnostic definition , they often manifest very different symptoms and comorbidities . according to grizenko et al , the two subtypes appear as almost separate diagnoses.24 while looking at comorbidities , these authors found that the hyperactive group displayed symptoms such as conduct disorder , while the inattentive group manifested different ones ( mainly depression ) . another example of a difference in comorbidities is that the inattentive group often shows comorbid obesity , whereas the hyperactive group does not.25 other comorbidities associated with the hyperactive / impulsive subtypes are drug abuse and addictive behavior.26 the literature from various sources ( neurological , genetic , and behavioral disciplines ) seems to concur that there is a close link between adhd and present - oriented behavioral patterns both in children and adults . this present - oriented behavior includes a wide array of behaviors which are hedonistically oriented : alcohol and drug abuse , compulsive gambling , dependence on electronic media , poor eating habits , higher rates of obesity and overall body weight in people , nicotinism , and others.27,28 analyses of populations of alcohol abusers and alcoholics indicate that up to 71% of the population have adhd and in the case of other drug abusers , the number is up to 25%.29,30 similarly , predominantly present hedonistically minded individuals were found to suffer from higher rates of drug and alcohol abuse.31 interestingly , one genetic expression the dopamine receptor genes ( drd2 , drd4 ) associated with adhd and impulsivity32 was also associated with alcoholism.33 in a prospective study , higher rates of alcohol consumption were found among present hedonistic and past negative individuals.34 impulsive drinking and drug abuse were directly correlated with adhd diagnosis in various studies.35,36 individuals with a diagnosis of childhood adhd and conduct disorder were found to be at much higher risk than controls for becoming drug abusers in adolescence.37 patients suffering from adhd as well as present hedonists often manifest very poor dietary choices . these include high consumption and often dependence on fatty foods , sweets , and fast food.38 a prospective study carried out in australia on 2868 children found a correlation between adhd and a western type diet that included fast food ; this was compared with a healthy diet that included adequate amounts of fruits and vegetables . howard et al noted that impulsive eating can also become an addictive behavioral pattern39 often seen in adhd.22 one major predictor of binge eating in adolescence was found to be childhood adhd.40 furthermore , a study by wilhelm et al analyzed the difference between overweight and adhd individuals and found more impulsiveness within the adhd group , especially at the beginning of the meal.41 binge eating itself may be addictive or it is conjointly linked with other addictions , nutritional or behavioral.24 a high correlation between binge eating / food addiction and cocaine abuse has been found , specifically for high fatty food intake.42 swanson et al43 and nigg26 attest that both binge eating and bulimia nervosa are connected with adhd , and the common comorbidity of eating disorders in adolescence is substance abuse . a great amount of research has been done on adhd and dietary habits , whereas the literature on present hedonism and lifestyle is just starting to emerge . piu has been a topic of interest in psychiatric and clinical settings since the late 90s ; this diagnosis had been proposed , but was later rejected by the dsm . the impulsive and addictive use of the internet has been deemed problematic for certain individuals and indeed clinically significant . the disorder met all the criteria for being identified as an impulse control disorder , which is marked by impulsive behavior.44 adhd is often associated with piu.45 a correlation was found between piu and alcohol abuse / dependence among adolescent populations in germany and korea.46 although internet use can well serve educational and recreational purposes , increasing evidence shows its excessive and addictive abuse . the incidence of piu has been estimated at 2%20% among young people and has been increasing.47 zimbardo and coloumbe highlight a wide range of negative consequences among boys and young men from excessive , socially isolated video gaming coupled with internet pornography viewing.48 in a korean study , adhd and depression turned out to be the biggest risk factors for developing online gaming addictions.49 in a turkish study , individuals with adhd were also more likely to use the internet impulsively.50 both chinese and korean research on internet addiction among adolescents found boys to be more likely to suffer from the condition than girls ; risk factors for developing such dependence were adhd and impulsivity.51,52 this is congruent with the previous studies on the classical forms of addictions ( ie , alcohol and other drugs ) . one possible shortcoming of the study was that there was no distinction between the subtypes of adhd . the authors of the study did not attempt to discover the differences between the adhd and depressed groups to see what the reasons for impulsive gaming were ( such as for lowering depression or quenching the impulsivity ) . an american cross - sectional study on nearly 70,000 children has indicated the protective factors against adhd , which are : watching less than 1 hour of television / gaming a day , being active in sports teams , and a solid family structure.53 the unhealthy lifestyles learned from parents , especially from lower socioeconomic status households , should not be underestimated when looking at adhd and impulsiveness . it is very likely that the impulsive behaviors such as piu and excessive gaming are directly related to parenting styles and also inadequate care by school teachers . deficits in social learning as a direct effect of parenting styles seem to play a very significant role in development of late - onset adhd . high emotional expression in parenting ( particularly negative - reactive instead of positive parenting strategies ) was associated with adhd54 as well as higher rates of aggressive behavior , fighting , and later impulsivity.55 similarly , research has found a direct correlation with the behaviors and parenting roles in time perspective studies . specifically , when teenagers perceived their parents as psychologically controlling , the result was that they displayed predominantly present hedonistic behaviors . on the other hand , future - oriented teenagers perceived their parents as adequately responsive and giving ample autonomy.56 worrel et al reported the present hedonistic mindset to be closely associated with authoritarian and permissive parenting.57 furthermore , authoritarian parenting styles were correlated with disorders such as anxiety58 and drug abuse / addiction.59 incidentally , both are major , well - documented comorbidities associated with adhd.60,61 however , deeper analysis of these phenomena is methodologically challenging . correlational studies tend to describe the vicious circle within the family instead of providing a deeper insight into the causality . in contrast to authoritarian and permissive parenting styles , authoritarian parenting , which is associated with clear roles and boundaries as well as healthy levels of communication , was found to have positive effects . authoritative parenting styles were found to be the most advisable in managing adhd in family setting . maintaining the communication and clear borders were also found to be essential along with medication in managing the symptoms and having healthy family relationships among those suffering from adhd.62 parenting styles , besides neurological and genetic disorders , are often related to aggressiveness among adhd patients as well as among present - oriented hedonists . in a 2016 study , stolarski et al administered the zimbardo time perspective inventory ( ztpi ) as well as the aggression questionnaire to 300 individuals and found that individuals with present hedonistic perspective were more likely to show aggressive and impulsive tendencies.63 the study did not assess for adhd symptoms , but it would most likely yield similar results with adhd being linked to both present hedonism and aggression . likewise , during a neurological mapping study , adolescents with adhd have been observed to show low inhibition along with high levels of aggression and impulsivity . the study included 18 adhd diagnosed children and 18 controls , individuals from both groups had their brains scanned by fmri machines while playing a game specifically designed to elicit aggressive responses to fictitious opponents . the comorbidities in the adhd group included disruptive behavior disorder and conduct disorder.64 the study did not inquire about drug usage or any other lifestyle habits of the participants . again , there was no differentiation between hyperactive impulsive individuals and generally inattentive subjects in this study . we can assume from the comorbidities and aggressiveness that this was most likely the hyperactive / impulsive group that we are associating with present hedonism . generally , the respective behaviors tend to be related . both in adhd and in present hedonists , overconsumption of fast food , alcohol as well as unhealthy media habits and internet use are not only signs of possible disorders but of poor discipline and inadequate parenting . impulsivity and hedonism are often tied together by various behavioral addictions ( ie , gambling , video games , nicotine ) . presently , we see new forms of addictions emerge , which both the adhd group as well as present hedonists are likely to suffer from . zimbardo developed a time perspective theory with a corresponding standardized questionnaire known as the ztpi . in this , subjects answer how much of their thoughts are spent in the past , present , and future and whether their time perspective tends to have a positive or negative accent . past positive dimension relates to positive reminiscence , for example , certain stimuli bring forward pleasant memories of the past . past negative dimension assesses the degree to which unpleasant and possibly traumatic past experiences are influencing the current life of the individual . present fatalism expresses conviction that rather than by free will , individual lives are influenced by unexpected and uncontrollable forces , fate , luck , and so on . future dimension assesses to what degree individuals are goal oriented , focusing on accomplishments and responsibilities to other people , and display what is known as a traditional protestant work ethic.30 these dimensions can be assessed individually or in their mutual relationship and balance . examples include anxiety or posttraumatic stress when past negative orientation takes over , or risky behaviors and risk - taking in present hedonistic orientation.31 from the above - mentioned literature review of risky lifestyle habits and adhd comorbidities , we hypothesize a clear and definite correlation between adhd and present hedonistic time orientation . we hypothesize that adhd sufferers , especially from the hyperactive impulsive subtype , are most likely to be overwhelmingly anchored in the present hedonistic mindset . they will more likely engage in behaviors representative of present hedonistic time perspective which often leads to substance abuse , poor eating habits , and currently most popular modern addictions , especially piu , and overall dependence on the electronic media . as follows from the zimbardo theory , people with a prevalence of present hedonistic orientation are generally very focused on enjoying the present , feeling excited in their lives , and are therefore driven by what freud called the pleasure principle . although healthy and well - adapted individuals may enjoy the present moments as well , the present hedonism in their case tends to be balanced with a realistic level of future orientation and reminiscence of the past . if present hedonism becomes the guiding principle , individuals may be more likely to engage in risky behaviors in order to seek strong sensations ; this can possibly put the person at risk of developing addictions , whether classical ( ie , alcoholism , overeating , nicotinism ) or the newer , electronic versions , such as gaming or internet addiction.65,48 high levels of present hedonistic orientation are usual , especially among young males . empirical data show that present hedonism sharply declines with age and it is lower in women . these phenomena are illustrated by the present hedonism ztpi scores of a national survey which we conducted in the czech republic . the target population included czech - speaking residents , almost exclusively czechs , seldom slovaks , and rarely other nationalities . the fieldwork was executed by the sociological institute of the czech academy of sciences ( center for public opinion research ) . the sample involved n=2,201 respondents , consisting of 48.8% males and 51.2% females , aged 1589 years ( 44 years on average ) . the sample was proportional to the population regions and urban rural areas of the country . the ztpi data were collected by face - to - face ( pen and paper ) interview using quota - sampling methodology in two subsequent waves in 2003 and 2008 . the relationship between age and the hedonism scale was calculated from n=2,118 valid responses to the hedonism scale ( ie , 96.2% of the total sample ) ; almost all ( n=2,199 ) respondents were included in the latent class analysis ( lca ) . this study focused on ztpi and its 56 items and did not include any adhd indices at that point . the overall decline with age was statistically significant ( high eta 0.367 for interval and cramer s v=0.248 for categorized age : beta=208 in multivariate regression ) . there were significant differences among all age groups , except the middle - age category of 3544 years ; in the age group of 3544 years , the decline in present hedonism was not apparent . higher present hedonism in men was confirmed by two - tailed non parametric statistics : both median and kolmogorov smirnov test of independent samples confirmed the difference on asymptotic significance level of 0.023 and 0.008 , respectively . lca used on the same czech sample yielded an even more dramatic depiction of the decline of present hedonism with age . lca is able to identify subgroups of really existing respondents of ztpi who share similar patterns of time perspective . the age composition of this pattern / class is interesting : half of this group was represented by the youngest respondents aged 1524 years ; older ages contributed to this group with radically declining frequency . figure 2 shows the percentage of respondents from various age categories who constituted a latent class of present hedonists . men predominated in the present hedonists lca class , as it consisted of 60% men and only 40% women . this corresponds to the gender difference observed in present hedonism illustrated in figure 1 and in other studies that find men more present hedonistic than women . as mentioned earlier , adhd symptoms , as well as present hedonism , are also most pronounced in the young age group.66 likewise , gender is a major variable to take into account for addictions , as males are more likely to show addictive behavior such as alcoholism . for example , 58% of men reported binge drinking on a monthly basis.5 binge drinking is an example of a present hedonistic behavior . our article introduces both indicators and pieces of evidence showing that there is a substantial overlap between adhd and present hedonistic orientation . this leads us to hypothesize that present hedonism itself in all its various forms may well serve as a telling indicator of adhd or proneness to adhd . furthermore , psychometric methods devised for assessment of present hedonism , such as ztpi by zimbardo , may prove particularly useful for this purpose . even more importantly , zimbardo s temporal theory was elaborated into a systematic therapeutic approach time perspective therapy ( tpt ) . so far , this approach has been used mainly to treat clients who are pathologically focused on their past negative experiences due to their traumatic past . these include especially war veterans who suffer from posttraumatic stress disorder , but also include survivors of abuse , accidents , assault , and neglect . in their case , tpt is being used to restore the temporal balance , to switch negativity to positivity , and to shift from the past to present and to future . zimbardo et al2 provide a detailed description of the procedure in the time cure : overcoming ptsd with the new psychology of time perspective therapy , which is praised as a landmark book . similar time perspective principles that take into account the ratio of the past , present , and future have been traditionally used in gerontology and geriatrics.67,68 similarly , patients suffering from depression and dwelling in negative past may profit from reframing their time perspective , which is an observation by bitsko et al who worked with depressive adolescent cancer patients.69 in contrast to the above - mentioned groups , most of which consist of patients stuck to the past , the adhd patients seem to suffer from being stuck in the present . coincidentally , this is the core of the title that gruber et al used for their article about mania and its association with the present oriented time perspective.70 refugees are another group of clients who have been known to focus on the present to the relative exclusion of past and future71 or to suffer from atomism of a single time orientation.72 also , prisoners may suffer from a similar exclusive focus.73 and then , of course , intense orientation in the present time has been observed in a wide array of people who suffer from dependencies that are very often linked to adhd and that have been described above . only sporadic reports mention the usefulness of time perspective as a useful framework or even as a possible direction of treatment . for example , henik and domino published an article entitled , alterations in future time perspective in heroin addicts.74 excessive players and pathological gamblers time perspective and the possible therapeutic uses thereof are mentioned in the reports of hodgins and engel75 and lukavska.76 finally , irrespective of diagnoses , stolarski et al63 point out the significance of balanced time perspective for a general satisfaction with life . overall , it is surprising that although adhd tends to be viewed as a disorder which generally responds well to behavioral therapy,77 tpt in general or , specifically , the tpt approach by zimbardo and sword2 is not discussed and utilized more . this article reviews significant relationships between adhd and various factors from genetic and neurological to developmental and social . attention is also paid to a range of manifestations of typical adhd lifestyle and comorbidities . it is concluded that zimbardo s time perspective approach suggests an important relationship between adhd and present hedonism . although literature is available both on adhd and , to a lesser degree , on time perspective , many of the studies are methodologically limited . many are based mostly on correlations , without a deeper concern for the direction of causality . furthermore , the adhd studies typically do not differentiate between the two main types of adhd disorder . although our focus is mostly on the hyperactive subtype of adhd , the differentiation between inattentive and hyperactive is not always properly recorded by clinicians and , as such , can create some confusion in the findings . carelli and wiberg compared 30 adhd participants and 30 controls in a swedish study and found that adhd was associated with predominantly future positive time perspective.78 however , a major confounding factor was that the patients were already being pharmacologically treated for adhd . perhaps , they had measured the effects of treatment rather than adhd itself . in any case , our hypothesis is that adhd individuals seem to live in a different time perspective than the general population . in principles of psychology , william james argued that time is a sensation.79 it appears that adhd individuals spend their waking state ( possibly dream states as well ) more often in present hedonism than other individuals to the detriment of their future . the modern era poses a particular challenge for adhd - prone individuals by the new electronic environment that is both widespread and intensive . at the same time , it is pointed out that modern psychology , namely , zimbardo s time perspective theory , provides a new paradigm which is useful as a new insight into the substance of adhd , the methodology of assessment of proneness to adhd , and finally , the possible therapeutic interventions based on the concept of balanced time perspective . it is surprising that all of the relevant literature only seldom mentions about the therapeutic use of time perspective . we hope this article will contribute to the eventual implementation of temporal interventions in therapy . still , we realize that although the relationship between adhd and excessive focus on present hedonism seems to be straightforward , the relationship itself and the therapeutic interventions may not be that simple . let us take an example from lukavska.76 she found in her study of online game players among others that present fatalistic tp [ time perspective ] was demonstrated to be a stronger predictor of extensive playing than present hedonistic tp . such finding calls for replacing of simple correlational studies by more complex models which take into account mediating roles of multiple variables . the stressors leading to adhd are increasing , along with the enticing incentives of the brave new electronic world ( even electronic schools ) and with fast food , cigarette , pornography , and other industries . these industries are particularly focused on targeting ever - younger consumers.80 the instant gratification principle is facilitated by advertising to young people , appealing to adults not to deprive their loved ones , seducing young consumers by credit cards , and so on . the peer pressure is increased by use of electronic social media such as facebook and others . all this poses a grave threat to the public health not just in the developed countries but also in the developing countries . thus , while adhd is one of the most diagnosed disorders , it is also one of the most misdiagnosed disorders . after all , the adhd lifestyle may often be just a symptom of novelty seeking , an expression of conforming social mimicry , or a defense mechanism to the overstimulated electronic state of consciousness in children and young adults . the search for new effective approaches to assessment and treatment of adhd in the current era is particularly topical . zimbardo s time perspective perception approach seems to provide both a welcome theoretical basis as well as a practical tool . it becomes not only a medical and educational issue , but also a philosophical question as to how to utilize the current technology and at the same time promote sanity , harmony , and freedom , both individual and collective .
the article draws primarily from the behavioral findings ( mainly psychiatric and psychological observations ) and points out the important relationships between attention - deficit / hyperactivity disorder ( adhd ) symptoms and time orientation . specifically , the authors argue that there is a significant overlap between the symptoms of adhd and present hedonism . present hedonism is defined by zimbardo s time perspective theory and assessed by zimbardo time perspective inventory . developmental data on present hedonism of males and females in the czech population sample ( n=2201 ) are also presented . the hypothesis of relationship between adhd and present hedonism is mainly derived from the prevalence of addictive behavior ( mainly excessive internet use , alcohol abuse , craving for sweets , fatty foods , and fast foods ) , deficits in social learning , and increased aggressiveness both in adhd and in the population scoring high on present hedonism in the zimbardo time perspective inventory . we conclude that zimbardo s time perspective offers both : 1 ) a potential diagnostic tool the zimbardo time perspective inventory , particularly its present hedonism scale , and 2 ) a promising preventive and/or therapeutic approach by the time perspective therapy . time perspective therapy has so far been used mainly to treat past negative trauma ( most notably , posttraumatic stress disorder ) ; however , it also has value as a potential therapeutic tool for possible behavioral compensation of adhd .
Introduction The incidence, characteristics, comorbidities, and categories of ADHD Correspondence in lifestyles of ADHD and Present Hedonistic individuals Zimbardo time perspective ADHD and Present Hedonism Time perspective paradigm as a potential diagnostic and therapeutic tool Discussion
attention - deficit / hyperactivity disorder ( adhd ) is a widespread neurodevelopmental disorder , the prevalence and significance of which is rapidly growing . the connection between adhd and time perception specifically using the methods laid out by zimbardo and sword and the zimbardo time perception inventory can bring a new and helpful tool in diagnosis and therapy of adhd.1,2 adhd is a disorder which is widely known and classically recognized in children but that is only recently getting studied in the adult population.3 simon et al report that prevalence of adult adhd is 2%3%.3 other sources state that adhd affects between 5%4 and 11%5 of the population aged 417 years in the us . adhd used to be classified as a childhood syndrome , but it has recently become a chronic , lifelong disorder with adhd , childhood adhd , and adult adhd defined as three different categories under diagnostic and statistical manual of mental disorders , fifth edition ( dsm - v).4 in the dsm system , the child or teenager can be labeled with the diagnosis of predominantly inattentive , predominantly hyperactive impulsive , or a mix of the two categories ( dsm ) ; the symptoms can later be carried on to adulthood . another example of a difference in comorbidities is that the inattentive group often shows comorbid obesity , whereas the hyperactive group does not.25 other comorbidities associated with the hyperactive / impulsive subtypes are drug abuse and addictive behavior.26 the literature from various sources ( neurological , genetic , and behavioral disciplines ) seems to concur that there is a close link between adhd and present - oriented behavioral patterns both in children and adults . this present - oriented behavior includes a wide array of behaviors which are hedonistically oriented : alcohol and drug abuse , compulsive gambling , dependence on electronic media , poor eating habits , higher rates of obesity and overall body weight in people , nicotinism , and others.27,28 analyses of populations of alcohol abusers and alcoholics indicate that up to 71% of the population have adhd and in the case of other drug abusers , the number is up to 25%.29,30 similarly , predominantly present hedonistically minded individuals were found to suffer from higher rates of drug and alcohol abuse.31 interestingly , one genetic expression the dopamine receptor genes ( drd2 , drd4 ) associated with adhd and impulsivity32 was also associated with alcoholism.33 in a prospective study , higher rates of alcohol consumption were found among present hedonistic and past negative individuals.34 impulsive drinking and drug abuse were directly correlated with adhd diagnosis in various studies.35,36 individuals with a diagnosis of childhood adhd and conduct disorder were found to be at much higher risk than controls for becoming drug abusers in adolescence.37 patients suffering from adhd as well as present hedonists often manifest very poor dietary choices . these include high consumption and often dependence on fatty foods , sweets , and fast food.38 a prospective study carried out in australia on 2868 children found a correlation between adhd and a western type diet that included fast food ; this was compared with a healthy diet that included adequate amounts of fruits and vegetables . both in adhd and in present hedonists , overconsumption of fast food , alcohol as well as unhealthy media habits and internet use are not only signs of possible disorders but of poor discipline and inadequate parenting . examples include anxiety or posttraumatic stress when past negative orientation takes over , or risky behaviors and risk - taking in present hedonistic orientation.31 from the above - mentioned literature review of risky lifestyle habits and adhd comorbidities , we hypothesize a clear and definite correlation between adhd and present hedonistic time orientation . our article introduces both indicators and pieces of evidence showing that there is a substantial overlap between adhd and present hedonistic orientation . so far , this approach has been used mainly to treat clients who are pathologically focused on their past negative experiences due to their traumatic past . overall , it is surprising that although adhd tends to be viewed as a disorder which generally responds well to behavioral therapy,77 tpt in general or , specifically , the tpt approach by zimbardo and sword2 is not discussed and utilized more . it is concluded that zimbardo s time perspective approach suggests an important relationship between adhd and present hedonism . at the same time , it is pointed out that modern psychology , namely , zimbardo s time perspective theory , provides a new paradigm which is useful as a new insight into the substance of adhd , the methodology of assessment of proneness to adhd , and finally , the possible therapeutic interventions based on the concept of balanced time perspective . still , we realize that although the relationship between adhd and excessive focus on present hedonism seems to be straightforward , the relationship itself and the therapeutic interventions may not be that simple .
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motor dysfunction is the major neurological deficits after stroke , with 50% of patients suffering from hemiparesis and 30% remaining unable to walk independently . transsynaptic degeneration of lower motor neurons secondary to upper motor neurons injury plays a role in muscle mass loss . it has been demonstrated that transsynaptic degeneration occurred wildly in nervous system after cerebral cortex injury , such as cerebellum , thalamus , and substantial nigra , whereas transsynaptic degeneration in motor system remains controversial . clinical electrophysiological studies have shown that neurogenic injury could be detected in paretic limbs of patients with stroke , suggesting that there is lower motor neuron injury . abnormal spontaneous activity ( sa , including fibrillation potentials , and positive sharp waves ) could be observed in hemiplegic muscles 23 weeks after stroke , lasting up to 1 year . hara and associates reported that motor unit number estimation ( mune ) and the mean compound muscle action potential ( cmap ) amplitude on the paretic side was significantly lower in a period of 9 days to several months , after a unilateral cerebrovascular accident . observed that mune and the maximal cmap amplitude were decreased as early as 4 hours after cerebral infarction . these studies indicate that upper motor neuron lesions may lead to lower motor neuron abnormality . pathological evidence of anterior motoneurons degeneration in patients or rats after stroke is insufficient , and few studies had directly proved the degeneration of lower motor neurons . they observed inflammation and degeneration of myelinated corticospinal axons in the spinal cord . but neither study showed the number of anterior horn cells decreased . experiments of wu and ling reported that in rats undergoing middle cerebral artery occlusion ( mcao ) , all ventral horn motoneurons remained structurally intact in 3 and 5 days after mcao . our work combined the electrophysiological and pathological assessments to investigate the existence transsynaptic degeneration in the motor system , and the relationship between electrophysiology and pathology . this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the peking union medical college hospital ( pumch ) . sprague - dawley male rats were obtained from the animal center of pumch , weighing between 250 g and 380 g. animals were specific pathogen free , and raised in 2025c room temperature . seventeen subjects were died of anesthesia accidents and cerebral hernia after mcao , and another nine subjects were found had subarachnoid hemorrhage and intracranial hemorrhage after sacrifice . every 10 mcao rats were sacrificed for pathology tests , at 24 hours , 7 days , and 14 days after operation separately . all surviving subjects were evaluated by electrophysiology assessments and modified neurological severity score ( mnss ) at 0 day , 24 hours , 7 days , and 14 days after surgery . the permanent mcao procedure was based on the longa mcao logi after anesthesia with 34 ml / kg i.p . the internal carotid artery was also isolated and separated carefully from the adjacent vagus nerve , and the external carotid artery was ligated . monofilament nylon suture ( diameter : 2.63.0 mm ) was introduced into the common carotid artery lumen through a puncture and advanced gently to the internal carotid artery lumen . after about 18 mm length of nylon suture had been inserted into artery lumen , resistance was felt , indicating that the suture had passed the middle cerebral artery origin and occluded the artery . all electrophysiology tests were performed using a portable electromyography ( emg ) machine ( 10ch medelec synergy , natus europe gmbh , germany ) . rats were placed on the operation table after anesthesia , with bilateral lower limbs spread at an approximately 45 angle to the spine [ figure 1a ] , under lamplight to stay warm . we chose the internal posterior tibial muscle group and sciatic nerve for both emg and motor nerve conduction studies ( mncs ) . r1 : the active recording electrode ; r2 : the reference recording electrode ; s1 : the cathode of the stimulating electrode ; s2 : the anode of stimulating electrode ; g : the ground electrode . ( b and c ) compound muscle action potential and motor unit number estimation examples . four points in the muscle group were selected to investigate sa , with more than 10-second observation at each point . sweep duration was 10 ms / division , sensitivity was 100 v / division , and filter settings were 20 hz10 khz . the sciatic nerve was stimulated at the root of the hind limb , and the anode of the stimulating electrode was placed at anterior superior iliac spine . the active electrode was placed over the belly of the internal posterior tibial muscle group , while the reference electrode was positioned on the ankle . sweep speed was 2 ms / division , and sensitivity was 20 mv / division . mune [ figure 1c ] was performed by the standard incremental method after careful training . mune was equal to the value of the maximal cmap amplitude divided the average of the 10 potentials amplitudes . sweep speed was 2 ms / division , and sensitivity was 100 v to 20 mv / division . the brain and spinal cord were removed . gross observation and hematoxylin and eosin staining were performed to identify the cerebral infarction . the lower lumbar spinal cord ( l4s1 ) was cut into 3-m thick slides at l4 , l5 and s1 levels . neuron - specific nuclear protein ( neun ) that is nuclear protein specifically expressed by mature neuron was used here to mark neurons . b - cell lymphoma / leukaemia-2 ( bcl-2 ) protein can prevent cell apoptosis , while bcl-2 associated x ( bax ) protein promotes apoptosis and has an antagonistic effect on bcl-2 . neurons were stained with bcl-2 and bax to express their conditions , trend to alive or apoptosis . only anterior motoneurons with visible staining nucleus and nucleolus were counted . three nonoverlapping regions in the posterior horn were chosen to count neun positive cells , and the average number represented the number of neurons unit area in the posterior horn . the gfap positive astrocytes in anterior horn were counted in the same way of posterior horn neurons counting . we performed the statistical analysis of our data using spss for windows , version 17.0 ( spss inc . , all data were expressed in the form of mean standard deviation ( sd ) . the data were detected with shapiro - wilk test and levene test for normality and homogeneity of the variance respectively first . if the normality or homogeneity was not met , we adopted nonparametric statistical tests . otherwise , we used paired t - test to compare bilateral electrophysiological and pathologic data , and independent - sample t - test for pairwise comparison in groups of pathologic data . analysis of variance ( anova ) for repeated measurement data , or one - way anova was used for comparison of data over time . group t - test was used to analyze the difference of mcao and control groups . the frequency of sa in different mnss groups was analyzed by the fisher 's exact test . sprague - dawley male rats were obtained from the animal center of pumch , weighing between 250 g and 380 g. animals were specific pathogen free , and raised in 2025c room temperature . seventeen subjects were died of anesthesia accidents and cerebral hernia after mcao , and another nine subjects were found had subarachnoid hemorrhage and intracranial hemorrhage after sacrifice . every 10 mcao rats were sacrificed for pathology tests , at 24 hours , 7 days , and 14 days after operation separately . all surviving subjects were evaluated by electrophysiology assessments and modified neurological severity score ( mnss ) at 0 day , 24 hours , 7 days , and 14 days after surgery . the permanent mcao procedure was based on the longa mcao logi after anesthesia with 34 ml / kg i.p . the internal carotid artery was also isolated and separated carefully from the adjacent vagus nerve , and the external carotid artery was ligated . monofilament nylon suture ( diameter : 2.63.0 mm ) was introduced into the common carotid artery lumen through a puncture and advanced gently to the internal carotid artery lumen . after about 18 mm length of nylon suture had been inserted into artery lumen , resistance was felt , indicating that the suture had passed the middle cerebral artery origin and occluded the artery . after all possible bleedings were stopped , all electrophysiology tests were performed using a portable electromyography ( emg ) machine ( 10ch medelec synergy , natus europe gmbh , germany ) . rats were placed on the operation table after anesthesia , with bilateral lower limbs spread at an approximately 45 angle to the spine [ figure 1a ] , under lamplight to stay warm . we chose the internal posterior tibial muscle group and sciatic nerve for both emg and motor nerve conduction studies ( mncs ) . r1 : the active recording electrode ; r2 : the reference recording electrode ; s1 : the cathode of the stimulating electrode ; s2 : the anode of stimulating electrode ; g : the ground electrode . ( b and c ) compound muscle action potential and motor unit number estimation examples . four points in the muscle group were selected to investigate sa , with more than 10-second observation at each point . sweep duration was 10 ms / division , sensitivity was 100 v / division , and filter settings were 20 hz10 khz . the sciatic nerve was stimulated at the root of the hind limb , and the anode of the stimulating electrode was placed at anterior superior iliac spine . the active electrode was placed over the belly of the internal posterior tibial muscle group , while the reference electrode was positioned on the ankle . sweep speed was 2 ms / division , and sensitivity was 20 mv / division . mune [ figure 1c ] was performed by the standard incremental method after careful training . mune was equal to the value of the maximal cmap amplitude divided the average of the 10 potentials amplitudes . sweep speed was 2 ms / division , and sensitivity was 100 v to 20 mv / division . the brain and spinal cord were removed . gross observation and hematoxylin and eosin staining were performed to identify the cerebral infarction . the lower lumbar spinal cord ( l4s1 ) was cut into 3-m thick slides at l4 , l5 and s1 levels . neuron - specific nuclear protein ( neun ) that is nuclear protein specifically expressed by mature neuron was used here to mark neurons . b - cell lymphoma / leukaemia-2 ( bcl-2 ) protein can prevent cell apoptosis , while bcl-2 associated x ( bax ) protein promotes apoptosis and has an antagonistic effect on bcl-2 . neurons were stained with bcl-2 and bax to express their conditions , trend to alive or apoptosis . only anterior motoneurons with visible staining nucleus and nucleolus were counted . three nonoverlapping regions in the posterior horn were chosen to count neun positive cells , and the average number represented the number of neurons unit area in the posterior horn . the gfap positive astrocytes in anterior horn were counted in the same way of posterior horn neurons counting . we performed the statistical analysis of our data using spss for windows , version 17.0 ( spss inc . , all data were expressed in the form of mean standard deviation ( sd ) . the data were detected with shapiro - wilk test and levene test for normality and homogeneity of the variance respectively first . if the normality or homogeneity was not met , we adopted nonparametric statistical tests . otherwise , we used paired t - test to compare bilateral electrophysiological and pathologic data , and independent - sample t - test for pairwise comparison in groups of pathologic data . analysis of variance ( anova ) for repeated measurement data , or one - way anova was used for comparison of data over time . group t - test was used to analyze the difference of mcao and control groups . the frequency of sa in different mnss groups was analyzed by the fisher 's exact test . abnormal sa were detected in the paretic hind limb of 16.7% ( 5/30 ) mcao rats , including 3 at 24 hours after mcao ( mnss : 9 , 10 , 11 ) , 1 at 7 days after mcao ( mnss : 4 ) , and 1 at 14 days after mcao ( mnss : 9 ) . we divided each group into two subgroups according to mnss ( mnss 7 and mnss > 7 ) . the frequency of sa in mnss 7 group was 7.1% ( 1/14 ) , while the frequency in mnss > 7 group was 25.0% ( 4/16 ) ( p = 0.209 ) . these results showed sa was more likely to occur in rats with a higher mnss , although there was no significant difference between the two groups . changes of cmap amplitude are showed in table 1 and figure 2a . in the control group , there was no significant difference between bilateral cmap amplitudes and among cmap amplitudes over time . in mcao groups , cmap amplitude on paretic side was significantly lower than nonparetic side , at 24 hours and 7 days after operation ( 24 hours : 61.9 10.4 vs. 66.6 8.9 , p < 0.05 ; 7 days : 60.9 8.4 vs. 67.3 9.6 , p < 0.05 ) [ table 1 and figure 2a ] . the cmap amplitudes over time showed no significant difference , neither on the paretic side nor the nonparetic side . at the four given time points , we further divided the 7 days group into two subgroups ( paretic cmap amplitude > 60.60 mv and 60.60 mv ) . the average mnss was 5.7 in group with cmap amplitude > 60.60 mv ( n = 11 ) , and 6.9 in group with cmap amplitude 60.60 mv ( n = 10 ) . the summary of cmap amplitude data in control and mcao rats ( mean sd ) * wilcoxon test . sd : standard deviation ; cmap : compound muscle action potential ; mcao : middle cerebral artery occlusion . ( a and b ) the max difference of bilateral compound muscle action potential ( cmap ) amplitude and motor unit number estimation ( mune ) showed at 7 days after the operation . ( c - e ) the number of anterior / posterior horn neurons was decreasing over the time . the mune data are showed in table 2 and figure 2b . in the control group , there was no significant difference in mune between bilateral limbs at different duration . in mcao groups , however , mune on paretic side was significantly less than that on nonparetic side , at 7 days after operation ( 379.0 84.6 vs. 445.0 89.5 , p there was no significant difference in munes of paretic limbs among four given time points , as well as a nonparetic side . the 7-day group was also divided into two subgroups ( paretic mune > 381 and 381 ) . the average mnss was 5.6 in group with mune > 381 ( n = 10 ) , and 6.9 in group with mune 381 ( n = 10 ) . the summary of mune data in control and mcao rats ( mean sd ) mcao : middle cerebral artery occlusion ; sd : standard deviation ; mune : motor unit number estimation . cmap amplitude on paretic side was significantly lower than on the nonparetic side , at both 24 hours and 7 days after mcao , and mune on paretic side was significantly less than on nonpareticside at 7 days after mcao . these results revealed that cerebral infarction could cause spinal motor neurons abnormalities , and loss of functional anterior motor neurons . compared with control group , the number of neun - positive anterior motor neurons [ figure 3a ] declined over time , and was significantly decreased in 14-day mcao group ( 5.3 0.7 vs. 7.3 1.8 , p < 0.05 ) [ table 3 and figure 2c ] . the expression of bcl-2 and bax in anterior motor neurons [ figure 3b and 3c ] at 24 hours after mcao was significantly increased ( bcl-2 : 5.6 0.8 vs. 2.8 1.5 , p < 0.05 ; bax : 5.0 1.1 vs. 2.1 0.8 , p < 0.05 ) , while in 14 days mcao group , bax expression increased significantly again ( 4.3 0.7 vs. 2.1 0.8 , p < 0.05 ) , with bcl-2 expression remained normal [ table 3 and figure 2c ] . these results revealed that in the early time after stroke , apoptosis of the spinal motor neurons were induced and seemed to be reversible for high expression of bcl-2 . after 14 days , high expression of bax alone and significantly decreased a number of anterior neurons indicated that anterior motor neurons were degenerated . ( a - c ) motoneurons with neuron - specific nuclear protein ( immunohistochemical [ ihc ] staining , original magnification 40 ) , b - cell lymphoma / leukaemia-2 ( ihc , 100 ) , b - cell lymphoma / leukaemia-2 associated x expressions ( ihc , 100 ) , respectively . neurons of rexed layers i iii ( white rome number ) in three nonoverlapping areas ( white rectangle ) were counted ( ihc , 100 ) . ( e ) glial fibrillary acidic protein positive astrocytes ( red arrow ) ( ihc , 200 ) . the summary of histological data in control and mcao rats ( mean sd ) mcao : middle cerebral artery occlusion ; sd : standard deviation ; neun : neuron - specific nuclear protein ; bcl-2 : b - cell lymphoma / leukaemia-2 ; bax ; bcl-2 associated x ; gfap : glial fibrillary acidic protein . the number of neun positive posterior horn neurons [ figure 3d ] in 7 days and 14 days mcao group was significantly less than control ( control vs. 7 days : 50.4 4.7 vs. 44.7 5.4 , p < 0.05 ; control vs. 14 days : 50.4 4.7 vs. 43.0 3.1 , p < 0.05 ) [ table 3 and figure 2d ] . there were more gfap positive astrocytes [ figure 3e ] in 24 hours group than in control ( p = 0.050 ) [ table 3 and figure 2e ] . astrocytes were activated at 24 hours , suggesting that the injury and inflammation occurs in the early time . abnormal sa were detected in the paretic hind limb of 16.7% ( 5/30 ) mcao rats , including 3 at 24 hours after mcao ( mnss : 9 , 10 , 11 ) , 1 at 7 days after mcao ( mnss : 4 ) , and 1 at 14 days after mcao ( mnss : 9 ) . we divided each group into two subgroups according to mnss ( mnss 7 and mnss > 7 ) . the frequency of sa in mnss 7 group was 7.1% ( 1/14 ) , while the frequency in mnss > 7 group was 25.0% ( 4/16 ) ( p = 0.209 ) . these results showed sa was more likely to occur in rats with a higher mnss , although there was no significant difference between the two groups . changes of cmap amplitude are showed in table 1 and figure 2a . in the control group , there was no significant difference between bilateral cmap amplitudes and among cmap amplitudes over time . in mcao groups , cmap amplitude on paretic side was significantly lower than nonparetic side , at 24 hours and 7 days after operation ( 24 hours : 61.9 10.4 vs. 66.6 8.9 , p < 0.05 ; 7 days : 60.9 8.4 vs. 67.3 9.6 , p < 0.05 ) [ table 1 and figure 2a ] . the cmap amplitudes over time showed no significant difference , neither on the paretic side nor the nonparetic side . at the four given time points , we further divided the 7 days group into two subgroups ( paretic cmap amplitude > 60.60 mv and 60.60 mv ) . the average mnss was 5.7 in group with cmap amplitude > 60.60 mv ( n = 11 ) , and 6.9 in group with cmap amplitude 60.60 mv ( n = 10 ) . the summary of cmap amplitude data in control and mcao rats ( mean sd ) * wilcoxon test . sd : standard deviation ; cmap : compound muscle action potential ; mcao : middle cerebral artery occlusion . ( a and b ) the max difference of bilateral compound muscle action potential ( cmap ) amplitude and motor unit number estimation ( mune ) showed at 7 days after the operation . ( c - e ) the number of anterior / posterior horn neurons was decreasing over the time . the mune data are showed in table 2 and figure 2b . in the control group , there was no significant difference in mune between bilateral limbs at different duration . in mcao groups , however , mune on paretic side was significantly less than that on nonparetic side , at 7 days after operation ( 379.0 84.6 vs. 445.0 89.5 , p there was no significant difference in munes of paretic limbs among four given time points , as well as a nonparetic side . the 7-day group was also divided into two subgroups ( paretic mune > 381 and 381 ) . the average mnss was 5.6 in group with mune > 381 ( n = 10 ) , and 6.9 in group with mune 381 ( n = 10 ) . the summary of mune data in control and mcao rats ( mean sd ) mcao : middle cerebral artery occlusion ; sd : standard deviation ; mune : motor unit number estimation . cmap amplitude on paretic side was significantly lower than on the nonparetic side , at both 24 hours and 7 days after mcao , and mune on paretic side was significantly less than on nonpareticside at 7 days after mcao . these results revealed that cerebral infarction could cause spinal motor neurons abnormalities , and loss of functional anterior motor neurons . compared with control group , the number of neun - positive anterior motor neurons [ figure 3a ] declined over time , and was significantly decreased in 14-day mcao group ( 5.3 0.7 vs. 7.3 1.8 , p < 0.05 ) [ table 3 and figure 2c ] . the expression of bcl-2 and bax in anterior motor neurons [ figure 3b and 3c ] at 24 hours after mcao was significantly increased ( bcl-2 : 5.6 0.8 vs. 2.8 1.5 , p < 0.05 ; bax : 5.0 1.1 vs. 2.1 0.8 , p < 0.05 ) , while in 14 days mcao group , bax expression increased significantly again ( 4.3 0.7 vs. 2.1 0.8 , p < 0.05 ) , with bcl-2 expression remained normal [ table 3 and figure 2c ] . these results revealed that in the early time after stroke , apoptosis of the spinal motor neurons were induced and seemed to be reversible for high expression of bcl-2 . after 14 days , high expression of bax alone and significantly decreased a number of anterior neurons indicated that anterior motor neurons were degenerated . ( a - c ) motoneurons with neuron - specific nuclear protein ( immunohistochemical [ ihc ] staining , original magnification 40 ) , b - cell lymphoma / leukaemia-2 ( ihc , 100 ) , b - cell lymphoma / leukaemia-2 associated x expressions ( ihc , 100 ) , respectively . neurons of rexed layers i iii ( white rome number ) in three nonoverlapping areas ( white rectangle ) were counted ( ihc , 100 ) . ( e ) glial fibrillary acidic protein positive astrocytes ( red arrow ) ( ihc , 200 ) . the summary of histological data in control and mcao rats ( mean sd ) mcao : middle cerebral artery occlusion ; sd : standard deviation ; neun : neuron - specific nuclear protein ; bcl-2 : b - cell lymphoma / leukaemia-2 ; bax ; bcl-2 associated x ; gfap : glial fibrillary acidic protein . the number of neun positive posterior horn neurons [ figure 3d ] in 7 days and 14 days mcao group was significantly less than control ( control vs. 7 days : 50.4 4.7 vs. 44.7 5.4 , p < 0.05 ; control vs. 14 days : 50.4 4.7 vs. 43.0 3.1 , p < 0.05 ) [ table 3 and figure 2d ] . there were more gfap positive astrocytes [ figure 3e ] in 24 hours group than in control ( p = 0.050 ) [ table 3 and figure 2e ] . astrocytes were activated at 24 hours , suggesting that the injury and inflammation occurs in the early time . this study showed that abnormal sa , decreased mune and cmap appeared on the paretic side in mcao rats , consistently with previous clinical studies , suggesting that lower motor neurons injury happened in the early time after stroke . and the pathology results showed that anterior motor neurons significantly decreased , indicating transsynaptic degeneration happened . however , we also found the asynchronization of electrophysiology and pathology changes . at 24 hours after cerebral infarction , sa appeared , cmap amplitude on paretic side declined , while bcl-2 , bax , and gfap expressions were increased , with normal anterior motor neurons counting . had reported that abnormal electrophysiology could be appeared in the paretic limb as early as 4 h after stroke , which may due to the cut - off of corticospinal nerve fibers and inflammation . our study provided pathology evidence to this speculation . at 7 days after cerebral infarction , electrophysiology showed a significant decrease in paretic mune and cmap , while pathology results showed almost normal after the early stress reaction . at 14 days after cerebral infarction , the electrophysiology was back to normal , while anterior horn neurons decreased significantly with high expression of bax . our study provided the pathology evidence of lower motor neurons dysfunction and degeneration after stroke , and the variation characteristics of electrophysiology and pathology . these results revealed that abnormal electrophysiology could be detected in the early time , when motor neurons were functionally impaired , suggesting electrophysiology was more sensitive than pathology . however , when lower motor neurons degenerated in the later stage , the abnormal electrophysiological changes could not be detected . the reason for this phenomenon might be complicated . we suspected that , the rats have strong self - repair ability and quick recovering from cerebral infarction , although there was decrease in motor neurons due to transsynaptic degeneration in pathological studies at the later stage , the denervated muscle fibers of those motor neuron may be reinnervated by the survived motor neurons , which may lead to the increase in cmap amplitude and mune . clinical symptoms presented only when the number of lower motor neurons loss over 40% , while our study showed only about 20% loss of motor neurons . these results also indicated that electrophysiology may be more liable to be affected by dysfunction of motor neurons instead of the number of motor neurons at an early stage after mcao . we also found , significantly higher expression of bcl-2 and bax in anterior motoneurons , and gfap in astrocytes was observed at 24 hours after cerebral infarction . these results revealed that in the early time after stroke , apoptosis of the spinal motor neurons were induced and astrocytes were activated , suggesting that injury and inflammation have occurred , which may initiate the lower motor neurons impairments . posterior horn neurons also declined , and even faster than anterior neurons , illustrating that transsynaptic degeneration also existed in the sensory system , which was consistent with the previous study . it was speculated that the anterior motor neurons received multiple inputs from other interneurons postsynaptic , which abating the influence from impaired corticospinal projections . posterior horn neuron did not have many contacts with other cells , which made it more sensitive . however , some results need to be further discussed : ( 1 ) previous studies showed that sa appeared in hemiparetic stroke patients as early as 2 weeks after stroke , while in our experiment , sa appeared at 24 hours after mcao , which could be related to the short length of peripheral nerve in rats . the onset duration of sa is dependent on the distance of peripheral nerve lesion and the target muscle . it has been reported that sa could be observed at 36 h after cutting the sciatic nerve at the sciatic notch in rats . in previous research , when anterior motor neurons were functional impaired , the neuromuscular junctions were abnormal , which might lead to spontaneous exciting of the muscle fiber . ( 2 ) there was no significant difference in changes of cmap amplitude and mune among different duration after mcao , neither in cmap amplitude and mune between control and mcao groups . the electrophysiology could be affected by multiple factors , such as individual differences , electrode differences , and environmental temperature difference , as well as good recovering capacity in rats . fortunately , bilateral sides will not be affected by these factors , that was more reasonable and powerful for detecting the electrophysiological changes . in conclusion , in the early time after cerebral infarction , functional disturbance in the lower motor neurons caused the abnormal electrophysiology results . in the later stage , anterior motor neurons declined , which provide the evidence of transsynaptic degeneration in the motor system . our study combined the electrophysiology and pathology to provide the evidence of transsynaptic degeneration in the motor system . more researches were necessary for further exploring the mechanisms of the transsynaptic degenerations in the motor system . this study was funded by a grant of national natural science foundation of china ( no . this study was funded by a grant of national natural science foundation of china ( no .
background : motor dysfunction is common in stroke patients . clinical electrophysiological studies suggest that transsynaptic degeneration occurred in the lower motor neurons , while pathological evidence is lacked . this study aimed to combine the electrophysiological and pathological results to prove the existence of transsynaptic degeneration in the motor system after stroke.methods:modified neurologic severity score , electrophysiological , and pathological assessments were evaluated in rats before middle cerebral artery occlusion ( mcao ) , and at 24 hours , 7 days , and 14 days after mcao . paired and independent - sample t - tests were applied to assess the changes of electrophysiological and pathological data.results:compound motor action potential amplitude in the paretic side was significantly lower than the nonparetic side at both 24 hours ( 61.9 10.4 vs. 66.6 8.9 , p < 0.05 ) and 7 days ( 60.9 8.4 vs. 67.3 9.6 , p < 0.05 ) after mcao . motor unit number estimation of the paretic side was significantly less than the nonparetic side ( 379.0 84.6 vs. 445.0 89.5 , p < 0.05 ) at 7 days after mcao . until 14 days after stroke , the pathological loss of motor neurons was detected . motor neurons in 14-day mcao group were significantly decreased , compared with control group ( 5.3 0.7 vs. 7.3 1.8 , p < 0.05).conclusions : both electrophysiological and pathological studies showed transsynaptic degeneration after stroke . this study identified the asynchronization in changes of electrophysiology and pathology . the abnormal physiological changes and function impairment can be detected in the early stage and recovered quickly , while the pathological loss of motor neuron can be detected only in a later stage .
I M Animals Surgical procedures Electrophysiological assessments Histological and immunohistochemical assessments Statistical analysis R Electrophysiological assessments Histological and immunohistochemical assessment D Financial support and sponsorship Conflicts of interest
in mcao groups , cmap amplitude on paretic side was significantly lower than nonparetic side , at 24 hours and 7 days after operation ( 24 hours : 61.9 10.4 vs. 66.6 8.9 , p < 0.05 ; 7 days : 60.9 8.4 vs. 67.3 9.6 , p < 0.05 ) [ table 1 and figure 2a ] . in mcao groups , however , mune on paretic side was significantly less than that on nonparetic side , at 7 days after operation ( 379.0 84.6 vs. 445.0 89.5 , p there was no significant difference in munes of paretic limbs among four given time points , as well as a nonparetic side . cmap amplitude on paretic side was significantly lower than on the nonparetic side , at both 24 hours and 7 days after mcao , and mune on paretic side was significantly less than on nonpareticside at 7 days after mcao . compared with control group , the number of neun - positive anterior motor neurons [ figure 3a ] declined over time , and was significantly decreased in 14-day mcao group ( 5.3 0.7 vs. 7.3 1.8 , p < 0.05 ) [ table 3 and figure 2c ] . the expression of bcl-2 and bax in anterior motor neurons [ figure 3b and 3c ] at 24 hours after mcao was significantly increased ( bcl-2 : 5.6 0.8 vs. 2.8 1.5 , p < 0.05 ; bax : 5.0 1.1 vs. 2.1 0.8 , p < 0.05 ) , while in 14 days mcao group , bax expression increased significantly again ( 4.3 0.7 vs. 2.1 0.8 , p < 0.05 ) , with bcl-2 expression remained normal [ table 3 and figure 2c ] . the number of neun positive posterior horn neurons [ figure 3d ] in 7 days and 14 days mcao group was significantly less than control ( control vs. 7 days : 50.4 4.7 vs. 44.7 5.4 , p < 0.05 ; control vs. 14 days : 50.4 4.7 vs. 43.0 3.1 , p < 0.05 ) [ table 3 and figure 2d ] . in mcao groups , cmap amplitude on paretic side was significantly lower than nonparetic side , at 24 hours and 7 days after operation ( 24 hours : 61.9 10.4 vs. 66.6 8.9 , p < 0.05 ; 7 days : 60.9 8.4 vs. 67.3 9.6 , p < 0.05 ) [ table 1 and figure 2a ] . in mcao groups , however , mune on paretic side was significantly less than that on nonparetic side , at 7 days after operation ( 379.0 84.6 vs. 445.0 89.5 , p there was no significant difference in munes of paretic limbs among four given time points , as well as a nonparetic side . cmap amplitude on paretic side was significantly lower than on the nonparetic side , at both 24 hours and 7 days after mcao , and mune on paretic side was significantly less than on nonpareticside at 7 days after mcao . compared with control group , the number of neun - positive anterior motor neurons [ figure 3a ] declined over time , and was significantly decreased in 14-day mcao group ( 5.3 0.7 vs. 7.3 1.8 , p < 0.05 ) [ table 3 and figure 2c ] . the expression of bcl-2 and bax in anterior motor neurons [ figure 3b and 3c ] at 24 hours after mcao was significantly increased ( bcl-2 : 5.6 0.8 vs. 2.8 1.5 , p < 0.05 ; bax : 5.0 1.1 vs. 2.1 0.8 , p < 0.05 ) , while in 14 days mcao group , bax expression increased significantly again ( 4.3 0.7 vs. 2.1 0.8 , p < 0.05 ) , with bcl-2 expression remained normal [ table 3 and figure 2c ] . the number of neun positive posterior horn neurons [ figure 3d ] in 7 days and 14 days mcao group was significantly less than control ( control vs. 7 days : 50.4 4.7 vs. 44.7 5.4 , p < 0.05 ; control vs. 14 days : 50.4 4.7 vs. 43.0 3.1 , p < 0.05 ) [ table 3 and figure 2d ] .
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auditory verbal hallucinations ( avhs ) are among the core symptoms used in the diagnosis of schizophrenia , occurring in 6080% of schizophrenia patients . characterizing the brain structural and functional features of specific symptoms can enhance our understanding of the etiology of schizophrenia and provide objective indictors for precision medicine . some neuroimaging data regarding the avhs of schizophrenia have been obtained from structural magnetic resonance imaging ( mri ) and functional mri ( fmri ) studies . structural imaging studies have found that schizophrenia patients with avhs usually exhibit gray matter volume reductions in the superior temporal gyrus . complementary diffusion tensor imaging studies have found that the avhs of schizophrenia are associated with disruptions of white matter integrity in the left arcuate fasciculus bundle and interhemispheric auditory fiber bundles . fmri studies have found that the avhs of schizophrenia are associated with abnormal activation within the default mode network ( dmn ) and auditory- and visual - associated resting - state ( rs ) networks . all the aforementioned findings advanced our understanding of the pathological traits of schizophrenic avhs to some extent . however , considering the high heterogeneity and complexity of avhs , for a comprehensive understanding of avhs , we must use multiple techniques to characterize the pathological features of avhs from different angles . regional homogeneity ( reho ) measures the similarity of the time series of blood - oxygen - level - dependent ( bold ) signals of a given voxel to those of its nearest neighbors in a voxel - wise way , which reflects the local synchronization in the functional cluster . a specific region in the brain that is abnormal can be identified by reho ; aberrant reho represents aberrant regional brain spontaneous activity in a specific region . in recent years , reho has been used to investigate functional modulations in the rs of patients with schizophrenia and other mental disorders . in the current study , we were interested in whether schizophrenic patients with avhs would demonstrate specific reho alterations and , if so , whether the brain regions ( rs networks ) with abnormal reho were specific to avhs . a total of 126 right - handed individuals were enrolled in the present study , including 76 schizophrenia patients and fifty healthy controls . the diagnosis of schizophrenia was determined by the consensus of two professional psychiatrists using the structured clinical interview for diagnostic and statistical manual of mental disorders , 4 edition ( scid ) . all healthy controls were screened using the nonpatient edition of the scid to confirm a lifetime absence of psychiatric illnesses . exclusion criteria for all subjects were a history of head trauma with consciousness disturbances lasting more than 5 min , a history of drug or alcohol abuse , pregnancy , and any physical illnesses , such as cardiovascular disease or neurological disorders , as diagnosed by an interview and medical records review . in addition , all healthy controls were interviewed to exclude individuals with a known history of psychiatric illness in first - degree relatives . avh group ( n = 35 ) included patients who experienced avhs at least once daily , and the non - auditory verbal hallucinations ( navh ) group ( n = 41 ) included patients who had never experienced avhs or had not experienced avhs within 12 months before mri . clinical symptoms of psychosis were quantified using the positive and negative syndrome scale ( panss ) . the auditory hallucination rating scale was used to assess avh with seven characteristics such as frequency , reality , loudness , number of voices , length , attention dedicated to the hallucinations , and hallucination - induced arousal . the daily antipsychotic dosages ( chlorpromazine equivalents ) for two schizophrenia patient groups are listed in table 1 . the medical research ethics committee of tianjin medical university general hospital approved this study . after receiving a complete description of the study , demographic and clinical characteristics of the sample * one - way anova was used to test the difference in age across the three groups ; chi - square test was used to test the difference in gender across the three groups ; two - sample t - test was used to compare the differences in antipsychotic dosage , duration of illness and panss scores between two patient groups . na : not applicable ; panss : positive and negative syndrome scale ; ahrs : auditory hallucination rating scale ; avh : schizophrenia patients with auditory verbal hallucinations ; navh : schizophrenia patients without auditory verbal hallucinations ; sd : standard deviation . mri data were acquired using a 3.0-tesla mr system ( discovery mr750 , general electric , milwaukee , wi , usa ) . tight but comfortable foam padding was used to minimize head motion , and earplugs were used to reduce scanner noise . sagittal three - dimensional t1-weighted images were acquired using a brain volume sequence with the following parameters : repetition time ( tr ) = 8.2 ms ; echo time ( te ) = 3.2 ms ; inversion time = 450 ms ; flip angle ( fa ) = 12 ; field of view ( fov ) = 256 mm 256 mm ; matrix = 256 256 ; slice thickness = 1 mm , no gap ; 188 sagittal slices ; and acquisition time = 250 s. rs functional bold images were acquired using a gradient - echo single - shot echo planar imaging sequence with the following parameters : tr / te = 2000/45 ms , fov = 220 mm 220 mm , matrix = 64 64 , fa = 90 , slice thickness = 4 mm , gap = 0.5 mm , 32 interleaved transverse slices , 180 volumes , and acquisition time = 370 s. all subjects were instructed to keep their eyes closed , relax , move as little as possible , think of nothing in particular , and not fall asleep during the scans . all mri were visually inspected to ensure that only images without visible artifacts were included in subsequent analyses . the first 10 volumes for each participant were discarded to allow the signal to reach equilibrium and the participants to adapt to the scanning noise . all participants bold data were within the defined motion thresholds ( i.e. , translational or rotational motion parameters < 2 mm or 2 ) . we also calculated frame - wise displacement ( fd ) , which indexes the volume - to - volume changes in the head position . several nuisance covariates ( six motion parameters , their 1 time derivatives , the global brain signal , the white matter signal , and the cerebrospinal fluid signal ) were regressed out from the data . the signal spike caused by head motion significantly contaminates the final rs fmri results even after regressing out the linear motion parameters . therefore , we further regressed out spike volumes when the fd of the specific volume exceeded 0.5 . the datasets were then band - pass filtered in a frequency range of 0.010.08 hz . in the normalization step , individual structural images were linearly coregistered with the mean functional image ; then , the transformed structural images were segmented into gray matter , white matter , and cerebrospinal fluid . the gray matter maps were linearly coregistered to the tissue probability maps in the montreal neurological institute ( mni ) space . finally , each filtered functional volume was spatially normalized to the mni space using the parameters estimated during the linear coregistration and resampled into a 3-mm cubic voxel . reho was defined as the kendall correlation coefficient ( kcc ) of the time series of a given voxel with those of its nearest neighbors ( 26 voxels ) on a voxel - wise basis . the kcc can be computed by the following formula : where w is the kcc among given voxels , ranging from 0 to 1 ; ri is the sum rank of the i time point ; is the mean of ri ; k is the number of time series within a measured cluster ( k = 27 , one given voxel plus its 26 neighbors ) , and n is the number of ranks ( n = 240 ) . then , each reho map was spatially smoothed with a gaussian kernel of 6 mm 6 mm 6 mm full width at half maximum . finally , we normalized the reho of each voxel by dividing it by the mean reho value of the whole brain . group differences in reho among the three groups were tested using a voxel - wise one - way analysis of covariance ( ancova ) with age and gender as covariates followed by post hoc intergroup comparisons . the post hoc intergroup comparisons were conducted within a mask showing reho differences from the ancova analysis . in general , gender could not be considered as a covariance in the ancova analysis ; however , in our current study , our ancova analysis was performed using the general linear model ( glm ) implemented in spm8 . for glm , categorical variable including gender should also be taken as independent variable . hence , in this study , we used the gender as a covariance in our current ancova analysis . multiple comparisons were corrected using a false discovery rate ( fdr ) method with a significance threshold of p < 0.05 . a total of 126 right - handed individuals were enrolled in the present study , including 76 schizophrenia patients and fifty healthy controls . the diagnosis of schizophrenia was determined by the consensus of two professional psychiatrists using the structured clinical interview for diagnostic and statistical manual of mental disorders , 4 edition ( scid ) . all healthy controls were screened using the nonpatient edition of the scid to confirm a lifetime absence of psychiatric illnesses . exclusion criteria for all subjects were a history of head trauma with consciousness disturbances lasting more than 5 min , a history of drug or alcohol abuse , pregnancy , and any physical illnesses , such as cardiovascular disease or neurological disorders , as diagnosed by an interview and medical records review . in addition , all healthy controls were interviewed to exclude individuals with a known history of psychiatric illness in first - degree relatives . avh group ( n = 35 ) included patients who experienced avhs at least once daily , and the non - auditory verbal hallucinations ( navh ) group ( n = 41 ) included patients who had never experienced avhs or had not experienced avhs within 12 months before mri . clinical symptoms of psychosis were quantified using the positive and negative syndrome scale ( panss ) . the auditory hallucination rating scale was used to assess avh with seven characteristics such as frequency , reality , loudness , number of voices , length , attention dedicated to the hallucinations , and hallucination - induced arousal . the daily antipsychotic dosages ( chlorpromazine equivalents ) for two schizophrenia patient groups are listed in table 1 . the medical research ethics committee of tianjin medical university general hospital approved this study . after receiving a complete description of the study , demographic and clinical characteristics of the sample * one - way anova was used to test the difference in age across the three groups ; chi - square test was used to test the difference in gender across the three groups ; two - sample t - test was used to compare the differences in antipsychotic dosage , duration of illness and panss scores between two patient groups . na : not applicable ; panss : positive and negative syndrome scale ; ahrs : auditory hallucination rating scale ; avh : schizophrenia patients with auditory verbal hallucinations ; navh : schizophrenia patients without auditory verbal hallucinations ; sd : standard deviation . mri data were acquired using a 3.0-tesla mr system ( discovery mr750 , general electric , milwaukee , wi , usa ) . tight but comfortable foam padding was used to minimize head motion , and earplugs were used to reduce scanner noise . sagittal three - dimensional t1-weighted images were acquired using a brain volume sequence with the following parameters : repetition time ( tr ) = 8.2 ms ; echo time ( te ) = 3.2 ms ; inversion time = 450 ms ; flip angle ( fa ) = 12 ; field of view ( fov ) = 256 mm 256 mm ; matrix = 256 256 ; slice thickness = 1 mm , no gap ; 188 sagittal slices ; and acquisition time = 250 s. rs functional bold images were acquired using a gradient - echo single - shot echo planar imaging sequence with the following parameters : tr / te = 2000/45 ms , fov = 220 mm 220 mm , matrix = 64 64 , fa = 90 , slice thickness = 4 mm , gap = 0.5 mm , 32 interleaved transverse slices , 180 volumes , and acquisition time = 370 s. all subjects were instructed to keep their eyes closed , relax , move as little as possible , think of nothing in particular , and not fall asleep during the scans . all mri were visually inspected to ensure that only images without visible artifacts were included in subsequent analyses . the first 10 volumes for each participant were discarded to allow the signal to reach equilibrium and the participants to adapt to the scanning noise . all participants bold data were within the defined motion thresholds ( i.e. , translational or rotational motion parameters < 2 mm or 2 ) . we also calculated frame - wise displacement ( fd ) , which indexes the volume - to - volume changes in the head position . several nuisance covariates ( six motion parameters , their 1 time derivatives , the global brain signal , the white matter signal , and the cerebrospinal fluid signal ) were regressed out from the data . the signal spike caused by head motion significantly contaminates the final rs fmri results even after regressing out the linear motion parameters . therefore , we further regressed out spike volumes when the fd of the specific volume exceeded 0.5 . the datasets were then band - pass filtered in a frequency range of 0.010.08 hz . in the normalization step , individual structural images were linearly coregistered with the mean functional image ; then , the transformed structural images were segmented into gray matter , white matter , and cerebrospinal fluid . the gray matter maps were linearly coregistered to the tissue probability maps in the montreal neurological institute ( mni ) space . finally , each filtered functional volume was spatially normalized to the mni space using the parameters estimated during the linear coregistration and resampled into a 3-mm cubic voxel . reho was defined as the kendall correlation coefficient ( kcc ) of the time series of a given voxel with those of its nearest neighbors ( 26 voxels ) on a voxel - wise basis . the kcc can be computed by the following formula : where w is the kcc among given voxels , ranging from 0 to 1 ; ri is the sum rank of the i time point ; is the mean of ri ; k is the number of time series within a measured cluster ( k = 27 , one given voxel plus its 26 neighbors ) , and n is the number of ranks ( n = 240 ) . then , each reho map was spatially smoothed with a gaussian kernel of 6 mm 6 mm 6 mm full width at half maximum . finally , we normalized the reho of each voxel by dividing it by the mean reho value of the whole brain . group differences in reho among the three groups were tested using a voxel - wise one - way analysis of covariance ( ancova ) with age and gender as covariates followed by post hoc intergroup comparisons . the post hoc intergroup comparisons were conducted within a mask showing reho differences from the ancova analysis . in general , gender could not be considered as a covariance in the ancova analysis ; however , in our current study , our ancova analysis was performed using the general linear model ( glm ) implemented in spm8 . for glm , categorical variable including gender should also be taken as independent variable . hence , in this study , we used the gender as a covariance in our current ancova analysis . multiple comparisons were corrected using a false discovery rate ( fdr ) method with a significance threshold of p < 0.05 . the three groups were well - matched in gender ( chi - square test , = 0.308 , p = 0.857 ) and age ( one - way anova , f = 0.100 , p = 0.905 ) . there were no significant differences in antipsychotic dosage ( two - sample t - test , t = 1.163 , p = 0.248 ) , duration of illness ( two sample t - test , t = 0.916 , p = 0.363 ) , panss negative score ( two sample t - test , t = 0.594 , p = 0.555 ) , panss general score ( two sample t - test , t = 0.275 , p = 0.784 ) , or panss total score ( two sample t - test , t = 0.893 , p = 0.375 ) between the avh and the navh patients . a voxel - wise ancova revealed that the intergroup differences in reho were mainly located in the bilateral postcentral gyrus and thalamus , the left precuneus and putamen , and the right caudate , inferior temporal gyrus and inferior occipital gyrus ( fdr corrected , p < 0.05 ) [ figure 1 ] . specifically , the avh group exhibited significantly increased reho in the left precuneus relative to the navh group . compared with the healthy controls , the avh patients showed significantly increased reho in the left precuneus and putamen and the right caudate and inferior temporal gyrus and decreased reho in the bilateral postcentral gyrus and thalamus and the right inferior occipital gyrus ( fdr corrected , p < 0.05 ) [ figure 1 ] . in addition , the navh group had significantly increased reho in the right caudate and inferior temporal gyrus and decreased reho in the bilateral postcentral gyrus and thalamus and the right inferior occipital gyrus compared with the healthy controls ( fdr corrected , p < 0.05 ) [ figure 1 ] . one - way analysis of covariance and post hoc two - sample t - tests were used for intergroup comparisons ( p < 0.05 , false discovery rate corrected ) . avh : schizophrenia patients with auditory verbal hallucinations ; navh : schizophrenia patients without auditory verbal hallucinations ; reho : regional homogeneity . unfortunately , in brain regions demonstrating avh - specific reho alterations , we did not find any statistical correlation between reho and arhs score in the schizophrenia patients with avhs . the three groups were well - matched in gender ( chi - square test , = 0.308 , p = 0.857 ) and age ( one - way anova , f = 0.100 , p = 0.905 ) . there were no significant differences in antipsychotic dosage ( two - sample t - test , t = 1.163 , p = 0.248 ) , duration of illness ( two sample t - test , t = 0.916 , p = 0.363 ) , panss negative score ( two sample t - test , t = 0.594 , p = 0.555 ) , panss general score ( two sample t - test , t = 0.275 , p = 0.784 ) , or panss total score ( two sample t - test , t = 0.893 , p = 0.375 ) between the avh and the navh patients . a voxel - wise ancova revealed that the intergroup differences in reho were mainly located in the bilateral postcentral gyrus and thalamus , the left precuneus and putamen , and the right caudate , inferior temporal gyrus and inferior occipital gyrus ( fdr corrected , p < 0.05 ) [ figure 1 ] . specifically , the avh group exhibited significantly increased reho in the left precuneus relative to the navh group . compared with the healthy controls , the avh patients showed significantly increased reho in the left precuneus and putamen and the right caudate and inferior temporal gyrus and decreased reho in the bilateral postcentral gyrus and thalamus and the right inferior occipital gyrus ( fdr corrected , p < 0.05 ) [ figure 1 ] . in addition , the navh group had significantly increased reho in the right caudate and inferior temporal gyrus and decreased reho in the bilateral postcentral gyrus and thalamus and the right inferior occipital gyrus compared with the healthy controls ( fdr corrected , p < 0.05 ) [ figure 1 ] . brain regions with altered reho across the avh , navh , and control groups . one - way analysis of covariance and post hoc two - sample t - tests were used for intergroup comparisons ( p < 0.05 , false discovery rate corrected ) . avh : schizophrenia patients with auditory verbal hallucinations ; navh : schizophrenia patients without auditory verbal hallucinations ; reho : regional homogeneity . unfortunately , in brain regions demonstrating avh - specific reho alterations , we did not find any statistical correlation between reho and arhs score in the schizophrenia patients with avhs . to the best of our knowledge , the current study is the first study to investigate the alteration of local brain spontaneous neural activity specific to avhs in patients with schizophrenia by rs - fmri using reho as an index . we found that reho in the right caudate and inferior temporal gyrus was higher in both schizophrenic patient groups than in the healthy controls whereas reho in the bilateral postcentral gyrus and thalamus and the right inferior occipital gyrus was lower in both schizophrenic patient groups ( fdr corrected , p < 0.05 ) . however , the key and novel finding of our current study is that the avh group demonstrated significantly increased reho in the left precuneus compared with the navh group . we found aberrant reho distributed in several brain regions in both schizophrenic patient groups , providing further evidence that local synchronization disturbances in the somatosensory cortex , visual processing related cortices , and some components of the limbic system may be the pathological features of schizophrenia . these synchronization disturbances were irrespective of the presence of avh and were mostly consistent with our previous meta - analysis and large - sample study findings . our finding that schizophrenia patients with avh demonstrated higher reho in the left precuneus than the navh patients and the healthy controls , suggests that increased reho in the left precuneus may be a pathological feature exclusive to avh in schizophrenia . the precuneus is the key component of the dmn , and there is growing evidence that the dmn plays a pivotal role in cognitive , memory retrieval , and self - referential processing and also participates in the processing of monitor inner speech . all of the aforementioned processes modulated by dmn are involved in the generation and monitoring of speech and have been associated with the experience of avh . more importantly , converging evidence suggests that functional alterations in the dmn play a key role in the generation of avh . recently , a new hypothesis that intrinsic dmn instability can account for the emergence of hallucination was proposed by jardri et al . according to this hypothesis , the dmn 's spatial and temporal instability is related to the severity of the hallucination ; however , spatial instability exists only in the course of hallucination activity and is negatively correlated with the severity of the hallucination . conversely , temporal instability exists in both the symptom - active and symptom - free course . more importantly , hyperpower spectral density ( an index of the default mode component 's time course ) in dmn is positively correlated with the severity of the hallucination . the functional disability of dmn subsequent to the intrinsic instability influences the transition between resting and active conscious sensory states , hence the emergence of hallucination . our finding of increased reho in the left precuneus , one component of dmn , supports the hypothesis that intrinsic instability of the dmn participates in the generation of avhs . we did not find a correlation between the reho value of the left precuneus and the severity of auditory hallucinations in the avh patients . this finding likely indicates that aberrant reho is a pathological feature of avh schizophrenia , irrespective of its severity . this finding is partly consistent with the previous finding that the intrinsically temporal instability of dmn exists in both hallucination - active and -alleviated states . first , the majority of the participating patients received antipsychotic drug treatment , and its effect on the reho in schizophrenia remain unclear . however , there was no significant difference in antipsychotic dosage between the schizophrenia patients with and without avh , which can control for this confounder to some extent . future studies focusing on first - episode drug - naive schizophrenia patients are needed to thoroughly control for this confounder . second , we did not assess the status of hallucination symptoms during the mri , which means that some patients likely experienced avh , whereas others may not have , during the mri course . instead , we assessed avh and other psychosis symptoms in all of the patients before the mri procedure to improve our ability to precisely characterize the exclusive features of avhs third , we enrolled patients who had not experienced avh within 12 months before mri scanning in the present study . these patients experienced auditory hallucinations only earlier in their psychotic illness or were in complete remission after treatment , which means that the findings of the present study probably do not comprehensively characterize the features of active hallucination when scanning . fourth , we did not find a correlation between the reho value and the severity of avh , likely due to the complexity of the relationship between local spontaneous neural activity and avh severity beyond a simple linear correlation . thus , more complex models , such as a quadratic regression model , can help us to clarify the relationship between them in a future study . first , we did not collect the panss score of healthy subjects ; hence , unfortunately , we could not take the positive score as a covariate in the process of ancova analysis . second , similar to many previous studies , healthy controls did not accept antipsychotic administration ; hence , we also did not take the psychotics dosage as a covariate in the ancova analysis . in the future study , we will consider these factors in the process of statistics and provide more accurate information for enhancing the understanding of brain function alteration of schizophrenia . collectively , in this study , we found that schizophrenia patients with and without avhs share common local spontaneous neural activity alterations in distributed brain regions that participate in somatosensory and visual processing and some regions of the limbic system . more importantly , we found that only schizophrenia patients with avh demonstrate increased reho in the pivotal component of the dmn ( left precuneus ) . this finding provides new data for the development of hypotheses regarding schizophrenic avhs and suggests that the intrinsic instability in the dmn is associated with the generation of avhs . this work was supported by grants from the natural science foundation of china ( no . 14zczdsy00018 ) , the national key clinical specialty project and the china postdoctoral science foundation funded project ( no . this work was supported by grants from the natural science foundation of china ( no . 81425013 , no . 91332113 and no . 81271551 ) , the tianjin key technology r&d program ( no . 14zczdsy00018 ) , the national key clinical specialty project and the china postdoctoral science foundation funded project ( no .
background : auditory verbal hallucinations ( avhs ) of schizophrenia have been associated with structural and functional alterations of some brain regions . however , the brain regional homogeneity ( reho ) alterations specific to avhs of schizophrenia remain unclear . in the current study , we aimed to investigate reho alterations specific to schizophrenic avhs.methods:thirty-five schizophrenic patients with avh , 41 schizophrenic patients without avhs , and fifty healthy subjects underwent resting - state functional magnetic resonance imaging . reho differences across the three groups were tested using a voxel - wise analysis.results:compared with the healthy control group , the two schizophrenia groups showed significantly increased reho in the right caudate and inferior temporal gyrus and decreased reho in the bilateral postcentral gyrus and thalamus and the right inferior occipital gyrus ( false discovery rate corrected , p < 0.05 ) . more importantly , the avh group exhibited significantly increased reho in the left precuneus compared with the non - avh group . however , using correlation analysis , we did not find any correlation between the auditory hallucination rating scale score and the reho of brain regions.conclusions:our results suggest that increased reho in the left precuneus may be a pathological feature exclusive to schizophrenic avhs .
I M Subjects Data acquisition Data preprocessing Regional homogeneity calculation Statistical analysis R Demographic and clinical characteristics Regional homogeneity differences across groups D Financial support and sponsorship Conflicts of interest
in the current study , we were interested in whether schizophrenic patients with avhs would demonstrate specific reho alterations and , if so , whether the brain regions ( rs networks ) with abnormal reho were specific to avhs . group differences in reho among the three groups were tested using a voxel - wise one - way analysis of covariance ( ancova ) with age and gender as covariates followed by post hoc intergroup comparisons . avh group ( n = 35 ) included patients who experienced avhs at least once daily , and the non - auditory verbal hallucinations ( navh ) group ( n = 41 ) included patients who had never experienced avhs or had not experienced avhs within 12 months before mri . na : not applicable ; panss : positive and negative syndrome scale ; ahrs : auditory hallucination rating scale ; avh : schizophrenia patients with auditory verbal hallucinations ; navh : schizophrenia patients without auditory verbal hallucinations ; sd : standard deviation . group differences in reho among the three groups were tested using a voxel - wise one - way analysis of covariance ( ancova ) with age and gender as covariates followed by post hoc intergroup comparisons . a voxel - wise ancova revealed that the intergroup differences in reho were mainly located in the bilateral postcentral gyrus and thalamus , the left precuneus and putamen , and the right caudate , inferior temporal gyrus and inferior occipital gyrus ( fdr corrected , p < 0.05 ) [ figure 1 ] . specifically , the avh group exhibited significantly increased reho in the left precuneus relative to the navh group . compared with the healthy controls , the avh patients showed significantly increased reho in the left precuneus and putamen and the right caudate and inferior temporal gyrus and decreased reho in the bilateral postcentral gyrus and thalamus and the right inferior occipital gyrus ( fdr corrected , p < 0.05 ) [ figure 1 ] . in addition , the navh group had significantly increased reho in the right caudate and inferior temporal gyrus and decreased reho in the bilateral postcentral gyrus and thalamus and the right inferior occipital gyrus compared with the healthy controls ( fdr corrected , p < 0.05 ) [ figure 1 ] . unfortunately , in brain regions demonstrating avh - specific reho alterations , we did not find any statistical correlation between reho and arhs score in the schizophrenia patients with avhs . a voxel - wise ancova revealed that the intergroup differences in reho were mainly located in the bilateral postcentral gyrus and thalamus , the left precuneus and putamen , and the right caudate , inferior temporal gyrus and inferior occipital gyrus ( fdr corrected , p < 0.05 ) [ figure 1 ] . specifically , the avh group exhibited significantly increased reho in the left precuneus relative to the navh group . compared with the healthy controls , the avh patients showed significantly increased reho in the left precuneus and putamen and the right caudate and inferior temporal gyrus and decreased reho in the bilateral postcentral gyrus and thalamus and the right inferior occipital gyrus ( fdr corrected , p < 0.05 ) [ figure 1 ] . in addition , the navh group had significantly increased reho in the right caudate and inferior temporal gyrus and decreased reho in the bilateral postcentral gyrus and thalamus and the right inferior occipital gyrus compared with the healthy controls ( fdr corrected , p < 0.05 ) [ figure 1 ] . unfortunately , in brain regions demonstrating avh - specific reho alterations , we did not find any statistical correlation between reho and arhs score in the schizophrenia patients with avhs . we found that reho in the right caudate and inferior temporal gyrus was higher in both schizophrenic patient groups than in the healthy controls whereas reho in the bilateral postcentral gyrus and thalamus and the right inferior occipital gyrus was lower in both schizophrenic patient groups ( fdr corrected , p < 0.05 ) . however , the key and novel finding of our current study is that the avh group demonstrated significantly increased reho in the left precuneus compared with the navh group . our finding that schizophrenia patients with avh demonstrated higher reho in the left precuneus than the navh patients and the healthy controls , suggests that increased reho in the left precuneus may be a pathological feature exclusive to avh in schizophrenia . we did not find a correlation between the reho value of the left precuneus and the severity of auditory hallucinations in the avh patients . fourth , we did not find a correlation between the reho value and the severity of avh , likely due to the complexity of the relationship between local spontaneous neural activity and avh severity beyond a simple linear correlation . more importantly , we found that only schizophrenia patients with avh demonstrate increased reho in the pivotal component of the dmn ( left precuneus ) .
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in patients with liver metastases from colorectal cancer hepatic resection has proven survival benefits and is curative in a small proportion of cases . patients whose metastases are small , few in number and metachronous have the best prognosis , but there is now good evidence that patients with more extensive disease can benefit from resection . the number , size and distribution of lesions are no longer limiting factors , provided all lesions are removed with adequate tumour - free margins and there is sufficient normal liver to maintain liver function postoperatively . survival benefits are also well established in patients with neuroendocrine metastases and in a minority of patients with localised metastases from other primary sites which have a more indolent course . treatment success , however , depends on the removal of all sites of intra - hepatic disease and the absence of disease outside the liver ; incomplete resection does not prolong survival . consequently , more patients are being referred for preoperative assessment and , since many have multiple small lesions , the role of imaging is increasingly challenging . almost all metastases larger than 1 cm are demonstrated with current imaging techniques but the detection of smaller lesions is still relatively poor . although the primary modalities for liver imaging are ultrasound and ct , recent studies have suggested that contrast - enhanced mri is the most sensitive method for detecting small metastases and mri is now considered the preoperative standard . moreover , recent developments in mri hardware and software and the availability of novel mri contrast agents have improved small lesion detection . this article considers the current status of mr imaging techniques in the detection and characterisation of liver metastases and discusses the technical requirements for optimum performance . the accuracy and relative capability of various mr techniques for detecting sub - cm lesions is emphasised . mr performance is primarily dependent on technique and the optimisation of pulse sequences . to date , liver mr has been most successful at a field strength of 1.5 t using high performance gradients , phased array multicoils and optimised imaging parameters . signal - to - noise ratio ( snr ) is influenced by field strength and surface coil configuration whilst imaging speed and spatial resolution are influenced by gradient performance . with stronger gradient systems fast breathhold sequences with t1 and t2 weighting have become routine and motion - induced artifacts are no longer a significant problem . we recommend using breathhold sequences wherever possible since they are the most effective means of eliminating motion artifact . moreover , breathhold versions of traditionally used sequences have been shown to have superior image quality and to be at least as good as non - breathhold versions for lesion detection . lesion signal intensity ( si ) on unenhanced t1w and t2w images often provides an indication of the type of lesion and guides the selection of contrast media for a more definitive diagnosis . t2w images determine the fluid content of lesions and are invaluable for distinguishing solid and non - solid tumours . we currently use a single shot fast spin echo ( fse ) sequence with half - fourier reconstruction ( haste ) because motion artifacts are consistently absent even during free breathing and small cysts and fluid collections are well defined . in - phase ( ip ) and opposed - phase ( op ) t1w gre imaging ( chemical shift imaging ) provides a definitive diagnosis of focal or diffuse fatty infiltration . this is particularly important when patients are being considered for hepatic resection since fatty change the fatty liver has a clearly reduced si on opt1w compared with ipt1w images , so whilst the detection and characterisation of liver lesions by ultrasound and ct is handicapped by fatty change , chemical shift mr imaging allows accurate differentiation of metastases and focal fatty change or focal sparing in a fatty liver . in terms of lesion detection , low lesion - to - liver contrast on unenhanced sequences has resulted in rather poor detection rates for sub - cm metastases . low - contrast lesions are particularly inconspicuous on breathhold t2w fse sequences and are often better depicted on t1w images ( fig . fse is insensitive to susceptibility so liver signal is slightly higher than with conventional spin echo imaging ; magnetisation transfer ( mt ) effects which cause solid lesions to lose si are particularly marked on multi - slice fse sequences and become more pronounced with increasing echo train length ; and fse is also subject to blurring of short t2 tissues because most of the high spatial frequencies are collected at the later echoes when there is relatively little remaining signal . of the unenhanced sequences , breathhold stir is probably the most sensitive for depicting metastases although image quality is variable . the additive effects of t1 and t2 provide strong image contrast which improves the conspicuousness of small lesions ( fig . rapid sequential imaging with extra - cellular space ( ecs ) gadolinium ( gd)-based contrast agents has been shown to be superior to unenhanced imaging and helical ct for detecting metastatic disease . conventional 2d sequences have been widely used for dgei but they are handicapped by relatively thick sections and inter - section gaps , which limits the diagnosis of sub - cm lesions . the recent introduction of fast 3d t1w gre sequences has overcome many of these problems . current 3d sequences result in a higher snr and thinner effective slice thickness than 2d methods without inter - slice gap or cross - talk and comfortably allow thin section coverage of the whole liver in a single breathhold ( fig . 2 ) . such sequences are now available from most manufacturers ( siemens vibe , philips wave , ge fame ) . use of a short repetition time ( 4 ms ) , and echo time ( 1.6 ms ) combined with interpolation algorithms , allows higher resolution matrices and a thinner effective slice thickness with minimal time penalties . the intermittent application of a chemically selective fat saturation ( fs ) pulse before each partition loop achieves homogeneous fat suppression . this is particularly important for dgei because effective fs increases the dynamic range within the image and accentuates gadolinium enhancement . a flip angle ( fa ) of approximately 15 is chosen to minimise the saturation of stationary tissues for the simultaneous display of liver parenchyma and hepatic vessels . flexible parameters allow scan times to be adjusted to accommodate the breathhold capacity of individual patients , and can also be manipulated to produce isotropic voxels for optimum 3d reconstruction . isotropic voxels are achieved at the expense of anatomic coverage but this is largely overcome by parallel imaging techniques . in most cases a parallel imaging factor of two enables isotropic imaging of the whole liver in approximately 20 s. imaging with gd should include baseline pre - contrast images and sequential acquisitions at arterial , portal and equilibrium phases . the arterial phase is the most crucial acquisition and the timing should be optimised by test bolusing wherever possible . best results are obtained when the contrast is administered by a power injector at a rate of 24 ml per second followed by a saline flush . hypervascular metastases are most conspicuous at the arterial phase when there is only minimal enhancement of background liver and many are only visible at this time . most hypovascular lesions are best demonstrated at the portal phase when liver enhancement is maximum but these lesions usually exhibit a transient rim of enhancement which is highly specific for metastases and often only visible at the earlier arterial phase ( fig . an optimised arterial phase is also important for identifying sub - cm metastases and distinguishing them from benign lesions . compared with simple cysts which exhibit non - progressive enhancement and haemangiomas which show discontinuous and persistent enhancement , metastases have a progressive enhancement pattern and become either less conspicuous over time or more conspicuous as a result of delayed central enhancement due to non - specific accumulation of contrast within the lesion s extra - cellular space . most small metastases become less distinct and apparently smaller on subsequent acquisitions . by the equilibrium phase contrast between normal and abnormal tissue delayed gd - enhanced imaging with fs is the technique of choice for demonstrating small metastases on the liver surface . fat suppression not only accentuates gadolinium enhancement but also suppresses the competing high signal of intra - abdominal fat adjacent to the liver surface . surface deposits are usually best seen on delayed images acquired 510 min after injection when they become hyperintense due to slow accumulation of contrast within the tumour ( fig . 4 ) . in patients with metastatic disease , however , the main role of dgei is probably to differentiate benign and malignant lesions . dgei is the most reliable method for characterising lesions and is recommended in all surgical candidates with equivocal lesions in a location which is likely to influence the surgical approach . the perfusion and extraction characteristics of tissues at the different phases of enhancement allow the differentiation of cysts , haemangiomas , fnh and metastases and a specific diagnosis is possible in most patients . liver - specific contrast agents were developed to increase and prolong lesion - liver contrast beyond that of the ecs agents . a minority of lesions have perfusion characteristics similar to normal liver and are occult on dgei . also many small lesions are no longer visible by the equilibrium phase when contrast is evenly distributed between intravascular and extra - cellular spaces . all liver - specific agents produce high tissue contrast and have been shown significantly to improve the detection of metastases compared with unenhanced mr , with enhanced mr using ecs agents with 2d gre sequences and with contrast - enhanced ct . agents which target the hepatocytes and produce positive enhancement on t1w images ( gadobenate , gadoxetic acid , mangafodipir ) and superparamagnetic iron oxide ( spio ) agents ( ferumoxides , ferucarbotran ) , which target the kupffer cells and cause a marked signal loss on t2w gre images , are currently available . in terms of lesion detection , metastases are more conspicuous after contrast enhancement because malignant lesions lack functioning hepatocytes or kupffer cells ; whilst lesion signal intensity is unchanged the surrounding liver becomes either hyperintense ( t1 agents ) or hypointense ( spio ) . gadobenate ( gd - bopta , multihance , bracco ) and gadoxetic acid ( gd - eob - dpta , primovist , schering ) have a biphasic enhancement profile . both behave like ecs gd in the first few minutes after injection and exhibit hepatocyte selectivity on delayed phase images . both phases are recommended to maximise sensitivity but the detection of small metastases is better on delayed images than on the early vascular phases ( fig . maximum contrast between normal and abnormal tissue occurs 40120 min and 1040 min after injection of gadobenate and gadoxetic acid respectively . compared with unenhanced images mangafodipir trisodium ( mn - dpdp , teslascan , nycomed amersham ) also improves lesion detection . lesion - to - tumour contrast is maximal between 15 min and 2 h after injection but some metastases may be most conspicuous on 24 h images due to delayed washout around their periphery ( fig . 6 ) . t1w gre imaging is recommended for all three hepatocyte agents . at the time of writing no comparative studies have evaluated the accuracy of these agents using high - resolution 3d t1w gre imaging but it is likely that the better spatial resolution provided by this sequence will improve the detection of sub - cm lesions compared with 2d sequences . mri enhanced with spio is probably the most sensitive method for detecting hepatic metastases . in the few studies which have compared the different liver specific agents against each other , spio - enhanced mri has demonstrated varying degrees of superiority , particularly for small lesions . two spio agents are available for liver mr , ferumoxides ( ami-25 , endorem , guerbet ) and ferucarbotran ( shu 555a , resovist , schering ) . both agents have a particle size of 30200 nm which results in approximately 80% of the injected dose being taken up by the normal liver . when the particles are clustered within the kupffer cells they induce local field inhomogeneities which lead to rapid spin dephasing and a reduction in signal on both t1w and t2w images although the effect is most pronounced on t2w due to a high r2 /r1 ratio . while metastases retain their high si on t2w images the signal of the background liver is dramatically reduced , so tumour - to - liver contrast is markedly increased . signal loss after spio increases with field strength and depends on the type of sequence used . the effect of spio is more pronounced on gre than fse sequences due to the greater sensitivity of gre to susceptibility effects . conversely spio enhancement is less with fse sequences because multiple closely spaced refocusing pulses diminish the local field inhomogeneities induced by the spio particles . mt effects which reduce the si of solid lesions are also a feature of fse but not gre sequences . although the effect of spio is maximised with gre imaging , best results are obtained when parameters are optimised to minimise noise and maximise the signal from solid lesions . in a recent multi - observer study optimised t2w breathhold spio - enhanced fse and gre sequences were compared with unenhanced images , for the detection of surgically confirmed metastases . whilst the best gre sequence achieved accuracies of 93% for all lesions and 82% for sub - cm lesions , enhanced fse was no better than unenhanced images and achieved accuracies of 82% and 64% for all lesions and sub - cm lesions respectively . on the basis of these results the authors now use only this optimised gre sequence with spio . however , in an attempt to improve the detection of sub - cm and surface lesions the sequence has been further refined by reducing the slice thickness from 10 to 6 mm and applying fat suppression ( fig . 7 ) the details of this sequence are as follows : tr 148 ms , te14 ms , fa 30 , bw 6580 hz /pixel , 65% phase resolution combined with a 68%75% rectangular 280400 mm field of view to achieve a 132256 matrix and flow compensation . the low bandwidth is used to minimise noise and increase snr whilst flow compensation gradients minimise flow artifact . the flip angle is based on the ernst angle ( the fa at which maximum signal for any tissue occurs ) for hepatic metastases at 1.5 t and a t1 of 1000 ms . more recently , this spio - enhanced sequence was compared with high - resolution 3d fs t1w dgei and thin - slice contrast - enhanced helical ct for detecting hepatic metastases using histopathology and surgery with ious as the reference standard . overall the two mr techniques showed similar accuracies and both were significantly more accurate than ct , but the detection of lesions 1 cm or smaller was substantially improved with spio . ferucarbotran is given by bolus injection and provides the opportunity to obtain dynamic t1w images in the first few minutes after injection . at this time , because the iron oxide particles are distributed within the intravascular space and less concentrated they produce t1w enhancement on t1w images . liver - to - lesion contrast is maximum on delayed t2w images when the particles are clustered within the re cells and more concentrated , but we have found this early t1 enhancement on 3d fs t1w gre images to be particularly valuable for depicting small tumours . the t1 effect is usually considerably less than occurs with ecf gd agents but this is often beneficial in the context of metastatic disease . liver and vessels often have a similar si which produces a virtual blank canvas against which small metastases are extremely conspicuous and reliably distinguished from vessels ( fig . experience the combination of thin - slice 3d t1w and t2w imaging after spio increases diagnostic confidence and is more accurate for small lesion detection than delayed t2w imaging alone , although there are currently no published data to support this view . we have also found that optimised fs t2w gre imaging after spio is of value in depicting extra - hepatic and peritoneal tumour deposits which are well seen against the suppressed signal of fat and the reduced liver signal after spio ( fig . the most efficient method of fs involves the selective excitation of water protons using a binomial pulse sequence . compared with the standard frequency selective method of fs this approach is less sensitive to magnetic field inhomogeneities and allows more slices to be obtained for a given acquisition time . we have found that , regardless of the position of the imaging slab , manual shimming close to the isocentre achieves homogeneous fs even at more peripheral levels where the main magnetic field is less homogeneous . all liver metastases start out as microscopic seedlings which eventually grow to a size where they become visible on imaging . according to surveillance studies the mean age of synchronous metastases at the time of surgery for the primary tumour is approximately 23 years . most lesions larger than a centimetre are depicted on all imaging techniques but the detection of sub - cm metastases is still disappointing . using optimum technique , high contrast lesions of 23 mm can be detected but metastases ( which have low contrast ) in this size range are rarely visualised . although there is no expectation that current imaging techniques will depict metastases of millimetre size , detection rates of 85%90% are consistently reported for ct and mr . the literature on liver imaging is generally limited by inadequate methods for verifying findings and in most studies false negative lesions are not assessed . this inevitably means that reported sensitivities are overestimated and that the true incidence of disease is underestimated . moreover , in more recent studies investigators have attempted to judge their results against more rigorous reference standards so there has been little if any improvement in apparent sensitivities despite continuing improvements in imaging techniques . it is also likely that these results continue to underestimate the problem of metastases in the millimetre size range and reported sensitivities remain falsely elevated . even when histological examination of the resected liver is used as the gold standard the verification of very small lesions is questionable since most specimens are sectioned at 1 cm intervals . furthermore , recent follow - up studies have confirmed that a proportion of small metastases are undetected by preoperative imaging and surgery with ious . in two studies referred to previously approximately 15% of patients were found to have it is likely that these metastases were present at the time of surgery but missed by preoperative imaging and by surgical inspection with ious . a meticulous correlation with surgery and histology of the resected specimen sectioned at 3 mm intervals showed that 24% and 18% of lesions 1 cm or smaller were not detected by any imaging technique . moreover for the detection of small lesions , the results of both studies compared favourably with those of earlier studies in which a specific analysis of sub - cm lesions was performed . on state - of - the - art systems 3d sequences can achieve full liver coverage with isotropic voxels of 1 mm in a breathhold . however , snr decreases with decreasing voxel size so lesion contrast is degraded by noise . the higher snr provided by 3.0 t technology ( snr at 3.0 t is twice that at 1.5 t ) has the potential to improve contrast resolution but increased power deposition , decreased rf field uniformity and distortion artifacts due to increased susceptibility are problematic . hepatocyte - specific agents used with 3d t1w sequences combine high tissue contrast with improved spatial resolution and may rival spio - enhanced t2w imaging for the detection of small metastases but they are unlikely to outperform spio when high - resolution t1w and t2w images are combined . data on the use of diffusion weighted imaging ( dwi ) for liver lesion detection are limited . variable image quality caused by motion artifacts and reduced snr have largely restricted applications to differentiating benign and malignant lesions ( benign lesions have higher apparent diffusion coefficients than malignant lesions ) . also , most diffusion weighted sequences are acquired with a slice thickness of 78 mm so the detection of small metastases is limited . however , recent work suggests that dwi using small b - values to produce black blood images improves the differentiation of small metastases and vessels on t2w images and is more sensitive than t2w fse sequences for lesion detection . clearly , continuing improvements in imaging are allowing metastases to be identified at an earlier stage but a different approach is needed to improve the detection of metastases smaller than 2 mm . hepatic perfusion indexing ( hpi ) using nuclear medicine and ultrasound techniques has had success in identifying metastases before they become evident on conventional imaging . patients with liver metastases have increased arterial blood flow and a higher arterial /portal ratio ( the hpi ) than those with a normal liver . several animal studies indicate that the increase in hepatic arterial fraction occurs shortly after metastatic seeding and reliably predicts the development of overt metastases . early results in patients were promising but subsequent studies produced varied findings and showed substantial intra - observer variability so neither technique has found acceptance in routine practice . more recently mr has been used to measure hpi but the technique remains developmental and the best measurement method is still to be determined . once the methodology is established rigorous multi - observer studies will be required to validate the technique and determine its impact on patient management . the author s experience has evolved in a centre which provides a supra - regional service for liver surgery . in patients who are candidates for hepatic resection our current practice is to perform unenhanced ip and opt1w gre and haste sequences followed by dynamic spio - enhanced 3d fs t1w gre imaging and delayed t2w gre sequences . if a lesion has benign characteristics on haste and a location which may influence the surgical approach we perform dgei immediately after spio - enhanced imaging for more reliable characterisation . in non - surgical patients or patients with rising tumour the author thanks professor p. j. robinson for his review of the manuscript and helpful comments and mrs s boyes for her help in preparing the manuscript . ( b ) in a second patient also with multiple small metastases , two left lobe lesions ( arrows ) are highly conspicuous on bh stir ( ( c ) and ( d ) ) . both lesions are visible on bh fse ( ( e ) and ( f ) ) and ipt1w ( ( g ) and ( h ) ) but with reduced liver - to - lesion contrast compared with stir . two additional sub - cm lesions ( arrows ) are well seen on spio - enhanced 3d fs t1w ( i ) and t2w gre images ( j ) but only one is seen on the corresponding stir image ( k ) . several metastases larger than 1 cm are well seen on bh t2 fse ( a ) and ipt1w ( b ) images . adjacent thin - slice ( 2.5 mm ) portal phase post - gd 3d fs t1w gre images ( ( c ) and ( d ) ) obtained at the same level as ( ( a ) and ( b ) ) clearly show several additional previously undetected sub - cm metastases . characteristic continuous rim enhancement is clearly seen on arterial phase post - gd 3d fs t1w gre images ( a ) . the lesions are still conspicuous by the portal phase ( b ) but the enhancing rim is less apparent and the lesions appear smaller . role of gd - enhanced delayed imaging with fat suppression for detecting small metastases on the liver surface . in a patient with colorectal cancer small surface deposits ( arrows ) are well seen on 3d fs t1w gre images obtained approximately 10 min after gd ( ( a ) and ( b ) ) . the lesions are not visible on the earlier portal phase images ( ( c ) and ( d ) ) . surface lesions are also highly conspicuous on fs t2w gre images following spio ( ( e ) and ( f ) ) . multiple metastases improved detection with gd - eob - dtpa at the hepatocyte phase of enhancement . compared with non - contrast t1w images ( a ) liver - to - lesion contrast is improved on 20 min post - contrast t1w images ( b ) . additional surgically confirmed sub - cm lesions ( arrows ) were only visible on hepatocyte phase images ( ( c ) and ( d ) ) . adapted with permission from robinson pja , ward j ( 2006 ) mri of the liver : a practical guide . multiple metastases ( arrows ) are seen with high lesion - to - liver contrast on 20 min post - mangafodipir t1w 2d gre images ( a ) . however several additional lesions are only visible on the corresponding images obtained 24 h after contrast ( b ) when the background liver signal has returned to normal , due to retained contrast in the compressed liver tissue at the periphery of the lesions . improved detection of small metastases with optimised spio - enhanced t2w gre imaging . in a patient with colorectal metastases and a fatty liver , right and left lobe lesions ( arrows ) are well seen on haste ( a ) and ipt1w ( b ) images . the lesions are isointense against the reduced signal of the adjacent fatty liver on opt1w ( c ) . additional small metastases ( arrows ) not seen on ( a c ) are clearly seen on spio - enhanced t2w gre images ( d ) acquired with a 6 mm slice thickness and fat suppression . multiple small metastases ( many not visible on unenhanced images ) are highly conspicuous on 3d fs t1w gre imaging ( effective slice thickness 2.5 mm ) obtained 45 s after bolus injection of ferucarbotran ( ( a ) and ( b ) ) . note the relatively weak t1 effect resulting in isointensity of the background liver and vessels . the lesions are also well seen on corresponding t2w gre images ( c e ) obtained 10 minutes after ( a ) and ( b ) .
it is well established that hepatic resection improves the long - term prognosis of many patients with liver metastases . however , incomplete resection does not prolong survival , so knowledge of the exact extent of intra - hepatic disease is crucially important in determining patient management and outcome . mr imaging is well recognised as one of the most sensitive methods for detecting metastases . recent developments in gradient coil design , the use of body phased array coils and the availability of novel mr contrast agents have resulted in mr being recognised as the pre - operative standard in this group of patients . however , diagnostic efficacy is extremely dependent on the choice and optimisation of pulse sequences and the appropriate use of mr contrast agents . this article reviews current mr imaging techniques for the detection and characterisation of metastases and discusses the relative capability of different techniques for detecting small lesions .
Introduction Unenhanced sequences Dynamic gadolinium-enhanced imaging (DGEI) Tissue-specific contrast agents The true accuracy of imaging and future developments Recommendations Acknowledgements Figures and Tables
in patients with liver metastases from colorectal cancer hepatic resection has proven survival benefits and is curative in a small proportion of cases . survival benefits are also well established in patients with neuroendocrine metastases and in a minority of patients with localised metastases from other primary sites which have a more indolent course . treatment success , however , depends on the removal of all sites of intra - hepatic disease and the absence of disease outside the liver ; incomplete resection does not prolong survival . consequently , more patients are being referred for preoperative assessment and , since many have multiple small lesions , the role of imaging is increasingly challenging . although the primary modalities for liver imaging are ultrasound and ct , recent studies have suggested that contrast - enhanced mri is the most sensitive method for detecting small metastases and mri is now considered the preoperative standard . moreover , recent developments in mri hardware and software and the availability of novel mri contrast agents have improved small lesion detection . this article considers the current status of mr imaging techniques in the detection and characterisation of liver metastases and discusses the technical requirements for optimum performance . the accuracy and relative capability of various mr techniques for detecting sub - cm lesions is emphasised . mr performance is primarily dependent on technique and the optimisation of pulse sequences . this is particularly important when patients are being considered for hepatic resection since fatty change the fatty liver has a clearly reduced si on opt1w compared with ipt1w images , so whilst the detection and characterisation of liver lesions by ultrasound and ct is handicapped by fatty change , chemical shift mr imaging allows accurate differentiation of metastases and focal fatty change or focal sparing in a fatty liver . fse is insensitive to susceptibility so liver signal is slightly higher than with conventional spin echo imaging ; magnetisation transfer ( mt ) effects which cause solid lesions to lose si are particularly marked on multi - slice fse sequences and become more pronounced with increasing echo train length ; and fse is also subject to blurring of short t2 tissues because most of the high spatial frequencies are collected at the later echoes when there is relatively little remaining signal . of the unenhanced sequences , breathhold stir is probably the most sensitive for depicting metastases although image quality is variable . fat suppression not only accentuates gadolinium enhancement but also suppresses the competing high signal of intra - abdominal fat adjacent to the liver surface . in patients with metastatic disease , however , the main role of dgei is probably to differentiate benign and malignant lesions . all liver - specific agents produce high tissue contrast and have been shown significantly to improve the detection of metastases compared with unenhanced mr , with enhanced mr using ecs agents with 2d gre sequences and with contrast - enhanced ct . mri enhanced with spio is probably the most sensitive method for detecting hepatic metastases . both agents have a particle size of 30200 nm which results in approximately 80% of the injected dose being taken up by the normal liver . while metastases retain their high si on t2w images the signal of the background liver is dramatically reduced , so tumour - to - liver contrast is markedly increased . more recently , this spio - enhanced sequence was compared with high - resolution 3d fs t1w dgei and thin - slice contrast - enhanced helical ct for detecting hepatic metastases using histopathology and surgery with ious as the reference standard . the literature on liver imaging is generally limited by inadequate methods for verifying findings and in most studies false negative lesions are not assessed . it is also likely that these results continue to underestimate the problem of metastases in the millimetre size range and reported sensitivities remain falsely elevated . even when histological examination of the resected liver is used as the gold standard the verification of very small lesions is questionable since most specimens are sectioned at 1 cm intervals . moreover for the detection of small lesions , the results of both studies compared favourably with those of earlier studies in which a specific analysis of sub - cm lesions was performed . data on the use of diffusion weighted imaging ( dwi ) for liver lesion detection are limited . however , recent work suggests that dwi using small b - values to produce black blood images improves the differentiation of small metastases and vessels on t2w images and is more sensitive than t2w fse sequences for lesion detection . patients with liver metastases have increased arterial blood flow and a higher arterial /portal ratio ( the hpi ) than those with a normal liver . in non - surgical patients or patients with rising tumour the author thanks professor p. j. robinson for his review of the manuscript and helpful comments and mrs s boyes for her help in preparing the manuscript . role of gd - enhanced delayed imaging with fat suppression for detecting small metastases on the liver surface . however several additional lesions are only visible on the corresponding images obtained 24 h after contrast ( b ) when the background liver signal has returned to normal , due to retained contrast in the compressed liver tissue at the periphery of the lesions .
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prostate cancer is the second leading cause of cancer - related deaths in american men.1 , 2 although hormonal and radiation therapy can be very effective for local disease , patients usually become refractory to hormonal therapy ( castration resistant ) within 13 years . this is usually associated with the transition to a more aggressive form of the disease , leading to the development of bone and organ metastases . the addition of combination chemotherapy in the late stages of the disease has limited benefit , increasing survival for only several months . these therapeutic strategies have employed oncolytic viruses , vaccines , adjuvant immune modulation therapies ( checkpoint inhibitors ) ( slovin et al . , 2012 , j. clin . oncol , abstract ) , and adoptive immune cell ( t cell ) therapies . during the past 15 years , genetic engineering has been applied to more effectively direct t cells to tumor - expressing antigens , through the expression of specific chimeric antigen receptors ( cars ) on an individual patient s t cells.8 , 9 , 10 cars consist of a tumor antigen - binding domain that is fused to an intracellular signaling domains and costimulatory receptors capable of activating t cells.10 , 11 , 12 therefore , antigen - recognition is not mhc - restricted , as is the case for t cell receptor ( tcr)-mediated antigen recognition . in vivo efficacy of car - modified effector human and murine t cells has been demonstrated,13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 and prostate - specific membrane antigen ( psma ) is a promising molecular marker for targeted therapy of prostate cancer . psma is a glycosylated type - ii membrane protein that is upregulated during malignant transformation in more aggressive prostate cancer , resulting in abnormally high levels of it on the cell surface . we24 , 25 and others21 , 26 , 27 , 28 ( slovin et al . , 2012 , j. clin . oncol , abstract ) have participated in developing imaging approaches to visualize anti - hpsma car t cells and optimization of the structure of cars to achieve more profound effects in the targeting of tumors bearing the corresponding antigen ( hpsma ) . car t cell therapy in solid tumors has not achieved the clinical success that has been observed in hematologic malignancies.29 , 30 , 31 one reason for the poor treatment response is the failure of car t cells to accumulate and expand in the hostile tumor microenvironment.32 , 33 the failure of a substantial car - mediated t cell response in solid tumors relates to a number of factors including car t cell inactivation and possible exclusion from the tumor mass , the reciprocal interactions between tumor and stromal cells,34 , 35 , 36 and propensity of cancer like prostate to disseminate preferentially to bone . thus , preclinical studies that incorporate imaging to monitor t cell trafficking and activation are necessary to adequately explore the biology and efficacy of different treatment strategies designed to enhance t cell targeting and penetration of solid tumors . several strategies have been employed to optimize the migration , survival , and effector functions of adoptive cell therapy ( act ) using tils ( tumor - infiltrated lymphocytes ) . for example , ( 1 ) increasing cxcr2 expression resulted in the improvement of t cell migration to tumors , ( 2 ) genetic manipulation of il-12 production by transferred t cells extends t cell survival , and ( 3 ) the generation of novel car - based engineered t cells to improve tumor recognition and t cell activation39 , 40 as well as the delivery of car t cells through specific polymer implants or local injection . recently , it was demonstrated that car t cell therapy and programmed cell death protein 1 ( pd-1 ) checkpoint blockade are a rational combination in a solid tumor model . programmed death - ligand 1 ( pd - l1)/pd-1-mediated t cell inhibition is involved in immune evasion in prostate cancer . although only 1% pd - l1 cells were detected in prostate tumor samples,42 , 43 immune cells ( pd-1- and pd - l1-positive ) were observed to surround prostate tumor nodules . more recently , a novel monoclonal rabbit antibody to pd - l1 revealed that 62% human prostate tumors stain positive for pd - l1 expression . the possibility that pd - l1/pd1-mediated t cell inhibition might be involved in immune evasion in prostate cancer is being explored in several clinical trials . patients with metastatic castration - resistant prostate cancer ( mcrpc ) are being treated with anti - pd-1 antibody in two phase ii clinical trials , involving pembrolizumab ( keytruda ) and ct-011 ( anti - pd-1 antibody ; curetech).46 , 47 the goal of this study was to study the efficacy of human hpsma car - directed t cell therapy in an appropriate animal model . we show that anti - hpsma car - directed t cell therapy of myc - cap : psma(+ ) tumors alone was unsuccessful , whereas the combination of anti - hpsma car - directed t cells plus anti - hpd1 mab immune modulation therapy provided a partial , short - duration and sub - optimal response . several prostate tumor models , both human and murine origins , have been described and used to study the targeting of hpsma - car t cells , but some limitations have been identified.21 , 24 , 48 , 49 we focused on murine cell lines , with prospective to utilize them in a syngeneic mouse model using immunocompetent mice . we chose a well established and studied murine prostate cancer cell line , myc - cap , for our initial studies . myc - cap tumors are composed of sheets and indistinct lobules / nests of polygonal to oval cells separated by fine fibrovascular straie , as described previously . necrosis is rare in small tumors ; larger tumors have more extensive necrosis . the percentage of necrosis varied from 2% to 25% , depending on the overall tumor mass ( figure 1 ) . to generate an appropriate murine model for the anti - hpsma car t cell therapy , wild - type ( wt ) myc - cap cancer cells were stably transduced with a hpsma - containing retroviral vector and sorted as described in materials and methods ( figure s1a).24 , 51 , 52 we compared the growth profiles of wild - type and hpsma(+ ) myc - cap tumors in mice , and found no significant difference in tumor doubling time , morphology , extent of necrosis ( figure 1a ) , or level of tumor hypoxia ( figure 1b ) . the only difference was that myc - cap : hpsma(+ ) tumors stained strongly positive for hpsma , whereas wt myc - cap : hpsma( ) tumors did not ( figure 1b ) . t cells derived from human blood were isolated , activated , and transduced with a newly developed retroviral second - generation anti - hpsma car plg28z vector and with sfg - tdrfp / cbrluc vector . reproducible levels of hpsma car expression on transduced human t cells were observed ( figure s1b).24 , 53 cytotoxicity studies revealed that hpsma targeted car t cells have a 13.8 + 5.9% cytolytic efficacy against myc - cap : hpsma(+ ) cells , when the ratio of the effector to the target is 40 to 1 . however , the same hpsma - targeted car t cells showed 2-fold higher cytotoxic activity toward a human prostate cancer cell line pc3/psma - ires - puromycin ( pc3-pip ) ( figure s1c ) . using two distinct luciferase reporter systems , we were able to image the location of myc - cap tumors with the renilla luciferase ( rluc ) reporter and image the trafficking of anti - hpsma car t cells with the tdrfp / cbrluc reporter . first , we assessed the capacity of systemically administered anti - hpsma car t cells to traffic and accumulate within established subcutaneous myc - cap : hpsma(+ ) tumors . when tumors reached 50100 mm size , mice were injected intravenously with 20 10 anti - hpsma car t cells ( 87% of t cells had anti - hpsma car expression , and 67% of car - positive t cells were also positive for the tdrfp / cbrluc fusion reporter ) . injection of anti - hpsma car t cells was performed on 0 , 1 , 5 , 9 , and 12 days after administration . initially , car t cells sequestered in the lungs for up to 910 days , as visualized by bli ( figure s2a ) . their presence in lungs was confirmed by cd3 immunohistochemistry ( ihc ) staining ( figure s2b ) . a minimal bli signal was observed within myc - cap : hpsma(+ ) tumors during the first 48 hr , suggesting that some car t cells trafficked to the tumor . however , only a minimal ( non - statistically - significant ) response of anti - hpsma car t cell therapy was observed on tumor growth / volume , compared with control ( no car - t cells treated ) tumors ( figure s2c ) . to further evaluate in vivo therapeutic efficacy of anti - hpsma car - transduced t cells anti - hpsma car t cells alone or mixed with myc - cap : hpsma(+ ) or myc - cap : hpsma( ) target cells were prepared just prior to subcutaneous inoculation into nod.scid il2rg/ ( nsg ) mice ; the inoculum included a 1:10 tumor - to - t cell ratio , mixed in growth media and matrigel matrix ( 1:1 ) at 4c . the location of the tumor was identified using renilla luciferase bli ( figure 2a ) . we also monitored tumor growth by bli ( figure 2c ) ( in addition to caliper measurements ; figure 2d ) until the point where the tumors reached a size where the bli renilla luciferase signal was saturated and tumors showed evidence of necrosis ( day 15 ; figure 2c ) . progressive tumor growth was observed in the two control groups of mice : ( 1 ) mice injected with myc - cap : hpsma(+ ) tumors alone ( without car t cells ) and ( 2 ) myc - cap : hpsma( ) tumors ( without hpsma expression ) but mixed with anti - hpsma car t cells ( figures 2a , 2c , 2d ) . a marked reduction in tumor growth rate was observed in the test group of mice : tumors that developed from the mixture of myc - cap : hpsma(+ ) cells and anti - hpsma car t cells ( figures 2a , 2c , 2d ) . by day 26 , the test group tumor size was 224 240 mm , whereas the size of control tumors without anti - hpsma car t cell inclusion was significantly larger , 1,676 406 mm ( p < 0.05 ) . ( winn assay ) experiments clearly demonstrate that anti - hpsma car t cells can inhibit myc - cap : hpsma(+ ) tumor growth and that this inhibition is hpsma dependent . the persistence of anti - hpsma - targeted car t cells in the same winn assay described above ; t cells were monitored by the cbrluc / luciferin bli signal generated from the reporter - transduced car t cells ( figures 2b and 2e ) . we observed that anti - hpsma car t cells injected alone in matrigel matrix ( control , in the absence of any tumor cells ) were localized at the site of injection and were visualized over 8 days . however , the presence myc - cap tumor cells ( with or without human psma / antigen ) notably influenced the bli signal intensity at the injection site . since the intensity of the bli signal has been used as indicator of the injected car t cells , the difference in bli signal suggested that the survival of car t cells was different in the three study groups . the bli signal from car t cells was visualized longest ( 8 days ) when they were injected alone ( non - tumor group ) , less in the hpsma( ) tumor group ( 6 days ) , and least in the hpsma(+ ) tumor group ( 4 days ) . these data suggest that bli detection and the survival of car t cells was shortened by the presence of hpsma(+ ) on tumor cells . it is known that t cells undergo a transition from a quiescent to a highly active effector phenotype upon stimulation / activation and transduction with the anti - hpsma car vector . this transition also involves a significant shift from oxidative to glycolytic metabolism . to study the metabolic status of t cells during activation , transduction , and proliferation , we measured the extracellular acidification rate ( ecar ) and the oxygen consumption rate ( ocr ) of these cells . the rate of glycolysis ( ecar ) , as well as basal and maximal ocrs ( an indicator of mitochondrial respiration ) , was assessed on days 2 , 8 , 10 , and 15 of car t cell preparation ( figure 3 ) . human t cells from three donors were isolated from buffy coat using ficoll separation . forty - eight hours after phytohemagglutinin ( pha ) stimulation , cells were transduced with a retroviral vector bearing a car targeting hpsma . we obtained a measure of ecar in naive t cells , t cells stimulated by pha and hpsma car - transduced t cells ( figure 3a ) . naive non - stimulated t cells demonstrated low levels of glycolysis , whereas non - specific pha stimulation of t cells results in a significant ( p < 0.05 ) increase in glycolysis , that is maintained at high levels over the course of t cell transduction and expansion ( figure 3a ) . the initial increase in a basal mitochondrial respiration after non - specific pha stimulation is followed by a progressive decline in mitochondrial function during subsequent procedures ( figure 3b ) . fccp ( carbonyl cyanide p - trifluoromethoxy phenylhydrazone ) was added to uncouple oxidative phosphorylation from the electron transport chain to measure the maximum respiratory capacity . the maximum respiratory rate ( ocr ) was significantly higher at day 2 after pha stimulation but declined by day 8 ( figure 3c ) . similar results were obtained for car - transduced t cells , showing low mitochondrial function following anti - hpsma car transduction ( figures 3b and 3c ) . to assess the associations between pd - l1/pd-1 signaling and t cell targeting of myc - cap tumors , and the impact on tumor progression , we performed immunofluorescence staining for pd - l1 in ( 1 ) myc - cap : hpsma(+ ) tumors growing in nod.scid mice and ( 2 ) myc - cap wt tumors growing in immune - competent fvb / n . both tumors stained positive for pd - l1 ( figure 4a ) . the spatial distribution of cd3 t cells in myc - cap wt tumors ( growing in fvb / n mice ) was also examined ; a predominance of t cells was observed along the invasive tumor margin ( stromal - tumor edge ) , with reduced numbers in the center of the tumor ( figures 4b and 5c , upper panel ) . we then evaluated whether anti - murine programmed death - ligand 1 ( mpd-1 ) mab treatment increased the number of tumor - infiltrating lymphocytes in subcutaneous myc - cap tumors ( fvb / n mice ) . we administrated 5 doses of 200 g of anti - mpd1 mab per mouse ( on days 9 , 11 , 13 , 15 , and 17 after tumor cell injection ) and observed a significant delay ( p < 0.05 ) in tumor growth ( figure 5a ) , which continued even after mice stopped receiving anti - mpd1 therapy . interestingly , tumors < 50 mm responded to the anti - mpd1 mab treatment , whereas larger tumors failed to respond to the treatment ( figure 5a ) . the density of cd3 t cells per square millimeter of tumor area ( figures 5b and 5d ) , t cell size ( figure 5d ) and the correlation between these two parameters and t cells distribution along the tumor edge was evaluated ( figure 5c ) . a marked increase ( 13-fold ) in cd3 t cell density was observed in central tumor areas following anti - mpd1 therapy ( figure 5d ) . we also noticed that t cell size increased in responder tumors from 40 15 m ( igg treated ) to 51 23 m ( anti - mpd1 treated ) but not in the non - responding tumor ( 42 15 m ) . a comparison of cd3 t cell distribution over the tumor sections showed that cd3 t cells predominantly localized along the rim of tumor nodules prior to and following anti - pd1 treatment , even though the restriction of cd3 t cells from the center of the tumor nodules was reversed ( figure 5c ) . no significant difference was observed in cd31 blood vessel staining of the tumors ( data not shown ) . encouraged by the response of myc - cap wt tumors to anti - mpd1 mab treatment in fvb / n mice , we evaluated the effect of anti - hpd1 treatment combined with anti - hpsma car t cell adoptive therapy in the myc - cap : hpsma(+ ) and myc - cap : hpsma( ) tumor models . anti - hpsma car t cells ( transduced with the cbrluc reporter ) were monitored by bli at different time points after i.v . administration ( days 0 , 1 , and 6 ) ( figure 6b ) . ten minutes following car t cell injection , the t cells were localized largely in the lungs of all animals , with a variable low intensity signal appearing in the area of the tumors ( figure 6b ) . interestingly , the highest tumor - associated t cell bli signal was observed in the myc - cap : hpsma( ) tumors ( anti - hpd1-treated control group ) , and the lowest signal was observed in the myc - cap : hpsma(+ ) tumors ( anti - hpd1-treated test group ) . nevertheless , myc - cap : hpsma(+ ) tumor - bearing mice receiving the combined treatment ( both anti - hpsma car t cells ( 10 10 ) and anti - hpd-1 mab ; test group ) had a significant ( p = 0.03 , p = 0.04 ) treatment response ( tumor volume ) , when compared to the two control groups ( figures 6c and 6d ) . the absence of a treatment response in myc - cap : psma( ) tumors ( anti - hpd1 mab - treated control group ) , despite the more robust t cell trafficking - to and bli signal - in the tumor region is of particular interest . a comparison of car t cell bli signals from tumors and lungs during the first 24 hr showed that the bli signal in the tumors was more stable ( figure s3a ) compared with that in the lungs ( figure s3b ) , where a decline in signal intensity was observed in all treatment groups . by day 6 after anti - hpsma car t cell injection , there was near total fading of the bli signal from the lungs , whereas a longer - lasting bli signal in the tumor areas was observed ( figure 6b ) . following the cessation of anti - hpd1 treatment ( day 20 ) , we observed an increase in tumor growth of hpsma(+ ) positive tumors treated with both anti - hpd1 mab ( five doses ) and anti - hpsma car t cells ( one dose injection ) , comparable with other groups of mice ( figure 6c ) . staining of one tumor from each treatment group shows different levels of necrosis 24 hr after initiation of treatment ( figure 7 ) . hpsma(+ ) tumors treated with both anti - hpd1 mab and anti - hpsma car t cells ( test ) showed the most necrosis ( figure 7a ) , whereas both control tumors showed considerably less necrosis ( figures 7b and 7c ) . the density of tunel positive cells showed a similar pattern ; 54 tunel ( + ) cells / mm were detected for the test group ( hpsma(+ ) , anti - hpd1 ) , whereas 29 and 26 tunel cells were observed in the anti - igg , hpsma(+ ) and anti - hpd1 , psma( ) control groups , respectively ( figure 7 , far right column ) . the distribution and density of anti - hpsma car t cells was higher in the center of a hpsma(+ ) tumor treated with anti - hpd1 mab at day 6 compared to controls ( figures s4 and s5 ) , although more car t cells were localized along the periphery of these tumors ( figure s4 ) . in contrast , cd31 staining of the tumors following 6 days of combined treatment showed no difference in microvessel density ( figure s5 ) . car t cell therapy for hematologic malignancies has shown some remarkable success in recent years.29 , 30 , 39 , 57 , 58 we focused on hpsma targeting in prostate tumors using human anti - hpsma car t cells , because of an existing clinical trial at our institution ( mskcc ) ( clinical trial # nct01140373 , https://clinicaltrials.gov ) ( slovin et al . , 2013,j . it has been shown that adoptive transfer of t cells modified with the anti - hpsma car could specifically mediate regression of pulmonary experimental lung metastasis in scid mice.21 , 24 , 49 however , this approach was ineffective against established subcutaneous tumors , unless the car t cells were injected locally . to evaluate the effectiveness of second - generation anti - hpsma car t cells in a pre - clinical setting , we developed the myc - cap ( hpsma ) tumor model in immune deficient mice ( nod.scid il2rg , nsg ) . the myc - cap murine prostate cancer model was chosen to investigate hpsma - directed adoptive car t cell therapy for the following reasons : ( 1 ) myc - cap s.c . tumors are slow - growing ( allowing time for immune targeted therapy ) and can be used in a syngeneic mouse model using immune competent mice ; ( 2 ) moderate size tumors have little necrosis ( reducing confounding factors in the interpretation of results ) ; ( 3 ) a genetically modified myc - cap cell line with high cell - membrane localized psma expression was developed to allow direct comparisons between psma(+ ) and psma( ) tumors ; ( 4 ) both psma(+ ) and psma( ) tumors had very similar growth and morphological characteristics ( figure 1 ) . this study focuses on the potential for enhancing the efficacy of hpsma car - directed t cell therapy using an immune checkpoint inhibitor ( pd-1l / pd1 blockade ) just prior to i.v . our initial efforts to evaluate the effectiveness of second - generation anti - hpsma car t cells24 , 53 were comparable to that of others ( figure s2 ) . namely , anti - hpsma car t cell therapy alone was not effective against established subcutaneous myc - cap hpsma(+ ) tumors . nevertheless , a winn assay demonstrated that anti - hpsma car t cells can inhibit myc - cap : hpsma(+ ) tumor growth in a scarce nutrient microenvironment , and that this inhibition is hpsma dependent . however , myc - cap : hpsma(+ ) tumors re - grew in 3 weeks after implantation ( figures 2c and 2d ) . the finding that myc - cap tumors are pd - l1 positive in both an immunocompromised and immunocompetent mice raised the question whether the restriction of cd3 t cells from the interior of myc - cap wt tumors in fvb / n mice was related to pd - l1/pd1 engagement ( figure 4b ) . a similar exclusion of cd3 t cells was described in metastatic melanoma and other types of cancer.60 , 61 given that pd-1 receptor expression is increased on activated t cells following engagement with pd - l1 ligand and high pd - l1/pd1 expression is associated with t cell exhaustion,59 , 62 we treated myc - cap wt tumors in fvb / n with anti - mpd1 mab and observed a significant , but only a partial delay in tumor growth ( figure 5a ) . although based on a small number of animals ( n = 5 ) , we observed a 13-fold increase in intratumoral cd3 t cell density following anti - pd1 inhibition ( figure 5b ) , consistent with releasing the pd-1 immune checkpoint and leading to t cell proliferation , intratumoral infiltration and increased effector function . we also observed that t cells changed their morphology and increased in size following anti - mpd1 mab treatment , consistent with cell cycle activation and proliferation.64 , 65 although this suggests that anti - mpd1 mab treatment resulted in increased cd3 t cell activation , proliferation , and effector functions , the response was limited and indicates other factors are involved . nevertheless , the encouraging response of wild - type myc - cap tumors to anti - mpd1 mab treatment in immune competent fvb / n mice led us to evaluate the effect of anti - hpd1 mab treatment in combination with hpsma - targeted car t cells in the myc - cap : hpsma(+ ) tumor model . in these adoptive car t cell experiments , we first showed that combined treatment with anti - hpd1 antibody significantly inhibited myc - cap : hpsma(+ ) tumor growth ( figure 6c ) and that the antitumor response mediated by this combined therapy was both anti - hpd1 and hpsma specific . namely , the igg - treated control group did not show an inhibitory effect , nor did tumors that were hpsma( ) . the blocking of hpd-1 enhanced the anti - tumor effect of hpsma - targeted car t cells ( figure 6c ) , even in the presence of murine pd - l1 in the target tumors ( figure 4 ) . the treatment response was confirmed by h&e and tunel staining of the tumor 24 hr after initiation of treatment ( figure 7a ) . these results suggest that there is a direct impact of pd-1 blockade on car t cell cytolytic function in these tumors . anti - pd1 mab treatment of myc - cap wt tumors growing in immune - competent fvb / n mice had a more profound and prolonged effect , even following treatment withdrawal ( figure 5a ) . this effect was greater than a single injection of anti - hpsma car t cells and five doses of anti - hpd1 mab treatment of myc - cap : hpsma(+ ) tumors growing in nsg mice ( tumor growth was delayed for only 10 days ) . we suggest that anti - hpd1 antibody treatment combined with anti - hpsma car human t cells had enhanced killing capacity and cytokine function against myc - cap : hpsma(+ ) tumors , although the treatment response was comparatively short in duration . recent literature40 , 66 demonstrates that adoptive immunotherapy using genetically modified t cells in combination with pd-1 checkpoint blockade can enhance the antitumor effects of car t cells against established subcutaneous tumors in an immune compromised host . nevertheless , our results indicate that other immune modulation mechanisms exist and restrict car t cell targeting , function , and persistence in hpsma expressing myc - cap tumors administered car t cells is well known.49 , 67 nevertheless , the persistence of car t cells is well documented in the treatment of many liquid tumors,68 , 69 , 70 whereas the persistence of car t cells is not a common finding in the treatment of solid tumors . previous studies have shown that the therapeutic efficacy of adoptive t cell transfer is correlated with the ability of t cells to proliferate and survive in vivo . to address this issue , we used bioluminescence reporter - gene imaging ( bli ) and immunofluorescence staining to track adoptively administered car t cells . in our bli studies , there was a clear difference in anti - hpsma car t cell trafficking to and initial persistence in myc - cap tumors ( with and without the presence of the target antigen - hpsma ) , even during the first minutes after t cells injection ( figures 6a and 6b ) . this difference in bli signal was present over days 0 , 1 , and 6 following car t cells injection ( figure 6b ) . the seemingly contradictory t cell bli and treatment response observations ( figures 2b , 2e , 6b , and 6c ) and inconsistencies between t cell bli ( figure 6 ) and cd3 staining for t lymphocytes ( figure s5 ) , can be explained in several ways . we performed our experiments thinking that cbrluciferase ( cbrluc ) bli could be used as a readout of car t cell number and persistence . to explain the above observations , we assumed the anti - hpsma car t cells were undergoing more rapid destruction in hpsma(+ ) myc - cap tumors compared to hpsma( ) tumors , since the t cells were bearing a second generation car . to confirm this hypothesis , we detected more apoptotic car t cells in co - culture experiments with hpsma(+ ) , than with hpsma( ) myc - cap tumor cells ( data not shown ) . anti - hpsma car t cells in the absence of the hpsma are likely to remain in a non - activated ( anergic ) state and not be subject to psma antigen - induced activation leading to t cell death / destruction . quiescent t cells could survive for a longer period of time and be visualized by bli . an additional and more plausible explanation is that following contact with tumor cell membrane - localized hpsma , the anti - hpsma car t cells undergo a transition from a quiescent to a highly active effector phenotype . this transition also leads to a significant shift in the metabolism of hpsma car t cells , from a tca cycle and oxidative phosphorylation oxyphos - based metabolism , to a more glycolysis - dependent metabolism to support macromolecule synthesis , proliferation , and effector function ) . these changes in the metabolism of car t cells transitioning from a quiescent to an activated state could directly affect cbrluc bli signal intensity . recently , it was observed that activated tumor - infiltrating t cells display a phenotype of metabolic insufficiency , characterized by a persistent loss of mitochondrial function and mass . additionally , it was shown that chronic activation of t cells ( associated with an anti - cancer response ) represses oxidative metabolism , concomitant with a loss of mitochondrial mass and function , and that a significant decrease / loss of atp was observed in activated cd8 t cells . in light of these observations and the data provided in figure 3 showing lower mitochondrial activity in car t cells , we suggest that activated t cells can be associated with a low bli readout ( photons ) using luciferin - luciferase - based reporter systems . the click beetle red luciferase ( cbrluc ) reporter is dependent on the presence of intra - cellular atp , since this reporter belongs to the class of oxidative enzymes catalyzing the reaction of luciferin+atp leading to formation of oxyluciferin , amp and light . therefore , the cbrluc reporter ( and other atp - dependent luciferases , including firefly luciferase ) is dependent on the presence of saturable amounts of atp for reliable bli measurements of reporter - cell number and persistence . interestingly , several other investigators have found that firefly luciferase is not an optimal reporter for tracking activated t cells78 , 79 ( as well as unpublished conversations with other investigators ) . first is the very short life of anti - hpsma car t cells in the presence of hpsma(+ ) myc - cap cells / tumors compared with hpsma( ) cells / tumors , which leads to their longer persistence in the winn assay ( control group , hpsma( ) tumors ) . second is a modest cytotoxicity of car t cells ( figure s1 ) to murine cancer cells bearing hpsma , which leads to myc - cap : hpsma(+ ) tumor progression after 3 weeks in the winn assay ( figures 2c and 2d ) compared with total eradication of pc3/pip tumors ( a human prostate tumor genetically modified to overexpress hpsma ) . the combination human car t cells ( injected i.v . ) and a murine prostate tumor transduced to express human psma may not be an optimal model to explore car t cell therapy . however , there is the potential for interaction between anti - hpd1 abs with mpdl-1 , since pd1 shares 64% of protein identity between murine and human species , and pd - l1 shares 77% identity.80 , 81 murine pd-1 binds in vitro to both murine and human pd - l1 , and human pd-1 binds to the pd - l1 of each species . the structures of the murine pd-1 and human pd - l1 complexes and that of the murine pd-1 and murine pd - l2 have been published . despite the high homology and the ability for binding between different species , interspecies differences are responsible for the variation in the affinity of human and mouse pd-1 for their ligands . since human pd-1 is relatively flexible , consistent with prior studies,40 , 85 we were able to enhance car t cells therapy when combined with pd-1 blockade . however , the treatment response was only partial , of short - duration and sub - optimal . other second generation cars have been shown to be as or more effective , including the cd28- and 4 - 1bb - based mesothelin - targeted car t cells that achieved tumor eradication , but only following direct intra - pleural administration . we suggest that the limited response we observed also suggests that there are other mechanisms restricting car t cell trafficking and function in prostate solid tumors , which may extend to other solid tumors as well . we show that anti - hpsma - directed car t cell monotherapy of subcutaneous myc - cap : psma(+ ) tumors is ineffective , whereas the combination of anti - hpsma - directed car t cells plus anti - hpd1 mab immune modulation provides a short - duration , sub - optimal treatment response . these results also suggest that other immune modulation mechanisms need to be brought into play to further reverse the restriction to car t cell targeting and persistence in hpsma expressing myc - cap tumors and to provide optimal car t cell therapy . the results also suggest that atp - dependent luciferin - luciferase bioluminescence reporters should be used with caution in the monitoring of t cell trafficking and persistence , particularly when t cells transition to an activated state . the myc - cap androgen - dependent prostate cancer cell line , derived from a c - myc transgenic mouse with prostate cancer , was provided by dr . charles sawyers and was cultured in dmem media supplemented with 10% fbs , 4 mm glutamine , and 5 mm glucose . myc - cap cancer cells were transduced with a newly generated vector sfg - hpsma . a transgene containing human psma complementary dna ( cdna ) was amplified from total mrna derived from human prostate cancer cell line lncap using 5hpsma 5-acatgtggaatctccttcacgaaac-3 and 5-ggatcctcgagcttaggctacttcactcaaag-3 primers set . human psma cdna was cloned into the sfg - based retroviral vector.24 , 51 , 53 human psma expression was assessed using anti - human psma rat antibody as described previously and cells were sorted using the fluorescence - activated cell sorter ( facs ) ( bd bioscience , ca , usa ) several times to achieve a 100% hpsma - positive population . additionally , myc - cap : hpsma(+ ) and myc - cap : hpsma( ) cells were transduced with a sfg - rluc - ires - gfp vector to detect tumor location and its relative borders . a new sfg - tdrfp / cbrluc ( rfp / cbr ) retroviral vector was obtained by subcloning click beetle red luciferase ( cbrluc ) cdna from the pcbr basic vector ( promega ) into the sfg - tdrfp / renilla luciferase ( rfp / rluc ) retroviral vector by replacing the renilla luciferase gene . a new hpsma - specific car retroviral vector named sfg - pig28z was developed by inserting a ch2-ch3 domain from the human igg heavy chain in the noti restriction site between the anti - hpsma scfv and cd28 signaling motif in the sfg - p28z vector . it was performed for better detectability by facs staining with anti - human igg antibody which is specific for the inserted region ( # 2040 - 08 ; southern biotechnology associates ) . for transduction we have used the pg13 producer cell lines , bearing anti - hpsma car and sfg - tdrfp / cbrluc vectors . retroviral particles were obtained using the gpg29 ( h29 ) producer cell line and were used to infect target cells . cells were stably transduced by incubating 50% confluent cell cultures with virus - containing medium for 12 hr in presence of polybrene ( 8 g / ml ; sigma - aldrich ) . cells were sorted using facs ( bd biosciences ) using gfp or tdrfp as fluorescence markers . sfg - pig28z- and sfg - tdrfp / cbrluc- retroviral supernatants were produced as described above . monocyte - depleted pbmcs were activated with anti - cd3/cd28 beads ( dynabeads ; thermo fisher scientific ) in a 3:1 bead : cell ratio with 20 iu / ml il-2 for 7 days . activated t cells were then retrovirally transduced on days 3 and 4 , supernatants from the different vectors were mixed on transduction days at a 1:1 ratio . transduction efficacy was confirmed by facs after staining with anti - human igg antibody ( # 2040 - 08 ; southern biotechnology associates ) for the detection of cells bearing anti - hpsma vector and detection of tdrfp / cbrluc . to assess car t cell function we decided to follow the clinical protocol of car t cell preparation . two sets of car t cells ( from different donors ) were obtained for the current study . one set of car t cells was utilized for the first car t cell trafficking experiment ( figure s2 ) and a winn assay . to perform anti - hpd1 mab and anti - hpsma car t cell treatment we obtained another set of car t cells . transduction efficiencies varied from 87% to 99.8% for the anti - hpsma marker after cell sorting , and between 67% and 34% for cells that were double - positive for both anti - hpsma marker and tdrfp / cbrluc . 2 cryopreserved medium composed of 7% plasma - lyte , 20% of rimso-50 ( dmso ; mylan institutional ) , 40% of albumin ( human ; grifols ) , and 33% ( hespan [ hetastarch ] ) . target tumor cells were loaded with 100 ci of cr for 1 hr , and then 10,000 tumor cells were co - incubated with car t cells for 6 hr at effector - to - target ( e : t ) ratios ranging from 40:1 to 1:1 . percent lysis was calculated as follows : percent lysis = ( experimental lysis spontaneous lysis)/(maximal lysis spontaneous lysis ) 100% , where maximal lysis was induced by incubation in a 2% triton x-100 solution . the metabolic profiles were determined at steps of t cell stimulation and transduction to assess glycolytic function and mitochondrial respiration of t cells during stimulation , transduction and expansion . we performed a series of real - time measurements of the extracellular acidification rate ( ecar ) and ocr using a seahorse xf96 extracellular flux analyzer ( agilent ) . the xf analyzer ( seahorse biosciences ) simultaneously measures energy producing pathways non - invasively in real - time . for the assessment of glycolysis , the first injection is a saturating concentration of glucose ( 25 mm ) , where glucose is taken up by the cells and catabolized through the glycolytic pathway to lactate , producing atp and protons . the extrusion of protons into the surrounding medium produces a rapid increase in ecar . in the mitochondrial respiration assay , basal ocr is measured under full media growing conditions followed by sequential addition of oligomycin , fccp , and antimycin a / rotenone with a measurement of changes in ocr . the resulting profiles show the relative contribution of non - respiratory chain oxygen consumption , atp - linked oxygen consumption and the maximal respiration after the addition of fccp . data presented here represent two independent experiments at different days and were normalized to 350,000 cells . nod.scid il2rg ( nsg ) mice were obtained from jackson laboratories and fvb / n male mice were from charles river laboratories . all mice were 56 weeks of age and all mice groups consisted of n = 5 per group . the animal protocol was approved by the memorial sloan kettering institutional animal care and use committee . myc - cap : hpsma(+ ) or myc - cap : hpsma( ) cells in 1:1 ratio of matrigel to media in the right flank subcutaneously . myc - cap wild - type cells ( 1 10 ) were injected into fvb / n mice . we reduced the number of car t cells for injection from 20 10 per mouse ( figure s2 ) to 10 10 , in later experiments . the first experiment included three mice per group , whereas the second experiment included five mice per group . the data are presented for the second and most representative experiment . for the combined treatment studies , anti - hpsma car t cells were thawed and reconstituted in standard t cell media for 24 hr . anti - hpd1 mab or igg - control mab were administrated to three groups of mice 3 hours before i.v . infusion of car t cells , and then every other day ( between days 10 and 20 of tumor growth ) . three groups of mice consisted of myc - cap : hpsma(+ ) igg group n = 5 ; myc - cap : hpsma( ) anti - pd1 group n = 5 ; and myc - cap : hpsma(+ ) anti - pd1 groups n = 5 and n = 3 . myc - cap : hpsma(+ ) or myc - cap : hpsma( ) tumors in nod.cg-prkdc(scid)il2rg mice were treated with anti - hpd-1 ( human , j110 ) and isotype mopc-21 ( human , igg control ) antibodies . myc - cap wild - type tumors in fvb / n mice were treated with anti - mpd-1 ( murine , clone rmp1 - 14 ) , and isotype 2a3 ( murine , igg control ) antibodies when tumors reached a size of 50 mm , which occurred within 10 days after implantation of the tumor cells . bli was performed with an ivis spectrum imaging system ( perkin elmer ) and analyzed as described previously . tumor and lung tissue processing and staining was performed at molecular cytology core facility of memorial sloan kettering cancer center . the stained sections were digitized and scanned using a panoramic viewer ( 3dhistech ) and analyzed with metamorph image analysis software ( molecular devices ) . a fluorescence threshold was used to include only cell - specific signals and exclude background . size and morphology filters have been applied to ensure only cells are counted ( not cellular debris ) , and the number of immune cells were recorded . cell sizes were obtained by applying the above thresholds and obtaining the area of each identified t cell . the data ( area of individual t cels ) were combined for group statistics ( n = 3,621 t cells for the control group ; n = 14,531 t cells for anti - pd1 group ) . detailed information for cd31/pdl1 , cd3 , and tunel staining of tissues is provided in the supplemental material and methods . all data presented for t cells assessment using ifc staining were analyzed using graphpad prism ( version 6.0 ; graphpad software ) and are presented as mean sd . results were analyzed using the unpaired student s t test , and statistical significance was defined as p < 0.05 . conception / design , i.s . , e.m . , v.p . , and r.b . ; acquisition of data , e.m . , i.s . , m. mane , and m. moroz ; analysis and interpretation of data , e.m . , i.s . ; pathological diagnosis , analysis , and interpretation of the immunohistochemical data , i.s . and i.c . ; and carrying out experiments and analyzing data , e.m . , i.s . , m. moroz , and i.c
chimeric antigen receptor ( car ) t cell therapy in hematologic malignancies has shown remarkable responses , but the same level of success has not been observed in solid tumors . a new prostate cancer model ( myc - cap : psma(+ ) ) and a second - generation anti - hpsma human car t cells expressing a click beetle red luciferase reporter ) were used to study hpsma targeting and assess car t cell trafficking and persistence by bioluminescence imaging ( bli ) . we investigated the antitumor efficacy of human car t cells targeting human prostate - specific membrane antigen ( hpsma ) , in the presence and absence of the target antigen ; first alone and then combined with a monoclonal antibody targeting the human programmed death receptor 1 ( anti - hpd1 mab ) . pdl-1 expression was detected in myc - cap murine prostate tumors growing in immune competent fvb / n and immune - deficient scid mice . endogenous cd3 + t cells were restricted from the centers of myc - cap tumor nodules growing in fvb / n mice . following anti - programmed cell death protein 1 ( pd-1 ) treatment , the restriction of cd3 + t cells was reversed , and a tumor - treatment response was observed . adoptive hpsma - car t cell immunotherapy was enhanced when combined with pd-1 blockade , but the treatment response was of comparatively short duration , suggesting other immune modulation mechanisms exist and restrict car t cell targeting , function , and persistence in hpsma expressing myc - cap tumors . interestingly , an inverse pattern of car t cell bli intensity was observed in control and test tumors , which suggests car t cells undergo changes leading to a loss of signal and/or number following hpsma - specific activation . the lower bli signal intensity in the hpsma test tumors ( compared with controls ) is due in part to a decrease in t cell mitochondrial function following t cell activation , which may limit the intensity of the atp - dependent luciferin - luciferase bioluminescence signal .
Introduction Results Discussion Materials and Methods Author Contributions Conflicts of Interest
car t cell therapy in solid tumors has not achieved the clinical success that has been observed in hematologic malignancies.29 , 30 , 31 one reason for the poor treatment response is the failure of car t cells to accumulate and expand in the hostile tumor microenvironment.32 , 33 the failure of a substantial car - mediated t cell response in solid tumors relates to a number of factors including car t cell inactivation and possible exclusion from the tumor mass , the reciprocal interactions between tumor and stromal cells,34 , 35 , 36 and propensity of cancer like prostate to disseminate preferentially to bone . we show that anti - hpsma car - directed t cell therapy of myc - cap : psma(+ ) tumors alone was unsuccessful , whereas the combination of anti - hpsma car - directed t cells plus anti - hpd1 mab immune modulation therapy provided a partial , short - duration and sub - optimal response . to generate an appropriate murine model for the anti - hpsma car t cell therapy , wild - type ( wt ) myc - cap cancer cells were stably transduced with a hpsma - containing retroviral vector and sorted as described in materials and methods ( figure s1a).24 , 51 , 52 we compared the growth profiles of wild - type and hpsma(+ ) myc - cap tumors in mice , and found no significant difference in tumor doubling time , morphology , extent of necrosis ( figure 1a ) , or level of tumor hypoxia ( figure 1b ) . to assess the associations between pd - l1/pd-1 signaling and t cell targeting of myc - cap tumors , and the impact on tumor progression , we performed immunofluorescence staining for pd - l1 in ( 1 ) myc - cap : hpsma(+ ) tumors growing in nod.scid mice and ( 2 ) myc - cap wt tumors growing in immune - competent fvb / n . the spatial distribution of cd3 t cells in myc - cap wt tumors ( growing in fvb / n mice ) was also examined ; a predominance of t cells was observed along the invasive tumor margin ( stromal - tumor edge ) , with reduced numbers in the center of the tumor ( figures 4b and 5c , upper panel ) . encouraged by the response of myc - cap wt tumors to anti - mpd1 mab treatment in fvb / n mice , we evaluated the effect of anti - hpd1 treatment combined with anti - hpsma car t cell adoptive therapy in the myc - cap : hpsma(+ ) and myc - cap : hpsma( ) tumor models . interestingly , the highest tumor - associated t cell bli signal was observed in the myc - cap : hpsma( ) tumors ( anti - hpd1-treated control group ) , and the lowest signal was observed in the myc - cap : hpsma(+ ) tumors ( anti - hpd1-treated test group ) . the absence of a treatment response in myc - cap : psma( ) tumors ( anti - hpd1 mab - treated control group ) , despite the more robust t cell trafficking - to and bli signal - in the tumor region is of particular interest . nevertheless , the encouraging response of wild - type myc - cap tumors to anti - mpd1 mab treatment in immune competent fvb / n mice led us to evaluate the effect of anti - hpd1 mab treatment in combination with hpsma - targeted car t cells in the myc - cap : hpsma(+ ) tumor model . nevertheless , our results indicate that other immune modulation mechanisms exist and restrict car t cell targeting , function , and persistence in hpsma expressing myc - cap tumors administered car t cells is well known.49 , 67 nevertheless , the persistence of car t cells is well documented in the treatment of many liquid tumors,68 , 69 , 70 whereas the persistence of car t cells is not a common finding in the treatment of solid tumors . in our bli studies , there was a clear difference in anti - hpsma car t cell trafficking to and initial persistence in myc - cap tumors ( with and without the presence of the target antigen - hpsma ) , even during the first minutes after t cells injection ( figures 6a and 6b ) . we show that anti - hpsma - directed car t cell monotherapy of subcutaneous myc - cap : psma(+ ) tumors is ineffective , whereas the combination of anti - hpsma - directed car t cells plus anti - hpd1 mab immune modulation provides a short - duration , sub - optimal treatment response . these results also suggest that other immune modulation mechanisms need to be brought into play to further reverse the restriction to car t cell targeting and persistence in hpsma expressing myc - cap tumors and to provide optimal car t cell therapy .
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large - scale clinical trials have created a robust evidence base to inform much of what is now standard acute - stroke practice.13 the classical clinical trial is designed to test efficacy of a particular intervention over a comparator , for example , placebo or usual care . to facilitate comparison between the groups requires a standard measure of outcome that is relevant and suited to the clinical question , valid for the population studied , and meaningful to the research team . in those trials that describe interventions designed to impact on quantifiable physiological variables , such as glycemia or blood pressure , choice of end - point assessment choice of assessment strategy is more challenging for a chronic , nonprogressive , or variably progressive disorder with potential multisystem effects such as cerebrovascular disease . hard clinical end points such as stroke mortality or stroke recurrence are useful , but do not fully capture the potential devastating effect of a disabling but survivable stroke . as stroke represents the leading global cause of adult disability,4 an important consideration for any study of stroke interventions this is recognized by regulatory authorities , who now recommend a measure of functional recovery / disability as primary or coprimary end point for stroke intervention trials . although the focus of this review will be functional assessment tools for stroke trials , these instruments also have utility in clinical practice . as functional assessment scales give a numerical value to abstract concepts such as disability , they can be used to objectively quantify deficits and track change over time . in clinical practice , an appreciation of how to describe stroke recovery in terms of common stroke scales allows for development of a common language between professionals caring for stroke survivors that facilitates comparisons of patients and services . within a single review , it would be impossible to review all stroke - specific and generic scales that may be needed in a stroke survivor s journey ( figure 1 ) . for the interested reader , we recommend a number of reference works.57 we recognize there is also extensive literature on assessment strategies for cognitive function poststroke . we will not review cognitive testing , suffice to say that there are a multitude of tools available with little consistency in choice of assessment.8 a large number of stroke - assessment scales are described , with novel scales frequently appearing ( and often subsequently disappearing ) in the literature . for those who are new to functional assessment , the large and varied nature of available scales and tools may seem daunting . the world health organization s international classification of functioning , disability and health ( who - icf)9 gives a conceptual framework that can aid classification of the scales and help decide on the appropriate measure for a particular purpose . who - icf describes levels of pathology ( in this case , the stroke lesion ) , impairments ( the direct loss of function ) , activity limitation ( formerly called disability ) , and societal participation ( formerly called handicap ) . the who - icf grades do not exist in isolation ; they interact and often create feedback loops . for example , an ischemic stroke ( pathology ) may cause a hemianopia ( impairment ) ; this may lead to poor mobility ( activity limitation ) and may restrict the stroke survivor from driving ( societal participation limitation ) . these problems may result in a fall with soft - tissue injury ( impairment ) , and fear of falling may cause the stroke survivor to forgo usual hobbies and activities ( societal participation limitation ) ( figure 2 ) . measures of pathology ( for example , size of infarct on imaging ) or impairment ( for example the medical research council motor assessment scale ) are straightforward to perform and interpret , but give little useful information on how stroke affects the individual . for this reason , impairment scales are often used in early phase trials . for phase iii studies , activity measures or measures of participation although not part of the who - icf , a further concept of quality of life ( qol ) is also described , and tools exist for its measurement . measures of qol give a far more detailed assessment , but as a result can be more burdensome to the patient and are often more difficult to interpret ( figure 3 ) . clinimetrics is the study of properties of clinical assessment tools;10 the term is derived from the theory of psychometrics.11 classical test theory describes important properties such as validity and reliability.12 other important factors for clinical scales are acceptability , both to patient and to assessor , and responsiveness to change . although in psychometrics , classical test measures are increasingly being superseded by contemporary theories of item response , the measures of validity , reliability , and responsiveness remain important for understanding clinical scales , and we will discuss them in turn . the clinimetric property of validity seeks to assess whether a scale measures the concept it purports to measure . adequate validity is essential for a stroke scale to have clinical utility , as a functional assessment tool that does not measure function is meaningless . validity can be assessed in various complementary ways.57 there is no gold standard for poststroke function , so assessment of criterion validity , where a scale is compared to a reference standard , is not possible . however , concurrent validity can be applied to a stroke scale by comparing it with another measure that purports to measure a similar construct ; for example , comparing a novel impairment scale with an established scale . face validity is an assessment of whether a priori the scale should measure the concept of interest , usually assessed by experts in the field . content validity asks whether the various items of a scale can adequately describe the concept of interest . prognostic or predictive validity for a stroke scale may be examined by , for example , studying if an impairment scale is associated with longer - term stroke outcomes . important reliability measures include the reproducibility of repeat scoring by the same observer ( intraobserver reliability or test retest reliability ) and between scorers ( interobserver variability ) . whether all items within a scale measure the same construct is a further measure of reliability , usually termed internal consistency . in contemporary stroke trials , where many thousands of stroke survivors may be assessed by hundreds of international research teams , reliability of assessment is clearly paramount . whereas validity of a scale is inherent , reliability of assessment may be modified . various methods to improve consistency of assessment are employed in large - scale trials , including training in use of scales , certification exams , and use of standardized protocols . while validity is relative , reliability can be objectively described . there is no consensus on the optimal method to measure reliability , although kappa statistics are frequently used in the biomedical literature to assess agreement . a kappa of 0 would imply no agreement other than that expected by chance , and perfect agreement is scored as 1.0 . traditionally , a kappa greater than 0.6 is taken as sufficient agreement to justify use of a scale . various forms of statistical weighting of kappa values can be used to give a measure of the degree of difference between raters.13,14 increasingly , more sophisticated analyses , such as that of bland altman , are being used to assess reliability.15 responsiveness can be thought of as the ability to detect meaningful change over time . meaningful change is clearly a subjective term , and will vary with the context in which the scale is used . the issue of responsiveness and the ability to detect small but meaningful change is especially important for a condition with high incidence and prevalence , such as stroke . if a scale does not pick up change in function , treatment effects that are modest for the individual but potentially important at a population level could be missed . the ideal scale would be easy and quick to administer , acceptable to patients and researchers , valid for its chosen purpose , reliable , and responsive to meaningful clinical change . there is no ideal stroke measure that fulfills all these criteria ( nor is there ever likely to be ) . although some guidance on stroke assessment for trials is emerging , debate continues as to the relative strengths and limitations of differing assessment strategies , and there is no consensus as to the optimal outcome measure(s ) for use . the stroke literature describes a variety of instruments , generic and specific to stroke , for functional assessment of recovery . a recent analysis of tools used in stroke trials suggests substantial heterogeneity in choice of assessment measure and in method of application.16 use of bespoke , nonvalidated assessments is still seen , although less commonly than previously . certain assessments are used more frequently than others and are increasingly recommended by specialist societies.17 for the non - stroke specialist , a basic knowledge of the more prevalent stroke scales will allow for improved understanding and critical analysis of stroke studies . we will describe three common stroke assessments : the national institutes of health stroke scale ( nihss ) , the modified rankin scale ( mrs ) and the barthel index ( bi ) . for each scale , we will discuss history , development , and application , and use the scales to further discuss the importance of clinimetric properties . the scientific study of assessment scales , particularly stroke assessment , is rapidly expanding and it would be impossible to comprehensively cover all areas . in this review , we will not describe the optimal analysis of functional scales for stroke trials . debate continues as to the relative merits of various statistical techniques , including dichotomization and use of the complete range of a scale , with the differing approaches having vocal proponents.18,19 equally , we will not consider the literature on outcome assessment in animal models of stroke.20,21 the nihss is a 15-item scale that standardizes and quantifies the basic neurological examination , paying particular attention to those aspects most pertinent to stroke . the nihss provides an ordinal , nonlinear measure of acute stroke - related impairments by assigning numerical values to various aspects of neurological function.22 the scale incorporates assessment of language , motor function , sensory loss , consciousness , visual fields , extraocular movements , coordination , neglect , and speech.22 it is scored from 0 ( no impairment ) to a maximum of 42 . a standardized approach to assessment , starting with fundamental assessments such as level of consciousness , is recommended , and guidance is given on how to score where the stroke survivor is not able to respond to commands . the nihss was developed in the early 1980s as a research tool to allow consistent reporting of neurological deficits in acute - stroke studies , particularly the early trials of thrombolysis and putative neuroprotectants.22 the nihss was developed through a robust consensus approach , taking the most informative measures from existent stroke - examination scales ( toronto stroke scale , oxbury initial severity scale , and cincinnati stroke scale ) and creating a composite scale that was further reviewed by a panel of stroke researchers and amended ( further items were added to ensure the assessment was as comprehensive as possible ) . the resulting scale was piloted and refined in a controlled trial of naloxone in acute stroke . it has since been used as primary or coprimary end point in landmark trials of thrombolytic agents and is commonly used in clinical acute - stroke practice . using a factor - analysis process , utility of individual components of the nihss has been assessed.23 this work has formed the basis for development of the modified nihss or mnihss , which removed components deemed unreliable.24 the resulting 11-point mnihss has been prospectively assessed , and improved reliability is described.25 as the standard nihss is already fairly quick to perform and reliable , it is debatable whether a shorter scale is needed , and at present the mnihss is not frequently used in trials or practice . further amendments to the nihss have been described to facilitate use of the scale in prehospital settings.26 a pediatric nihss ( pednihss ) is also described.27 the nihss has many advantages as a stroke outcome - assessment tool . it is relatively straightforward and takes around 6 minutes to perform , with no need for additional equipment . in the acute - stroke environment , it has been suggested that a change in the nihss of more than 2 points represents clinically relevant early improvement or deterioration.28 nihss scores are reliable across observers , and this has been demonstrated both in cohorts of neurology - trained and non - neurologist raters . the availability of a reliable method for neurological exam that is suitable for nonspecialists is a particular strength of the nihss . reliability and validity has also been demonstrated for remote nihss assessment via telemedicine.29 the interobserver reliability of the nihss is further improved by the various training materials available . training resources now exist , such as dvds and online educational aids , as well as pocket - sized nihss summary scales . practitioners can undergo certification to demonstrate their proficiency in assessment and interpretation of the nihss.30 content validity of the nihss has been demonstrated , although high internal consistency suggests that certain items of the nihss may be redundant . the nihss has predictive validity , as initial score is a robust predictor of in - hospital complication and outcome at 3 months.31,32 correlations with objective measures of stroke severity , such as size of infarct on imaging , provide further evidence of nihss validity.23,33,34 compared with bi and mrs , the nihss is the more sensitive outcome score , requiring potentially smaller sample sizes to detect relevant therapeutic effects.23,35 the nihss is responsive to change and can measure impairment throughout the expected range of stroke severity.36 a criticism of the nihss relates to its validity in certain nondominant - hemisphere stroke syndromes . it is well recognized that an individual can score 0 on the nihss , despite having evidence of ischemic stroke , particularly in the posterior circulation territory.37 examination of the component subscales of the nihss reveals a focus on limb and speech impairments and relatively little attention to , for example , cranial nerve lesions . similarly , when the nihss is used to predict dependent living , lower scores are seen in posterior circulation events compared to anterior circulation.38 there are radiological correlates , when quantifying extent of cerebral damage for a specified nihss score , the median volume of right - hemisphere strokes is larger than the volume of left - hemisphere strokes , suggesting nondominant strokes are required to be more severe to reach the same grading on the nihss.39 as an impairment scale , the nihss can give only limited information on how stroke has affected the individual stroke survivor . excellent outcome from stroke ; a hemianopia that precludes driving and may necessitate loss of employment would score nihss 1 , but for the individual this may not seem an excellent result . adapted from the maryland disability index , the bi authors florence i mahoney and dorothea w barthel intended their scale for use as a simple index of independence , useful in scoring improvement in rehabilitation.40 first described in the 1950s and published in 1965 , the bi was developed to assist in discharge planning from long - term care wards . with time , the bi has been adopted by other disciplines and is a recommended assessment in older adult care.41 the bi is the most commonly used functional measure in stroke - rehabilitation settings and the second most commonly used functional outcome measure across stroke trials.16,42 many scales have been described that take the name some authors have sought to modify or adapt the bi from the original ; these include reducing the number of items,43 extending it with the addition of cognitive and social domains,44 and attempts to further subdivide the outcomes to include different degrees of assistance.45 however , each of these requires independent validation , as it is known that even comparatively minor adaptations alter the validity of a tool and accuracy of responses.46 for consistency , it is recommended that a single bi measure is used ; the scale as described by wade and collin47 has been used in many trials . scores range from 0 and 100 , with a higher score indicating greater independence ( table 1 ) . while this can be useful for statistical analysis , it is more informative in practice to present the scores for the individual domains . an unresolved issue for trials is how to define a good bi outcome , with significant heterogeneity within the published literature48 and attempts to subcategorize , based on total score.49,50 a popular interpretation of bi scores is that subjects with bi > 80 are generally independent and should be able to return home ; while subjects with bi < 40 are very dependent.51 other interpretations of favorable and unfavorable bi outcomes have been described : statistical modeling looking at differing bi scores as a trial end point suggested a score of 95/100 was the optimal descriptor of an excellent outcome and that 75/100 was the best cut point for defining a poor outcome.52 validity of bi is well described . the scale is recognized as a valid prognostic tool following stroke , in particular as predictor of recovery , level of care required,53 and duration of rehabilitation required following stroke.54 bi scores correlate with other stroke - assessment scales,55 including other more detailed adl scales.56 interobserver reliability is usually quoted as a strength of the bi , and reliability has been demonstrated in nonstroke populations.57 systematic review of reliability of bi in stroke also suggests reasonable reliability , although few multicenter reliability studies are available.58 the original bi is not without its limitations . as a scale of primarily physical function , it does not reflect the burden on the individual of communication and cognitive deficits that can result from a stroke event.59 for clinical trials , the bi lacks a result to represent stroke mortality , and this can complicate analysis of results . however , the major limitation of the bi for clinical trial use is its responsiveness to change . although in certain stroke - care settings , the bi is as sensitive to change as other scales,60 a score must be able to represent changes throughout the entire spectrum of potential functional outcomes . it is in this regard that the foor and ceiling effects of the bi become apparent.50 floor and ceiling describes the phenomenon by which the score does not change from minimum or maximum despite clinical change.61 for example , a stroke patient in a neurointensive care setting can make significant gains but still score a total 0 on the bi ; conversely , a patient who is discharged from hospital and independent may still have substantial functional problems but will score 100 on the bi . given this limitation , the bi may be best suited to stroke survivors requiring inpatient rehabilitation , while other scales may be needed to assess functional change in those with more major or minor stroke symptoms.62 the bi can be considered as a measure of basic adl ( self - care and mobility ) . extended activities of daily living ( e - adl ) measures.63 the term instrumental adl was first used in lawton and brody s work , and a lawton i - adl scale is described.64 a validated measure that has been used with stroke survivors is the nottingham extended adl scale , which asks participants to reflect their actual activities over the preceding weeks , rather than simply what they have the capability to do.65,66 the nottingham extended adl scale compares favorably to the bi , and is less susceptible to the ceiling effects described.67 the mrs is a 6-point , ordinal hierarchical scale that describes global disability with a focus on mobility ( table 2 ) . the original rankin scale was developed by the scottish physician john rankin to describe the positive outcomes he was achieving in his prototypic stroke unit.68 although not originally intended as an assessment for clinical trials , a slightly modified version of rankin s eponymous scale was used as end point in the first multicenter stroke trial ( the uk tia study).69 since this time , the mrs has grown in popularity and is now the most commonly used functional measure in stroke trials , and has been the primary or coprimary outcome in most recent large - scale stroke trials.16 a further variation of the mrs , the oxford handicap scale , has been described but is not commonly used by trialists . in contemporary stroke studies , the mrs is often used both as a measure of premorbid ability to assist in selection of patients and as final outcome measure . the mrs has many potential strengths , and it is acceptable to patient and assessor , with nonstandardized interviews taking around 5 minutes to complete.70 concurrent validity is demonstrated by strong correlation with measures of stroke pathology ( for example , infarct volumes ) and agreement with other stroke scales.71,72 the six potential scores on the mrs ( 05 ) describe a full range of stroke outcomes , with a score of 6 usually added to denote death . with a limited number of scores , the mrs may be less responsive to change than some other scales ; however , a single - point change on the mrs will always be clinically relevant . the principle limitation of the mrs is its reliability , with the potential for substantial interobserver variability . a study describing the interobserver variability of the mrs is available ; indeed , in the first clinical studies that used the mrs as end point , the trialists described interobserver variability for a third of subjects interviewed by paired assessors.73 a systematic review and meta - analysis of studies describing interobserver variability of the mrs reports pooled reliability across ten published studies ( n = 587 patients ) of kappa = 0.46.74 those studies that assessed mrs reliability with multiple raters and centers ( ie , similar to a contemporary clinical trial ) revealed a worryingly low agreement of kappa = 0.21.75 this level of inconsistency will impact on the validity of the trial results and conclusions . the statistical noise created by the interobserver variability will increase the possibility of a type ii error , ie , a beneficial treatment effect is missed . it has been postulated that problems with mrs reliability may have partly explained a series of unexpected neutral results in large - scale neuroprotectant studies . there are published examples of nonstroke studies whose results were fundamentally altered when statistical analysis accounted for observer variation.76 recognizing the problems of reliability in standard mrs assessments , trialists have explored various interventions to improve consistency in scoring . usual mrs interviews are unstructured , and researchers vary considerably in their length of interview and number of questions asked . more structured approaches to assessment have been described , from a comprehensive scripted interview75 to use of anchoring questions that require a yes / no answer.77 the groups that developed these assessments describe substantial improvements in reliability . however , improvements have not been seen when the structured interviews have been tested by independent centers.78 training in use of the mrs can also offer potential to improve consistency . as with the nihss and bi , an online training resource is available with an accompanying certification exam.79 a further trial modification that may improve reliability is to record mrs interviews and have a remote consensus grading by experienced stroke trialists . two other modifications to mrs assessment are commonly used and deserve some discussion : using proxies to substitute for stroke survivors in the mrs interview and calculating a prestroke mrs . stroke survivors often have physical , language , or cognitive impairments that may complicate a standard face - to - face interview . in this situation , an informant who knows the patient often supplements the interview or substitutes it completely . while this approach makes intuitive sense , we should not assume validity , and clinimetric analysis is still required . a recent systematic review of proxy stroke scales ( the mrs was not included ) suggested that the properties of certain proxy - based assessments may differ from equivalent standard assessments.80 a study of proxy mrs described suboptimal reliability and validity , and recommended that direct mrs interview with the patient should be the preferred assessment if possible.81 in stroke trials , traditional statistical analyses assess numbers achieving a good functional outcome . to improve trial power , prestroke mrs has been used in many landmark stroke trials , where prestroke mrs > 2 is used as the exclusion criterion during participant selection . the wording of the mrs grades is not suited to such prestroke assessment , and it is perhaps unsurprising that when formally assessed , prestroke mrs had only moderate reliability and validity.82 in view of improving longer - term survival and functional outcomes following stroke,83 it could be argued that assessments against participation or qol will become increasingly important.84 certainly evaluations of health - related qol in stroke survivors can provide a rich description of the multifaceted effects of a stroke , providing insights above those recorded with traditional impairment and activity measures.85 measuring health - related qol in stroke presents particular challenges . important predictors and components of qol following stroke will vary at different periods following the event.86 thus , we must balance having a suitably comprehensive assessment that is sensitive to the nuances of qol against the time and burden required for this assessment . carer / family - based assessments of the patient s qol are often biased , with the proxies reporting poorer outcomes than the subject.87 various qol scales have been proposed , some generic and some specific to stroke / brain injury . qol scales should be subject to the same rigor of clinimetric assessment as any other scale . it is evident from the published literature that for qol there is a propensity to generate new scales rather than validating existing ones.88,89 it has been argued that qol can be assessed by asking just two questions assessing dependency and problems.90 an alternative approach is to apply existing health - related qol scales or to use disease - specific scores . the short form 36 ( sf-36 ) is a generic scale intended for patient completion that assesses eight domains of health - related qol derived from the medical outcomes study ( table 3).91 although the sf-36 is validated for stroke patients,92 noncompletion bias and marked floor and ceiling effects may limit its utility.93,94 the generic qol scale , euro - qol , was developed based on the findings of an international postal survey.95 the self - completion questionnaire requires assessment across five domains complemented by a visual analog scale ( table 3).96 euroqol has been validated in stroke populations.97 however , noncompletion bias is recognized : in one study , only 61% of stroke survivors could complete the scale without external assistance.97 the stroke - specific qol scale was developed based on interviews with stroke survivors.98 it is based on twelve domains ( table 3).93 the scale is validated in stroke populations , and values for minimal detectable change and clinically important difference99 are established . many assessment tools , spanning various functional domains , are available to clinicians and researchers working with stroke survivors . we have given a favor of the marked heterogeneity in use of assessment scales . lack of consistency in outcome assessment has hindered comparative research and meta - analysis , and so we would recommend that future researchers use a common set of outcome assessments . no perfect stroke - assessment scale exists , and in this review we have deliberately avoided suggestions that one scale is better than an other . we have focused on the three most commonly used stroke scales ( mrs , bi , nihss ) as exemplars . these scales have been validated , are familiar to many , and have proven utility , with each suited to differing assessment scenarios . assessment , we would recommend continuing use of the three core assessment scales : the mrs as an outcome if the study is describing global disability , the nihss for studies looking at neurological impairment , and the bi for studies looking at basic adl . trialists and clinicians can supplement these core assessments with specific tools suited to the clinical scenario / research question . increasing awareness of the importance of clinimetric properties has highlighted deficiencies and potential limitations with stroke functional assessment . clinicians and researchers should always select their assessment tool(s ) based on the question of interest and the evidence base around clinimetric properties . where , as is often the case , the research around clinimetric properties of a scale is sparse , we would encourage researchers to design and conduct their own clinimetric studies .
as stroke care has developed , there has been a need to robustly assess the efficacy of interventions both at the level of the individual stroke survivor and in the context of clinical trials . to describe stroke - survivor recovery meaningfully , more sophisticated measures are required than simple dichotomous end points , such as mortality or stroke recurrence . as stroke is an exemplar disabling long - term condition , measures of function are well suited as outcome assessment . in this review , we will describe functional assessment scales in stroke , concentrating on three of the more commonly used tools : the national institutes of health stroke scale , the modified rankin scale , and the barthel index . we will discuss the strengths , limitations , and application of these scales and use the scales to highlight important properties that are relevant to all assessment tools . we will frame much of this discussion in the context of clinimetric analysis . as they are increasingly used to inform stroke - survivor assessments , we will also discuss some of the commonly used quality - of - life measures . a recurring theme when considering functional assessment is that no tool suits all situations . clinicians and researchers should chose their assessment tool based on the question of interest and the evidence base around clinimetric properties .
Why measure functional outcomes in stroke trials? Which functional measure to use Clinimetric properties of scales National Institutes of Health Stroke Scale Barthel Index Modified Rankin Scale Quality of life Conclusion
although in psychometrics , classical test measures are increasingly being superseded by contemporary theories of item response , the measures of validity , reliability , and responsiveness remain important for understanding clinical scales , and we will discuss them in turn . we will describe three common stroke assessments : the national institutes of health stroke scale ( nihss ) , the modified rankin scale ( mrs ) and the barthel index ( bi ) . for each scale , we will discuss history , development , and application , and use the scales to further discuss the importance of clinimetric properties . debate continues as to the relative merits of various statistical techniques , including dichotomization and use of the complete range of a scale , with the differing approaches having vocal proponents.18,19 equally , we will not consider the literature on outcome assessment in animal models of stroke.20,21 the nihss is a 15-item scale that standardizes and quantifies the basic neurological examination , paying particular attention to those aspects most pertinent to stroke . similarly , when the nihss is used to predict dependent living , lower scores are seen in posterior circulation events compared to anterior circulation.38 there are radiological correlates , when quantifying extent of cerebral damage for a specified nihss score , the median volume of right - hemisphere strokes is larger than the volume of left - hemisphere strokes , suggesting nondominant strokes are required to be more severe to reach the same grading on the nihss.39 as an impairment scale , the nihss can give only limited information on how stroke has affected the individual stroke survivor . with time , the bi has been adopted by other disciplines and is a recommended assessment in older adult care.41 the bi is the most commonly used functional measure in stroke - rehabilitation settings and the second most commonly used functional outcome measure across stroke trials.16,42 many scales have been described that take the name some authors have sought to modify or adapt the bi from the original ; these include reducing the number of items,43 extending it with the addition of cognitive and social domains,44 and attempts to further subdivide the outcomes to include different degrees of assistance.45 however , each of these requires independent validation , as it is known that even comparatively minor adaptations alter the validity of a tool and accuracy of responses.46 for consistency , it is recommended that a single bi measure is used ; the scale as described by wade and collin47 has been used in many trials . the scale is recognized as a valid prognostic tool following stroke , in particular as predictor of recovery , level of care required,53 and duration of rehabilitation required following stroke.54 bi scores correlate with other stroke - assessment scales,55 including other more detailed adl scales.56 interobserver reliability is usually quoted as a strength of the bi , and reliability has been demonstrated in nonstroke populations.57 systematic review of reliability of bi in stroke also suggests reasonable reliability , although few multicenter reliability studies are available.58 the original bi is not without its limitations . the original rankin scale was developed by the scottish physician john rankin to describe the positive outcomes he was achieving in his prototypic stroke unit.68 although not originally intended as an assessment for clinical trials , a slightly modified version of rankin s eponymous scale was used as end point in the first multicenter stroke trial ( the uk tia study).69 since this time , the mrs has grown in popularity and is now the most commonly used functional measure in stroke trials , and has been the primary or coprimary outcome in most recent large - scale stroke trials.16 a further variation of the mrs , the oxford handicap scale , has been described but is not commonly used by trialists . the short form 36 ( sf-36 ) is a generic scale intended for patient completion that assesses eight domains of health - related qol derived from the medical outcomes study ( table 3).91 although the sf-36 is validated for stroke patients,92 noncompletion bias and marked floor and ceiling effects may limit its utility.93,94 the generic qol scale , euro - qol , was developed based on the findings of an international postal survey.95 the self - completion questionnaire requires assessment across five domains complemented by a visual analog scale ( table 3).96 euroqol has been validated in stroke populations.97 however , noncompletion bias is recognized : in one study , only 61% of stroke survivors could complete the scale without external assistance.97 the stroke - specific qol scale was developed based on interviews with stroke survivors.98 it is based on twelve domains ( table 3).93 the scale is validated in stroke populations , and values for minimal detectable change and clinically important difference99 are established . assessment , we would recommend continuing use of the three core assessment scales : the mrs as an outcome if the study is describing global disability , the nihss for studies looking at neurological impairment , and the bi for studies looking at basic adl . clinicians and researchers should always select their assessment tool(s ) based on the question of interest and the evidence base around clinimetric properties .
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children born before week 32 with very - low - birth weight ( vlbw : birth weight 1500 g ) are more likely to need medical treatment during the perinatal period , and their immature nervous and cardiovascular systems render these children prone to focal brain injuries such as intraventricular hemorrhages and periventricular leukomalacia ( pvl ) ( volpe , 2009 ) . volpe ( 2009 ) has suggested that the complex of encephalopathy of prematurity includes both destructive and developmental disturbances , and primary white matter injury could have secondary effects on cortical and gray matter nuclei development . although perinatal care and medical treatment in the neonatal intensive care unit ( nicu ) have improved radically during the last decades with reduced incidence of focal brain injury , the immature brain and exposure to the harsh extra - uterine environment in the nicu are still believed to increase the risk of disrupted brain development in very preterm born survivors . the consequences of such developmental disruptions in an extremely sensitive period of brain growth may be profound alterations of subcortical and cortical morphology that may affect brain function . previous studies have reported abnormal cerebral white matter in vlbw infants as the most common pathological finding , manifested as reduced fractional anisotropy on diffusion tensor images at 2746 weeks of gestation ( ball et al . , 2013 ) , in adolescents ( skranes et al . , 2007 ) and in young adults aged 1822 years ( eikenes et al . , 2011 ) . however , changes in cortical and subcortical gray matter have also been demonstrated in children with vlbw as reduced brain cortical surface area at term - equivalent age ; ( ajayi - obe et al . , 2000 ; 2006 ) , in toddlers 1822 months old ( phillips et al . , 2011 ) , in children at the age of 10 years ( grunewaldt et al . , 2014 ) , and in adolescents and young adults ( frye et al . , 2010 ; skranes et al . , both regional thinning and thickening of the cerebral cortex have been reported in children 712 years of age ( grunewaldt et al . , 2014 ; mrner - lavanchy et al . , 2014 ) and in adolescents at the age of 19 ( bjuland et al . , 2014 ) . possible mechanisms underlying these cerebral changes in the vlbw population may include injuries that affect neuronal migration and thereby cortical development ( volpe , 2009 ) . the aim of the present study was to investigate cortical thickness and cortical surface area in 510 year old children born preterm with vlbw and term - born controls . to our knowledge , no previous study has explored regional cortical morphology using continuous cortical surface maps in vlbw children as young as 510 years of age . using continuous maps of cortical thickness and surface area increases both sensitivity and specificity compared to volumetric methods ( rimol et al . , 2012 ) . in addition , the present study explores the relationship between regional measures of cortical morphology and full iq as an overall measure of cognitive functioning . cognitive abilities have been shown to be reduced in the vlbw population ( aarnoudse - moens et al . , 2009 ; anderson et al . , 2004 ; lohaugen et al . , 2010 ; nosarti et al . , 2007 ; 2004 ) , and reduced cognitive performance has been related to reduced cortical volume in 1415 year old vlbw adolescents ( nosarti et al . , 2014 ) . finally , previous studies of vlbw young adults have shown negative correlations between iq and cortical thickness ( bjuland et al . , 2013 ) , and positive correlations with surface area ( skranes et al . , 2013 ) . however , these vlbw young adults were born in 19861988 , and it is unclear whether the same relationships between cortical morphology and cognitive function exist for school aged vlbw children born after year 2000 , who have received modern neonatal intensive care . the children born prematurely with very low birth weight ( vlbw ) ( birth weight 1500 g ) were recruited based on admittance to the neonatal intensive care unit ( nicu ) at st . sixty - three non - cp children were invited and 57 agreed to participate in the study ( 31 females ) . one child ( a twin sibling to a vlbw child ) with birth weight at 2090 g was included in the data analysis , and post - hoc analysis showed similar brain morphology and iq scores for this child as for the vlbw cohort . the control subjects were recruited from the national norwegian mother and child cohort study ( moba ) managed by the norwegian institute of public health ( magnus et al . , 2006 ) , with ages ranging between 4 and 11 years ( n = 143 , 70 females ) . the participants included in the current analysis were living in the same geographical area as the vlbw participants ( nord- and sr - trndelag ) and had normal vision and hearing . the exclusion criteria were a history of injury or disease known to affect the central nervous system ( cns ) function , including neurological or psychiatric illness and serious head trauma . furthermore , if the child was under psychiatric treatment , used psychoactive drugs known to affect cns functioning , had a birth weight below 2500 g , or had any known mri contraindications , they were excluded from participation in the current study . mri data were collected using a 12-channel head coil on a 1.5 t siemens avanto scanner ( siemens medical solutions ) . the pulse sequence used for morphometric analyses was one 3d t1-weighted magnetization prepared rapid acquisition gradient echo ( mprage ) scan with the following parameters : repetition time ( tr ) , 2400 ms ; echo time ( te ) , 3.61 ms ; inversion time ( ti ) , 1000 ms ; flip angle , 8 , fov 240 240 and acquisition duration of 4 min and 18 s. each volume consisted of 160 sagittal slices with voxel sizes of 1.25 1.25 1.20 mm . raw datasets were de - identified and transferred to linux work - stations for processing . each mprage was visually inspected and only scans with no or minimal movement artifacts were included in the analyses . cortical reconstruction was performed with the freesurfer 5.3.0 image analysis suite , which is documented and freely available for download online ( http://surfer.nmr.mgh.harvard.edu/ ) . the technical details of these procedures are described in other publications ( dale et al . , 1999 ; dale and sereno , 1993 ; fischl et al . , 2004a ; fischl and dale , 2000 ; fischl et al . , 2001 ) . briefly , this includes motion correction and averaging ( reuter et al . , 2010 ) of multiple volumetric t1 weighted images , removal of non - brain tissue using a hybrid watershed / surface deformation procedure ( sgonne et al . , 2004 ) , automated talairach transformation , intensity normalization ( sled and pike , 1998 ) , tessellation of the gray and white matter boundary , automated topology correction ( fischl et al . , 2001 ; sgonne et al . , 2007 ) , and surface deformation following intensity gradients to optimally place the gray / white and gray / cerebrospinal fluid ( csf ) borders at the location where the greatest shift in intensity defines the transition to the other tissue class ( dale et al . , 1999 ; dale and sereno , 1993 ; fischl and dale , 2000 ) . once the cortical models are complete , a number of deformable procedures can be performed for further data processing and analysis including surface inflation ( fischl et al . , 1999 ) , registration to a spherical atlas which is based on individual cortical folding patterns to match cortical geometry across subjects ( fischl et al . , 1999 ) , parcellation of the cerebral cortex into units with respect to the gyral and sulcal structures ( desikan et al . , 2006 ; fischl et al . , 2004b ) , and creation of a variety of surface based data . this method uses both intensity and continuity information from the entire three - dimensional mr volume in the segmentation and deformation procedures to produce representations of cortical thickness , calculated as the closest distance from the gray / white boundary to the gray / csf boundary at each vertex on the tessellated surface ( fischl and dale , 2000 ) . the maps are created using spatial intensity gradients across tissue classes and are therefore not simply reliant on absolute signal intensity . the surfaces were smoothed with a full - width - half - maximum gaussian kernel of 30 mm ( 662 iterations ) . each surface consisted of approximately 160,000 vertices arranged in a triangular grid , and estimates of the cortical area were obtained by computing the area of each triangle in the standardized , spherical atlas space surface tessellation when mapped into the individual subject space . vertex - wise estimates of cortical area were then computed by assigning one - third of the area of each triangle to each of its vertices ( rimol et al . , 2012 ) . the cortical surface of each subject was automatically parcellated using defined gyri and sulci as landmarks , and the surface was divided into 34 anatomical regions for each brain hemisphere defined in freesurfer ( desikan et al . , 2006 ; fischl et al . , 2004a ) , which were used to anatomically identify the affected regions after significance testing . in the vlbw group , analyses were conducted based on mr - images from 37 children ( 21 females ) . of the 57 who were eligible for mr - scanning , 10 children did not want to be scanned and had cognitive assessment only , and 10 images were excluded due to movement artifacts or disrupted scanning . in the control group we were able to attain 104 mprage images of good quality ( 54 females ) . a total of 143 children were invited to mr imaging , 22 children did not want to participate and 17 of the images had to be excluded due to movement artifacts or disrupted scanning . the youngest participants ( 56 years of age ) in both groups were most likely to decline mri scanning or be excluded due to movement artifacts . in the vlbw group , children < 6 years of age were assessed with the age - appropriate , complete version of the wechsler preschool and primary scale of intelligence , 3rd edition ( wppsi - iii ) ( wechsler , 2002 ) , whereas children 6 years were assessed with wechsler intelligence scale for children , 4th edition ( wisc- iv ) ( wechsler , 2003 ) . wppsi - iii provides three iq indices : full scale iq , verbal iq and performance iq , while wisc - iv comprises four indices : verbal comprehension index , perceptual reasoning index , working memory index and processing speed index , and full scale iq . cognitive abilities in the controls who were 6.5 years of age were assessed with the wechsler abbreviated scale of intelligence ( wasi ) ( wechsler , 1999 ) . the wasi is a validated screening test that is used to assess the following aspects of intelligence : verbal knowledge , visual information processing , spatial and nonverbal reasoning , and general intelligence . three iq scores can be extracted using the wasi : a verbal iq ( viq ) score ( subtests : vocabulary and similarities ) and a performance iq ( piq ) score ( subtests : block design and matrices ) , which when combined provide an estimated full - scale iq ( fsiq ) score . the controls who were younger than 6.5 years of age completed a short form of the wechsler preschool and primary scale of intelligence , 3rd edition ( wppsi - iii ) ( wechsler , 2003 ) , including similar subtests : vocabulary , similarities , block design and matrices , and verbal iq ( viq ) , performance iq ( piq ) and full - scale iq ( fsiq ) were calculated . ibm spss statistics 19 edition was used for the analysis of the clinical and cognitive measurements by independent samples t - tests and non - parametric tests . matlab 2011b was used for statistical analyses of morphometry data . to examine group differences , a general linear model was fitted with cortical surface area or cortical thickness as dependent variable and group , sex and age at mri scan as independent variables in each vertex across the cortical surface . the regression of iq on cortical morphology was tested with the same glm with full iq as an added continuous predictor . the hemispheres were analyzed separately , and effect size and p - maps were generated . effect size is reported as cohen 's d for group comparisons and r = f / ( f + df ) for the continuous predictors ( iq and birth weight ) . the p - maps were thresholded and multiple comparisons were corrected for with a 5% false discovery rate ( fdr ) that was applied co - jointly across the hemispheres . significance tests were performed to investigate differences in cortical morphology between the groups . for the clinical variables birth weight , gestational age , and days on ventilator general linear models were fitted in each vertex across the surface , with cortical surface area or cortical thickness as the dependent variable and one of the clinical variables as a covariate , and adjusted for sex and age at mri scan . missing data in the independent variables ( full iq and birth weight ) were dealt with by multiple imputations . pattern analysis was performed , showing that we had below 5% missing data and that we could assume that data were missing at random . seven full iq data and two birth weights were imputed and pooled imputations were used in further analyses . hollingshead 's ( 1975 ) two factor index of social position based on education and occupation of one parent or the mean index of both was used to calculate socio - economic status ( ses ) . the regional committee for medical research ethics approved the study protocol ( project number : 2010/2359 ) , and written informed consent was obtained from the parent / guardian of all participants . demographic and clinical characteristics of the study groups are shown in table 1 . in the vlbw group , mean birth weight was 1048 g and mean gestational age was 28.5 weeks . there was no significant group difference in age at examination ; however , the controls had higher mean socioeconomic status ( ses ) than the vlbw group . the vlbw group had significantly lower scores than controls on full iq , also after adjusting for socioeconomic status ( n = 34/85 ) . there were significant differences in cortical surface area between the vlbw and the control group . the vlbw group showed bilateral reduction in cortical surface area in the frontal , temporal , and parietal lobes ( fig . the effect size of the group difference ranged from d = 0.4 to 0.8 in most cortical regions ( see appendix fig . table 2 lists all cortical regions with significant group differences in surface area as determined by the glm ( after 5% fdr correction ) , where 20 out of 35 regions had 90% reduction of cortical surface area in the vlbw group compared with controls . in the control group , there were widespread cortical regions in both hemispheres showing a significant relationship between full iq scores and cortical surface area ( see appendix fig . the relationship between full iq scores and cortical surface area did not reach statistical significance . however , the correlation coefficients were 0.20.4 in several cortical regions in both groups , and as high as 0.6 in some regions in the vlbw group ( fig . the lack of statistical significance in these analyses is readily explained by loss of statistical power due to the smaller sample size . the vlbw group showed significantly thicker cortex in the frontal ( medial orbitofrontal gyrus , rostral anterior cingulate , frontal pole ) and occipital regions ( pericalcarine sulcus ) bilaterally , and a thinner cortex in the right posterior parietal lobe compared with controls ( fig . 3 ) . moderate to large effect sizes ( d = 0.60.8 ) were observed in the frontal and occipital regions ( appendix fig . a3 ) . proportion ( % ) of cortical regions with significant differences in thickness between the vlbw and the control groups is displayed in table a3 ( appendix ) with > 90% involvement of the frontal poles , medial orbitofrontal gyri , left rostral anterior cingulate and right pericalcarine sulcus . a4 ( appendix ) demonstrates the degree of spatial overlap between the observed between - group differences in cortical surface area and cortical thickness . there were no significant correlations between cortical thickness and full iq in either group . demonstrate a trend - level negative relationship between cortical thickness and full iq in both groups in widespread cortical regions , i.e. the thinner the cortex , the higher the iq scores ( fig . some temporal and parietal regions showed a positive relationship to full iq in the vlbw group , i.e. thinner cortex was related to lower iq scores ( fig . there were no significant associations between birth weight or gestational age and cortical area or cortical thickness in the vlbw group . there was , however , a significant effect of days on ventilator on surface area bilaterally in the dorsal frontal regions , including the superior and medial frontal gyrus , precentral gyrus , and orbitofrontal cortex , as well as the left supramarginal and posterior superior temporal gyrus , and the right precuneus and superior parietal gyrus . there were two subjects with extreme scores on days on ventilator , i.e. more than 3 weeks on ventilator ( 35 and 47 days ) , and these subjects also had low gestational age ( 23.5 , 26 weeks ) . region - of - interest based examination of cortical surface area and cortical thickness showed that the child with 47 days on ventilator was an outlier on 5 of 36 cortical parcellations ( > 2 sd from the mean in the control group ) , and the child with 35 days on ventilator was an outlier in one parcellation . excluding these two subjects from the analysis of group differences between vlbw and controls did not affect the results . finally , in order to check the effect of prolonged exposure to the ventilator , we excluded children who had spent more than 10 days on ventilator . since this reduces the statistical power to detect , because the sample is smaller than that in the full analysis , we compared maps of effect size ( cohen 's d ) . we report significant reduction of cortical surface area in 510 year old vlbw children , relative to a term - born control group . cortical area was reduced in frontal , temporal , parietal , and occipital regions , and cortical thickness was increased in the medial frontal and occipital lobes , in the vlbw group . moreover , there were medium sized to large correlations between reduced surface area and thicker cortex and poorer iq scores , in both the vlbw and the control group . however , only in the control group did correlations between reduced surface area and iq reach statistical significance . the vlbw children showed significantly reduced cortical surface area in frontal , temporal , posterior parietal and medial occipital regions , as well as the right anterior cingulate ( see fig . 1 ) . cortical surface area expands significantly during preschool years and into adolescence in normally developing children , with the greatest changes occurring in higher order regions such as the prefrontal cortex and temporal association cortex ( brown and jernigan , 2012 ) . however , by the age of 10 , the occipital and superior parietal lobes start to show a decrease in surface area , most probably due to pruning ( brown and jernigan , 2012 ) . hence , the reduced surface area observed in the vlbw children could reflect altered maturation of cortical surface area . reduced cortical surface area has previously been reported in extremely low birth weight ( elbw ) children at the age of 10 ( grunewaldt et al . , 2014 ) , vlbw adolescents at 1516 years of age ( frye et al . , 2010 ) , and vlbw late adolescents at 19 years of age ( skranes et al . , 2013 ) . the magnitudes of reduction , and precisely which gyri / sulci are affected , differ somewhat between these studies . however , skranes et al . ( 2013 ) reported reductions in cortical surface area similar to the reductions observed in the present study , both in terms of magnitude and localization of the affected regions . the fact that similar cortical regions are affected in cohorts aged 510 and 1820 , suggests that the morphological abnormalities observed in the present study may not simply reflect delayed maturation but rather aberrant development leading to permanently altered cortical architecture . these results also demonstrate that similar cortical changes are found both in vlbw survivors born in the late 80s and after year 2000 , in spite of the advances in perinatal medicine . we speculate whether the explanation for this has to do with prenatal factors , such as fetal growth restriction , or that immature birth exposes the neonate to environmental factors such as inflammation that exert an epigenetic influence on the genes controlling normal cortical development . performing the analyses of group differences in cortical surface area without the two children who had extreme scores on days on ventilator ( 32 and 45 days ) , did not affect the results . excluding children who had been on ventilator for more than 10 days , in order to exclude a possible effect of prolonged respiratory support on cortical surface area , we observed similar group differences in cortical morphometry as for the full sample , albeit with reduced effect sizes in most regions . however , excluding children with more than 10 days on ventilator implies excluding many of the most immature and sickest individuals , and leaves us with a sample that is not representative of the premature birth population . nonetheless , it is worth noting that even when subjects with prolonged respiratory support were excluded , a number of cortical regions still showed group differences . one could speculate whether this may reflect adverse prenatal factors , since these regions show cortical deviations even in the individuals who require the least amount of neonatal care . the vlbw children showed thicker cortex in the frontal and occipital lobes bilaterally , which is consistent with previous studies reporting increased cortical thickness in both children and adolescents born prematurely ( bjuland et al . , 2005 ; mrner - lavanchy et al . , 2014 ; phillips et al . , 2011 ) . with normal development of the cerebral cortex , thickness will increase during early childhood due to late arriving interneurons then decrease , due to pruning , as neural connectivity improves ( raznahan et al . sowell et al . ( 2004 ) reported that the pattern of progressive cortical thinning varies across development , in a longitudinal study of normally developing children 511 years old , observing a significant cortical thinning in the dorsolateral frontal regions and bilateral parietal occipital regions , and cortical thickening in perisylvian regions of the ventral frontal lobe and superior temporal lobe with increasing age . children develop at varying paces and one possible explanation for the group differences in cortical thickness in our study is delayed maturation in the vlbw group . this would be consistent with mrner - lavancy 's ( 2014 ) study of vlbw children and term - born controls , 712 years old , which reported thicker frontal and parietal cortices in the youngest vlbw children compared to controls but no such group difference in the oldest children . ( 2014 ) found cortical thickness differences exclusively in the occipital lobe at 10 years of age in a cohort of elbw . on the other hand , bjuland et al . ( 2013 ) found increased cortical thickness in frontal and occipital regions , but also thinner cortex in frontal , parietal and temporal regions , in 19 year old vlbw adolescents . thus , it is unclear whether the differences observed in the present study reflect aberrant development and permanent cortical changes or , rather , divergent developmental cortical trajectories that converge with increasing age . longitudinal studies are needed to answer such questions , in order to conclusively determine whether vlbw children born after 2000 have permanent changes in cortical thickness similar to what has been reported for children born in the late 80s . 2009 ) demonstrated that cortical area and cortical thickness reflect at least two distinct sources of genetic influence , consistent with the developmental origin of cortical architecture described by the radial unit hypothesis ( rakic , 1988 ) , and other studies have suggested independent and divergent developmental trajectories for area and thickness ( raznahan et al . , 2011 ; shaw et al . , we found that regions displaying group differences in surface area and cortical thickness overlapped to a limited extent ( as shown in fig . a4 ) . regions displaying overlapping effects were mainly located on the mesial aspect of the hemispheres ; anteriorly in the anterior portion of the sfg , medial orbitofrontal cortex , and anterior cingulate , and posteriorly in the pericalcarine sulcus and cuneus . the vlbw children in the present study were born between 23 and 35 weeks of gestation , which is a particularly sensitive period of neural migration and rapid cortical development . disorders of migration are more likely to occur in the second trimester ( zhang et al . , 2013 ) by either under - migration or over - migration of neurons , and preterm birth may affect processes like neuronal migration , synaptogenesis and apoptosis late in the 2nd and early 3rd trimesters ( tau and peterson , 2010 ) resulting in the kind of deviant cortical thickness and reduced surface area observed here . the migration of neuroprogenitor cells may be hindered in preterm children by germinal matrix hemorrhages that can destroy neuronal precursors , or by injury to guiding glial cells ( volpe , 2009 ) . a reduced pool of neuroprogenitor cells and deficient migration can lead to a reduced number of founder cells in the ventricular zone and number of cerebral columns , which may result in decreased surface area ( rakic , 1995 ) . however , in our study only three out of 37 vlbw children had intraventricular hemorrhages and none had focal pvl , suggesting that focal perinatal brain injury is probably not the cause of the cortical deviations seen in our vlbw group . within 2832 weeks of gestation a fast emergence of short - range connectivity , in addition to the long - range association pathways , is observed ( takahashi et al . , 2012 ) . in the vlbw population , reduced fractional anisotropy has been reported in the inferior longitudinal and the longitudinal occipito - frontal fascicles ( eikenes et al . , 2011 ; skranes et al . , 2007 ) . whether diffuse white matter injury causing disrupted connectivity and cortical reorganization leads to reductions in surface area and increased cortical thickness in the vlbw is not known , but can not be excluded as an explanation for the presently observed cortical changes . the frontal , parietal and occipital regions with deviant cortical surface area and/or increased thickness in the vlbw children are all regions involved in networks receiving long - range association tracts . we therefore speculate that the deviations seen in cortical morphology in the vlbw group may be both primary changes due to cortical maldevelopment as well as secondary to altered white matter microstructure and connectivity . a positive association between cortical surface area and iq was observed in both vlbw and control subjects , albeit as a non - significant trend in the vlbw group . however , the magnitude of the effect was larger in frontal , temporal and medial parietal regions in the vlbw group than that in the control group , although these structure function associations survived significance testing only in the control group due to its larger sample size . the frontal regions in which surface area was related with iq included the caudal middle frontal gyrus , lateral orbitofrontal gyrus , medial orbitofrontal gyrus , pars orbitalis , rostral anterior cingulate , frontal pole and insula . these are regions where the vlbw children have significantly reduced surface area in comparison with controls , and are believed to be important for cognitive functions such as decision making , executive functions , semantics , attention , and working memory . previous studies have consistently shown poorer executive abilities in individuals born with vlbw than term - born peers , as well as problems with attention and working memory ( aarnoudse - moens et al . , 2009 ; bayless and stevenson , 2007 ; anderson , 2014 ; lohaugen et al . , 2010;anderson et al . , 2004 ) , and in the present study , the vlbw group had lower iq scores than those of the controls . our results indicate that a larger surface area is positively correlated to higher iq , consistent with skranes et al . ( 2013 ) , and it is tempting to speculate that reduced cognitive function in the vlbw group may be caused , at least in part , by the observed reduction in surface area . there was a negative association , albeit not significant , between iq and cortical thickness in both groups . in the vlbw group , the regions with the strongest negative associations between iq and cortical thickness were also the regions where the vlbw children displayed thicker cortex than controls . this is consistent with previous studies of the relationship between cortical thickness and cognitive functions in normally developing 511 year old children , where cortical thinning in the left dorsal frontal and parietal lobes was correlated with improved verbal performance ( sowell et al . , 2004 ) . moreover , in vlbw adolescents with low iq , full iq and cortical thickness were negatively correlated in the frontal and positively correlated in the parietal lobes ( bjuland et al . , 2013 ) . taken together , our findings suggest that altered cortical development in vlbw children seems to affect their cognitive abilities ; however , longitudinal studies are needed to determine whether these deviations persist during further brain maturation throughout school age , adolescence and into early adulthood also for these recent year cohorts of vlbw children . the present study demonstrates altered development of cortical surface and cortical thickness in vlbw children born in 20032007 , and these deviations are associated with poorer cognitive abilities . the present brain morphological deviations are evident even in vlbw children without cerebral palsy who have received state of the art medical treatment in the perinatal period , and who did not present with focal brain injuries on neonatal ultrasonography .
children born prematurely with very low birth weight ( vlbw : bw 1500 g ) have an increased risk of preterm perinatal brain injury , which may subsequently alter the maturation of the brain , including the cerebral cortex . the aim of study was to assess cortical thickness and surface area in vlbw children compared with term - born controls , and to investigate possible relationships between cortical morphology and full iq . in this cross - sectional study , 37 vlbw and 104 term children born between the years 20032007 were assessed cognitively at 510 years of age , using age appropriate wechsler tests . the freesurfer software was used to obtain estimates of cortical thickness and surface area based on t1-weighted mri images at 1.5 tesla . the vlbw children had smaller cortical surface area bilaterally in the frontal , temporal , and parietal lobes . a thicker cortex in the frontal and occipital regions and a thinner cortex in posterior parietal areas were observed in the vlbw group . there were significant differences in full iq between groups ( vlbw m = 98 , sd = 9.71 ; controls m = 108 , sd = 13.57 ; p < 0.001 ) . there was a positive relationship between iq and surface area in both groups , albeit significant only in the larger control group . in the vlbw group , reduced iq was associated with frontal cortical thickening and temporo - parietal thinning.we conclude that cortical deviations are evident in childhood even in vlbw children born in 20032007 who have received state of the art medical treatment in the perinatal period and who did not present with focal brain injuries on neonatal ultrasonography . the cortical deviations were associated with reduced cognitive functioning .
Introduction Material and methods Results Discussion
children born before week 32 with very - low - birth weight ( vlbw : birth weight 1500 g ) are more likely to need medical treatment during the perinatal period , and their immature nervous and cardiovascular systems render these children prone to focal brain injuries such as intraventricular hemorrhages and periventricular leukomalacia ( pvl ) ( volpe , 2009 ) . although perinatal care and medical treatment in the neonatal intensive care unit ( nicu ) have improved radically during the last decades with reduced incidence of focal brain injury , the immature brain and exposure to the harsh extra - uterine environment in the nicu are still believed to increase the risk of disrupted brain development in very preterm born survivors . the aim of the present study was to investigate cortical thickness and cortical surface area in 510 year old children born preterm with vlbw and term - born controls . however , these vlbw young adults were born in 19861988 , and it is unclear whether the same relationships between cortical morphology and cognitive function exist for school aged vlbw children born after year 2000 , who have received modern neonatal intensive care . the children born prematurely with very low birth weight ( vlbw ) ( birth weight 1500 g ) were recruited based on admittance to the neonatal intensive care unit ( nicu ) at st . there were significant differences in cortical surface area between the vlbw and the control group . the vlbw group showed bilateral reduction in cortical surface area in the frontal , temporal , and parietal lobes ( fig . table 2 lists all cortical regions with significant group differences in surface area as determined by the glm ( after 5% fdr correction ) , where 20 out of 35 regions had 90% reduction of cortical surface area in the vlbw group compared with controls . in the control group , there were widespread cortical regions in both hemispheres showing a significant relationship between full iq scores and cortical surface area ( see appendix fig . the vlbw group showed significantly thicker cortex in the frontal ( medial orbitofrontal gyrus , rostral anterior cingulate , frontal pole ) and occipital regions ( pericalcarine sulcus ) bilaterally , and a thinner cortex in the right posterior parietal lobe compared with controls ( fig . demonstrate a trend - level negative relationship between cortical thickness and full iq in both groups in widespread cortical regions , i.e. some temporal and parietal regions showed a positive relationship to full iq in the vlbw group , i.e. cortical area was reduced in frontal , temporal , parietal , and occipital regions , and cortical thickness was increased in the medial frontal and occipital lobes , in the vlbw group . the vlbw children showed significantly reduced cortical surface area in frontal , temporal , posterior parietal and medial occipital regions , as well as the right anterior cingulate ( see fig . the vlbw children showed thicker cortex in the frontal and occipital lobes bilaterally , which is consistent with previous studies reporting increased cortical thickness in both children and adolescents born prematurely ( bjuland et al . this would be consistent with mrner - lavancy 's ( 2014 ) study of vlbw children and term - born controls , 712 years old , which reported thicker frontal and parietal cortices in the youngest vlbw children compared to controls but no such group difference in the oldest children . however , in our study only three out of 37 vlbw children had intraventricular hemorrhages and none had focal pvl , suggesting that focal perinatal brain injury is probably not the cause of the cortical deviations seen in our vlbw group . the frontal , parietal and occipital regions with deviant cortical surface area and/or increased thickness in the vlbw children are all regions involved in networks receiving long - range association tracts . a positive association between cortical surface area and iq was observed in both vlbw and control subjects , albeit as a non - significant trend in the vlbw group . however , the magnitude of the effect was larger in frontal , temporal and medial parietal regions in the vlbw group than that in the control group , although these structure function associations survived significance testing only in the control group due to its larger sample size . in the vlbw group , the regions with the strongest negative associations between iq and cortical thickness were also the regions where the vlbw children displayed thicker cortex than controls . this is consistent with previous studies of the relationship between cortical thickness and cognitive functions in normally developing 511 year old children , where cortical thinning in the left dorsal frontal and parietal lobes was correlated with improved verbal performance ( sowell et al . the present study demonstrates altered development of cortical surface and cortical thickness in vlbw children born in 20032007 , and these deviations are associated with poorer cognitive abilities . the present brain morphological deviations are evident even in vlbw children without cerebral palsy who have received state of the art medical treatment in the perinatal period , and who did not present with focal brain injuries on neonatal ultrasonography .
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the fgf family consists of 18 receptor - binding members that regulate a broad spectrum of cellular activities 1 . the fgf elicits its regulatory signals via activating the fgfr tyrosine kinases encoded by four highly homologous genes . frs2 , also called snt1 for suc13-associating neurotropic factor target 1 , is a broadly expressed membrane - anchored adaptor protein that is required for the fgf to activate the map and pi3 kinase pathways , the two major pathways in the fgf signaling cascade 2 - 4 . the frs2 family has two highly homologous members , frs2 and frs2. frs2 is broadly expressed in adult and fetal tissues , whereas frs2 is more restrictively expressed 5 - 7 . frs2 null mouse embryos die between embryonic ( e ) 7.0 - 7.5 days 5 . although frs2 can compensate for the loss of frs2 with respect to mapk activation in mouse embryonic fibroblast ( mef ) cells 8 , recent reports demonstrate that frs2 and frs2 do not always mediate the same signals 9 - 12 . frs2 and frs2 share similar structure domains , which include the n - terminal myristylation site that anchors frs2 to the cell membrane , the phosphotyrosine - binding ( ptb ) domain required for binding to the fgfr , and the c - terminal sequence that contains multiple tyrosine and serine / threonine phosphorylation sites . the ptb - binding domain of the fgfr does not include a phosphorylated tyrosine residue , which is different from the ptb - binding sites of other growth factor receptors 13 . the ptb domain of frs2 also binds cks1 , a molecule that triggers degradation of cell cycle regulatory protein p27 during the g1/s transition in the cell cycle 14 . although not essential 15 , a vt ( valine - threonine ) motif encoded by alternatively spliced sequences in the intracellular juxtamembrane domain of fgfr1 and fgfr2 is important for association with the ptb domain 3,13,16 - 18 . interestingly , the binding of fgfr1 to frs2 , but not frs2 , is enhanced by receptor autophosphorylation 15 . frs2 has six tyrosine phosphorylation sites . among them , tyr196 , tyr306 , tyr349 , and tyr392 are grb2-binding sites that have been shown to be important for transmitting the signals to the pi3k / akt pathway . tyr436 and tyr471 are shp2-binding sites that have been shown important for transmitting the signals to the map kinase pathway 19 - 21 . mice expressing a frs2 mutant that lacks the shp2-binding sites exhibit severe developmental defects ; those that lack the grb2-binding sites have less severe defects 19,22 . fgf stimulation also causes phosphorylations on multiple serine / threonine residues of frs2 , which provides a negative feedback for the fgf signaling activity 23 . frs2 phosphorylation appears to be fgfr isoform - specific , which may result in differential recruitments of downstream signaling molecules and , thus , contribute to signaling specificity of the fgfr 24,25 . we reported previously that the four grb2-binding , but not the two shp2-binding sites , are essential for fgfr1 to activate the fgf - inducible response element ( fire ) of the mouse syndecan 1 gene 15 , which is an enhancer required for the fgf to promote syndecan 1 expression and has been widely used as a reporter for the fgf signaling pathway 15,24,26 . process for degrading and recycling various cellular constituents , such as long - lived proteins and entire organelles . autophagy initiates with the formation of autophagosomes in which the isolation membrane engulfs cellular constituents 27 . autophagosomes then fuse with lysosomes to form autolysosomes where the contents are degraded by acidic lysosomal hydrolases . as a self - digestion system , autophagy may influence cell survival , proliferation , and differentiation by accelerating turnover of old protein or organelles 28 . however , whether autophagy can be regulated by the fgf signaling axis is currently unknown . here , we show that frs2 is required for the fgf signaling axis to activate the mtor pathway and to suppress the autophagy activity in mefs . ablation of frs2 , as well as inhibition of fgfr or pi3k , but not erk , kinase activity suppressed fgf to activate the mtor pathway and to inhibit autophagy . thus , the results , for the first time , demonstrate that the frs2-mediated mtor pathway is required for the fgf signaling axis to regulate autophagy , and suggest a new mechanism by which the fgf elicits its regulatory signals . all animals were housed in the program for animal resources of the institute of biosciences and technology , and handled in accordance with the principles and procedures of the guide for the care and use of laboratory animals . the embryos were harvested at embryonic day 14.5 for establishing mef cultures as described 15 . briefly , e14.5 embryos carrying homozygous frs2 were minced in 3 ml ice - cold 0.25% trypsin - edta solution ( sigma - aldrich , st . louis , mo ) , incubated at 4 c overnight , and then 37 c for 30 minutes in the same solution . the isolated cells were propagated at a ratio of 1:3 in 6-well plates and maintained in 5% fetal bovine serum / dmem until being used . the mefs were then immortalized by transfection with prsv - tag plasmid that carried the sv40 t antigens . ad5-cmv - cre - gfp and ad5-cmv - gfp viruses from the center for cell and gene therapy ( houston , tx ) were used to delete the floxed fragment . overnight cultured mefs ( 1x10 cells in 6-well plates ) were transfected with 5 g of the indicated frs2 mutants in pcdnazeo plasmids ( invitrogen corporation , la jolla , ca ) and 5 l lipofectamine ( invitrogen corporation ) . ( camarillo , ca ) , and pi3k ( ly294002 ) was from cell signaling ( beverly , ma ) . the cells were incubated at 37 c for 24 hours , and the culture media were then changed to a serum - free dmem containing 10 g / ml heparin for serum starvation . after incubation at 37 c for 12 hours , the cells were stimulated with fgf2 at the indicated concentrations for the indicated times before being lysed with 200 l ripa buffer containing the protease - phosphatase inhibitor cocktail ( sigma - aldrich , inc . the cell lysates equivalent to 50 g total proteins were separated on sds - pages and blotted onto pvdf membranes for western analyses with the indicated antibodies . the sources and dilution of the antibodies are : phosphorylated frs2 tyr196 ( 1:1000 ) , phosphorylated erk1/2 ( 1:1000 ) , phosphorylated akt thr308 ( 1:1000 ) , phosphorylated akt ser473 ( 1:1000 ) , and phosphorylated s6k1 thr389 ( 1:1000 ) from cell signaling technology inc . ( danvers , ma ) ; human lc3 ( 1:500 ) from novus biologicals , llc ( littleton , co ) ; and -actin ( 1 : 5000 ) from santa cruz biotechnology , inc . , ( santa cruz , ca ) . the specifically bound primary antibodies were detected with the horseradish peroxidase conjugated secondary antibodies and visualized with the ecl - plus chemoluminescent reagents . we previously reported that the mef cells derived from frs2 floxed embryos are able to respond to fgf signals 15 . since the frs2-mediated pi3k / akt pathway is an upstream regulator of the mtor pathway , we investigated whether fgf activated mtor . mef cells were treated with fgf2 at the final concentration of 5 ng / ml , and lysed at various time points . activation of the fgf signaling axis and mtor pathway was analyzed by western blot . treating with akt and erk1/2 , the major downstream molecules of the fgf signaling axis , were also strongly phosphorylated within 5 minutes , which were then gradually reduced . s6k1 , the major substrate of the mtor c1 complex , was also strongly phosphorylated , although the phosphorylation was slightly delayed . consistently , phosphorylation of mtor s2448 , an s6k1 phosphorylation site 30 , was increased at a lagging mode . together , the results demonstrate that the fgf signaling axis induces activation of the mtor pathway . frs2 is an adaptor protein in the fgf signaling pathway , which recruits multiple downstream pathways , including the erk1/2 and pi3k / akt pathways , to the fgfr kinase . to investigate whether frs2 was required for fgf2 to activate the mtor pathway , mefs bearing homozygous frs2 floxed alleles were treated with adenovirus - cre / gfp to delete the floxed fragment unlike mefs bearing floxed frs2 alleles that exhibited strong activation of erk1/2 , akt , and s6k1 by fgf2 , mefs bearing frs2 null alleles failed to respond to fgf2 with respect to erk1/2 , akt , and s6k1 phosphorylation ( fig . 2b ) . the weak activation of erk activation in frs2 null mefs were likely due to frs2-independent pathways . the results indicated that frs2 was required for the fgf signaling axis to activate the mtor pathway . interestingly , the background phosphorylation of akt s473 , an mtor - rictor phosphorylation site 31 , was significantly increased in frs2 null mef . this is not surprising since frs2 has been shown to play important roles in cell signaling feedback regulation 32 . to confirm that the results were not associated with the cloning procedure , primary mefs were isolated from frs2 floxed embryos and infected either with adenovirus - gfp or adenovirus - cre / gfp to repeat the experiments . the results demonstrated that ablation of the frs2 alleles significantly impaired activation of akt , erk , and mtor by fgf2 ( fig . 2c ) , which confirmed that frs2 was required for fgf2 to activate the mtor pathway . in addition to the frs2-independent pathway , incomplete disruption of frs2 in primary mefs might also contribute to compromised phosphorylation of erk1/2 induced by fgf2 . frs2 has 6 tyrosine phosphorylation sites of which 4 are grab2 binding that are important for pi3k / akt activation and 2 are shp2 binding that are important for map kinase activation 19 . both pi3k / akt and map kinase pathways can be linked to the mtor pathway 33 . to determine which pathway is required for fgf2 to activate mtor in mefs , inhibitor specific for either pi3k or mek1/2 inhibition of pi3k , but not mek1/2 , abolished the phosphorylation of s6k1 , indicating that only the pi3k / akt pathway was required for fgf2 to activate the mtor pathway ( fig . 3 ) . interestingly , inhibition of the map kinase pathway significantly increased the phosphorylation of akt t308 and s473 , which were catalyzed by the pi3k / pdpk1 ( phosphoinositide dependent kinase 1 ) and mtor c2 , respectively . thus , the results suggested that inhibition of the map kinase pathway sustained or promoted activation of the akt - mtor signaling axis . the results are in line with the reports that sustained activation of the map kinase provides a feedback control mechanism of the fgf signaling axis 23 . the mtor pathway is a major signaling pathway that inhibits autophagy 34 . to investigate whether fgf signaling regulated autophagy , we then assessed the influence of fgf2 on lc3 ii abundance . lc3 , also called microtubule associated light chain 3 , is a broadly used autophagy indicator . the full - length prolc3 is processed to its cytosolic form , lc3 i , which is activated by conversion to its lipidated form , lc3 ii . the lipidated lc3 ii is translocated to preautophagosomes and autophagosomes , which are then degraded after fusing with lysosomes . thus , the increased abundance of lc3 ii reflects enhanced autophagic activity or reduced autophagosome turnover 35,36 . to determine whether the fgf signaling axis regulated autophagy , the mefs with either frs2 floxed or null alleles were treated with fgf2 , and the abundance of lc3 was assessed by western blot . treating the frs2 floxed mefs with fgf2 enhanced s6k1 phosphorylation and reduced the abundance of the lc3 ii isoform , suggesting that fgf2 regulated autophagy , likely through the mtor pathway . consistent with the result that fgf2 treatment did not induce s6k1 phosphorylation in frs2 null mefs ( fig . 2b ) , fgf2 treatment also did not reduce lc3 ii abundance in frs2 null mefs ( fig . 4a ) , indicating that fgf2 regulated the autophagy activity through frs2-mediated pathway(s ) . as the abundance of the lc3 ii isoform was dynamically controlled , the variation of lc3 ii abundance alone may reflect either an inhibition of lc3 i to lc3 ii conversion or an activation of lc3 ii turnover . atg1 and atg13 are substrates of the mtor c1 kinase that is activated by the pi3k / akt pathway . phosphorylation of atg1 and atg13 by mtor c1 changes the conformation of the atg1 complex to an conformation , resulting in suppression of autophagy 34 . as s6k1 is a substrate of the mtor c1 kinase , which is often used as readout for mtor c1 activity , the data of enhanced s6k1 phosphorylation in fgf2 treated mefs inclines toward the possibility that autophagic activity is repressed by increased mtor c1 activity . to clarify how fgf2 regulated autophagy , the mefs were treated with bafilomycin a1 although blocking the degradation of lc3 ii by suppression of fusions between autophagosomes and lysosomes increased the abundance of lc3 ii either in the presence or absence of fgf2 , treating the cells with fgf2 still significantly reduced the abundance of the lc3 ii isoform , indicating less lc3 ii formation ( fig . 4b ) . therefore , the results indicate fgf2 suppresses lc3 i to lc3 ii conversion , and thus , the autophagy initiation in cells . to determine whether the mtor pathway was required for fgf2 to suppress autophagy , rapamycin , an mtor c1 inhibitor , was added to the mef cultures . treating with rapamycin suppressed the phosphorylation of s6k1 and increased the abundance of the lc3 ii in fgf2 treated mefs ( fig . 4c ) , although it did not affect akt and erk1/2 phosphorylation ( data not shown ) . separate experiments revealed that treating with rapamycin alone did not increase lc3 ii abundance in mefs ( fig . the results suggest that activation of mtor c1 is required for fgf2 to suppress the autophagy activity . further experiments with other autophagy markers , including expression of p62 , atg5 , atg12 , etc . reinstatement of full length frs2 expression in frs2 null mefs by transfection restored regulation of fgf2 on the autophagy activity ( fig . 5 ) . to determine whether the grb2- or shp2-binding sites were required for regulating autophagy , mutants lacking the four grb2- or two shp2-binding sites were expressed in frs2 null mefs as described 15 . to better demonstrate the difference in lc3 ii abundance , only lightly exposed films were shown . consistent with the chemical inhibition experiments demonstrating that the pi3k / akt , but not erk , pathway was required for fgf2 to suppress autophagy , expression of the frs2 mutant lacking the grb2-binding sites ( y196 , y306 , y349 , and y392 ) failed to restore the activity , whereas expression of frs2 lacking the shp2 binding sites ( y436 and y471 ) restored the response to fgf2 with respect to autophagy inhibition . the results again demonstrate that the fgf signaling pathway regulates autophagy through the grb2-mediated pi3k / akt pathway . there are four grb2-binding sites on frs2. to further investigate which grb2-binding phosphorylation sites were required for frs2 to mediate the autophagy regulation signals , mutant frs2 carrying an individual grb2-binding site mutation were expressed in frs2 null mefs . expression of mutants with a substitution of either y196 or y306 with phenylalanine rescued the fgf activity to inhibit autophagy activity , whereas expression of the mutant with a substitution of either y349 or y392 with phenylalanine failed to rescue such defects ( fig . the results indicate that y349 and y392 , but not y196 and y306 , are required for the fgf to suppress autophagy , and imply that each grb2-binding site may mediate a subset of fgf signals . activation of pi3-kinase is mediated by assembly of gab1 to the grb2/frs2 complex , which enables tyrosine phosphorylation of gab1 by fgfr to generate binding sites for p85 , the regulatory subunit of pi3-kinase , thus resulting in recruitment and activation of the pi3-kinase 38 . although all four grb2-binding sites are involved in activation of the pi3k / akt pathway in 3t3 cells 2 , only two grb2-binding sites are involved in autophagy regulation in mefs . the molecular mechanism underlying the results suggest that the autophagy - suppressing signals of the fgf signaling axis are different from the fire - activating signals that are mediated by the four grb2-binding sites additively , since substitution of each grb2-binding site only partially reduces fire activation activity 15 . the fire consists of a 170-bp array of five dna motifs that bind two fgf - inducible fos - jun heterodimers , one inducible ap-2-related protein , one constitutively expressed upstream stimulatory factor , and one constitutive 46-kda transcription factor . it has been shown that fire is selectively activated by the fgf , but not by other tyrosine kinase receptor - activating growth factors 26 . furthermore , the fgf activates fire independent of its mitogenic activity in prostate cancer cells 24 . here we showed that the signals of fgf to regulate the mtor / autophagy pathway was different from those for fire activation , and further demonstrate mechanistic diversity of the fgf signaling axis . furthermore , it has been reported that the lc3 antibody exhibits different detection sensitivities toward lc3 i and lc3 ii 40 . therefore , future efforts to monitor expression of other autophagic markers , such as p62 , atg5 , and atg12 , etc . , will be needed to confirm the findings and to elucidate the molecular mechanism underlying how fgf signaling regulates autophagic activity . autophagy is a major cellular pathway to degrade bulky subcellular organelles and macromolecules , and plays important roles in development , metabolism , tumorigenesis , and diseases 41 . however , how autophagy contributes to development is not understood , although it has been proposed that autophagy may influence cell differentiation either by impairing new protein or organelle formation or by accelerating turnover of old proteins or organelles . in this study , we provided the first in vitro evidence that autophagy can be negatively regulated by the fgf signaling axis . recently , we also discovered that the fgf regulates cardiac progenitor cell and cardiomyocyte differentiation via controlling the mtor pathway - regulated autophagy , and that the frs2-mediated pathways are required for the fgf to suppress premature differentiation of heart progenitor cells . inhibition of autophagic activity suppresses cardiomyocyte differentiation both in the second heart field progenitors and in embryoid body ( eb ) cultures ( 42 ) . in summary , the fgf signaling axis activated the mtor kinase and repressed autophagy by activating the frs2-mediated pi3k / akt pathway . suppression of mtor activation abolished the repression activity of fgf2 on autophagy , indicating that fgf regulated autophagy via the mtor pathway . this is the first report that the fgf signaling axis plays a crucial role in autophagy regulation , thus , sheding new light on cell signaling mechanisms . we previously reported that the mef cells derived from frs2 floxed embryos are able to respond to fgf signals 15 . since the frs2-mediated pi3k / akt pathway is an upstream regulator of the mtor pathway , we investigated whether fgf activated mtor . mef cells were treated with fgf2 at the final concentration of 5 ng / ml , and lysed at various time points . activation of the fgf signaling axis and mtor pathway was analyzed by western blot . treating with akt and erk1/2 , the major downstream molecules of the fgf signaling axis , were also strongly phosphorylated within 5 minutes , which were then gradually reduced . s6k1 , the major substrate of the mtor c1 complex , was also strongly phosphorylated , although the phosphorylation was slightly delayed . consistently , phosphorylation of mtor s2448 , an s6k1 phosphorylation site 30 , was increased at a lagging mode . together , the results demonstrate that the fgf signaling axis induces activation of the mtor pathway . frs2 is an adaptor protein in the fgf signaling pathway , which recruits multiple downstream pathways , including the erk1/2 and pi3k / akt pathways , to the fgfr kinase . to investigate whether frs2 was required for fgf2 to activate the mtor pathway , mefs bearing homozygous frs2 floxed alleles were treated with adenovirus - cre / gfp to delete the floxed fragment . 2a ) . unlike mefs bearing floxed frs2 alleles that exhibited strong activation of erk1/2 , akt , and s6k1 by fgf2 , mefs bearing frs2 null alleles failed to respond to fgf2 with respect to erk1/2 , akt , and s6k1 phosphorylation ( fig . 2b ) . the weak activation of erk activation in frs2 null mefs were likely due to frs2-independent pathways . the results indicated that frs2 was required for the fgf signaling axis to activate the mtor pathway . interestingly , the background phosphorylation of akt s473 , an mtor - rictor phosphorylation site 31 , was significantly increased in frs2 null mef . this is not surprising since frs2 has been shown to play important roles in cell signaling feedback regulation 32 . to confirm that the results were not associated with the cloning procedure , primary mefs were isolated from frs2 floxed embryos and infected either with adenovirus - gfp or adenovirus - cre / gfp to repeat the experiments . the results demonstrated that ablation of the frs2 alleles significantly impaired activation of akt , erk , and mtor by fgf2 ( fig . 2c ) , which confirmed that frs2 was required for fgf2 to activate the mtor pathway . in addition to the frs2-independent pathway , incomplete disruption of frs2 in primary mefs might also contribute to compromised phosphorylation of erk1/2 induced by fgf2 . frs2 has 6 tyrosine phosphorylation sites of which 4 are grab2 binding that are important for pi3k / akt activation and 2 are shp2 binding that are important for map kinase activation 19 . both pi3k / akt and map kinase pathways can be linked to the mtor pathway 33 . to determine which pathway is required for fgf2 to activate mtor in mefs , inhibitor specific for either pi3k or mek1/2 inhibition of pi3k , but not mek1/2 , abolished the phosphorylation of s6k1 , indicating that only the pi3k / akt pathway was required for fgf2 to activate the mtor pathway ( fig . 3 ) . interestingly , inhibition of the map kinase pathway significantly increased the phosphorylation of akt t308 and s473 , which were catalyzed by the pi3k / pdpk1 ( phosphoinositide dependent kinase 1 ) and mtor c2 , respectively . thus , the results suggested that inhibition of the map kinase pathway sustained or promoted activation of the akt - mtor signaling axis . the results are in line with the reports that sustained activation of the map kinase provides a feedback control mechanism of the fgf signaling axis 23 . the mtor pathway is a major signaling pathway that inhibits autophagy 34 . to investigate whether fgf signaling regulated autophagy , we then assessed the influence of fgf2 on lc3 ii abundance . lc3 , also called microtubule associated light chain 3 , is a broadly used autophagy indicator . the full - length prolc3 is processed to its cytosolic form , lc3 i , which is activated by conversion to its lipidated form , lc3 ii . the lipidated lc3 ii is translocated to preautophagosomes and autophagosomes , which are then degraded after fusing with lysosomes . thus , the increased abundance of lc3 ii reflects enhanced autophagic activity or reduced autophagosome turnover 35,36 . to determine whether the fgf signaling axis regulated autophagy , the mefs with either frs2 floxed or null alleles were treated with fgf2 , and the abundance of lc3 was assessed by western blot . treating the frs2 floxed mefs with fgf2 enhanced s6k1 phosphorylation and reduced the abundance of the lc3 ii isoform , suggesting that fgf2 regulated autophagy , likely through the mtor pathway . consistent with the result that fgf2 treatment did not induce s6k1 phosphorylation in frs2 null mefs ( fig . 2b ) , fgf2 treatment also did not reduce lc3 ii abundance in frs2 null mefs ( fig . 4a ) , indicating that fgf2 regulated the autophagy activity through frs2-mediated pathway(s ) . as the abundance of the lc3 ii isoform was dynamically controlled , the variation of lc3 ii abundance alone may reflect either an inhibition of lc3 i to lc3 ii conversion or an activation of lc3 ii turnover . atg1 and atg13 are substrates of the mtor c1 kinase that is activated by the pi3k / akt pathway . phosphorylation of atg1 and atg13 by mtor c1 changes the conformation of the atg1 complex to an conformation , resulting in suppression of autophagy 34 . as s6k1 is a substrate of the mtor c1 kinase , which is often used as readout for mtor c1 activity , the data of enhanced s6k1 phosphorylation in fgf2 treated mefs inclines toward the possibility that autophagic activity is repressed by increased mtor c1 activity . to clarify how fgf2 regulated autophagy , the mefs were treated with bafilomycin a1 to suppress the fusion between autophagosomes and lysosomes 37 . although blocking the degradation of lc3 ii by suppression of fusions between autophagosomes and lysosomes increased the abundance of lc3 ii either in the presence or absence of fgf2 , treating the cells with fgf2 still significantly reduced the abundance of the lc3 ii isoform , indicating less lc3 ii formation ( fig . 4b ) . therefore , the results indicate fgf2 suppresses lc3 i to lc3 ii conversion , and thus , the autophagy initiation in cells . to determine whether the mtor pathway was required for fgf2 to suppress autophagy , rapamycin , an mtor c1 inhibitor , was added to the mef cultures . treating with rapamycin suppressed the phosphorylation of s6k1 and increased the abundance of the lc3 ii in fgf2 treated mefs ( fig . 4c ) , although it did not affect akt and erk1/2 phosphorylation ( data not shown ) . separate experiments revealed that treating with rapamycin alone did not increase lc3 ii abundance in mefs ( fig . the results suggest that activation of mtor c1 is required for fgf2 to suppress the autophagy activity . further experiments with other autophagy markers , including expression of p62 , atg5 , atg12 , etc . reinstatement of full length frs2 expression in frs2 null mefs by transfection restored regulation of fgf2 on the autophagy activity ( fig . 5 ) . to determine whether the grb2- or shp2-binding sites were required for regulating autophagy , mutants lacking the four grb2- or two shp2-binding sites were expressed in frs2 null mefs as described 15 . to better demonstrate the difference in lc3 ii abundance , only lightly exposed films were shown . therefore , lc3 i bands in this figure are too weak to be seen . consistent with the chemical inhibition experiments demonstrating that the pi3k / akt , but not erk , pathway was required for fgf2 to suppress autophagy , expression of the frs2 mutant lacking the grb2-binding sites ( y196 , y306 , y349 , and y392 ) failed to restore the activity , whereas expression of frs2 lacking the shp2 binding sites ( y436 and y471 ) restored the response to fgf2 with respect to autophagy inhibition . the results again demonstrate that the fgf signaling pathway regulates autophagy through the grb2-mediated pi3k / akt pathway . there are four grb2-binding sites on frs2. to further investigate which grb2-binding phosphorylation sites were required for frs2 to mediate the autophagy regulation signals , mutant frs2 carrying an individual grb2-binding site mutation were expressed in frs2 null mefs . expression of mutants with a substitution of either y196 or y306 with phenylalanine rescued the fgf activity to inhibit autophagy activity , whereas expression of the mutant with a substitution of either y349 or y392 with phenylalanine failed to rescue such defects ( fig . the results indicate that y349 and y392 , but not y196 and y306 , are required for the fgf to suppress autophagy , and imply that each grb2-binding site may mediate a subset of fgf signals . activation of pi3-kinase is mediated by assembly of gab1 to the grb2/frs2 complex , which enables tyrosine phosphorylation of gab1 by fgfr to generate binding sites for p85 , the regulatory subunit of pi3-kinase , thus resulting in recruitment and activation of the pi3-kinase 38 . although all four grb2-binding sites are involved in activation of the pi3k / akt pathway in 3t3 cells 2 , only two grb2-binding sites are involved in autophagy regulation in mefs . the molecular mechanism underlying the results suggest that the autophagy - suppressing signals of the fgf signaling axis are different from the fire - activating signals that are mediated by the four grb2-binding sites additively , since substitution of each grb2-binding site only partially reduces fire activation activity 15 . the fire consists of a 170-bp array of five dna motifs that bind two fgf - inducible fos - jun heterodimers , one inducible ap-2-related protein , one constitutively expressed upstream stimulatory factor , and one constitutive 46-kda transcription factor . it has been shown that fire is selectively activated by the fgf , but not by other tyrosine kinase receptor - activating growth factors 26 . furthermore , the fgf activates fire independent of its mitogenic activity in prostate cancer cells 24 . here we showed that the signals of fgf to regulate the mtor / autophagy pathway was different from those for fire activation , and further demonstrate mechanistic diversity of the fgf signaling axis . although widely used , lc3 lipidation is not a perfect marker for autophagy 39 . furthermore , it has been reported that the lc3 antibody exhibits different detection sensitivities toward lc3 i and lc3 ii 40 . therefore , future efforts to monitor expression of other autophagic markers , such as p62 , atg5 , and atg12 , etc . , will be needed to confirm the findings and to elucidate the molecular mechanism underlying how fgf signaling regulates autophagic activity . autophagy is a major cellular pathway to degrade bulky subcellular organelles and macromolecules , and plays important roles in development , metabolism , tumorigenesis , and diseases 41 . however , how autophagy contributes to development is not understood , although it has been proposed that autophagy may influence cell differentiation either by impairing new protein or organelle formation or by accelerating turnover of old proteins or organelles . in this study , we provided the first in vitro evidence that autophagy can be negatively regulated by the fgf signaling axis . recently , we also discovered that the fgf regulates cardiac progenitor cell and cardiomyocyte differentiation via controlling the mtor pathway - regulated autophagy , and that the frs2-mediated pathways are required for the fgf to suppress premature differentiation of heart progenitor cells . inhibition of autophagic activity suppresses cardiomyocyte differentiation both in the second heart field progenitors and in embryoid body ( eb ) cultures ( 42 ) . in summary , the fgf signaling axis activated the mtor kinase and repressed autophagy by activating the frs2-mediated pi3k / akt pathway . suppression of mtor activation abolished the repression activity of fgf2 on autophagy , indicating that fgf regulated autophagy via the mtor pathway . this is the first report that the fgf signaling axis plays a crucial role in autophagy regulation , thus , sheding new light on cell signaling mechanisms .
although the fibroblast growth factor ( fgf ) signaling axis plays important roles in cell survival , proliferation , and differentiation , the molecular mechanism underlying how the fgf elicits these diverse regulatory signals is not well understood . by using the frs2 null mouse embryonic fibroblast ( mef ) in conjunction with inhibitors to multiple signaling pathways , here we report that the fgf signaling axis activates mtor via the fgf receptor substrate 2 ( frs2)-mediated pi3k / akt pathway , and suppresses autophagy activity in mefs . in addition , the pi3k / akt pathway regulated mtor is crucial for the fgf signaling axis to suppress autophagy in mefs . since autophagy has been proposed to play important roles in cell survival , proliferation , and differentiation , the findings suggest a novel mechanism for the fgf signaling axis to transmit regulatory signals to downstream effectors .
Introduction Materials and Methods Results and Discussion FGF2 activates the mTOR pathway in a time-dependent manner FRS2 is required for FGF2 to activate the mTOR pathway FGF2 activates the mTOR pathway via the PI3K/Akt pathway FRS2 is required for FGF2 to inhibit autophagy through the mTOR pathway Two Grb2 binding sites of FRS2, Y349 and Y392, are required for FGF signals to suppress autophagy
although frs2 can compensate for the loss of frs2 with respect to mapk activation in mouse embryonic fibroblast ( mef ) cells 8 , recent reports demonstrate that frs2 and frs2 do not always mediate the same signals 9 - 12 . among them , tyr196 , tyr306 , tyr349 , and tyr392 are grb2-binding sites that have been shown to be important for transmitting the signals to the pi3k / akt pathway . as a self - digestion system , autophagy may influence cell survival , proliferation , and differentiation by accelerating turnover of old protein or organelles 28 . here , we show that frs2 is required for the fgf signaling axis to activate the mtor pathway and to suppress the autophagy activity in mefs . thus , the results , for the first time , demonstrate that the frs2-mediated mtor pathway is required for the fgf signaling axis to regulate autophagy , and suggest a new mechanism by which the fgf elicits its regulatory signals . frs2 is an adaptor protein in the fgf signaling pathway , which recruits multiple downstream pathways , including the erk1/2 and pi3k / akt pathways , to the fgfr kinase . this is not surprising since frs2 has been shown to play important roles in cell signaling feedback regulation 32 . consistent with the chemical inhibition experiments demonstrating that the pi3k / akt , but not erk , pathway was required for fgf2 to suppress autophagy , expression of the frs2 mutant lacking the grb2-binding sites ( y196 , y306 , y349 , and y392 ) failed to restore the activity , whereas expression of frs2 lacking the shp2 binding sites ( y436 and y471 ) restored the response to fgf2 with respect to autophagy inhibition . the results again demonstrate that the fgf signaling pathway regulates autophagy through the grb2-mediated pi3k / akt pathway . the molecular mechanism underlying the results suggest that the autophagy - suppressing signals of the fgf signaling axis are different from the fire - activating signals that are mediated by the four grb2-binding sites additively , since substitution of each grb2-binding site only partially reduces fire activation activity 15 . here we showed that the signals of fgf to regulate the mtor / autophagy pathway was different from those for fire activation , and further demonstrate mechanistic diversity of the fgf signaling axis . , will be needed to confirm the findings and to elucidate the molecular mechanism underlying how fgf signaling regulates autophagic activity . in summary , the fgf signaling axis activated the mtor kinase and repressed autophagy by activating the frs2-mediated pi3k / akt pathway . this is the first report that the fgf signaling axis plays a crucial role in autophagy regulation , thus , sheding new light on cell signaling mechanisms . frs2 is an adaptor protein in the fgf signaling pathway , which recruits multiple downstream pathways , including the erk1/2 and pi3k / akt pathways , to the fgfr kinase . this is not surprising since frs2 has been shown to play important roles in cell signaling feedback regulation 32 . consistent with the chemical inhibition experiments demonstrating that the pi3k / akt , but not erk , pathway was required for fgf2 to suppress autophagy , expression of the frs2 mutant lacking the grb2-binding sites ( y196 , y306 , y349 , and y392 ) failed to restore the activity , whereas expression of frs2 lacking the shp2 binding sites ( y436 and y471 ) restored the response to fgf2 with respect to autophagy inhibition . the results again demonstrate that the fgf signaling pathway regulates autophagy through the grb2-mediated pi3k / akt pathway . the molecular mechanism underlying the results suggest that the autophagy - suppressing signals of the fgf signaling axis are different from the fire - activating signals that are mediated by the four grb2-binding sites additively , since substitution of each grb2-binding site only partially reduces fire activation activity 15 . here we showed that the signals of fgf to regulate the mtor / autophagy pathway was different from those for fire activation , and further demonstrate mechanistic diversity of the fgf signaling axis . , will be needed to confirm the findings and to elucidate the molecular mechanism underlying how fgf signaling regulates autophagic activity . in summary , the fgf signaling axis activated the mtor kinase and repressed autophagy by activating the frs2-mediated pi3k / akt pathway . this is the first report that the fgf signaling axis plays a crucial role in autophagy regulation , thus , sheding new light on cell signaling mechanisms .
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the present analysis was prespecified within the simvastatin ezetimibe in aortic stenosis ( seas ) study analysis plan . study design , baseline characteristics , and main outcome results of the seas study have previously been published . in short , 1873 men and women aged 45 to 85 years with asymptomatic mild - to - moderate as having a peak aortic jet velocity between 2.5 and 4.0 m / s by echocardiography were randomized to placebo or to combination treatment with simvastatin 40 mg and ezetimibe 10 mg daily . patients with known coronary heart disease , heart failure , diabetes mellitus , history of stroke or peripheral vascular disease , clinically significant mitral valve disease , severe or predominant aortic regurgitation , rheumatic valvular disease , aortic valve prosthesis , or renal insufficiency and patients already on lipid - lowering therapy or with a guideline indication for lipid - lowering therapy were not included in the seas study . core laboratory readings of peak aortic jet velocity and lv mass were available from baseline and at least 1 follow - up echocardiogram in 1656 patients ( 88% of the study population ) , who comprise the present study population . hypertension was defined as history of hypertension , use of antihypertensive drug treatment , or elevated blood pressure at the baseline clinic visits . all patients gave written informed consent , and the study was approved by ethics committees in all participating countries . echocardiograms were performed at baseline , annually and before planned aortic valve surgery following a standardized protocol at 173 study centers in 7 european countries . echocardiographic images were stored on videotapes , compact discs , or magnetic optical discs and forwarded for blinded interpretation at the seas echocardiography core laboratory at haukeland university hospital , bergen , norway , as previously published . lv mass was measured by an autopsy - validated method and indexed to body height in the allometric power of 2.7 . lv hypertrophy was defined using the prognostically validated cutoff values lv mass index > 46.7 g / m in women and relative wall thickness was assessed from 2lv posterior wall thickness / lv end - diastolic diameter ratio and considered increased if > 0.43 ( concentric lv geometry ) . pressure recovery was assessed at the aortic sinotubular junction and used for calculation of energy loss index as prognostically validated . the prespecified primary end point of seas was major cardiovascular events , a composite end point , including aortic valve related events ( combined aortic valve replacement , hospitalization for heart failure because of aortic stenosis , and death from cardiovascular causes ) and ischemic cardiovascular events ( combined death from cardiovascular causes , nonfatal myocardial infarction , hospitalization for unstable angina , coronary revascularization , and nonhemorrhagic stroke ) . all end points were classified by an independent end point classification committee whose members were unaware of study group assignments . we also assessed the post hoc defined composite end point of total mortality and hospitalization for heart failure because of aortic stenosis . statistical analysis was performed using ibm spss version 22.0 ( ibm corporation , armonk , ny ) . comparison between groups was performed by paired and unpaired t test , test , and general linear model with post hoc test and bonferroni adjustment as appropriate . cumulative incidences of cardiovascular events during follow - up were estimated by kaplan meier . meier plots were used to compare event - free survival in groups of patients with and without lv hypertrophy at baseline . correlates of the prespecified primary and secondary composite end points were identified by cox regression analysis in univariable and multivariable models and presented as hazard ratio and 95% confidence intervals . in the primary analyses , lv mass index was used as the continuous variable . in secondary models , lv hypertrophy as a dichotomous variable was used . age , sex , body mass index , peak aortic jet velocity , lv ejection fraction , concentric lv geometry , hypertension , and valvuloarterial impedance were included as covariates in all multivariable models . aortic valve replacement was included as a time - varying covariate in models assessing cardiovascular death and total mortality . in subsequent models , concentric lv geometry was replaced by stress - corrected midwall shortening , and the presence of aortic regurgitation was added . to take the progressive increase in lv mass during progression of as into account , time - varying cox regression analysis was used . two - tailed p<0.05 was regarded as statistically significant both in univariable and multivariable analyses . the present analysis was prespecified within the simvastatin ezetimibe in aortic stenosis ( seas ) study analysis plan . study design , baseline characteristics , and main outcome results of the seas study have previously been published . in short , 1873 men and women aged 45 to 85 years with asymptomatic mild - to - moderate as having a peak aortic jet velocity between 2.5 and 4.0 m / s by echocardiography were randomized to placebo or to combination treatment with simvastatin 40 mg and ezetimibe 10 mg daily . patients with known coronary heart disease , heart failure , diabetes mellitus , history of stroke or peripheral vascular disease , clinically significant mitral valve disease , severe or predominant aortic regurgitation , rheumatic valvular disease , aortic valve prosthesis , or renal insufficiency and patients already on lipid - lowering therapy or with a guideline indication for lipid - lowering therapy were not included in the seas study . core laboratory readings of peak aortic jet velocity and lv mass were available from baseline and at least 1 follow - up echocardiogram in 1656 patients ( 88% of the study population ) , who comprise the present study population . hypertension was defined as history of hypertension , use of antihypertensive drug treatment , or elevated blood pressure at the baseline clinic visits . all patients gave written informed consent , and the study was approved by ethics committees in all participating countries . echocardiograms were performed at baseline , annually and before planned aortic valve surgery following a standardized protocol at 173 study centers in 7 european countries . echocardiographic images were stored on videotapes , compact discs , or magnetic optical discs and forwarded for blinded interpretation at the seas echocardiography core laboratory at haukeland university hospital , bergen , norway , as previously published . lv mass was measured by an autopsy - validated method and indexed to body height in the allometric power of 2.7 . relative wall thickness was assessed from 2lv posterior wall thickness / lv end - diastolic diameter ratio and considered increased if > 0.43 ( concentric lv geometry ) . pressure recovery was assessed at the aortic sinotubular junction and used for calculation of energy loss index as prognostically validated . the prespecified primary end point of seas was major cardiovascular events , a composite end point , including aortic valve related events ( combined aortic valve replacement , hospitalization for heart failure because of aortic stenosis , and death from cardiovascular causes ) and ischemic cardiovascular events ( combined death from cardiovascular causes , nonfatal myocardial infarction , hospitalization for unstable angina , coronary revascularization , and nonhemorrhagic stroke ) . all end points were classified by an independent end point classification committee whose members were unaware of study group assignments . we also assessed the post hoc defined composite end point of total mortality and hospitalization for heart failure because of aortic stenosis . statistical analysis was performed using ibm spss version 22.0 ( ibm corporation , armonk , ny ) . comparison between groups was performed by paired and unpaired t test , test , and general linear model with post hoc test and bonferroni adjustment as appropriate . cumulative incidences of cardiovascular events during follow - up were estimated by kaplan meier . meier plots were used to compare event - free survival in groups of patients with and without lv hypertrophy at baseline . correlates of the prespecified primary and secondary composite end points were identified by cox regression analysis in univariable and multivariable models and presented as hazard ratio and 95% confidence intervals . in the primary analyses , lv mass index was used as the continuous variable . in secondary models , lv hypertrophy as a dichotomous variable was used . age , sex , body mass index , peak aortic jet velocity , lv ejection fraction , concentric lv geometry , hypertension , and valvuloarterial impedance were included as covariates in all multivariable models . aortic valve replacement was included as a time - varying covariate in models assessing cardiovascular death and total mortality . in subsequent models , concentric lv geometry was replaced by stress - corrected midwall shortening , and the presence of aortic regurgitation was added . to take the progressive increase in lv mass during progression of as into account , time - varying cox regression analysis was used . two - tailed p<0.05 was regarded as statistically significant both in univariable and multivariable analyses . compared with patients with normal lv mass index at baseline , the group with lv hypertrophy was older , had higher body mass index , lower lv midwall function , and included more patients with hypertension ( all p<0.01 ; tables 1 and 2 ) . during a median of 4.3-year follow - up , lv mass indexed to height ( lv mass index ) and concentricity increased , whereas lv endocardial and myocardial function declined ( all p<0.001 ) . the prevalence of lv hypertrophy increased from 36% at baseline to 60% at the last study visit ( p<0.01 ) . the annual as progression rate did not differ between groups of patients with and without lv hypertrophy at baseline , whether calculated based on change in peak aortic jet velocity ( 0.210.39 versus 0.200.27 m / s per year ) , mean gradient ( 47 versus 45 mm hg / y ) , or aortic valve area ( 0.030.25 versus 0.030.29 cm / y , all p>0.3 ) . the average time between the baseline and the last follow - up study was 3.61.2 years . the average time between the follow - up study and an aortic valve event , an ischemic cardiovascular event , and death from any cause was on average 0.590.03 , 0.940.06 , and 0.800.55 years , respectively . clinical patient characteristics in the total population and in patients with or without lv hypertrophy at baseline echocardiographic findings in the total study population and in patients with or without lv hypertrophy at baseline during follow - up , each sd higher unindexed lv mass , lv mass / height , and lv mass / body surface area was associated with comparable 21% , 23% , and 25% higher rates of the primary study end point , respectively , in univariable analyses ( all p<0.001 ) . the rates of aortic valve events , ischemic cardiovascular events , cardiovascular death , and combined death from any cause and hospitalization for heart failure because of progression of as all increased progressively with increasing quartile of baseline lv mass index and were 1.5 , 1.8 , 3.2 , and 2.5 times higher in the upper lv mass index quartile than in the lowest quartile ( figure 1 ) . in multivariable cox regression , higher lv mass index was associated with higher rates of aortic valve events , ischemic cardiovascular events , cardiovascular death , and combined death from any cause and hospitalization for heart failure when adjusted for known prognosticators in as patients like age , sex , body mass index , as severity , lv ejection fraction , concentric lv geometry , and concomitant hypertension ( table 3 ) . similar results were found in a second model , replacing concentric geometry by stress - corrected midwall shortening ( hazard ratio , 1.13 for primary end point per 1 sd higher lv mass index [ 95% confidence interval , 1.021.24 ] ; p=0.017 ) . adding the presence of aortic regurgitation or type of antihypertensive drug among the covariates did not influence results . impact of baseline left ventricular mass index ( per 1 sd [ 15 g / m2.7 ] higher ) on the rates of the primary and secondary study end points , hospitalization for heart failure , cardiovascular death , all - cause death , and combined all - cause death and hospitalization for heart failure during > 4.3 years of follow - up in patients with initially asymptomatic as cumulative incidences of aortic valve events ( ave ) , ischemic cardiovascular ( cv ) events ( ice ) , cv death ( cvd ) , and combined death from any cause and hospitalization for heart failure because of progression of aortic stenosis ( death&chf ) during > 4.3 years of follow - up in relation to quartile of baseline left ventricular ( lv ) mass index in mild - to - moderate asymptomatic aortic stenosis . in a secondary set of models , having lv hypertrophy on the baseline echocardiogram was associated with higher rates of the primary and secondary composite study end points and combined all - cause death and hospitalization for heart failure , consistent with the outcome association demonstrated for lv mass index ( table 4 ; figure 2 ) . impact of baseline left ventricular hypertrophy on the rates of the primary and secondary study end points , hospitalization for heart failure , cardiovascular death , all - cause death , and combined all - cause death and hospitalization for heart failure during > 4.3 years of follow - up in patients with initial asymptomatic as survival free from major cardiovascular ( cv ) events ( a ) , aortic valve events ( b ) , ischemic cv events ( c ) , and combined death from any cause and hospitalization for heart failure because of progression of aortic stenosis ( as ; d ) in groups of patients with ( ) and without ( - ) left ventricular hypertrophy ( lvh ) on the baseline echocardiogram . in multivariable linear regression higher lv mass / height at the last study echocardiogram was associated with male sex ( =0.06 ) , and higher mean aortic gradient ( =0.15 ) , systolic blood pressure ( =0.04 ) , body mass index ( =0.14 ) , initial lv mass / height ( =0.54 ) , and presence of normal midwall shortening ( =0.05 , all p<0.05 ) at the baseline echocardiogram . to take into account the progressive increase in lv mass during progression of as , higher lv mass index during follow - up was associated with a 16% higher rate of the primary study end point , 13% higher rate of aortic valve events , 25% higher rate of ischemic cardiovascular events , 63% higher cardiovascular mortality , and 44% higher combined death from any cause and hospitalization for heart failure ( all p<0.01 ; table 5 ) . in subsequent models replacing energy loss index by peak aortic jet velocity , mean aortic valve gradient , or aortic valve area as measure of as severity , or lv ejection fraction by midwall shortening or stress - corrected midwall shortening , the results did not change ( data not shown ) . impact of in - study left ventricular mass index ( per 1 sd [ 15 g / m2.7 ] higher ) on the rates of study end points during > 4.3 years of follow - up in patients with initial asymptomatic aortic stenosis this is the first large prospective study to assess the prognostic impact of lv mass and hypertrophy assessed by echocardiography in patients with asymptomatic mild - to - moderate as without known coronary heart disease or diabetes mellitus . as demonstrated by our results , higher lv mass at baseline or during follow - up was associated with higher rates of both the primary and secondary prespecified composite study end points in the seas study , resulting in a considerably increased overall cardiovascular morbidity and mortality . these findings were also independent of documented prognosticators in asymptomatic as , including as severity , hypertension , body mass index , sex , lv ejection fraction , and concentric lv geometry . of note , patients in the highest versus lowest quartile of lv mass index at baseline had a 13% higher 4.3-year cumulative incidence of aortic valve events and a 11% higher incidence of combined death from any cause and hospitalization for heart failure , corresponding to absolute differences of 3.0% and 2.6% per year , respectively . traditionally , development of lv hypertrophy in as has been considered a physiological , compensatory process taking advantage of laplace s law to sustain normal systolic function during chronically elevated systolic stress . however , as recently demonstrated , concomitant hypertension , obesity , and the presence of the metabolic syndrome have been associated with increased lv mass in patients with asymptomatic nonsevere as , suggesting that development of lv hypertrophy is multifactorial also in patients with as . the relatively larger impact of hypertension on lv wall volume in mild - to - moderate as than of as itself has also been demonstrated in experimental simulation models by garcia et al . however , having increased lv mass on the baseline echocardiogram was associated with increased event rates in the present study independent of the prognostic impact of concomitant hypertension and increased body mass index previously demonstrated in asymptomatic mild - to - moderate as . risk prediction in asymptomatic as remains a challenge , including identification of as patient with high risk for development of congestive heart failure , the most prognostically severe complication of as . both american and european guidelines recommend aortic valve replacement in patients with severe as irrespective of symptoms if lv dysfunction defined as lv ejection fraction < 50% is present . population - based studies have demonstrated that increased lv mass was associated with incident heart failure independent of lv ejection fraction and independent of incident myocardial infarction . the present findings expands this knowledge by demonstrating that also in patients with mild - to - moderate as , increased lv mass index is associated with higher rate of combined death and heart failure independent of lv systolic function . current european guidelines suggest excessive lv hypertrophy unless because of hypertension among indications for aortic valve replacement in as , as this has been associated with increased perioperative morbidity and mortality and may be less reversible after delayed surgery , precluding an optimal long - term prognosis . the present results from the large seas study document the association of increased lv mass with increased cardiovascular morbidity and mortality also in patients with asymptomatic mild - to - moderate as independent of the presence of concomitant hypertension . our findings contrast with previous reports from smaller studies in patients with moderate to - severe as . stewart et al following 183 patients with initially asymptomatic moderate or severe as for a median of 31 months found that neither lv mass nor tissue doppler measures of lv systolic and diastolic function predicted outcome independent of as severity . similar findings were reported by monin et al in a study of 107 patients with moderate - to - severe as , who therefore did not include lv mass in the suggested risk assessment score for asymptomatic patients with moderate - to - severe as based on their findings . electrocardiographic lv strain pattern was recently suggested as a strong correlate of mortality and hospitalization for heart failure by greve et al in a seas substudy . of note , electrocardiographic strain pattern was not significantly associated with either cardiovascular death or all - cause mortality when mean aortic gradient was included as covariate in their multivariable models , in contrast to the strong independent association with echocardiographic lv mass and hypertrophy reported in the present article . however , shah et al documented that electrocardiographic strain pattern as a highly specific marker of midwall myocardial fibrosis , reflecting more advanced myocardial injury , lv decompensation , and impaired prognosis . concentric lv geometric patterns have been demonstrated to carry individual risk of cardiovascular morbidity and mortality in hypertension . furthermore , an association with reduced coronary flow reserve as a substrate for reduced myocardial function in hypertensive subjects with lv concentric geometry free from coronary artery disease has been reported by galderisi et al . in patients operated for as , both concentric lv geometry and excessive lv hypertrophy have been associated with higher postoperative mortality . cioffi et al previously demonstrated that excessive lv hypertrophy was the strongest correlate of combined death , congestive heart failure , and nonfatal myocardial infarction in 218 patients with asymptomatic severe as . of note , these findings were independent of patient age , extent of aortic valve calcification , renal dysfunction , or the presence of concomitant diabetes mellitus , all factors that have been associated with worsened prognosis in previous studies in asymptomatic severe as . the present results expand this knowledge by demonstrating the independent prognostic importance of higher lv mass in a large prospective study of patients with initially asymptomatic mild - to - moderate as . patients with known coronary heart disease , heart failure , diabetes mellitus , history of stroke or peripheral vascular disease , other clinically significant valve disease , rheumatic valve disease , or renal insufficiency and patients with a guideline indication for lipid - lowering therapy were not included in the seas study . thus , projection of study results to these patient groups should be done with caution . in the seas study , referral for aortic valve replacement was left to the decision of the attending cardiologist at the 173 participating centers , and the basis for referral of individual patients for surgery was not captured in the study database . we can not exclude that presence of extreme lv hypertrophy may have influenced the decision to refer for surgery in individual cases . however , an independent impact of higher lv mass index was also found with the more objective end points cardiovascular and total mortality . several studies have found speckle strain imaging , in particular 2-dimensional ( 2d ) global longitudinal strain , useful for detecting asymptomatic as patients with more advanced lv injury despite normal lv ejection fraction . lower global longitudinal strain in these patients has been associated with higher lv mass , concentric lv geometry , more severe as , concomitant hypertension , and with impaired prognosis . recently , nagata et al reported the superior performance of 3d compared with 2d global longitudinal strain for risk prediction in such patients , also when adjusting for lv mass in multivariate analysis . however , speckle tracking echocardiography was not included in the large seas study , where the majority of echocardiogram were recorded on video tapes during the period 2002 to 2008 . marchaux et al have demonstrated the usefulness of exercise stress echocardiography for risk stratification in asymptomatic patients with severe as , and current european guidelines include exercise testing for additional risk assessment in patients with asymptomatic severe as . however , exercise testing was not included in the large seas study , which was undertaken in 173 study centers during the years 2002 to 2008 . in patients with asymptomatic as , higher lv mass index patients with known coronary heart disease , heart failure , diabetes mellitus , history of stroke or peripheral vascular disease , other clinically significant valve disease , rheumatic valve disease , or renal insufficiency and patients with a guideline indication for lipid - lowering therapy were not included in the seas study . thus , projection of study results to these patient groups should be done with caution . in the seas study , referral for aortic valve replacement was left to the decision of the attending cardiologist at the 173 participating centers , and the basis for referral of individual patients for surgery was not captured in the study database . we can not exclude that presence of extreme lv hypertrophy may have influenced the decision to refer for surgery in individual cases . however , an independent impact of higher lv mass index was also found with the more objective end points cardiovascular and total mortality . several studies have found speckle strain imaging , in particular 2-dimensional ( 2d ) global longitudinal strain , useful for detecting asymptomatic as patients with more advanced lv injury despite normal lv ejection fraction . lower global longitudinal strain in these patients has been associated with higher lv mass , concentric lv geometry , more severe as , concomitant hypertension , and with impaired prognosis . recently , nagata et al reported the superior performance of 3d compared with 2d global longitudinal strain for risk prediction in such patients , also when adjusting for lv mass in multivariate analysis . however , speckle tracking echocardiography was not included in the large seas study , where the majority of echocardiogram were recorded on video tapes during the period 2002 to 2008 . marchaux et al have demonstrated the usefulness of exercise stress echocardiography for risk stratification in asymptomatic patients with severe as , and current european guidelines include exercise testing for additional risk assessment in patients with asymptomatic severe as . however , exercise testing was not included in the large seas study , which was undertaken in 173 study centers during the years 2002 to 2008 . in patients with asymptomatic as , higher lv mass index is independently associated with increased cardiovascular morbidity and mortality during progression of valve stenosis . the simvastatin ezetimibe in aortic stenosis ( seas ) echocardiography core laboratory was supported by the msp singapore company , llc , singapore , a partnership between merck & co. , inc . and the schering - plough corporation . drs pedersen , rosseb , and gerdts were members of the scientific steering committee for the simvastatin ezetimibe in aortic stenosis ( seas ) study in the years 2002 to 2008 and received honoraria for this work . it is well known that the presence of left ventricular ( lv ) hypertrophy by echocardiography predicts increased cardiovascular morbidity and mortality both in general and hypertensive populations . in patients with aortic valve stenosis ( as ) , lv hypertrophy has traditionally been considered as an adaptive response that keeps lv wall stress close to normal , offsetting the hemodynamic load . recent publications have demonstrated that the presence of concomitant hypertension , obesity , and metabolic syndrome significantly modulates lv mass and geometry in patients with asymptomatic as independent of as severity . furthermore , excessive lv hypertrophy in severe as has been associated with incident heart failure and increased mortality . the present study is the first to demonstrate the prognostic impact of lv mass and hypertrophy in a large , prospective study in asymptomatic mild - to - moderate as . higher lv mass at baseline or during follow - up was associated with considerable increased overall cardiovascular morbidity and mortality . patients in the highest versus the lowest quartile of baseline lv mass index had 2.6% higher incidence per year of death and hospitalization for heart failure . emerging data suggest that speckle strain echocardiography may be used for further identification of as patients with more advanced lv injury . whether asymptomatic as patients with lv hypertrophy however , lv hypertrophy in asymptomatic as should not be regarded as purely compensatory , and referral to a heart valve center for further evaluation may be indicated .
background the prognostic importance of left ventricular ( lv ) mass in nonsevere asymptomatic aortic stenosis has not been documented in a large prospective study.methods and results cox regression analysis was used to assess the impact of echocardiographic lv mass on rate of major cardiovascular events in 1656 patients ( mean age , 67 years ; 39.6% women ) with mild - to - moderate asymptomatic aortic stenosis participating in the simvastatin ezetimibe in aortic stenosis ( seas ) study . patients were followed during 4.3 years of randomized treatment with combined simvastatin 40 mg and ezetimibe 10 mg daily or placebo . at baseline , lv mass index was 45.9 + 14.9 g / m2.7 , and peak aortic jet velocity was 3.09 + 0.54 m / s . during follow - up , 558 major cardiovascular events occurred . in cox regression analyses , 1 sd ( 15 g / m2.7 ) higher baseline lv mass index predicted increases in hazards of 12% for major cardiovascular events , 28% for ischemic cardiovascular events , 34% for cardiovascular mortality , and 23% for combined total mortality and hospitalization for heart failure ( all p<0.01 ) , independent of confounders . in time - varying models , taking the progressive increase in lv mass index during follow - up into account , 1 sd higher in - study lv mass index was consistently associated with 13% to 61% higher hazard for cardiovascular events ( all p<0.01 ) , independent of age , sex , body mass index , valvuloarterial impedance , lv ejection fraction and concentricity , and the presence of concomitant hypertension.conclusionshigher lv mass index is independently associated with increased cardiovascular morbidity and mortality during progression of aortic stenosis.clinical trial registration url : http://www.clinicaltrials.gov . unique identifier : nct00092677 .
Clinical Trial Registration Methods Patient Population Echocardiography End Points Statistical Analysis Results Discussion Limitations Conclusions Sources of Funding Disclosures CLINICAL PERSPECTIVE
in short , 1873 men and women aged 45 to 85 years with asymptomatic mild - to - moderate as having a peak aortic jet velocity between 2.5 and 4.0 m / s by echocardiography were randomized to placebo or to combination treatment with simvastatin 40 mg and ezetimibe 10 mg daily . to take the progressive increase in lv mass during progression of as into account , time - varying cox regression analysis was used . in short , 1873 men and women aged 45 to 85 years with asymptomatic mild - to - moderate as having a peak aortic jet velocity between 2.5 and 4.0 m / s by echocardiography were randomized to placebo or to combination treatment with simvastatin 40 mg and ezetimibe 10 mg daily . to take the progressive increase in lv mass during progression of as into account , time - varying cox regression analysis was used . the rates of aortic valve events , ischemic cardiovascular events , cardiovascular death , and combined death from any cause and hospitalization for heart failure because of progression of as all increased progressively with increasing quartile of baseline lv mass index and were 1.5 , 1.8 , 3.2 , and 2.5 times higher in the upper lv mass index quartile than in the lowest quartile ( figure 1 ) . in multivariable cox regression , higher lv mass index was associated with higher rates of aortic valve events , ischemic cardiovascular events , cardiovascular death , and combined death from any cause and hospitalization for heart failure when adjusted for known prognosticators in as patients like age , sex , body mass index , as severity , lv ejection fraction , concentric lv geometry , and concomitant hypertension ( table 3 ) . impact of baseline left ventricular mass index ( per 1 sd [ 15 g / m2.7 ] higher ) on the rates of the primary and secondary study end points , hospitalization for heart failure , cardiovascular death , all - cause death , and combined all - cause death and hospitalization for heart failure during > 4.3 years of follow - up in patients with initially asymptomatic as cumulative incidences of aortic valve events ( ave ) , ischemic cardiovascular ( cv ) events ( ice ) , cv death ( cvd ) , and combined death from any cause and hospitalization for heart failure because of progression of aortic stenosis ( death&chf ) during > 4.3 years of follow - up in relation to quartile of baseline left ventricular ( lv ) mass index in mild - to - moderate asymptomatic aortic stenosis . impact of baseline left ventricular hypertrophy on the rates of the primary and secondary study end points , hospitalization for heart failure , cardiovascular death , all - cause death , and combined all - cause death and hospitalization for heart failure during > 4.3 years of follow - up in patients with initial asymptomatic as survival free from major cardiovascular ( cv ) events ( a ) , aortic valve events ( b ) , ischemic cv events ( c ) , and combined death from any cause and hospitalization for heart failure because of progression of aortic stenosis ( as ; d ) in groups of patients with ( ) and without ( - ) left ventricular hypertrophy ( lvh ) on the baseline echocardiogram . to take into account the progressive increase in lv mass during progression of as , higher lv mass index during follow - up was associated with a 16% higher rate of the primary study end point , 13% higher rate of aortic valve events , 25% higher rate of ischemic cardiovascular events , 63% higher cardiovascular mortality , and 44% higher combined death from any cause and hospitalization for heart failure ( all p<0.01 ; table 5 ) . impact of in - study left ventricular mass index ( per 1 sd [ 15 g / m2.7 ] higher ) on the rates of study end points during > 4.3 years of follow - up in patients with initial asymptomatic aortic stenosis this is the first large prospective study to assess the prognostic impact of lv mass and hypertrophy assessed by echocardiography in patients with asymptomatic mild - to - moderate as without known coronary heart disease or diabetes mellitus . however , having increased lv mass on the baseline echocardiogram was associated with increased event rates in the present study independent of the prognostic impact of concomitant hypertension and increased body mass index previously demonstrated in asymptomatic mild - to - moderate as . the present results from the large seas study document the association of increased lv mass with increased cardiovascular morbidity and mortality also in patients with asymptomatic mild - to - moderate as independent of the presence of concomitant hypertension .
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obesity is a growing epidemic worldwide ; its prevalence has been rising tremendously over the last 30 years ( who , 2013 ) . excess adiposity is an established risk factor for metabolic diseases including insulin resistance , type 2 diabetes ( t2d ) , hypertension , nonalcoholic fatty liver disease ( nafld ) , polycystic ovarian diseases , and several types of cancer . obesity is a proinflammatory condition in which hypertrophied adipocytes and adipose tissue - resident immune cells ( primarily lymphocytes and macrophages ) both contribute to increased circulating levels of proinflammatory cytokines . metabolic inflammation , is considered a focal point in the pathogenesis of insulin resistance and t2d in humans and rodent animal models [ 25 ] . although liver and muscle show obesity - induced mild inflammatory responses , white adipose tissue ( wat ) is the key site mediating systemic inflammation . adipose tissue primary function is to store excess nutrients as triacylglycerols and to release free fatty acids during fasting . a major step forward to the recognition of the major secretory and endocrine role of wat occurred in the 1990 's with the demonstration that adipocytes synthesize and secrete the proinflammatory cytokine tumor necrosis factor alpha ( tnf- ) and the hormone leptin which regulates appetite and energy balance . evidence shows that the adipose tissue secretes more than 50 hormones and signaling molecules , collectively called adipokines , which exert their biological roles in an autocrine , paracrine , or systemic manner and influence several physiological processes concerning energy , glucose metabolism , and immunity . more specifically , adipokines can exhibit either proinflammatory or anti - inflammatory properties , thereby contributing to insulin resistance . adipose tissue from lean individuals preferentially secretes anti - inflammatory adipokines such as adiponectin , transforming growth factor beta ( tgf ) , interleukin ( il)-10 , il-4 , il-13 , il-1 receptor antagonist ( il-1ra ) , and apelin . in contrast , obese adipose tissue mainly releases proinflammatory cytokines among which are tnf- , il-6 , leptin , visfatin , resistin , angiotensin ii , and plasminogen activator inhibitor 1 . in lean individuals , anti - inflammatory adipokines mediate physiological functions , whilst in states of metabolic diseases , the proinflammatory adipokines modulate insulin resistance either directly by affecting the insulin signaling pathway or indirectly via stimulation of inflammatory pathways . indeed , serine phosphorylation of insulin receptor substrate ( irs ) by various adipokines directly or via inflammatory pathways including the c - jun n - terminal kinase ( jnk ) pathway and i - kappa b kinase ( ikk)/nfb pathway disrupts the insulin signaling pathways , possibly giving rise to insulin resistance . adipokines enlisted in regulation of insulin resistance are adiponectin , leptin , resistin , visfatin , chemerin , tnf- , il-1 , il-6 , il-8 , il-10 , plasminogen activator inhibitor 1 , monocyte chemoattractant protein-1 , and retinol binding protein-4 ( tables 1 and 2 ) . because this topic has been the subject of recent reviews [ 12 , 13 ] it will not be discussed in detail . we will rather focus on the prototypical adipokines ( tnf- , il-6 , leptin , adiponectin , and resistin ) highlighting their roles in the development of insulin resistance as well as in immunity and inflammation . tnf- is a potent proinflammatory cytokine , primarily secreted from myeloid cells via activation of mapk and nfb signaling pathways , resulting in the release of other inflammatory cytokines , such as il-1 and il-6 . it was the first wat - derived inflammatory cytokine reported to be implicated in the initiation and progression of insulin resistance [ 8 , 15 ] . although originally thought to be mainly secreted by adipocytes , it is now admitted that the majority of tnf- is secreted by adipose tissue - resident macrophages . in rodents tnf- is overexpressed in adipose tissue from obese animals , and obese mice lacking either tnf- or its receptor show protection against the development of insulin resistance . in humans tnf- levels are higher in plasma and adipose tissue of obese individuals , and circulating levels reduce with weight loss . tnf- levels were also found to be positively correlated with other markers of insulin resistance ; nonetheless , acute treatment with tnf- inhibitor in obese subjects with type 2 diabetes reduced other systemic inflammatory markers without reducing insulin resistance , fueling lingering uncertainty about the biological relevance of this pathway in human insulin resistant states . more recently , the long - term assessment of anti - tnf- inhibitor treatment to subjects diagnosed with metabolic syndrome has been shown to improve fasting blood glucose and to increase adiponectin levels , confirming a role for tnf- in obesity - related insulin resistance in humans . a key mechanism by which tnf- induces insulin resistance involved phosphorylation of irs-1 . beside its direct negative interference with the insulin signaling pathway , tnf- is known to promote lipolysis and the secretion of free fatty acids , which contribute to an increase in hepatic glucose production . moreover , tnf- inhibits the conversion of preadipocytes to mature adipocytes notably through downregulating adipogenic genes such as peroxisome proliferator - activated receptor gamma ( ppar ) and ccaat / enhancer binding protein ( c / ebp)allowing further recruitment of uncommitted cells and thus possible expansion of adipose tissue mass . tnf--activated nf-b suppressed genes involved in lipid uptake and storage as well as many adipocyte - specific genes . tnf- also downregulates the mrna levels of adiponectin , an adipocyte - derived hormone which contributes to the maintenance of peripheral glucose and lipid homeostasis . nevertheless , the influence of tnf- on immune response mostly results from its enhancing effect on the production of other cytokines , such as il-6 , rather than from a direct effect . il-6 is a multifaceted , pleiotropic cytokine that is a central player in the regulation of inflammation , hematopoiesis , immune responses , and host defense mechanisms . il-6 is secreted by wat , skeletal muscle , and liver [ 16 , 29 ] . because one - third of circulating il-6 in healthy individuals is estimated to originate from adipose tissue , il-6 is considered an adipokine . in wat , only a fraction of il-6 is secreted by adipocytes , the other part being produced by other cells , particularly macrophages . similarly to tnf- , wat and plasma il-6 expression correlate with increased body mass , waist circumference , and free fatty acid levels , with reduction in circulating il-6 following weight loss . il-6 has been implicated as a marker for visceral adiposity because visceral adipose tissue releases more il-6 than subcutaneous adipose tissue . nevertheless , data regarding the role of il-6 in both obesity and insulin resistance are controversial and unresolved . while several studies indicate that increased il-6 levels correlate with adiposity and fat mass , and not necessarily with insulin action or responsiveness [ 30 , 33 ] , another study has pointed to higher il-6 levels in patients with obesity - related insulin resistance . it can be inferred that relentless increase in systemic levels of il-6 may lead to insulin resistance , whereas a transient increase in il-6 may assist in normal glucose homeostasis . in fact , il-6 appears to have dual functions depending on the tissue and metabolic state . during exercise , il-6 increases glucose uptake in the skeletal muscle , leading to muscle hypertrophy and myogenesis and ampk - mediated fatty acid oxidation , as well as having an anti - inflammatory effect . in adipose tissue and liver , however , il-6 will exert proinflammatory activities , increasing insulin resistance by upregulating socs3 ( suppressor of cytokine signaling 3 ) which , in turn , impairs insulin - induced insulin receptor and irs1 phosphorylation . il-6 may promote dysregulation of fatty acid metabolism in wat as it enhanced mesenchymal stem cell proliferation , maintaining the cells in an undifferentiated state and inhibiting adipogenesis additionally , il-6 was recently shown to stimulate insulin secretion via enhanced glp-1 ( glucagon - like peptide-1 ) expression in pancreatic cells . thus , obesity - induced il-6 secretion may reflect a mechanism to increase insulin production in the obese insulin resistant state . however , while elevated il-6 secretion from wat and liver is unfavorable , the opposite is true for skeletal muscle . on the other hand , a number of in vitro and in vivo studies demonstrate that il-6 is capable of inducing insulin resistance . in cultured murine adipocytes , il-6 production is strongly increased by tnf- and induces insulin resistance by inhibiting glucose uptake and impairing insulin signaling and action . whether or not il-6 impairs insulin action in adipose tissue in vivo has yet to be clearly determined . like tnf- , il-6 can directly affect lipid metabolism and activate pathways to promote increased energy turnover . il-6 stimulates lipolysis in humans , increases free fatty acid ( ffa ) concentrations and whole body fat oxidation . notably , il-6 can decrease the expression and secretion of adiponectin in human adipocytes , as well as other markers of adipocyte differentiation . therefore , understanding and clarifying its role in the regulation of metabolism is of utmost importance . as stated above , leptin was one of the first proteins shown to be secreted from adipose tissue , through the identification and sequencing of the ob gene from the ob / ob mouse . leptin is primarily secreted by adipocytes proportionally to fat cell mass and is well known for its key contribution to energy metabolism . leptin exerts its effect on energy balance mainly by acting on the brain , either directly or indirectly by activating specific centers in the hypothalamus to decrease food intake , to increase energy expenditure , to influence glucose and lipid metabolism , or to alter neuroendocrine function . daily injection of leptin in ob / ob mice resulted in a rapid reduction in food intake , body mass , and percentage of body fat but maintained lean muscle mass , increased energy expenditure , and restored euglycemia , confirming its important role in energy homeostasis and storage . however , leptin levels are increased in obese subjects , with little or no impact to regulate energy homeostasis , which coined the well - established phrase leptin resistance in obesity . indeed , preclinical and clinical experiments showed that obese rodents and humans displayed leptin resistance that may directly contribute to the reduction of lipid oxidation in insulin - sensitive organs , leading to accumulation of lipids and insulin resistance [ 44 , 45 ] . recently , it has been proposed that socs3 could be involved in negative regulation of leptin - induced intracellular signal transduction in the brain . moreover , neuronal deletion as well as whole - body knock - out of protein tyrosine phosphatase 1b ( ptp1b ) increased leptin and insulin sensitivity , preventing body weight gain in a diet - induced obesity animal model [ 47 , 48 ] , hence suggesting that , likewise socs3 , ptp1b also orchestrates leptin resistance control . on the other hand , the role of leptin on insulin resistance is still not fully understood . leptin is decreased in low insulin states , such as experimentally induced diabetes , and increases after insulin treatment . in humans , insulin resistance is associated with elevated plasma leptin levels independently of body fat mass . however , in patients with lipodystrophy , a condition characterized by almost complete lack of adipose tissue , leptin levels are very low and correlate significantly with markers of insulin resistance . leptin therapy in lipodystrophic patients improves their metabolic state with remarkable improvements in insulin sensitivity , suggesting that leptin acts as a signal that contributes to regulation of total body sensitivity to insulin . leptin has proinflammatory functions : it stimulates t - cell proliferative responses , polarized nave cd4 t - cell proliferation towards the th1 phenotype , promotes a marked increase in th1-type cytokine production , induces the expression of proinflammatory cytokines by macrophages and monocytes , and acts directly on hepatocytes to promote c - reactive protein expression . the proinflammatory nature of leptin has been noted in several studies , with intravenous injection of endotoxin inducing a sudden rise in leptin levels , as well as endotoxin - induced fever and anorexia in rats , again inducing an increase in leptin levels as part of the inflammatory response . the importance of leptin in immunity was confirmed in obese mice with homozygous mutation in leptin ( ob / ob mice ) or leptin receptor ( db / db mice ) , in which high levels of lymphocyte atrophy and significant reduced thymus cortex were evidenced . replacement of leptin in the ob / ob mice or in congenital leptin - deficient children is able to restore normal thymic function , to increase the number of cd4/cd8 t - cells , to promote th1 differentiation , and to reduce thymic apoptosis . we also reported impaired functionality of t - lymphocytes , dendritic cells , and macrophages in ob / ob and high - fat ( hf ) diet - fed mice [ 59 , 60 ] . more recently , leptin has also been shown to activate human b lymphocytes to secrete tnf- , il-6 , and il-10 via the jak2 , stat3 , p38mapk , and erk signaling pathways . besides acting on adaptive immunity , leptin also regulates innate immune cells such as polymorphonuclear neutrophils , monocytes , and natural killer ( nk ) cells . leptin can induce chemotaxis of neutrophils , is involved in the development and maintenance of a functional nk ( natural killer ) pool , and induces the production of il-6 and tnf- from macrophages . unlike leptin , the circulating levels of adiponectin , a hormone produced predominantly by adipocytes , are decreased in obesity . adiponectin has important insulin - sensitizing effect : adiponectin - deficient transgenic mouse showed improved insulin sensitivity and association studies have consistently linked plasma adiponectin levels to insulin sensitivity in rodent models and in humans . among the three major adiponectin isoforms , high - molecular weight ( hmw ) adiponectin is the most biologically active form and best reflective of the reduction in total adiponectin levels associated with obesity . indeed , hmw adiponectin levels have been identified as an independent risk factor for insulin resistance . adiponectin can suppress the production of tnf- and ifn ( interferon gamma ) and is a negative regulator of t cells , notably through its effect on the t - cell presenting function of dendritic cells . adiponectin maintains a mutual antagonistic action to tnf- : as mentioned above tnf- inhibits the expression of adiponectin , and conversely adiponectin suppresses lipopolysaccharide- ( lps- ) induced tnf- production . resistin is another unique adipocyte - derived signaling cysteine - rich molecule that was first identified in obese mice , deriving its name because of its resistance to the action of insulin . in rodents , resistin is secreted primarily from adipose tissue , whereas in humans resistin can be detected in other tissues like placenta , skeletal muscle , small intestine , spleen , stomach , thymus , thyroid gland , and uterus , being predominantly expressed in macrophages . in rodents , demonstrated that circulating resistin levels are elevated and positively concordant with rising levels of insulin , glucose , and lipids in ob / ob mice and that leptin administration improved insulin sensitivity associated with a decrease in resistin gene expression . moreover , transgenic mice overexpressing a dominant negative form of resistin showed increased adiposity , possibly owing to enhanced adipose tissue differentiation and adipocyte hypertrophy . resistin appears to interfere with normal insulin signaling by decreasing insulin receptor and insulin receptor substrate ( irs1 and 2 ) protein expression and phosphorylation level in preadipose 3t3-l1 cells . in addition , resistin has been showed to decrease ampk activation which is known to be implicated as a potential insulin sensitizing molecule . however , the role of resistin in the development of insulin resistance in humans is not as clear as in rodents . since resistin is preferentially expressed by macrophages in humans , it suggests a proinflammatory role of resistin rather than a role in regulating glucose metabolism . resistin mrna expression level is higher in obese subjects , likely resulting from increased infiltration of macrophages in the adipose tissue . several studies have reported positive correlations between resistin levels and insulin resistance in vivo and in vitro . moreover , genetic studies showed that two single nucleotide polymorphisms ( snps : 537a > c and 420c > g ) were associated with increased resistin levels in diabetic patients , but not in control subjects . recently , associations have been reported between resistin and metabolic syndrome components on one hand and early atherosclerosis in obese children on the other hand . finally , resistin has been demonstrated to stimulate the secretion of several inflammatory factors ( e.g. , tnf- , il-6 , il-8 , and mcp-1 ) known to play a role in the induction of insulin resistance . therefore , resistin may have an indirect effect on insulin resistance in humans through exacerbating inflammation , which has been shown to disturb insulin sensitivity . during the past decades , il-7 has been identified as the major homeostatic cytokine supporting the survival of and t cells , nkt cells , innate lymphoid cells , and regulatory t cells ( tregs ) . il-7 is predominantly produced by stromal and vascular endothelial cells , with very low levels of il7 transcripts detectable in adult animals , consistent with the concept that under basal states there are limited amounts of il-7 available for lymphocytes in vivo . in a homeostatic animal , il-7 amount is thought to be constant yet stroma - derived il-7 production can be induced by overt inflammation . il-7 receptor ( il-7r ) is composed of the private il-7r chain ( cd127 ) combined with the common gamma ( c ; cd132 ) chain and is expressed mainly by t lymphocytes but also by nk cells , macrophages , dendritic cells , lymphoid tissue inducer cells , and certain subsets of b cells . one central characteristic of il-7r expression is its dynamic regulation by cytokines and by the overall metabolic and differentiation state of the cells . for example , tnf- has been reported to upregulate il-7r expression and il-6 to be a critical effector of il-7r signaling . without il-7 the lymphoid system can not be built and maintained . interestingly , the role of il-7 on lymphocyte homeostasis was shown to partly rely on its control of basal lymphocyte glucose metabolism through the expression of the glucose transporter glut-1 , which promotes glucose uptake and increases metabolic activity as well as cell size . recently , we and others identified il-7 as a new secretory product of the adipose tissue , mostly produced by cells of the stromal vascular fraction [ 82 , 83 ] . furthermore , we reported that il-7 also contributes to body weight regulation via both hypothalamic and adipose tissue control . regarding the latter , we showed that il-7 modulates the adipose tissue through acting on its mass and function . in fact , a single administration of il-7 was sufficient to decrease adipose tissue inflammation and to protect mice from obesity in three different models of experimentally induced obesity ( i.e. , monosodium glutamate - induced hypothalamic obesity , gold thioglucose - induced hypothalamic obesity ( wolowczuk i , unpublished data ) , and hf diet- ( hfd- ) induced obesity ) . strikingly , we showed that il-7 overexpressing mice presented a lipodystrophy - like phenotype : reduced wat mass is associated with impaired adipocyte differentiation and intolerance to glucose and insulin resistance , these traits being commonly associated with lipodystrophy in both animals and humans . the first part of our review showed that the apparent metabolic simplicity of the adipose tissue is illusory ; this is also true regarding its cellular composition . besides lipid - filled mature adipocytes , the tissue is also composed of various stromal cells , including preadipocytes , endothelial cells , fibroblasts , and immune cells . during the progression of obesity , both the adipocyte and the stroma vascular fractions are changed : adipocytes grow larger , secrete predominantly proinflammatory cytokines , and are insulin resistant ; coincidently , the nature of wat immune cells is also modified . notably , proinflammatory macrophage infiltration and inflammation - related gene expression precede the development of insulin resistance and appear to be a cardinal feature of obesity in rodents and humans . adipose tissue macrophages ( atms ) accumulate in both the subcutaneous and visceral expanding fat depots , even though macrophage infiltration appears to be more prominent in the latter . apart from increasing in numbers , adipose tissue macrophages are also phenotypically changed during obesity : while anti - inflammatory m2 macrophages reside in wat of lean mice , obese wat predominantly contains proinflammatory m1 macrophages . activated m1 atms are a prominent source of proinflammatory cytokines such as tnf- and il-6 , which can block insulin action in adipocytes via autocrine / paracrine signaling causing systemic insulin resistance via endocrine signaling ( cf . thus , both recruitment and proinflammatory polarization of atms are required for the development of insulin resistance . in both humans and rodents , atms content positively correlates with inflammation and insulin resistance . despite its importance in adipose tissue inflammatory responses and systemic insulin sensitivity the recent discovery of micrornas ( mirnas ) provides a new opportunity to understand this complicated but crucial network for macrophage activation and adipose tissue function . mirnas , which correspond to a group of highly conserved , small ( i.e. , approximately 22 nucleotides in length ) noncoding rnas , can trigger either a block in translation and/or mrna degradation [ 88 , 89 ] . numerous studies have provided compelling evidence that mirnas are key regulators of cell fate determination and significantly contribute to the pathogenesis of complex diseases , including obesity - associated metabolic diseases [ 9092 ] . recently identified mirna-223 ( mir-223 ) as a potent regulator of macrophage polarization and provided strong evidence supporting the functional significance of this new pathway in metabolic homeostasis . the authors showed a suppressive effect of mir-223 on macrophage proinflammatory activation ( m1 ) and a stimulatory effect on anti - inflammatory activation ( m2 ) : high - fat diet - fed mir-223-deficient mice displayed increased adipose tissue inflammation and were more insulin resistant . at the molecular level , a major target of mir-223 in macrophages is pknox1 , which itself favors the proinflammatory activation pathway . however , a key question still unanswered by now is how the mir-223/pknox1 pathway interacts with known regulatory pathways that control macrophage activation . the identification of mechanisms underlying functional polarization of macrophages into m1 or m2 might provide new insights into a basis for macrophage - centered therapeutic strategies for metabolic diseases . similarly to any immune and inflammatory response , macrophage infiltration in the obese adipose tissue results from blood monocyte influx , mainly attracted by the chemokine monocyte chemoattractant protein-1 ( mcp-1 ) which is secreted by hypertrophic adipocytes . it has been reported that mcp-1 secretion is markedly enhanced locally and in plasma of obese rodents and humans . overexpression , deficiency , or mutation - induced dysfunction of mcp-1 in different mouse models were shown to interfere with atms accumulation , along with insulin - resistance development [ 95 , 96 ] . however , the role of mcp-1 in promoting atm recruitment and insulin resistance has recently been challenged by the absence of noticeable impact on macrophage accumulation and glucose intolerance resulting from mcp-1 genetic disruption . furthermore , hfd - fed mcp-1 receptor i.e. , ccr2-deficient mice ( namely , ccr2 mice ) do not normalize atm content and insulin resistance to the levels of lean animals , suggesting that atm recruitment and insulin resistance are also regulated by mcp-1/ccr2 independent signaling pathways . recently identified and characterized a critical role for ccr5 , another c - c motif chemokine receptor , in the regulation of obesity - induced wat inflammatory response and insulin resistance . , ccr5 mice were protected from insulin resistance induced by hf feeding through both reduction in atm accumulation and induction of anti - inflammatory m2 shift in those cells . additionally , a bone marrow transplantation study revealed that lack of ccr5 expression in macrophages alone could protect mice from the hfd - induced insulin resistance , this being associated with a significant reduction in atm infiltration . in humans , recent studies have also shown upregulation of ccr5 in the visceral fat of morbidly obese individuals in whom macrophage infiltration has been confirmed . however , further studies are needed to evaluate whether ccr5 inhibitor treatment ( e.g. , maraviroc ) affects macrophage activation and other aspects of adipose tissue biology in obese patients . also , it remains to be established whether the two c - c chemokine receptors , ccr2 and ccr5 , play common or unique roles in obesity - induced adipose tissue inflammation and insulin resistance . alterations in atm content and polarization state occur fairly late in the progression of obesity and probably are not initiating events of inflammation and development of sustained insulin resistance . evidence has accumulated showing that other changes in adipose - resident immune cells may precede these events . under this scenario , atms will be effectors of a coordinated inflammatory response that includes the accumulation of proinflammatory t cells ( cd8 and th1 cd4 t cells ) and the loss of anti - inflammatory regulatory t cells ( tregs ) , as well as the appearance of b cells , nk cells , nkt cells , eosinophils , neutrophils , and mast cells . adipocytes in lean adipose tissue produce factors such as il-4 and il-13 that induce m2 activation of macrophages and th2 activation of cd4 t cells and maintain treg cell and eosinophil numbers . in obesity , the progressive accumulation of adipose tissue is accompanied by early increased infiltration of proinflammatory cd8 t cells and a shift towards a higher cd8/cd4 ratio . cd8 infiltration appears to be a key event preceding the depletion of adipose tregs and the increased cd4 th1 cell activation observed in murine models of diet - induced obesity [ 101 , 102 ] . increased adipose - resident cd8 t - cell activation also potentiates adipocyte expression of il-6 and tnf- in mice , while cd8 t - cell depletion reverses this effect . neutrophils are known to play a role in the early stages of inflammatory responses , and it has been recently reported a sustained increased in adipose tissue neutrophil content in hfd - induced obesity with neutrophil secreted elastase being a key effector in this process . the enhanced release of il-8 , a factor involved in neutrophil chemotaxis , by hypertrophic adipocytes , may partly explain neutrophil recruitment . adipose tissue neutrophils produce chemokines and cytokines , facilitating macrophage infiltration , which could contribute to development of insulin resistance . in the 1980s , a new cell population known as natural suppressor cells , distinct from t and nk cells , was described in the bone marrow and spleen of tumor - bearing mice [ 104 , 105 ] . myeloid - derived suppressor cells ( mdsc ) because of their myeloid origin and their ability to suppress immune responses . in fact , mdscs represent a heterogeneous and metabolically plastic population of immature myeloid cells in different stages of differentiation , having in common the capacity to inhibit effector immune responses and to accumulate under conditions of inflammation . the term plasticity here refers to the ability of mdscs to change both their expression of various mediators of suppression ( e.g. , inos , arginase i ) in response to environmental influences ( e.g. , local il-4/13 or ifn concentration ) and also their differentiation state ( e.g. , becoming more / less neutrophil or myeloid cells ) . these cells are also defined by their immature state of macrocytic / monocytic , granulocytic / neutrophilic , and dendritic cell precursors and are characterized by the increased production of extracellular degradative enzymes , cytokines , and reactive oxygen and nitrogen species . in mice , since there are several subpopulations within gr-1cd11b cells , several groups further subcategorized mdsc into monocytic mdsc ( cd11bly6gly6c ) and granulocytic / neutrophil - like mdsc ( cd11bly6gly6c ) , based on the expression of ly6c and gr-1/ly6 g . there is no human marker equivalent to mouse gr-1 , human mdsc being typically defined as cd11bcd33cd34cd14hla - dr cells . in addition to heterogeneity , discrepancies exist in cell surface expression of certain activation / maturation markers , such as mhc ii and costimulatory molecules , and of lineage markers ( e.g. , f4/80 ) between mdsc . this heterogeneity supports the notion that mdsc include multiple subpopulations of myeloid - derived cells that are at various stages of maturity . in the steady state , mdscs are predominantly present in the bone marrow and participate in the normal process of myelopoiesis . however , under various pathological inflammatory conditions such as cancer , infection , sepsis , graft - versus - host disease , and bone marrow transplantation , a variety of cytokines and soluble factors released induce rapid expansion of mdsc that will accumulate in peripheral lymphoid organs and blood , as well as in tumors , where they have been described to block cd4 and cd8 t - cell responses thus favoring cancer development . in cancer , one key factor controlling mdsc expansion and tumor progression is ppar . vascular endothelial growth factor ( vegf ) , macrophage colony - stimulating factor ( m - csf ) , or il-6 it has been suggested that mdscs contribute to tumor progression by both facilitating neoangiogenesis and metastasis and by inhibiting antitumor responses . in addition to their recognized role in tumor tolerance , mdscs may also be involved in the induction and maintenance of transplant tolerance . recently , an exciting observation has been described by xia et al . , showing that mdscs and m2 macrophage induction may be a physiological response to promotion of insulin sensitivity . these authors showed that obese ob / ob mice , as well as wild - type mice fed on high - fat diet , have marked accumulation of anti - inflammatory mdscs and m2 macrophages in adipose tissue . furthermore , the increase in mdscs and m2 macrophage number was associated with higher response to insulin . adoptive transfer of mdscs ( e.g. , gr-1 cells ) into high - fat diet - fed mice improved the response of the recipient mice to insulin while , in contrast , mdscs depletion ( after treatment with anti - gr-1 antibody ) increased their susceptibility to obesity and further worsened their resistance to insulin . have also described the ability of mdscs to delay onset of type 1 diabetes and insulin resistance , through inducing expansion of antigen - specific tregs and suppressing t - cell proliferation . the mechanisms by which obesity - associated chronic inflammation induces expansion / accumulation of mdscs are arguably stepwise . however , the initial proinflammatory state created in early obesity may induce the accumulation of mdsc in an attempt to curtail overt inflammation , as described in other well - described models of inflammation , and may improve insulin sensitivity . in addition , we recently showed that the mechanistic target of rapamycin ( mtor ) signaling pathway might be involved in the expansion of mdscs ( makki , ms submitted ) , as well as in myelopoiesis . although mechanisms by which mdscs enhance insulin sensitivity are unknown , it has been proposed that upregulation of insulin growth factor-1 ( igf-1 ) in the setting of insulin resistance may lead to the accumulation of mdscs or m2 macrophages . suggestions have been made that not only does insulin resistance induce physiological response for mdsc and m2 macrophage expansion , but insulin may also modulate direct gene transcriptional control of these cells . therefore , pharmacological enhancement of insulin sensitivity in obese individuals may preemptively hinder the development of mdscs . the complex alterations in adipose tissue secretion of cytokines , adipokines , and chemokines and immune cell composition observed in adipose tissue - related pathologies such as obesity ( figure 1 ) have been , and still are , an active research area . as the proportion of overweight and obese ( even among the youngest ) continues to rise worldwide , understanding the role of adipose tissue in the pathogenesis of obesity and its metabolic and immune - based complications will be critical to optimize long - term health outcomes . as summarized in the present review , there might be a potential therapeutic value of targeting certain immune resident cells ( such as m2 , tregs , or mdsc ) and/or certain cytokines , adipokines , or chemokines ( such as mcp-1/ccr2 or ccr5 ) to improve insulin resistance and restrain organ damage in type 2 diabetic obese patients by limiting the proinflammatory milieu .
adipose tissue is a complex organ that comprises a wide range of cell types with diverse energy storage , metabolic regulation , and neuroendocrine and immune functions . because it contains various immune cells , either adaptive ( b and t lymphocytes ; such as regulatory t cells ) or innate ( mostly macrophages and , more recently identified , myeloid - derived suppressor cells ) , the adipose tissue is now considered as a bona fide immune organ , at the cross - road between metabolism and immunity . adipose tissue disorders , such as those encountered in obesity and lipodystrophy , cause alterations to adipose tissue distribution and function with broad effects on cytokine , chemokine , and hormone expression , on lipid storage , and on the composition of adipose - resident immune cell populations . the resulting changes appear to induce profound consequences for basal systemic inflammation and insulin sensitivity . the purpose of this review is to synthesize the current literature on adipose cell composition remodeling in obesity , which shows how adipose - resident immune cells regulate inflammation and insulin resistance notably through cytokine and chemokine secretion and highlights major research questions in the field .
1. Adipose Tissue Inflammation Is Crucial in the Development of Obesity-Induced Insulin Resistance 2. Adipose Tissue Cellular Remodeling in Obesity 3. Concluding Remarks
obesity is a proinflammatory condition in which hypertrophied adipocytes and adipose tissue - resident immune cells ( primarily lymphocytes and macrophages ) both contribute to increased circulating levels of proinflammatory cytokines . evidence shows that the adipose tissue secretes more than 50 hormones and signaling molecules , collectively called adipokines , which exert their biological roles in an autocrine , paracrine , or systemic manner and influence several physiological processes concerning energy , glucose metabolism , and immunity . adipose tissue from lean individuals preferentially secretes anti - inflammatory adipokines such as adiponectin , transforming growth factor beta ( tgf ) , interleukin ( il)-10 , il-4 , il-13 , il-1 receptor antagonist ( il-1ra ) , and apelin . we will rather focus on the prototypical adipokines ( tnf- , il-6 , leptin , adiponectin , and resistin ) highlighting their roles in the development of insulin resistance as well as in immunity and inflammation . tnf- is a potent proinflammatory cytokine , primarily secreted from myeloid cells via activation of mapk and nfb signaling pathways , resulting in the release of other inflammatory cytokines , such as il-1 and il-6 . more recently , the long - term assessment of anti - tnf- inhibitor treatment to subjects diagnosed with metabolic syndrome has been shown to improve fasting blood glucose and to increase adiponectin levels , confirming a role for tnf- in obesity - related insulin resistance in humans . moreover , tnf- inhibits the conversion of preadipocytes to mature adipocytes notably through downregulating adipogenic genes such as peroxisome proliferator - activated receptor gamma ( ppar ) and ccaat / enhancer binding protein ( c / ebp)allowing further recruitment of uncommitted cells and thus possible expansion of adipose tissue mass . nevertheless , the influence of tnf- on immune response mostly results from its enhancing effect on the production of other cytokines , such as il-6 , rather than from a direct effect . however , in patients with lipodystrophy , a condition characterized by almost complete lack of adipose tissue , leptin levels are very low and correlate significantly with markers of insulin resistance . adiponectin can suppress the production of tnf- and ifn ( interferon gamma ) and is a negative regulator of t cells , notably through its effect on the t - cell presenting function of dendritic cells . during the past decades , il-7 has been identified as the major homeostatic cytokine supporting the survival of and t cells , nkt cells , innate lymphoid cells , and regulatory t cells ( tregs ) . besides lipid - filled mature adipocytes , the tissue is also composed of various stromal cells , including preadipocytes , endothelial cells , fibroblasts , and immune cells . during the progression of obesity , both the adipocyte and the stroma vascular fractions are changed : adipocytes grow larger , secrete predominantly proinflammatory cytokines , and are insulin resistant ; coincidently , the nature of wat immune cells is also modified . also , it remains to be established whether the two c - c chemokine receptors , ccr2 and ccr5 , play common or unique roles in obesity - induced adipose tissue inflammation and insulin resistance . alterations in atm content and polarization state occur fairly late in the progression of obesity and probably are not initiating events of inflammation and development of sustained insulin resistance . evidence has accumulated showing that other changes in adipose - resident immune cells may precede these events . under this scenario , atms will be effectors of a coordinated inflammatory response that includes the accumulation of proinflammatory t cells ( cd8 and th1 cd4 t cells ) and the loss of anti - inflammatory regulatory t cells ( tregs ) , as well as the appearance of b cells , nk cells , nkt cells , eosinophils , neutrophils , and mast cells . adipocytes in lean adipose tissue produce factors such as il-4 and il-13 that induce m2 activation of macrophages and th2 activation of cd4 t cells and maintain treg cell and eosinophil numbers . in obesity , the progressive accumulation of adipose tissue is accompanied by early increased infiltration of proinflammatory cd8 t cells and a shift towards a higher cd8/cd4 ratio . the complex alterations in adipose tissue secretion of cytokines , adipokines , and chemokines and immune cell composition observed in adipose tissue - related pathologies such as obesity ( figure 1 ) have been , and still are , an active research area . as the proportion of overweight and obese ( even among the youngest ) continues to rise worldwide , understanding the role of adipose tissue in the pathogenesis of obesity and its metabolic and immune - based complications will be critical to optimize long - term health outcomes .
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degeneration of the dopaminergic system is still considered to be the pathological hallmark of parkinson s disease ( pd ) , although the serotonergic , cholinergic , noradrenergic and gamma - amino - butyric acid systems are affected as well ( hirsch et al . 2003 ) . importantly , when compared to the dopaminergic circuitry , these systems may actually be involved to a larger degree in the early stages of pd ( braak et al . abnormalities in non - dopaminergic systems have now been implicated in the diversity of part of the motor and non - motor symptomatology in pd ( chaudhuri 2006 ; de rijk et al . in particular , due to the widespread serotonergic innervations , serotonin ( 5-ht ) may play a role in regulating other neurotransmitter activities ( fink et al . post - mortem neurochemical studies in pd have detected up to a 50% 5-ht loss in both the cortex and the basal ganglia brain regions ( birkmayer et al . . moreover , neuronal loss and lewy body formation not only affect the dopaminergic neurons ( forno 1996 ) but also the serotonergic system ( jellinger 1987 ) . whereas , loss of 5-ht in post - mortem pd studies is well established , its degeneration in vivo and particularly its potential role in affecting clinical pd presentation still awaits elucidation . various lines of evidence have suggested that 5-ht may be involved in the etiology of resting tremor in pd . loss of striatal dopamine transporter ( dat ) binding has been shown to correlate with rigidity / bradykinesia , but not with tremor ( spiegel et al . 2006 ) and , unlike rigidity and bradykinesia the response of resting tremor to levodopa therapy is variable at best ( koller et al . these findings would argue against a pure dopaminergic etiology of resting tremor . since the late 1960s there has been continuous debate over the genesis of tremor . lesion studies in animal models of parkinsonian tremor have mainly targeted midbrain and cerebellar areas ( wilms et al . 1999 ) , thereby mainly lesioning ascending serotonergic projections to the forebrain . tremor induced by harmaline in rats and chickens can be effectively antagonized by 5-ht supplementation ( bowman et al . it has been hypothesized that this tremorgenic compound may act indirectly by inhibiting the inhibitory serotonergic projections arising from the raphe nuclei ( headley et al . finally , decrease in midbrain 5ht-1a receptors in pd along with an association with resting tremor has been recently reported in a c - way 100635 pet study ( doder et al . selective neurodegeneration within the thalamus may well contribute to the motor and non - motor symptoms of pd ( henderson et al . 2000a , b ) . noteworthy is thalamic involvement in the extensive cerebral oscillatory network that is postulated to determine resting tremor in pd , as reported in various magnetoencephalografic studies ( schnitzler et al . 2006 ; timmermann et al . 2003 ; volkmann et al . based on primate studies , experimental tremor generated by a thalamo - cortical mechanism has been defined as a parkinson - like tremor , whereas , the olivo - cerebellar system was assessed to be responsible for the faster physiological tremor ( delong 1978 ; lamarre et al . 1975 ) . in the 1980s deep brain stimulation ( dbs ) was first applied to the thalamus in patients with severe tremor ( benabid et al . a multicenter european study reported thalamic dbs to be effective on pd tremor , while the other parkinsonian hallmarks did not receive consistent benefits ( limousin et al . indeed , thalamic dbs is recommended for patients disabled only by tremor ( limousin - dowsey et al . several post - mortem studies as well as biochemical studies of breakdown products of 5-ht in the cerebral spinal fluid ( csf ) have shown reduced 5-ht activity in the brain of pd patients ( birkmayer et al . moreover , there is a functional polymorphism in the 5-ht transporter ( 5-htt ) synthesis , and the short allele ( which synthesizes less 5-htt ) is associated with pd depression ( mossner et al . treatment with selective 5-ht reuptake inhibitors ( ssri ) , e.g. , citalopram , improves pd depressive symptoms ( antonini et al . 2006 ) , although a role for dopaminergic drugs has also been suggested ( barone et al . 2006 ) . interestingly , a [ i]-cit spect study showed the capability of citalopram to block thalamic 5-htt in depressed patients ( pirker et al . 1995 ) , thus demonstrating the thalamus to be an anatomic site of action of an ssri . in vivo displacement studies in primates have shown striatal [ i]-cit binding to be predominantly associated with dat , whereas , binding in the diencephalon was mainly associated with 5-htt ( innis et al . [ i]-cit has been shown to be useful in monitoring the dopaminergic degeneration of the nigrostriatal pathway in pd ( innis 1994 ; tissingh et al . 1998 ) , but it may be also used to assess the integrity of thalamic 5-htt ( de win et al . 2005 ) . in the light of these data we chose to investigate 5-htt binding in the thalamic area by means of [ i]-cit spect . the primary aim of this study was to assess whether in pd thalamic 5-htt binding is decreased in vivo . consequently , we investigated thalamic 5-htt binding as well as striatal dat binding in 32 unrelated drug - nave early pd patients and in 13 healthy subjects using [ i]-cit spect . additionally , we hypothesized that 5-htt binding declines as disease progresses . in order to verify this hypothesis we repeated scans on 26 patients in an average of 17 months later . we postulated that 5-htt loss may be associated with the presence of tremor and depression at disease onset . we studied 32 drug - nave early - stage pd patients as well as 13 healthy volunteers . diagnosis of pd was made according to uk brain bank criteria ( hughes et al . 1992 ) . patients were recruited regardless of their phenotype , i.e. , presence of tremor or depression , as the primary aim of the study was to investigate whether 5-htt binding has decreased in vivo in an early drug - nave pd population . a [ i]-cit follow - up scan was performed on 26 patients out of the initial group over a period ( mean sd ) of 17 9 months . at baseline , all patients were drug - nave for dopaminergic treatment and two patients used benzodiazepines , whereas , at follow - up , they had initiated dopaminergic medication ( levodopa or a d2 agonist ) . pd patients with dementia were not included : a mini - mental state examination ( mmse ) score below 26 was used as an exclusion criterion . at baseline , pd severity was assessed using the motor part of the unified parkinson s disease rating scale ( updrs - iii ) . based on updrs tremor scores ( total score on item 20 ) , we categorized patients in two subgroups : pd patients with moderate / severe tremor at onset ( pdt subgroup ; tremor score 2 in at least one limb ) and pd patients without tremor at onset ( pdwt subset ; tremor score of 0 ) . note that all patients with tremor had a score equal to zero on item 21 : they revealed resting but no action tremor during clinical examination . the two groups displayed no significant difference in rigidity and bradykinesia updrs scores ( items 18 , 19 , 22 , 27 , 28 , 29 , 30 and 31 ) . in contrast , patients ( n = 12 ) who had a updrs tremor score of 1 were not included in any of the subset . at the time of the first scan , the beck depression inventory scale ( bdi ) was used to assess depressive symptoms ( visser et al . see table 1 for details on demographic and clinical data of the participants . table 1demographic and clinical data of the participantsbaseline17 months follow - upco ( n = 13)pd ( n = 32)pdt ( n = 8)pdwt ( n = 12)pd ( n = 26)pdt ( n = 6)pd wt ( n = 11)sex ( male)822551745age ( years , mean sd)53 854 1055 956 1056 954 958 7disease duration ( years , mean sd)2 12 12 14 14 14 1updrs motor score18 716 619 8updrs tremor score ( item 20)1 13 10updrs bradykinesia / rigidity score ( items:18 + 19 + 22 + 27 + 28 + 29 + 30 + 31)13 69 613 6bdi score8 67 78 6pd parkinson s disease , pdt pd patients subgroup with moderate / severe tremor at onset , pdwt pd patients subgroup without tremor at onset , sd standard deviation data not available demographic and clinical data of the participants pd parkinson s disease , pdt pd patients subgroup with moderate / severe tremor at onset , pdwt pd patients subgroup without tremor at onset , sd standard deviation data not available patients were recruited at the vu university medical center ( vumc ) , movement disorders clinic . the hospital ethics committee approved the study and all patients signed informed consent forms . spect studies were performed using a brain - dedicated system , the strichmann medical equipment 810. the strichmann camera system consists of 12 individual crystals , each equipped with a focusing collimator . the transaxial resolution is 7.6 mm full - width at half - maximum ( fwhm ) ( vermeulen et al . [ i]-cit ( specific activity of > 185 mbq / nmol ; radiochemical purity > 99% ) was injected intravenously as a bolus , at a dose of approximately 110 mbq . [ i ] labeling of -cit was performed as described earlier ( tissingh et al . spect image acquisition was performed 24 h after injection , a time point at which [ i]-cit binding to striatal dat and thalamic 5-htt is in an equilibrium state ( pirker et al . slices were acquired during 300 s periods from the orbitomeatal line to the vertex with an interslice distance of 10 mm . data acquisition took place in a 128 128 matrix . attenuation correction and reconstruction of the images analysis of the [ i]-cit binding was performed on two contiguous transverse slices representing the most intense striatal and thalamic binding . a standard region of interest ( roi ) template was manually drawn with the aid of a stereotactic atlas . in order to distinguish the forebrain from the brainstem , we defined the striatal inferior level to be the anatomic limit between the midbrain and the thalamic region . specific striatal [ i]-cit binding to dat in whole striatum , putamen and caudate nucleus and specific thalamic [ i]-cit binding to 5-htt were calculated using the formula : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ { { \left [ { { \left ( { { \text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{specific}}\,{\text{roi } } } \right ) } - { \left ( { { \text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non } } - { \text{specific}}\,{\text{roi } } } \right ) } } \right ] } } \mathord{\left/ { \vphantom { { { \left [ { { \left ( { { \text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{specific}}\,{\text{roi } } } \right ) } - { \left ( { { \text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non } } - { \text{specific}}\,{\text{roi } } } \right ) } } \right ] } } { { \left ( { { \text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non } } - { \text{specific}}\,{\text{roi } } } \right ) } } } } \right . \kern-\nulldelimiterspace } { { \left ( { { \text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non } } - { \text{specific}}\,{\text{roi } } } \right ) } } $ $ \end{document}this formula is referred as the specific to non - specific [ i]-cit binding ratio ( sns binding ratio ) . the occipital region was chosen as reference region because of negligible density for both dat and 5-htt ( laruelle et al . difference in mean [ i]-cit binding ratio between the various groups was expressed as a percentage and calculated as follows : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ { \left [ { { \left ( { { \text{groupx}}\,{\text{sns } } - { \text{groupy}}\,{\text{sns } } } \right ) } - { \text{groupx}}\,{\text{sns } } } \right ] } \times 100\% . $ $ \end{document } the investigator performing roi analysis was blind to the subjects diagnosis as well as demographics . an independent sample t test with equal variances not assumed was used to investigate the variation in mean [ i]-cit binding values between pd patients and healthy control subjects . we compared [ i]-cit binding values between pdt and pdwt cohorts using one - way analysis of variance ( anova ) . a paired simple two tailed t test was used to examine the change between the same group imaging results analyzed at baseline and at follow - up ( i.e. , pd patients , pdt and pdwt subgroup ) . the kolmogrov smirnov and the levene s test were applied to screen for normality and equal variance , respectively . additionally , to increase statistical power , we attempted to normalize pd putamen and pdwt striatum binding values with an algorithmic [ log10(x + 1 ) ] and an inverse ( 1/x ) transformation , respectively . conversely , putamen and thalamic binding values of the pdt cohort at follow - up could not be normalized using these transformations , thus were compared to the respective values of the pdt cohort at baseline using the mann whitney test . the latter was also used to compare putamen and thalamic binding values of the pdt and pdwt subgroup at follow - up . pearson bivariate correlation was used to correlate bdi scores with thalamic [ i]-cit binding values . one - way anova was applied to examine variations in mean bdi score between pdt and pdwt subgroups . differences between groups were considered significant at a level of p 0.05 . we studied 32 drug - nave early - stage pd patients as well as 13 healthy volunteers . diagnosis of pd was made according to uk brain bank criteria ( hughes et al . 1992 ) . patients were recruited regardless of their phenotype , i.e. , presence of tremor or depression , as the primary aim of the study was to investigate whether 5-htt binding has decreased in vivo in an early drug - nave pd population . a [ i]-cit follow - up scan was performed on 26 patients out of the initial group over a period ( mean sd ) of 17 9 months . at baseline , all patients were drug - nave for dopaminergic treatment and two patients used benzodiazepines , whereas , at follow - up , they had initiated dopaminergic medication ( levodopa or a d2 agonist ) . pd patients with dementia were not included : a mini - mental state examination ( mmse ) score below 26 was used as an exclusion criterion . at baseline , pd severity was assessed using the motor part of the unified parkinson s disease rating scale ( updrs - iii ) . based on updrs tremor scores ( total score on item 20 ) , we categorized patients in two subgroups : pd patients with moderate / severe tremor at onset ( pdt subgroup ; tremor score 2 in at least one limb ) and pd patients without tremor at onset ( pdwt subset ; tremor score of 0 ) . note that all patients with tremor had a score equal to zero on item 21 : they revealed resting but no action tremor during clinical examination . the two groups displayed no significant difference in rigidity and bradykinesia updrs scores ( items 18 , 19 , 22 , 27 , 28 , 29 , 30 and 31 ) . in contrast , patients ( n = 12 ) who had a updrs tremor score of 1 were not included in any of the subset . at the time of the first scan , the beck depression inventory scale ( bdi ) was used to assess depressive symptoms ( visser et al . see table 1 for details on demographic and clinical data of the participants . table 1demographic and clinical data of the participantsbaseline17 months follow - upco ( n = 13)pd ( n = 32)pdt ( n = 8)pdwt ( n = 12)pd ( n = 26)pdt ( n = 6)pd wt ( n = 11)sex ( male)822551745age ( years , mean sd)53 854 1055 956 1056 954 958 7disease duration ( years , mean sd)2 12 12 14 14 14 1updrs motor score18 716 619 8updrs tremor score ( item 20)1 13 10updrs bradykinesia / rigidity score ( items:18 + 19 + 22 + 27 + 28 + 29 + 30 + 31)13 69 613 6bdi score8 67 78 6pd parkinson s disease , pdt pd patients subgroup with moderate / severe tremor at onset , pdwt pd patients subgroup without tremor at onset , sd standard deviation data not available demographic and clinical data of the participants pd parkinson s disease , pdt pd patients subgroup with moderate / severe tremor at onset , pdwt pd patients subgroup without tremor at onset , sd standard deviation data not available patients were recruited at the vu university medical center ( vumc ) , movement disorders clinic . the hospital ethics committee approved the study and all patients signed informed consent forms . spect studies were performed using a brain - dedicated system , the strichmann medical equipment 810. the strichmann camera system consists of 12 individual crystals , each equipped with a focusing collimator . the transaxial resolution is 7.6 mm full - width at half - maximum ( fwhm ) ( vermeulen et al . [ i]-cit ( specific activity of > 185 mbq / nmol ; radiochemical purity > 99% ) was injected intravenously as a bolus , at a dose of approximately 110 mbq . [ i ] labeling of -cit was performed as described earlier ( tissingh et al . spect image acquisition was performed 24 h after injection , a time point at which [ i]-cit binding to striatal dat and thalamic 5-htt is in an equilibrium state ( pirker et al . slices were acquired during 300 s periods from the orbitomeatal line to the vertex with an interslice distance of 10 mm . data acquisition took place in a 128 128 matrix . attenuation correction and reconstruction of the images analysis of the [ i]-cit binding was performed on two contiguous transverse slices representing the most intense striatal and thalamic binding . a standard region of interest ( roi ) template was manually drawn with the aid of a stereotactic atlas . in order to distinguish the forebrain from the brainstem , we defined the striatal inferior level to be the anatomic limit between the midbrain and the thalamic region . specific striatal [ i]-cit binding to dat in whole striatum , putamen and caudate nucleus and specific thalamic [ i]-cit binding to 5-htt were calculated using the formula : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ { { \left [ { { \left ( { { \text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{specific}}\,{\text{roi } } } \right ) } - { \left ( { { \text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non } } - { \text{specific}}\,{\text{roi } } } \right ) } } \right ] } } \mathord{\left/ { \vphantom { { { \left [ { { \left ( { { \text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{specific}}\,{\text{roi } } } \right ) } - { \left ( { { \text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non } } - { \text{specific}}\,{\text{roi } } } \right ) } } \right ] } } { { \left ( { { \text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non } } - { \text{specific}}\,{\text{roi } } } \right ) } } } } \right . \kern-\nulldelimiterspace } { { \left ( { { \text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non } } - { \text{specific}}\,{\text{roi } } } \right ) } } $ $ \end{document}this formula is referred as the specific to non - specific [ i]-cit binding ratio ( sns binding ratio ) . the occipital region was chosen as reference region because of negligible density for both dat and 5-htt ( laruelle et al . difference in mean [ i]-cit binding ratio between the various groups was expressed as a percentage and calculated as follows : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ { \left [ { { \left ( { { \text{groupx}}\,{\text{sns } } - { \text{groupy}}\,{\text{sns } } } \right ) } - { \text{groupx}}\,{\text{sns } } } \right ] } \times 100\% . $ $ \end{document } the investigator performing roi analysis was blind to the subjects diagnosis as well as demographics . statistical analysis was performed using the spss 13.0 software package . an independent sample t test with equal variances not assumed was used to investigate the variation in mean [ i]-cit binding values between pd patients and healthy control subjects . we compared [ i]-cit binding values between pdt and pdwt cohorts using one - way analysis of variance ( anova ) . a paired simple two tailed t test was used to examine the change between the same group imaging results analyzed at baseline and at follow - up ( i.e. , pd patients , pdt and pdwt subgroup ) . the kolmogrov smirnov and the levene s test were applied to screen for normality and equal variance , respectively . additionally , to increase statistical power , we attempted to normalize pd putamen and pdwt striatum binding values with an algorithmic [ log10(x + 1 ) ] and an inverse ( 1/x ) transformation , respectively . conversely , putamen and thalamic binding values of the pdt cohort at follow - up could not be normalized using these transformations , thus were compared to the respective values of the pdt cohort at baseline using the mann whitney test . the latter was also used to compare putamen and thalamic binding values of the pdt and pdwt subgroup at follow - up . pearson bivariate correlation was used to correlate bdi scores with thalamic [ i]-cit binding values . one - way anova was applied to examine variations in mean bdi score between pdt and pdwt subgroups . differences between groups were considered significant at a level of p 0.05 . there was no difference with regard to demographic features ( age and disease duration ) between pd patients and healthy controls and between the pdt and pdwt cohorts ( table 1 ) . pd patients had an average of 61% lower putaminal sns [ i]-cit binding ratios than healthy controls . in addition , average caudate nucleus and whole striatum sns binding ratios were also significantly lower in pd patients compared to healthy controls ( table 2 ) . we found a significant reduction of 22% in mean thalamic sns [ i]-cit binding ratio in pd patients versus healthy controls ( table 2 ) . interestingly , mean sns thalamic binding ratio was significantly lower in the pdt as compared to the pdwt cohort by 19% ( table 2 , fig . 2 ) . conversely , there was no significant difference in whole striatum , putamen and caudate nucleus average sns binding ratios between pdt and pdwt ( table 2 ) . table 2mean specific to non - specific [ i]-cit binding ratios ( mean sd ) assessed at baselineregion of interestbaselinecontrols ( n = 13)pd patients ( n = 32)pdt ( n = 8)pdwt ( n = 12)striatum , whole8.65 2.614.39 1.10 * 4.38 0.954.21 1.45caudate , whole9.43 2.556.34 1.55 * 5.60 0.986.93 1.93putamen , whole7.72 2.653.02 0.85 * 3.08 0.792.87 1.10thalamus , whole1.98 0.561.55 0.34 * 1.37 0.371.70 0.33 * * significant difference between controls and pd patients ( * p 0.02 ) and between pdt and pdwt patients ( p = 0.05)pd parkinson s disease , pdt pd patients subgroup with moderate / severe tremor at onset , pdwt pd patients subgroup without tremor at onset , sd standard deviation mean specific to non - specific [ i]-cit binding ratios ( mean sd ) assessed at baseline * significant difference between controls and pd patients ( * p 0.02 ) and between pdt and pdwt patients ( p = 0.05 ) pd parkinson s disease , pdt pd patients subgroup with moderate / severe tremor at onset , pdwt pd patients subgroup without tremor at onset , sd standard deviation average putaminal sns [ i]-cit ratio in repeated scans showed a significant decline of 17% from the baseline ( table 3 ) . notably , we found that thalamic sns [ i]-cit binding ratio was significantly further decreased at follow - up as compared to the pd baseline measurement by 29% ( table 3 ; fig . 1 ) . note that there was no significant difference in thalamic sns binding ratios between pdt and pdwt subgroups at the time of the second [ i]-cit spect ( fig . 2 ) . whereas , there was no significant difference in putaminal and thalamic radiotracer binding between pdt cohort at baseline , and at follow - up putaminal and thalamic [ i]-cit binding was significantly decreased in the pdwt subgroup at the time of the second as compared to the first scan ( 19 and 39% , respectively ) ( table 3 ; fig . 2 ) . table 3mean specific to non - specific [ i]-cit binding ratio ( mean sd ) at baseline and at follow - up ( 17 9 months later)region of interestbaseline pd ( n = 26)follow - up pd ( n = 26)baseline pdt ( n = 6)follow - up pdt ( n = 6)baseline pdwt ( n = 12)follow - up pdwt ( n = 11)striatum , whole4.17 0.953.86 0.754.16 0.983.79 0.423.91 1.13.64 0.73caudate , whole6.13 1.475.60 1.365.88 1.365.65 0.685.76 1.585.52 1.16putamen , whole2.84 0.712.36 0.51 * 2.83 0.522.37 0.292.74 0.932.22 0.40*thalamus , whole1.50 0.351.07 0.39 * 1.26 0.341.16 0.291.65 0.311.00 0.45 * * significant difference between pd patients at baseline and at follow - up ( p < 0.01 ) and between pdwt subgroup at baseline and at follow - up ( p < 0.05)pd parkinson s disease , pdt pd patients subgroup with moderate / severe tremor at onset , pdwt pd patients subgroup without tremor at onset , sd standard deviationfig . 1scatterplot showing the rate of change in thalamic specific to non - specific [ i]-cit binding ratio in healthy controls and in pd patients at baseline and at follow - up . at the time of the first imaging series , mean thalamic [ i]-cit binding ratio was significantly lower in pd patients as compared to controls ( p = 0.02 ) . moreover , mean thalamic ratio obtained in pd patients was significantly lower at the follow - up as compared to the baseline ( p < 0.01 ) . pd baseline and pd follow - up indicate pd patients at baseline and at the follow - up , respectivelyfig . 2scatterplot showing the rate of change in thalamic specific to non - specific [ i]-cit binding ratio in the pdwt as compared to the pdt subgroup over time . mean thalamic [ i]-cit binding ratio was significantly lower in the pdt versus the pdwt subgroup at baseline ( p = 0.05 ) . no difference in mean thalamic ratio between the pdt and the pdwt subset was detected at the time of the second imaging series . moreover , mean thalamic ratio in the pdwt subgroup was significantly lower at follow - up than at baseline ( p < 0.01 ) . pdt and pdwt indicate pd patients subgroups with moderate / severe tremor and without tremor at onset , respectively mean specific to non - specific [ i]-cit binding ratio ( mean sd ) at baseline and at follow - up ( 17 9 months later ) * significant difference between pd patients at baseline and at follow - up ( p < 0.01 ) and between pdwt subgroup at baseline and at follow - up ( p < 0.05 ) pd parkinson s disease , pdt pd patients subgroup with moderate / severe tremor at onset , pdwt pd patients subgroup without tremor at onset , sd standard deviation scatterplot showing the rate of change in thalamic specific to non - specific [ i]-cit binding ratio in healthy controls and in pd patients at baseline and at follow - up . at the time of the first imaging series , mean thalamic [ i]-cit binding ratio was significantly lower in pd patients as compared to controls ( p = 0.02 ) . moreover , mean thalamic ratio obtained in pd patients was significantly lower at the follow - up as compared to the baseline ( p < 0.01 ) . pd baseline and pd follow - up indicate pd patients at baseline and at the follow - up , respectively scatterplot showing the rate of change in thalamic specific to non - specific [ i]-cit binding ratio in the pdwt as compared to the pdt subgroup over time . mean thalamic [ i]-cit binding ratio was significantly lower in the pdt versus the pdwt subgroup at baseline ( p = 0.05 ) . no difference in mean thalamic ratio between the pdt and the pdwt subset was detected at the time of the second imaging series . moreover , mean thalamic ratio in the pdwt subgroup was significantly lower at follow - up than at baseline ( p < 0.01 ) . conversely no difference was found in the pdt subset . pdt and pdwt indicate pd patients subgroups with moderate / severe tremor and without tremor at onset , respectively thalamic sns binding ratios did not correlate with bdi scores and there was no difference in bdi score between pdt and pdwt ( table 1 ) . we found that mean [ i]-cit spect putaminal binding was reduced in all early pd patients at baseline as well as at follow - up . moreover , average thalamic 5-htt binding was decreased in early drug - nave pd patients compared to controls and declined as the disease proceeded . the pdt cohort displayed a lower 5-htt thalamic binding when compared to the pdwt cohort at baseline , while no difference could be detected during follow - up . finally , in the pdwt subgroup a further decline in both putaminal and thalamic binding was detected at follow - up , which was not true for the pdt subgroup . to our knowledge , this is the first longitudinal study to investigate 5-htt loss in pd . van dyck et al . ( 2000 ) reported a mean 4.2% per decade age - related decline of 5-htt in the diencephalon of healthy humans by means of [ i]-cit spect . however , this decay rate is much lower that the one we presently reported in pd patients ( 29% over 17 months ) . therefore , whereas 5-htt loss may occur with age on a physiological basis , consistent with our results its decay appears more severe in pd patients . these data confirm the first two hypotheses we stated , according to our results 5-htt is decreased in pd and further declines along with the disease . noteworthy , it has been argued that [ i]-cit radiotracer in the thalamus may bind to monoaminergic transporters other than 5-htt . in particular , in a monkey pet study ( farde et al . 1994 ) citalopram as well as desipramine , a norepinephrine transporter ( net ) blocker , was able to displace thalamic [ i]-cit binding by 50 and 40% , respectively . ( 1993 ) demonstrated that the selective net inhibitor maprotiline failed to affect [ i]-cit binding in monkey diencephalon . ( 2005 ) validated [ i]-cit to assess in vivo 5-htt in the thalamic area . importantly , in this study , a double - blind , placebo - controlled , crossover design with citalopram ( the most selective 5-htt blocker ) was performed . moreover , it has been demonstrated that citalopram blocks approximately 50% of thalamic [ i]-cit binding in depressed patients ( pirker et al . 2007 ) , venlafaxine ( a 5-htt and net inhibitor ) proved to decrease [ i]-cit spect binding by 54% , an outcome similar to the one obtained by pirker et al . therefore , it may be argued that in vivo only a small part of the [ i]-cit binding to human thalamus is due to net binding . finally , it must be taken into account that in the human brain 5-htt is much higher than net thalamic concentration ( tong et al . 2007 ) , while the affinity of [ i]-cit ( or rti-55 ) for net is lower than for 5-htt ( scheffel et al . 1997 ) . to summarize , although we can not exclude that in vivo [ i]-cit binding in the human thalamus , may be partly due to net binding ; the largest part likely represents 5-htt binding . nonetheless , further studies with selective radiotracers for 5-htt are needed to reproduce our findings . in this context , two previous pet studies with selective 5-htt tracers found no significant thalamic 5-htt decrease in pd patients when compared to controls ( guttman et al . in particular , kerenyi et al . describe a trend for lower thalamic 5-htt binding with a decrease of approximately 25% , a range loss similar to the one we presently report ( 22% ) . importantly , the small number of participants investigated in the pet studies mentioned above ( 9 and 13 , respectively ) may have played a role for a non - significant thalamic decreased binding outcome . this may be relevant to the present study where a thalamic 5-htt decline over time was demonstrated in a larger cohort of 26 pd patients . at baseline , we found lower thalamic 5-htt density in pd patients with mild to severe tremor compared to patients without tremor . the two cohorts ( pdt and pdwt ) differed in terms of updrs tremor scores but were comparable in terms of rigidity and bradykinesia . in fact , pdt had a updrs resting tremor score 2 in at least one limb , whereas , the pdwt presented no tremor at all . resting tremor was the only one out of the cardinal motor features that clinically characterize pd ( tremor , bradykinesia , rigidity and postural instability ) to differentiate the two patient subsets . moreover , the absence of significant differences in striatal dat binding between the two subgroups confirms the assumption we made based on previous studies : bradykinesia and rigidity but not tremor correlates with striatal dat binding ( spiegel et al . 2006 ) . these data suggest that 5-ht may play a role in the etiology of resting tremor in early pd stages . in this context , haapaniemi et al . reported a lower thalamic [ i]-cit binding in pd compared to controls , although , in contrast with our results , no correlation to tremor scores was detected ( haapaniemi et al . we obtained data 24 h post - injection , whereas they acquired scans at 4 h post - injection . although there is still considerable debate as to the best timing to acquire spect images ( brucke et al . 1994b ) , scans obtained between 20 and 24 h showed to be closer to a state of transient equilibrium ( pirker et al . 2000 ) . recently , doder et al . ( 2003 ) reported a reduction of in vivo raphe 5-ht1a binding in pd patients and an association with the severity of updrs tremor scores using c - way 100635 pet . the authors suggested that this reduction expresses loss of 5-ht cell bodies , possibly due to lewy body degeneration . our present finding indicating thalamic [ i]-cit binding decline is in agreement with this hypothesis , since the 5-htt is located exclusively on the 5-ht neuronal membrane . alternatively , 5-ht and dopaminergic activity may decline in parallel in the striatum and in the midbrain of parkinsonian patients , particularly in patients with tremor onset . reduced serotonergic function would result in less inhibition and in turn facilitate striatal dopamine release ( de et al . we investigated pd patients at onset , and did not investigate radiotracer binding within raphe nuclei ; whereas doder et al . examined participants at an advanced stage and did not analyze the thalamic area . note that unlike their study , we chose not to correlate thalamic binding to updrs total tremor scores . in fact , patients we enrolled were at an early stage and thus presented a very low total tremor score ( mean items 20 total score sd = 1 1 ) . due to such a low variance in total tremor score , a correlation between these values and sns thalamic binding could not have been evaluated adequately . also note that both studies did not take into account 5-htt levels in striatal and cortical regions , which express low to moderate 5-htt concentrations . according to braak et al . ( 2003 ) midbrain stem is the first component of the somato - motor and emotional motor system to be affected , whereas , the degeneration of cells in the substantia nigra , thalamic nuclei and neocortical areas follows . in the light of this evidence , a comparison between 5-htt availability among brain areas presenting 5-htt in subsequent pd stages by means of selective 5-htt tracers is warranted to better assess the role of 5-ht in pd . in fact , although in pd alterations of striatal and cortical 5-htt may be associated with tremor , in the present study we were not able to assess striatal 5-htt binding , as [ i]-cit binds predominantly to dat in the striatum ( laruelle et al . additionally , although we provided some evidence that [ i]-cit spect may be used to measure 5-htt in 5-htt - low cortical areas , these measurements must be interpreted with caution ( de win et al . unlike baseline imaging , the repeated scans showed no statistical difference in thalamic 5-htt binding between the pdwt and pdt group . furthermore , the pdwt subgroup revealed a faster 5-htt thalamic decline as compared to the pdt group . it has been demonstrated that pd patients , who manifest symptoms at a relatively [ i]-cit high density , progress substantially faster than those who manifest symptoms at a lower transporter density ( seibyl et al . 1999 ) . while we do not have an explanation for this phenomenon , a progressive loss of surviving neurons could be postulated . both lewy body degeneration and reduced 5-htt inhibiting activity over the striatum might have a major effect in those patients still presenting with a high transporter density . additionally , we were not able to assess whether tremor had eventually revealed in the pdwt subgroup at the time of the second scan , as patients were under dopaminergic treatment . indeed the lack of follow - up updrs clinical examinations represents a limitation of our study . 5-ht neurotransmission is impaired in pd , but its causal relationship with tremor remains controversial and it is unclear what the clinical relevance of a 22% thalamic 5-htt loss is . due to the design of the present study , our findings do not allow a definitive answer on pd tremor but certainly supports the role of 5-htt function . as of yet , [ i]-cit spect imaging correlating 5-htt binding decline to depressive symptoms in pd has revealed inconsistent results . kim et al . ( 1999 ) reported no significant correlation between hypothalamic / midbrain 5-htt in pd and updrs - i ( depression items ) . a further study showed no relationship between 5-htt binding and updrs rating of mood in thalamus , but detected a significant correlation in dorsal midbrain ( murai et al . finally , haapianemi and colleagues demonstrated that 5-htt frontal region binding in pd correlates to updrs - i ( haapaniemi et al . , we found no correlation between bdi scores and thalamic 5-htt and , even more importantly , there was no difference in bdi between the pdt and pdwt subgroups suggesting no contribution of depressive symptoms to these findings . whereas , we used bdi , a reliable self - assessment scale to assess depression in pd ( visser et al . 2006 ) , other studies used updrs rating for mood , which is a less specific screening tool . we did not assess bdi at the follow - up , therefore depressive symptoms may not have been revealed until a more advanced stage , along with thalamic 5-htt decline . in conclusion , in this study we show loss of thalamic 5-htt binding in pd and its progressive loss over an average of 17 months . at baseline , a decreased thalamic 5-htt density in patients presenting tremor at onset was found , while no correlation could be detected between thalamic [ i]-cit binding ratios and bdi scores . in this context , further studies with selective 5-htt tracers are needed to reproduce our findings in early pd . additionally , new research is warranted to gain a better insight into the causal relation that links 5-ht dysfunction and tremor and depression in pd . finally , pharmacological challenges with serotonergic agents may be performed to test their potential capacity in counteracting pd tremor .
in vitro studies revealed serotonin transporter ( 5-htt ) decline in parkinson s disease ( pd ) . yet , few studies investigated thalamic 5-htt in vivo and its effect on pd heterogeneity . we analyzed thalamic [ 123i]-cit binding ( mainly reflecting 5-htt binding ) in 32 drug - nave pd patients and 13 controls with spect . twenty - six patients were examined twice ( 17 months apart ) . based on updrs scores , we identified subgroups of patients with moderate / severe tremor ( pdt ) and without tremor ( pdwt ) at the time of clinical diagnosis . additionally , depressive symptoms were evaluated using the beck depression inventory ( bdi ) at baseline . mean thalamic specific to non - specific [ 123i]-cit binding ratio was lower in patients when compared to controls , and further decreased during follow - up . at baseline , average thalamic ratio was significantly lower in the pdt than in the pdwt subgroup . no correlation was found between bdi scores and thalamic binding ratios . our findings show decline of [ 123i]-cit binding to thalamic 5-htt in pd and its possible contribution to tremor onset .
Introduction Materials and methods Subjects SPECT procedure Data processing Statistical analysis Results Discussion
table 2mean specific to non - specific [ i]-cit binding ratios ( mean sd ) assessed at baselineregion of interestbaselinecontrols ( n = 13)pd patients ( n = 32)pdt ( n = 8)pdwt ( n = 12)striatum , whole8.65 2.614.39 1.10 * 4.38 0.954.21 1.45caudate , whole9.43 2.556.34 1.55 * 5.60 0.986.93 1.93putamen , whole7.72 2.653.02 0.85 * 3.08 0.792.87 1.10thalamus , whole1.98 0.561.55 0.34 * 1.37 0.371.70 0.33 * * significant difference between controls and pd patients ( * p 0.02 ) and between pdt and pdwt patients ( p = 0.05)pd parkinson s disease , pdt pd patients subgroup with moderate / severe tremor at onset , pdwt pd patients subgroup without tremor at onset , sd standard deviation mean specific to non - specific [ i]-cit binding ratios ( mean sd ) assessed at baseline * significant difference between controls and pd patients ( * p 0.02 ) and between pdt and pdwt patients ( p = 0.05 ) pd parkinson s disease , pdt pd patients subgroup with moderate / severe tremor at onset , pdwt pd patients subgroup without tremor at onset , sd standard deviation average putaminal sns [ i]-cit ratio in repeated scans showed a significant decline of 17% from the baseline ( table 3 ) . table 3mean specific to non - specific [ i]-cit binding ratio ( mean sd ) at baseline and at follow - up ( 17 9 months later)region of interestbaseline pd ( n = 26)follow - up pd ( n = 26)baseline pdt ( n = 6)follow - up pdt ( n = 6)baseline pdwt ( n = 12)follow - up pdwt ( n = 11)striatum , whole4.17 0.953.86 0.754.16 0.983.79 0.423.91 1.13.64 0.73caudate , whole6.13 1.475.60 1.365.88 1.365.65 0.685.76 1.585.52 1.16putamen , whole2.84 0.712.36 0.51 * 2.83 0.522.37 0.292.74 0.932.22 0.40*thalamus , whole1.50 0.351.07 0.39 * 1.26 0.341.16 0.291.65 0.311.00 0.45 * * significant difference between pd patients at baseline and at follow - up ( p < 0.01 ) and between pdwt subgroup at baseline and at follow - up ( p < 0.05)pd parkinson s disease , pdt pd patients subgroup with moderate / severe tremor at onset , pdwt pd patients subgroup without tremor at onset , sd standard deviationfig . pdt and pdwt indicate pd patients subgroups with moderate / severe tremor and without tremor at onset , respectively mean specific to non - specific [ i]-cit binding ratio ( mean sd ) at baseline and at follow - up ( 17 9 months later ) * significant difference between pd patients at baseline and at follow - up ( p < 0.01 ) and between pdwt subgroup at baseline and at follow - up ( p < 0.05 ) pd parkinson s disease , pdt pd patients subgroup with moderate / severe tremor at onset , pdwt pd patients subgroup without tremor at onset , sd standard deviation scatterplot showing the rate of change in thalamic specific to non - specific [ i]-cit binding ratio in healthy controls and in pd patients at baseline and at follow - up .
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dna sequences coding for the huj591 variable regions were kindly provided by dr . the mb ( 80 kda ) is a bivalent homodimer with each monomer consisting of a single - chain variable fragment ( scfv ) linked to a human igg1 hinge and ch3 domain . the mb exists as a stable dimer due to the natural association between the ch3 domains as well as the formation of disulfide bonds within the hinge regions . the cys - db ( 50 kda ) is a bivalent homodimer formed from two cross - paired scfv fragments . each chain consists of the variable regions linked together by a gly - ser linker . a cys tail , consisting of the sequence gly - gly - cys , is fused to the c - terminus to enable the stabilization of the diabody complex by formation of a covalent disulfide bond . purified huj591 mb and cys - db proteins were provided by imaginab , inc . ( inglewood , ca ) . a 3-fold equivalence of dfo - bz - scn ( macrocylics , inc . ) in dmso ( < 5% total volume ) was added to a solution of mb or cys - db . huj591 was reacted with dfo - bz - scn with a 1:5 mab : chelate ratio . all reactions were incubated at 37 c for 45 min with occasional mild stirring . subsequent purification using a pd10 size exclusion column removed any unreacted dfo - bz - scn and dmso with 0.9% saline as the mobile phase . the purified product was concentrated to a volume of 500 l using a vivaspin 500 ( mwco : 10 kda ) centrifugal filter . sds gel electrophoresis was performed to analyze purified dfo conjugates of the mb and cys - db ( see the supporting information and si figure 1 in the supporting information ) . zr - oxalate ( 37 mbq ) was produced as previously described ( see the supporting information ) . the ph of zr was adjusted to 7.07.2 with 1 m na2co3 . to each separate reaction , approximately 125140 g of mb / cys - db - dfo ( mb - dfo , 1.561.75 nmol ; cys - db - dfo , 2.52.8 nmol ) or 250 g ( 1.7 nmol ) of huj591-dfo was added and incubated at room temperature with intermittent mild shaking . after 11.5 h , the reaction was quenched with 10 l of 50 mm dtpa ( ph 7 ) to remove any nonspecifically bound zr . crude radiolabeling yields were determined to be > 95% using itlc with the zr - labeled proteins remaining close to the origin at rf = 0.30 while free zr-89 is found near the solvent front at rf = 0.65 . purification of zr - proteins was performed using a pd10 size exclusion column with saline as the eluting buffer . the final radiochemical purity was > 99% based on itlc analysis . to address retained affinity for the antigen , both radiolabeled constructs zr - mb and zr - cys - db lncap ( psma(+ ) ) and pc3 ( psma( ) ) human prostate cancer ( pc ) cell lines ( american type culture collection ) were cultured in a sterile environment with 5% co2 at 37 c and grown as described . all animal experiments were conducted in accordance with the guidelines set by mskcc animal care and use committee and research animal resource center . for imaging experiments , male athymic nude ( nu / nu ) mice ( 68 week old , taconic ) lncap cells ( 3 10 ) in 1:1 medium : matrigel ( bd sciences ) were implanted on the left shoulder . pc3 cells ( 3 10 in 1:1 medium : matrigel ) were injected on the right shoulder . tumor growth was monitored weekly and measured using vernier calipers with the volume calculated using the formula length width height 0.52 . internalization of zr - mb , zr - cys - db , and fully human zr - huj591 was investigated on lncap and pc3 cells . approximately 1 10 cells were seeded in a 12-well plate and incubated overnight . a volume of 2 ml of radiolabeled protein ( 37 kbq / ml ) was added to each well . the plates were incubated at 37 and 4 c for 0.524 h. following each incubation period , the medium was collected and the cells were rinsed with 1 ml of phosphate buffered saline ( pbs ) twice . surface - bound activity was collected by washing the cells in 1 ml of 100 mm acetic acid + 100 mm glycine ( 1:1 , ph 3.5 ) at 4 c . the adherent cells were then lysed with 1 ml of 1 m naoh . each wash was collected and counted for activity . the % internalized activity was calculated as the ratio of the activity of the lysate and the total activity from the medium , pbs , acid , and base washes . in 12-well plates , 1 ( 100 g , 0.67 nmol ) was either coadministered with the radioactive probes or preincubated for 1 h at 37 c . after addition of zr - mb ( 12 g , 12.525 pmol ) and zr - cys - db ( 12 g , 2040 pmol ) in separate wells , the cells were incubated at 37 c for 1 h and then carefully washed with medium to remove any excess unbound activity . the level of bound radioligands was calculated as % bound , normalized to the amount of activity added . to determine the dissociation constant ( kd ) of each of the zr - mabs , saturation binding studies briefly , varying concentrations of the zr - mabs ( 0.033 g / ml , 0.260 pmol / ml ) were added to 5 10 cells in pbs containing 1% bovine serum albumin . the same experiment was repeated with additions of cold mabs ( 140 g / ml for the mb ( 1.75 nmol / ml ) and cys - db ( 2.8 nmol / ml ) and 300 g / ml for huj591 ( 2 nmol / ml ) ) to determine nonspecific binding ( nsb ) . after 1 h incubation at room temperature , the cells were washed twice with 1 pbs . specific binding ( sb , nm ) , derived by subtracting nsb from total bound activity , was plotted against the amount of zr - mabs added . the dissociation constant , kd , was calculated by nonlinear regression fitting of the resulting plot using graphpad prism v. 6.02 . mice ( n = 34 ) bearing lncap xenografts were administered intravenously ( iv ) with 7.410.2 mbq of either zr - mb ( 2838 g , 0.350.48 nmol ) or zr - cys - db ( 2534 g , 0.500.68 nmol ) in saline . small - animal pet studies were conducted using micropet - r4 and focus 120 scanners ( concorde microsystems ) . using asipro vm software ( concorde microsystems ) , volumes of interest ( vois ) were measured on various planar sections of the acquired image by manually drawing on the tumor site and on select organs . the mean voi was calculated and expressed as % injected dose per gram of tumor tissue ( % id / g ) . single tumor bearing mice were administered intravenously with 370555 kbq of either zr - mb ( 12 g , 12.525 pmol ) or zr - cys - db ( 12 g , 2040 pmol ) . competitive inhibition studies were performed with coadministration of 200500 g ( 2.510 nmol ) of nonradioactive mb or cys - db in lncap tumor - bearing mice ( n = 35 ) . in a separate cohort of mice ( n = 4 ) bearing the psma(+ ) tumor , the parent huj591 ( 500 g , 3.3 nmol ) was administered 36 h prior to dosing with zr - mb . mice were euthanized by co2 asphyxiation after 1 , 4 , 12 , and 24 h p.i . ( n = 45 per group ) . select tissues including the tumor were harvested and weighed with bound activity measured using a gamma counter ( perkinelmer ) . the tissue uptake ( % id / g ) was calculated against the total net activity injected . for autoradiographic studies , separate animals were administered with zr - mb , zr - cys - db , and zr - huj591 ( 7.410.2 mbq ) as described above , 24 h before sacrifice . five minutes prior to sacrifice , animals were injected with hoechst 33342 ( 1 mg / ml pbs , iv ) . immediately following sacrifice , tumors were removed and frozen in oct mounting medium ( sakura , europe ) and a series of 10 m tissue sections obtained . autoradiographic distribution of zr at 25 m pixel resolution was obtained by exposing the sections to a bas ms2025 phosphor plate ( fujifilm ) , followed by reading on a ge typhoon 7000ip plate reader . the same sections were then used to obtain high - magnification whole - mount images of hoechst 33342 distribution and h&e staining as previously described . following autoradiography , sections were fixed in 4% paraformaldehyde and stained for psma expression using rabbit anti - psma antibody ( clone epr6253 , abcam , 1:100 dilution ) overnight at 4 c , followed by goat anti - rabbit - alexa-568 ( invitrogen , 1:100 ) for 1 h at room temperature . finally , sections were stained with h&e , and digital microsopic images obtained in the same manner . statistical analysis was performed using a one - way anova test followed by dunnett s multiple comparison test when applicable for biodistribution experiments . a two - tail student s t test was employed in in vitro assays and tumor uptake comparison . following production and purification of the mb and cys - db , the proteins were analyzed to confirm their purity and identity prior to conjugation . the mb and cys - db migrated primarily as covalent dimers of 80 kda and 50 kda , respectively , under nonreducing sds page conditions , but migrated as monomers of 40 kda and 25 kda , respectively , under reducing sds a low level of product - related impurities ( likely partially clipped fragments ) of the mb was also detected under the denaturing sds tof - ms confirmed the identity of the mb and cys - db by molecular mass . facile dfo - bz - scn conjugation and zr - radiolabeling of mb , cys - db , and huj591 were achieved . sds page gel confirmed purity of both dfo - attached fragments compared to unmodified proteins ( si figure 1 in the supporting information ) . table 1 summarizes radiolabeling yield , purity , and specific activities of zr - mb and zr - cys - db , which compared favorably to the previously reported values for zr - huj591 . both zr - mb and zr - cys - db demonstrated excellent radiolabeling yields ( > 70% ) and radiochemical purities ( > 99% ) . specific activities of 222296 mbq / mg ( 17.823.7 mbq/mol ) and 259333 mbq / mg ( 12.916.6 mbq/mol ) were attained for zr - mb ( n = 8) and zr - cys - db ( n = 5 ) respectively . both radiolabeled fragments sufficiently retained their affinity for psma with immunoreactivities established at 72.4 1.7% for zr - mb and 72.8 1.4% for zr - cys - db ( n = 3 for each ) . with reports showing psma possessing a putative endocytic functionality , we conducted in vitro internalization studies using lncap cells to compare the rates of internalization of zr - mb , zr - cys - db , and zr - huj591 . the internalization patterns of all three radiotracers appear to be rapid , however , initial slow uptake was displayed by zr - mb at 4 h at 37 c with lower fractions internalized ( 3.7 0.5% , p = 0.002 ) ( figure 1a , left panel ) . this was then followed by rapid internalization , peaking at 12 h with 15.1 1.0% ( p = 0.027 ) , which reached a plateau at 24 h ( 16.6 1.0% ) . in figure 1a ( middle panel ) , zr - cys - db showed consistent linear internalization , spanning all time points ( 1.0 0.1% at 0.5 h , 3.5 0.2% at 1 h , 4.5 0.5% at 4 h , 8.9 0.7% at 12 h , and 11.9 0.1% at 24 h ) at 37 c . at 37 c , zr - huj591 demonstrated internalization with 1.4 0.1% at 0.5 h , 1.8 0.8% at 1 h , 7.3 0.6% at 4 h , 12.1 1.3% at 12 h , and 10.1 1.3% at 24 h ( figure 1a , right panel ) . minimal internalization was demonstrated by all three imaging probes at 4 c , evidence of an endocytic uptake process . in vitro assays in lncap prostate cancer cells . both antibody fragment radiotracers were added to lncap prostate cancer cells and incubated at 37 and 4 c at 0.5 , 1 , 4 , 12 , and 24 . ( a ) the internalization rates of zr - mb ( left ) and zr - huj591 ( right ) follow a nonlinear trend with slow uptake rates after 4 h of incubation ; a subsequent increase in intracellular radioactivity with higher internalized fractions was observed for zr - mb after 12 h. zr - cys - db ( middle ) displays a linear rate of internalization . all three radiotracers display minimal intracellular accumulation at all time periods at 4 c . ( b ) in lncap prostate cancer cells , 1 h preincubation or coaddition of huj591 ( 100 g ) with the radiotracer diminished binding of zr - mb ( 12 g , left ) and zr - cys - db ( 12 g , right ) . the specificities of both zr - mb and zr - cys - db to psma were evaluated by competitive inhibition with nonradioactive huj591 . in figure 1b ( left panel ) , zr - mb demonstrated as much as 4.96 0.73% binding to lncap cells with no added huj591 . this level of bound mb was satisfactorily blocked as much as 10-fold with both preincubated ( 0.55 0.19% , p < 0.0001 ) and coadded ( 0.56 0.3% , p < similar outcomes were observed with zr - cys - db ( figure 1b , right panel ) where , in the absence of huj591 , the radiotracer displayed 4.77 0.21% psma - binding activity ; this uptake was significantly diminished by preincubation ( 0.16 0.08% , p < 0.0001 ) and coaddition ( 0.15 0.07% , p < 0.0001 ) of huj591 . zr - huj591 , zr - mb , and zr - cys - db demonstrated specific binding and high affinities for psma localized on the surface of lncap cells . the saturation binding studies showed apparent kd values that are similar for all three radiotracers : zr - huj591 ( kd = 2.31 0.27 nm ) , zr - mb ( kd = 2.18 0.50 nm ) , and zr - cys - db ( kd = 2.59 0.41 nm ) . tissue distributions of zr - mb were analyzed in mice with single implants of psma(+ ) lncap ( figure 2a ; si table 1 in the supporting information ) or psma( ) pc3 tumors ( si table 2 in the supporting information ) . tumor uptake of zr - mb at 1 h ( 2.3 0.5% id / g ) was observed to steadily increase at 4 h p.i . accumulation of the probe in the tumor increased at 12 h p.i . with 6.2 2.5% id / g and at 24 h p.i . ( 12.1 3.6% id / g ) . to establish the specificity of zr - mb for psma in vivo , we performed blocking assays with various doses of nonradioactive mb ( si table 3 in the supporting information ) . at 12 h p.i . , the uptake of zr - mb was moderately decreased by 2-fold at 200 g of coinjected cold mb with 3.73 1.27% id / g ( p = 0.21 ) . increasing the nonradioactive dose to 500 g did not significantly block the uptake either with 3.82 0.70% id / g ( p = 0.19 ) . a 500 g huj591 dose was administered iv 36 h prior to injection of zr - mb with the mice ( n = 4 ) euthanized 24 h p.i . of the radiotracer , which mitigated tumor uptake ( 5.0 2.4% id / g , p = 0.025 ) . the in vivo specificity of this probe was further strengthened with differences between zr - mb accumulation in psma( ) pc3 tumors against lncap tumors in ( figure 2a , inset ) . the pc3 tumor sustained a constant accretion of 2.3 0.4% id / g at 1 h ( p = 0.28 ) , 2.1 0.3% id / g at 4 h ( p = 0.03 ) , 1.7 0.5% id / g at 12 h ( p = 0.04 ) , and 2.4 0.3% id / g at 24 h. this nonspecific tumor uptake is attributed to the tumor s leaky vasculature . ex vivo tissue distribution studies of zr - mb and zr - cys - db administered intravenously in lncap ( psma(+ ) ) and pc3 ( psma( ) ) prostate tumor - bearing mice . ( a ) biodistribution of zr - mb at different time points ( 124 h ) in select tissues including the tumor ; a blocking dose of 500 g of huj591 36 h prior to intravenous injection of zr - mb effectively mitigated tumor uptake at 24 h p.i . ( a , inset ) direct comparison between lncap and pc3 tumors displays cumulative zr - mb uptake in the psma(+ ) xenograft , which was significantly higher as early as 4 h p.i . , whereas nonspecific binding in the negative tumor is observed . ( b ) zr - cys - db exhibits selective tumor tissue targeting , which peaks at 12 h p.i . a blocking dose of 200 g of nonradioactive cys - db decreases the radiotracer tumor uptake at 12 h p.i . ( b , inset ) higher uptake of zr - cys - db in lncap tumors compared to pc3 implants at 12 and 24 h p.i . assessment of zr - cys - db localization in the psma(+ ) tumor demonstrated uptake with 4.1 1.0% id / g at 1 h , 7.0 3.0% id / g at 4 h , and 12.3 2.5% id / g at 12 h ( figure 2b ; si table 4 in the supporting information ) . a decrease in tumor accretion at 24 h ( 6.5 1.0% id / g ) was observed . blocking with 200 g of cold cys - db at 12 h p.i . effectively lowered the tumor accumulation to 5.64 1.75% id / g ( p = 0.044 ) . pc3 tumor uptake ( figure 2b inset ; si table 5 in the supporting information ) displayed lower accretion across all time points with 2.36 0.48% id / g ( 1 h , p = 0.068 ) , 3.65 1.54% id / g ( 4 h , p = 0.12 ) , 2.75 0.51% id / g ( 12 h , p = 0.007 ) , and 3.44 0.85% id / g ( 24 h , p = 0.0011 ) . we performed in vivo pet imaging in mice bearing psma(+ ) lncap tumors from 1 to 24 h on all three radiotracers ( si figure 2 in the supporting information ) . in figure 3a , planar sections of acquired images revealed accumulation of zr - mb at 12 and 24 h p.i . maximal intensity projection ( mip ) at 12 h showed tumor delineation with nonspecific binding observed in the liver and kidneys . similar results were achieved with zr - cys - db where localized delivery of the radiotracer to the tumor provided high contrasts at earlier time points ( figure 3b ) . in contrast to zr - huj591 ( figure 3c ) , very minimal to no activity is present in the lung and heart in both zr - antibody fragments , proof that the circulating probes were eliminated faster than the intact mab . quantification of tumor uptake is presented in figure 4a ( si table 6 in the supporting information ) , which displays an increasing trend in tumor uptake for all three radiotracers . at 12 h p.i , the accumulation of both zr - mb ( 6.85 0.87% id / g ) and zr - cys - db ( 9.84 2.54% id / g ) plateaued , whereas zr - huj591 ( 15.84 1.79% id / g ) steadily increased at 12 h p.i . after 24 h p.i . , the tumor uptake of the intact mab was 3-fold higher compared to the scfv proteins . this lower tumor delivery of the engineered mab fragments most likely stemmed from low availability of both fragments in the blood pool . as shown in the vois drawn on the heart ( figure 4b ; si table 7 in the supporting information ) , a sharp decreasing trend in radiotracer activity was displayed by zr - cys - db followed by zr - mb across all time points such that , at 12 h , only 2.73 0.61% id / g zr - cys - db and 5.06 2.84% id / g zr - mb remained . at 24 h , residual cardiac activity from both tracers was determined ( i.e. , 2.00 0.18% id / g for zr - cys - db and 3.26 1.20% id / g for zr - mb ) . zr - huj591 heart activity remained consistently higher at all time points ( i.e. , 16.36 1.57% id / g at 12 h and 12.13 1.26% id / g at 24 h ) . representative maximum intensity projections ( mips ) and planar pet images of ( a ) zr - mb , ( b ) zr - cys - db , and ( c ) zr - huj591 acquired in male athymic nude mice bearing lncap ( left shoulder , t ) prostate tumors xenografts at 12 and 24 h postinjection . kidney ( k ) and liver ( l ) accumulations were observed for both zr - mb and zr - cys - db and rationalized as routes of excretion . all three probes localize in the tumor , however , only zr - huj591 remains in circulation between 12 and 24 h. time activity curves of zr - mb , zr - cys - db , and zr - huj591 demonstrating ( a ) tumor volumes of interest ( vois ) , ( b ) blood clearance through heart vois , ( c ) tumor - to - muscle ratios , and ( d ) tumor - to - heart ratios from 1 to 24 h. scrutiny of tumor - to - muscle ( t / m ) ratios from the pet scans disclosed no significant differences among all three radiotracers at all time points ( p > 0.05 ) ( figure 4c ; si table 8 in the supporting information ) . tumor - to - heart ( t / h ) ratios ( figure 4d ; si table 9 in the supporting information ) showed no distinctions between zr - mb and zr - huj591 ; however , analysis of t / h ratios for zr - cys - db provided notably higher values at all time points against the intact mab , for example , 3.58 0.19 vs 0.98 0.19 ( p = 0.0002 ) at 12 h and 4.91 0.26 vs 2.22 0.46 ( p = 0.014 ) at 24 h. distribution of zr - mb ( figure 5a ) , zr - cys - db ( figure 5b ) , and zr - huj591 ( figure 5c ) at the microscopic level was evaluated using combined digital autoradiography ( right panels ) and whole - mount brightfield microscopy . the tumor tissues were viable with minimal necrosis observed as shown in the h&e stain ( si figure 3a c in the supporting information ) . psma expression ( red stain , left panels ) appeared to be essentially uniform in all lncap tumor sections examined . binding was generally absent from regions of stromal infiltration . slightly increased immunoreactivity was observed in tumor necrotic regions , but was assumed to represent nonspecific antibody binding . for each construct , focal uptake of zr was observed in central tumor regions , with seemingly less around the tumor periphery . regions of high zr uptake were generally centered on areas of high vascular perfusion ( as indicated by hoechst 33342 staining in blue , left panels ) . however , not all perfused regions , in particular those observed around the tumor rim , were associated with zr uptake . it is possible that vascular integrity or leakiness may play a role in determining the distribution of the zr - labeled constructs in this tumor model . this data may serve to indicate that the relative tumor distribution of each of the zr constructs appears to be equivalent over the time course of these experiments . ex vivo autoradiography and histology . registered whole - mount micrographs of lncap tumor sections with psma staining ( red ) coregistered with hoechst 33342 ( blue ) for perfusion ( left ) and digital autoradiography ( right ) of ( a ) zr - mb , ( b ) zr - cys - db , and ( c ) zr - huj591 . here in this study , we developed psma - targeting pet imaging probes using antibody fragments of huj591 as carriers . we rationalize that these smaller derivatives are necessary to enable lower dose exposures and faster imaging , which can potentially decrease the burden on prostate cancer patients . we opted to utilize zr as the radionuclide of choice for labeling these fragments to provide a systematic and parallel comparison to huj591-pet . these results demonstrated significantly improved pk attributes of both zr - labeled antibody fragments with retained tumor targeting properties and shorter tracer clearance , thus rendering both probes as excellent markers of psma(+ ) cancer with the advantage of same - day imaging . facile conjugation of dfo - bz - scn and zr - radiolabeling of mb and cys - db were achieved with high radiochemical yields and purities and with minimal loss of immunoreactivities . consistently high specific activities for both zr - constructs were produced after numerous labeling experiments . for uniformity , huj591 was also conjugated with dfo - bz - scn , which offers the benefit of shorter and more straightforward conjugation compared to an earlier protocol used by our group . all three probes were shown to internalize albeit with varying kinetics underpinning the critical use of radiometals in labeling internalizing proteins to capitalize on longer retention within the tumor for better imaging contrasts . we further demonstrated the specificity of both zr - mb and zr - cys - db by coaddition or prior incubation of cold huj591 in lncap cells . we next asked whether the binding affinities of zr - mb and zr - cys - db are comparable to zr - huj591 . from our saturation binding assays , all radiolabeled proteins displayed comparable kds , supporting the hypothesis that smaller antibody platforms offer stable association to psma in a similar fashion to the parent mab . our observed affinities for all zr - mabs are within the range reported for huj591 labeled with i ( kd = 1.83 1.21 nm ) and in ( kd = 3 nm ) . two reports of new anti - psma antibody fragments were recently published . of particular interest is a diabody developed from the same parent huj591 and labeled with tc ( tc - j591cdia ) with a kd of 5.0 0.5 nm . two chimeric scfv fragments derived form the parent murine mab 3/f11 ( kd 4.0 nm ) established kds of 8.3 nm for d7-fc and 9.6 nm for d7-ch3 . moreover , a previous study investigating a single - chain variable fragment of another psma - targeting mab , d2b , reported a 4-fold lower affinity ( 8.6 nm ) via biacore analysis , thus rendering our antibody fragments potentially superior . pet imaging visualized tumor uptake and rapid whole body clearance of the zr - mb and zr - cys - db in murine xenografts . tissue distribution studies showed minimal nonspecific binding of both radiotracers in healthy tissues except in the hepatic and renal organs , which are routes of elimination . this nonspecific binding observed for both liver and kidneys is significantly lower when compared to , for example , tc - j591cdia with > 13 1.8% id / g and > 29.2 3.5% id / g respectively , albeit at 8 h p.i . however , the uptake of our fragments , in particular , zr - cys - db , in the liver and the kidneys is significantly lower . retention of activity in the kidneys is likely due to a combination of clearance of the antibody fragments and radiolabeled metabolites , in addition to antigen - specific binding , as the proximal renal tubules have been reported to express psma ; only the latter would be reduced by blocking studies . specific tumor delivery of both probes zr - cys - db and zr - mb to psma was validated by addition of excess cold cys - db and huj591 respectively . the competitive inhibition of zr - mb , however , was not straightforward with excess doses of mb inefficiently attenuated . a plausible explanation can be attributed to the nonspecific clearance kinetics of each fragment , whereby competitive inhibition of the mb should be examined at longer time periods ( i.e. , 24 h p.i . ) when tumor uptake has stabilized . the results from autoradiography and histology ( figure 5b ) reinforce this rationale as zr - mb was found to colocalize with areas of vasculature even at 24 h p.i . ; this , however , does not definitively discriminate between the vascular contributions of each construct . stronger direct evidence is found in the blood activity at 12 and 24 h obtained from the ex vivo tissue distribution wherein accretion of zr - mb is 2-fold higher than that of the smaller zr - cys - db . may likely stem from the fact that the internalization of psma is critically enhanced by ligand or antibody binding to the antigen . as we have shown in our internalization assays , the kinetics vary with the minibody internalizing at a higher rate after 12 h p.i . with the psma recycling to the cell surface , more binding sites are available for zr - mb delivery with 3% id / g circulating in the bloodpool . in our study , engineered smaller fragments are postulated as viable imaging surrogates of therapeutic antibodies within the context of curtailing wait times between administration and imaging . clear distinction between the three imaging probes is attained upon analysis of each tracer s in vivo pharmacokinetics due primarily to varying molecular size . blood pool activity of all radiotracers demonstrated obvious disparities ( figure 4b ) in clearance kinetics with zr - cys - db clearly showing faster elimination during earlier time points . zr - mb showed parallel blood residencies at 12 and 24 h with zr - cys - db while zr - huj591 still remained in circulation at higher activities after 24 h. moreover , the measured tumor uptake from the pet images demonstrated significantly higher radiolocalization of zr - huj591 at later time points ( i.e. , 24 h ) . we ascribe this observation as a factor of systemic elimination of the fragments rather than loss of affinity . intact mabs possess innate prolonged blood residencies , significantly increasing its availability for tumor delivery . this extended circulation may benefit immunotherapies , but for diagnostic purposes , faster clearance with retained tumor localization is key to perform imaging within the same day of dosing . faster blood pool clearance considerably enhances the image quality and resolution , which puts these zr - engineered mabs at a critical advantage . moreover , the lower tumor delivery should not be taken as a major drawback as contrast or tumor - to - background ratios is considered one of the critical parameters not just in nuclear imaging but across all modalities . from the tumor - to - muscle ratios ( figure 4c ) , no significant disparities in contrast were observed between the antibody fragments and intact huj591 , supporting the notion that these intermediate - sized antibody fragments provide similar tumor delineating properties at the time points studied . one of the benefits of using engineered fragments is arguably the lower dose toxicity due to shortened blood residencies and minimized nonspecific healthy tissue accumulation . blood activities of both antibody fragments are far shorter than that of the intact huj591 with residencies decreasing by 2-fold within 12 h postadministration . thus , we surmise that , with such rapid kinetics , nontrivial absorbed whole body radioactive doses especially in hematopoietic tissues will be minimized proportionally . certainly , with smaller antibody fragments , a radioisotope with a half - life matching the pk of the fragment ( i.e. , cu , t1/2 = 12 h ) will afford better dosimetry profiles , especially if the probes are envisioned for repeat monitoring of treatment response in the same patient . although immunopet potentially holds a vital role in molecular and functional diagnosis , the lengthened circulation of intact antibodies and their nonspecific accumulation in normal tissues can impact radiotoxicity in normal organs , for example , the liver , the spleen , and the rest of the hematopoietic system , upon repeated pet scans . we believe that the use of engineered fragments derived from the intact antibody provides such benefit and advantage with observed terminal half - lives of 311 h in mice and with maintained radiotracer target specificity . recognizing that imaging tools require rapid clearance of the probe at shorter time periods for optimum contrast and in order to minimize patient burden , the smaller cys - db and mb fragments of huj591 for pet imaging are obvious choices as companion diagnostics of psma - targeted therapy . in summary , small engineered antibody fragments of huj591 have high potential as alternative noninvasive imaging probes for the detection and staging of psma - positive prostate tumors . both zr - mb and zr - cys - db offer rapid tumor delineation and background clearance , essentially establishing these scfv - based antibody fragments as faster alternatives to the intact mab .
engineered antibody fragments offer faster delivery with retained tumor specificity and rapid clearance from nontumor tissues . here , we demonstrate that positron emission tomography ( pet ) based detection of prostate specific membrane antigen ( psma ) in prostatic tumor models using engineered bivalent antibodies built on single chain fragments ( scfv ) derived from the intact antibody , huj591 , offers similar tumor delineating properties but with the advantage of rapid targeting and imaging . 89zr - radiolabeled huj591 scfv ( dimeric scfv - ch3 ; 89zr - mb ) and cysteine diabodies ( dimeric scfv ; 89zr - cys - db ) demonstrated internalization and similar kds ( 2 nm ) compared to 89zr - huj591 in psma(+ ) cells . tissue distribution assays established the specificities of both 89zr - mb and 89zr - cys - db for psma(+ ) xenografts ( 6.2 2.5% id / g and 10.2 3.4% id / g at 12 h p.i . respectively ) , while minimal accumulation in psma( ) tumors was observed . from the pet images , 89zr - mb and 89zr - cys - db exhibited faster blood clearance than the parent huj591 while tumor - to - muscle ratios for all probes show comparable values across all time points . ex vivo autoradiography and histology assessed the distribution of the probes within the tumor . imaging psma - expressing prostate tumors with smaller antibody fragments offers rapid tumor accumulation and accelerated clearance ; hence , shortened wait periods between tracer administration and high - contrast tumor imaging and lower dose - related toxicity are potentially realized .
Experimental Section Results Discussion
the pc3 tumor sustained a constant accretion of 2.3 0.4% id / g at 1 h ( p = 0.28 ) , 2.1 0.3% id / g at 4 h ( p = 0.03 ) , 1.7 0.5% id / g at 12 h ( p = 0.04 ) , and 2.4 0.3% id / g at 24 h. this nonspecific tumor uptake is attributed to the tumor s leaky vasculature . ex vivo tissue distribution studies of zr - mb and zr - cys - db administered intravenously in lncap ( psma(+ ) ) and pc3 ( psma( ) ) prostate tumor - bearing mice . assessment of zr - cys - db localization in the psma(+ ) tumor demonstrated uptake with 4.1 1.0% id / g at 1 h , 7.0 3.0% id / g at 4 h , and 12.3 2.5% id / g at 12 h ( figure 2b ; si table 4 in the supporting information ) . at 12 h p.i , the accumulation of both zr - mb ( 6.85 0.87% id / g ) and zr - cys - db ( 9.84 2.54% id / g ) plateaued , whereas zr - huj591 ( 15.84 1.79% id / g ) steadily increased at 12 h p.i . as shown in the vois drawn on the heart ( figure 4b ; si table 7 in the supporting information ) , a sharp decreasing trend in radiotracer activity was displayed by zr - cys - db followed by zr - mb across all time points such that , at 12 h , only 2.73 0.61% id / g zr - cys - db and 5.06 2.84% id / g zr - mb remained . representative maximum intensity projections ( mips ) and planar pet images of ( a ) zr - mb , ( b ) zr - cys - db , and ( c ) zr - huj591 acquired in male athymic nude mice bearing lncap ( left shoulder , t ) prostate tumors xenografts at 12 and 24 h postinjection . all three probes localize in the tumor , however , only zr - huj591 remains in circulation between 12 and 24 h. time activity curves of zr - mb , zr - cys - db , and zr - huj591 demonstrating ( a ) tumor volumes of interest ( vois ) , ( b ) blood clearance through heart vois , ( c ) tumor - to - muscle ratios , and ( d ) tumor - to - heart ratios from 1 to 24 h. scrutiny of tumor - to - muscle ( t / m ) ratios from the pet scans disclosed no significant differences among all three radiotracers at all time points ( p > 0.05 ) ( figure 4c ; si table 8 in the supporting information ) . tumor - to - heart ( t / h ) ratios ( figure 4d ; si table 9 in the supporting information ) showed no distinctions between zr - mb and zr - huj591 ; however , analysis of t / h ratios for zr - cys - db provided notably higher values at all time points against the intact mab , for example , 3.58 0.19 vs 0.98 0.19 ( p = 0.0002 ) at 12 h and 4.91 0.26 vs 2.22 0.46 ( p = 0.014 ) at 24 h. distribution of zr - mb ( figure 5a ) , zr - cys - db ( figure 5b ) , and zr - huj591 ( figure 5c ) at the microscopic level was evaluated using combined digital autoradiography ( right panels ) and whole - mount brightfield microscopy . zr - mb showed parallel blood residencies at 12 and 24 h with zr - cys - db while zr - huj591 still remained in circulation at higher activities after 24 h. moreover , the measured tumor uptake from the pet images demonstrated significantly higher radiolocalization of zr - huj591 at later time points ( i.e. from the tumor - to - muscle ratios ( figure 4c ) , no significant disparities in contrast were observed between the antibody fragments and intact huj591 , supporting the notion that these intermediate - sized antibody fragments provide similar tumor delineating properties at the time points studied .
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antrodia cinnamomea , a taiwan - specific mushroom , has been reported to have numerous biological activities including hepatoprotection , anti - inflammation , antihepatitis c virus activity , and anticancer activity . liver cancer ranks fourth in cancer - related mortality around the world ( who 2008 ) and is the second leading cause of cancer deaths in taiwan . twenty years ago , the major cause of liver cancer in taiwan was hepatitis virus infection , such as hepatitis b virus ( hbv ) infection . the incidence of hbv infection is very low now , mostly due to the hbv vaccine policy of taiwan government . however , the mortality of liver cancer is still high , which may be due to social culture and unhealthy living habits such as drugs abuse , drinking , and overworking . there are three common types of cell death : apoptosis , autophagy , and necrosis . apoptosis , also called type i programmed cell death , involves dna fragmentation , caspase induction , and phosphatidylserine translocation from the inner side of the cell membrane to its outer side ; autophagy , also recognized as type ii programmed cell death , forms autophagosome as the major phenomenon ; and necrosis would cause inflammation and disrupt organelles . there is no study on the determination of major mechanism of antcin k - induced cell death in human liver cancer cells . proteins must go through a series of post - translational modifications and be fully folded in order to be transported from the endoplasmic reticulum ( er ) . proteins with incomplete or incorrect folding will remain in the er or be degraded by the proteasome in the cytoplasm . many physiological and pathological circumstances such as hypoxia , oxidative damage , deficiency in er calcium ion , and viral infection may cause disorders of protein folding in the er , in which proteins with incomplete folding will accumulate on the er surface and cause er stress . in order to resist er stress , cells will start a series of reactions to adapt to the circumstance and regulate the er stress , which include promotion of protein degradation , inhibition of protein translation , and activation of relevant genes . however , if the er stress persists , apoptosis or autophagy would be induced , which will eventually lead to cell death . the unfolded protein response is primarily a survival response , acting to resolve dysregulation of protein - folding pathways . if the normal luminal environment can not be restored , the response to er stress would be switched from survival to apoptosis . perk , ire1 , and atf-6 are all involved in the induction of proapoptotic as well as prosurvival pathways . both perk and atf-6 induce expression of the transcription factor c / ebp homologous protein ( chop ) , which in turn leads to reduced expression of b - cell lymphoma 2 ( bcl - xl ) and increased expression of a number of proapoptotic proteins , including bh3-only protein bim and its downstream protein b - cell lymphoma extra - large ( bax)/bak.9 , 10 , 11 furthermore , bcl-2 family proteins at the er also regulate apoptosis through both direct modulation of signaling pathways leading to caspase activation and modulation of ca signaling of er.12 , 13 calcium is a common metal ion , which plays a dominant role in cell death signaling transduction . it has been reported that the concentration of calcium ion can regulate the stability of organelles such as mitochondria and er . here , we will demonstrate how antcin k modulates cell death through the change of calcium distribution in human liver cancer cells . , it would induce reactive oxygen species ( ros ) production , and the ros may attack some organelles such as mitochondria . next , the permeability transition pore of mitochondria would be opened by ros or other stimulation , and then by some proapoptotic protein bax / bak overexpression , which could release cytochrome c into the cytoplasm . finally , cytochrome c would increase the cleavage caspase-9 protein expression , which triggers caspase-3 activation . there are other caspase - independent death pathways in mitochondria , such as htra2/omi , endonuclease g ( endo g ) , and apoptotic - induced factor ( aif ) released from mitochondria.15 , 16 basswood cultivated a. cinnamomea ( bcrc930103 ) mycelial powder was supplied by po - zo co. , ltd ( taipei , taiwan ) . ethyl acetate ( ea ) was added to 500 g dry power of a. cinnamomea to a total volume of 4 l and stirred for 3 days . the extract was decanted , and the solvent was removed using a rotary evaporator at 50c three times for each sample . the column was consecutively eluted with 10% , 15% , 20% , 30% , 50% , 70% , and 100% ea / hexane . the fraction with 100% ea / hexane contained the highest amount of antcin k. it was further purified by high - performance liquid chromatography to obtain antcin k with > 90% purity ( fig . antimycotic , 2,7-dihydrodichlorofluorescein diacetate , dulbecco 's modified eagle 's medium , fetal bovine serum , fluo-3-acetoxymethyl ester , nonessential amino acids , rhod-2-acetoxymethyl ester , and 3,3-dihexyloxacarbocyanine iodide were purchased from invitrogen ( carlsbad , ca , usa ) . anti--actin antibody , anti - aif antibody , anti - bcl - xl antibody , anti - bax antibody , anti - bak ( d4e4 ) rabbit mab antibody , anti - caspase-9 antibody , anticleaved caspase-3 rabbit mab ( asp175)(5a1e ) antibody , anti - chop ( l63f7 ) mouse mab antibody , anti - cytochrome c antibody , anti - endo g antibody , anti - htra2/omi antibody , anti - parp antibody , antirabbit igg hrp - linked antibody , and antimouse igg hrp - linked antibody were obtained from cell signaling technology ( beverly , ma , usa ) . caspase-3 assay kit and annexin v - fitc apoptosis detection kit were purchased from bd biosciences ( san jose , ca , usa ) . anti - lc3b antibody was purchased from genetex ( irvine , ca , usa ) . adenosine diphosphate / adenosine triphosphate ( adp / atp ) ratio assay kit and lactate dehydrogenase ( ldh ) cytotoxicity assay kit were purchased from biochain institute ( hayward , ca , usa ) . all other chemicals were of analytical or reagent grade and obtained from sigma - aldrich ( st louis , mo , usa ) . human hepatoma hep 3b cell line was a kind gift from professor ming - shi shiao ( medical research and education department , taipei veterans general hospital , taipei , taiwan ) . hep 3b cells were cultured in dulbecco 's modified eagle 's medium supplemented with 10% fetal bovine serum , 1.5 g / l sodium bicarbonate , 1% nonessential amino acids , and 1% antibiotic antcin k was diluted in dimethyl sulfoxide ( dmso ) prior to being added to cultures . liver cancer cells at a concentration of 5 10 cells / well were seeded in 96-well plates and incubated for 24 hours , followed by treatment with 0m ( 0.3% dmso ) , 80m , 100m , and 125m antcin k and incubated further for 24 hours and 48 hours . at the end of the stipulated period , 100 l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide ( mtt ) solution ( 0.5 mg / ml ) was added , and the cells were incubated at 37c for 4 hours . the resulting mtt formazan was dissolved in 100 l dmso and the absorbance recorded at 570 nm using a powerwave ht microplate spectrophotometer ( bio - tek , winooski , vt , usa ) . hep3b cells ( 1 10 cells / well ) were seeded in 96-well plates for 24 hours , followed by treatment with 200 l medium containing 0m , 80m , 100m , and 125m antcin k and lysis solution ( as a positive control ) , and incubated for another 48 hours . the plates were then centrifuged at 250 g for 10 minutes , and 100 l of supernatant was transferred to corresponding cells of a new 96-well plate . following this , 45 l of assay mixture containing lactate , nicotinamide adenine dinucleotide , iodonitrotetrazolium , and diaphorase was added to each well , protected from light , and incubated for 60 minutes . the absorbance was recorded at 490 nm using a bio - tek powerwave ht microplate spectrophotometer . the cells ( 1.75 10 cells / well ) were seeded in a four - well plate for 24 hours . for the 4,6-diamidini-2-phenylindole ( dapi ) staining , after 24 hours of incubation , the cells were treated with 350 l medium containing 0m , 80m , 100m , and 125m antcin k for 24 hours . after 24 hours of treating , the cells were washed and fixed by 4% paraformaldehyde for 30 minutes , and then washed twice . after washing , the cells were blocked for 1 hour by 5% bovine serum albumin and 0.1% triton x-100 . after washing , the slides were mounted and examined under fluorescence microscopy ( olympus ix51 ; olympus , tokyo , japan ) . for the mitochondria calcium staining , after 24 hours of incubation , the cells were treated with 350 l medium containing 0m , 80m , 100m , and 125m antcin k for 30 minutes . after 30 minutes of treatment , the cells were incubated with 350 l medium containing 5m rhod-2-acetoxymethyl ester for 1 hour and protected from light . after washing , the cells were incubated with 5% bovine serum albumin at 37c for 30 minutes . the slides were mounted and examined under a leica tcs sp5 ii confocal fluorescence microscope ( leica , solms , germany ) . the cells ( 5 10 cells / well ) were seeded in 96-well plates for 24 hours . after 24 hours of incubation , the cells were treated with 100 l medium containing 0m , 80m , 100m , and 125m antcin k for 24 hours . after 24 hours of treatment , 90 l lysis solution , which contains luciferin and luciferase , was added to each well and incubated for 10 minutes . the luminescence was integrated for 5 seconds using a beckman coulter dtx-880 microplate reader ( beckman coulter , brea , ca , usa ) . the cells ( 3 10 cells / dish ) were seeded in 6 cm dishes for 24 h hours . after 24 hours of incubation , the cells were treated with 6 ml medium containing 0m , 80m , 100m , and 125m antcin k for 30 minutes to detect ca , 24 hours to detect ros , 48 hours to detect autophagosome , 48 hours to determine mitochondrial membrane potential , and 24 hours to determine the percentage of cells undergoing apoptosis , using an annexin v - fitc / propidium iodide ( pi ) assay kit ( bd biosciences ) . at the end of the stipulated period , the cells were harvested and washed . for the detection of ca , ros , and mitochondrial membrane potential , the cells were separately stained by 4m fluo-3-acetoxymethyl ester , 5m dihydrodichlorofluorescein diacetate , and 4m 3,3-dihexyloxacarbocyanine iodide at 37c for 30 minutes . for the detection of autophagosome , the cells were permeabilized with 0.25 mg / ml digitonin for 5 minutes . following this , the cells were washed twice , pelleted by centrifugation at 1000 g , and incubated with anti - lc3-ii antibody ( 1:2000 ) for 30 minutes . after two washes , the cells were stained by alexa fluor 488 anti - rabbit igg antibody ( 1:500 ) for 1 hours and protected from light . all these cells were washed and filtered prior to being analyzed by a becton - dickinson facscan flow cytometer ( becton - dickinson , hercules , ca , usa ) . a total of 10,000 cells per sample were collected , and the mean fluorescence intensity and percentage of mitochondrial membrane potential detected by fl1-h ( 530 15 nm ) were analyzed using winmdi 2.8 software . for the detection of the percentage of apoptosis cells , the cells were resuspended in binding buffer [ 10 mmol / l hepes / naoh ( ph 7.4 ) , 140 mmol / l nacl , 2.5 mmol / l cacl2 ] and stained with annexin v - fitc and pi at room temperature for 15 minutes in the dark . cells were washed and filtered prior to being analyzed by a beckman coulter fc500 flow cytometer ( beckman coulter , pembroke pines , fl , usa ) . a total of 10,000 cells per sample were collected , and the apoptotic cells were defined as annexin v - fitc - positive and pi - negative cells.19 , 21 , 22 for western blotting , the cells ( 5 10 cells/10 ml / dish ) were seeded in 10 cm dishes for 24 hours . after 24 hours of incubation , the cells were treated with 10 ml medium containing 0m , 80m , 100m , and 125m antcin k for 48 hours . after 48 hours of treating , total cell extracts were prepared in protein extraction solution , which contained 1 mm phenylmethanesulfonylfluoride , 1 mm edta , 1m pepstatin a , 1m leupeptin , and 0.1m aprotinin . the cell lysates were sonicated and cleared by centrifugation , and the protein concentration in the lysates was measured by lowry 's method . polyacrylamide gel electrophoresis gels , transferred to polyvinylidene fluoride membranes , blotted with specific primary antibodies , and then labeled by horseradish peroxidase - conjugated secondary antibody according to the manufacturer 's instructions . the membranes were performed using the enhanced chemiluminescence and the ec3 300 corning uvp biospectrum ac system ( corning , ny , usa ) , and the relative density of each band after normalization for -actin was analyzed using image j 1.45 software . for the comet assay , the cells ( 5 10 cells/10 ml / dish ) were seeded in 6 cm dishes for 24 hours . after 24 hours of incubation , the cells were treated with 6 ml medium containing 0m , 80m , 100m , and 125m antcin k for 24 hours . after 24 hours of treatment , cells were examined for dna damage using the comet assay described previously . all results are reported as mean standard deviation ( sd ) , and the differences between the antcin k - treated group and the control group were analyzed by one - way analysis of variance ( anova ) and duncan 's multiple comparison tests ( sas institute inc . , cary , nc , usa ) to determine significant differences among treatments ( p < 0.05 ) . after treating hep 3b cells with 0m , 80m , 100m , and 125m antcin k for 24 hours or 48 hours , cell viability was analyzed by mtt assay . 1b shows that after being treated with 80m , 100m , and 125m antcin k for 24 hours , compared to the negative control ( 0.2% dmso , cell survival rate set at 100% ) , the cell survival rates were 85.7 5.0% , 63.8 5.8% , and 53.8 5.3% , respectively ; after 48 hours of treatment , cell survival rates decreased to 76.7 2.1% , 65.0 8.6% , and 46.0 4.2% , respectively . after treating hep 3b cells with 0m , 80m , 100m , and 125m antcin k , for 48 hours , the autophagosome formation was analyzed by flow cytometry . 1c shows that after being treated with 80m , 100m , and 125m antcin k , the lc3-ii contents were significantly decreased , and not increased . after treating hep 3b cells with 0m , 80m , 100m , and 125m antcin k and lysis solution for 48 hours 1d shows that after being treated with 80m , 100m , and 125m antcin k for 48 hours , compared with the positive control ( lysis solution , lactate dehydrogenase leakage rate set at 100% ) and negative control ( 0.2% dmso , lactate dehydrogenase leakage rate set at 0% ) , the lactate dehydrogenase leakage rates were 8.56 5.24% , 1.46 7.85% , and 7.21 7.48% , respectively , which were all low . we assessed the effect of antcin k on the induction of dna aggregation in hep 3b cells by dapi staining . the immunofluorescence at 24 hours showed that 80m , 100m , and 125m antcin k treatment resulted in the formation of chromatin condensation in hep 3b cells . the nuclei of control cells were round and dim , whereas those became condensed and bright after antcin k treatment ( fig . higher concentrations of antcin k led to a longer dna migration smear ( comet tail ) ( fig . a quantitative evaluation was sought using annexin v - fitc dye to detect the translocation of phosphatidylserine from the inner ( cytoplasmic ) leaflet of the plasma membrane to the outer side ( cell surface ) . compared with negative control cells , 80m , 100m , and 125m antcin k induced , respectively , 11.25 0.51% , 33.28 4.67% , and 39.87 3.83% of apoptotic cells in hep 3b cells in 24 hours ( fig . 3a ) . after treating hep 3b cells with 0m , 80m , 100m , and 125m antcin k for 24 hours , ros generation was analyzed by flow cytometry ; in addition , after treating hep 3b cells with the same concentrations of antcin k for 48 hours , the adp / atp ratio was analyzed with bioluminescence . 3b shows that as the antcin k dose increased , ros generation in cells increased significantly ( the maximum value being 1.49 times greater than the minimum ) and had a dose - dependent effect . 3d shows that as the antcin k dose increased , the adp / atp ratio in cells increased significantly . the adp / atp ratios were 0.9 0.9% , 5.9 1.5% , 14.7 2.8% , and 16.1 2.0% , respectively , for 0m , 80m , 100m , and 125m antcin k treatment . using fluo-3-acetoxymethyl ester , a cell - permeable ca indicator , we assessed the effect of antcin k treatment on cytosolic ca . the level of cytosolic ca induced significantly by 100m and 125m antcin k at 30 minutes . in addition , treatment with 125m antcin k similarly induced a 1.74-fold increase in ca level ( fig . mitochondrial free calcium levels were quantitated in cells that were still viable with the cell - permeability acetoxymethyl ester of the fluorescent marker rhod-2 . using rhod-2-acetoxymethyl ester , a selective indicator for mitochondrial ca , the distribution of calcium in 0m and 125m antcin k - treated hep 3b cells was revealed by confocal immunofluorescence microscopy ( fig . hep 3b cells were treated with 0m , 80m , 100m , and 125m antcin k for 48 hours , after which the mitochondrial membrane potential was analyzed and quantified by flow cytometry . 3c shows that as the antcin k dose increased , the mitochondrial membrane potential in cells decreased significantly ( the maximum potential being 0.34 times greater than the minimum ) and had a dose - dependent effect . after treating hep 3b cells with 0m , 80m , 100m , and 125m antcin k for 48 hours , unfolded protein response - related protein expression 4a shows that as the concentration of antcin k increased , the chop expressions increased in dose - dependent manners , compared with the negative control ( 0.2% dmso ) . as bcl-2 family proteins play an important regulatory role in calcium flux and mitochondrial apoptosis , we next studied the effect of antcin k on the protein expressions of bcl - xl , bax , and bak in hep 3b cells . the results showed that the bcl - xl expressions were decreased in dose - dependent manners and the protein expressions of bax and bak were increased in dose - dependent manners compared with the negative control , after treatment with various concentrations of antcin k in 48 hours . after treating hep 3b cells with 0m , 80m , 100m , and 125m antcin k for 48 hours , caspase - independent and caspase - dependent apoptosis - related protein expression was analyzed by western blotting . 4b shows that antcin k significantly increased the protein expression of cytochrome c in a dose - dependent manner , and the protein expressions of cleaved caspase-9 , parp , and caspase-3 were also increased . in addition , 4c shows that antcin k truly increased the caspase - independent apoptosis - related protein expressions of aif , htra2 , and endo g at 48 hours in hep 3b cells . currently known active ingredients of a. cinnamomea include polysaccharides , benzenoids , triterpenoids , and steroids . among them , triterpenoids have received more attention due to their potent anticancer effects . out of three artificial cultivation methods for a. cinnamomea , basswood cultivation is most valuable and yields the highest content of triterpenoids , followed by solid culture , and liquid fermentation material , which is almost free of triterpenoids . antcin k is a triterpenoid from fruiting bodies of basswood cultivated a. cinnamomea and accounts for 0.5% of a. cinnamomea . furthermore , it has previously been reported that antcin k can inhibit ros generation in human neutrophils to attain anti - inflammatory effects and inhibit na / k - atpase . our current study demonstrated that antcin k inhibited the proliferation of human liver cancer cells . treatment of hep 3b cells with antcin k caused the cells to undergo apoptotic cell death through the mitochondrial and er stress signaling pathway . er and mitochondria interact both physiologically and functionally , and one of the most critical aspects of this interaction is calcium signaling between the two organelles . as evident from ros production and the results of atp level analysis ( fig . 3b and d ) , antcin k can increase ros generation in human hepatoma hep 3b cells and also reduce the atp level of the cells , suggesting that er may be damaged by oxidation and in a hypoxic state . 4a ) , when er stress was induced , the protein expression of chop increased , downregulated bcl - xl , and activated bax / bak channel to promote intracellular calcium ion release from the er then transfer into the mitochondria . we studied the interaction of er and the mitochondrial pathway in our ongoing efforts to determine the apoptotic mechanism of antcin k against hep 3b cells . apoptosis occurs upon the perturbation of cellular ca homeostasis , such as cytosolic ca overload , er ca depletion , and mitochondrial ca increase.33 , 34 ca overload can induce mitochondrial depolarization and , subsequently , the release of apoptosis - inducing factors ( such as cytochrome c ) , which leads to the sequential activation of caspase-9 and caspase-3.35 , 36 as evident from the results of the calcium ion signal analysis ( fig . 2c and d ) , mitochondrial calcium ion signals were increased obviously after antcin k treatment ; antcin k might cause release of calcium ions from the er into the cytoplasm , leading to the mitochondrial uptake of these ions . in addition , as shown by the results of the mitochondrial membrane potential ( fig . 3c ) , antcin k can affect the cytosolic ca homeostasis and induce mitochondrial depolarization . 4b and c ) , caspase - independent and caspase - dependent apoptosis - related proteins were released , including htra2 , aif , endo g , and cytochrome c ; cytochrome c activated caspase-9 and caspase-3 , and cut downstream protein parp , ultimately leading to cell apoptosis . su et al have shown that eburicoic acid , a triterpenoid from a. cinnamomea , induced autophagy in human hepatoma hep 3b cells of the er stress pathway and increased the calcium ion concentration as well as the protein expressions of bcl-2 and beclin-1 . this study reported that antcin k could cause er stress in hep 3b cells and also increase calcium ion concentration in the cytoplasm , which were consistent with the findings of the previous studies conducted in our laboratory . however , the most interesting issue is that antcin k could not induce autophagy in hep 3b cells ; instead , it caused apoptosis , decreased the protein expression of bcl - xl , and changed mitochondrial membrane potential . why did eburicoic acid induce autophagy whereas antcin k cause apoptosis in hep 3b cells , although both these active components ( eburicoic acid and antcin k ) were from a. cinnamomea ? the reason for different cell death modes may be the different protein expressions of bcl-2 and bcl - xl . persisting er pressure will cause apoptosis or autophagy , and eventually lead to cell death ; the cell death mode , on the other hand , depends on factors such as cell types , growth conditions , and drug structures . after comparing the protein expressions of apoptosis and autophagy , we observed that apoptosis would inhibit bcl-2 and bcl - xl , while autophagy could activate bcl-2 and bcl - xl , which happened to be opposite effects . previous studies revealed that bcl-2 and bcl - xl exert two separate functions , depending on their subcellular localization . on the one hand , they induce apoptosis by ( directly or indirectly ) changing mitochondrial membrane potential . on the other hand , they can activate autophagy by liberating beclin-1 from its inhibition by bcl-2 or bcl - xl , allowing it to activate the lipid kinase activity of vps34 at the level of the er . however , the cell death mechanism of triterpenoids from a. cinnamomea was not the same . it may be caused by the different chemical structures , cell lines , environments , bioactivities , and bioavailabilities ; even some pure components had the effect of coordination . although we have limited references to clarify the cell death mechanism of the triterpenoids from a. cinnamomea , future studies will certainly reveal the anticancer mechanism of a. cinnamomea . several studies have demonstrated that a. cinnamomea has a good inhibitory effect on cancer.3 , 41 , 42 this study is the first of its kind to show that antcin k has an antiproliferative effect in liver cancer cells . antcin k treatment induced hep 3b cell death via promotion of intracellular ros generation and atp depletion , leading to er stress . moreover , the detail mechanism are followed by antcin k treatment induced er stress and mitochondria membrane permeability change through the induction of ca release , chop and bax / bak protein expression but downregulation of bcl - xl . in addition , it also induced caspase - dependent apoptosis by activating caspase-9 and caspase-3 , and caspase - independent apoptosis by inducing protein expressions of aif , endo g , and htra2 .
liver cancer is the second leading cause of cancer deaths in taiwan as per the 2011 statistics and ranks fourth in cancer - related mortality in the world . recent researches have shown that antrodia cinnamomea , a taiwan - specific medicinal mushroom , has biological activities , including hepatoprotection , anti - inflammation , antihepatitis b virus activity , and anticancer activity . in the present study , the antiproliferative activity and molecular mechanisms of antcin k , the most abundant ergostane triterpenoid from the fruiting bodies of basswood cultivated a. cinnamomea , were investigated using human hepatoma hep 3b cells . the results showed that antcin k effectively reduced hep 3b cells viability within 48 hours . antcin k induced phosphatidylserine exposure , chromatin condensation , and dna damage , but did not significantly increase autophagosome content or cause cell expansion and cell lysis . thus , the principal mode of hep 3b cells death induced by antcin k was apoptosis , rather than autophagy or necrosis . in - depth investigation of the molecular mechanisms revealed that antcin k first promoted reactive oxygen species generation and adenosine triphosphate depletion , leading to endoplasmic reticulum stress and resulting in mitochondrial membrane permeability changes . after losing the mitochondrial membrane potential , caspase - independent and caspase - dependent apoptosis - related proteins were released , including htra2 , apoptotic - induced factor , endonuclease g , and cytochrome c. cytochrome c activated caspase-9 and caspase-3 , and cut downstream protein parp , ultimately leading to cell apoptosis . these results suggested that antcin k induced mitochondrial and endoplasmic reticulum stress - mediated apoptosis in human hepatoma cells . coupled with these findings , antcin k has a potential to be a complementary agent in liver cancer therapy .
Introduction Materials and methods Results Discussion Conclusion Conflicts of interest
antrodia cinnamomea , a taiwan - specific mushroom , has been reported to have numerous biological activities including hepatoprotection , anti - inflammation , antihepatitis c virus activity , and anticancer activity . liver cancer ranks fourth in cancer - related mortality around the world ( who 2008 ) and is the second leading cause of cancer deaths in taiwan . there are other caspase - independent death pathways in mitochondria , such as htra2/omi , endonuclease g ( endo g ) , and apoptotic - induced factor ( aif ) released from mitochondria.15 , 16 basswood cultivated a. cinnamomea ( bcrc930103 ) mycelial powder was supplied by po - zo co. , ltd ( taipei , taiwan ) . after 24 hours of incubation , the cells were treated with 6 ml medium containing 0m , 80m , 100m , and 125m antcin k for 30 minutes to detect ca , 24 hours to detect ros , 48 hours to detect autophagosome , 48 hours to determine mitochondrial membrane potential , and 24 hours to determine the percentage of cells undergoing apoptosis , using an annexin v - fitc / propidium iodide ( pi ) assay kit ( bd biosciences ) . the immunofluorescence at 24 hours showed that 80m , 100m , and 125m antcin k treatment resulted in the formation of chromatin condensation in hep 3b cells . after treating hep 3b cells with 0m , 80m , 100m , and 125m antcin k for 24 hours , ros generation was analyzed by flow cytometry ; in addition , after treating hep 3b cells with the same concentrations of antcin k for 48 hours , the adp / atp ratio was analyzed with bioluminescence . hep 3b cells were treated with 0m , 80m , 100m , and 125m antcin k for 48 hours , after which the mitochondrial membrane potential was analyzed and quantified by flow cytometry . after treating hep 3b cells with 0m , 80m , 100m , and 125m antcin k for 48 hours , unfolded protein response - related protein expression 4a shows that as the concentration of antcin k increased , the chop expressions increased in dose - dependent manners , compared with the negative control ( 0.2% dmso ) . after treating hep 3b cells with 0m , 80m , 100m , and 125m antcin k for 48 hours , caspase - independent and caspase - dependent apoptosis - related protein expression was analyzed by western blotting . in addition , 4c shows that antcin k truly increased the caspase - independent apoptosis - related protein expressions of aif , htra2 , and endo g at 48 hours in hep 3b cells . antcin k is a triterpenoid from fruiting bodies of basswood cultivated a. cinnamomea and accounts for 0.5% of a. cinnamomea . 3b and d ) , antcin k can increase ros generation in human hepatoma hep 3b cells and also reduce the atp level of the cells , suggesting that er may be damaged by oxidation and in a hypoxic state . apoptosis occurs upon the perturbation of cellular ca homeostasis , such as cytosolic ca overload , er ca depletion , and mitochondrial ca increase.33 , 34 ca overload can induce mitochondrial depolarization and , subsequently , the release of apoptosis - inducing factors ( such as cytochrome c ) , which leads to the sequential activation of caspase-9 and caspase-3.35 , 36 as evident from the results of the calcium ion signal analysis ( fig . 4b and c ) , caspase - independent and caspase - dependent apoptosis - related proteins were released , including htra2 , aif , endo g , and cytochrome c ; cytochrome c activated caspase-9 and caspase-3 , and cut downstream protein parp , ultimately leading to cell apoptosis . su et al have shown that eburicoic acid , a triterpenoid from a. cinnamomea , induced autophagy in human hepatoma hep 3b cells of the er stress pathway and increased the calcium ion concentration as well as the protein expressions of bcl-2 and beclin-1 . however , the most interesting issue is that antcin k could not induce autophagy in hep 3b cells ; instead , it caused apoptosis , decreased the protein expression of bcl - xl , and changed mitochondrial membrane potential . several studies have demonstrated that a. cinnamomea has a good inhibitory effect on cancer.3 , 41 , 42 this study is the first of its kind to show that antcin k has an antiproliferative effect in liver cancer cells . in addition , it also induced caspase - dependent apoptosis by activating caspase-9 and caspase-3 , and caspase - independent apoptosis by inducing protein expressions of aif , endo g , and htra2 .
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risk benefit analysis with or without concern for legal issues has always been the mainstay of clinical practice . beneficence , insofar as it is a principle in medicine , has to a greater or lesser degree always been based on this issue . even under the accusation ( and hopefully this argument is resolved and exhausted now ) , whatever decisions doctors used to take was based on what in their opinion was in the patient s best interests and for the patient s good . leaving patients ignorant of their disease and outside the realm of decision - making was considered a therapeutic privilege and done in order to protect the patient from news which he or she did not or should not know in order to keep them from grief which could cause them more harm than good . the term values is used in order to emphasize that it is not merely a question of relativism or simply because we understand human nature better . it was indeed the philosophical debate which contributed considerably to increasing the principle ( in the kantian sense ) of respecting autonomy and trumping it over beneficence , until beauchamp and childress1 came around to give us ( or rather , lay down what was in the air ) the four principles . these evolved into an ethical framework and indeed doctors and health care providers were invoked to use these principles in their decision - making , calling them mid - level principles . principlism came under attack2 and the defenders went on to invoke methods of using principles , such as specifying and balancing . specification and balancing were meant to address the particular situation , yet steering away from situational ethics . assessing there is overlap also with the principle of justice because these decisions invariably also involve the use of resources that can be allocated elsewhere ( without , of course , bowing to utilitarianism , as indeed even a deontologist must consider her duty to society ) . in the meantime , the field of gerontology and geriatric medicine has taken on new life , and rightly so ; we are not only concerned with the elderly as a specific group , needing special needs with special attention to differences in medical care , just as children need a different approach to adults , but also because our populations , and perhaps cultures , are aging.3 making decisions concerning risk benefit for the elderly invariably therefore takes on a completely different dimension than for other adults , not only because where the elderly are concerned there is a different physiology and an aging body , but also because we are dealing with perhaps an aging culture , and more persistently so because we have to face the bottom line question : is it worth treating this patient at this stage of life ? these may be blunt questions , but it is a reality from which discussions of end - of - life decision - making ensue . the issues of cardiopulmonary resuscitation , advance directives , and research into longevity have arisen . because we are discussing an ethical concept , and not merely a clinical algorithm , what we have at base is our attitude , in philosophical terms , towards our elderly populations . the first discusses the reality of principles as an ethical framework and whether in essence it is our values towards the above questions which define outcomes . secondly , it discusses three real - world situations , the third of which discusses how we view elderly in this postmodern society . decisions are usually based on inherent cultural beliefs from which emanate virtues and general principles . inherently we are what we are because we have learned basic concepts throughout life which set the scene for our principles . but both the use of and the foundation of these principles are inherently attached to our concept of what is or should be right and wrong . a difference at this level , and you invoke a different reasoning on how the same principles are used to arrive at another different answer . indeed when we deliberate a moral problem we start with an empathic feeling of what is right and what is wrong . rational thought about a situation may allow us to change our course of action and perhaps the final decision , but our initial value system remains essentially unchanged . indeed , it is probable that we have conflict amongst principles because we have different value systems . gregory tillett perhaps best explains this conflict.4 tillett , in his analyses of resolving conflict distinguishes three concepts , or categories , of differences between ideas , humans , relationships , and between internal ( within oneself ) issues , ie , problems , disputes , and conflicts . a problem can be resolved by management , ie , by the agreement on how something can or should be done . we can easily conceive of a difficult clinical situation in which one consultant or family practitioner asks the advice of another , and doctors within a team discuss courses of action between themselves and with patients , especially within a context where there is refusal of a particular kind of treatment . tillett points out that these problems can indeed become disputes and can generate conflict.4 a dispute occurs when two ( or more ) people ( or groups ) perceive that their interests , needs , or goals are incompatible and they seek to maximize fulfilment of their own interests or needs , or achievement of their own goals ( often at the expense of others ) . consider , for example , the situation of an elderly woman who is being taken care of at her daughter s home . this daughter has three brothers , one of whom had distanced himself from his sister due to a dispute with her husband . the main contention was that this brother convinced the others that their mother should be put in a home because he refused to visit her at their sister s home . of course , prima facie , the best interests of the patient were for her to remain at home . however , it transpired that the woman was very depressed because this son , who was evidently her favorite , was not visiting her . the undue pressure was indeed coercive and the woman conceded to go to the home in order to be able to see all her children , not at all a voluntary choice . in the meantime after eight months in hospital and no remedy in finding a place at an institution , the decision had to be reversed to take into consideration again her best interests , ie , avoidance of a hospital infection . because the daughter was still willing to take her back , this course of action was taken , and the necessary legal procurements arranged . at each stage however a value - laden decision was taken , which brings us to the third category , ie , conflict . conflict , again as defined by tillett , arises when two ( or more ) people ( or groups ) perceive that their values or needs are incompatible whether or not they propose , at present or in the future , to take any action on the basis of those values.4 whilst problems and disputes arise within specific situations , conflicts do not need one . two parties can be in conflict because of beliefs or because of the values they uphold , and hence the heated debates on such issues as abortion and euthanasia . but , as in the case described here , we may find that superficially , at least , the people involved had the same values in that they all loved their mother , but when it came to taking a decision which was in the best interest of their mother , it was rather a decision in their own best interests which had become evident , until a situation arose which gave enough strength to override these different values . the difference in value was between one in which x would rather have his mother in a home and be able to see her ( and perhaps that she would be able to enjoy him ) , and the same person admitting that notwithstanding that his mother would not be able to see him , his sister was in a better position to take care of her and that given the state of existing homes , she may be better off . the conflict was that between x and the husband of x s sister , and not the situation . admittedly there can be a hazy line between a dispute and a conflict , as in this case . one has to delve into the narrative of each participant in order to reveal a conflict and understand that the matter is not simply one of disputes . in this case , the brothers agreed that the mother be allowed to go to their sister s home on the condition that the sister use her pension only and not the mother s savings in order to take care of her , ie , if she needed a new bed or a commode , they were not willing to contribute to either . this coercion reveals the underlying conflict between the daughter s husband and the dominant brother , notwithstanding a resolution of the conflict . it seems that the needs or values of the children trumped the needs of the mother or the value to respect and meet her needs . certainly this was not a dispute any more but a conflict , which unfortunately the medical practitioner or team may not be able to handle , unless by enforcing legal processes which may not be in the interests of all parties and not so conducive to conflict resolution . justice is conflict.5 doctors make decisions in the light of beneficence and at the same time doing justice to the person(s ) and the situation at hand . by doing justice , we obviously do not merely mean that the situation is handled well and correctly ( within , that is , the boundaries of standards of care ) , but we seek that justice is done morally . deciding whose justice and which rationality is certainly not easy in the modern sense , especially in dealing with end - of - life situations , whether to treat or not to treat , whether to leave elderly for acute treatment at their home , or incur a more expensive , perhaps better treatment , but in a hospital setting . certainly there can be hardly a better exposition of this philosophical reality than that put forward by macintyre.5 whether he does justice to the topic is probable but whether he resolves the dilemma is uncertain . certainly he speaks extensively of tradition , which is an argument extended through time , in that it can be a medical tradition or a religious tradition or simply a tradition of horse - riding . solomon6 believes that morality can not be reduced to any strict kantian system of principles . the very fact that the four principles of health care require further specification and balancing shows that a deeper sense of the real situation is needed , and what this usually boils down to is the narrative of the parties involved who bring forth their values and needs . solomon does not particularly like macintyre s use of traditions , in the sense that it still leaves open the nostalgia for religion and a sense of community , but goes on to admit that aristotle did invoke a sense of community when speaking about virtues . neither does he associate virtue with feminist ethics , values not being supposed to be held captive to gender , even though feminist ethics may have a point in contrasting male principles which are hard , oppressive , and impersonal , with the female virtues of warmth and caring . colloquially , by having principles , we are actually talking about the values a person holds as well . if one has a so - called value for life and is not in favor of euthanasia , one invokes principles to lead one to this conclusion . conversely , if one s value system of life includes terminating suffering even by allowing physician - assisted suicide , then one will use the same four principles to reach the opposite goal . having summarized the philosophical foundations on which risk benefit analysis is based , various relevant clinical situations faced in health care are grouped . jonsen et al7 classify three forms of disease and goals of medicine that are summarized here with particular reference to care and decisions for the elderly . these authors say that in the first , the patient suffers from an acute illness that , once diagnosed , can be readily treated and cured . in the second , the patient experiences a process that causes serious disabilities and which , while some relief can be provided , will progress despite treatment and eventually case death . in the third , the patient suffers a chronic disease that can be effectively treated so as to relieve many of the most debilitating effects . for simplicity they use the acronyms acure ( acute , critical , unexpected , responsive , and easily diagnosed and treated ) , care ( critical , active , recalcitrant , and eventual ) , and cope ( chronic , outpatient , palliative , and efficacious ) . perhaps using simply acute , critical , and chronic would have been quite an accurate substitute , but these acronyms serve the purpose just as well and are applied here . these authors point out that physicians habitually approach medical problems by attempting to determine the indications for or against medical intervention . in fact , assessing risk and benefit takes both medical intervention and ethical dilemmas to task , especially when considering elderly patients and when one asks oneself whether one should treat or not in a particular case . medicine remains a science of uncertainty ; even these acronyms are understood as being probability statements rather than absolute designations . indeed , uncertainty makes for good decision - making in the face of risk benefit analysis , and not simply one of clinical competence but also one which is value - laden . these goals apply to acute and relatively straightforward conditions , especially once a diagnosis has been made . the situations may be critical and unexpected , such as meningitis , reversible and easily treated ( by easy one of course does not mean easy in the strict sense , but that one knows what to do and has an immediate plan of action based on evidence - based medicine and/or standards of care ) . ethical issues in this situation may range from simply refusal of treatment to families not wishing the elderly patient to know . recent literature suggests a more culturally sensitive approach in these clinical situations,810 respecting wishes which may be culturally sensitive . in japan for example , it may be considered disrespectful to let an elderly person know of the medical condition , such that the sons and daughters take on the responsibility . the second category ( care ) describes patients with active , progressive , and deleterious conditions . one can easily see many elderly people in such situations , although this is by no means restricted to this section of the population . under this category lie most of the ethical dilemmas facing end - of - life decisions , such as withdrawing of life support , decisions not to intubate , decisions not to resuscitate , and irreversible coma / brain death . so are the mainline ethical discourses of ordinary versus extraordinary treatment , killing versus allowing to die , treating versus tender loving care , etc . what is in fact ordinary and extraordinary treatment ? standards of care weigh in heavily . even in invoking the principle of double effect by using high doses of morphine , for example , to treat palliatively , knowing that there is a risk that the patient may die of the dose of morphine and that this was a foreseen but unintended consequence , one must understand the nature of increasing doses of morphine and not be caught in a dispute of hastening death by going about the principle of double effect . indeed , even though the principle has four basic rules to it , it is heavily burdened on the moral agent to ascertain that it is morally and correctly invoked ( just as it takes a moral agent , for example , not to abuse the abortion act in the uk ) . there is always a standard of care in increasing dose strengths , although this is a patient - sensitive decision and hence can be externally seen as subjective . there is no duty to treat when treatment is judged to be useless , nor a duty to treat when treatment is deemed extraordinary . although today proportionate and disproportionate are terms which may be used instead of ordinary versus extraordinary , the latter are not only still the most commonly used terms , but they are probably the best guideline to make a judgment on risk benefit . however , one must clearly understand the meaning of extraordinary to make best use of the term , and for this one must look at its original catholic origins . extraordinary means of preserving life are all medicine , treatments , and operations , which can not be obtained or used without excessive expense , pain or other inconvenience for the patient or for others , or which , if used , would not offer reasonable hope of benefit to the patient.11 a closer look at this definition shows how this covers most , if not all , legal issues that may arise when making moral choices on risk benefit . when assessing risk , we are indeed facing the particular and singular clinical picture of this patient , including fitness for surgery , side effects and interaction of drugs , wishes of the patient , and physical state of the patient . the definition clearly does not take into account the state - of - the - art of medicine and technology , and something that is considered extraordinary today may by no means remain so tomorrow . conversely , something that is quite ordinary treatment in one country , either because of culture or economic status , may indeed be extraordinary in another . family need not be put to excessive burden , especially if they are required to pay or to go through extraordinary measures to meet the patient s needs . clearly , where insurance and state can not or will not pay , the family can not morally be held responsible to take all measures necessary ( in malta , it has almost become a societal pressure to go to the uk for care when one finds no hope on the island , even if consultants say that no further treatment can be found there ) . of particular note is that even medicines and excessive expense are mentioned in the definition , along with pain and inconvenience to the patient or family . hence a treatment which can not be afforded or which will cause prolonged agony need not be given . these issues are mostly found in outpatient and community settings of family practice , and deal with the chronic and palliative . although jonsen et al lament that the ethical issues here may be less evident , they in fact may represent the same issues discussed above . cope is a good acronym because in fact we are working with the patient to although the same goals of preserving life , preserving function , and reducing pain and suffering are used , they take on a more patient - centered approach and reach compromises with the patient that allow him or her to participate in the treatment . whilst there may be no drama as in life - and - death situations , there is a quality - of - life perspective and also end - of - life decision - making to face . the american college of family physicians have recently paid much attention to helping families cope with end - of - life and also address the cultural aspects of individuals and families.12 this departs considerably from the earlier days of bioethics when autonomy meant revealing everything to the patient and almost burdening the patient with information , giving details in order to allow them to feel they were making a choice , or preferably making the choice themselves . this renewed cultural sensitivity brings back the onus on the practitioner to be truly patient - centered and to share the burden with the patient who usually indeed seeks the doctors advice . the bigger burden lies with difficult patients , ie , those who are noncompliant and those who pose problems such as never being fully satisfied . in these situations , the doctor may be impelled to transfer the care to someone else , because a breakdown of the relationship is perceived to have occurred . yet patient relationship as well as compromise , and this may in turn translate into breakdown occurring less often . legal issues may arise with the family , who may not be satisfied with the care imparted to their parents . however , we need to acknowledge in this regard that the involvement of the family as a community - based approach to treating the elderly is still compatible with the patient - centered approach.13 of course the concept of aging has changed over time . today , with discourse on aging , one may find less difficulty with , eg , an elderly man seeking help for erectile dysfunction . yet many bioethics committees recommend against women having fertility treatment beyond their menopausal age . with advancing technology , the right to treatments , perhaps with aging populations , will probably give ground to the yearning of the elderly spirit to continue enjoying life beyond the social boundaries thus far accepted . leon kass , former chairman of the us council on bioethics , found considerable opposition to his general wariness about reproductive technology and efforts to forestall aging.14 therefore , risk benefit analysis can not look at the social concept of aging in a postmodern and perhaps posthuman society . the concern for us now lies in making the same analysis of legality in risk - benefit not only with regard to medical care but also in terms of research , this being imperative to the advancement of medicine . we have to decide which research is moral and which not to finance with public funds . there are strong arguments for the moral imperative to research elderly subjects further , even though research in this realm is both ethically required and ethically suspect.15 do we carry out research in order to increase longevity , to curb the aging process , or even perhaps to stop aging all together ? whereas better cures and medical advances have already brought this out , it was never really a medical imperative . it was , at most , a welcome , although perhaps foreseen consequence of treatment and care . yet the line is quickly drawing a distinction between a rise in the elderly population due to better survival and a deliberate search for survivorship and longevity , although such research into the genome and molecular cell biology is rapidly becoming hazy . in discussing aging and the sociology of embodiment , featherstone and hepworth16 give an interesting account worth reflecting upon . at the end of the day , the body is the bottom line , when we survive to advanced old age.17 elias , in his old age acknowledged , with an air almost of awe , that there is a simple reality of a finite life.18 therefore , the body in decline , for featherstone and hepworth,19 is a central issue for contemporary society to come to terms with . they assert that this does not mean that historically , when life expectancy was much shorter , people did not strive to prolong their life . fear of death , intensity of bereavement , and the longing for earthly pleasures are all issues to be faced . it is precisely in the struggle to reconstruct this cultural inheritance of pessimism that the element of difference between past and present attitudes towards aging through the later period of the life course may be found.19,20 this reconstruction , according to these social authors , lies in moving away from the pessimistic and melancholic processes of aging to one of optimism and a period enriched with distinctive creative possibilities.16 only in this way can the structure of feeling and attitudes towards aging be changed , especially with regard to the aging body . this social reconstruction ( or better , change in social acceptance ) , will then perhaps illuminate our attitude towards caring for our elderly in a new way and not simply as seeing them one of the vulnerable populations , even though they may be . we may see new light in research into the elderly and perhaps even in some form of longevity and prevention of body decline , which would bring with it a continuation of activity , be this sexual , educational , or occupational . we are already seeing countries prolong the retirement age due to improved standards of living and quality of life , and this not merely because elderly populations are on the rise and we need to continue collecting taxes from them . in understanding our ethical and legal attitudes to the risk and benefits of treatment , we must indeed be wary therefore of any desire by groups , such as practitioners of geriatric medicine , to make claims of a specialized form of knowledge and to legitimize the imposition of controls over aging members of the population.21 one should not take this to mean that geriatric medicine should not exist , but to entail a change in concept even of the discipline itself . , much as family doctors are advocates for seeing the family more consistently in the biopsychosocial dimension , acting as family counselors and advocates for patient rights . notwithstanding the discipline of geriatrics and the vulnerability experienced by an increased percentage of this sector of the population , we can not continue to see old age as a form of pathology and the body in decline as a medicalized entity , which according to katz , is an accusation often leveled at medicine . perhaps we need to understand that the stages model of life is a cultural clich , as are expressions like ticking of the clock.22 the implications for the sociology of ageing are clear : it is no longer possible to make adequate generalizations about the aging process that are grounded on biological assumptions about the ages of life . nor is it particularly useful to adopt schemata of the life course based upon loosely conceptualized models of unspecified processes of interaction between the ontologically distinctive entities , body , self and society . as a consequence contemporary models of ageing into old age must be increasingly post - modern , by which we mean they must anticipate even more advanced forms of biocultural destabilization.16 these authors eloquently argue how our struggle is not really with biologic decline , but with the social construction of aging that has been the center of debate . hormone replacement therapy , even if it had an element of success , was more about remaining young and active , hidden behind the excuse of better heart and skin outcomes , until the reality of the cancer issue came to light , ie , something which many were very skeptical about from the outset . the social pressure to take hormone replacement therapy can not be denied , in that many women may have taken it seeking a better skin complexion and less wrinkling , or at least have been encouraged to take the medication for these reasons . it is interesting that postmodern feminist studies of the interpretation of aging were associated with wisdom . before patriarchal societies , the female crone was seen as a naturalistic and matriarchal part of life , with wrinkles being badges of honor.23 history has brought about a separation of the self from the body and society , and the seeking of youth is found at the base of this force . whether this is the result of patriarchal influence which feminism blames is beyond the scope of this paper . the profound reality is that it is an existential issue and an entity to be reckoned with . this dualism , ie , the separation of the self from the body , lies in the very nature of the fear of aging in terms of a declining body . is an expression that perhaps was less heard in primitive society , which adorned their elders with a wisdom and respect much less seen today . this lies at the heart and concept of the recent change towards end - of - life decision - making . western culture is learning yet again from populations which lagged behind the materialistic taint and preserve many of their traditional values , and such is the case for eastern countries . featherstone and hepworth continue to make the argument that whilst accepting the limits of the body and the aging process as having its own rewards , they adopt a negative attitude to technologic advances whereby medical intervention , such as hormone replacement therapy , is a possibility . nowadays , in ethical circles , words like thus , the main question would be whether hormone replacement therapy is unnatural and a threat to womanhood ? indeed , there is much sense in not seeing technology as an external factor to human nature , but as a relationship between nature and the human bodily nature ( the creator of the technology itself ) , which is a culturally dynamic process . we have stopped seeing technology as part of our nature , because it is human nature which creates it , just as a monkey uses a stick , and perhaps moral discourse is more based on a fear of progress rather than a balancing of moral choices . when seen in this light , we are be forced to accept a change in balancing risk as opposed to benefit . whilst medicine has been attacked for being paternalistic and indeed domineering , in a foucaultian sense it is the world in general that has strived for technology and improvement of the human condition . medicine has certainly taken advantage of new technologies for better cure rates , but research is often not carried out by medicine as such , but by corporations who strive to satisfy the thirst of society , and profit of course in the process . there is a delicate , and sometimes controversial , balance with cultural and religious values . there is nostalgia , ie , a longing to remain attached to certain roots.24 the reality of the new field of bioethics , as opposed to the centuries old hippocratic tradition , emerged at a time when technology was also blooming . therefore , we may still be tied to concepts of not treating the elderly because of their age . the balance between cultural nontelling , and indeed giving a treatment adequate to what the body can withstand ( it would be unwise to opt for surgery , but certainly a long - term course of tamoxifen , even if palliation is not needed ) is not that difficult to conceive . however , accepting cultural criteria , such as the children of this woman , indeed in their late 60s themselves , of not giving her the bad news , even if she had never explicitly expressed a wish not to know , is the morally ( and perhaps legally ) correct thing to do . what is probably wrong in our approach to the elderly is to see the changing body as a pathologic process . even if we can come to accept change in body parts , better appearance , etc , as socially acceptable , there will always be a time of reckoning with death and the human condition . decline begins very early on in life , perhaps immediately after peak physical growth is reached . there are certainly problems that affect the elderly more , as there are problems that affect children more . two years for a 70-year - old is a larger proportion of one s remaining lifespan than the same number of years for a 40-year - old . certainly balancing risks and benefits may not work out in favor of the elderly person . a new social awareness towards aging helps cultivate an attitude that old age is worthwhile for what it is rather than for what the elderly do or what they have . whilst looking at the risk and benefit of treatment , we can not ignore the larger risk benefit picture of what research and medicine hold in the future . advanced research must be respected as part of the human need to ask , invent , and discover . however , a balance must be struck so as not to uproot people from their cultures because this is what gives people their identities . simone weil warns against uprooting people , cautioning that uprootedness leads to misery and spiritual lethargy on one hand and to violent efforts to adapt and uproot those not already uprooted .
the ethical dimension of treating the elderly , including risk benefit analysis , focuses mainly on quality of life and end - of - life issues . these include arguments on advance directives and the concept of extraordinary treatments . this paper looks more closely at the philosophical approach to aging in order to address questions on the direction of research and issues such as longevity and social construction of the aging process . it is the way society moves to understand the value - laden choices on aging that directs the goals of treatment and research . whilst these vary culturally , one has to reckon with a postmodern view of aging which may , in turn , reflect on the course of action of future care and research in aging . the paper canvasses how , in reality , four principles act as guidelines for moral discourse , and discusses how changing values in society decide this course of action .
Introduction Why principles and why conflict? Clinical decision-making Social understanding of aging Should we strive for technologic advancement of aging? Treatment and medicalization Conclusion
in the meantime , the field of gerontology and geriatric medicine has taken on new life , and rightly so ; we are not only concerned with the elderly as a specific group , needing special needs with special attention to differences in medical care , just as children need a different approach to adults , but also because our populations , and perhaps cultures , are aging.3 making decisions concerning risk benefit for the elderly invariably therefore takes on a completely different dimension than for other adults , not only because where the elderly are concerned there is a different physiology and an aging body , but also because we are dealing with perhaps an aging culture , and more persistently so because we have to face the bottom line question : is it worth treating this patient at this stage of life ? these may be blunt questions , but it is a reality from which discussions of end - of - life decision - making ensue . because the daughter was still willing to take her back , this course of action was taken , and the necessary legal procurements arranged . one has to delve into the narrative of each participant in order to reveal a conflict and understand that the matter is not simply one of disputes . indeed , uncertainty makes for good decision - making in the face of risk benefit analysis , and not simply one of clinical competence but also one which is value - laden . under this category lie most of the ethical dilemmas facing end - of - life decisions , such as withdrawing of life support , decisions not to intubate , decisions not to resuscitate , and irreversible coma / brain death . however , one must clearly understand the meaning of extraordinary to make best use of the term , and for this one must look at its original catholic origins . whilst there may be no drama as in life - and - death situations , there is a quality - of - life perspective and also end - of - life decision - making to face . the american college of family physicians have recently paid much attention to helping families cope with end - of - life and also address the cultural aspects of individuals and families.12 this departs considerably from the earlier days of bioethics when autonomy meant revealing everything to the patient and almost burdening the patient with information , giving details in order to allow them to feel they were making a choice , or preferably making the choice themselves . however , we need to acknowledge in this regard that the involvement of the family as a community - based approach to treating the elderly is still compatible with the patient - centered approach.13 of course the concept of aging has changed over time . leon kass , former chairman of the us council on bioethics , found considerable opposition to his general wariness about reproductive technology and efforts to forestall aging.14 therefore , risk benefit analysis can not look at the social concept of aging in a postmodern and perhaps posthuman society . there are strong arguments for the moral imperative to research elderly subjects further , even though research in this realm is both ethically required and ethically suspect.15 do we carry out research in order to increase longevity , to curb the aging process , or even perhaps to stop aging all together ? it is precisely in the struggle to reconstruct this cultural inheritance of pessimism that the element of difference between past and present attitudes towards aging through the later period of the life course may be found.19,20 this reconstruction , according to these social authors , lies in moving away from the pessimistic and melancholic processes of aging to one of optimism and a period enriched with distinctive creative possibilities.16 only in this way can the structure of feeling and attitudes towards aging be changed , especially with regard to the aging body . perhaps we need to understand that the stages model of life is a cultural clich , as are expressions like ticking of the clock.22 the implications for the sociology of ageing are clear : it is no longer possible to make adequate generalizations about the aging process that are grounded on biological assumptions about the ages of life . as a consequence contemporary models of ageing into old age must be increasingly post - modern , by which we mean they must anticipate even more advanced forms of biocultural destabilization.16 these authors eloquently argue how our struggle is not really with biologic decline , but with the social construction of aging that has been the center of debate . before patriarchal societies , the female crone was seen as a naturalistic and matriarchal part of life , with wrinkles being badges of honor.23 history has brought about a separation of the self from the body and society , and the seeking of youth is found at the base of this force . this lies at the heart and concept of the recent change towards end - of - life decision - making . featherstone and hepworth continue to make the argument that whilst accepting the limits of the body and the aging process as having its own rewards , they adopt a negative attitude to technologic advances whereby medical intervention , such as hormone replacement therapy , is a possibility . whilst medicine has been attacked for being paternalistic and indeed domineering , in a foucaultian sense it is the world in general that has strived for technology and improvement of the human condition .
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dementia , a syndrome usually resulting from a chronic or progressive brain disorder such as alzheimer s disease , currently affects 18 million people worldwide . dementia is characterized by a decline of cognitive functions such as memory , language , recognition , reasoning , and judgment . the risk of developing dementia increases with age ; thus , the number of patients with dementia will increase worldwide over the coming decades along with the demographic aging of the population . a considerable number of patients suffering from dementia at moderate to advanced stages are institutionalized . factors contributing to institutionalization vary and can include behavioral and psychological symptoms of dementia [ 2 , 3 ] as well as physical performance or level of independence in activities of daily living ( adl ) . indeed , dependence in adl is a critical risk factor for institutionalization [ 4 , 5 ] . furthermore , dependence for adl causes direct and indirect costs related to the care of this population to increase in parallel . recent data about the relationship between cognitive functioning and physical performance suggest that regular physical activity could provide cognitive benefits for patients with alzheimer s disease [ 7 , 8 ] . a high - intensity physical activity program may delay adl decline in older nursing home residents with severe cognitive impairment . however , sound evidence on which form of physical activity has the highest impact is still lacking . knowing what can be done , in which form , at what time , how long , and at what stage of the illness is of crucial importance to caregivers , to healthcare professionals , and to public health decision makers this is especially important at moderate to severe stages of the illness when adl ability decreases . the objectives of this review are ( 1 ) to describe the different types of physical activity programs designed for patients with moderate to severe dementia and ( 2 ) to identify the impact of these activities on functional independence in adl . a computerized search strategy was applied using the following mesh terms : ( motor activity [ mesh ] or exercise [ mesh ] or physical activity ) and dementia [ mesh ] and ( activities of daily living [ mesh ] or functional independence ) . the consulted databases were medline via pubmed , cinahl , cochrane library , isi social sciences citation index , otseeker , and pedro . we completed our computerized search by checking the reference lists of the included articles , of five systematic reviews [ 7 , 1013 ] , and one meta - analysis . studies were included if they investigated physical activity interventions applied to moderately to severely demented persons ( defined as an mini - mental state examination ( mmse ) score of 17 or less ) [ 15 , 16 ] . additional inclusion criteria were the presence of outcome measures for adl , the randomized controlled trial design , and the language of the article ; only articles written in english , french , german , or italian were considered . quality assessment was carried out using the scale developed by downs and black which is appropriate for assessing randomized and non - randomized studies . we adapted the last criterion of this scale , i.e. , the power of the study , and attributed one point if the authors did a prior sample size calculation . two authors ( eb , nk ) independently assessed the quality of the included papers after having completed the training of the scale application on one study which had not been included in the review . disagreement was resolved through discussion , and if necessary , the judgment of a third person ( avg ) was retained . we summarized the characteristics of each study sample , the physical activity interventions and their modalities of application , the measured adl outcomes , the assessment tools used to measure adl capacities , and the effect on the patients capacities to perform adl . for missing data , the original author of the trial was contacted in order to receive the required information . three meta - analyses were conducted in order to combine the results of the different studies included in our review . the first analysis compared the pre- and post - treatment values of the experimental group , the second analysis compared the pre- and post - treatment values of the control group , and the last analysis compared the post - treatment difference of both groups . since the number of included studies was small and since different exercise programs were proposed across these studies , the use of additional meta - analyses for the comparison of the different intervention modes was not feasible . published results were first converted into a common effect size measure : the standardized mean difference which is defined as the mean difference divided by the corresponding standard deviation . bias was removed using hedges g method , and the homogeneity between studies was evaluated by the q test . here , the homogeneity was rejected for all analyses , meaning that the true effect size in each study can be considered as similar , but not as identical . the solution was then to compute the overall effect size under the assumption of a random effect model . in practice , each study was weighted by the inverse of a variance composed of two terms : the variance of the study and a constant computed from q and representing the variability of the effect size across studies [ 21 , 22 ] . the study by steinberg et al . could not be included in the meta - analyses because the results were in a form incompatible with the computation of standardized mean differences . for similar reasons , the study by stevens and killeen so , to be coherent between the analyses , we decided to perform twice each of the first two analyses : with and without the stevens and killeen s study . since the results with only three studies included did not significantly differ from those with the four studies included , we reported results for the latter case only . results were reported as forest plots including the effect sizes and the 95% confidence intervals . we summarized the characteristics of each study sample , the physical activity interventions and their modalities of application , the measured adl outcomes , the assessment tools used to measure adl capacities , and the effect on the patients capacities to perform adl . for missing data , the original author of the trial was contacted in order to receive the required information . three meta - analyses were conducted in order to combine the results of the different studies included in our review . the first analysis compared the pre- and post - treatment values of the experimental group , the second analysis compared the pre- and post - treatment values of the control group , and the last analysis compared the post - treatment difference of both groups . since the number of included studies was small and since different exercise programs were proposed across these studies , the use of additional meta - analyses for the comparison of the different intervention modes was not feasible . published results were first converted into a common effect size measure : the standardized mean difference which is defined as the mean difference divided by the corresponding standard deviation . bias was removed using hedges g method , and the homogeneity between studies was evaluated by the q test . here , the homogeneity was rejected for all analyses , meaning that the true effect size in each study can be considered as similar , but not as identical . the solution was then to compute the overall effect size under the assumption of a random effect model . in practice , each study was weighted by the inverse of a variance composed of two terms : the variance of the study and a constant computed from q and representing the variability of the effect size across studies [ 21 , 22 ] . the study by steinberg et al . could not be included in the meta - analyses because the results were in a form incompatible with the computation of standardized mean differences . for similar reasons , the study by stevens and killeen could not be included in the third meta - analysis . so , to be coherent between the analyses , we decided to perform twice each of the first two analyses : with and without the stevens and killeen s study . since the results with only three studies included did not significantly differ from those with the four studies included , we reported results for the latter case only . results were reported as forest plots including the effect sizes and the 95% confidence intervals . further reasons for exclusion comprised other or missing interventions ( 125 ) , other outcomes ( 49 ) , other languages ( 12 ) , other populations ( 51 ) , and other designs ( 16 ) . after reading the full text , we excluded two additional studies with samples whose mmse score was higher than 17 ( fig . 1 ) . the results of the quality assessment ( table 1 ) showed that only one of the five randomized clinical trials included demonstrated a high - quality score ( 25 of 27 ) .fig . the first column represents the number of articles identified on the consulted databases ( medline , cinahl , pedro , isi social sciences citation index , cochrane , otseeker ) and those found by hand search . the second column refers both to the articles excluded after reading the title or the abstract and to the reason of exclusion . the last column contains the number of the articles that were assessedtable 1results of the quality assessmentitemsdescriptionscorereportingkwak et al . hypothesisis the hypothesis / aim / objective of the study clearly described?11111main outcomesare the main outcomes to be measured clearly described in the introduction or methods section?01111included patients characteristicsare the characteristics of the patients included in the study clearly described?01100interventions of interestare the interventions of interest clearly described?11100distributionare the distributions of principal confounders in each group of subjects to be compared clearly described?12211main findingsare the main findings of the study clearly described?11111random variabilitydoes the study provide estimates of the random variability in the data for the main outcomes?11101adverse eventshave all important adverse events that may be a consequence of the intervention been reported?01100characteristics of patients lost to follow - uphave the characteristics of patients lost to follow - up been described?00101probability valueshave actual probability values been reported ( e.g. 0.035 rather than < 0.05 ) for the main outcomes except where the probability value is less than 0.001?01111external validityrepresentativity of the participantswere the subjects asked to participate in the study representative of the entire population from which they were recruited?00111representativity of the prepared subjectswere those subjects who were prepared to participate representative of the entire population from which they were recruited?00000representativity of the staff , places and facilitieswere the staff , places , and facilities where the patients were treated , representative of the treatment the majority of patients receive?00100internal validity biasblind study subjectswas an attempt made to blind study subjects to the intervention they have received?00000blind staffwas an attempt made to blind those measuring the main outcomes of the intervention?01100data dredgingif any of the results of the study were based on data dredging , was this made clear?11101adjustment of the analysesin trials and cohort studies , do the analyses adjust for different lengths of follow - up of patients , or in case control studies , is the time period between the intervention and outcome the same for cases and controls?11111statistical testswere the statistical tests used to assess the main outcomes appropriate?11110compliancewas compliance which the intervention / s reliable?01100validity and reliability of the main outcomeswere the main outcome measures used accurate ( valid and reliable)?01001internal validity confoundingsubjects recruited from the same populationwere the patients in different intervention groups ( trials and cohort studies ) or were the cases and controls ( case control studies ) recruited from the same population?01111subjects recruited over the same period of timewere the patients in different intervention groups ( trials and cohort studies ) or were the cases and controls ( case control studies ) recruited over the same period of time?00111randomizationwere study subjects randomized to intervention groups?11111randomized intervention assignmentwas the randomized intervention assignment concealed from both patients and health care staff until was complete and irrevocable?00100adequate adjustment for confoundingwas there adequate adjustment for confounding in the analyses from which the main findings were drawn?11100losses of patients to follow - upwere losses of patients to follow - up taken into account?00100powerclinically important effectdid the study have sufficient power to detect a clinically important effect where the probability value for a difference being due to chance is less than 5%?00110total score1019251214 flowchart of identified and included studies . the first column represents the number of articles identified on the consulted databases ( medline , cinahl , pedro , isi social sciences citation index , cochrane , otseeker ) and those found by hand search . the second column refers both to the articles excluded after reading the title or the abstract and to the reason of exclusion . the last column contains the number of the articles that were assessed results of the quality assessment the main weaknesses of the included articles were the lack of information concerning the following three criteria : external validity , description of randomization , and description of the patients lost to follow - up . blinding of therapists and participants is not feasible when applying physical activity interventions , and hence , item 14 was not retained as a quality criterion . the agreement between the two raters when applying the downs and black scale was good with a chance corrected kappa coefficient of 0.67 . we checked the internal validity of the included studies by applying the cochrane criteria that are specific for randomized trials . the results confirmed that none of the studies was free of bias . according to the cochrane criteria , the main weaknesses consisted of ( 1 ) the unclear process of randomization for four [ 23 , 24 , 26 , 27 ] out of the five included studies , ( 2 ) the unequal treatment time in two studies [ 25 , 26 ] , ( 3 ) the lack of blinding of the outcome assessors in three studies [ 24 , 26 , 27 ] , and ( 4 ) the missing report of withdrawals in three studies [ 24 , 26 , 27 ] . the extracted information is summarized in tables 2 and 3.table 2main characteristics of the selected studiesauthorsdesignpopulationprogram referenceintervention : experimental groupintervention : control groupparticipants ratedropoutkwak et al . experimental group : age 79.7 ( 6.6 ) years , mmse 14.5 ( 5.3 ) . exercise intensity was gradually increased from 30% to 60% of expected maximal oxygen consumptionno informationno information0/30rolland et al . experimental group : age 82.8 ( 7.8 ) years , mmse 9.7 ( 6.8 ) . control group : age 83.1 ( 7.0 ) years , mmse 7.9 ( 6.4)experience and based on two other references [ 6 , 7]aerobic , strength ( lower extremity ) , balance training , fast walking , flexibility , program accompanied by music . intensity : at the beginning light intensity and was gradually increased over the first month . no restriction in nursing , physiotherapy , medical care , advice , or any other healthcare support134/42911/67 ( eg)13/67 ( cg)steinberg et al . randomized controlled trial stratified for gender and age over 7527 community dwelling persons with alzheimer disease . experimental group : age 74 ( 8.1 ) years , mmse 15.5 ( 5.4 ) . control group : age 76.5 ( 3.9 ) years , mmse 20.1 ( 5.1)graduate program at the johns hopkins bloomberg school of public healthaerobic fitness : brisk walking , strength training , balance and flexibility training , intensity : compliance measurehome visit , with recommendations27/300/27stevens and killeen randomized controlled trial120 nursing home residents from 6 different nursing homes . control group 2 : 80.5 years , mmse scores > 9 < 23program was designed based on knowledge concerning physiological adaptation in older frail people and in consultation with the school of sport and exercise science , southern cross university , lismoreaerobic by moving joint and large muscle groups . control group 2 : social visits equivalent in duration and frequency as those undertaking the exercise program in the experimental groupno information45/120francese et al . experimental design12 severely demented residents of a medicare nursing facilityprogram was based on previous interventions for frail or impaired residentsactivities such as catching , throwing , and kicking balls , leg weight exercises , parachute reaches , program accompanied by music , intensity : gentle aerobic exertionsing - along video12/300/6 ( eg)1/6 ( cg)mean age ( in years ) and mean mini - mental state examination scores and the corresponding standard deviation are reported for the experimental and the control group . ( data not available ) , and stevens and killeen reported a significant improvement of adl scores . . showed significant delay of adl deterioration , only after 12 months of program duration . post - treatment adl scores of the control group of stevens and killeen s study ( data not available ) decreased significantly compared to baseline assessment . adl scores of both groups deteriorated significantly in the study of rolland et al . compared to baseline assessmenteg experimental group , cg control groupindication of sd of mean age as well as mean mmse scores and the corresponding sd for each group is missinginformation related to age and mmse scores is missingtable 3intervention modalities of the selected studiesauthorsprogram duration ( months)session duration ( minutes)frequency per weekexercise leadergroup size participants ( number)outcomeskwak et al . 3no information6 ( aerobic ) , 4 ( strength , balance and flexibility training)exercise physiologistindividualhand activity relevant for adlstevens and killeen 3303researchersno informationadlfrancese et al . ( data not available ) , and stevens and killeen reported a significant improvement of adl scores . rolland et al . showed significant delay of adl deterioration , only after 12 months of program duration . post - treatment adl scores of the control group of stevens and killeen s study ( data not available ) decreased significantly compared to baseline assessment . adl scores of both groups deteriorated significantly in the study of rolland et al . compared to baseline assessment main characteristics of the selected studies mean age ( in years ) and mean mini - mental state examination scores and the corresponding standard deviation are reported for the experimental and the control group . ( data not available ) , and stevens and killeen reported a significant improvement of adl scores . . showed significant delay of adl deterioration , only after 12 months of program duration . post - treatment adl scores of the control group of stevens and killeen s study ( data not available ) decreased significantly compared to baseline assessment . adl scores of both groups deteriorated significantly in the study of rolland et al . compared to baseline assessment eg experimental group , cg control group indication of sd of mean age as well as mean mmse scores and the corresponding sd for each group is missing information related to age and mmse scores is missing intervention modalities of the selected studies kwak et al . , ( data not available ) , and stevens and killeen reported a significant improvement of adl scores . . showed significant delay of adl deterioration , only after 12 months of program duration . post - treatment adl scores of the control group of stevens and killeen s study ( data not available ) decreased significantly compared to baseline assessment . adl scores of both groups deteriorated significantly in the study of rolland et al . compared to baseline assessment the included studies were conducted between 2006 and 2009 , except for one publication dating back to 1997 . three studies [ 24 , 25 , 27 ] included nursing home residents and two studies [ 23 , 26 ] community - dwelling people . the participants were 75 years or older and , according to the mmse scores or population description , moderately to severely demented . the physical activity programs differed in each study , and in one study , references for the activity programs were missing . the most frequently mentioned interventions were strengthening exercises , balance , and gait training [ 23 , 25 , 26 ] as well as endurance training [ 2325 ] . the frequency varied between biweekly and daily and the duration of each session between 20 and 75 min . the intensity of exercises was inconsistently reported with few information : oxygen consumption , aerobic exertion [ 24 , 25 , 27 ] , and compliance measures [ 23 , 25 ] . each study used a different assessment tool . among them , the katz index was the most common adl assessment tool . one article indicated the applied assessment method and the considered adl items . in three studies [ 23 , 24 , 26 ] , the physical activity program significantly improved adl performance in the experimental group by the end of the intervention . it was a small but clinically meaningful difference in the experimental group compared to the control group after 12 months of intervention ; the intermediate result after 6 months was not significant . physical activity practice showed a significant effect already after 6 months , although modalities of the physical activity program were quite similar between these two studies . one very small study including only six participants in the experimental group did not show any significant effect on adl performance . among the controls , adl performance deteriorated over the short observational period ( table 4).table 4outcomes : mean score and standard deviation of adl assessmentsbaseline scoretimepost - intervention scorep valueegcgegcge / creferenceassessment toolmax scoremeansdmeansdmonthsmeansdmeansdadl deteriorationkwak et al . acsm24617.535.4612.074.52**change scoreassessment toolmax scoretimedifferences pre-/post - interventionp valuemonthsegcgadlcgadle / cstevens and killeen self - help skill2434.2922.8**0.524ns**jebsen total time test assesses hand function which is relevant for adl performance . american college of sports medicine method assesses adl according to the seven following categories : clothing , eating , moving , urinating , bathing , controlling feces , and washing hands . ( data not available ) , and stevens and killeen reported a significant improvement of adl scores . . showed significant delay of adl deterioration , only after 12 months of program duration . post - treatment adl scores of the control group of stevens and killeen s study ( data not available ) decreased significantly compared to baseline assessment . adl scores of both groups deteriorated significantly in the study of rolland et al . compared to baseline assessmentbi barthel index , self - help skill is an adl item of the revised elderly persons disability scale , jtt jebsen total time test , cads changes in advanced dementia scale , acsm american college of sports medicine , na data non - available , time time since baseline assessment , adl decline of adl performance , max score maximum score , eg experimental group , cg control group , e / c in - between group comparison , ns not significant*p < 0.05 ; * * p < 0.01control group 1 = no intervention ; control group 2 = social intervention outcomes : mean score and standard deviation of adl assessments jebsen total time test assesses hand function which is relevant for adl performance . american college of sports medicine method assesses adl according to the seven following categories : clothing , eating , moving , urinating , bathing , controlling feces , and washing hands . ( data not available ) , and stevens and killeen reported a significant improvement of adl scores . . showed significant delay of adl deterioration , only after 12 months of program duration . post - treatment adl scores of the control group of stevens and killeen s study ( data not available ) decreased significantly compared to baseline assessment . adl scores of both groups deteriorated significantly in the study of rolland et al . compared to baseline assessment bi barthel index , self - help skill is an adl item of the revised elderly persons disability scale , jtt jebsen total time test , cads changes in advanced dementia scale , acsm american college of sports medicine , na data non - available , time time since baseline assessment , adl decline of adl performance , max score maximum score , eg experimental group , cg control group , e / c in - between group comparison , ns not significant * p < 0.05 ; * * p < 0.01 control group 1 = no intervention ; control group 2 = social intervention figures 2 , 3 , and 4 summarized the main results of each meta - analysis . figure 2 showed that the adl status did not significantly change between the pre- and post - measurements among treated patients ( aggregate effect size 0.30 , 95% confidence interval [ 0.49 ; 1.09 ] ) . we examined the effect of including or not including stevens and killeen s study in the meta - analysis , and it did not change our conclusion . when performed on the control group ( fig . 3 ) , the same analysis indicated a significant decline of the adl functioning between the pre- and post - measurements ( 0.63 [ 1.02 ; 0.23 ] ) . 4 ) did not show any real difference in post - treatment values of both groups ( 0.43 [ 0.63 ; 1.50]).fig . 2meta - analysis conducted for the comparison of the pre- and post - treatment values in the experimental groupfig . 3meta - analysis conducted for the comparison of the pre- and post - treatment values in the control groupfig . 4meta - analysis conducted for the comparison of the pre- and post - treatment values in the both groups meta - analysis conducted for the comparison of the pre- and post - treatment values in the experimental group meta - analysis conducted for the comparison of the pre- and post - treatment values in the control group meta - analysis conducted for the comparison of the pre- and post - treatment values in the both groups thus , the decline in adl performance in demented subjects may be due not only to disease progression but also to physical inactivity . one study showed that the effect on adl performance disappeared after cessation of the physical activity programs . this observation may explain why three [ 23 , 24 , 26 ] out of five trials showed a significant effect on adl performance , but only one reported clinically meaningful results despite differences in their physical activity programs . the negative evolution of adl performance in all control groups except one tends to support the relationship between physical activity and functional abilities . the most severe adl degradation was observed in the study which included the patients with the lowest mmse scores . delaying adl deterioration in nursing home residents with moderate or severe cognitive impairment through a physical activity program may be both clinically and economically relevant as adl care in this patient group accounts for two thirds of the total care time . the descriptive results were supported by our meta - analyses and allowed us to conclude as forbes et al . did that even if the proposed treatments have not proven their efficiency in improving the adl status of the patients , they could nevertheless limit the decline in adl functioning . however , the physical activity programs varied among the included studies . in line with yu and kolanowski as well as taylor et al . , we confirm that there are no clinical practice guidelines for aerobic exercises for persons with dementia . reasons for the lack of both studies on the matter and practice guidelines may include the individual variability of the aging process and the limitation of physical activity practice due to the disabilities of the very old . nonetheless , endurance can positively influence the physiological aging process of the cardiovascular system at a central and peripheral level . aging per se causes loss of muscle strength , and regular strengthening exercises can counteract to some degree this loss in the very old . based on this knowledge , the content of the physical activity programs covering endurance , gait , and strength training proposed in the included studies seemed to be appropriate . questions related to the duration and intensity of the physical activity program , the duration of each session , or their frequency remain unanswered . one high - quality study applied a physical activity program with moderate to high intensity and showed a significant delay of adl decline only after 12 months duration , but not after 6 months . a high intensity strengthening program during 3 months achieved a similar result in dementia nursing home residents . paterson and warburton suggest that the intensity of physical activity should be at least moderate in order to improve adl performance in the elderly . the literature on the topic does not either give clear indications on how long an ideal activity program should last . yu and kolanowski , based on their summary of current knowledge concerning the prevention of alzheimer s disease , suggest an ideal program duration of 2 months and the ideal session frequency of three times per week for an aerobic exercise program designed for a medically stable population with dementia and aimed at improving their adl performance . the most important parameter was regularity of exercising with high exercise adherence significantly preventing adl decrease . the result of the study of littbrand et al . including residents with less severe dementia showed no lasting effect of physical activity training on adl . demented people are likely to need some encouragement to stay physically active and to slow the decrease of their adl performance especially in those with moderate to severe dementia . overall , the optimal frequency and intensity of physical activities which provide a satisfactory long - term effect are still unknown and their determination highly relevant . physical activity programs were accompanied by music in only three studies [ 24 , 25 , 27 ] , although there is a growing body of evidence that music in advanced stages of dementia could help improve the performance of patients [ 33 , 34 ] . according to a qualitative study , demented patients claimed that programs should respond to their psychological and social needs , whereas their caregivers considered maintenance of functional independence as the most important goal . thus , physical activity in groups accompanied by music could more easily respond to the expectations of demented patients and increase their adherence to a physical activity program . in addition , a further criterion to respect is the meaningfulness of the proposed activities . the reduced communication skills , the frailty of this population , and the increased risk of falling may require limited group sizes . an individual approach , as opposed to group sessions , allows meeting a patient s needs more specifically as community - dwelling participants may prefer exercising at home . the need for individual adaptation of physical activity programs has been stressed by yu and kolanowski as patients with moderate to severe dementia do not express reliably exertion and need guidance to exercise at the targeted level . variability in content and modalities of physical activity programs ( session frequency and duration ) impede any precise recommendation for clinical practice . the variety of applied adl assessment tools hampered comparison of the program effect on the adl performance across all studies . any recommendation to use objective measurement tools for moderately to severely demented subjects was altogether missing from the literature . however , it has little or no sensitivity to small changes and is therefore inappropriate for longitudinal studies . only one study applied a population specific measure , i.e. , the changes in advanced dementia scale , to assess adl performance in demented persons . assessing mobility is crucial as it is a key capacity among elderly with moderate to severe dementia . mobile patients decrease caregiver burden , and their quality of life may be better . the majority of items in the changes in advanced dementia scale deal with cognitive abilities to perform adl and not physical abilities . however , while physical activity programs act primarily on motor tasks , they may also have an impact on cognition . given the preponderance of items related to cognition , physical components of adl capacities may be underestimated when applying the changes in advanced dementia scale . the preceding considerations may explain the clinicians and researchers difficulties in selecting the appropriate adl assessment tool . higher levels of physical activity were beneficial and decreased the number of falls in a mildly to moderately demented population . no study reported serious adverse events related to physical activity , but steinberg et al . found trends of poorer quality of life and increased depression in their exercise group and discussed the possibility that physical activity may cause distress . however , the finding of higher depression scores was not corroborated by other studies [ 43 , 44 ] . . the methodological quality of the majority of the included studies is low , and information regarding the reliability , validity , and sensitivity to change ( i.e. , the clinically minimal important change ) of the various assessment tools applied to a moderate or severely demented population is missing . the scores obtained for external validity were low which limits the generalizability of the results and the formulation of recommendations . biases were due to inappropriate randomization methods in four out of the five included studies [ 23 , 24 , 26 , 27 ] , to the lack of blinding of the outcome assessors in three studies [ 24 , 26 , 27 ] and two studies [ 23 , 26 ] used adl assessment tools which were not validated for the moderately and severely demented . the clinical importance of the results was threatened by the insufficient power of three out of the five included studies [ 23 , 26 , 27 ] . the absence of interventions for the control group in two trials [ 25 , 26 ] represented a further weakness and prevented interpretation of the impact of physical activity programs on adl in these studies . the small number of studies corresponding to the selection criteria of our review represents an important limitation of this paper . although we tried to identify all significant studies , we do not pretend to have conducted a comprehensive review . defining dementia severity using mean mmse scores was a pragmatic decision as mmse scores are the most frequently reported severity measures . however , although populations were described as being moderately to severely demented in the included studies , not all authors reported dementia severity scores [ 24 , 27 ] . we are aware of the confounding education levels by interpreting mmse scores as well as the questionability of the cutoff scores which define moderate and severe dementia . while a uniform definition of severity is still lacking [ 47 , 48 ] , the relevant mmse scores are still debated . evidence for efficacy of physical activity programs on adl performance in the elderly with moderate to severe dementia remains very limited . one high - quality study showed a small but statistically significant and clinically meaningful effect . further investigations determining ideal physical activity program content as well as appropriate session duration and frequency are warranted . the variable program content between studies is of concern since it provides little guidance to clinicians as to what protocols may be the most suitable in patient care . gaining a more complete picture of the impact and limits of physical activity programs in this domain is urgent . while the number of people with dementia is growing , reducing adl dependence contributes to a better control of costs and alleviates family and caregiver burdens . in this context , the issue of the most effective dose for physical activity programs ( nature , frequency , duration ) is of crucial importance to healthcare quality and costs .
the objectives of this study were to describe the different modalities of physical activity programs designed for moderate to severe dementia and to identify their impact on functional independence in activities of daily living ( adl ) . a critical review of randomized controlled trials related to the impact of physical activity programs in moderately to severely demented persons on adl performance and meta - analysis of the identified studies were performed . among the 303 identified articles , five responded to the selection criteria . four out of the five studies demonstrated limited methodological quality . in one high - quality study , physical activity programs significantly delayed deterioration of adl performance . the program components and adl assessment tools vary widely across studies . although the proposed treatments have not proven their efficiency in improving the adl status of the patients , they were able to limit the decline in adl functioning . future research is warranted in order to identify clinically relevant modalities for physical activity programs for people with moderate to severe dementia .
Introduction Methods Data extraction and analysis Results Discussion Conclusion
factors contributing to institutionalization vary and can include behavioral and psychological symptoms of dementia [ 2 , 3 ] as well as physical performance or level of independence in activities of daily living ( adl ) . the objectives of this review are ( 1 ) to describe the different types of physical activity programs designed for patients with moderate to severe dementia and ( 2 ) to identify the impact of these activities on functional independence in adl . studies were included if they investigated physical activity interventions applied to moderately to severely demented persons ( defined as an mini - mental state examination ( mmse ) score of 17 or less ) [ 15 , 16 ] . 0.035 rather than < 0.05 ) for the main outcomes except where the probability value is less than 0.001?01111external validityrepresentativity of the participantswere the subjects asked to participate in the study representative of the entire population from which they were recruited?00111representativity of the prepared subjectswere those subjects who were prepared to participate representative of the entire population from which they were recruited?00000representativity of the staff , places and facilitieswere the staff , places , and facilities where the patients were treated , representative of the treatment the majority of patients receive?00100internal validity biasblind study subjectswas an attempt made to blind study subjects to the intervention they have received?00000blind staffwas an attempt made to blind those measuring the main outcomes of the intervention?01100data dredgingif any of the results of the study were based on data dredging , was this made clear?11101adjustment of the analysesin trials and cohort studies , do the analyses adjust for different lengths of follow - up of patients , or in case control studies , is the time period between the intervention and outcome the same for cases and controls?11111statistical testswere the statistical tests used to assess the main outcomes appropriate?11110compliancewas compliance which the intervention / s reliable?01100validity and reliability of the main outcomeswere the main outcome measures used accurate ( valid and reliable)?01001internal validity confoundingsubjects recruited from the same populationwere the patients in different intervention groups ( trials and cohort studies ) or were the cases and controls ( case control studies ) recruited from the same population?01111subjects recruited over the same period of timewere the patients in different intervention groups ( trials and cohort studies ) or were the cases and controls ( case control studies ) recruited over the same period of time?00111randomizationwere study subjects randomized to intervention groups?11111randomized intervention assignmentwas the randomized intervention assignment concealed from both patients and health care staff until was complete and irrevocable?00100adequate adjustment for confoundingwas there adequate adjustment for confounding in the analyses from which the main findings were drawn?11100losses of patients to follow - upwere losses of patients to follow - up taken into account?00100powerclinically important effectdid the study have sufficient power to detect a clinically important effect where the probability value for a difference being due to chance is less than 5%?00110total score1019251214 flowchart of identified and included studies . 4meta - analysis conducted for the comparison of the pre- and post - treatment values in the both groups meta - analysis conducted for the comparison of the pre- and post - treatment values in the experimental group meta - analysis conducted for the comparison of the pre- and post - treatment values in the control group meta - analysis conducted for the comparison of the pre- and post - treatment values in the both groups thus , the decline in adl performance in demented subjects may be due not only to disease progression but also to physical inactivity . did that even if the proposed treatments have not proven their efficiency in improving the adl status of the patients , they could nevertheless limit the decline in adl functioning . one high - quality study applied a physical activity program with moderate to high intensity and showed a significant delay of adl decline only after 12 months duration , but not after 6 months . demented people are likely to need some encouragement to stay physically active and to slow the decrease of their adl performance especially in those with moderate to severe dementia . the need for individual adaptation of physical activity programs has been stressed by yu and kolanowski as patients with moderate to severe dementia do not express reliably exertion and need guidance to exercise at the targeted level . the variety of applied adl assessment tools hampered comparison of the program effect on the adl performance across all studies . biases were due to inappropriate randomization methods in four out of the five included studies [ 23 , 24 , 26 , 27 ] , to the lack of blinding of the outcome assessors in three studies [ 24 , 26 , 27 ] and two studies [ 23 , 26 ] used adl assessment tools which were not validated for the moderately and severely demented . the absence of interventions for the control group in two trials [ 25 , 26 ] represented a further weakness and prevented interpretation of the impact of physical activity programs on adl in these studies . evidence for efficacy of physical activity programs on adl performance in the elderly with moderate to severe dementia remains very limited . gaining a more complete picture of the impact and limits of physical activity programs in this domain is urgent .
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patients with functional bowel disorders ( fbds ) manifest variable combinations of intestinal symptoms without structural and/or biochemical abnormalities . the latter concept has been challenged by growing evidence showing low - grade inflammatory changes in the gut and altered gut - brain axis signaling.1,2 according to the rome iii classification , fbds include the irritable bowel syndrome ( ibs ) , functional bloating ( fb ) , functional constipation , functional diarrhea , and unspecified fbd , and they are attributed to abnormalities likely originating from the small bowel , colon , and rectum.3,4 since fbds lack objective biomarkers , their diagnosis is based on the clinical symptoms reported by patients , physical examination , and the exclusion of alarm symptoms / signs ( eg , blood in stools , anemia , weight loss , and others ) . although fbds are not regarded as life threatening , these conditions can significantly worsen the patient s quality of life . indeed , fbds are responsible for prolonged absenteeism from work as well as for suboptimal performance in the workplace with relevant social costs.5,6 amongst fbds , ibs is certainly the most common clinical entity affecting up to 20% of the general population.7 classically , an ibs diagnosis revolves around abdominal pain / discomfort in conjunction with altered bowel habits . the clinical phenotypes include ibs with constipation , with diarrhea ( ibs - d ) , alternating bowel or mixed ( the most frequent pattern in western industrialized countries ) , and unsubtyped according to stool frequency and consistency.3,8,9 the pathogenesis underlying ibs is only partly understood and notoriously referred to as multifactorial being attributable to dysfunction of the gut - brain axis . in this context , recognized mechanisms in ibs span a wide spectrum including gut dysmotility , low - grade inflammation , visceral hypersensitivity , changes of gut microbiome , infections , altered gut barrier function , and genetic and psychosocial factors.1014 the role of dietary factors in ibs pathogenesis is a topic of great interest.1517 indeed , more than 60% of patients with ibs relate the occurrence of bloating and abdominal pain to the ingestion of certain foods . the majority of these patients report worsening of symptoms between 15 minutes to a few hours after meal intake.18 however , only recent animal and human studies have focused on the key role of specific foods in altering gut physiology . the aim of the present review is to provide an overview highlighting the major aspects of the complex interplay existing between foods and gut function with relevance to ibs . specifically , the reader will have an update on the role of gluten / wheat sensitivity as potential dietary triggers evoking gut dysfunction and symptoms in ibs . the pathophysiology of ibs is still not well understood , limiting the capacity to effectively treat the disorder.19 enteric infections are the strongest environmental triggers for ibs , constituting the well - characterized subgroup of post - infective ibs,20 which is associated with dysbiosis , low - grade inflammation and altered intestinal permeability.21 these mechanisms have also been proposed in the general ibs population , but results are not as consistent as in post - infective ibs.22 in addition to enteric infection , other environmental and psychosocial triggers have been linked with ibs . interestingly , many of these triggers induce visceral hypersensitivity , changes in gut microbiota , and altered levels of enteric hormones and neurotransmitters which may explain symptom generation.19,22,23 alterations in gastrointestinal transit , which may be caused by stress,24 have also been reported in ibs patients . although ibs is considered to affect mainly the colon , several studies have reported motility alterations also in the esophagus , stomach and small intestine , which often correlate with patients symptoms.25 several studies and a recent meta - analysis2628 have demonstrated bile acid ( ba ) malabsorption , at least in a sub - population of ibs - d . in a study of 119 patients with ibs,28 32% had abnormal colonic transit measured by scintigraphy at 24 or 48 hours , with accelerated transit in 48% of ibs - d patients ; the causes of abnormal transit are unclear . ba sequestrants have been proposed as a possible therapeutic option , however , the mechanism of action of these ba sequestrants to ameliorate bowel function is not yet clearly demonstrated.29 of all the possible non - infectious environmental triggers , food is a likely candidate that can affect a variety of physiologic parameters important in ibs such as motility , visceral perception , brain - gut interactions , microbiota composition , permeability , immune activation , and neuro - endocrine function.30 among the foods reported to associate with ibs symptoms , those rich in carbohydrates,19 gluten and wheat31,32 are common . however , food sensitivity or allergy can not be always confirmed in patients that recognize a specific food , as a trigger of their symptoms.31 therefore , a better understanding of the dietary factors involved in ibs and their underlying mechanisms is key to determine the real benefit of exclusion diets in ibs . ingestion of a meal activates an array of complex mechanisms enabling the digestive system to perform the complex task of digestion and absorption of nutrients as well as expulsion of waste . under normal circumstances , our gut has adapted to respond with a tightly regulated system of neuro - immune interactions necessary to maintain proper gut function and homeostasis . dietary factors , however , can be harmful in certain circumstances and can cause intolerance , allergy , or hypersensitivity via a number of different mechanisms . food intolerance , such as lactose intolerance , classically relates to a reaction to food components based on some metabolic deficiency.3335 food allergy and hypersensitivity instead refer to undesirable immune - based reactions that cause symptoms . the most common food sensitivities have been related to proteins in nuts , wheat , and milk.36 some of these reactions are typically ige mediated ( allergy ) , while others recognize non - ige immune pathways . patients with ibs often report worsening of symptoms by a wheat containing diet.37 gluten is a group of immunogenic proteins in wheat , which causes celiac disease , an inflammatory and autoimmune disease , in genetically susceptible people.38 this led to the hypothesis that a subgroup of ibs patients could develop mild immune or functional alterations in the absence of celiac disease.39 gluten proteins are indeed insufficiently degraded by gut proteases , leaving undigested peptides that could trigger innate immune mechanisms that may be of importance in ibs . a mouse study determined that gluten sensitization causes altered smooth muscle contractility and altered barrier function , which are mechanisms commonly associated with ibs . it is important to stress that other proteins in wheat , such as -amylase / trypsin inhibitors ( atis)40 and wheat lectin agglutinin,41 have recently shown to induce innate immune pathways . atis have been shown to trigger a toll - like - receptor ( tlr)-4 mediated activation40 and it remains to be determined whether this is associated with gut functional changes . in addition to protein fractions , wheat contains a group of carbohydrates , called fructans . these are members of fermentable oligosaccharide , disaccharide , mono - saccharides , and polyols ( fodmaps ) that are poorly absorbed in the small intestine.37,40,42 these substrates are very important for gut health , supporting microbiota diversity and short chain fatty acid production43 but under certain circumstances could cause , through excessive bacterial colonic fermentation , symptoms such as bloating and altered bowel habits.19,44 regardless of the offending component , the withdrawal of wheat from the diet in a subgroup of patients with ibs has indeed been shown to improve symptoms.45 although unselected ibs patients display clinical improvement after wheat withdrawal , only a proportion may truly reflect underlying gluten sensitivity ( table 1).4656 the absence of specific biomarkers and the great proportion of patients with placebo effect observed in ibs trials , are 2 barriers for identifying the specific role of wheat components in generating ibs symptoms . therefore , double - blind - placebo - controlled - crossover ( dbpcc ) trials are a reliable way to establish whether wheat and its fractions are involved in ibs symptom induction.50,51,53,56 different mechanisms have been proposed to explain how gluten may trigger gastrointestinal symptoms in the absence of celiac disease ( figure ) . in vitro studies have demonstrated that digests of gliadin increase the expression of co - stimulatory molecules and the production of proinflammatory cytokines in monocytes and dendritic cells.40,57,58 certain toxic ( that only stimulates the innate immune response ) gliadin - derived peptides such as the 3143mer , may evoke epithelial cell dysfunction , increased il-15 production and enterocyte apoptosis.59 recent studies have demonstrated increased expression of tlr-2 in the intestinal mucosa of non - celiac compared to celiac patients , suggesting a role of the innate immune system in the pathogenesis of non - celiac reactions to gluten or other wheat components.49 other studies have shown that monocytes from hla - dq2 + non - celiac individuals spontaneously release 23 fold more il-8 than monocytes from hla - dq2 negative patients . this suggests that patients without celiac disease ( no enteropathy and negative specific serology ) , but with positive hla - dq2 status , may represent a subpopulation reacting mildly to gluten.60 in terms of gut dysfunction , gluten sensitization in mice has been shown to induce acetylcholine release , one of the main excitatory neurotransmitters in the gut , from the myenteric plexus.57 this correlates with increased smooth muscle contractility and a hypersecretory status with increased ion transport and water movements.57 these functional effects induced by gluten were not accompanied by mucosal atrophy , and were not observed after sensitization with non - gluten proteins . interestingly gluten - induced gut dysfunction was particularly notable in mice transgenic for the human celiac gene hla - dq8.57 atis , a group of wheat proteins that confer resistance of the grain to pests , are strong inducers of innate immune responses via tlr4 and via the myeloid differentiation factor 88-dependent and -independent pathway.40 this activation occurs both in vitro and in vivo after oral ingestion of purified atis or gluten , while gluten - free cereals display no or minimal activities.61 the role of atis in ibs is not yet known , however there is clear description of a mechanism that could be involved in the generation of gut dysfunction and symptoms . these mechanisms are different from those proposed for gluten and thus it is conceivable that they could co - exist in given patients or have a synergistic effect . ibs and celiac disease are common conditions , and they may overlap by chance . however , celiac disease is 34 times more common in ibs patients compared to controls suggesting that both disorders could be mechanistically associated.62 active screening by celiac serology and biopsy reveals that 4% of patients labeled as ibs have underlying celiac disease.6265 this overlap between the 2 conditions is different from the issue of wheat components , including gluten , causing functional symptoms in patients without celiac disease.39 clinical studies have revealed that a subgroup of ibs patients in whom celiac disease and wheat allergy were ruled out , displayed intestinal and extra - intestinal symptoms after wheat ingestion . three consensus conferences defined this new syndrome as non - celiac gluten sensitivity ( ncgs ) or , alternatively , non - celiac wheat sensitivity ( ncws ) given the possibility for immune reactions or intolerances to other wheat components as explained above.6668 it is important to bear in mind the complexity of food hypersensitivities and intolerances , many of which could coexist in the same patient . this constitutes a confounder that we must be aware of for the design and interpretation of clinical trials.69 the prevalence of ncgs / ncws is still not clearly established mainly due to the hitch in standardizing international diagnostic criteria . in united states this condition seems to be identified more frequently in tertiary referral centers than in primary care . data obtained in primary care practice , from the national health and nutrition examination survey ( nhanes ) , indicate that the estimated prevalence of ncgs / ncws was 0.6% over 7762 subjects.70 on the other hand , data published from the celiac disease center in baltimore ( university of maryland ) , showed a prevalence of suspected ncgs / ncws of 6% over 5896 subjects.66 moreover , an italian multicenter study prospectively evaluated the prevalence of ncgs / ncws and celiac disease in pediatric and adult centers for gluten related disorders.71 among 12 255 subjects consecutively investigated during a one - year survey , 391 ( 3.2% ) cases of suspected ncgs / ncws and 340 ( 2.8% ) celiac patients were identified . based on these results , it is possible to estimate a ratio between ncgs / ncws and celiac disease of 1.15:1 , data in line with the nhanes survey . although ncgs / ncws can occur at any age , this condition seems to occur more frequently in adulthood with a mean age at diagnosis of 40 years . the female gender , similarly to ibs , is more affected by this sensitivity with a ratio up to 5:1.71 some clinical trials have attempted to investigate underlying functional and immune abnormalities in patients with a clinical picture of ncgs / ncws . initial results showed that there is increased expression of tlr2 in the small intestine of patients with ncgs/ ncws.49 this was in contrast with the normal expression of il-17a , il-6 , ifn- , il-17 , and il-21 in the duodenal mucosa of these patients , which seemed to rule out the contribution of adaptive immunity.72 recent work has suggested increased ifn- levels in small intestinal biopsies of ncgs / ncws patients following a short - term gluten challenge.73 although this cytokine is a key mediator of t - helper 1 adaptive immune responses , it could also be produced by innate cells such as intraepithelial lymphocytes ( iels).73 another relevant pathogenic aspect that has been investigated in ncgs / ncws pertains to possible functional changes , such as the increase of intestinal permeability . using the lactulose / mannitol test , ibs patients with self - reported sensitivity to gluten did not exhibit significant alterations in sugar permeability.49 this may be related to the population involved or technical difficulties in the interpretation of clinical permeability tests . however , in contrast to this study , vazquez - roque et al,52 using the same method , found increased intestinal permeability in a subgroup of hla - dq2/dq8 + ncgs/ ncws patients with ibs - d , once again raising the possibility of a particularly vulnerable subpopulation . this underscores the importance of careful patient phenotyping in studies involving functional symptoms and adverse reactions to food components . it also raises the concept of a subgroup of genetically predisposed individuals carrying celiac markers , but without active celiac disease , that may be more sensitive to low - grade inflammation or functional changes induced by gluten . an initial study enrolling ibs patients fulfilling criteria for ncgs / ncws showed symptom recurrence following gluten reintroduction.48 in a second trial from the same group,51 ibs patients that responded to a gluten - free diet were challenged with low dose of gluten ( 2 g / day ) , high dose of gluten ( 16 g / day ) or whey protein ( 16 g / day ) after 2 weeks of low dose fodmaps diet . it is worthwhile to note that patients increased their symptoms with all challenges , including placebo , and that gluten challenge did not show a dose response . carroccio et al50 demonstrated that ibs patients randomized to receive whole wheat vs placebo , had a significant worsening of their intestinal and extra - intestinal symptoms after wheat ingestion . a recent dbpcc trial used pure gluten vs rice starch ( control)-containing capsules to clarify the exact role played by gluten in symptom generation in patients with highly suspected ncgs / ncws.54 the results showed that pure gluten ingestion induced recurrence of a variety of symptoms including , bloating , abdominal pain , foggy mind , aphthous stomatitis , headache and depression , but not in all of ncgs patients . two recent dbpcc trials55,56 performed in gluten / wheat sensitive strongly suggested that gluten / wheat was responsible for symptom generation in up to one - third of ibs patients . overall the results indicated that gluten and other wheat proteins cause symptoms in the absence of celiac disease in a subset of the ibs population ( table 2 ) . the dilemma is that a gluten - free diet may also be low in fodmaps . some studies have assessed the efficacy of low - fodmaps diet in ibs with variable results ( table 3 ) . however , none of these trials included a placebo group , thus introducing a possible bias hampering the actual value of a low - fodmaps diet both in terms of diagnosis and as a therapeutic intervention . a recent dbpc trial ( without crossover ) comparing a low - fodmap diet with traditional dietary advice in ibs , showed no difference among the 2 strategies , thus renewing controversy of the specificity of therapeutic effect of fodmaps exclusion in the general ibs population.74 moreover , this study indicated that patients are more likely to react positively to fodmaps restriction , when already reducing fodmaps from their diet before initiation of the trial . finally , the data on the effect of fodmaps in ibs were pooled in three systematic reviews , and one of them included a meta - analysis . in the systematic reviews from rao et al75 and moayyedi et al76 the authors showed that the data could not be combined for meta - analysis and concluded that low - fodmaps is of uncertain benefit in ibs . in a third review,77 with less rigorous inclusion criteria , the authors were able to pool the data and found that low fodmap diet was effective in ibs . however , the results from this meta - analysis should be taken cautiously due to a great heterogeneity in population included and the comparator for the intervention ( table 3 ) . ncgs / ncws is often considered a self - diagnosis , usually reported by the patient . for the physician , it is based on the thorough evaluation of the clinical features according to the indications proposed by the consensus conferences on this syndrome.6668 identification of biomarkers that allow us to diagnose ncgs / ncws with more specificity and differentiate it from the unselected ibs population will be key to define and manage this condition.78,79 the clinical picture of patients with ncgs / ncws is characterized by symptoms occurring shortly after consumption of wheat/ gluten containing meals and disappearing or recurring in a few hours / days after specific withdrawal or challenge.6668 symptoms that characterize ibs , such as bloating , abdominal discomfort / pain , altered bowel habits and tiredness , are present in ncgs / ncws . it has been suggested that a clinical distinction can be made between general ibs and ncgs / ncws because of more extra - intestinal manifestations involving the central and/or peripheral nervous system , joint / muscle ( fibromyalgia - like ) and skin manifestations in the latter.71 due to the lack of biomarkers , the diagnosis of ncgs/ ncws remains highly presumptive being based only on clinical and exclusion criteria.80,81 the improvement of symptoms after a gluten - free diet , which is regarded as the major diagnostic criteria for ncgs / ncws , might be due to a placebo effect which often follows the elimination of some foods from the diet.82,83 in this context it is important to stress the negative potential influence ( nocebo effect ) generated by media on the deleterious effect of wheat consumption . there is no scientific evidence to support that gluten/ wheat consumption is deleterious to the overall population and such sensitivity seems to be limited to a subpopulation of patients that present with ibs symptoms . despite some disproportionate press on one hand , and some healthy skepticism on the other , there is no doubt that awareness and interest in ncgs / ncws continues to grow . antibodies to native gliadin ( aga ) have been suggested as a potential diagnostic marker for ncgs / ncws diagnosis , in the absence of specific celiac serology such as tissue transglutaminase , endomysial and deamidated gliadin antibodies.84 aga have been detected in the sera of about half of ncgs / ncws patients being predominantly of the igg class . although aga are not specific for ncgs / ncws being detectable in various conditions , ie , autoimmune disorders , connective tissue diseases and even in healthy controls , their positivity , especially at high titers , in patients with a clinical picture suggestive of gluten / wheat sensitivity may be regarded as a diagnostic adjunct . in parallel to symptom resolution , aga normalized in almost all patients with ncgs / ncws within 6 months of gluten - free diet.85 in cases with suspected ncgs / ncws , the most important clinical issue is to rule out celiac disease while the patient is on a gluten - containing diet . patients with ncgs / ncws have normal duodenal mucosal histology , although an increased number of iels ranging from 25 to 40/100 epithelial cells are found in at least 40% of cases , suggesting an accompanying low - grade inflammation.71 there is no increase of t - cell receptor / iels in biopsies of patients with ncgs / ncws . some studies found that hla - dq2 and/or hla - dq8 genes were present in ~50% of ncgs / ncws patients , which is slightly higher than in the general population.84 on the other hand , some studies have suggested that ibs patients who carry celiac susceptibility genes are more likely to respond to the gluten - free diet.52 this apparent controversy may be explained by the fact that overall the ncgs / ncws population may be heterogeneous and responsive to multiple stimuli . it could be speculated that those with celiac susceptibility genes may be more prone to develop mild immune responses and gut dysfunction to gluten . up to 20% of ncgs / ncws show mild laboratory abnormalities , such as low levels of ferritin , folic acid , vitamin d and b12 , most likely related to a minimal inflammatory state in the intestinal mucosa.86 evidence of osteopenia detected by bone densitometry has been found in about 50% of ncgs / ncws.87 finally , recent data reported a high prevalence of serum autoantibodies ( antinuclear antibodies , ana ) and a frequent association with autoimmune disorders ( ie , hashimoto s thyroiditis ) in patients with ncgs / ncws.88,89 no specific guidelines are yet available for the treatment of ibs patients with ncgs / ncws . gluten is a common ingredient of many food items and its complete removal is almost impossible to achieve . exposure to as little as 1050 mg of gluten ( a breadcrumb ) can cause intestinal lesions and symptoms in celiac patients , although the threshold for gluten tolerance is unknown in the ncgs / ncws population . moreover , there seems to be an individual level of tolerance in ncgs / ncws.6668 experts in the field recommend that investigations to rule out celiac disease and wheat allergy should be performed in people who consider themselves as gluten / wheat sensitive before starting a gluten - free diet.66 patients should be warned that an inappropriate dietary restriction can cause nutritional deficiencies . because gluten - rich grains are important sources of nutrients in the general diet , their exclusion could potentially have major effects on nutritional status . lower caloric9093 and fiber intake,90 but a higher intake of total and saturated fat91,92 was observed in the diet of celiac patients compared to healthy control subjects on a gluten containing diet . lower levels of folate , niacin , vitamin b12 , vitamin e , vitamin a , phosphorus , calcium , zinc , and selenium were described in the diet of celiac individuals compared to control subjects.9093 currently , it is still difficult to draw a conclusion on the nutritional adequacy of a gluten - free diet because of discordant results noticed from the studies regarding macro- and micro - nutrient intake . however , the evidence suggests that following a gluten - free diet may be detrimental if not properly evaluated and medically indicated . dietary restriction has been shown to affect the richness and composition of small intestinal and fecal microbiota , reducing beneficial bacterial groups such as firmicutes.94,95 it is necessary to consider that after any restriction diet , adaptations in gut physiology and microbial metabolism could increase sensitivity to the subsequent re - introduction of gluten / wheat or fodmaps . the persistence of symptoms after a period of gluten - free diet ( at least 6 weeks ) in patients with suspected ncgs / ncws suggests that other food sensitivities / intolerances may be responsible for symptom generation.37 in this respect , the patient may benefit from a low - fodmaps trial excluding rapidly absorbable carbohydrates such as those naturally contained in legumes , onions , honey , pears , water melon , dry fruits , fennel , and dairy products . foods can be triggers of gastrointestinal and extra - intestinal symptoms in a proportion of ibs patients . inside the spectrum of the so - called food hypersensitivity , ncgs / ncws has been recognized as a newly identified gluten - related disorder which presents clinically with overlapping symptoms of ibs.6668 along with gluten , other wheat and food components , have emerged as functional digestive symptom triggers . within wheat , a potential culprit of symptom generation is ati , a still poorly investigated soluble protein fraction of wheat.40 a role for fodmaps , detectable not only in gluten - containing cereals ( wheat , rye , and barley ) , but also in milk , honey , and legumes , has been proposed , although a recent dbpc trial did not find this restriction more efficient than traditional dietary advice for ibs patients.74 we have discussed the evidence for some specific dietary components , such as gluten and other wheat components , to cause functional digestive symptoms and extra - intestinal manifestations matching the current criteria for ibs . as with overall ibs , the mechanisms underlying symptom generation in the subgroup of patients with ncgs / ncws are still poorly understood . our review focused on clinical features with the intent to expand current knowledge in this area of gastroenterology . a better understanding of food hypersensitivity and intolerances as well as the development of biomarkers will enable physicians to design tailored dietary approaches to treat patients with food - related functional bowel disorders .
a tight link exists between dietary factors and irritable bowel syndrome ( ibs ) , one of the most common functional syndromes , characterized by abdominal pain / discomfort , bloating and alternating bowel habits . amongst the variety of foods potentially evoking food sensitivity , gluten and other wheat proteins including amylase trypsin inhibitors represent the culprits that recently have drawn the attention of the scientific community . therefore , a newly emerging condition termed non - celiac gluten sensitivity ( ncgs ) or non - celiac wheat sensitivity ( ncws ) is now well established in the clinical practice . notably , patients with ncgs / ncws have symptoms that mimic those present in ibs . the mechanisms by which gluten or other wheat proteins trigger symptoms are poorly understood and the lack of specific biomarkers hampers diagnosis of this condition . the present review aimed at providing an update to physicians and scientists regarding the following main topics : the experimental and clinical evidence on the role of gluten / wheat in ibs ; how to diagnose patients with functional symptoms attributable to gluten / wheat sensitivity ; the importance of double - blind placebo controlled cross - over trials as confirmatory assays of gluten / wheat sensitivity ; and finally , dietary measures for gluten / wheat sensitive patients . the analysis of current evidence proposes that gluten / wheat sensitivity can indeed represent a subset of the broad spectrum of patients with a clinical presentation of ibs .
Introduction Pathogenesis of Irritable Bowel Syndrome Dietary Factors in Irritable Bowel Syndrome Experimental Evidence for a Role of Wheat Components in Irritable Bowel Syndrome Clinical Evidence for a Role of Wheat Components in Irritable Bowel Syndrome Can We Diagnose Non-celiac Reactions to Wheat/Gluten? Dietary Options in Irritable Bowel Syndrome Conclusions
the latter concept has been challenged by growing evidence showing low - grade inflammatory changes in the gut and altered gut - brain axis signaling.1,2 according to the rome iii classification , fbds include the irritable bowel syndrome ( ibs ) , functional bloating ( fb ) , functional constipation , functional diarrhea , and unspecified fbd , and they are attributed to abnormalities likely originating from the small bowel , colon , and rectum.3,4 since fbds lack objective biomarkers , their diagnosis is based on the clinical symptoms reported by patients , physical examination , and the exclusion of alarm symptoms / signs ( eg , blood in stools , anemia , weight loss , and others ) . specifically , the reader will have an update on the role of gluten / wheat sensitivity as potential dietary triggers evoking gut dysfunction and symptoms in ibs . these are members of fermentable oligosaccharide , disaccharide , mono - saccharides , and polyols ( fodmaps ) that are poorly absorbed in the small intestine.37,40,42 these substrates are very important for gut health , supporting microbiota diversity and short chain fatty acid production43 but under certain circumstances could cause , through excessive bacterial colonic fermentation , symptoms such as bloating and altered bowel habits.19,44 regardless of the offending component , the withdrawal of wheat from the diet in a subgroup of patients with ibs has indeed been shown to improve symptoms.45 although unselected ibs patients display clinical improvement after wheat withdrawal , only a proportion may truly reflect underlying gluten sensitivity ( table 1).4656 the absence of specific biomarkers and the great proportion of patients with placebo effect observed in ibs trials , are 2 barriers for identifying the specific role of wheat components in generating ibs symptoms . in vitro studies have demonstrated that digests of gliadin increase the expression of co - stimulatory molecules and the production of proinflammatory cytokines in monocytes and dendritic cells.40,57,58 certain toxic ( that only stimulates the innate immune response ) gliadin - derived peptides such as the 3143mer , may evoke epithelial cell dysfunction , increased il-15 production and enterocyte apoptosis.59 recent studies have demonstrated increased expression of tlr-2 in the intestinal mucosa of non - celiac compared to celiac patients , suggesting a role of the innate immune system in the pathogenesis of non - celiac reactions to gluten or other wheat components.49 other studies have shown that monocytes from hla - dq2 + non - celiac individuals spontaneously release 23 fold more il-8 than monocytes from hla - dq2 negative patients . this suggests that patients without celiac disease ( no enteropathy and negative specific serology ) , but with positive hla - dq2 status , may represent a subpopulation reacting mildly to gluten.60 in terms of gut dysfunction , gluten sensitization in mice has been shown to induce acetylcholine release , one of the main excitatory neurotransmitters in the gut , from the myenteric plexus.57 this correlates with increased smooth muscle contractility and a hypersecretory status with increased ion transport and water movements.57 these functional effects induced by gluten were not accompanied by mucosal atrophy , and were not observed after sensitization with non - gluten proteins . three consensus conferences defined this new syndrome as non - celiac gluten sensitivity ( ncgs ) or , alternatively , non - celiac wheat sensitivity ( ncws ) given the possibility for immune reactions or intolerances to other wheat components as explained above.6668 it is important to bear in mind the complexity of food hypersensitivities and intolerances , many of which could coexist in the same patient . overall the results indicated that gluten and other wheat proteins cause symptoms in the absence of celiac disease in a subset of the ibs population ( table 2 ) . for the physician , it is based on the thorough evaluation of the clinical features according to the indications proposed by the consensus conferences on this syndrome.6668 identification of biomarkers that allow us to diagnose ncgs / ncws with more specificity and differentiate it from the unselected ibs population will be key to define and manage this condition.78,79 the clinical picture of patients with ncgs / ncws is characterized by symptoms occurring shortly after consumption of wheat/ gluten containing meals and disappearing or recurring in a few hours / days after specific withdrawal or challenge.6668 symptoms that characterize ibs , such as bloating , abdominal discomfort / pain , altered bowel habits and tiredness , are present in ncgs / ncws . although aga are not specific for ncgs / ncws being detectable in various conditions , ie , autoimmune disorders , connective tissue diseases and even in healthy controls , their positivity , especially at high titers , in patients with a clinical picture suggestive of gluten / wheat sensitivity may be regarded as a diagnostic adjunct . as with overall ibs , the mechanisms underlying symptom generation in the subgroup of patients with ncgs / ncws are still poorly understood .
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medical research has increased greatly in many developing countries during the recent decade , motivated by the need to improve health in these countries . since medical research involves human participants , such research needs to be guided by fundamental ethical principles to ensure the protection of their rights and welfare . furthermore , international standards mandate the review of research by research ethics committees ( recs ) [ 2 , 3 ] . however , research regulations do not exist in many developing countries , and commentators have expressed concerns regarding the extent of individual and institutional research ethics capacity , including the existence of functioning ethics review systems [ 4 , 5 ] . accordingly , several studies have demonstrated that research ethics review is not optimal in the developing world , including the middle east . for example , abou - zeid and colleagues found that 28% of researchers in the middle east region did not obtain ethical clearance for their research proposals submitted for funding [ 6 , 7 ] . in many of these proposals these investigators also showed that many basic elements of informed consent were omitted from the submitted informed consent forms . concerns have also been expressed regarding the capacity building efforts for recs as well as the challenges that prevent the optimal functioning of recs [ 1014 ] . for example , sleem and colleagues showed that barriers to the effective functioning of recs in egypt include insufficient training of members , lack of diverse membership , and limited resources . less than optimal functioning recs have been highlighted by recent research - related scandals with occasionally tragic consequences [ 15 , 16 ] . these results demonstrating less than optimal individual and institutional research ethics capacity may be explained by the relative novelty of research ethics regulations and the recent requirement of ethics review of research in the developing world , including the middle east . as such , little is also known regarding academics ' attitudes towards recs , their practices in research ethics ( e.g. , informed consent ) , and training opportunities in research ethics . recently , asem and colleagues assessed the knowledge and attitudes of the faculty at cairo university towards research informed consent . their results showed that many academics lacked training in research ethics and that their attitudes towards several practices in the research setting were not optimal . however , these investigators also showed acceptance of the faculty towards the establishment of recs and a desire for educational programs in research ethics . the field of dentistry is committed to ongoing research investigating the causes and treatment of dental diseases and adheres to the same ethical standards embraced by the fields of medicine . however , little research has investigated the attitudes of dental faculty towards concepts of research ethics , including the acceptability of recs and their desire for training in research ethics . recently , commentators have expressed concerns in dental research related to aspects of scientific misconduct [ 19 , 20 ] . accordingly , our objectives were to assess the knowledge , awareness , and attitudes of dental faculty regarding recs and training in research ethics , as well as potential independent variables associated with our findings . our results will help institutional officials understand better how well recs are accepted in their institutions and also help them develop relevant educational programs in research ethics directed towards dental faculty . we conducted a cross - sectional survey study performed during the period between april and june 2007 . we recruited members of the dental faculty ( demonstrators , assistant lecturers , lecturers , assistant professors , associate professors , and professors ) at king abdulaziz university ( kau ) , jeddah , saudi arabia and at ain shams university ( asu ) , cairo , egypt . defined as faculty with a status of demonstrators or assistant lecturers at asu and those with a status of demonstrators or lecturers at kau . demonstrators at both asu and kau cared for patients under supervision , worked on their research projects for their master 's theses , and assisted upper level faculty members in students ' clinical sessions . assistant lecturers at asu and lecturers at kau held similar academic roles ( the rank of assistant lecturers did not exist at kau ) : both had obtained their master 's degree , worked on their research projects for their phds , and attended all students ' clinical sessions . defined as faculty with a status of lecturers and assistant professors at asu and a status of assistant and associate professors at kau . lecturers at asu and assistant professors at kau held similar academic roles : both faculty types had obtained their phds , were cosupervisors for students ' theses , worked on their research projects for their promotions , and gave lectures to undergraduate students . assistant professors at asu and associate professors at kau held similar academic roles ( the rank of associate professor did not exist at asu ) : both worked on their research projects for their promotions , supervised theses , and gave lectures to both under- and postgraduate students . defined as faculty who achieved the status of professors . at both universities , professors conducted and supervised research projects / theses and gave lectures to both under- and postgraduate students . ain shams university and king abdulaziz university are among the most important universities in their respective countries . king abdulaziz university was founded in 1967 and its faculty of dentistry and its four departments ( oral basic and clinical sciences , oral / maxillofacial rehabilitation , preventive dental sciences , and conservative dental sciences ) were established in 1985 . at king abdulaziz university , one of the coauthors h. f. el - dessouky , distributed the surveys by placing them into the mailboxes of the faculty in the dental school . at ain shams university , another coauthor r. a. fadl , faculty was asked to return the surveys back anonymously by placing it in a general mailbox in the department 's office . we developed a questionnaire based on our study objectives , taking guidance from the previous literature regarding research ethics in the developing world . the first part collected demographic information of the participants : age , gender , academic position , prior participation in human subjects research ( e.g. , research involving human subjects and/or human biological samples ) , number of research projects involved in , and prior training in research ethics ( e.g. , having attended a course or a workshop in research ethics ) . we did not ask for any details regarding any courses or workshops the faculty had attended . the second part of the survey assessed the participants ' self - awareness of research ethics principles and functions of research ethics committees . are you familiar with ethical principles that govern conducting research involving human subjects ? are you fully aware of the functions of ethics committees ? the first part of the knowledge section consisted of several case scenarios involving the ethics of clinical research in dentistry and asking the respondents to answer questions based on these cases . case 1 ( informed consent describing risks and benefits ) thirty patients from the outpatient clinic of the faculty of dentistry were enrolled in a study that aimed to evaluate the flexible denture base material as compared with conventional denture base . one of the most serious disadvantages of the resilient denture liners is colonization and infection of the material surface by candida albicans . an oral consent has been taken from the patients without full description of the risks and benefits . which of the following best describes obligations of informed consent?the investigators can conduct the research without any ethical responsibility.a written consent with a brief description of the procedures must be taken.a full description of the risks and benefits should be stated in the informed consent.there is no need for informed consent , as the patients were enrolled from the outpatient clinic . thirty patients from the outpatient clinic of the faculty of dentistry were enrolled in a study that aimed to evaluate the flexible denture base material as compared with conventional denture base . one of the most serious disadvantages of the resilient denture liners is colonization and infection of the material surface by candida albicans . an oral consent has been taken from the patients without full description of the risks and benefits . which of the following best describes obligations of informed consent?the investigators can conduct the research without any ethical responsibility.a written consent with a brief description of the procedures must be taken.a full description of the risks and benefits should be stated in the informed consent.there is no need for informed consent , as the patients were enrolled from the outpatient clinic . the investigators can conduct the research without any ethical responsibility . a written consent with a brief description of the procedures must be taken . a full description of the risks and benefits should be stated in the informed consent . there is no need for informed consent , as the patients were enrolled from the outpatient clinic . case 2 ( research involving children)one hundred children of both sexes , age range from 6 to 15 years , were randomly selected from the outpatient clinic of the faculty of dentistry . one group will have their extensively carious teeth extracted , while the other group will go through pulpotomy in an attempt to keep the tooth as long as possible in their mouth.a clear description of the procedure should be explained to the child 's parent / guardian.an assent ( oral approval ) should be taken from the child.an assent should be taken from the child as well as a written informed consent from the child 's parent / guardian.no need to have an assent or consent as the children were already enrolled in the outpatient clinic and ready to receive any type of treatment . one hundred children of both sexes , age range from 6 to 15 years , were randomly selected from the outpatient clinic of the faculty of dentistry . one group will have their extensively carious teeth extracted , while the other group will go through pulpotomy in an attempt to keep the tooth as long as possible in their mouth.a clear description of the procedure should be explained to the child 's parent / guardian.an assent ( oral approval ) should be taken from the child.an assent should be taken from the child as well as a written informed consent from the child 's parent / guardian.no need to have an assent or consent as the children were already enrolled in the outpatient clinic and ready to receive any type of treatment . a clear description of the procedure should be explained to the child 's parent / guardian . an assent should be taken from the child as well as a written informed consent from the child 's parent / guardian . no need to have an assent or consent as the children were already enrolled in the outpatient clinic and ready to receive any type of treatment . case 3 ( retrospective research on stored samples originally collected for clinical purposes)fifty patients from the outpatient clinic of the faculty of dentistry were diagnosed as having lichen planus . biopsies were taken from the patients after their approval to confirm the clinical diagnosis ( patients were not charged any money ) . a month later , a research on lichen planus is planned by the faculty involving all biopsies that were previously obtained from the patients . this research can not be done without the approval of the patients.the biopsies belong to the faculty of dentistry , so no patient approval is needed.it is for the researchers to decide whether to take the patient 's consent or not.it is up to the dean or head of department to decide what to do with the biopsies without patient 's interference . fifty patients from the outpatient clinic of the faculty of dentistry were diagnosed as having lichen planus . biopsies were taken from the patients after their approval to confirm the clinical diagnosis ( patients were not charged any money ) . a month later , a research on lichen planus is planned by the faculty involving all biopsies that were previously obtained from the patients . this research can not be done without the approval of the patients.the biopsies belong to the faculty of dentistry , so no patient approval is needed.it is for the researchers to decide whether to take the patient 's consent or not.it is up to the dean or head of department to decide what to do with the biopsies without patient 's interference . the biopsies belong to the faculty of dentistry , so no patient approval is needed . it is for the researchers to decide whether to take the patient 's consent or not . it is up to the dean or head of department to decide what to do with the biopsies without patient 's interference . case 4 ( confidentiality in medical research)eighty patients from the outpatient clinic were enrolled in a research . the aim of the research was to differentiate between two different treatment modalities in the management of periodontal intraosseous bony defects . patients ' research files should be coded to ensure patients ' confidentiality.no need for confidentiality as the procedures are common in dental practice.it is left to the investigator to decide whether to keep the research data confidential or not.the dean or head of the department is the one to decide regarding the provisions of confidentiality . the correct answer for cases 1 and 2 is c , while for cases 3 and 4 the correct answer is a.these answers were based on concepts of research ethics drawn from research ethics guidelines . the second part of the knowledge section consisted of the following two questions . which of the following are considered guidelines in research ethics ? nuremberg code , declaration of helsinki , belmont report , council of the international organizations of the medical sciences ( cioms ) , orall of the above . review the ethical aspects of the research , determine whether informed consent is needed , review the scientific design of the research , protect the welfare and rights of the subjects in the research , ormake research more difficult to perform , other . 1 and either ( a , b , c , d ) or ( a , b , and d ) for question no . 2 , because both universities in this study have scientific committees that review the scientific design of the research , and therefore , it is conceivable that faculty might have thought that their rec does not review the scientific design of the research . the cioms guidelines recognize this possibility , as they state in their guidelines that a research ethics committee must either carry out or arrange for a proper scientific review or verify that a competent expert body has determined that the research is scientifically sound . respondents were required to choose from a 5-point likert scale ranging from 1 to 5 ( 1-strongly agree , 2-agree , 3-not sure , 4-disagree and 5-strongly disagree ) . the fifth part of the questionnaire assessed respondents ' attitudes towards certain practices in the conduct of research . these practices included those involved with informed consent , enrolment of vulnerable individuals , confidentiality , and responsible conduct of research . respondents were required to answer yes , no , or uncertain . the aim of the research was to differentiate between two different treatment modalities in the management of periodontal intraosseous bony defects . patients ' research files should be coded to ensure patients ' confidentiality.no need for confidentiality as the procedures are common in dental practice.it is left to the investigator to decide whether to keep the research data confidential or not.the dean or head of the department is the one to decide regarding the provisions of confidentiality . patients ' research files should be coded to ensure patients ' confidentiality . no need for confidentiality as the procedures are common in dental practice . it is left to the investigator to decide whether to keep the research data confidential or not . the dean or head of the department is the one to decide regarding the provisions of confidentiality . the correct answer for cases 1 and 2 is c , while for cases 3 and 4 the correct answer is a. these answers were based on concepts of research ethics drawn from research ethics guidelines . the second part of the knowledge section consisted of the following two questions . which of the following are considered guidelines in research ethics ? nuremberg code , declaration of helsinki , belmont report , council of the international organizations of the medical sciences ( cioms ) , orall of the above . what do you think is the role of a research ethics committee ? review the ethical aspects of the research , determine whether informed consent is needed , review the scientific design of the research , protect the welfare and rights of the subjects in the research , ormake research more difficult to perform , other . which of the following are considered guidelines in research ethics ? nuremberg code , declaration of helsinki , belmont report , council of the international organizations of the medical sciences ( cioms ) , orall of the above . declaration of helsinki , council of the international organizations of the medical sciences ( cioms ) , or what do you think is the role of a research ethics committee ? review the ethical aspects of the research , determine whether informed consent is needed , review the scientific design of the research , protect the welfare and rights of the subjects in the research , ormake research more difficult to perform , other . review the ethical aspects of the research , determine whether informed consent is needed , review the scientific design of the research , protect the welfare and rights of the subjects in the research , or make research more difficult to perform , correct answers were e for question no . 1 and either ( a , b , c , d ) or ( a , b , and d ) for question no . 2 , because both universities in this study have scientific committees that review the scientific design of the research , and therefore , it is conceivable that faculty might have thought that their rec does not review the scientific design of the research . the cioms guidelines recognize this possibility , as they state in their guidelines that a research ethics committee must either carry out or arrange for a proper scientific review or verify that a competent expert body has determined that the research is scientifically sound . . respondents were required to choose from a 5-point likert scale ranging from 1 to 5 ( 1-strongly agree , 2-agree , 3-not sure , 4-disagree and 5-strongly disagree ) . the fifth part of the questionnaire assessed respondents ' attitudes towards certain practices in the conduct of research . these practices included those involved with informed consent , enrolment of vulnerable individuals , confidentiality , and responsible conduct of research . we entered data from completed questionnaires into microsoft excel and then converted it to spss version 13.0 ( statistical package for social sciences , 2009 ) . for purposes of analysis , we collapsed the categories of strongly agree and agree . we report in percentages the positive responses of the available choices ( yes as opposed to no and do not know and the strongly agree and agree responses for those questions employing a likert scale ) . we used chi - square tests to determine , in bivariate analyses , the association of each of the independent variables ( gender , academic position , prior ethics training , and prior involvement with research ) with each of the main outcome of interest ( dependent responses involving knowledge , awareness , and attitudes ) . we used covariates that were significant in a multivariate logistic regression analysis to determine independent predictors ( covariates ) of the dependent responses . we report in the text the odds ratios and confidence intervals for those associations that were significant . this study was approved by the recs at king abdulaziz university , ain shams university , and the university of maryland . potential participants received a cover letter attached to the survey tool that included the following elements of informed consent : the purpose of the research study , potential benefits and risks , and that participation was voluntary and refusal to participate would not be associated with any academic penalty . to ensure anonymity , the recs waived the requirement of signed written consent ; completion of the survey implied participants ' provision of informed consent . of the 100 questionnaires distributed at each university ( total of 200 ) , we received responses from 125 individuals . there were 75 surveys from king abdul aziz university ( kau ) and 50 from ain shams university ( asu ) , representing response rates of 50% and 75% , respectively , and a total response rate of 62.5% . table 1(a ) shows the demographic data of the faculty from the universities , both separately and combined . there were no significant differences between the two universities regarding the demographic data . the combined results showed that there were slightly more women faculty compared to men ( 55.2% versus 43.2% ) , and there was an almost even distribution among the different faculty types . regarding prior research experience , almost three quarters of the respondents from both universities ( 71.2% ) had performed research involving human subjects or human tissue samples or both . regarding prior ethics training , a minority ( 36.8% ) of the respondents had prior training in ethics , whereas a majority ( 63.2% ) had no such prior training . of those faculty who received ethics training , 16 ( 35% ) had attended both a course and a workshop , whereas 30 ( 65% ) had attended either a course or a workshop , but not both ( data not shown in table ) . table 1(b ) shows the association between gender and academic position with prior research experience ( percentage of faculty performing research and number of projects / faculty ) and prior ethics training . research experience was higher with men compared with women , both in terms of the percentage of faculty ( 85.2% versus 61.2% , p < .01 ) and in the mean number of research projects per faculty ( 7.56 versus 3.31 , p < .01 ) . regarding research experience , while the percentage of faculty performing human subjects research was similar among the different faculty types , the mean number of research projects per faculty was higher for the more senior faculty . faculty at asu and kau performed similar mean number of projects per faculty ( 5.34 versus 5.20 projects / faculty , p = ns ; data not shown in table ) . regarding prior ethics training , men and women showed similar percentages , while there was a significantly higher percentage of mid - level faculty who had prior ethics training ( 57.5% versus 28.2% and 26.2% for professors and juniors , the percentage of faculty with prior ethics training was similar between those with and without prior research experience involving human subjects ( 37.1% versus 32.4% , resp . table 2 shows the respondents ' responses regarding their awareness of research ethics principles and the functions of recs . less than half of the respondents stated that they were familiar with research ethical principles , and less than a third stated that they were familiar with the functions of recs . a higher percentage of faculty at kau compared with those at asu stated their familiarity with research ethics principles ( p < .05 ) . table 2 also shows the association between the responses and demographic ( independent ) variables . professors were significantly more likely to state that they were familiar with research ethics principles ( p < .01 ) ; mid - level faculty was significantly more likely to state they were familiar with the functions of an rec ( p < .01 ) . were significantly more likely to state they were familiar with research ethics principles and with the functions of recs compared with those who had no such prior training ( both p < .01 ) . there was a tendency for faculty with prior research experience involving human subjects to more likely state they were familiar with research ethics principles and functions of recs compared with faculty without such research experience , but these differences did not reach statistical significance . we performed a multiple logistic regression analysis to determine which independent variables were the strongest predictors of the responses in table 2 . our analysis showed that prior ethics training was a strong predictor for stating a familiarity with research ethics principles ( p < .001 , or 10.10 ; 95% ci , 2.4541.67 ) and for stating a familiarity with the functions of recs ( p < .001 ; or 5.95 ; 95% ci 2.4114.68 ) . 14 ) and questions ( items no . 5 and 6 ) that assess respondents ' knowledge in research ethics . more than half of the respondents gave the correct answer for the first case involving an informed consent issue , while less than half of the respondents gave correct answers for the other three case scenarios . a small number of respondents ( 12.0% ) knew the guidelines in research ethics ( item no . 5 ) and less than a third knew the roles of recs ( item no . the average score was 40.2% ; 15.2% of the respondents gave correct answers to at least five of the questions , while approximately half ( 56.0% ) knew the correct answers to at most two of the questions . table 3 also shows the association between the responses and the demographic ( independent ) variables . faculty at kau were significantly more likely to give the correct response to the 4th and 6th items ( both p < .05 ) . mid - level faculty were significantly more likely to give the correct answers to several of the knowledge questions ( 2nd , 3rd , and 6th items ; p < .01 , p < .01 , and p < .05 , resp . ) compared with the other faculty types . there was a tendency for faculty with any prior ethics training or prior research experience to more likely give correct answers to all of the questions compared with those without prior ethics training or prior research experience ; but these differences only reached statistical significance for item no . 5 for faculty with prior ethics training ( p < .01 ) we performed a multiple logistic regression analysis to determine which independent variables were the strongest predictors of the responses in table 3 . our analysis showed that mid - level faculty was a strong predictor of knowing the correct responses to the 2nd and 3rd cases ; ( both p < .001 ; or 8.62 ; 95% ci 2.8725.64 ; or 10.75 ; 95% ci 3.1637.03 ; resp . ) ; and that prior ethics training was a strong predictor for knowing the guidelines in research ethics ( item no . 5 ; p < .001 ; or 10.55 ; 95% ci , 2.6042.86 ) . regarding the overall score , faculty at kau compared with those at asu achieved a higher average score ( 47.0% versus 37.0% , p < .01 ) , whereas mid - level faculty had a higher average score compared with that obtained by the professor and junior faculty ( 55.8% versus 33.8% and 32.2% , resp . , p < .01 ) . kau faculty , mid - level faculty , and those with prior ethics training were significantly more likely to give correct answers to at least five of the questions ( p < .01 , p < .01 , and p < .05 , resp . ) . logistic regression revealed that kau faculty and mid - level faculty were strong predictors for knowing the correct answers to at least five of the questions ( p < .05 ; or 4.69 ; 95% ci , 1.1319.53 ; p < .01 ; or 9.52 ; 95% ci , 1.6655.56 , resp . ) . table 4 shows the respondents ' attitudes to recs and research ethics education . greater than 90% of the respondents agreed that an rec would be helpful , there is a need for an rec in each institution , and that human subject research must be reviewed by an rec . furthermore , less than 20% believed that ethical review is only necessary for international collaborative research and less than 10% thought that the presence of scientific committees made the existence of an rec unnecessary . however , almost half ( 44% ) thought that recs would delay research and would make research harder to perform . a large majority of the respondents ( greater than 90% ) were in favor of research ethics education for postgraduates , investigators , and members of recs . table 4 also shows the association between these attitudes and the demographic variables . of note , those without any prior ethics training were significantly more likely to think that an rec would be helpful ( p < .01 ) . were significantly more likely to believe that an rec would delay research ( p < .05 ) . professors compared with the other faculty were significantly more likely to agree that ethical review of research is only necessary for international research ( p < .01 ) . multiple logistic regression analysis showed that none of the independent variables were strong predictors for any of these attitudes . a significant majority of the respondents ( > 90% ) believed in the need for confidentiality protections of research participants ' data ( question no.1 ) . a large majority ( > 85% ) also held strong opinions regarding the importance of informed consent , as indicated by their responses to questions no . less than 10% believed that patients should not be told about the risks of research because they may not enroll in the study . however , almost a third of the respondents thought it was not necessary to obtain research informed consent for blood samples that were obtained for clinical tests ( question no . 7 ) . almost 40% of the respondents thought that vulnerable groups such as children and the mentally ill could provide informed consent ( question no . 8) . a small minority ( < 10% ) of the respondents thought that if surrogates are not available to give informed consent for vulnerable individuals , it would still be proper to enroll such individuals in research ( item no . slightly more than 10% of the respondents thought it is proper to fabricate data to improve the outcome of the research if such an act did not cause harms to patients ( question no . table 5 also shows the association between these attitudes and the demographic variables . of note , men and those with prior research experience were significantly more likely to agree that vulnerable groups could provide informed consent ( both p < .01 ) . research informed consent is not necessary for blood samples obtained for clinical test and that it is okay to fabricate data prior research experience was a strong predictor for agreeing that vulnerable groups could provide informed consent ( p < .01 ; or 4.55 ; 95% ci 1.5413.41 ) . this survey study showed several key findings that should be of interest to educators and policy makers . first , the surveyed dental faculty indicated a high endorsement for the existence of recs , as most thought that such committees should review research , that they would be helpful , and that they should exist in universities . previous studies observed similar results regarding the acceptance of recs among academics in sudan and egypt [ 17 , 23 ] . however , while the faculty in this study endorsed the existence of recs , almost half of them held the opinion that such committees would delay research and make it more difficult to perform research . commentators from western countries , where recs have been in existence for more than twenty years , have recently written about concerns with excessive bureaucratic details that cause costly delays in research approval [ 2426 ] . in our study , respondents who are more likely to harbor the belief regarding delays in research by recs were mid - level faculty and those with prior ethics training . first , faculty , in general , might not understand the extent of the processes needed for an adequate review of research . indeed , less than a third of all of the respondents stated their familiarity with the functions of recs , underscoring that efforts are needed to enhance faculty awareness of the operations of the recs . second , the finding that faculty with prior ethics training were more likely to believe that recs would delay research raises the question as to whether the prior training gave a mistaken impression about recs by emphasizing the many processes of recs without stressing the benefits of rec review . third , that mid - level faculty was more likely to believe that recs would delay research can be explained by them having had more unfavorable experiences with their recs . indeed , mid - level faculty was shown to have conducted more human subjects research projects than the junior faculty and might have had more interactions with recs compared with professors , who probably did the majority of their research when recs were not in existence . finally , it is possible that these results regarding the associations between mid - level faculty and prior ethics training with the belief that recs delay research might represent false positives , since these independent variables were not significant on multiple logistic testing . future qualitative research involving in - depth interviews is warranted to further explore the basis of faculty attitudes regarding the process of research review by recs . our survey also yielded interesting results regarding the attitudes of the dental faculty towards certain research ethics practices . a large majority of the faculty appears to be aware of the accepted practices regarding confidentiality protections and several aspects regarding the informed consent process . these results contrast with the concerns mentioned regarding informed consent practices by commentators working in egypt . however , approximately a third of the respondents held the attitude that research performed on blood samples obtained for clinical purposes do not require informed consent . professors were more likely to agree with this practice compared with the other faculty . asem and colleagues observed similar results regarding this issue among the faculty at cairo university . another concern we discovered regarding informed consent is that almost 40% of the faculty believed that certain vulnerable subjects ( e.g. , children and the mentally ill ) could provide informed consent to participate in research . this result raises the underlying issue of how informed is informed consent for subjects who might lack decision making capacity . this result is made more significant by the findings of another study showing that research participants who participated in studies on oral health in nigeria had poor understanding of several key elements of the informed consent process . accordingly , we suggest future training efforts for dental faculty to be focused on informed consent issues , including those issues involved with concepts of understanding and vulnerability . a small but significant minority ( approximately 10% ) thought it is permissible to fabricate data and professors were more likely to hold this opinion compared with the other faculty types . for example , eastwood and colleagues found that approximately 2% of respondents were willing to fabricate data for a grant application or a paper , whereas about 27% would be willing to select or omit data to improve the results . similarly , a study of biomedical trainees showed that 15% admitted of personal misconduct , and they were willing to fabricate , select , or omit data for publishing a paper or obtaining a research grant . finally , a recent meta - analysis of surveys involving scientists ' self - report of research misconduct yielded a pooled weighted estimate of almost 2% of the scientists replying that they had fabricated , falsified , or modified data at least once , with a range from 0.3% to 4.9% . these numbers reflecting self - report are lower than those in the previously mentioned studies reporting what individuals would be willing to do , thus highlighting the difference between perception and actual practice . finally , martinson and colleagues also showed that scientists late in their careers admitted to fabrication at rate higher than those early in their careers . reasons that may account for differences between the different faculty levels regarding fabrication include ( a ) senior faculty have had more opportunities to engage in misconduct , ( b ) perceptions of being caught might change during one 's career , and ( c ) senior faculty received their education and work habits at a time when there were different behavior standards . specifically , less than half ( 36.8% ) of the 125 respondents from both universities received prior training in research ethics . furthermore , the overall average score achieved on the knowledge questions was only 40.2% , and only about 15% of the faculty was able to give correct answers to at least five of the six questions . thus , lack of training and knowledge gaps on research ethics exist among the academic dental faculty . interestingly , while the variable prior ethics training was shown to be an independent predictor for awareness of ethical principles and the functions of recs , it was not an independent predictor for achieving a high score on the knowledge - type questions . these results raise the issue that current training programs in research ethics might be insufficient . interestingly , mid - level faculty compared with the other faculty types were more likely to score well on the knowledge - type questions . several reasons can be offered to explain these results regarding the mid - level faculty . first , as shown in table 1(b ) , mid - level faculty was more likely to have received prior ethics training compared with the other faculty types . however , as explained previously , prior ethics training was not an independent predictor for knowledge . but , ethics training coupled with ample research experience might have led these faculty to obtain the amount of theoretical and practical knowledge necessary to answer the questions on our survey . indeed , mid - level faculty had a combination of ethics training and research experience ( defined by number of projects / faculty ) that together was greater than observed for the other faculty types . alternatively , mid - level faculty might have had more recent opportunities to travel abroad for their academic education and thus had more exposures with research ethics issues from their experiences at these other universities . further research should probe for the factors that account for any differences in knowledge gaps between the different faculty types . although knowledge gaps exist in research ethics , our findings showed that all faculty levels were favorable towards research ethics training for postgraduates , investigators , and rec members . previous studies have had mixed results regarding the effects of ethics education on knowledge . a 3-day workshop in research ethics involving clinicians and scientists in a nigerian university improved participants ' knowledge and application of the principles of research ethics , international guidelines and regulations , and operations of recs . other investigators have shown similar findings regarding the effects of ethics training [ 3436 ] . however , other studies have demonstrated the lack of an effect of prior ethics instruction on the extent of research ethics knowledge [ 37 , 38 ] . also , previous studies have yielded inconclusive results regarding positive relationships between ethics education and moral conduct [ 29 , 30 , 3941 ] . these findings do not necessarily call into question the value of ethics education , but rather the quality of the educational experiences , as well as the long - range effects of short training in research ethics . further research is needed to determine the teaching methods ( e.g. , face - to - face or distance learning ) that are most effective in addressing the existing knowledge gaps in research ethics . first , our study was based on convenience sampling , thus the professionals who completed the survey may not reflect the awareness , knowledge , and attitudes of the entire membership of the dental faculty of the two universities . second , this study involved one university in each of two countries in the middle east , thus further limiting the generalizability of our results . future studies are warranted to investigate the knowledge , awareness , and attitudes of academics from other faculties in other universities . third , the knowledge type questions we used in our study do not reflect the broad range of topics in research ethics . however , the questions on the survey represent basic information that academics should be expected to know in the area of research ethics . finally , we did not obtain detailed information regarding the types of courses and workshops that the faculty had attended , as well as the methods of instructions used in their training . despite these limitations second , there seems to be an acceptance of the need for education in research ethics among all faculty levels . since , as revealed in our study , prior research ethics training was not an independent predictor for the extent of knowledge in research ethics , attention should be directed towards the type and extent of training needed to further enhance faculty knowledge on research ethics issues . we therefore recommend further development of educational instruction in research ethics for all university faculty , with special emphasis on vulnerable participants , responsible conduct of research , and the roles and functions of recs . recently , educational initiatives have been organized in many regions in the developing world [ 42 , 43 ] . such efforts can lead to enhanced knowledge and acceptance of research ethics principles among investigators . finally , we recommend qualitative studies to further explore the attitudes of faculty towards recs and certain practices in research ethics .
objective . to assess the knowledge , awareness , and attitudes of dental faculty regarding research ethics and research ethics committees ( recs ) . design . through convenience sampling , we distributed a survey to academics at dental faculties at two universities in the middle east . we used descriptive , chi - square , and logistic regression statistics to analyze the data . results . our response rate was 62.5% . a large majority ( > 90% ) held positive attitudes towards recs ; however , almost half ( 44.0% ) thought that recs would delay research . less than half ( 36.8% ) had received prior training in research ethics , and the average score they achieved on the questions on research ethics was only 40.2% . most ( > 90% ) , however , were favorable towards research ethics education . finally , some faculty held attitudes regarding certain research ethics practices that were not optimal . conclusions . we conclude that among the dental faculties participating in our study , there is broad - based acceptance of recs and training in research ethics , while there are knowledge gaps in research ethics . we recommend further studies to determine the generalizability of our findings to other institutions .
1. Introduction 2. Methods 3. Results 4. Discussion
furthermore , international standards mandate the review of research by research ethics committees ( recs ) [ 2 , 3 ] . accordingly , several studies have demonstrated that research ethics review is not optimal in the developing world , including the middle east . these results demonstrating less than optimal individual and institutional research ethics capacity may be explained by the relative novelty of research ethics regulations and the recent requirement of ethics review of research in the developing world , including the middle east . , informed consent ) , and training opportunities in research ethics . their results showed that many academics lacked training in research ethics and that their attitudes towards several practices in the research setting were not optimal . however , these investigators also showed acceptance of the faculty towards the establishment of recs and a desire for educational programs in research ethics . however , little research has investigated the attitudes of dental faculty towards concepts of research ethics , including the acceptability of recs and their desire for training in research ethics . accordingly , our objectives were to assess the knowledge , awareness , and attitudes of dental faculty regarding recs and training in research ethics , as well as potential independent variables associated with our findings . , research involving human subjects and/or human biological samples ) , number of research projects involved in , and prior training in research ethics ( e.g. we used chi - square tests to determine , in bivariate analyses , the association of each of the independent variables ( gender , academic position , prior ethics training , and prior involvement with research ) with each of the main outcome of interest ( dependent responses involving knowledge , awareness , and attitudes ) . regarding prior ethics training , a minority ( 36.8% ) of the respondents had prior training in ethics , whereas a majority ( 63.2% ) had no such prior training . the average score was 40.2% ; 15.2% of the respondents gave correct answers to at least five of the questions , while approximately half ( 56.0% ) knew the correct answers to at most two of the questions . however , almost half ( 44% ) thought that recs would delay research and would make research harder to perform . a large majority of the respondents ( greater than 90% ) were in favor of research ethics education for postgraduates , investigators , and members of recs . however , while the faculty in this study endorsed the existence of recs , almost half of them held the opinion that such committees would delay research and make it more difficult to perform research . second , the finding that faculty with prior ethics training were more likely to believe that recs would delay research raises the question as to whether the prior training gave a mistaken impression about recs by emphasizing the many processes of recs without stressing the benefits of rec review . our survey also yielded interesting results regarding the attitudes of the dental faculty towards certain research ethics practices . specifically , less than half ( 36.8% ) of the 125 respondents from both universities received prior training in research ethics . furthermore , the overall average score achieved on the knowledge questions was only 40.2% , and only about 15% of the faculty was able to give correct answers to at least five of the six questions . thus , lack of training and knowledge gaps on research ethics exist among the academic dental faculty . interestingly , while the variable prior ethics training was shown to be an independent predictor for awareness of ethical principles and the functions of recs , it was not an independent predictor for achieving a high score on the knowledge - type questions . although knowledge gaps exist in research ethics , our findings showed that all faculty levels were favorable towards research ethics training for postgraduates , investigators , and rec members . a 3-day workshop in research ethics involving clinicians and scientists in a nigerian university improved participants ' knowledge and application of the principles of research ethics , international guidelines and regulations , and operations of recs . first , our study was based on convenience sampling , thus the professionals who completed the survey may not reflect the awareness , knowledge , and attitudes of the entire membership of the dental faculty of the two universities . second , this study involved one university in each of two countries in the middle east , thus further limiting the generalizability of our results . future studies are warranted to investigate the knowledge , awareness , and attitudes of academics from other faculties in other universities . third , the knowledge type questions we used in our study do not reflect the broad range of topics in research ethics . however , the questions on the survey represent basic information that academics should be expected to know in the area of research ethics . since , as revealed in our study , prior research ethics training was not an independent predictor for the extent of knowledge in research ethics , attention should be directed towards the type and extent of training needed to further enhance faculty knowledge on research ethics issues . we therefore recommend further development of educational instruction in research ethics for all university faculty , with special emphasis on vulnerable participants , responsible conduct of research , and the roles and functions of recs . finally , we recommend qualitative studies to further explore the attitudes of faculty towards recs and certain practices in research ethics .
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while the beneficial effects of exercise for physical and mental health are well recognized , average physical activity is below recommended levels , and it remains a challenge to support the adoption of active behaviours . virtual reality environments are increasingly being used to encourage individuals to exercise more regularly , for the prevention of sedentary lifestyles among relatively healthy individuals , including , and as part of treatment , in those with mental health or neurological disorders . when exercising in virtual environments for the benefit of mental health , there is a need to consider the theoretical sense of presence . sense of presence is defined as a perceived feeling of immersion when exposed to a virtual environment ; it is perfected when one is completely unaware of their real surroundings [ 47 ] . beyond subjective assessments to explore a sense of presence , there is increasing reliance on more objective physiological measurements , for example , heart rate monitoring . only a few studies have investigated the neural responses that underlie the sense of presence perception . there has been a particular focus on the neural activation patterns that accompany and may regulate the sense of presence during the process of habituation to virtual environments [ 9 , 10 ] . recently , frontal brain regions ( i.e. , dorsolateral prefrontal cortex ) were identified to form a key node for a sense of presence network ( spn ; ) . participants who are highly engaged and most attentive during imagination and movement representation studies show strong spn activation . electroencephalography ( eeg ) frequency analyses have also been used to explore the neural activation patterns associated with sense of presence perception . alterations in alpha oscillations , particularly over the frontoparietal brain regions , have been associated with perceived sense of presence . notwithstanding continuous debates on inconsistent findings , alpha and beta oscillations are also associated with mental state , for example , physical and psychological strain [ 10 , 13 ] . it has been shown that sense of presence perception depends largely on the characteristics of the virtual environment and differs with screen size , duration of exposure , and the realism of the presentation [ 10 , 1416 ] . whether the addition of a real exercise ( e.g. , cycling on an ergometer ) within an immersive virtual environment alters sense of presence perception , or the accompanying changes in neural activation patterns or mental state , is not known . in healthy participants , the beneficial effects of exercise on mental state and mental well - being are reflected by neurophysiological and behavioural adaptations [ 17 , 18 ] . these effects of exercise on mental well - being are suggested to be largely dependent on the volume or dose of exercise [ 19 , 20 ] and are also influenced by a preference bias towards self - paced ( moderate - intensity ) exercise . studies of brain activity and the alterations in alpha and beta oscillations with exercise suggest the presence of a general model of cortical arousal ( mca ; ) . according to this , a reduction in alpha activity and an increase in beta activity reflect cortical arousal ; and the reverse is associated with relaxed cortical states that may be recorded immediately after self - paced ( moderate - intensity ) exercise . the relationship between exercise - induced changes in mental state and neural activation patterns may also be described on the basis of the transient hypofrontality theory ( tht ; ) . transient hypofrontality would suggest that the eeg changes that accompany exercise reflect redistribution of limited cortical resources away from less required brain regions ( e.g. , decreased activity in frontal brain regions ) towards more required brain regions ( e.g. , increased activity in motor regions ) . thus , based upon the dominant role of frontal brain regions in mental state , this exercise - induced redistribution is in favour of well - being ( i.e. , decreased activity in frontal brain regions as a refreshment of mental state capacity ) . transient hypofrontality is particularly evident in response to self - paced ( moderate - intensity ) exercise , and similar responses are seen across a diverse range of participants including younger and older participants and those with and without a mental impairment [ 2426 ] . with this , combining traditional interpretations of alterations in eeg frequency bands ( i.e. , mca ) with the latest understanding of cortical redistribution ( i.e. , tht ) seems reasonable . it is not known whether these same neural responses occur during exercise or movement representation in a virtual environment , and this is important to establish if virtual environments are to be used to facilitate exercise as a therapy for mental well - being . therefore , the objective of this study was to investigate the interactive effects of virtual environment exposure and exercise on physiological and perceptual responses in healthy adults . specifically , we aimed to examine the influence of moderate - intensity exercise ( i.e. , self - paced cycling ) , compared with movement representation , within three levels of virtual environment exposure ( a complex three - screen mode , a simple one - screen mode , and a no - screen control ) on cortical neural oscillations ( i.e. , frontal alpha and beta activity ) , perceived sense of presence , and mental state ( i.e. , perceived physical state , motivational state , and psychological strain ) . participants were eighteen healthy volunteers ( 7 females , 11 males ) with no known history of neurological or musculoskeletal disorders ( age : 28.78 5.19 years ; height : 177.50 10.15 cm ; weight : 75.44 13.23 kg ) . participants considered themselves recreationally active with no particular experience of being exposed to virtual environments . this study was approved by the institutional human research ethics committee and was conducted in accordance with the declaration of helsinki . participants attended a single experimental test session , following familiarisation with the cycle ergometer and the test environment on the same day . experimental procedures were arranged in a randomized and controlled study design . after baseline measurements , exercise or no - exercise trials were conducted within three levels of virtual environment exposure . each trial was 5 minutes in duration and was followed by posttrial assessments of heart rate , perceived sense of presence , and eeg as well as mental state ( figure 1 ) . an immersion square system ( bonn - rhein - sieg university of applied sciences ; ) back projection ( 8 gb ram , amd radeon hd 5800 series ) on three screens ( 260.0 cm width , 195.0 cm height ) provided 4200 1050 pixel resolution ( each 1400 1050 pixels ) . screens were arranged in a square surrounding the participant 's visual field ( figure 2 ) . screen configuration allowed for three levels of virtual environment exposure : ( 1 ) one - screen mode ( osm ) with only the front screen switched on , ( 2 ) three - screen mode ( tsm ) with all three screens switched on , and ( 3 ) control with all screens switched off . during each trial , participants were seated on a fivis bicycle simulator ( bonn - rhein - sieg university of applied sciences ; ) . during both the osm and tsm trials , 3d content was identical and comprised an endless two - lane road ( straight ) in an urban setting . the 3d video display was synchronised with the actions of the bicycle , including steering ( i.e. , handlebar movement ) , velocity ( i.e. , pedal cadence and torque ) , and braking ( i.e. , backpedal or handbrake ) . with respect to dose [ 19 , 20 ] and preference , exercise trials were performed at a self - paced moderate intensity that was defined by consistent heart rate monitoring and verbal instructions prior to each exercise trial ( i.e. , please pace your pedalling consistently to meet your perceived moderate intensity ) . during no - exercise trials , participants sat on the cycle ergometer without moving and the virtual bicycle ( 3d video ) travelled the course at a fixed virtual speed of 22.0 km / h . heart rate was monitored using a polar rcx5 portable heart rate monitor ( polar electro oy , finland ) . average heart rate ( bpm ) at rest and during the period immediately after each trial was used for analysis . perceived sense of presence was assessed immediately after each trial using a simple verbal query / response - questionnaire [ 28 , 29 ] . participants were asked , without hesitating , how strongly do you feel connected to the virtual environment at this moment ? responses were anchored to an 11-step scale from 0 ( not at all ) to 10 ( totally ) . the bodyfinder module is sensitive to short - term mood alterations and has been developed to determine current perceived physical state ( peps ) based on the subdomains of physical energy , physical fitness , physical flexibility , and physical health . this module has been used and validated in various biomedical settings , including exercise physiology and internal medicine studies . the feelfinder module comprises a shortened version of the ez - scale ( eigenzustandsskala ; ) . in comparison to other psychological adjective scales ( e.g. , poms ) the ez - scale assesses motivational state ( mot ) , in addition to the commonly assessed psychological strain ( psych ) . this study used a 16-item ez - scale , comprising eight subdomains to generate assessments of mot ( self - confidence , willingness to seek contact , social acceptance , and readiness for strain ) and psych ( relaxation , positive mood , calmness , and recovery ) . for this study , the moodmeter was configured with 32 adjectives ( 16 peps , 8 mot , and 8 psych ) that were stored and presented to participants on a handheld axim x50 pocket pc ( dell , usa ) in a quasi - random sequence . upon the presentation of each adjective , participants were asked to indicate how well that adjective described their current physical or mental state by selecting one of six options from 0 ( not at all ) to 5 ( totally ) . the response time for each adjective was limited to five seconds so as to discourage rational deliberation , and this time was shown with a progress bar at the bottom of the screen . with this , the moodmeter was specifically designed to detect short - term alterations that are of particular relevance for exercise - related research . on each occasion , completion of the mood assessment ( all 32 adjectives ) took less than two minutes . for further details on the development and operation of the moodmeter , please refer to kleinert . to record cortical neural oscillations , brainvision recorder 1.20.0701 together with a portable acticap system was used ( brainproducts gmbh , germany ) . a permeable - to - air eeg - cap that is adapted to individual head size the eeg - cap consisted of 64 ag / agcl electrodes , arranged in the international 10:20 system : fp1 , fp2 , af7 , af3 , af4 , af8 , f7 , f5 , f3 , f1 , fz , f2 , f4 , f6 , f8 , ft9 , ft7 , fc5 , fc3 , fc1 , fc2 , fc4 , fc6 , ft8 , ft10 , t7 , c5 , c3 , c1 , cz , c2 , c4 , c6 , t8 , tp9 , tp7 , cp5 , cp3 , cp1 , cpz , cp2 , cp4 , cp6 , tp8 , tp10 , p7 , p5 , p3 , p1 , pz , p2 , p4 , p6 , p8 , po7 , po3 , poz , po4 , po8 , o1 , oz , o2 , and po10 . fcz ( reference ) and afz ( ground ) were added and po9 served as horizontal electrooculogram ( eog ) to detect eye movements . supervisc electrode gel ( easycap gmbh , germany ) was added to each electrode to optimize conductivity . distances between electrodes were > 25 mm to avoid bridging ( though not measured , perspiration was not noticeably profuse in any participant ) . a 60-second resting eeg was recorded ( sampling rate of 500 hz ) with eyes closed , while seated on the cycle ergometer before ( rest ) and after each trial . analogue eeg data were amplified and digitally converted for analyses using brainvision analyser 2.1.0.327 ( brainproducts gmbh , germany ) . low and high cutoff filter frequencies ranged between 7.5 and 45.0 hz ( time constant 0.02 seconds , 48 db / octave ) . based on the eog , gratton 's standard ocular correction was performed to reduce eye - moving artifacts . after segmentation and an automatic artifact rejection ( gradient < 35 v , min / max amplitudes 100 v ) , a minimum of twelve 4-second segments remained for fast fourier transformation ( spectral analysis : resolution at 0.24 hz , hanning window of 10% ) . averaged segments were pooled into frontal ( fp1 , fp2 , af7 , af3 , af4 , af8 , f7 , f5 , f3 , f1 , fz , f2 , f4 , f6 , f8 , ft9 , ft7 , fc5 , fc3 , fc1 , fc2 , fc4 , fc6 , ft8 , and ft10 ) , central ( c5 , c3 , c1 , cz , c2 , c4 , c6 , cp5 , cp3 , cp1 , cpz , cp2 , cp4 , and cp6 ) , parietal ( p7 , p5 , p3 , p1 , pz , p2 , p4 , p6 , and p8 ) , and occipital ( po7 , po3 , poz , po4 , po8 , o1 , oz , o2 , and po10 ) electrode sites before exporting prominent frequency bands alpha ( 7.512.5 hz ) and beta ( 12.535.0 hz ) as mean activity in v . for statistical comparisons , frontal , central , parietal , and occipital pools were each referenced to a global pool ( export of all respective other electrode sites ) . all statistical analyses were performed using the software statistica 7.1 ( statsoft , tulsa , usa ) . repeated - measures analysis of variance ( anova ) was used to detect condition effects and interactions between exercise ( exercise , no - exercise ) and virtual environment exposure ( control , osm , and tsm ) for heart rate , cortical neural oscillations ( eeg : alpha , beta ) , and mental state ( peps , mot , and psych ) . fisher 's least significant difference ( lsd ) was applied post hoc where interactions and main effects were identified . friedman 's anova followed by wilcoxon paired samples test was used to identify changes in the perceived sense of presence . possible correlations between cortical neural oscillations ( eeg : alpha , beta ) , mental state ( peps , mot , and psych ) , sense of presence , and heart rate in both no - exercise and exercise trials were determined using pearson 's correlation coefficient . data ( n = 18 ) in figures are presented as mean confidence interval ( 0.95 ) and as mean standard error of mean in the text and tables . heart rates showed significant increases from rest to no - exercise and exercise trials ( f(3,51 ) = 39.63 , p < 0.05 ) , and these changes were consistent across the three levels of virtual environment exposure ( table 1 ) . ancova revealed no significant effect of gender on heart rate ( = 0.58 , f(7,9 ) = 0.92 , p > 0.05 ) . sense of presence increased from no - exercise to exercise trials , with increasing levels of virtual environment exposure from control to osm and tsm ( chi(18,5 ) = 83.98 , p < 0.05 ; table 2 ) . ancova revealed no significant gender effect for perceived sense of presence ( = 0.52 , f(6,10 ) = 1.52 , p > 0.05 ) . there were significant main effects where mental state differed between exercise and no - exercise trials and between the different levels of virtual environment exposure ( f(4,68 ) = 3.37 , p < 0.05 ) . post hoc analysis revealed increases in peps and psych from control to osm and tsm during the exercise trial , whereas mot increased from control to osm and tsm in the no - exercise trial ( table 3 ) . there was no significant gender effect ( ancova : = 0.52 , f(15,1 ) = 1.22 , p > 0.05 ) . frontal alpha activity showed significant differences between no - exercise and exercise trials across the various virtual environment exposures ( f(2,34 ) = 5.90 , p < 0.05 ) . post hoc analysis revealed frontal alpha activity increased from control to tsm during the no - exercise condition , whereas the respective global alpha activity did not change with levels in virtual environment exposure . the reverse was shown during the exercise trial where the respective global alpha activity increased from control to tsm , whereas frontal alpha activity did not change significantly with levels in virtual environment exposure ( figure 3 ) . ancova revealed no significant effect of gender for alpha activity ( = 0.12 , f(12,4 ) = 2.45 , p > 0.05 ) . central ( f(2,34 ) = 1.60 , p > 0.05 ) , parietal ( f(2,34 ) = 2.16 , p > 0.05 ) , and occipital ( f(2,34 ) = 2.86 , p > 0.05 ) alpha activity revealed no significant changes compared to their respective global alpha activity . frontal beta activity showed significant differences between no - exercise and exercise trials during the different levels of virtual environment exposure ( f(2,34 ) = 5.11 , p < 0.05 ) . post hoc analysis revealed that , during the no - exercise trial , frontal beta activity increased from control to tsm by trend , whereas the respective global beta activity did not change significantly . these findings were reversed during the exercise trial , where the respective global beta activity increased from control to tsm by trend , and frontal beta activity did not change significantly ( figure 4 ) . there was no significant gender effect ( ancova : = 0.15 , f(12,4 ) = 1.92 , p > 0.05 ) . central ( f(2,34 ) = 1.59 , p > 0.05 ) , parietal ( f(2,34 ) = 0.78 , p > 0.05 ) , and occipital ( f(2,34 ) = 1.55 , p > 0.05 ) beta activity revealed no significant changes compared to their respective global beta activity . for the no - exercise trial , positive correlations could be obtained between cortical neural oscillations and mental state as well as between cortical neural oscillations and heart rate ( table 4 ) . for the exercise trial , a positive correlation could be obtained between mental state and heart rate ( table 5 ) . this study aimed to investigate the interactive effects of virtual environment exposure and exercise on neurophysiological and perceptual responses in healthy adults . we verified that exposure to increasing levels of virtual environment exposure led to increases in perceived sense of presence , and a key finding was that this change in perception was further enhanced by the inclusion of exercise . measures of mental state responded to increasing levels of virtual environment exposure , and the addition of exercise led to increases in physical state ( peps ) and psychological strain ( psych ) and a reduction in motivational state ( mot ) . the addition of exercise to the virtual environment had a significant influence on the eeg responses , with a shift from strong frontal alpha and beta activity responses during the no - exercise condition to strong global alpha and beta responses when exercise was added to the virtual environment exposure . perceived sense of presence increased with increasing levels of virtual environment exposure , from control to one - screen mode ( osm ) and three - screen mode ( tsm ) , and in each condition sense of presence was further amplified with the addition of exercise . though not correlated , these changes in sense of presence were underlined by changes in heart rate . the increases in heart rate during the no - exercise condition are consistent with previous studies , and collectively these findings support the use of heart rate monitoring as an objective physiological measure that may reflect sense of presence during exposure to virtual environments . there was a significant increase in peps and psych during exercise in the virtual environments compared with the no - exercise condition . the increase in peps ( perceived physical state ) reflects the increased heart rate and effort during exercise , and such a response is well documented in the literature [ 1720 ] . the strong psych ( psychological strain ) response to exercise in the virtual environments appears to be at odds with evidence of the beneficial and calming effects of exercise , although exercise alone in the present study ( during control ) did in fact lead to a slight ( not significant ) reduction in psych . the increase in psych during exercise in the virtual environment was accompanied by a reduction in motivation ( mot ) . though surprising , it seems reasonable that exercising in virtual environments may lead to less motivation , relative to an increasing virtual environment exposure ( i.e. , from control to osm to tsm ) that is coexistent with an increase in psych ( e.g. , increased discomfort ) . also , it has previously been suggested that virtual reality increases the psychological demands of an environment , and these demands increase with increased duration of exposure . our findings suggest that the addition of exercise increases the demands of a virtual environment and this results in a pronounced psychological strain . in contrast , it appears that less demanding virtual environments ( i.e. , without exercise ) result in a stronger motivational state , thus , in line with the above suggested less motivation while exercising in the virtual environment . the increase in global beta activity after exercise in the virtual environment is consistent with the hypothesis of a generalised model of cortical arousal ( mca ) . according to the mca , the increase in beta activity is consistent with cortical arousal and has previously been associated with the incidence of cybersickness , akin to motion sickness , and this corresponds with the elevated psychological strain and the decreased motivation during this trial . rather than a corresponding decrease in alpha activity , as would be expected according to the mca , global alpha activity also increased during exercise in the virtual environment . this is suggestive of a relaxed cortical state , which is consistent with the low motivational state in the current trial , but at odds with the elevated psychological strain . assessment of cortical current density allows for the localisation of neural changes to specific regions of the brain . the transient hypofrontality theory ( tht ) reflects redistribution of cortical activity away from the frontal regions [ 23 , 25 ] , thus allowing for greater cortical resources to maintain mental state . while there were significant increases in frontal cortical neural oscillations in the virtual environment during the no - exercise condition , the addition of exercise led to reductions in both alpha and beta frontal activity , relative to global activity . this is consistent with the tht and presumably is driven largely by the increase in physical and psychological strain . from a sense of presence perspective , the neural activation patterns are in line with previous neuroimaging studies , suggesting that frontal brain regions form a key sense of presence node . though documented in a nonexercise fmri study ( i.e. , rollercoaster scenarios ) , baumgartner et al . suggest lesser cortical activation in the dorsolateral prefrontal cortex accompanied by a stronger sense of presence perception . in addition , it is well accepted that frontoparietal brain regions are strongly involved in movement control . even the imagination of movements or motor representation is likely to shape the sense of presence perception in virtual environments . with this and previous evidence that postural changes enhance sense of presence in virtual environments , it seems reasonable that the inclusion of real exercise ( i.e. , self - paced cycling on an ergometer ) similarly fosters and possibly intensifies sense of presence perception compared to motor representation ( i.e. , automatic drive ) . this is underlined by increased sense of presence perception from no - exercise to exercise trials as well as from control to osm and tsm . despite plausible findings , we are aware that the present study is limited by a rather small number of participants . additionally , lack of data pertaining to exercise intensity ( e.g. , watt / rpm , vo2 ) makes it difficult to verify the standardisation of exercise conditions ; however , the heart rate responded as would be expected to further support the moderate exercise definition , which is mainly owing to available infrastructure . the primary aim of this study was to examine cortical neural oscillations and related alterations in mental state in response to exercise in immersive virtual environments compared to movement representation . changes in psychological strain and physical state were generally mirrored by neural activation patterns of frontal and global alpha and beta activity . furthermore , these changes are likely to indicate , based on the model of cortical arousal and , in particular , the transient hypofrontality theory , that exercise augments the demands of virtual environment exposures and this possibly contributes to enhanced sense of presence .
virtual reality environments are increasingly being used to encourage individuals to exercise more regularly , including as part of treatment those with mental health or neurological disorders . the success of virtual environments likely depends on whether a sense of presence can be established , where participants become fully immersed in the virtual environment . exposure to virtual environments is associated with physiological responses , including cortical activation changes . whether the addition of a real exercise within a virtual environment alters sense of presence perception , or the accompanying physiological changes , is not known . in a randomized and controlled study design , moderate - intensity exercise ( i.e. , self - paced cycling ) and no - exercise ( i.e. , automatic propulsion ) trials were performed within three levels of virtual environment exposure . each trial was 5 minutes in duration and was followed by posttrial assessments of heart rate , perceived sense of presence , eeg , and mental state . changes in psychological strain and physical state were generally mirrored by neural activation patterns . furthermore , these changes indicated that exercise augments the demands of virtual environment exposures and this likely contributed to an enhanced sense of presence .
1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions
virtual reality environments are increasingly being used to encourage individuals to exercise more regularly , for the prevention of sedentary lifestyles among relatively healthy individuals , including , and as part of treatment , in those with mental health or neurological disorders . electroencephalography ( eeg ) frequency analyses have also been used to explore the neural activation patterns associated with sense of presence perception . whether the addition of a real exercise ( e.g. , cycling on an ergometer ) within an immersive virtual environment alters sense of presence perception , or the accompanying changes in neural activation patterns or mental state , is not known . it is not known whether these same neural responses occur during exercise or movement representation in a virtual environment , and this is important to establish if virtual environments are to be used to facilitate exercise as a therapy for mental well - being . , self - paced cycling ) , compared with movement representation , within three levels of virtual environment exposure ( a complex three - screen mode , a simple one - screen mode , and a no - screen control ) on cortical neural oscillations ( i.e. , frontal alpha and beta activity ) , perceived sense of presence , and mental state ( i.e. after baseline measurements , exercise or no - exercise trials were conducted within three levels of virtual environment exposure . each trial was 5 minutes in duration and was followed by posttrial assessments of heart rate , perceived sense of presence , and eeg as well as mental state ( figure 1 ) . with respect to dose [ 19 , 20 ] and preference , exercise trials were performed at a self - paced moderate intensity that was defined by consistent heart rate monitoring and verbal instructions prior to each exercise trial ( i.e. repeated - measures analysis of variance ( anova ) was used to detect condition effects and interactions between exercise ( exercise , no - exercise ) and virtual environment exposure ( control , osm , and tsm ) for heart rate , cortical neural oscillations ( eeg : alpha , beta ) , and mental state ( peps , mot , and psych ) . friedman 's anova followed by wilcoxon paired samples test was used to identify changes in the perceived sense of presence . possible correlations between cortical neural oscillations ( eeg : alpha , beta ) , mental state ( peps , mot , and psych ) , sense of presence , and heart rate in both no - exercise and exercise trials were determined using pearson 's correlation coefficient . heart rates showed significant increases from rest to no - exercise and exercise trials ( f(3,51 ) = 39.63 , p < 0.05 ) , and these changes were consistent across the three levels of virtual environment exposure ( table 1 ) . sense of presence increased from no - exercise to exercise trials , with increasing levels of virtual environment exposure from control to osm and tsm ( chi(18,5 ) = 83.98 , p < 0.05 ; table 2 ) . there were significant main effects where mental state differed between exercise and no - exercise trials and between the different levels of virtual environment exposure ( f(4,68 ) = 3.37 , p < 0.05 ) . we verified that exposure to increasing levels of virtual environment exposure led to increases in perceived sense of presence , and a key finding was that this change in perception was further enhanced by the inclusion of exercise . measures of mental state responded to increasing levels of virtual environment exposure , and the addition of exercise led to increases in physical state ( peps ) and psychological strain ( psych ) and a reduction in motivational state ( mot ) . the addition of exercise to the virtual environment had a significant influence on the eeg responses , with a shift from strong frontal alpha and beta activity responses during the no - exercise condition to strong global alpha and beta responses when exercise was added to the virtual environment exposure . perceived sense of presence increased with increasing levels of virtual environment exposure , from control to one - screen mode ( osm ) and three - screen mode ( tsm ) , and in each condition sense of presence was further amplified with the addition of exercise . the increases in heart rate during the no - exercise condition are consistent with previous studies , and collectively these findings support the use of heart rate monitoring as an objective physiological measure that may reflect sense of presence during exposure to virtual environments . our findings suggest that the addition of exercise increases the demands of a virtual environment and this results in a pronounced psychological strain . with this and previous evidence that postural changes enhance sense of presence in virtual environments , it seems reasonable that the inclusion of real exercise ( i.e. , self - paced cycling on an ergometer ) similarly fosters and possibly intensifies sense of presence perception compared to motor representation ( i.e. changes in psychological strain and physical state were generally mirrored by neural activation patterns of frontal and global alpha and beta activity . furthermore , these changes are likely to indicate , based on the model of cortical arousal and , in particular , the transient hypofrontality theory , that exercise augments the demands of virtual environment exposures and this possibly contributes to enhanced sense of presence .
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pancreatic neuroendocrine tumors ( pnets ) are a relatively rare and heterogeneous group of neoplasms comprising 12% of all pancreatic neoplasms and 2.6% of pancreatic cancers . due to the widespread use of high - quality imaging techniques , the incidence of pnets has remarkably increased from 0.17 to 0.43/100,000 over the past three decades in the united states . furthermore , autopsy studies indicate that the prevalence of pnets may be even higher . depending on whether clinical symptoms are related to excessive secretion of endocrine hormones , pnets are divided into functional and nonfunctional pnets ( nf - pnets ) . nf - pnets are defined by the absence of a hormone hypersecretion syndrome and account for approximately 90% of all pnets . nf tumors are asymptomatic or present with nonspecific symptoms related to local mass effect or metastatic disease and are associated with a poorer prognosis than functional tumors , probably due to delayed diagnosis and higher malignant potential . compared to pancreatic ductal adenocarcinoma , nf - pnets often have an indolent outcome but postoperative recurrence is not rare . lymph node metastasis ( lnm ) regarding nf - pnets , lymph node status is regarded as an important prognostic factor in both the european neuroendocrine tumor society ( enets ) tnm staging system and the american joint committee on cancer ( ajcc ) cancer staging system . in current guidelines , the national comprehensive cancer network ( nccn ) guidelines do not advocate routine lymphadenectomy in tumors < 2 cm , while this procedure is recommended for tumors that are 12 cm because of the risk of lnm . moreover , previous reports have demonstrated that only 3040% of patients with nf - pnets present with lnm at diagnosis , which suggests the importance of preoperative recognition of patients at high - risk of lnm , who may benefit from regional lymphadenectomy . therefore , the identification of reliable predictors of lnm is important in guiding clinical management decisions and avoiding an unnecessary lymphadenectomy in low - risk patients . the aims of this study were : ( 1 ) to evaluate the impact of lnm on postoperative recurrence of patients with surgically treated nf - pnet ; ( 2 ) to evaluate the feasibility of preoperative prediction of lnm in nf - pnets using preoperatively available variables . this was a mono - institutional retrospective cohort study of the clinical records of 100 patients who underwent pancreatic surgery with curative intent for nf - pnet between january 2004 and december 2014 . all patients who were diagnosed with nf - pnet were included ( n = 111 ) , whereas syndromic patients ( n = 1 ) , patients lost to the postoperative follow - up ( n = 6 ) and patients with distal metastasis ( n = 4 ) , were excluded from this study . nonfunctioning neoplasms were defined by the lack of any clinical syndrome caused by excess hormonal secretion . in total , information about clinical presentation , demographics , data regarding surgical procedures , postoperative course and complications , pathologic findings , and follow - up was collected . all patients underwent presurgical computed tomography ( ct ) evaluation of the dimensions , local invasiveness , and the presence of lymph node or distant metastasis . the pathological diameter of neoplasms was defined as the largest diameter of the surgical specimens . standard or parenchyma - preserving resection was selected for according to tumor size and anatomical location . all patients in this study had at least one lymph node sampled on resected specimens . the extent of regional lymphadenectomy was the same as that performed in cases of pancreatic ductal adenocarcinoma . for tumors located in the pancreatic head , regional nodes consisted of those located along the common bile duct , common hepatic artery , portal vein , superior mesenteric vein , posterior and anterior pancreatic head , and the right lateral wall of the superior mesenteric artery . for tumors located in the pancreatic body or tail , regional nodes included those along the common hepatic artery , celiac axis , splenic artery , and splenic hilum . the surgical specimens of all cases were classified according to the world health organization ( who ) classification criteria ( 2010 ) . all pnets were divided according to a grading scheme based on mitotic count or ki67 index into g1 ( mitotic count < 2/10 high - power fields ( hpf ) and/or 2% ki67 index ) , g2 ( mitotic count 220/10 hpf and/or 320% ki67 index ) , and g3 ( mitotic count > 20/10 hpf and/or > 20% ki67 index ) . the enets recommended tnm staging system was used for tumor staging . primary tumors ( t stage ) were classified into four categories : t1 , tumor limited to the pancreas and size < 2 cm ; t2 , tumor limited to the pancreas and size 24 cm ; t3 , tumor limited to the pancreas and size > 4 cm or invading the duodenum or bile duct ; and t4 , tumor invading adjacent organs ( stomach , spleen , colon , and adrenal gland ) or the wall of large vessels ( celiac axis or superior mesenteric artery ) . all patients enrolled in this study underwent a postoperative clinical and radiological follow - up . all patients underwent a radiological examination by ct scans every 612 months after surgery , and magnetic resonance imaging was performed if necessary . if patients had any symptoms suspected to be associated with tumor progression during follow - up , a radiological examination was performed immediately to rule out recurrence or distant metastasis . disease - free survival ( dfs ) was calculated as the months between surgery and 30 jun 2015 or the first documented disease recurrence . an acute postoperative mortality was defined as death which occurred within 30 days after surgery . the data on the operation and postoperative morbidity was collected from the electronic patient records of our institution . phone interviews were conducted for all patients with a response rate of 95% ( 105 patients ) and data on survival status , date of death , and tumor recurrence were collected . for all living patients , data were expressed as a mean standard deviation for continuous variables and as number and percentage for categorical variables . the comparison between subgroups was performed by the analysis of variance for quantitative variables and by the chi - square test or fisher exact test for categorical variables , when necessary . the cox regression model was used in univariate and multivariate analyses to evaluate the independent predictive factors of postoperative recurrence . hazard ratios ( hr ) and 95% confidence intervals ( ci ) were also calculated . variables with p 0.05 in univariate analysis were included in the multivariate model . logistic regression analysis performed in a stepwise fashion with backward selection was used to evaluate the value of clinical factors for predicting lnm and to establish the preoperative predictive model . receiver operating characteristic ( roc ) curve analysis was performed to assess the predictive power of the models through calculating the area under the curve ( auc ) . spss version 20.0 ( ibm , usa ) was used to perform all the data analysis . this was a mono - institutional retrospective cohort study of the clinical records of 100 patients who underwent pancreatic surgery with curative intent for nf - pnet between january 2004 and december 2014 . all patients who were diagnosed with nf - pnet were included ( n = 111 ) , whereas syndromic patients ( n = 1 ) , patients lost to the postoperative follow - up ( n = 6 ) and patients with distal metastasis ( n = 4 ) , were excluded from this study . nonfunctioning neoplasms were defined by the lack of any clinical syndrome caused by excess hormonal secretion . in total , information about clinical presentation , demographics , data regarding surgical procedures , postoperative course and complications , pathologic findings , and follow - up was collected . all patients underwent presurgical computed tomography ( ct ) evaluation of the dimensions , local invasiveness , and the presence of lymph node or distant metastasis . the pathological diameter of neoplasms was defined as the largest diameter of the surgical specimens . standard or parenchyma - preserving resection was selected for according to tumor size and anatomical location . all patients in this study had at least one lymph node sampled on resected specimens . the extent of regional lymphadenectomy was the same as that performed in cases of pancreatic ductal adenocarcinoma . for tumors located in the pancreatic head , regional nodes consisted of those located along the common bile duct , common hepatic artery , portal vein , superior mesenteric vein , posterior and anterior pancreatic head , and the right lateral wall of the superior mesenteric artery . for tumors located in the pancreatic body or tail , regional nodes included those along the common hepatic artery , celiac axis , splenic artery , and splenic hilum . the surgical specimens of all cases were classified according to the world health organization ( who ) classification criteria ( 2010 ) . all pnets were divided according to a grading scheme based on mitotic count or ki67 index into g1 ( mitotic count < 2/10 high - power fields ( hpf ) and/or 2% ki67 index ) , g2 ( mitotic count 220/10 hpf and/or 320% ki67 index ) , and g3 ( mitotic count > 20/10 hpf and/or > 20% ki67 index ) . the enets recommended tnm staging system was used for tumor staging . primary tumors ( t stage ) were classified into four categories : t1 , tumor limited to the pancreas and size < 2 cm ; t2 , tumor limited to the pancreas and size 24 cm ; t3 , tumor limited to the pancreas and size > 4 cm or invading the duodenum or bile duct ; and t4 , tumor invading adjacent organs ( stomach , spleen , colon , and adrenal gland ) or the wall of large vessels ( celiac axis or superior mesenteric artery ) . all patients enrolled in this study underwent a postoperative clinical and radiological follow - up . all patients underwent a radiological examination by ct scans every 612 months after surgery , and magnetic resonance imaging was performed if necessary . if patients had any symptoms suspected to be associated with tumor progression during follow - up , a radiological examination was performed immediately to rule out recurrence or distant metastasis . disease - free survival ( dfs ) was calculated as the months between surgery and 30 jun 2015 or the first documented disease recurrence . an acute postoperative mortality was defined as death which occurred within 30 days after surgery . the data on the operation and postoperative morbidity was collected from the electronic patient records of our institution . phone interviews were conducted for all patients with a response rate of 95% ( 105 patients ) and data on survival status , date of death , and tumor recurrence were collected . for all living patients , data were expressed as a mean standard deviation for continuous variables and as number and percentage for categorical variables . the comparison between subgroups was performed by the analysis of variance for quantitative variables and by the chi - square test or fisher exact test for categorical variables , when necessary . the cox regression model was used in univariate and multivariate analyses to evaluate the independent predictive factors of postoperative recurrence . hazard ratios ( hr ) and 95% confidence intervals ( ci ) were also calculated . variables with p 0.05 in univariate analysis were included in the multivariate model . logistic regression analysis performed in a stepwise fashion with backward selection was used to evaluate the value of clinical factors for predicting lnm and to establish the preoperative predictive model . receiver operating characteristic ( roc ) curve analysis was performed to assess the predictive power of the models through calculating the area under the curve ( auc ) . a two - sided p < 0.05 was considered to indicate statistical significance . spss version 20.0 ( ibm , usa ) was used to perform all the data analysis . the clinical and pathological data of the 100 nf - pnets are shown in table 1 . among the 100 patients , more than 50% ( n = 53 ) were symptomatic at the time of diagnosis . overall , 81 patients ( 81% ) underwent standard pancreatic resection , while 19 patients ( 19% ) underwent parenchyma - preserving pancreatic resection . postsurgical complications occurred in 55 patients ( 55% ) , among which , postoperative pancreatic fistula ( popf ) ( 50.9% ) was the most common . approximately , 40% of popfs were classified as grade a according to the international study group of pancreatic fistula criteria . clinical and pathological data of patients with nonfunctioning pnets * who 2010 classification ; enets recommended tnm staging system . pnets : pancreatic neuroendocrine tumors ; enets : european neuroendocrine tumor society ; tnm : tumor - node - metastasis ; who : world health organization . according to the 2010 who classification , among the 100 nf - pnets , 61 patients ( 61% ) were diagnosed as g1 tumors , 24 ( 24% ) patients as g2 tumors , and 15 ( 15% ) as g3 tumors . among the 85 patients with g1 or g2 tumors , 44 ( 51.8% ) were asymptomatic . in contrast , among the patients with g3 tumors , 12 ( 80% ) had symptoms before surgery . compared to g1 neoplasms , g2 or g3 neoplasms had larger diameter ( g2 vs. g1 : 4.4 cm 2.6 cm vs. 2.8 cm 2.1 cm , p = 0.001 ; g3 vs. g1 : 4.6 cm 2.0 cm vs. 2.8 cm 2.1 cm , p = 0.004 ) . tumor size in ln+ patients was larger than that in ln - patients ( 4.7 cm 2.6 cm vs. 3.2 cm 2.2 cm , p = 0.01 ) . according to the enets recommended tnm staging system , 29 ( 29% ) patients had t1 tumors , 35 ( 35% ) had t2 , 31 ( 31% ) had t3 , and 5 ( 5% ) had t4 . among the 100 patients , 27 patients ( 27% ) had stage i neoplasms , 32 ( 32% ) had stage iia , 22 ( 22% ) had stage iib , and 19 ( 19% ) had stage iiib . pathological examination revealed the presence of angioinvasion and perineural invasion in 15 patients ( 15% ) and 7 patients ( 7% ) , respectively . malignant behavior was found in 31 ( 31% ) of all nf - pnets and four ( 8.5% ) of 47 patients with radiological tumor size < 2.5 cm with lnm identified in 3 ( 75% ) of these patients . the 1 , 5 , and 8-year dfs was 90.2 , 64.147.1% , respectively [ figure 1 ] . during the follow - up period , variables associated with dfs in the univariate analysis are shown in table 2 . in the multivariate analysis , lymph node positive ( hr = 3.995 , 95% ci : 1.58510.06 , p = 0.003 ) , angioinvasion ( hr = 4.049 , 95% ci : 1.472 - 11.135 , p = 0.007 ) , and high tumor grading ( g3 vs. g1 + g2 : hr = 7.286 , 95% ci : 2.779718.980 , p = 0.000048 ) were significantly associated with decreased dfs in patients with resected nf - pnet [ table 2 ] . the 5- and 8-year dfs was 79.4% and 71.4% , respectively , for ln - patients compared to 24.9% and 8.3% , respectively , for patients with ln+ disease ( p = 0.000001 ) [ figure 2 ] . disease - free survival of patients with nonfunctional pancreatic neuroendocrine tumors . the 1 , 5 , and 8-year disease - free survival was 90.2% , 64.1% and 47.1% , respectively . univariate and multivariate analysis of risk factors of dfs * size on resected specimens ; who 2010 classification ; enets recommended tnm staging system . dfs : disease - free survival ; sd : standard deviation ; hr : hazard ratio ; 95% ci : 95% confidence interval ; ln : lymph node ; enets : european neuroendocrine tumor society ; tnm : tumor - node - metastasis ; who : world health organization . the 5-year disease - free survival was 79.4% for lymph node - patients compared to 24.9% for patients with ln + disease ( p = 0.000001 ) , ln : lymph node . among these 100 patients with nf - pnets , 92 who underwent regional lymphadenectomy variables that could be measured preoperatively were selected for the univariate analysis of the feasibility of preoperative prediction of ln status . in the univariate analysis , factors associated with ln metastasis were radiological tumor diameter > 2.5 cm ( odds ratio [ or ] = 5.667 , p = 0.010 ) , elevated ca199 ( or = 4.714 , p = 0.017 ) , high tumor grading ( g2:g1 , or = 6.125 , p = 0.007 ; g3:g1 , or = 14.000 , p = 0.000322 ) , and presence of symptoms ( or = 3.545 , p = 0.026 ) [ table 3 ] . in the multivariate analysis , tumor grading ( g2 vs. g1 : or = 6.287 , p = 0.008 ; g3 vs. g1 : or = 12.407 , p = 0.001 ) was an independent predictor of lnm [ table 3 ] . the rate of lnm progressively increased from g1 to g3 ( g1 , g2 vs. g3 : 7.5% , 33.3% vs. 53.3% ) . considering the difficulty of preoperative retrievability of tumor grade , we excluded tumor grade from the analysis , and as a result , radiological tumor size > 2.5 cm ( or = 5.430 , p = 0.013 ) and presence of symptoms ( or = 3.366 , p = 0.039 ) were independently associated with lnm [ table 3 ] . radiological diameter was consistent with pathological diameter ( 34.1 mm vs. 34.7 mm , p = 0.237 ) . when tumor size was treated as a continuous variable , the correlation with ln metastasis also reached significance ( or = 1.313 , p = 0.016 ) . roc analysis demonstrated radiological tumor diameter was a reliable and feasible predictor of lnm in patients with resectable nf - pnet with an auc of 0.693 [ figure 3a ] . compared to neoplasms with radiological size > 2.5 cm ( 32.1% ) , tumors 2.5 cm had an obviously lower risk of lnm ( 7.7% ) . the various clinicopathologic factors reviewed in this study stratified by a tumor size cut - off of 2.5 cm are summarized in table 4 . compared to tumors 2.5 cm , tumors > 2.5 cm had higher tumor grade and greater malignant potential . a cut - off of > 2.5 cm was associated with a sensitivity of 85% for the presence of ln+ disease . other cut - offs were also examined [ table 5 ] . with the purpose of promoting the predictive power , we constructed a preoperative predictive model of ln metastasis based on radiological tumor size combined with symptoms . the auc of this model was 0.747 [ figure 3b ] , and the probability of ln+ for every patient was calculated . for the patients with an ln+ risk of 20% , 89.6% of these patients were , actually , ln - negative . of the 21 incidentally discovered patients with tumors size 2.5 cm on ct scans , only one ( 4.8% ) had lnm and none presented a postoperative recurrence during follow - up . univariate and multivariate logistic regression analysis of ln metastasis * who 2010 classification ; multivariate analysis excluding tumor grade . or : odds ratio ; 95% ci : 95% confidence interval ; ln : lymph node ; who : world health organization . the area under the curve of radiological tumor size ( a ) is 0.693 , while the area under the curve of the constructed predictive model ( b ) is 0.747 . clinicopathologic factors stratified by radiological tumor size * standard resections include pancreaticoduodenectomy and distal pancreatectomy . atypical resections include middle pancreatectomy and enucleation ; laparoscopic and robotic surgery ; who 2010 classification ; enets recommended tnm staging system . sd : standard deviation ; ln : lymph node ; who : world health organization ; enets : european neuroendocrine tumor society ; tnm : tumor - node - metastasis . predictive values of different radiological size cut - offs for ln metastasis in patients with nf - pnets npv : negative predictive value ; ppv : positive predictive value ; auc : area under the curve ; nf - pnets : nonfunctional pancreatic neuroendocrine tumors ; ln : lymph node . the clinical and pathological data of the 100 nf - pnets are shown in table 1 . among the 100 patients , more than 50% ( n = 53 ) were symptomatic at the time of diagnosis . overall , 81 patients ( 81% ) underwent standard pancreatic resection , while 19 patients ( 19% ) underwent parenchyma - preserving pancreatic resection . postsurgical complications occurred in 55 patients ( 55% ) , among which , postoperative pancreatic fistula ( popf ) ( 50.9% ) was the most common . approximately , 40% of popfs were classified as grade a according to the international study group of pancreatic fistula criteria . clinical and pathological data of patients with nonfunctioning pnets * who 2010 classification ; enets recommended tnm staging system . pnets : pancreatic neuroendocrine tumors ; enets : european neuroendocrine tumor society ; tnm : tumor - node - metastasis ; who : world health organization . according to the 2010 who classification , among the 100 nf - pnets , 61 patients ( 61% ) were diagnosed as g1 tumors , 24 ( 24% ) patients as g2 tumors , and 15 ( 15% ) as g3 tumors . among the 85 patients with g1 or g2 tumors , 44 ( 51.8% ) were asymptomatic . in contrast , among the patients with g3 tumors , 12 ( 80% ) had symptoms before surgery . compared to g1 neoplasms , g2 or g3 neoplasms had larger diameter ( g2 vs. g1 : 4.4 cm 2.6 cm vs. 2.8 cm 2.1 cm , p = 0.001 ; g3 vs. g1 : 4.6 cm 2.0 cm vs. 2.8 cm 2.1 cm , p = 0.004 ) . tumor size in ln+ patients was larger than that in ln - patients ( 4.7 cm 2.6 cm vs. 3.2 cm 2.2 cm , p = 0.01 ) . according to the enets recommended tnm staging system , 29 ( 29% ) patients had t1 tumors , 35 ( 35% ) had t2 , 31 ( 31% ) had t3 , and 5 ( 5% ) had t4 . among the 100 patients , 27 patients ( 27% ) had stage i neoplasms , 32 ( 32% ) had stage iia , 22 ( 22% ) had stage iib , and 19 ( 19% ) had stage iiib . pathological examination revealed the presence of angioinvasion and perineural invasion in 15 patients ( 15% ) and 7 patients ( 7% ) , respectively . malignant behavior was found in 31 ( 31% ) of all nf - pnets and four ( 8.5% ) of 47 patients with radiological tumor size < 2.5 cm with lnm identified in 3 ( 75% ) of these patients . the 1 , 5 , and 8-year dfs was 90.2 , 64.147.1% , respectively [ figure 1 ] . during the follow - up period , variables associated with dfs in the univariate analysis are shown in table 2 . in the multivariate analysis , lymph node positive ( hr = 3.995 , 95% ci : 1.58510.06 , p = 0.003 ) , angioinvasion ( hr = 4.049 , 95% ci : 1.472 - 11.135 , p = 0.007 ) , and high tumor grading ( g3 vs. g1 + g2 : hr = 7.286 , 95% ci : 2.779718.980 , p = 0.000048 ) were significantly associated with decreased dfs in patients with resected nf - pnet [ table 2 ] . the 5- and 8-year dfs was 79.4% and 71.4% , respectively , for ln - patients compared to 24.9% and 8.3% , respectively , for patients with ln+ disease ( p = 0.000001 ) [ figure 2 ] . disease - free survival of patients with nonfunctional pancreatic neuroendocrine tumors . the 1 , 5 , and 8-year disease - free survival was 90.2% , 64.1% and 47.1% , respectively . univariate and multivariate analysis of risk factors of dfs * size on resected specimens ; who 2010 classification ; enets recommended tnm staging system . dfs : disease - free survival ; sd : standard deviation ; hr : hazard ratio ; 95% ci : 95% confidence interval ; ln : lymph node ; enets : european neuroendocrine tumor society ; tnm : tumor - node - metastasis ; who : world health organization . impact of lymph node status on disease - free survival . the 5-year disease - free survival was 79.4% for lymph node - patients compared to 24.9% for patients with ln + disease ( p = 0.000001 ) , ln : lymph node . among these 100 patients with nf - pnets , 92 who underwent regional lymphadenectomy were selected for analysis of predictors of lnm . of these patients , variables that could be measured preoperatively were selected for the univariate analysis of the feasibility of preoperative prediction of ln status . in the univariate analysis , factors associated with ln metastasis were radiological tumor diameter > 2.5 cm ( odds ratio [ or ] = 5.667 , p = 0.010 ) , elevated ca199 ( or = 4.714 , p = 0.017 ) , high tumor grading ( g2:g1 , or = 6.125 , p = 0.007 ; g3:g1 , or = 14.000 , p = 0.000322 ) , and presence of symptoms ( or = 3.545 , p = 0.026 ) [ table 3 ] . in the multivariate analysis , tumor grading ( g2 vs. g1 : or = 6.287 , p = 0.008 ; g3 vs. g1 : or = 12.407 , p = 0.001 ) was an independent predictor of lnm [ table 3 ] . the rate of lnm progressively increased from g1 to g3 ( g1 , g2 vs. g3 : 7.5% , 33.3% vs. 53.3% ) . considering the difficulty of preoperative retrievability of tumor grade , we excluded tumor grade from the analysis , and as a result , radiological tumor size > 2.5 cm ( or = 5.430 , p = 0.013 ) and presence of symptoms ( or = 3.366 , p = 0.039 ) were independently associated with lnm [ table 3 ] . radiological diameter was consistent with pathological diameter ( 34.1 mm vs. 34.7 mm , p = 0.237 ) . when tumor size was treated as a continuous variable , the correlation with ln metastasis also reached significance ( or = 1.313 , p = 0.016 ) . roc analysis demonstrated radiological tumor diameter was a reliable and feasible predictor of lnm in patients with resectable nf - pnet with an auc of 0.693 [ figure 3a ] . compared to neoplasms with radiological size > 2.5 cm ( 32.1% ) , tumors 2.5 cm had an obviously lower risk of lnm ( 7.7% ) . the various clinicopathologic factors reviewed in this study stratified by a tumor size cut - off of 2.5 cm are summarized in table 4 . compared to tumors 2.5 cm , tumors > 2.5 cm had higher tumor grade and greater malignant potential . a cut - off of > 2.5 cm was associated with a sensitivity of 85% for the presence of ln+ disease . other cut - offs were also examined [ table 5 ] . with the purpose of promoting the predictive power , we constructed a preoperative predictive model of ln metastasis based on radiological tumor size combined with symptoms . the auc of this model was 0.747 [ figure 3b ] , and the probability of ln+ for every patient was calculated . for the patients with an ln+ risk of 20% , 89.6% of these patients were , actually , ln - negative . of the 21 incidentally discovered patients with tumors size 2.5 cm on ct scans , only one ( 4.8% ) had lnm and none presented a postoperative recurrence during follow - up . univariate and multivariate logistic regression analysis of ln metastasis * who 2010 classification ; multivariate analysis excluding tumor grade . or : odds ratio ; 95% ci : 95% confidence interval ; ln : lymph node ; who : world health organization . the area under the curve of radiological tumor size ( a ) is 0.693 , while the area under the curve of the constructed predictive model ( b ) is 0.747 . clinicopathologic factors stratified by radiological tumor size * standard resections include pancreaticoduodenectomy and distal pancreatectomy . atypical resections include middle pancreatectomy and enucleation ; laparoscopic and robotic surgery ; who 2010 classification ; enets recommended tnm staging system . sd : standard deviation ; ln : lymph node ; who : world health organization ; enets : european neuroendocrine tumor society ; tnm : tumor - node - metastasis . predictive values of different radiological size cut - offs for ln metastasis in patients with nf - pnets npv : negative predictive value ; ppv : positive predictive value ; auc : area under the curve ; nf - pnets : nonfunctional pancreatic neuroendocrine tumors ; ln : lymph node . nf - pnets are relatively rare and heterogeneous pancreatic neoplasms with a remarkably increasing incidence . compared to functional neoplasms , nf neoplasms show a worse outcome in part due to the delay of diagnosis and higher malignant potential . the optimal management for pnet is still controversial . given its positive impact on survival , surgical resection has become the treatment choice for most patients with nf - pnets . in recent years , small nf - pnets are increasingly being discovered incidentally in cross - sectional imaging for other purposes and routine regional lymphadenectomy when surgical resection is considered in such cases remains controversial . for these reasons , we conducted a mono - institutional retrospective study of resectable nf - pnets with the purpose of ( 1 ) elucidating the clinical significance of lnm and ( 2 ) identifying reliable predictors of lnm to help surgeons to make informed treatment decisions . our clinical data demonstrated that the 5-year dfs of resectable nf - pnets was 64.1% . lnm is a significant prognostic predictor for most malignant tumors , although the impact of lnm on the survival of patients with nf - pnets remains open to debate . many previous studies have yielded conflicting evidence regarding the prognostic value of lnm for pnet . the difference in patient selection and low lymph node sampling rates during resection for pnets may account for these inconsistencies . in accordance with our results , several previous studies have demonstrated lnm is significantly associated with a poor prognosis . both the enets - tnm staging system and the ajcc cancer staging system regard lymph node status as an important prognostic factor . in our study , patients with lnm had a significantly higher risk ( nearly 4-fold ) of postoperative recurrence compared with those without lnm , which supports the necessity for regional lymphadenectomy in patients with high - risk of lymph node involvement . since lnm is apparently related to prognosis , the ability to distinguish patients with high - risk of lnm preoperatively is of great importance . nf - pnets classified , according to the criteria of the 2010 who classifications , were divided into three groups using a grading scheme based on the ki-67 index or mitotic count . our study confirmed the significant correlation between the 2010 who grading system and postoperative recurrence , which is consistent with recent studies . in addition , lnm occurred more frequently in patients with g2 or g3 nf - pnets than in those with g1 tumors ( g1 , g2 vs. g3 : 7.5% , 33.3% , 53.3% ) ; therefore , it was presumed that these patients would benefit from node clearance and routine lymphadenectomy was recommended . however , classification of tumor grade usually depends on pathological examination and the possibility of a preoperative evaluation of ki-67 is still questionable . endoscopic ultrasonography ( eus ) combined with fine - needle aspiration ( fna ) or tru - cut needle biopsy ( tcb ) can be used to evaluate tumor pathology preoperatively . preoperative evaluation of ki-67 index , combined with lesion size and imaging findings may help surgeons to decide on the best therapeutic approach and whether lymphadenectomy should be performed . however , there is a lack of data regarding the accuracy of fna cytology in the assessment of ki-67 value . all of the available reports describe studies with small sample sizes . in a recent study , use of eus tcb needles can overcome many of the problems that reduce the accuracy of fna , but they are not feasible for most patients as they are often performed only at high - volume centers , can be challenging for small masses , and are associated with a risk of pancreatitis and bleeding . moreover , intratumoral ki-67 heterogeneity limits the accurate preoperative evaluation of ki-67 value by eus fna . considering the limitations of preoperative evaluation of ki-67 index , can easily be obtained preoperatively by use of radiological techniques , it has been extensively studied to identify the patients with lnm . increasing tumor size reported that radiological tumor size 4 cm was an independent predictor of nodal metastasis in low and intermediate grade nf - pnets . in a retrospective study of 116 patients undergoing resection for nf - pnet , toste et al . demonstrated that radiological tumor size 2 cm predicted nodal metastasis with a sensitivity of 93.8% and only two ( 7.4% ) of 27 patients with tumor size < 2 cm had lnm . hashim et al . reported that patients with tumor diameter > 1.5 cm were 4.7 times more likely to have lnm compared to those with smaller tumor diameters . they also found two ( 12% ) of 17 patients with tumor size 1 cm and five ( 13% ) of 38 patients with tumor size 1.5 cm had lnm . in contrast , some studies indicate that tumor size is not an accurate predictor of lnm . reported that there was no difference in tumor size for patients with and without nodal metastasis ( 5.2 cm vs. 4.6 cm ) . <3 cm had nodal metastasis , while only 1 patient in their series with a tumor < 2 cm was ln positive . reported there was no association between decreasing tumor size and decreased percentage of cases presenting with regional nodal metastasis . our study suggests radiological tumor size is a sensitive predictor of lymph node status and demonstrates a strong correlation between increasing radiological tumor size and lnm . the incidence of lnm in patients with radiological size > 2.5 cm was more than 4 times greater than that in patients with a tumor size 2.5 cm . a cut - off of > 2.5 cm was associated with a sensitivity of 85% for the presence of positive lns . three of 39 ( 7.7% ) patients with a tumor size 2.5 cm had nodal metastasis and 2 patients ( 11.1% ) with a tumor size 1.5 cm had nodal metastasis . given that smaller tumors are associated with low rates of lnm , better histology , and a better outcome than larger tumors , it is unclear whether lymphadenectomy should be avoided for small nf - pnets . in a retrospective study of 1854 patients with nf - pnets 2 cm , gratian et al . demonstrated that there was no difference in 5-year overall survival in patients undergoing surgical resection between those who underwent lymphadenectomy and those who did not . interestingly , in this study , 29% of tumors 2 cm and 33% of tumors 0.5 cm presented with regional lnm with a median of eight lymph nodes sampled , which was unexpectedly higher than had been reported previously . however , patient selection bias might overestimate the malignant potential of these tumors and account for the high rate of lnm . nccn guidelines suggest that lymphadenectomy should not be performed routinely for tumors < 2 cm but should be considered in tumors that are 12 cm in size . the benefits of lymphadenectomy in patients with small tumors is still unknown and more clinical data or well - designed clinical trials are needed to resolve this problem ; however , such clinical trials are limited by the relative rarity and indolent behavior of small nf - pnets . in our study , patients with small tumors ( 2.5 cm ) has a very low - risk of lnm . in addition , recurrence occurred in only one of the patients with small tumors 84 months after surgery , and no deaths caused by these tumors were reported during the follow - up . nccn guidelines and current staging systems use the same cut - off of 2 cm as that used in pancreatic ductal adenocarcinoma , although patients with nf - pnets have a much better prognosis than those with pda . a cut - off of 2 cm has a low specificity for estimating the risk of lnm ; consequently , many patients undergo unnecessary lymphadenectomy under this criterion . based on our results , a cut - off of 2.5 cm was shown to be appropriate and safe . compared to 2 cm , a cut - off of 2.5 cm had a similar sensitivity , but higher specificity and negative predictive value , showing that a cut - off of 2.5 cm is more effective in distinguishing the patients who require ln resection . given the previously demonstrated strict correlation between tumor diameter and poor survival after resection of nf - pnets , raising the threshold may result in some tumors with malignant potential being considered benign . preoperative evaluation of ki-67 index , combined with lesion size and imaging findings , may help surgeons to decide the best therapeutic approach . since low - grade tumors are associated with a very low - risk of lnm and excellent survival , relaxing the indications for lymphadenectomy seems to be feasible . based on the results of this study , we identified radiological tumor size as a noninvasive and reliable factor to predict lnm in nf - pnets . a radiological diameter 2.5 cm yielded a powerful correlation with the risk of lnm . the presence of symptoms at diagnosis was also shown to be a predictor of lnm . although the symptoms of patients with nf - pnets are always unspecific , they are thought to be related to local mass effect or metastatic disease ; in other words , tumor burden . incidental detection of tumors is a strong prognostic factor for postoperative progression . in this study , incidentally discovered tumors were slightly smaller in size ( 3.2 cm vs. 3.7 cm ) and associated with a much lower risk of lnm ( 12.2% vs. 29.2% ) . reported only 6% of nf - pnets 2 cm were malignant when discovered incidentally . in our study , incidentally discovered small ( 2.5 cm ) nf - pnets had a very low - risk of lnm ( 4.2% ) and no cases of postoperative recurrence or death due to the disease occurred . considering the low - risk of ln involvement and the positive outcomes , routine lymphadenectomy is not recommended as an addition to pancreatic resection in cases of asymptomatic small nf - pnets when surgery is considered . in conclusion , preoperative prediction of ln involvement is feasible and radiological tumor size , which can be measured easily and accurately , was shown to be a useful and alternative variable correlated with ln involvement . our results suggest that parenchyma - sparing resection without regional lymphadenectomy is a reasonable option for selected patients with incidentally discovered nf - pnets 2.5 cm when surgical resection is considered because of the low probability of lnm and a good outcome . in contrast , lymphadenectomy should be performed routinely in patients with nf - pnets > 2.5 cm .
background : the optimal surgical management of nonfunctional pancreatic neuroendocrine tumors ( nf - pnets ) is still controversial . here , we evaluated the impact of lymph node status on postoperative recurrence in patients with nf - pnet and the potential of preoperative variables for predicting lymph node metastasis ( lnm).methods : in this mono - institutional retrospective cohort study conducted in 100 consecutive patients who underwent nf - pnet resection between january 2004 and december 2014 , we evaluated risk factors for survival using the kaplan meier method and the cox regression model . predictors of lnm were evaluated using the logistic regression model , and the power of predictive models was evaluated using receiver operating characteristic curve analysis.results:five-year disease - free survival of resected nf - pnet was 64.1% . lnm was independently associated with postoperative recurrence ( hazard ratio = 3.995 , p = 0.003 ) . multivariate analysis revealed tumor grade as an independent factor associated with lnm ( g2 vs. g1 : odds ratio [ or ] = 6.287 , p = 0.008 ; g3 vs. g1 : or = 12.407 , p = 0.001 ) . when tumor grade was excluded , radiological tumor diameter > 2.5 cm ( or = 5.430 , p = 0.013 ) and presence of symptoms ( or = 3.366 , p = 0.039 ) were significantly associated with lnm . compared to neoplasms with radiological diameter > 2.5 cm ( 32.1% ) , tumors 2.5 cm had an obviously lower risk of lnm ( 7.7% ) , indicating the reliability of this parameter in predicting lnm ( area under the curve , 0.693 ) . incidentally discovered nf - pnets 2.5 cm were associated with a low - risk of lnm and excellent survival.conclusions:lnm is significantly associated with postoperative recurrence . radiological tumor diameter is a reliable predictor of lnm in nf - pnets . our results indicate that lymphadenectomy in small ( 2.5 cm ) nf - pnets is not routinely necessary .
I M Patients Surgical treatment of nonfunctional pancreatic neuroendocrine tumors Pathological examination and staging system Follow-up Statistical analysis R Demographics, operative details, pathological findings Long-term outcomes Predictors of lymph node metastasis D Financial support and sponsorship Conflicts of interest
the aims of this study were : ( 1 ) to evaluate the impact of lnm on postoperative recurrence of patients with surgically treated nf - pnet ; ( 2 ) to evaluate the feasibility of preoperative prediction of lnm in nf - pnets using preoperatively available variables . this was a mono - institutional retrospective cohort study of the clinical records of 100 patients who underwent pancreatic surgery with curative intent for nf - pnet between january 2004 and december 2014 . this was a mono - institutional retrospective cohort study of the clinical records of 100 patients who underwent pancreatic surgery with curative intent for nf - pnet between january 2004 and december 2014 . in the multivariate analysis , lymph node positive ( hr = 3.995 , 95% ci : 1.58510.06 , p = 0.003 ) , angioinvasion ( hr = 4.049 , 95% ci : 1.472 - 11.135 , p = 0.007 ) , and high tumor grading ( g3 vs. g1 + g2 : hr = 7.286 , 95% ci : 2.779718.980 , p = 0.000048 ) were significantly associated with decreased dfs in patients with resected nf - pnet [ table 2 ] . in the univariate analysis , factors associated with ln metastasis were radiological tumor diameter > 2.5 cm ( odds ratio [ or ] = 5.667 , p = 0.010 ) , elevated ca199 ( or = 4.714 , p = 0.017 ) , high tumor grading ( g2:g1 , or = 6.125 , p = 0.007 ; g3:g1 , or = 14.000 , p = 0.000322 ) , and presence of symptoms ( or = 3.545 , p = 0.026 ) [ table 3 ] . in the multivariate analysis , tumor grading ( g2 vs. g1 : or = 6.287 , p = 0.008 ; g3 vs. g1 : or = 12.407 , p = 0.001 ) was an independent predictor of lnm [ table 3 ] . considering the difficulty of preoperative retrievability of tumor grade , we excluded tumor grade from the analysis , and as a result , radiological tumor size > 2.5 cm ( or = 5.430 , p = 0.013 ) and presence of symptoms ( or = 3.366 , p = 0.039 ) were independently associated with lnm [ table 3 ] . compared to neoplasms with radiological size > 2.5 cm ( 32.1% ) , tumors 2.5 cm had an obviously lower risk of lnm ( 7.7% ) . in the multivariate analysis , lymph node positive ( hr = 3.995 , 95% ci : 1.58510.06 , p = 0.003 ) , angioinvasion ( hr = 4.049 , 95% ci : 1.472 - 11.135 , p = 0.007 ) , and high tumor grading ( g3 vs. g1 + g2 : hr = 7.286 , 95% ci : 2.779718.980 , p = 0.000048 ) were significantly associated with decreased dfs in patients with resected nf - pnet [ table 2 ] . in the univariate analysis , factors associated with ln metastasis were radiological tumor diameter > 2.5 cm ( odds ratio [ or ] = 5.667 , p = 0.010 ) , elevated ca199 ( or = 4.714 , p = 0.017 ) , high tumor grading ( g2:g1 , or = 6.125 , p = 0.007 ; g3:g1 , or = 14.000 , p = 0.000322 ) , and presence of symptoms ( or = 3.545 , p = 0.026 ) [ table 3 ] . in the multivariate analysis , tumor grading ( g2 vs. g1 : or = 6.287 , p = 0.008 ; g3 vs. g1 : or = 12.407 , p = 0.001 ) was an independent predictor of lnm [ table 3 ] . considering the difficulty of preoperative retrievability of tumor grade , we excluded tumor grade from the analysis , and as a result , radiological tumor size > 2.5 cm ( or = 5.430 , p = 0.013 ) and presence of symptoms ( or = 3.366 , p = 0.039 ) were independently associated with lnm [ table 3 ] . compared to neoplasms with radiological size > 2.5 cm ( 32.1% ) , tumors 2.5 cm had an obviously lower risk of lnm ( 7.7% ) . in our study , incidentally discovered small ( 2.5 cm ) nf - pnets had a very low - risk of lnm ( 4.2% ) and no cases of postoperative recurrence or death due to the disease occurred .
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korea provides cash compensation to occupationally injured workers as a part of worker 's compensation insurance . interest in the return - to - work of occupationally injured workers has risen since 2001 , and policy to promote their return - to - work has been in full scale since 2005 ( 1 ) . supported by various businesses led by the korea workers ' compensation & welfare service , the return - to - work rate of occupationally injured workers in korea showed marked increases to 49.9% in 2007 and 70.4% in 2011 ( 2 ) . although this rate increased rapidly within a short period , related studies , have been limited , focusing on demographic characteristics , occupational injury - related characteristics , vocational rehabilitation , and correlation with business owners that influence return - to - work ( 1 , 3 - 6 ) . research in other countries has mostly focused on individual factors , such as the worker 's age , educational level , vocational characteristics , and psychosocial factors , or examined return - to - work programs or business owner factors . most studies investigated impairment severity as a factor influencing return - to - work ( 7 - 13 ) . however , impairment includes not only simple anatomical or functional problems but also limitations of activities or participation ( 14 ) . the world health organization considers impairment to be a comprehensive notion including body function and structure , limitations of an individual 's participation in society or in an occupation , and environmental factors ( 15 ) . the perspectives of different countries regarding impairment vary , and people accept even the same impairment differently ; thus , each country considers different limitations to occupations and activities according to impairment ( 16 ) . ( 17 ) analyzed impairment assessment and income reduction in california and found that income reduction of the same impairment grade differed according to impairment type . accordingly , california established standards to differently assess the disability rate according to impairment type . korea took these standards as a benchmark for assessing the disability rate according to occupation and impairment type ( 18 , 19 ) . therefore , because impairment type influences occupation and future income , it is believed to influence return - to - work . this article examines the effect of impairment type among occupationally injured workers on their return - to - work . this study was conducted with 109,746 participants with impairment grades who received medical treatment due to occupational injury during the 3 yr from january 1 , 2009 to december 31 , 2011 . the data on the subjects were acquired from the administrative data of the korea workers ' compensation & welfare service ( kcomwel ) . rehabilitation counselors in 55 kcomwel branches confirmed the status of return - to - work through the employment information acquired from the korea employment information services ( keis ) every december and from telephone interviews . we excluded from the study 20,655 persons who did not have clearly locatable injuries , 4,898 persons whose return - to - work could not be determined , and 4,072 persons without company information before injuries . we also excluded 140 persons with pneumoconiosis , 87 with oral function impairment , 118 with nasal function impairment , and 82 with bony deformity . return - to - work was classified for investigation into four types : " return to former work " , " work at a new firm " , " self - employment " , and " unemployment " . the first three categories were defined as completion of return - to - work , and " unemployment " as incompletion of return - to - work . the impairment grades and impairment types were decided by a physician according to the korean workers ' compensation act guidelines . the impairment classification has 14 grades , with the severest impairment as grade 1 , and the slightest as grade 14 . this article reclassified impairment severity into 4 groups : severe ( grades 1 - 3 ) , moderately severe ( grades 4 - 7 ) , moderate ( grades 8 - 10 ) , and mild ( grades 11 - 14 ) . the korean workers ' compensation act guidelines classify 26 types of impairment according to affected body parts and physiologic functions ; however , this article reclassified the impairment types into 9 groups . upper limbs , lower limbs , and spine disabilities were included as anatomical and functional disabilities . arm and finger disabilities were included with upper limb impairments ; and leg and foot disabilities were included with lower limb impairments . eyelid , auricle , and outside nose disabilities and scars were reclassified as figure impairments . we excluded masticatory disorder , speaking disorder , nasal function disorder , body deformity , pneumoconiosis , and thoracoabdominal disorder because they were difficult to combine with other disorders , and because they have low frequency . the pre - injury business size was classified into businesses with fewer than 30 workers , between 30 and 49 , between 50 and 99 , and over 100 . the pre - injury occupational category was largely classified into white collar , blue collar , and service workers . we performed chi - square tests to examine the factors that influence return - to - work , return - to - work according to impairment type , and characteristics that distinguish impairment grades . to investigate the characteristics of return - to - work according to impairment type , we performed logistic regression adjusted by age , sex , impairment grade , pre - injury business size , and pre - injury occupational category . logistic regression was used to analyze the association between the level of return - to - work , " return to former work " , " work at a new firm " , and " self - employment " , and impairment type , adjusted by age , sex , impairment severity , pre - injury businesses size , and pre - injury occupational category . examining for multicolinearity of independent variables , we found the variance inflation factor ( vif ) to be less than 10 , indicating that there was no multicolinearity . this study was approved by the institutional review board of human research of yonsei university ( approval no . this study was conducted with 109,746 participants with impairment grades who received medical treatment due to occupational injury during the 3 yr from january 1 , 2009 to december 31 , 2011 . the data on the subjects were acquired from the administrative data of the korea workers ' compensation & welfare service ( kcomwel ) . rehabilitation counselors in 55 kcomwel branches confirmed the status of return - to - work through the employment information acquired from the korea employment information services ( keis ) every december and from telephone interviews . we excluded from the study 20,655 persons who did not have clearly locatable injuries , 4,898 persons whose return - to - work could not be determined , and 4,072 persons without company information before injuries . we also excluded 140 persons with pneumoconiosis , 87 with oral function impairment , 118 with nasal function impairment , and 82 with bony deformity . return - to - work was classified for investigation into four types : " return to former work " , " work at a new firm " , " self - employment " , and " unemployment " . the first three categories were defined as completion of return - to - work , and " unemployment " as incompletion of return - to - work . the impairment grades and impairment types were decided by a physician according to the korean workers ' compensation act guidelines . the impairment classification has 14 grades , with the severest impairment as grade 1 , and the slightest as grade 14 . this article reclassified impairment severity into 4 groups : severe ( grades 1 - 3 ) , moderately severe ( grades 4 - 7 ) , moderate ( grades 8 - 10 ) , and mild ( grades 11 - 14 ) . the korean workers ' compensation act guidelines classify 26 types of impairment according to affected body parts and physiologic functions ; however , this article reclassified the impairment types into 9 groups . upper limbs , lower limbs , and spine disabilities were included as anatomical and functional disabilities . arm and finger disabilities were included with upper limb impairments ; and leg and foot disabilities were included with lower limb impairments . eyelid , auricle , and outside nose disabilities and scars were reclassified as figure impairments . we excluded masticatory disorder , speaking disorder , nasal function disorder , body deformity , pneumoconiosis , and thoracoabdominal disorder because they were difficult to combine with other disorders , and because they have low frequency . the pre - injury business size was classified into businesses with fewer than 30 workers , between 30 and 49 , between 50 and 99 , and over 100 . the pre - injury occupational category was largely classified into white collar , blue collar , and service workers . we performed chi - square tests to examine the factors that influence return - to - work , return - to - work according to impairment type , and characteristics that distinguish impairment grades . to investigate the characteristics of return - to - work according to impairment type , we performed logistic regression adjusted by age , sex , impairment grade , pre - injury business size , and pre - injury occupational category . logistic regression was used to analyze the association between the level of return - to - work , " return to former work " , " work at a new firm " , and " self - employment " , and impairment type , adjusted by age , sex , impairment severity , pre - injury businesses size , and pre - injury occupational category . examining for multicolinearity of independent variables , we found the variance inflation factor ( vif ) to be less than 10 , indicating that there was no multicolinearity . this study was approved by the institutional review board of human research of yonsei university ( approval no . 4 - 2013 - 0365 ) . the sample consisted of 67,182 males ( 84.7% ) and 12,146 females ( 15.3% ) , and the average age was 47.6 yr ( sd , 11.4 yr ) . the mild impairment group was the largest with 60,128 participants , and the most common impairment type was upper limbs impairment , which affected 31,292 participants ( 39.5% ) . in case of the pre - injury business size , the number of participants who returned to work was 55,154 ( 69.5% ) ; and those who did not were 24,174 ( 30.5% ) . with respect to the rate of return - to - work by age , workers in their 30s and 40s had the highest return - to - work rate ( 79.3% ) and those in their 60s or older had the lowest ( 52.0% ) . in general , the rate of return - to - work decreased with age ( p<0.001 ) . the rate of return - to - work in male was 71.2% , which was higher than the rate in female ( 60.5% ) ( p<0.001 ) . for the rate of return - to - work according to impairment type , figure impairments showed the highest rate of 78.0% , and neuropsychiatric impairments showed the lowest at 30.3% ( p<0.001 ) . for the rate of return - to - work according to impairment grade , the mild group ( grades 11 - 14 ) had the highest rate at 72.4% ; and the severe group ( grades 1 - 3 ) had the lowest at 16.8% ( p<0.001 ) . as a whole , pre - injury business size of over 100 workers had 72.8% of return - to - work , while those with less than 30 workers showed 68.2% ( p<0.001 ) . regarding pre - injury occupational category , white collar workers showed the highest rate of return - to - work at 80.1% , and service workers showed the lowest at 64.0% ( table 1 ; p<0.001 ) . for impairments of the upper and lower limbs and eyes , the mild group ( grades 11 - 14 ) showed the highest rate of return - to - work ; and the severe group ( grades 1 - 3 ) showed the lowest ( p<0.001 ) . for spine injuries , the moderately severe group ( grades 4 - 7 ) showed the highest rate of return - to - work at 72.2% , but the sample size was small . therefore , except for the group , the mild group showed the highest rate of return - to - work . for injuries of the oral cavity , ears , and figure , there were no statistically significant differences in return - to - work rates between the impairment grades . for neuropsychiatric impairment , the moderate group ( grades 8 - 10 ) showed the highest rate of return - to - work at 46.4% , and the severe group ( grades 1 - 3 ) was the lowest at 16.9% ( p<0.001 ) . for upper and lower limb , eyes , and neuropsychiatric impairments , the rate of return - to - work decreased with the severity grade . for injuries to the oral cavity , ears , and figure , there were no statistically significant differences in return - to - work rates between the impairment severity grades ( table 2 ) . logistic regression was used to analyze the association between return to work and impairment type , adjusted by age , sex , impairment severity , pre - injury businesses size , and pre - injury occupational category . compared to injuries of the upper limbs , the odds ratio of return to work was 0.63 ( 95% ci , 0.60 - 0.65 ) for injuries involving the lower limbs , 0.62 ( 95% ci , 0.59 - 0.66 ) for the spine , 0.75 ( 95% ci , 0.66 - 0.86 ) for the eyes , 0.44 ( 95% ci , 0.37 - 0.53 ) for the ears , 0.75 ( 95% ci , 0.72 - 0.79 ) for pain , and 0.36 ( 95% ci , 0.32 - 0.41 ) for neuropsychiatric impairment which were statistically significant ( table 3 ) . logistic regression was used to analyze the association between the level of return - to - work , " return to former work " , " work at a new firm " , and " self - employment " , and impairment type , adjusted by age , sex , impairment severity , pre - injury businesses size , and pre - injury occupational category . in case of return to former work , compared to injuries of the upper limbs , the odds ratio was 0.65 ( 95% ci , 0.62 - 0.68 ) for injuries involving the lower limbs , 0.64 ( 95% ci , 0.61 - 0.67 ) for the spine , 0.67 ( 95% ci , 0.59 - 0.76 ) for the eyes , 0.67 ( 95% ci , 0.54 - 0.83 ) for the oral cavity , 0.29 ( 95% ci , 0.23 - 0.36 ) for the ears , 0.76 ( 95% ci , 0.64 - 0.91 ) for the figure , 0.62 ( 95% ci , 0.60 - 0.65 ) for pain , and 0.38 ( 95% ci , 0.33 - 0.43 ) for neuropsychiatric impairment which were statistically significant . in case of work at a new firm , compared to injuries of the upper limbs , the odds ratio was 0.77 ( 95% ci , 0.65 - 0.91 ) for involving neuropsychiatric impairment , 1.49 ( 95% ci , 1.20 - 1.84 ) for the oral cavity , 1.51 ( 95% ci , 1.24 - 1.85 ) for the ears , 1.28 ( 95% ci , 1.06 - 1.54 ) for the figure , and 1.26 ( 95% ci , 1.21 - 1.32 ) for pain which were statistically significant . however , lower limbs , spine , and eyes impairment had no statistical significance . in case of self - employment , compared to injuries of the upper limbs , the odds ratio was 1.23 ( 95% ci , 1.11 - 1.35 ) for injuries involving the lower limbs , 1.38 ( 95% ci , 1.23 - 1.54 ) for the spine , 1.40 ( 95% ci , 1.07 - 1.82 ) for the eyes , and 1.17 ( 95% ci , 1.06 - 1.29 ) for pain which were statistically significant . oral cavity , ears , figure , and neuropsychiatric impairment had no statistical significance ( table 4 ) . various factors are related to the return - to - work of occupationally injured workers . among them prior studies on impairment and return - to - work have emphasized impairment in specific parts as well as severity of impairment ( 7 , 21 - 24 ) . ( 23 ) found that not only severity of impairment but also the locations and causes of impairment had important effects on return - to - work . however , chang et al . ( 25 ) reported that , even if impairment severity are the same in upper limbs , they showed differences in return - to - work according to impairment types . therefore , it was found that not only severity of impairment but also impairment type play important roles in return - to - work . the study results showed statistical significance relative to upper limb impairment , of the lower ors for involving the spine , eyes , ears , pain , and neuropsychiatric impairment on return - to - work . on the other hand it is believed that general opinions in korea about impairment are reflected in the results . differing from expert opinion , people in the general public tend to think that upper limb impairment is less serious than lower limb impairment , and that impairments of the eyes , ears , mental ability , intellect , or spine are more serious ( 26 ) . experts in impairment evaluation consider arm function to be 60% and leg function to be 40% of the whole body functioning ( 14 , 27 ) . however , korean people commonly believe that leg function equally or more important than arm function ( 26 ) . in korea , because social conditions such as transportation , road conditions , and building structures are unfavorable for the disabled , impairment related to walking and movement is regarded to be most important . therefore , we suggest that impairments involving the lower limbs , spine , or eyes lead to the greatest difficulties in finding jobs and returning to work because they are commonly considered to be more serious . when return - to - work is classified into return to the former work , work at a new firm , and self - employment to examine the return - to - work ors according to impairment types , we found that impairments of the lower limbs , spine , and eyes had a lower rate of return to the former work ( or 0.64 - 0.67 ) , but a higher rate of self - employment ( or 1.23 - 1.40 ) , compared to upper limbs impairment . we believe that this difference shows that the difficulty arises in finding a job in compliance with an individual 's personal situation rather than from a problem with return - to - work due to objective functional impairment . in korea , pain disorder had only one impairment grade , mild . the number in this group of injured workers was next highest after the group with upper limb impairment . the return - to - work or for impairment due to pain was lower than that of upper limb impairment , which we believe is not because pain disorder restrict physical function but because of avoidance of return to the original work , based on the worker 's subjective judgment . that is , pain disorder , compared to upper limb impairment , showed undesirable to return to the former work ( or 0.62 ) but desirable to work at a new firm ( or 1.26 ) and to have self - employment ( or 1.17 ) . therefore , it appears that pain disorder often activates a subjective avoidance of the former work . in fact , pain disorder is viewed not as clear impairment but as feigned illness ( 28 ) . in this study , neuropsychiatric impairment had lower or for the return - to - work compared to upper limb impairment . specifically , we found that return to the former work ( or , 0.38 ) and work at a new firm ( or , 0.77 ) were lower , and self - employment ( or , 0.95 ) was similar , to that of upper limb impairment . in the study cases of neuropsychiatric impairment , impairment grades ranged from severe to moderate , and thus , it is assumed that the reason for not returning to work is health restrictions . in fact , health was the reason given for unemployment by 87.1% of participants with neurological disorder who did not return - to - work . for impairments of the ears , compared to upper limbs impairment , the return - to - work or was low . specifically , return to the former work was lower ( or , 0.29 ) , work at a new firm was higher ( or , 1.51 ) , and self - employment ( or , 1.18 ) was similar to that of upper limb impairment . ears impairment differed from other disabilities in that the exposure period of the harmful factor ( noise ) increased impairment levels with increasing age . it appears that the work environment of the former work influenced the severity of the impairment . this suggests that these participants preferred to work in another place with less noise exposure . results showed that participants with impairments of the lower limbs , spine , and eyes and related to moving the body were more likely to become self - employed ; workers with impairments of the oral cavity and figure were most likely to work at a new firm ; those with pain disorder tended to return - to - work at a new firm or to become self - employed ; those with serious neuropsychiatric impairment mainly found self - employment ; and those with ears impairment usually returned to work at a new firm . it is significant that return - to - work ors according to these impairment types were statistically significant even after adjusting pre - injury occupational categories . because the return - to - work rates of blue collar and service workers , who do manual labor , were lower than for white collar workers . the different views of experts versus the general public about impairment , impairment severity , and impairment type are likely to affect return - to - work . this study minimized the effects of selection bias that may have been created by utilizing the national institution 's data through overall inspections of the return - to - work of occupationally injured workers . in addition , because this study was not based on a questionnaire about impairment types and levels but was instead based on evaluations by doctors , these have high accuracy . however , because this study concentrated on people who were recognized as occupationally injured workers and given impairment grades , it has the limitation that it did not include the disabled who were not recognized as occupationally injured workers . in conclusion , the study findings indicate that , in addition to individual characteristics and socioeconomic factors , the impairment severity , type , and patterns should be seriously considered in the planning by businesses and others to help injured workers return - to - work , such as via vocational rehabilitation .
this study examined the association between return - to - work and impairment type . database of the korea workers ' compensation and welfare service was used to identify disabled persons ; and return - to - work information was obtained from the korea employment information services database . the study participants were 79,328 persons who received workers ' compensation and who were confirmed as disabled during 2009 - 2011 . logistic regression was used to analyze the association between return - to - work and impairment type , adjusted by age , sex , impairment severity , pre - injury businesses size , and pre - injury occupational category . compared to injuries of the upper limbs , the odds ratio of return - to - work was 0.63 ( 95% ci , 0.60 - 0.65 ) for injuries involving the lower limbs , 0.62 ( 95% ci , 0.59 - 0.66 ) for the spine , 0.75 ( 95% ci , 0.66 - 0.86 ) for the eyes , 0.98 ( 95% ci , 0.77 - 1.25 ) for the oral cavity , 0.44 ( 95% ci , 0.37 - 0.53 ) for the ears , 1.02 ( 95% ci , 0.83 - 1.25 ) for the figure , 0.75 ( 95% ci , 0.72 - 0.79 ) for pain , and 0.36 ( 95% ci , 0.32 - 0.41 ) for neuropsychiatric impairment . these findings indicate that impairment type influences return - to - work rates .
INTRODUCTION MATERIALS AND METHODS Study participants Variables Data analysis Ethics statement RESULTS DISCUSSION
to investigate the characteristics of return - to - work according to impairment type , we performed logistic regression adjusted by age , sex , impairment grade , pre - injury business size , and pre - injury occupational category . logistic regression was used to analyze the association between the level of return - to - work , " return to former work " , " work at a new firm " , and " self - employment " , and impairment type , adjusted by age , sex , impairment severity , pre - injury businesses size , and pre - injury occupational category . to investigate the characteristics of return - to - work according to impairment type , we performed logistic regression adjusted by age , sex , impairment grade , pre - injury business size , and pre - injury occupational category . logistic regression was used to analyze the association between the level of return - to - work , " return to former work " , " work at a new firm " , and " self - employment " , and impairment type , adjusted by age , sex , impairment severity , pre - injury businesses size , and pre - injury occupational category . logistic regression was used to analyze the association between return to work and impairment type , adjusted by age , sex , impairment severity , pre - injury businesses size , and pre - injury occupational category . compared to injuries of the upper limbs , the odds ratio of return to work was 0.63 ( 95% ci , 0.60 - 0.65 ) for injuries involving the lower limbs , 0.62 ( 95% ci , 0.59 - 0.66 ) for the spine , 0.75 ( 95% ci , 0.66 - 0.86 ) for the eyes , 0.44 ( 95% ci , 0.37 - 0.53 ) for the ears , 0.75 ( 95% ci , 0.72 - 0.79 ) for pain , and 0.36 ( 95% ci , 0.32 - 0.41 ) for neuropsychiatric impairment which were statistically significant ( table 3 ) . logistic regression was used to analyze the association between the level of return - to - work , " return to former work " , " work at a new firm " , and " self - employment " , and impairment type , adjusted by age , sex , impairment severity , pre - injury businesses size , and pre - injury occupational category . in case of return to former work , compared to injuries of the upper limbs , the odds ratio was 0.65 ( 95% ci , 0.62 - 0.68 ) for injuries involving the lower limbs , 0.64 ( 95% ci , 0.61 - 0.67 ) for the spine , 0.67 ( 95% ci , 0.59 - 0.76 ) for the eyes , 0.67 ( 95% ci , 0.54 - 0.83 ) for the oral cavity , 0.29 ( 95% ci , 0.23 - 0.36 ) for the ears , 0.76 ( 95% ci , 0.64 - 0.91 ) for the figure , 0.62 ( 95% ci , 0.60 - 0.65 ) for pain , and 0.38 ( 95% ci , 0.33 - 0.43 ) for neuropsychiatric impairment which were statistically significant . in case of work at a new firm , compared to injuries of the upper limbs , the odds ratio was 0.77 ( 95% ci , 0.65 - 0.91 ) for involving neuropsychiatric impairment , 1.49 ( 95% ci , 1.20 - 1.84 ) for the oral cavity , 1.51 ( 95% ci , 1.24 - 1.85 ) for the ears , 1.28 ( 95% ci , 1.06 - 1.54 ) for the figure , and 1.26 ( 95% ci , 1.21 - 1.32 ) for pain which were statistically significant . in case of self - employment , compared to injuries of the upper limbs , the odds ratio was 1.23 ( 95% ci , 1.11 - 1.35 ) for injuries involving the lower limbs , 1.38 ( 95% ci , 1.23 - 1.54 ) for the spine , 1.40 ( 95% ci , 1.07 - 1.82 ) for the eyes , and 1.17 ( 95% ci , 1.06 - 1.29 ) for pain which were statistically significant . the study results showed statistical significance relative to upper limb impairment , of the lower ors for involving the spine , eyes , ears , pain , and neuropsychiatric impairment on return - to - work . when return - to - work is classified into return to the former work , work at a new firm , and self - employment to examine the return - to - work ors according to impairment types , we found that impairments of the lower limbs , spine , and eyes had a lower rate of return to the former work ( or 0.64 - 0.67 ) , but a higher rate of self - employment ( or 1.23 - 1.40 ) , compared to upper limbs impairment .
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the imgt / hla database was established to provide a locus - specific database ( lsdb ) for the allelic sequences of the genes in the hla system , also known as the human major histocompatibility complex ( mhc ) . this complex of over four megabases is located within the 6p21.3 region of the short arm of human chromosome 6 and contains in excess of 220 genes ( 1 ) . the core genes of interest in the hla system are 21 highly polymorphic hla genes that mediate the host response to infectious disease and influence the outcome of cell and organ transplants . with a nomenclature spanning over 50 genes and 3000 alleles , there is an obvious need for a lsdb to curate these highly polymorphic variants . the sequencing of hla alleles began in the late 1970s predominantly using protein - based techniques to determine the sequences of hla class i allotypes . the first complete hla class i allotype sequence , b7.2 now know as b*070201 , was published in 1979 ( 2 ) . the first hla class ii allele defined by dna sequencing , dra*0101 , followed in 1982 ( 3 ) . while the first hla antigens were named during the 1960s ( 4 ) , the first hla dna sequences or alleles were named by the who nomenclature committee for factors of the hla system ( 5 ) in 1987 . at that time 12 class two years later , in 1989 , the nomenclature committee assigned official allele names to 56 novel class i alleles and 78 class ii alleles ( 6 ) . advances and availability of sequencing technology meant that in 2007 the nomenclature committee was able to name over 400 new alleles , with the number of hla - b alleles exceeding 1000 in 2008 . the dissemination of new allele names and sequences is of paramount importance in the clinical setting . the importance of a single recognized source for this data led to the first incarnation of the database , the hla sequence databank ( hla - db ) ( 7 ) , which allowed the periodic publication of hla class i ( 811 ) and class ii ( 1217 ) sequence alignments in a variety of journals . by 1995 , the numbers of new alleles being reported warranted the publication of monthly nomenclature updates ( 18 ) , which continues to this day . that year also saw the first distribution of the hla sequence alignments online through the web pages of the tissue antigen laboratory at the imperial cancer research fund ( icrf ) , london , uk . this work transferred to the anthony nolan research institute ( anri ) in 1996 where it continues to this day . the latest incarnation , is the imgt / hla database ( 1922 ) , which began in 1997 as part of a collaboration involving the icrf , anri and the european bioinformatics institute ( ebi ) . the first public release of the imgt / hla database was made on the 16th december 1998 ( 23 ) . since then the database has been updated every 3 months , in a total of 40 releases , to include all the publicly available sequences officially named by the who nomenclature committee at the time of release . the imgt / hla database contains entries for all hla alleles , and alleles of some related genes , officially named by the nomenclature committee . these entries are derived from expertly annotated copies of the original embl - bank / genbank / ddbj entries . this means that the imgt / hla database may contain multiple entries for any single allele . these component entries are submitted to the database either by the original author , or by our curators , when sequences of interest have been identified by data - mining but have yet to be submitted to the database . to distinguish each imgt / hla entry from the component embl entries , each new allele the accession numbers follow the format hla00000 , where the 00000 represents a numerical code . it must be noted that all sequences within the imgt / hla database should also be available from the more general nucleotide sequence databases : embl - bank ( 24,25 ) , genbank ( 26 ) and the dna database of japan ( ddbj ) ( 27,28 ) . the main problem when accessing hla sequences from these databases lies in the definition of the sequence . despite the work of the members of the who nomenclature committee for factors of the hla system in monitoring hla allele designations and maintaining the sequences , they have no control of how sequences are defined in these generalist databases . readers should , therefore , be aware that entries in these generalist databases may be incorrectly named , contain unofficial designations or contain known , but uncorrected , sequencing errors . the main access point for the user is the world wide web ( www ) , which allows users to employ a number of search tools and other facilities to retrieve , manipulate and analyse hla data . the first area comprises information and help pages that provide background on the database and provide in - depth help on the tools and data available and documentation of the imgt / hla file formats . these core tools allow the users to perform sequence alignments , allele queries and sequence searches as well as queries more relevant to how the data are used and interpreted in a clinical setting . the third area comprises final pages that provide links to commonly used third - party applications such as the sequence - analysis tools at the ebi , including srs , blast and fasta . as the primary users of the database are members of the clinical hla community involved in transplantation of tissues and organs , the most commonly accessed tools have been written to aid in their common queries . all tools are written in perl as cgi scripts and access restricted views of the underlying oracle relational database . the transplant and tissue typing community have two main queries ; either to retrieve information on a particular allele or to view how a number of alleles differ in sequence . to answer these questions , the database provides a detailed report on any allele , as well as an interactive alignment tool to view how allelic sequences differ . the allele search tool provides a simple - to - use interface for retrieving allele information . the output , see figure 1 , for each allele includes the official allele designation , previously used designations and the unique imgt / hla accession number . other information provided includes the date that the allele was named , current status ( as some allele designations have been deleted ) and information on the individual or cell line from which the sequence was derived . links to all component embl - bank / genbank / ddbj entries are also included . recently , information from the hla dictionary ( 29 ) has also been added to some entries . the dictionary presents the serological equivalents of hla - a , -b , -c , -drb1 , -drb3 , -drb4 , -drb5 and -dqb1 allotypes . the data summarizes equivalents obtained by the who nomenclature committee for factors of the hla system , the international cell exchange ( ucla ) , the national marrow donor program ( nmdp ) , the 13th international histocompatibility workshop , recent publications and individual laboratories . any citations are also included with , wherever possible , a link to the pubmed entry for that citation . the pubmed link provides an online version of the abstract as well as links to other citations by the author and to similar papers . the final section of the output details the official nucleotide and protein sequence as well as any genomic sequence for the allele that is available . the report provides cross - references to a text flat - file in srs ( hla0001 ) , the omim entry for hla - a , the source entries in embl - bank ( aj278305-z93949 ) and to the seminal citations in pubmed . the full entry also contains the nucleotide sequence at both the cds and genomic level . the report provides cross - references to a text flat - file in srs ( hla0001 ) , the omim entry for hla - a , the source entries in embl - bank ( aj278305-z93949 ) and to the seminal citations in pubmed . other information provided includes links to the source material the full entry also contains the nucleotide sequence at both the cds and genomic level . hla allele sequences can differ from each other by as little a single nucleotide substitution , within a genomic sequence of 3300 bases . such nucleotide differences between the alleles of prospective transplant donors and recipients can make the difference between a successful transplant , graft failure and death . this means that the database must be able to quickly and easily display this information to the user . the hla community is interested in seeing the polymorphisms in terms of the changes to the sequence rather than as a list of individual single nucleotide polymorphisms ( snps ) . to this end , we have developed the alignment tool , rather than push the users into producing their own alignments for the sequences of interest or simply just reporting the polymorphic positions . these alignments allow a visual interpretation of sequence similarity so that polymorphic positions and motifs , found in multiple alleles , can easily be identified . the representation of hla sequences in this manner can be useful when designing reagents for hla typing , such as primers or oligonucleotide probes or comparing mismatches when looking at potential donors . the interface provided lets the user define a number of key variables for the alignments , these include the gene(s ) to be aligned , the alleles of interest and the reference sequence they are aligned against , as well as the type of sequence : nucleotide coding region , nucleotide genomic and the amino - acid sequence of the protein , to be aligned . the alignment tool uses standard formatting conventions for the display of sequence alignments and alignments adhere to standard conventions for displaying evolutionary events and numbering . an example of alignments specially tailored to the hla transplant community is in the presentation of alleles with an alternative splice site . for most alleles , the nucleotide sequence displayed as a coding sequence ( cds ) the sequence displayed will contain the spliced exons plus any alternatively spliced segment that lies within the traditional exon framework , when compared to a reference sequence . the otherwise missing sequence is also included and highlighted to emphasize the region of interest , rather than omit it , a feature important for the design of reagents that allow for typing of the alternatively spliced allele . figure 2 illustrates how an alternatively spliced allele ( a*0111n ) is represented in the sequence alignments . identity to the a*01010101 allele is shown by hyphens ( - ) and the exon borders are indicated by a pipe ( | ) . the single nucleotide mutation in the third base of codon 175 , introduces a new splice site and as a result the region shaded in green is spliced out and is not part of the cds . it is helpful to include the otherwise missing sequence and highlight it to emphasize the region of interest , rather than omit it , this feature is important for the design of reagents to allow for typing of this allele . identity to the a*01010101 allele is shown by hyphens ( - ) and the exon borders are indicated by a pipe ( | ) . the single nucleotide mutation in the third base of codon 175 , introduces a new splice site and as a result the region shaded in green is spliced out and is not part of the cds . it is helpful to include the otherwise missing sequence and highlight it to emphasize the region of interest , rather than omit it , this feature is important for the design of reagents to allow for typing of this allele . the previous text - only versions of the alignments are still requested and as a result , are available from the anri website and in a zipped file in the ftp directory . for users who prefer to use other existing software to produce their own alignments , then the ftp directory contains files in popular formats for them to download and import . recent developments to the website have seen the addition of a search tool for identifying primer and probe sequences . many hla typing laboratories who have designed their own reagents for hla typing have spreadsheets detailing probe - hit patterns for different alleles . these are used when typing samples to identify known alleles based on the reaction patterns seen . each time a new release of the database was made it was necessary to manually update these ever - expanding lists by cross - referencing the primer sequence with the sequence alignments , which with the rapidly increasing numbers of alleles was becoming a slow and laborious task . the new probe & primer search tool allows users to enter a list of primer sequences and the tool will search the known alleles for the presence of these sequences and report any matches in a file format suitable for cutting - and - pasting into existing spreadsheets . the tool is currently limited to coding sequences but as the number of genomic sequences in the database expands it will be modified to search these regions as well . the imgt / hla database is also involved in developing data format standards for hla information exchange between the reference database , hla typing laboratories and commercial typing - kit manufacturers . this work , which is being performed in collaboration with other immuno - informatics groups will provide both an xml output format for the imgt / hla database as well as xml reporting format for tissue typing laboratories . the xml output will contain similar information to that described for the flat files and allele output ( 30 ) . the rise of high - throughput genome typing has seen the expansion of genome browsers like ensembl ( 31 ) . these browsers have a different priority in how you view a gene , the alleles and any snps . the imgt / hla database is working with groups like ensembl , embl - bank and uniprot ( 32 ) to help define hla references to suit all parties at the different levels through the development of locus reference genomic sequences ( lrgs ) . a current project is to improve cross - referencing of the hla data with that from other systems . the aim is to make sure that when users find an entry referring to an hla allele in a third - party system they can also find a link back to the imgt / hla entry for that allele , which should be considered the primary reference for the sequence . the main access point for the user is the world wide web ( www ) , which allows users to employ a number of search tools and other facilities to retrieve , manipulate and analyse hla data . the first area comprises information and help pages that provide background on the database and provide in - depth help on the tools and data available and documentation of the imgt / hla file formats . these core tools allow the users to perform sequence alignments , allele queries and sequence searches as well as queries more relevant to how the data are used and interpreted in a clinical setting . the third area comprises final pages that provide links to commonly used third - party applications such as the sequence - analysis tools at the ebi , including srs , blast and fasta . as the primary users of the database are members of the clinical hla community involved in transplantation of tissues and organs , the most commonly accessed tools have been written to aid in their common queries . all tools are written in perl as cgi scripts and access restricted views of the underlying oracle relational database . the transplant and tissue typing community have two main queries ; either to retrieve information on a particular allele or to view how a number of alleles differ in sequence . to answer these questions , the database provides a detailed report on any allele , as well as an interactive alignment tool to view how allelic sequences differ . the allele search tool provides a simple - to - use interface for retrieving allele information . the output , see figure 1 , for each allele includes the official allele designation , previously used designations and the unique imgt / hla accession number . other information provided includes the date that the allele was named , current status ( as some allele designations have been deleted ) and information on the individual or cell line from which the sequence was derived . links to all component embl - bank / genbank / ddbj entries are also included . recently , information from the hla dictionary ( 29 ) has also been added to some entries . the dictionary presents the serological equivalents of hla - a , -b , -c , -drb1 , -drb3 , -drb4 , -drb5 and -dqb1 allotypes . the data summarizes equivalents obtained by the who nomenclature committee for factors of the hla system , the international cell exchange ( ucla ) , the national marrow donor program ( nmdp ) , the 13th international histocompatibility workshop , recent publications and individual laboratories . any citations are also included with , wherever possible , a link to the pubmed entry for that citation . the pubmed link provides an online version of the abstract as well as links to other citations by the author and to similar papers . the final section of the output details the official nucleotide and protein sequence as well as any genomic sequence for the allele that is available . the report provides cross - references to a text flat - file in srs ( hla0001 ) , the omim entry for hla - a , the source entries in embl - bank ( aj278305-z93949 ) and to the seminal citations in pubmed . the full entry also contains the nucleotide sequence at both the cds and genomic level . the report provides cross - references to a text flat - file in srs ( hla0001 ) , the omim entry for hla - a , the source entries in embl - bank ( aj278305-z93949 ) and to the seminal citations in pubmed . the full entry also contains the nucleotide sequence at both the cds and genomic level . hla allele sequences can differ from each other by as little a single nucleotide substitution , within a genomic sequence of 3300 bases . such nucleotide differences between the alleles of prospective transplant donors and recipients can make the difference between a successful transplant , graft failure and death . this means that the database must be able to quickly and easily display this information to the user . the hla community is interested in seeing the polymorphisms in terms of the changes to the sequence rather than as a list of individual single nucleotide polymorphisms ( snps ) . to this end , we have developed the alignment tool , rather than push the users into producing their own alignments for the sequences of interest or simply just reporting the polymorphic positions . these alignments allow a visual interpretation of sequence similarity so that polymorphic positions and motifs , found in multiple alleles , can easily be identified . the representation of hla sequences in this manner can be useful when designing reagents for hla typing , such as primers or oligonucleotide probes or comparing mismatches when looking at potential donors . the interface provided lets the user define a number of key variables for the alignments , these include the gene(s ) to be aligned , the alleles of interest and the reference sequence they are aligned against , as well as the type of sequence : nucleotide coding region , nucleotide genomic and the amino - acid sequence of the protein , to be aligned . the alignment tool uses standard formatting conventions for the display of sequence alignments and alignments adhere to standard conventions for displaying evolutionary events and numbering . an example of alignments specially tailored to the hla transplant community is in the presentation of alleles with an alternative splice site . for most alleles , the nucleotide sequence displayed as a coding sequence ( cds ) the sequence displayed will contain the spliced exons plus any alternatively spliced segment that lies within the traditional exon framework , when compared to a reference sequence . the otherwise missing sequence is also included and highlighted to emphasize the region of interest , rather than omit it , a feature important for the design of reagents that allow for typing of the alternatively spliced allele . figure 2 illustrates how an alternatively spliced allele ( a*0111n ) is represented in the sequence alignments . identity to the a*01010101 allele is shown by hyphens ( - ) and the exon borders are indicated by a pipe ( | ) . the single nucleotide mutation in the third base of codon 175 , introduces a new splice site and as a result the region shaded in green is spliced out and is not part of the cds . it is helpful to include the otherwise missing sequence and highlight it to emphasize the region of interest , rather than omit it , this feature is important for the design of reagents to allow for typing of this allele . identity to the a*01010101 allele is shown by hyphens ( - ) and the exon borders are indicated by a pipe ( | ) . the single nucleotide mutation in the third base of codon 175 , introduces a new splice site and as a result the region shaded in green is spliced out and is not part of the cds . it is helpful to include the otherwise missing sequence and highlight it to emphasize the region of interest , rather than omit it , this feature is important for the design of reagents to allow for typing of this allele . the previous text - only versions of the alignments are still requested and as a result , are available from the anri website and in a zipped file in the ftp directory . for users who prefer to use other existing software to produce their own alignments , then the ftp directory contains files in popular formats for them to download and import . recent developments to the website have seen the addition of a search tool for identifying primer and probe sequences . many hla typing laboratories who have designed their own reagents for hla typing have spreadsheets detailing probe - hit patterns for different alleles . these are used when typing samples to identify known alleles based on the reaction patterns seen . each time a new release of the database was made it was necessary to manually update these ever - expanding lists by cross - referencing the primer sequence with the sequence alignments , which with the rapidly increasing numbers of alleles was becoming a slow and laborious task . the new probe & primer search tool allows users to enter a list of primer sequences and the tool will search the known alleles for the presence of these sequences and report any matches in a file format suitable for cutting - and - pasting into existing spreadsheets . the tool is currently limited to coding sequences but as the number of genomic sequences in the database expands it will be modified to search these regions as well . the imgt / hla database is also involved in developing data format standards for hla information exchange between the reference database , hla typing laboratories and commercial typing - kit manufacturers . this work , which is being performed in collaboration with other immuno - informatics groups will provide both an xml output format for the imgt / hla database as well as xml reporting format for tissue typing laboratories . the xml output will contain similar information to that described for the flat files and allele output ( 30 ) . the rise of high - throughput genome typing has seen the expansion of genome browsers like ensembl ( 31 ) . these browsers have a different priority in how you view a gene , the alleles and any snps . the imgt / hla database is working with groups like ensembl , embl - bank and uniprot ( 32 ) to help define hla references to suit all parties at the different levels through the development of locus reference genomic sequences ( lrgs ) . a current project is to improve cross - referencing of the hla data with that from other systems . the aim is to make sure that when users find an entry referring to an hla allele in a third - party system they can also find a link back to the imgt / hla entry for that allele , which should be considered the primary reference for the sequence . the imgt / hla database provides a centralized resource for everybody interested , clinically or scientifically , in the hla system . the database and accompanying tools allow the study of all hla alleles from a single site on the world wide web . it should aid in the management and continual expansion of hla nomenclature , providing an ongoing resource for the who nomenclature committee . the earliest version of the imgt / hla database , december 1998 , included only 964 alleles , covering 24 genes and was limited to much simpler tools and interfaces . the latest release , july 2008 , contained over 3300 alleles for 34 genes , with this number set to grow as the database continues to receive and name over 450 new alleles a year . the expansion of the database content has been reflected in its use , in 1999 the website averaged just over 1500 visitors per month , in 2008 this had increased to over 7500 visitors viewing over 40 000 pages per month . the challenge for the database is to keep up with this increase in sequences , develop new tools for the visualization of the sequences whilst maintaining the high standards set in the presentation and quality of the hla sequences and nomenclature to the research community . this work was supported by histogenetics ; abbott laboratories inc . ; the american society for histocompatibility and immunogenetics ; the anthony nolan trust ; bag healthcare ; biotest ; the european federation for immunogenetics ; innogenetics ; invitrogen ; the marrow foundation ; the national marrow donor program ; one lambda inc . ; initial support for the imgt / hla database project was from the imperial cancer research fund ( now cancer research uk ) and an eu biotech grant ( bio4ct960037 ) .
it is 10 years since the imgt / hla database was released , providing the hla community with a searchable repository of highly curated hla sequences . the hla complex is located within the 6p21.3 region of human chromosome 6 and contains more than 220 genes of diverse function . many of the genes encode proteins of the immune system and are highly polymorphic . the naming of these hla genes and alleles , and their quality control is the responsibility of the who nomenclature committee for factors of the hla system . through the work of the hla informatics group and in collaboration with the european bioinformatics institute , we are able to provide public access to this data through the website http://www.ebi.ac.uk / imgt / hla/. the first release contained 964 sequences , the most recent release 3300 sequences , with around 450 new sequences been added each year . the tools provided on the website have been updated to allow more complex alignments , which include genomic sequence data , as well as the development of tools for probe and primer design and the inclusion of data from the hla dictionary . regular updates to the website ensure that new and confirmatory sequences are dispersed to the hla community , and the wider research and clinical communities .
INTRODUCTION IMGT/HLA CONTENT Retrieving allele information and displaying polymorphisms Recent developments and future applications CONCLUSIONS FUNDING
the imgt / hla database was established to provide a locus - specific database ( lsdb ) for the allelic sequences of the genes in the hla system , also known as the human major histocompatibility complex ( mhc ) . this complex of over four megabases is located within the 6p21.3 region of the short arm of human chromosome 6 and contains in excess of 220 genes ( 1 ) . while the first hla antigens were named during the 1960s ( 4 ) , the first hla dna sequences or alleles were named by the who nomenclature committee for factors of the hla system ( 5 ) in 1987 . the importance of a single recognized source for this data led to the first incarnation of the database , the hla sequence databank ( hla - db ) ( 7 ) , which allowed the periodic publication of hla class i ( 811 ) and class ii ( 1217 ) sequence alignments in a variety of journals . the latest incarnation , is the imgt / hla database ( 1922 ) , which began in 1997 as part of a collaboration involving the icrf , anri and the european bioinformatics institute ( ebi ) . the first public release of the imgt / hla database was made on the 16th december 1998 ( 23 ) . the imgt / hla database contains entries for all hla alleles , and alleles of some related genes , officially named by the nomenclature committee . it must be noted that all sequences within the imgt / hla database should also be available from the more general nucleotide sequence databases : embl - bank ( 24,25 ) , genbank ( 26 ) and the dna database of japan ( ddbj ) ( 27,28 ) . despite the work of the members of the who nomenclature committee for factors of the hla system in monitoring hla allele designations and maintaining the sequences , they have no control of how sequences are defined in these generalist databases . the first area comprises information and help pages that provide background on the database and provide in - depth help on the tools and data available and documentation of the imgt / hla file formats . the data summarizes equivalents obtained by the who nomenclature committee for factors of the hla system , the international cell exchange ( ucla ) , the national marrow donor program ( nmdp ) , the 13th international histocompatibility workshop , recent publications and individual laboratories . the interface provided lets the user define a number of key variables for the alignments , these include the gene(s ) to be aligned , the alleles of interest and the reference sequence they are aligned against , as well as the type of sequence : nucleotide coding region , nucleotide genomic and the amino - acid sequence of the protein , to be aligned . this work , which is being performed in collaboration with other immuno - informatics groups will provide both an xml output format for the imgt / hla database as well as xml reporting format for tissue typing laboratories . the imgt / hla database is working with groups like ensembl , embl - bank and uniprot ( 32 ) to help define hla references to suit all parties at the different levels through the development of locus reference genomic sequences ( lrgs ) . the first area comprises information and help pages that provide background on the database and provide in - depth help on the tools and data available and documentation of the imgt / hla file formats . the data summarizes equivalents obtained by the who nomenclature committee for factors of the hla system , the international cell exchange ( ucla ) , the national marrow donor program ( nmdp ) , the 13th international histocompatibility workshop , recent publications and individual laboratories . the interface provided lets the user define a number of key variables for the alignments , these include the gene(s ) to be aligned , the alleles of interest and the reference sequence they are aligned against , as well as the type of sequence : nucleotide coding region , nucleotide genomic and the amino - acid sequence of the protein , to be aligned . this work , which is being performed in collaboration with other immuno - informatics groups will provide both an xml output format for the imgt / hla database as well as xml reporting format for tissue typing laboratories . the imgt / hla database is working with groups like ensembl , embl - bank and uniprot ( 32 ) to help define hla references to suit all parties at the different levels through the development of locus reference genomic sequences ( lrgs ) . the earliest version of the imgt / hla database , december 1998 , included only 964 alleles , covering 24 genes and was limited to much simpler tools and interfaces . the challenge for the database is to keep up with this increase in sequences , develop new tools for the visualization of the sequences whilst maintaining the high standards set in the presentation and quality of the hla sequences and nomenclature to the research community .
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disturbances within the arachidonic acid ( aa ) cascade have been proposed as a pathophysiological mechanism in the regulation of mood and suggested to contribute to the underlying biological background for bipolar disorder ( bazinet , 2010 ) . this is corroborated by preclinical evidence pointing to the arachidonic cascade as a target of drugs used to manage bipolar disorder ( rapoport and bosetti , 2002 ; bazinet , 2009 ) , indicating that lithium ( galimberti et al . , 2014 ) , valproate ( kieseppa et al . , 2014 ) , carbamazepine ( lee et al . , 2012 ) , and antipsychotics ( cheon et al . , 2011 in contrast , the antidepressants imipramine and fluoxetine , which may induce mania , increase brain aa turnover , while bupropion , which may be at lower risk of inducing mania ( post et al . these findings have led to the formulation of the arachidonic acid theory of bipolar disorder ( bazinet , 2009 ) . additional evidence of downstream disturbances in the cascade , with increased levels of aa - derived prostaglandins in saliva ( ohishi et al . , 1988 ) , cerebrospinal fluid ( linnoila et al . , 1983 ) , and peripheral blood ( lieb et al . have supported a role for this pathway in bipolar disorder . in a recent genome - wide association study meta - analysis , one of three novel loci identified to be associated with bipolar disorder was near the ptgfr gene encoding the prostaglandin f receptor ( chen et al . , 2013 ) , which is highly expressed in the brain . arachidonic acid , particularly abundant in the brain , is a polyunsaturated fatty acid present in the cell membrane phospholipids , from which it is freed by cytosolic phospholipase a2 ( cpla2 ; rapoport , 2014 ) . the isoenzyme cpla2 iva is selective for aa hydrolysis and its activity is modulated by lithium and carbamazepine ( rapoport et al . the expression of cpla2 iva , as well as of the cyclooxygenase ( cox)-2 enzyme , has been found to be altered in the post - mortem brain tissue of bipolar disorder patients ( kim et al . , 2011 ) . aa acts as a precursor in the production of prostaglandin h2 ( pgh2 ) , mediated by cox , which , in turn , is converted to prostaglandins , thromboxanes , or prostacyclins ( funk , 2001 ) . the conversion of pgh2 to prostaglandin d2 ( pgd2 ) is catalyzed by prostaglandin d synthase ( ptgds ) , encoded by the ptgds gene , and preferentially expressed in the brain . pgd2 functions as a neuromodulator as well as a trophic factor in the central nervous system ( taniguchi et al . , 2007 ) and reduction of both pgh2 and pgd2 is catalyzed by the aldo - keto reductase family 1 member c3 ( akr1c3 ) enzyme , encoded by the akr1c3 gene , resulting in synthesis of prostaglandin f2 alpha ( figure 1 ) . the arachidonic acid cascade and prostaglandin metabolism pathway related to the function of ptgds and akr1c3 . akr1c3 , aldo - keto reductase family 1 member c3 ; pgd2 , prostaglandin d2 ; pgf2 , prostaglandin f2 ; pgh2 , prostaglandin h2 ; pla2 , phospholipase a2 ; ptgds , prostaglandin d synthase . evidence of aa cascade and prostaglandin pathway dysregulation on a transcriptional level in bipolar disorder is limited . two studies by the same group , a case study ( n = 1 ; begemann et al . , 2008 ) and an extended case series ( n = 4 ; gurvich et al . , 2014 ) of rapid - cycling bipolar disorder patients identified the ptgds and akr1c3 as differentially regulated between manic and depressive episodes and a case - control study of children and young adults with bipolar disorder ( n = 9 ) and adhd ( marn - mndez et al . , 2012 ) found the ptgds gene was differentially expressed between bipolar disorder patients and patients with adhd . in bipolar disorder , which is characterized by phenotypically distinct , recurrent episodes of various polarities , gene expression alterations have the potential to inform on pathophysiological processes related to illness activity and affective state and to the nature of the illness itself . 2014 ) , gene expression changes longitudinally between affective states have not been investigated , and no studies have included assessment of healthy control subjects of these specific genes . in a recent meta - analysis of 17 studies of gene expression alterations in peripheral blood in bipolar disorder patients , comprising 565 patients and 418 healthy control subjects ( munkholm et al . , 2012 ) , we showed that findings were limited overall by lack of replication across studies and limited control for possible confounders of gene expression levels . the present study is the first to investigate repeated measures over time of the gene expression of ptgds and akr1c3 in rapid - cycling bipolar disorder patients in a euthymic or current affective state and in healthy control subjects . we hypothesized that mrna expression of ptgds and akr1c3 was deregulated in patients in a euthymic or current affective state compared with healthy control subjects as well as in bipolar disorder patients between current affective states ( depressed , manic / hypomanic , or mixed ) compared with those in the euthymic state . a longitudinal , naturalistic design was employed , accommodating assessment of patients during multiple affective states of varying polarity . patients with a potential diagnosis of rapid - cycling bipolar disorder were recruited through referral by psychiatrists at hospitals or outpatient facilities throughout the region of zealand , denmark , with study recruitment taking place during the period of june 2010 to may 2012 . inclusion criteria were : adults aged 1870 years ; and a diagnostic and statistical manual of mental disorders , 4th version ( dsm - iv ) diagnosis of rapid - cycling bipolar disorder , defined by the occurrence of at least four mood episodes ( mania , hypomania , depression , or mixed ) during the preceding year in the context of bipolar disorder . exclusion criteria were : significant physical illness ( ie chronic heart disease , chronic pulmonary disease , inflammatory disease , chronic infectious disease , or neurodegenerative disease ) ; current drug abuse ; insufficient danish language skills ; and pregnancy . two bipolar patients declined further examination after one and three month follow - ups , respectively ; the remaining bipolar patients were followed for a minimum of six months with a mean ( standard deviation [ sd ] ) follow - up period of 11.9 ( 3.0 ) months . upon signs of new affective episodes , patients were evaluated with clinical assessments of mood and collection of blood samples which , when possible , were repeated after return to a subsequent euthymic state or change to an affective episode of opposite polarity . in cases of clinical signs of acute infection , any allergic symptoms , or any other acute medical condition , assessment and biochemical analysis blood samples were , on average , collected from bipolar patients 3.41.7 ( range , 110 ) times during the study . samples were obtained during euthymia in 34 patients ( mean 2.01.3 [ 06 ] ) , major depression in 26 patients ( mean 1.71.7 [ 05 ] ) , mania / hypomania in 11 patients ( mean 0.71.2 [ 05 ] ) , and in a mixed state in a total of 6 patients ( mean 0.20.4 [ 01 ] ) . forty healthy control subjects were recruited among blood donors affiliated with the blood bank at rigshospitalet , copenhagen . inclusion criteria were : adults aged 1870 years ; and no history of psychiatric disorder in the subjects or their first - degree relatives . healthy control subjects were evaluated with clinical assessments and collection of blood samples on two separate occasions approximately three months apart . assessment and biochemical analysis were postponed if there were clinical signs of acute infection , any allergic symptoms , or any other acute medical condition . mean ( sd ) follow - up time for the healthy control subjects was 2.9 ( 0.9 ) months . the study protocol was approved by the committee on health research ethics of the capital region of denmark ( protocol no . all participants were assessed by a specialist in psychiatry ( dr munkholm ) , using standardized semi - structured interviews . the schedules for clinical assessment in neuropsychiatry interview ( wing et al . , 1990 ) was used for diagnostic purposes and was based on available case material , referral reports , the interview with the participant , and the hypomania checklist ( angst et al . , 2005 ) , completed by the participant . a dsm - iv diagnosis of rapid - cycling bipolar disorder was established for the patients and comorbid psychiatric illness , if present , was recorded . for healthy control subjects , a clinical diagnosis according to dsm - iv , without applying duration criteria , was established at each study visit concurrently with the collection of samples for laboratory analysis . severity of depressive symptoms was assessed using the 17-item hamilton depression rating scale ( hamd-17 ; hamilton , 1967 ) , employing a structured interview guide ( williams , 1990 ) translated to danish , and manic symptoms were assessed using the young mania rating scale ( ymrs ; young et al . , 1978 ) , with a time period of three days applied . medication , alcohol intake , and smoking habits during the two weeks prior to assessment were recorded . categories of affective states were based on clinical evaluation according to the schedules for clinical assessment in neuropsychiatry interview combined with the hamd-17 and ymrs rating scales without applying duration criteria : euthymic ( hamd-17 and ymrs < 8) , depressive ( hamd-17 > 7 and ymrs < 8) , manic / hypomanic ( ymrs > 7 and hamd-17 < 8) , and mixed state blood samples were obtained in the fasting state between 0830 and 1030 hours , after a minimum period of 15 minutes rest , concurrently with the clinical evaluation . nine milliliters of blood were drawn by venipuncture into a citrate phosphate dextrose adenine containing vacuum tube ( vacuette ) , which was kept at room temperature before and after the blood draw . peripheral blood mononuclear cells ( pbmc ) were collected applying the standard ficoll - paque plus isolation procedure ( ge healthcare life sciences ) , within one hour of blood draw . total rna was extracted from pbmc by use of trizol reagent ( life technologies ) . rna quality and quantification was measured spectrophotometrically using a nanodrop ( nanodrop technologies ) spectrophotometer and software , applying the 260/280 and 260/230 ratio algorithms . cdna was synthesized from rna with a high capacity ddna reverse transcription kit ( life technologies ) . the cdna was subjected to quantitative real - time polymerase chain reaction ( pcr ) using the viia 7 real - time pcr system ( life technologies ) with sybr green pcr master mix ( life technologies ) . gene - specific sequence oligonucleotide primers ( ptgds , akr1c3 , gapdh , tbp , and sdha ) were purchased from tag copenhagen . a set of three genes , the glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) gene , the tata box binding protein ( tbp ) gene , and the succinate dehydrogenase complex , subunit a , flavoprotein ( sdha ) gene , were used as candidate reference genes for normalization . akr1c3 , aldo - keto reductase family 1 member c3 ; gapdh , glyceraldehyde-3-phosphate dehydrogenase ; ptgds , prostaglandin d synthase ; sdha , succinate dehydrogenase complex , subunit a , flavoprotein ; tbp , tata box binding protein . the stability of candidate reference genes was assessed using the normfinder software ( akhondzadeh et al . , 2009 ) . sdha exhibited the highest stability in comparisons between bipolar disorder patients and healthy control subjects , as well as between affective states in bipolar disorder patients ( sdha = 0.139 ; tbp = 0,322 ; gapdh = 0.271 ) , and no combination of two genes showed higher stability . semi - quantitative ptgds and akr1c3 mrna levels , assessed by cycle threshold ( ct ) were thus expressed relative to sdha . we measured the ct = ct ( each gene ) - ct ( sdha ) for each sample . relative levels of expression were determined using the comparative ct method ( ricken et al . , 2013 ) method , calculated by 2 t. all assays were performed in triplicate with laboratory personnel blinded to the clinical status of participants . in addition , standard clinical chemistry parameters were analyzed , including fasting blood glucose and fasting lipid parameters . independent t - tests were used to test differences in age between healthy control subjects and bipolar disorder patients , and the chi - squared test was used to examine differences in categorical demographic and clinical variables . for our main analyses we employed a two - level linear mixed effects model , accommodating both variation of the outcome variables within subjects ( intra - individual variation ) and between subjects ( inter - individual variation ) . level one represented repeated measures of ptgds and akr1c3 mrna levels and level two represented between - subject variation . we conducted two separate sets of analyses , one on comparisons between bipolar disorder patients and healthy control subjects ( set a ) and one on comparisons between affective states among bipolar disorder patients ( set b ) . in both sets , unadjusted mixed - model analyses with expression levels of each gene as the dependent variables were firstly conducted ( model a-1 and b-1 ) followed by several a priori models specified within each set of analyses ( a and b , models 24 ) . all models included a random intercept to accommodate correlations in the outcome variables over time within each participant . the assumptions of independence of errors , homoscedasticity , and normality were met . to evaluate the correlation between levels of ptgds and akr1c3 expression , a pearson s correlation analysis was performed , using residual values produced by our mixed model ( model a-2 ) . patients with a potential diagnosis of rapid - cycling bipolar disorder were recruited through referral by psychiatrists at hospitals or outpatient facilities throughout the region of zealand , denmark , with study recruitment taking place during the period of june 2010 to may 2012 . inclusion criteria were : adults aged 1870 years ; and a diagnostic and statistical manual of mental disorders , 4th version ( dsm - iv ) diagnosis of rapid - cycling bipolar disorder , defined by the occurrence of at least four mood episodes ( mania , hypomania , depression , or mixed ) during the preceding year in the context of bipolar disorder . exclusion criteria were : significant physical illness ( ie chronic heart disease , chronic pulmonary disease , inflammatory disease , chronic infectious disease , or neurodegenerative disease ) ; current drug abuse ; insufficient danish language skills ; and pregnancy . two bipolar patients declined further examination after one and three month follow - ups , respectively ; the remaining bipolar patients were followed for a minimum of six months with a mean ( standard deviation [ sd ] ) follow - up period of 11.9 ( 3.0 ) months . upon signs of new affective episodes , patients were evaluated with clinical assessments of mood and collection of blood samples which , when possible , were repeated after return to a subsequent euthymic state or change to an affective episode of opposite polarity . in cases of clinical signs of acute infection , any allergic symptoms , or any other acute medical condition , assessment and biochemical analysis blood samples were , on average , collected from bipolar patients 3.41.7 ( range , 110 ) times during the study . samples were obtained during euthymia in 34 patients ( mean 2.01.3 [ 06 ] ) , major depression in 26 patients ( mean 1.71.7 [ 05 ] ) , mania / hypomania in 11 patients ( mean 0.71.2 [ 05 ] ) , and in a mixed state in a total of 6 patients ( mean 0.20.4 [ 01 ] ) . forty healthy control subjects were recruited among blood donors affiliated with the blood bank at rigshospitalet , copenhagen . inclusion criteria were : adults aged 1870 years ; and no history of psychiatric disorder in the subjects or their first - degree relatives . healthy control subjects were evaluated with clinical assessments and collection of blood samples on two separate occasions approximately three months apart . assessment and biochemical analysis were postponed if there were clinical signs of acute infection , any allergic symptoms , or any other acute medical condition . mean ( sd ) follow - up time for the healthy control subjects was 2.9 ( 0.9 ) months . the study protocol was approved by the committee on health research ethics of the capital region of denmark ( protocol no . patients with a potential diagnosis of rapid - cycling bipolar disorder were recruited through referral by psychiatrists at hospitals or outpatient facilities throughout the region of zealand , denmark , with study recruitment taking place during the period of june 2010 to may 2012 . inclusion criteria were : adults aged 1870 years ; and a diagnostic and statistical manual of mental disorders , 4th version ( dsm - iv ) diagnosis of rapid - cycling bipolar disorder , defined by the occurrence of at least four mood episodes ( mania , hypomania , depression , or mixed ) during the preceding year in the context of bipolar disorder . exclusion criteria were : significant physical illness ( ie chronic heart disease , chronic pulmonary disease , inflammatory disease , chronic infectious disease , or neurodegenerative disease ) ; current drug abuse ; insufficient danish language skills ; and pregnancy . two bipolar patients declined further examination after one and three month follow - ups , respectively ; the remaining bipolar patients were followed for a minimum of six months with a mean ( standard deviation [ sd ] ) follow - up period of 11.9 ( 3.0 ) months . upon signs of new affective episodes , patients were evaluated with clinical assessments of mood and collection of blood samples which , when possible , were repeated after return to a subsequent euthymic state or change to an affective episode of opposite polarity . in cases of clinical signs of acute infection , any allergic symptoms , or any other acute medical condition , assessment and biochemical analysis blood samples were , on average , collected from bipolar patients 3.41.7 ( range , 110 ) times during the study . samples were obtained during euthymia in 34 patients ( mean 2.01.3 [ 06 ] ) , major depression in 26 patients ( mean 1.71.7 [ 05 ] ) , mania / hypomania in 11 patients ( mean 0.71.2 [ 05 ] ) , and in a mixed state in a total of 6 patients ( mean 0.20.4 [ 01 ] ) . forty healthy control subjects were recruited among blood donors affiliated with the blood bank at rigshospitalet , copenhagen . inclusion criteria were : adults aged 1870 years ; and no history of psychiatric disorder in the subjects or their first - degree relatives . healthy control subjects were evaluated with clinical assessments and collection of blood samples on two separate occasions approximately three months apart . assessment and biochemical analysis were postponed if there were clinical signs of acute infection , any allergic symptoms , or any other acute medical condition . mean ( sd ) follow - up time for the healthy control subjects was 2.9 ( 0.9 ) months . the study protocol was approved by the committee on health research ethics of the capital region of denmark ( protocol no . all participants were assessed by a specialist in psychiatry ( dr munkholm ) , using standardized semi - structured interviews . the schedules for clinical assessment in neuropsychiatry interview ( wing et al . , 1990 ) was used for diagnostic purposes and was based on available case material , referral reports , the interview with the participant , and the hypomania checklist ( angst et al a dsm - iv diagnosis of rapid - cycling bipolar disorder was established for the patients and comorbid psychiatric illness , if present , was recorded . for healthy control subjects , a clinical diagnosis according to dsm - iv , without applying duration criteria , was established at each study visit concurrently with the collection of samples for laboratory analysis . severity of depressive symptoms was assessed using the 17-item hamilton depression rating scale ( hamd-17 ; hamilton , 1967 ) , employing a structured interview guide ( williams , 1990 ) translated to danish , and manic symptoms were assessed using the young mania rating scale ( ymrs ; young et al . , 1978 ) , with a time period of three days applied . medication , alcohol intake , and smoking habits during the two weeks prior to assessment were recorded . categories of affective states were based on clinical evaluation according to the schedules for clinical assessment in neuropsychiatry interview combined with the hamd-17 and ymrs rating scales without applying duration criteria : euthymic ( hamd-17 and ymrs < 8) , depressive ( hamd-17 > 7 and ymrs < 8) , manic / hypomanic ( ymrs > 7 and hamd-17 < 8) , and mixed state ( hamd-17 > 7 and ymrs > 7 ) . blood samples were obtained in the fasting state between 0830 and 1030 hours , after a minimum period of 15 minutes rest , concurrently with the clinical evaluation . nine milliliters of blood were drawn by venipuncture into a citrate phosphate dextrose adenine containing vacuum tube ( vacuette ) , which was kept at room temperature before and after the blood draw . peripheral blood mononuclear cells ( pbmc ) were collected applying the standard ficoll - paque plus isolation procedure ( ge healthcare life sciences ) , within one hour of blood draw . total rna was extracted from pbmc by use of trizol reagent ( life technologies ) . rna quality and quantification was measured spectrophotometrically using a nanodrop ( nanodrop technologies ) spectrophotometer and software , applying the 260/280 and 260/230 ratio algorithms . cdna was synthesized from rna with a high capacity ddna reverse transcription kit ( life technologies ) . the cdna was subjected to quantitative real - time polymerase chain reaction ( pcr ) using the viia 7 real - time pcr system ( life technologies ) with sybr green pcr master mix ( life technologies ) . gene - specific sequence oligonucleotide primers ( ptgds , akr1c3 , gapdh , tbp , and sdha ) were purchased from tag copenhagen . a set of three genes , the glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) gene , the tata box binding protein ( tbp ) gene , and the succinate dehydrogenase complex , subunit a , flavoprotein ( sdha ) gene , were used as candidate reference genes for normalization . akr1c3 , aldo - keto reductase family 1 member c3 ; gapdh , glyceraldehyde-3-phosphate dehydrogenase ; ptgds , prostaglandin d synthase ; sdha , succinate dehydrogenase complex , subunit a , flavoprotein ; tbp , tata box binding protein . the stability of candidate reference genes was assessed using the normfinder software ( akhondzadeh et al . , 2009 ) . sdha exhibited the highest stability in comparisons between bipolar disorder patients and healthy control subjects , as well as between affective states in bipolar disorder patients ( sdha = 0.139 ; tbp = 0,322 ; gapdh = 0.271 ) , and no combination of two genes showed higher stability . semi - quantitative ptgds and akr1c3 mrna levels , assessed by cycle threshold ( ct ) were thus expressed relative to sdha . we measured the ct = ct ( each gene ) - ct ( sdha ) for each sample . relative levels of expression were determined using the comparative ct method ( ricken et al . , 2013 ) method , calculated by 2 t. all assays were performed in triplicate with laboratory personnel blinded to the clinical status of participants . in addition , standard clinical chemistry parameters were analyzed , including fasting blood glucose and fasting lipid parameters . independent t - tests were used to test differences in age between healthy control subjects and bipolar disorder patients , and the chi - squared test was used to examine differences in categorical demographic and clinical variables . for our main analyses we employed a two - level linear mixed effects model , accommodating both variation of the outcome variables within subjects ( intra - individual variation ) and between subjects ( inter - individual variation ) . level one represented repeated measures of ptgds and akr1c3 mrna levels and level two represented between - subject variation . we conducted two separate sets of analyses , one on comparisons between bipolar disorder patients and healthy control subjects ( set a ) and one on comparisons between affective states among bipolar disorder patients ( set b ) . in both sets , unadjusted mixed - model analyses with expression levels of each gene as the dependent variables were firstly conducted ( model a-1 and b-1 ) followed by several a priori models specified within each set of analyses ( a and b , models 24 ) . all models included a random intercept to accommodate correlations in the outcome variables over time within each participant . the assumptions of independence of errors , homoscedasticity , and normality were met . to evaluate the correlation between levels of ptgds and akr1c3 expression , a pearson s correlation analysis was performed , using residual values produced by our mixed model ( model a-2 ) . there were no significant differences between bipolar disorder patients and healthy control subjects with regard to age , gender , educational level , or bmi . more patients were smokers but alcohol consumption was higher among healthy control subjects ( table 2 ) . data are expressed as mean standard deviation ( range ) or n ( % ) . bipolar disorder patients were overall on stable medication for a month before study entry and during the course of the study , with few participants changing medication despite alterations of affective state , owing to the fact that the majority of patients received intensive outpatient treatment and , despite pharmacologically advanced treatment , continually experience affective episodes . four patients stopped and one patient started selective serotonin reuptake inhibitor treatment , two patients started lithium treatment , two started anticonvulsant treatment , and one started antipsychotic treatment during the study period . one bipolar disorder patient suffered from co - morbid obsessive - compulsive disorder ; no participants suffered from comorbid generalized anxiety disorder . four bipolar disorder patients reported mild , well - controlled hypertension and two reported mild , intermittent reflux esophagitis . one healthy control subject reported intermittent symptoms of allergic rhinitis , but not during the study period . the majority of the patients received specialized treatment at the mood disorders clinic , psychiatric center copenhagen , rigshospitalet , copenhagen , denmark , and all of the patients were outpatients at the time of inclusion . symptom severity of participants at the time of assessment and sampling are presented in table 3 . hamd-17 , hamilton rating scale , 17 items ; ymrs , young mania rating scale . * manic patients , n = 19/hypomanic patients , n = 5 . in an unadjusted analysis ( model a-1 ) , lower levels of ptgds mrna expression were observed in all affective states compared with healthy control subjects ; however , only between a euthymic state and healthy control subjects was this difference statistically significant ( b = -0.060 , 95% confidence interval [ ci ; -0.117 ; -0.002 ] , p = 0.041 ) . no difference was observed in an unadjusted analysis for akr1c3 mrna expression between bipolar disorder patients in any affective state and healthy control subjects [ f(4 , 184.86 ) = 0.343 , p = 0.8 ] . adjusting for age and gender ( model a-2 ) , a statistically significant down - regulation of ptgds mrna expression was present in bipolar disorder patients in both the euthymic state ( b = -0.073 , 95% ci [ -0.130 ; -0.017 ] , p = 0.012 ) , the depressive state ( b = -0.062 , 95% ci [ -0.120 ; -0.003 ] , p = 0.038 ) , and the manic / hypomanic state ( b = -0.076 , 95% ci [ -0.144 ; -0.008 ] , p = 0.028 ) , while the lower levels observed in a mixed state did not reach statistical significance ( b = -0.053 , 95% ci [ -0.143 ; 0.037 ] , p = 0.2 ; figure 2a ) . in an adjusted analysis ( model a-2 ) , no difference was observed in akr1c3 mrna expression between bipolar disorder patients in any affective state compared with healthy control subjects [ f(4 , 184.66 ) = 0.207 , p = 0.9 ; figure 2b ] . age , but not gender , was weakly but positively associated with both ptgds mrna expression ( b = 0.003 , 95% ci [ 0.001 ; 0.005 ] , p = 0.007 ) and akr1c3 mrna expression ( b = 0.001 , 95% ci [ 0.000 ; 0.001 ] , p = 0.01 ) . in an exploratory analysis of clinical and demographical variables ( bmi and alcohol intake ) possibly associated with ptgds and akr1c3 mrna expression ( model a-3 ) , where smokers were excluded because of uneven distribution between groups , alcohol intake was not associated with ptgds mrna expression ( p = 0.2 ) or akr1c3 mrna expression ( p = 0.5 ) and bmi was not associated with either ptgds mrna expression ( p = 0.8 ) or akr1c3 expression ( p = 0.2 ) . in this exploratory analysis , only among bipolar patients in a manic / hypomanic state was the down - regulated ptgds mrna expression compared with healthy control subjects statistically significant ( b = -0.100 , 95% ci [ -0.0.197 ; -0.003 ] , p = 0.044 ) with the non - significant findings for akr1c3 unaltered . ptgds ( a ) and akr1c3 ( b ) mrna expression in rapid - cycling bipolar disorder patients and healthy control subjects . levels represent back - transformed ct values based on a linear mixed - model analysis adjusted for age and gender ( model a-2 ) . ptgds mrna expression was down - regulated in rapid - cycling bipolar disorder patients in a euthymic ( p = 0.01 ) , depressive ( p = 0.04 ) , and manic / hypomanic ( p = 0.03 ) state compared with healthy control subjects ; no difference in ptgds mrna expression was observed between affective states . mrna expression did not differ between affective states or between bipolar disorder patients and healthy control subjects . akr1c3 , aldo - keto reductase family 1 member c3 ; bd , bipolar disorder ; ct , cycle threshold ; ns , not significant ; ptgds , prostaglandin d synthase . in a subgroup analysis including only bipolar disorder patients in a euthymic state of more than one month ( model a-4 ) , thus minimizing a possible effect of the previous episode on gene expression in a euthymic state , ptgds mrna expression remained significantly down - regulated in comparison to healthy control subjects ( b = -0.073 , 95% ci [ -0.145 ; -0.001 ] , p = 0.048 ) , while there was no difference between groups for akr1c3 ( p = 0.6 ) . in an unadjusted analysis ( model b-1 ) , there was no overall difference in mrna expression between any current affective state and the euthymic state of either ptgds [ f(3 , 139.820 ) = 0.208 , p = 0.9 ] or akr1c3 [ f(3 , 138.188 ) = 0.294 , p = 0.8 ] . there was also no difference in either ptgds mrna expression ( p = 0.6 ) or akr1c3 mrna expression ( p = 0.4 ) between the depressed and the manic / hypomanic state . adjusting for age and gender ( model b-2 ) and subsequently adding the covariates bmi , smoking status ( no / yes ) , alcohol intake , illness duration ( 10 years/<10 years ) and medication ( no / yes ) in an exploratory analysis ( model b-3 ) did not alter the results . in this analysis , none of the covariates entered into the model , including individual medications , were significantly associated with ptgds or akr1c3 mrna expression levels . specifically , there was no effect of either lithium ( b = -0.014 , 95% ci [ -0.067 ; 0.039 ] , p = 0.6 ) , anticonvulsant ( b = -0.034 , 95% ci [ -0.091 ; 0.023 ] , p = 0.2 ) , antipsychotic ( b = -0.001 , 95% ci [ -0.075 ; 0.075 ] , p = 0.9 ) , or antidepressant use ( b = -0.005 , 95% ci [ -0.065 ; 0.056 ] , p = 0.9 ) on ptgds mrna levels . non - smoking status compared with smoking status was similarly not associated with ptgds mrna levels ( b = 0.065 , 95% ci there was no association between hamd-17 scores or ymrs scores and mrna expression levels of ptgds and akr1c3 in depressive and manic / hypomanic bipolar disorder patients , respectively ( model b-4 ) . post hoc subgroup analysis of patients not taking acetylsalicylic acid ( n = 35 ) or statins ( n = 33 ) during the study period revealed no difference in either ptgds or akr1c3 mrna expression between affective states ( data not shown ) . ptgds and akr1c3 mrna expression levels were weakly correlated [ r(239 ) = 0.299 , p < 0.001 ] . there were no significant differences between bipolar disorder patients and healthy control subjects with regard to age , gender , educational level , or bmi . more patients were smokers but alcohol consumption was higher among healthy control subjects ( table 2 ) . data are expressed as mean standard deviation ( range ) or n ( % ) . bipolar disorder patients were overall on stable medication for a month before study entry and during the course of the study , with few participants changing medication despite alterations of affective state , owing to the fact that the majority of patients received intensive outpatient treatment and , despite pharmacologically advanced treatment , continually experience affective episodes . four patients stopped and one patient started selective serotonin reuptake inhibitor treatment , two patients started lithium treatment , two started anticonvulsant treatment , and one started antipsychotic treatment during the study period . one bipolar disorder patient suffered from co - morbid obsessive - compulsive disorder ; no participants suffered from comorbid generalized anxiety disorder . four bipolar disorder patients reported mild , well - controlled hypertension and two reported mild , intermittent reflux esophagitis . one healthy control subject reported intermittent symptoms of allergic rhinitis , but not during the study period . the majority of the patients received specialized treatment at the mood disorders clinic , psychiatric center copenhagen , rigshospitalet , copenhagen , denmark , and all of the patients were outpatients at the time of inclusion . symptom severity of participants at the time of assessment and sampling are presented in table 3 . hamd-17 , hamilton rating scale , 17 items ; ymrs , young mania rating scale . in an unadjusted analysis ( model a-1 ) , lower levels of ptgds mrna expression were observed in all affective states compared with healthy control subjects ; however , only between a euthymic state and healthy control subjects was this difference statistically significant ( b = -0.060 , 95% confidence interval [ ci ; -0.117 ; -0.002 ] , p = 0.041 ) . no difference was observed in an unadjusted analysis for akr1c3 mrna expression between bipolar disorder patients in any affective state and healthy control subjects [ f(4 , 184.86 ) = 0.343 , p = 0.8 ] . adjusting for age and gender ( model a-2 ) , a statistically significant down - regulation of ptgds mrna expression was present in bipolar disorder patients in both the euthymic state ( b = -0.073 , 95% ci [ -0.130 ; -0.017 ] , p = 0.012 ) , the depressive state ( b = -0.062 , 95% ci [ -0.120 ; -0.003 ] , p = 0.038 ) , and the manic / hypomanic state ( b = -0.076 , 95% ci [ -0.144 ; -0.008 ] , p = 0.028 ) , while the lower levels observed in a mixed state did not reach statistical significance ( b = -0.053 , 95% ci [ -0.143 ; 0.037 ] , p = 0.2 ; figure 2a ) . in an adjusted analysis ( model a-2 ) , no difference was observed in akr1c3 mrna expression between bipolar disorder patients in any affective state compared with healthy control subjects [ f(4 , 184.66 ) = 0.207 , p = 0.9 ; figure 2b ] . age , but not gender , was weakly but positively associated with both ptgds mrna expression ( b = 0.003 , 95% ci [ 0.001 ; 0.005 ] , p = 0.007 ) and akr1c3 mrna expression ( b = 0.001 , 95% ci [ 0.000 ; 0.001 ] , p = 0.01 ) . in an exploratory analysis of clinical and demographical variables ( bmi and alcohol intake ) possibly associated with ptgds and akr1c3 mrna expression ( model a-3 ) , where smokers were excluded because of uneven distribution between groups , alcohol intake was not associated with ptgds mrna expression ( p = 0.2 ) or akr1c3 mrna expression ( p = 0.5 ) and bmi was not associated with either ptgds mrna expression ( p = 0.8 ) or akr1c3 expression ( p = 0.2 ) . in this exploratory analysis , only among bipolar patients in a manic / hypomanic state was the down - regulated ptgds mrna expression compared with healthy control subjects statistically significant ( b = -0.100 , 95% ci [ -0.0.197 ; -0.003 ] , p = 0.044 ) with the non - significant findings for akr1c3 unaltered . ptgds ( a ) and akr1c3 ( b ) mrna expression in rapid - cycling bipolar disorder patients and healthy control subjects . levels represent back - transformed ct values based on a linear mixed - model analysis adjusted for age and gender ( model a-2 ) . ptgds mrna expression was down - regulated in rapid - cycling bipolar disorder patients in a euthymic ( p = 0.01 ) , depressive ( p = 0.04 ) , and manic / hypomanic ( p = 0.03 ) state compared with healthy control subjects ; no difference in ptgds mrna expression was observed between affective states . mrna expression did not differ between affective states or between bipolar disorder patients and healthy control subjects . akr1c3 , aldo - keto reductase family 1 member c3 ; bd , bipolar disorder ; ct , cycle threshold ; ns , not significant ; ptgds , prostaglandin d synthase . in a subgroup analysis including only bipolar disorder patients in a euthymic state of more than one month ( model a-4 ) , thus minimizing a possible effect of the previous episode on gene expression in a euthymic state , ptgds mrna expression remained significantly down - regulated in comparison to healthy control subjects ( b = -0.073 , 95% ci [ -0.145 ; -0.001 ] , p = 0.048 ) , while there was no difference between groups for akr1c3 ( p = 0.6 ) . in an unadjusted analysis ( model b-1 ) , there was no overall difference in mrna expression between any current affective state and the euthymic state of either ptgds [ f(3 , 139.820 ) = 0.208 , p = 0.9 ] or akr1c3 [ f(3 , 138.188 ) = 0.294 , p = 0.8 ] . there was also no difference in either ptgds mrna expression ( p = 0.6 ) or akr1c3 mrna expression ( p = 0.4 ) between the depressed and the manic / hypomanic state . adjusting for age and gender ( model b-2 ) and subsequently adding the covariates bmi , smoking status ( no / yes ) , alcohol intake , illness duration ( 10 years/<10 years ) and medication ( no / yes ) in an exploratory analysis ( model b-3 ) did not alter the results . in this analysis , none of the covariates entered into the model , including individual medications , were significantly associated with ptgds or akr1c3 mrna expression levels . specifically , there was no effect of either lithium ( b = -0.014 , 95% ci [ -0.067 ; 0.039 ] , p = 0.6 ) , anticonvulsant ( b = -0.034 , 95% ci [ -0.091 ; 0.023 ] , p = 0.2 ) , antipsychotic ( b = -0.001 , 95% ci [ -0.075 ; 0.075 ] , p = 0.9 ) , or antidepressant use ( b = -0.005 , 95% ci [ -0.065 ; 0.056 ] , p = 0.9 ) on ptgds mrna levels . non - smoking status compared with smoking status was similarly not associated with ptgds mrna levels ( b = 0.065 , 95% ci [ -0.016 ; 0.147 ] , p = 0.1 ) . there was no association between hamd-17 scores or ymrs scores and mrna expression levels of ptgds and akr1c3 in depressive and manic / hypomanic bipolar disorder patients , respectively ( model b-4 ) . post hoc subgroup analysis of patients not taking acetylsalicylic acid ( n = 35 ) or statins ( n = 33 ) during the study period revealed no difference in either ptgds or akr1c3 mrna expression between affective states ( data not shown ) . ptgds and akr1c3 mrna expression levels were weakly correlated [ r(239 ) = 0.299 , p < 0.001 ] . this study investigated alterations of mrna expression of ptgds and akr1c3 between bipolar disorder and healthy control subjects and is the first study in a larger cohort to explore possible state - related alterations in expression of ptgds and akr1c3 . we did this by employing a longitudinal design that allowed for assessment of gene expression in various affective states and incorporated both intra - individual and inter - individual alterations in mixed model analyses . in accordance with our hypothesis , ptgds mrna expression was altered in rapid - cycling bipolar disorder patients in euthymic , depressed , and manic / hypomanic states compared with healthy control subjects , with ptgds expression down - regulated in patients compared with healthy control subjects . no difference in akr1c3 mrna expression between patients and healthy control subjects contrary to our hypothesis , mrna expression of both ptgds and akr1c3 did not differ between affective states in bipolar disorder patients . the finding of down - regulated levels of ptgds mrna expression in peripheral blood in the current study is supported by findings in post - mortem brain tissue where both genes have been found down - regulated in frontal brain regions in bipolar disorder patients compared with healthy control subjects ( stanley medical research institute online genomics database ) . it would be of interest to elucidate possible single nuclear polymorphisms related to the ptgds locus , which could further lead to mapping of potential expression quantitative trait loci . this analysis could prove especially useful in investigating differentially - regulated genes of interest between affective states in bipolar disorder . our results of comparable expression levels of ptgds and akr1c3 across affective states in bipolar disorder patients are in contrast to the findings in two studies by the same group involving a total of five rapid - cycling disorder patients ( begemann et al . , 2008 ; gurvich et al . , 2014 ) , where ptgds and akr1c3 expression were found to be down - regulated in depressive episodes compared with manic episodes . our study consisted of a larger cohort of rapid - cycling disorder patients ( n = 37 ) , likely constituting a more representative sample of rapid - cycling bipolar disorder patients , involved a more rigorous methodological approach , such as transparent statistical analyses adjusted for relevant covariates , and used several reference genes that were tested for stability . our cohort also represents a heterogeneous population of patients , and it is possible that subgroups of patients could exhibit a different pattern of gene expression . in the study finding ptgds down - regulated in adhd patients compared with bipolar disorder patients ( marn - mndez et al . , 2012 ) , patients were both adolescents and adults and there was no information about the affective state or medication of participants . our finding of down - regulation of ptgds could possibly represent a compensatory mechanism in reaction to an activated aa cascade in rapid - cycling bipolar disorder . specifically , the down - regulation of ptgds activity could represent a counter reaction to up - regulated pla2 or cox activity , as increases in pla2 activity in in vivo ( noponen et al . , 1993 ) and of pla2 and cox-2 mrna and protein levels in post - mortem bipolar brain tissue ( rao , bazinet , et al . , 2007 ) this is in line with findings from a recent study of bipolar disorder patient post - mortem brain tissue , where decreased expression of cox-1 and cytosolic prostaglandin e synthase were speculated to be compensatory to the increased expression of cox-2 and membrane prostaglandin e synthase also demonstrated in the study ( kim et al . , 2011 ) . the relatively weak correlation observed between ptgds and akr1c3 mrna expression levels may indicate that the role of the ptgds and akr1c3 enzymes in the aa cascade are not closely interrelated or that they are possibly differentially influenced by medication on a transcriptional level . the finding of down - regulated ptgds expression in rapid - cycling bipolar disorder patients across all affective states is also consistent with preclinical evidence suggesting down - regulation of the aa cascade as a mechanism of action for these medications . the majority of patients in our study were treated with lithium , anticonvulsants , or antipsychotics that , in preclinical studies , have been demonstrated to down - regulate the aa cascade ( bazinet , 2009 ; rapoport et al . , 2009 ) , and it is possible that prolonged treatment with these medications not only results in normalizing an up - regulated aa cascade in these patients but even leads to down - regulation of the cascade below normal activity . while there are no studies of the effect of mood - stabilizing medications on the mrna expression of ptgds and akr1c3 specifically , medication may also down - regulate mrna expression of these genes . it is possible that under such circumstances , state - related alterations in ptgds and akr1c3 mrna expression do not occur . in the present study we did not find an effect of individual medication groups on either ptgds or akr1c3 mrna expression levels . however , the majority of the patients ( 78.4% ) were treated with two or more medication groups and it is therefore difficult to assess the impact of individual medications . along those lines , it can not be excluded that treatment with multiple medication groups adds to the possible down - regulating effect of these medications on mrna expression of ptgds and akr1c3 . investigation of larger cohorts may be necessary to elucidate the effect of individual medications on ptgds and akr1c3 mrna expression and of the aa cascade regulation in general . such an effect is not only suggested by post - mortem brain findings but also by in vivo preclinical studies demonstrating that antipsychotics down - regulate aa metabolism ( cheon et al . , 2011 ; modi et al . , the present finding of aberrations in the aa cascade is in line with the current hypothesis on the pathophysiological background of bipolar disorder involving disturbances within several inter - related pathways , such as inflammatory system dysregulation ( goldstein et al . , 2009 ) , oxidative and nitrosative stress pathways ( maes et al . , 2011 ) , impairments in neuroplasticity ( duman and monteggia , 2006 ) , and mitochondrial dysfunction ( clay et al . , 2011 ) . specifically , up - regulation of pla2 and cox-2 activity is observed upon activation of the inflammatory response system ( bauer et al . , 1997 ; adibhatla and hatcher , 2007 ) , which has been demonstrated to be activated in bipolar disorder ( munkholm et al . , 2013 ) , and elevated expression of pla2 and increased aa turnover have been found in animal models of neuroinflammation ( rao , ertley , et al . , 2007 ) . activation of the aa cascade can , conversely , also induce inflammatory responses and the production of pro - inflammatory cytokines ( munoz and costa , 2013 ) . accumulation of aa generates intracellular reactive oxygen species ( magder , 2006 ) , which can generate a state of oxidative stress that has been suggested to contribute to systemic toxicity ( kapczinski et al . , 2010 ) and neuroprogression in bipolar disorder . further , possible neuroprotective effects of mood - stabilizing treatment may be mediated through inhibition of the aa cascade ( rapoport et al . , 2009 ) and aa cascade dysregulation may be involved in the pathophysiology underlying the neuroprogressive changes suggested in bipolar disorder ( berk , 2009 ) with excess aa possibly inducing apoptosis by damaging mitochondria ( saitoh et al . , the mechanisms through which these pathways interact with disturbances in the aa cascade and relate to the clinically distinct affective states that constitute bipolar disorder , however , is unclear . genes expressed in the brain are to a large extent also expressed in peripheral blood , indicating that peripheral blood potentially could serve as a surrogate tissue ( liew et al . , 2006 ; le - niculescu et al . , 2009 ) the peripheral blood transcriptome may thus reflect system - wide biology , and it has further been demonstrated that a significant amount of single nuclear polymorphism expression relationships are conserved between the brain and peripheral blood lymphocytes ( iwamoto et al . , 2011 ) . it is unclear , however , to what extent gene expression in peripheral blood reflects gene expression changes in the brain , and ultimately whether peripheral blood can function as a neural probe ( chana et al . , 2013 ) . first , we specified relatively low cut - offs and waived duration criteria in defining affective states . it is possible that in these patients where ptgds and akr1c3 mrna expression was down - regulated in both a manic / hypomanic and a depressive state as well as a euthymic state , alterations between depressive and manic / hypomanic states would only be present in more severe episodes . second , it is possible that state - related alterations in the mrna expression of ptgds and akr1c3 could be observed in drug - nave patients . it is , however , likely not feasible to include a large cohort of unmedicated rapid - cycling bipolar disorder patients , due to the severity of illness of these patients . third , our sample size was relatively modest , and given the naturalistic design , not all patients experienced episodes of all polarities and the contribution of between - subject variation was therefore relatively large . since the number of hypotheses tested was relatively small and hypotheses , outcomes , and covariates were specified a priori and other tests were treated as hypothesis - generating , we did not correct statistical analyses for multiple testing , which may be considered appropriate ( streiner and norman , 2011 ; panda et al . , 2013 ) but may be regarded as a limitation . finally , the included rapid - cycling bipolar disorder patients had relatively long durations of illness and it is possible that the prolonged illness courses could have induced sustained down - regulation of these enzymes to a degree where more subtle expression changes between affective episodes do not occur . while the study could possibly suggest that ptgds mrna down - regulation in bipolar disorder patients is trait related , it is thus possible that the expression pattern could differ in patients with a more benign course or in patients in early stages of the disorder . therefore it would be of interest to study mrna alterations in a larger cohort including bipolar disorder patients in early , intermediary , and late stages of the disorder . in conclusion , we demonstrated down - regulation of mrna expression of ptgds in depressed , manic / hypomanic , and euthymic states in rapid - cycling bipolar disorder patients compared with healthy control subjects , with no alterations between affective states . the results suggest a role for aberrant regulation of ptgds and of aa cascade and prostaglandin pathway dysregulation in rapid - cycling bipolar disorder . lars vedel kessing has within the preceding three years been a consultant for lundbeck and astrazeneca . maj vinberg has been a consultant for lundbeck , astrazeneca , eli lilly and servier .
background : disturbances related to the arachidonic acid cascade and prostaglandin metabolism may be involved in the pathophysiology of bipolar disorder , as supported by a recent genome - wide association study meta - analysis ; however , evidence from clinical studies on a transcriptional level is lacking . two enzymes in the arachidonic acid cascade are the prostaglandin d synthase ( ptgds ) , which catalyzes the conversion of prostaglandin h2 to prostaglandin d2 ( pgd2 ) , and the aldo - keto reductase family 1 member c3 ( akr1c3 ) , which catalyzes the reduction of pgd2 . we aimed to test the hypothesis that mrna expression of ptgds and akr1c3 is deregulated in rapid - cycling disorder patients in a euthymic or current affective state compared with healthy control subjects , and that expression alters with affective states.methods : ptgds and akr1c3 mrna expression in peripheral blood mononuclear cells was measured in 37 rapid - cycling bipolar disorder patients and 40 age- and gender - matched healthy control subjects using reverse transcription quantitative real - time polymerase chain reaction . repeated measurements of ptgds and akr1c3 mrna expression were obtained in various affective states during 612 months and compared with repeated measurements in healthy control subjects.results:adjusted for age and gender , ptgds mrna expression was down - regulated in rapid - cycling bipolar disorder patients in a euthymic , depressive , and manic / hypomanic state compared with healthy control subjects . no difference in ptgds mrna expression was observed between affective states . akr1c3 mrna expression did not differ between bipolar disorder patients in any affective state or in comparison with healthy control subjects.conclusions:the results suggest a role for aberrantly - regulated ptgds mrna expression in rapid - cycling bipolar disorder . the sample size was limited ; replication of the findings in larger , independent samples is warranted to further explore the role of the arachidonic acid cascade and prostaglandin metabolism as a potential therapeutic target in bipolar disorder .
Introduction Methods and Materials Participants Bipolar Disorder Patients Healthy Control Subjects Clinical Assessments Blood Draw and Peripheral Blood Mononuclear Cell Isolation Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction Statistics Results Demographic and Clinical Characteristics State-Specific Alterations of Discussion Conclusion Statement of Interest
the present study is the first to investigate repeated measures over time of the gene expression of ptgds and akr1c3 in rapid - cycling bipolar disorder patients in a euthymic or current affective state and in healthy control subjects . we hypothesized that mrna expression of ptgds and akr1c3 was deregulated in patients in a euthymic or current affective state compared with healthy control subjects as well as in bipolar disorder patients between current affective states ( depressed , manic / hypomanic , or mixed ) compared with those in the euthymic state . adjusting for age and gender ( model a-2 ) , a statistically significant down - regulation of ptgds mrna expression was present in bipolar disorder patients in both the euthymic state ( b = -0.073 , 95% ci [ -0.130 ; -0.017 ] , p = 0.012 ) , the depressive state ( b = -0.062 , 95% ci [ -0.120 ; -0.003 ] , p = 0.038 ) , and the manic / hypomanic state ( b = -0.076 , 95% ci [ -0.144 ; -0.008 ] , p = 0.028 ) , while the lower levels observed in a mixed state did not reach statistical significance ( b = -0.053 , 95% ci [ -0.143 ; 0.037 ] , p = 0.2 ; figure 2a ) . ptgds mrna expression was down - regulated in rapid - cycling bipolar disorder patients in a euthymic ( p = 0.01 ) , depressive ( p = 0.04 ) , and manic / hypomanic ( p = 0.03 ) state compared with healthy control subjects ; no difference in ptgds mrna expression was observed between affective states . in a subgroup analysis including only bipolar disorder patients in a euthymic state of more than one month ( model a-4 ) , thus minimizing a possible effect of the previous episode on gene expression in a euthymic state , ptgds mrna expression remained significantly down - regulated in comparison to healthy control subjects ( b = -0.073 , 95% ci [ -0.145 ; -0.001 ] , p = 0.048 ) , while there was no difference between groups for akr1c3 ( p = 0.6 ) . adjusting for age and gender ( model a-2 ) , a statistically significant down - regulation of ptgds mrna expression was present in bipolar disorder patients in both the euthymic state ( b = -0.073 , 95% ci [ -0.130 ; -0.017 ] , p = 0.012 ) , the depressive state ( b = -0.062 , 95% ci [ -0.120 ; -0.003 ] , p = 0.038 ) , and the manic / hypomanic state ( b = -0.076 , 95% ci [ -0.144 ; -0.008 ] , p = 0.028 ) , while the lower levels observed in a mixed state did not reach statistical significance ( b = -0.053 , 95% ci [ -0.143 ; 0.037 ] , p = 0.2 ; figure 2a ) . ptgds mrna expression was down - regulated in rapid - cycling bipolar disorder patients in a euthymic ( p = 0.01 ) , depressive ( p = 0.04 ) , and manic / hypomanic ( p = 0.03 ) state compared with healthy control subjects ; no difference in ptgds mrna expression was observed between affective states . in a subgroup analysis including only bipolar disorder patients in a euthymic state of more than one month ( model a-4 ) , thus minimizing a possible effect of the previous episode on gene expression in a euthymic state , ptgds mrna expression remained significantly down - regulated in comparison to healthy control subjects ( b = -0.073 , 95% ci [ -0.145 ; -0.001 ] , p = 0.048 ) , while there was no difference between groups for akr1c3 ( p = 0.6 ) . in accordance with our hypothesis , ptgds mrna expression was altered in rapid - cycling bipolar disorder patients in euthymic , depressed , and manic / hypomanic states compared with healthy control subjects , with ptgds expression down - regulated in patients compared with healthy control subjects . no difference in akr1c3 mrna expression between patients and healthy control subjects contrary to our hypothesis , mrna expression of both ptgds and akr1c3 did not differ between affective states in bipolar disorder patients . the finding of down - regulated levels of ptgds mrna expression in peripheral blood in the current study is supported by findings in post - mortem brain tissue where both genes have been found down - regulated in frontal brain regions in bipolar disorder patients compared with healthy control subjects ( stanley medical research institute online genomics database ) . in conclusion , we demonstrated down - regulation of mrna expression of ptgds in depressed , manic / hypomanic , and euthymic states in rapid - cycling bipolar disorder patients compared with healthy control subjects , with no alterations between affective states .
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human investigations focusing on concentrated ambient particles have shown acute lung inflammation and changes in both blood indices and heart rate . it is an urgent priority to establish in vivo bioassays for the detection of hazards related to fine particles , which can be inhaled into deep lung tissue by humans . histopathologic observation of lung tissue from f344 rats treated with 4 mg / rat quartz i.t . on day 28 . arrows in the figures indicate typical findings . a , neutrophil infiltration in the walls ; b , neutrophil infiltration in the spaces of alveoli ; c , pulmonary edema ; d , pulmonary fibrosis ; e , histiocytic macrophage infiltration ; f , restructuring of walls ; g , granuloma . a , immunostaining of brdu on day 1 ; b , immunostaining of inos on day 28 after i.t . of 4 mg / rat quartz . in quartz dust - exposed construction workers , obstructive and restrictive loss of lung function has been detected , as has chronic obstructive pulmonary disease ( copd ) . these are associated with inflammatory cell responses characterized by alveolitis with recruitment of inflammatory cells , particularly neutrophils , and may result in pulmonary fibrosis and impaired lung function . intratracheal instillation ( i.t . ) of quartz into rats produces an inflammatory reaction followed by histological changes characteristic of lung fibrosis , similar to the above noted human conditions . reported a no observed adverse effect level ( noael ) for quartz of between 0.03 and 0.13 mg / m ( 40 year exposure ) based on an autopsy study for humans showing that lung burdens between 0.7 and 1.7 g of quartz are associated with macules only and not simple coal worker s pneumoconiosis . in order to establish an appropriate bioassay for detection of lung damage after particle inhalation , several experiments were performed in rats using quartz as a typical particle lung toxicant . histopathologically , neutrophil infiltration in the walls and in the spaces of alveoli , pulmonary edema , pulmonary fibrosis , macrophage infiltration in the alveoli , restructuring of walls of the alveoli and granuloma production were all assessed ( fig . 1 ) . these parameters of inflammatory change were scored as follows : 0 , no change ; 1 , weak ; 2 , moderate ; and 3 , severe . for objective assessment , 5-bromo-2-deoxyuridine ( brdu ) incorporation and expression of inducible no synthase ( inos ) were also immunohistochemically examined ( fig . antibodies specific for brdu provide a sensitive method for detecting dna replication for dna repair and cell proliferation in situ , while inos is associated with development of lung damage , inflammation , granulomas and fibrosis induced by inhalation of silica . these markers are generally associated with inflammation and may increase at different times after particle inhalation . in this review , we summarize some pilot data from a sequential analysis study , a dose response study and a vehicle assessment study conducted to establish a bioassay model featuring instilled quartz as the positive control . finally , the toxicity of a series of different particle types , including nanoparticles and diesel exhaust particles , was also tested with our new bioassay . in order to establish an appropriate bioassay for detection of lung damage after fine particle inhalation , sequential histopathological changes were examined after i.t . of quartz with a particle diameter of not more than 7 m ( dq-12 , deutzche montan technologie , gmbh , germany , 4 mg / rat ) , as a typical lung toxic agent , in f344 male rats . experimental design for the sequential analysis study . a total of 50 , 10-week - old animals were separated into two groups . , i.t . of 4 mg / rat quartz suspended in 0.2 ml saline . , a total of 50 , 10-week - old animals were separated into two groups ( fig . twenty - five rats were exposed to the material suspended in saline ( 0.2 ml ) using a specially designed aerolizer ( penn century , pa , usa ) , and subgroups were sacrificed 1 , 3 , 7 , 14 and 28 days thereafter . ( 0.2 ml ) as a control group and were sacrificed on the same days . both groups received intraperitoneal injections ( i.p . ) of 100 mg / kg b.w . brdu before sacrifice and underwent assessment of lung histopathology with immunohistochemical demonstration of brdu , inos and matrix metalloproteinase-3 ( mmp-3 , employed as a marker of fibrotic change ) . in this experiment , after quartz treatment , lungs on day 1 demonstrated acute inflammatory changes with neutrophil infiltration , while granulation - like changes with giant cells and macrophages in the alveoli were evident on day 28 . furthermore , the numbers of brdu positive cells were found to gradually decrease after the initial peak on day 1 . monoclonal antibodies specific for brdu provide a sensitive method for detecting dna replication for dna repair and cell proliferation in situ . the numbers and areas of inos positive cells , in contrast , increased with time up to day 28 , i. e. , throughout the experimental period . previous studies show that inos is expressed by many cells within the lung parenchyma after exposure to various inflammatory stimuli and is also associated with neovascularization and proliferation . furthermore , inos activity in primary tumor tissues in cases of fresh human gynecological and breast cancers correlates positively with the tumor grade . generation of oxidants and nitric oxide , in particular , is temporally and anatomically associated with development of lung damage , inflammation , granulomas and fibrosis induced by inhalation of silica . experimental design for validation of the sequential analysis study . a total of 108 , 10-week - old f344/ducrj male rats were randomly divided into 8 groups . , i.t . of 4 mg / rat test substances , quartz , hydrotalcite , potassium octatitanate , palladium oxide and carbon black , suspended in 0.2 ml saline or pg - cmc for carbon black . , the results suggest that days 1 and 28 after intratracheal instillation of fine test particles are the most appropriate day for detection of acute and subacute inflammatory changes , respectively . furthermore , brdu on day 1 and inos on day 28 proved to be suitable end - point markers for this purpose . the quartz - treated group demonstrated severe toxicity , while the other hydrotalcite , potassium octatitanate , palladium oxide and carbon black - treated groups all exhibited relatively mild toxicity . the toxicities of fine particles from various materials ( quartz , hydrotalcite , potassium octatitanate , palladium oxide and carbon black ) were examined using our in vivo bioassay with a special focus on the correlations between immunohistochemical and histopathological findings . a total of 108 , 10-week - old f344/ducrj male rats were randomly divided into 8 groups ( fig . of the 5 test particles ( 4 mg / rat ) , quartz , hydrotalcite , potassium octatitanate , palladium oxide and carbon black , respectively , suspended in 0.2 ml vehicle ( saline or 10% propylene glycol and 1% sodium carboxymethyl cellulose in saline : pg - cmc ) with an aerolizer , and subgroups of 7 rats were sacrificed on days 1 and 28 thereafter . groups 6 and 7 were similarly exposed to saline and pg - cmc , respectively , as vehicle controls , while group 8 was maintained untreated . histopathological changes and immunohistochemically assessed brdu labeling indices and inos levels were applied as end points . from the scoring indices generated by the comparative histopathological and immunohistochemical assessment ( fig . 5 ) , the quartz treated group demonstrated severe toxicity , while the other particle treated groups all exhibited relatively mild toxic effects . biochemical analyses at different time points following instillation of different materials earlier demonstrated that all of the exposed groups developed granulomas . alveolar lipoproteinosis and pulmonary fibrosis were most severe in the quartz treated lungs and progressed with time . the present results for brdu immunohistochemistry demonstrated that , in all groups , proliferation was enhanced on day 1 due to particle exposure but had returned to almost normal values by day 28 . in contrast , inos values were higher at the latter time point in the quartz and hydrotalcite groups . this time course of change in inos expression may be important in terms of toxicity assessment , and it may be necessary to examine a later time point ( estimated to be about 8 months ) after i.t . to clarify the influence of change with time . however , in this experiment , expression of mmp-3 was not associated with any of the subchronic changes , such as granulation , collagenization or fibrosis , after particle instillation . generally , bronchoalveolar lavage ( balf ) with markers of inflammation is often used to assess the lung toxicity of instilled test particles in rats . in our experiment , histopathological findings and 3 different immunohistochemical markers ( brdu , inos and mmp-3 ) were selected and were scored in order to provide an objective assessment . compared with balf , this approach has the advantage of allowing detailed investigation of lung damage ; it also has disadvantages , for example , in that rats must be sacrificed at each time point and sequential changes can not be followed at the individual animal level . the present study featured comparison of 5 different particles in one experiment using an in vivo bioassay model . there has hitherto been no data available concerning instillation or inhalation of hydrotalcite and palladium oxide . tismo has been evaluated after inhalation , and similarly , quartz and carbon black have been evaluated after inhalation and instillation . in this experiment , quartz exerted much stronger toxicity than the other test particles , which did not greatly differ in their effects . the dose of 4 mg / rat of quartz instilled directly into the trachea in this experiment was selected based on data reported previously , and dose dependence at higher levels is currently being investigated in our laboratory . there are biologically different responses to inhalation and instillation , but given the rapidity with which different particles can enter the lung , acute effects may be most important . the advantages of assessment of particle toxicity using in vivo bioassays such as i.t . have been stressed . method is clearly useful for detection of acute and subacute pulmonary particle effects using a histopathological scoring system and markers like brdu and inos . a total of 40 , 10-week - old male f344 rats were randomly separated into 4 groups of 10 rats . , i.t . of 4 mg , 2 mg or 1 mg / rat quartz suspended in 0.2 ml saline . , i.t . of 0.2 ml saline ( control ) . s(5 ) , sacrifice of 5 rats . the dose of 4 mg quartz used by mercer et al . ( 2003 ) is too large to allow for assessment of slight changes . in fact , the toxicities of the 5 materials in our validation experiment were found to be only high ( quartz only ) or low ( other particles ) , with no intermediate toxicity . in many reports of toxicity assessment of fine particles using quartz as a positive control , the doses employed have been less than 4 mg . in our study , a total of 40 , 10-week - old male f344 rats were randomly separated into 4 groups of 10 rats each ( fig . 6 ) and exposed by intratracheal instillation to 4 mg , 2 mg , 1 mg and 0 mg ( control ) quartz suspended in saline ( 0.2 ml ) using a specially designed aerosolizer . subgroups of 5 rats each were sacrificed on days 1 and 28 thereafter , and histopathological examination was performed on the lungs . in the quartz treated groups , granulomas were observed at 2 mg and 4 mg , with only mild inflammation was observed at 1 mg . a dose of 4 mg does not induce any novel extra alterations , and 1 mg is too weak to cause changes in histopathological parameters on day 28 . a dose of 2 mg quartz was thus suggested to be the most appropriate dose for sensitive detection of acute and subchronic inflammatory changes . experimental design for the vehicle assessment study ( dry powder assessment ) . a total of 20 , 10-week - old male f344 rats were randomly separated into 2 groups of 10 rats . , i.t . of 4 mg / rat quartz powder with 0.2 ml air using a dp-4 insufflator ( dry powder insufflator ) . , s(5 ) , sacrifice of 5 rats . in our experiments , test particles were suspended in saline , but this is associated with considerable agglutination . to prevent this as much as possible , we also tested the efficacy of a dry powder instillation method using a dry powder insufflator without any vehicle . a total of 20 , 10-week - old male f344 rats were randomly separated into 2 groups of 10 rats each ( fig . groups 1 and 2 were exposed to 4 mg and 0 mg ( air only control ) quartz powder , without suspension in any vehicle , and 2 ml air using another type of aerolizer , the dp-4 insufflator ( dry powder insufflator , penn century , pa , usa ) , and sacrificed on days 1 and 28 thereafter . histopathological examination revealed that 4 mg quartz dry powder also caused inflammation , but it was mild compared with the previous experiment result with the same dose suspended in 0.2 ml saline . while the dry powder instillation method may be suitable for experiments requiring dry powder formulations , for example , to increase stability in vaccine studies , instillation by this method may weaken the effects . because it is difficult to maintain precise dose and it is impossible to control loss due to expiration air after intratracheal instillation . there are also problems in regard to exposure of researchers since there is possibility of aspiration . the pilot experiments suggested that a dose of 2 mg quartz suspended in 0.2 ml saline was suggested to be most appropriate for sensitive detection of acute and subchronic inflammatory changes . using this as a control , examination of the toxicity of a series of particles these materials were selected to perform this validation study and for their variety of characteristics , including shape , composition and particle diameters with the nanometer order or not . histopathological and immunohistochemical analysis of brdu incorporation and inos were performed after exposure of the lungs . experimental design for the toxicity assessment of a series of different particle types ( experiments 13 ) . [ experiment 1 ] , i.t . of 2 mg / rat quartz , titanium dioxide , hydrotalcite or -cyclodextrin suspended in 0.2 ml saline or k2pdcl4 and na2pdcl4 , suspended in 0.2 ml distilled water . [ experiment 2 ] , i.t . of 2 mg / rat test substances , quartz , titanium dioxide , cuo , cuo nanoparticles , mno2 , nio or nio nanoparticles suspended in 0.2 ml saline . , i.t . of 0.2 ml saline ( control ) . [ experiment 3 ] , i.t . of 2 mg / rat quartz , diesel standard powder , diesel powder , c6h10o4pd or caco3 suspended in 0.2 ml saline or neutralized na2pdcl4 ( final ph was 6.5 ) in 20 mm phosphate buffer and 5 n nacl . experiment 1 : a total of 108 rats were randomly separated into 9 groups of 12 animals in each ( fig . mg / rat quartz , titanium dioxide , hydrotalcite and -cyclodextrin suspended in 0.2 ml saline , and groups 5 and 6 were exposed by i.t . to 2 mg / rat distilled water was employed as a vehicle because only the bases , na and k , of k2pdcl4 and na2pdcl4 differed . groups 79 were maintained as controls ( saline , distilled water or untreated ) . subgroups of 6 rats were sacrificed on days 1 and 28 . experiment 2 : a total of 106 rats were randomly separated into 9 groups ( 12 rats each in group 18 , 10 rats in group 9 ) . mg / rat quartz , titanium dioxide , cuo , cuo nano , mno2 , nio and nio nano suspended in 0.2 ml saline , and groups 8 and 9 were maintained as controls ( saline or untreated ) . some materials , including nanoparticles , demonstrated toxicity that was too strong for sensitive assessment . the ranking order is cuo > quartz > neutralized na2pdcl4 > nio > hydrotalcite > mno2 > diesel > titanium dioxide ( in experiment 2 ) > -cyclodextrin > diesel standard > titanium dioxide ( in experiment 1 ) > caco3 . experiment 3 : a total of 96 rats were randomly separated into 8 groups of 12 rats each . mg / rat quartz , diesel standard powder , diesel powder , c6h10o4pd and caco3 suspended in 0.2 ml saline , and group 5 was exposed to neutralized na2pdcl4 ( final ph was 6.5 ) in 20 mm phosphate buffer and 5 n nacl . groups 7 and 8 were maintained as vehicle controls ( saline or vehicle of group 5 ) . subgroups of 6 rats were sacrificed on days 1 and 28 . in order to assess toxicity to lungs for each fine particle material and to screen for hazard in a relatively simple way , toxicity points for each particle or chemical were calculated from histopathological and the immunohistochemical brdu and inos findings ( table 1 ) and placed in order . more detailed information on total toxicity points has previously been reported by yokohira et al .. the results of the evaluation of particle toxicity by total toxicity points are outlined below , and summarized results for fine particle toxicity are detailed in fig . only cuo caused greater toxicity than quartz ; the other particles had relatively minor effects . although some materials , including nanoparticles , demonstrated toxicity that was too strong for sensitive assessment , the ranking order could be clarified as follows : cuo > quartz > neutralized na2pdcl4 > nio > hydrotalcite > mno2 > diesel > titanium dioxide ( in experiment 2 ) > -cyclodextrin > diesel standard > titanium dioxide ( in experiment 1 ) > caco3 . the various particles employed in the present study , quartz , titanium dioxide , hydrotalcite and -cyclodextrin , k2pdcl4 , na2pdcl4 , cuo , cuo nano , mno2 , nio , nio nano , diesel standard powder , diesel powder , c6h10o4pd and caco3 , were chosen based on likelihood of human exposure . hydrotalcite is employed as an antacid , and -cyclodextrin is employed as a food additive and in the pharmaceutical field to improve dissolution , chemical stability and bioavailability . caco3 is calcium carbonate , is commonly referred to as chalk , and in industrial plants , workers may breathe in k2pdcl4 , na2pdcl4 , c6h10o4pd ( the preceding 3 particles are used as catalysers ) , cuo , cuo nanoparticles , mno2 , nio or nio nanoparticles . all rats instilled with k2pdcl4 or na2pdcl4 solution died from severe hemorrhage and edema of the lungs following intratracheal instillation in experiment a. the ph values of the k2pdcl4 and na2pdcl4 solutions were strongly acidic at 3.2 , and this presumably was a major contributor to lung damage . the lungs instilled with neutralized na2pdcl4 also demonstrated severe inflammatory changes on day 1 . the findings for lungs treated with c6h10o4pd were almost the same , severe hemorrhage and edema , as those for k2pdcl4 and na2pdcl4 . however , the ph value of the c6h10o4pd solution was 4.9 , which was not sufficiently acidic to lead to death . reported that pdso4 exerts significant effects on human epithelial lung cells , and we have previously confirmed pdo toxicity in rats . based on the available results , it is possible that not only strong acids but also palladium itself has toxic potential in the lung . in the case of cuo and nio , the diameters of the particles were in the micrometer and nanometer ranges . with the same volume of material , in general , it is likely that nanoparticles exert a greater impact because of the area and characteristics of the surfaces . for example , an oral administration study in mice indicated that titanium dioxide is retained in the liver , spleen , kidney and lung tissues after uptake through the gastrointestinal tract . however , no remarkable histopathological changes were seen in the liver and kidneys after treatment with nanoparticles of cuo or nio in the present study . also , nio nanoparticles induced only mild inflammatory change in the lungs on day 1 . similarly found limited histopathological effects on the lungs with 13.0 mg nio powder / kg b.w .. further examination is necessary to clarify the causes of death observed here . to establish a bioassay model for detection of lung toxicity due to fine particles for screening purposes , quartz is known as a typical lung toxic agent and provides a reliable positive control . titanium dioxide was classified as belonging to group 2b ( possibly carcinogenic to humans ) in a recent international agency for research on cancer ( iarc ) publication , but hext et al . concluded from an overview of epidemiology studies and toxicology studies in mice , rats and hamsters that titanium dioxide dust should not exert carcinogenic effects in the human lung . on the other hand , titanium dioxide has carcinogenic potential , and exposure may result in formation of dna adducts . with earlier assessment of acute or subacute findings , the severity of toxicity differed according to the instilled particles : quartz > 80:20 anatase : rutile and ultrafine titanium dioxide > fine - sized rutile titanium dioxide = ultrafine rutile titanium dioxide . use of titanium dioxide as a negative control should be considered , where possible , for experimental purposes . in both experiments 1 and 2 , however , based on the total points in these experiments , at least 3 rough grades of toxicity level , severe , moderate and mild , could be determined . it is expected that further investigations to obtain more particle toxicity data will allow more sensitive assessment of the toxicity of particles in the future . the results of several pilot experiments , performed in rats using quartz as a typical lung toxic particle , suggest that days 1 and 28 after i.t . of 2 mg of fine test particles suspended in 0.2 ml vehicle are the most appropriate for detection of acute and subacute inflammatory changes , respectively . furthermore , brdu on day 1 and inos on day 28 proved to be suitable end - point markers for this purpose . examination of the toxicity of a series of particles could be performed with the developed bioassay . although some materials , including nanoparticles , demonstrated toxicity that was too strong for sensitive assessment , a ranking order could be clarified . our bioassay does suffer from a weakness in that even low doses of some particles may lead to death . method has been proposed as the most reliable route for assessing the pulmonary toxicity of particles in rodents , although there are biologically different responses to inhalation and instillation . another limitation of this bioassay is that the toxicity of particles can only be detected at an early stage because the experimental period is limited to 28 days . in this experiment , a dose response study was performed for quartz , not for risk assessment , but for improvement of the bioassay , and this study was not performed with other test particles . for risk assessment , more experiments , including dose response studies for each test particle with hazard characterization , should be conducted . with in that said , the bioassay was originally designed to be used for hazard identification at an early stage and to rank the toxicities of various particles given at single representative concentrations . the approach adopted clearly is also useful for detection of acute and subacute pulmonary particle characteristics using a histopathological scoring system and markers like brdu and inos for screening purposes .
it is an urgent priority to establish in vivo bioassays for detection of hazards related to fine particles , which can be inhaled into deep lung tissue by humans . in order to establish an appropriate bioassay for detection of lung damage after particle inhalation , several experiments were performed in rats using quartz as a typical lung toxic particle . the results of pilot experiments suggest that days 1 and 28 after intratracheal instillation of 2 mg of fine test particles in vehicle are most appropriate for detection of acute and subacute inflammatory changes , respectively . furthermore , the brdu incorporation on day 1 and the inos level on day 28 proved to be suitable end - point markers for this purpose . an examination of the toxicity of a series of particles was performed with the developed bioassay . although some materials , including nanoparticles , demonstrated toxicity that was too strong for sensitive assessment , a ranking order could be clarified . the bioassay thus appears suitable for rapid hazard identification with a possible ranking of the toxicity of various particles at single concentrations .
Introduction Sequential Analysis Study Dose Response Study Vehicle Assessment Study (Dry Powder Assessment) Toxicity Assessment of a Series of Different Particle Types Conclusion
it is an urgent priority to establish in vivo bioassays for the detection of hazards related to fine particles , which can be inhaled into deep lung tissue by humans . in order to establish an appropriate bioassay for detection of lung damage after particle inhalation , several experiments were performed in rats using quartz as a typical particle lung toxicant . finally , the toxicity of a series of different particle types , including nanoparticles and diesel exhaust particles , was also tested with our new bioassay . in order to establish an appropriate bioassay for detection of lung damage after fine particle inhalation , sequential histopathological changes were examined after i.t . , the results suggest that days 1 and 28 after intratracheal instillation of fine test particles are the most appropriate day for detection of acute and subacute inflammatory changes , respectively . furthermore , brdu on day 1 and inos on day 28 proved to be suitable end - point markers for this purpose . a dose of 2 mg quartz was thus suggested to be the most appropriate dose for sensitive detection of acute and subchronic inflammatory changes . the pilot experiments suggested that a dose of 2 mg quartz suspended in 0.2 ml saline was suggested to be most appropriate for sensitive detection of acute and subchronic inflammatory changes . using this as a control , examination of the toxicity of a series of particles these materials were selected to perform this validation study and for their variety of characteristics , including shape , composition and particle diameters with the nanometer order or not . some materials , including nanoparticles , demonstrated toxicity that was too strong for sensitive assessment . although some materials , including nanoparticles , demonstrated toxicity that was too strong for sensitive assessment , the ranking order could be clarified as follows : cuo > quartz > neutralized na2pdcl4 > nio > hydrotalcite > mno2 > diesel > titanium dioxide ( in experiment 2 ) > -cyclodextrin > diesel standard > titanium dioxide ( in experiment 1 ) > caco3 . to establish a bioassay model for detection of lung toxicity due to fine particles for screening purposes , quartz is known as a typical lung toxic agent and provides a reliable positive control . it is expected that further investigations to obtain more particle toxicity data will allow more sensitive assessment of the toxicity of particles in the future . the results of several pilot experiments , performed in rats using quartz as a typical lung toxic particle , suggest that days 1 and 28 after i.t . of 2 mg of fine test particles suspended in 0.2 ml vehicle are the most appropriate for detection of acute and subacute inflammatory changes , respectively . furthermore , brdu on day 1 and inos on day 28 proved to be suitable end - point markers for this purpose . examination of the toxicity of a series of particles could be performed with the developed bioassay . although some materials , including nanoparticles , demonstrated toxicity that was too strong for sensitive assessment , a ranking order could be clarified . in this experiment , a dose response study was performed for quartz , not for risk assessment , but for improvement of the bioassay , and this study was not performed with other test particles . with in that said , the bioassay was originally designed to be used for hazard identification at an early stage and to rank the toxicities of various particles given at single representative concentrations .
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fxs patients exhibit neurological symptoms that include learning disabilities , social anxiety , attention deficits , hyperarousal , hypersensitivity , autism , and epilepsy . notwithstanding the complexity of neurophysiological and behavioral alterations , fxs is caused by the silencing , deletion , or loss - of - function mutation of a single gene , fmr1 . as a result , fmrp ( fragile x mental retardation protein ) , its protein product , is not expressed in the majority of cases or is non - functional in the rare cases with a point mutation [ 2 , 3 , 4 ] . fmrp is an mrna - binding protein that regulates several aspects of mrna metabolism such as nuclear export , transport to synaptic terminals , activity - dependent ribosome stalling and gene expression [ 6 , 7 , 8 ] . although much of fmrp activity is thought to be related to regulation of synaptic function [ 9 , 10 , 11 ] , little is known about the potential defects in neuronal function caused by the absence of fmrp , in particular how these neurophysiological alterations lead to impairment in neuronal computations and behavior in patients with fxs . initial studies revealed that dendritic spine number is increased in the cortex of fxs patients [ 12 , 13 ] . in fact , dendritic abnormalities are the most consistent anatomical correlates of intellectual disability . studies on animal models of fxs showed that fmrp regulates neuronal branching [ 15 , 16 , 17 ] as well as dendritic spine morphology and density [ 11 , 18 ] . in addition to defects in synaptic structure and axonal branching , impairments in animal behavior have been observed [ 11 , 16 ] . however , further studies showed that neuroanatomical and behavioral defects can be genetically uncoupled , suggesting that unknown impairments in neuronal circuit function may underlie behavioral deficits . fmrp regulates translation of mrnas at synapses , some of which encode proteins involved in synaptic plasticity [ 19 , 20 ] . importantly , the absence of fmrp leads to abnormally enhanced group 1 mglur ( metabotropic glutamate receptor ) signaling , which results in exaggerated long - term depression , with a net loss of ampa and nmda receptors [ 22 , 23 ] . additionally , enhanced group 1 mglur signaling contributes to the elongation of dendritic spines in rodent models of fxs [ 18 , 24 ] and leads to increased intrinsic neuronal excitability through the downregulation of potassium channels controlling resting membrane potential and action potential afterhyperpolarization [ 25 , 26 ] . moreover , fmrp directly influences neuronal excitability by regulating expression of potassium channels [ 27 , 28 ] and by interacting with potassium channels in a translation - independent manner . nevertheless , the recent failure of fxs clinical trials targeting group 1 mglur signaling has led the field to re - examine the group 1 mglur hypothesis . loss of fmrp was shown to increase network - level hyperexcitability in the rodent cortex [ 31 , 32 ] , which has been associated with the symptoms observed in fxs patients , such as hypersensitivity , hyperarousal , hyperactivity , anxiety , and epilepsy . interestingly , absence of fmrp downregulates gabaa receptor subunits in both mice and flies [ 34 , 35 ] . furthermore , the enzymes for gaba synthesis and degradation , gaba membrane transporters , a gaba receptor scaffolding protein , and a protein that regulates gabab receptor signaling are downregulated in the absence of fmrp [ 36 , 37 ] . these observations suggest a tantalizing , yet poorly understood , link between gabaergic signaling , network hyperexcitability , and behavioral deficits in fxs models and patients . in contrast to the group 1 mglur component of fxs , the potential effects of altered synaptic inhibition on neuronal circuit excitability and how these changes might impact sensory computations and animal behavior remain unexplored . in this study , we explore the changes in neuronal circuit function and connectivity underlying fxs by using a combination of behavioral assays , functional brain imaging , optogenetics , and electrophysiology in a fly fxs model . we focused on the drosophila melanogaster olfactory system , which is a well - understood and genetically tractable neuronal circuit . specifically , we evaluated olfactory computations in the antennal lobe , a circuit constituted by excitatory projection neurons , which receive synaptic input from their cognate olfactory receptor neurons , as well as inhibitory local interneurons involved in mediating lateral inhibition . we find that the absence of dfmrp , the fly homolog of the human fmrp , results in reduced olfactory attraction and aversion . calcium imaging data show that antennal lobe projection neurons have broader odor tuning in dfmr1 flies , leading to reduced specificity in odor coding and alterations in olfactory representations . consistent with these results , we observe that lateral inhibition across olfactory glomeruli , as well as the inhibitory connections between local interneurons and projection neurons , are impaired in dfmr1 flies . finally , downregulation of gaba receptors in projection neurons is sufficient to produce olfactory behavioral defects . we propose that absence of dfmrp leads to defective lateral inhibition across olfactory glomeruli , which , in turn , results in impaired odor coding and olfactory behaviors . dfmr1 flies were previously shown to have learning deficits in olfactory behavioral assays . the authors suggested that this was not due to a sensory deficit ; however , no detailed analysis of olfactory processing was performed . to evaluate whether the olfactory system of drosophila melanogaster is affected by the absence of fmrp , we conducted olfactory attraction and aversion assays ( figures 1a and s1a s1c ) . ethyl acetate is known to induce attraction in flies , whereas benzaldehyde induces aversion [ 40 , 41 ] . we presented these odors to starved flies and quantified attraction and aversion by counting the number of flies in odorized and non - odorized sections of the behavioral arena , before and during odor delivery . we found that dfmr1 flies exhibit significantly weaker olfactory attraction and aversion compared to wild - type ( wt ) flies ( figures 1b , 1c , 1h , 1i , 1l , and 1 m ) . we observed that dfmr1 flies spend less time exploring the quadrant odorized with the attractive odor ethyl acetate ( figures 1b , 1h , and 1l ) . similarly , dfmr1 flies were not repelled as much as wt flies by the aversive odor benzaldehyde ( figures 1c , 1i , and 1 m ) . furthermore , impaired olfactory performance in dfmr1 flies can be restored by the genomic construct of dfmrp ( figures 1d , 1e , 1h , 1i , 1l , and 1 m ) . to test whether reduced olfactory performance was due to the absence of dfmrp in the antennal lobe circuit , we knocked down dfmrp expression specifically in excitatory antennal lobe projection neurons . downregulation of dfmrp in the antennal lobe projection neurons led to a significant impairment of olfactory behaviors ( figures 1f , 1 g , and 1j1 m ) , confirming the role of dfmrp for antennal lobe circuit function and olfactory behaviors . similar results were obtained by knocking down dfmrp expression in inhibitory antennal lobe local interneurons ( figure s1e ) . reduced performance of dfmr1 flies in olfactory behaviors suggests that odor coding is compromised in these animals . to evaluate whether olfactory computations are affected by the absence of dfmrp , we measured the odor responses of antennal lobe projection neurons using calcium imaging ( figures 2a , 2b , and s2 ) in wt and dfmr1 flies ( figure 2d ) . we extracted the location of individual glomeruli using independent component analysis , which is effective in identifying even the sister glomeruli across antennal lobes with very similar locations and response profiles ( figures 2c and s3 ) . next , we investigated the glomerular activation patterns of projection neurons and compared the representations of 24 odors in wt and dfmr1 flies . we observed that overall responsiveness of olfactory glomeruli is significantly altered , with more excitatory and fewer inhibitory odor responses in dfmr1 flies ( figures s4a s4d ) . specifically , wt flies exhibited more inhibitory responses , more silent glomeruli , and more strong excitatory responses , whereas dfmr1 flies presented an increased number of weak excitatory responses ( figures s4a s4d ) , reflecting that not only are dfmr1 projection neurons hyperexcitable , but excitation of strongly responding neurons is also impaired . this indicates a deficit in contrast enhancement of olfactory representations , which might be a consequence of reduced lateral inhibition . to further evaluate olfactory coding , we carried out a pairwise comparison of odor - evoked glomerular activation patterns using two commonly used and complementary measures of similarity , cosine distance and euclidean distance . cosine distance compares odor responses regardless of amplitude , while euclidean distance takes the strength of odor responses into account . high cosine and euclidean distances indicate increased difference among odor representations and , hence , greater specificity in odor encoding . our results show significantly lower cosine and euclidean distances between pairs of odors in dfmr1 flies ( figures 3a3d ) . this indicates that loss of dfmrp causes odor - evoked glomerular activation patterns to become less distinct from each other and , therefore , harder to discriminate . reduced odor specificity of glomerular activation patterns could explain why dfmr1 flies are impaired in both attractive and aversive olfactory behavioral tasks . what underlies the increased similarity among odor representations in dfmr1 flies ? to answer this question , we visualized odor selectivity by plotting the responses of each glomerulus normalized to its maximum odor response . we observed that dfmr1 glomeruli have broader response profiles and , thus , reduced odor selectivity , represented by warmer colors ( figure 3e ) . to quantify this , we calculated the lifetime sparseness , a measure of response selectivity , of all glomeruli . we found that dfmr1 glomeruli have significantly lower lifetime sparseness values ( figure 3f ) , suggesting they are less odor selective . complementary to this , we computed the population sparseness , which is a measure of the number of glomeruli activated by a single odor . a high population sparseness value signifies that few glomeruli were activated by a given odor , whereas a low population sparseness value signifies that many glomeruli were similarly activated . we found that the antennal lobe of dfmr1 flies has significantly lower population sparseness ( figure 3 g ) , which is consistent with our complementary analysis showing reduced response to background ( signal - to - noise ) ratios ( figure s4d ) and increased correlations across antennal lobe glomeruli in dfmr1 flies ( figure s4e ) . our results reveal an impairment in olfactory coding and odor selectivity in dfmr1 flies due to broader tuning and reduced odor selectivity of antennal lobe projection neurons . this reduced odor selectivity can , in principle , arise from less selective glomerular innervation patterns of individual projection neurons in dfmr1 flies . however , we did not observe changes in glomerular morphology or size in any of the genetically identified projection neurons of dfmr1 flies ( figure s5 ) . the lack of any obvious morphological alterations in projection neurons , combined with our observations of increased excitatory and reduced inhibitory odor responses , suggests that defective lateral interactions among antennal lobe neurons might be responsible for the reduced specificity of olfactory representations in dfmr1 flies . in the fly antennal lobe , lateral interactions across olfactory glomeruli were shown to mediate the spread of both excitation , through gap junctions , and inhibition , through local interneurons . it has been shown that , when odors are mixed , lateral interactions across antennal lobe glomeruli can alter odor representations , both through lateral excitation and lateral inhibition . to compare the level of lateral interactions in wt and dfmr1 flies , we applied mixtures of odorants , in which the concentration of one of the components is kept constant while the concentration of the other mixture component is gradually increased ( figures 4a and s6a ) . this , in turn , will change the odor - evoked activity patterns , creating new odor representations depending on the degree of lateral interactions among all recruited neurons . we observed that the odor representation of the component with fixed concentration became progressively different with increasing concentrations of the second mixture component . these mixing - related changes in odor representations were more pronounced in wt flies than in dfmr1 flies ( figures 4a4c and s6a s6c ) . our results showed that , on average , wt flies exhibited significantly more mixture - related suppression , whereas dfmr1 flies exhibited significantly more mixture - related excitation ( figures 4d , 4e , s6d , and s6e ) . this suggests that , while lateral inhibition is impaired in dfmr1 flies , lateral excitatory interactions might be spared ( figure s7 ) . in line with this , our results suggest that populations of individual glomeruli in wt flies have a significantly larger variety of both inhibitory and excitatory effects at all mixture concentrations , when compared to dfmr1 flies ( figures 4f and s6f ) . altogether , these results support the idea that lateral inhibitory interactions are impaired in the antennal lobe of dfmr1 flies , which eventually results in reduced contrast across odor representations and , therefore , poorer performance in olfactory behaviors . our findings using odor mixtures point to reduced lateral inhibition among olfactory glomeruli in dfmr1 flies . in line with this , several components of the gabaergic transmission machinery are reported to be downregulated in mouse and fruit fly models of fxs [ 34 , 35 ] . moreover , gabaergic signaling appears to be disrupted in the brains of autistic patients , a recurrent phenotype in fxs . all this evidence led to the hypothesis that reduced inhibition may be a major mechanism underlying neuronal deficits in fxs . however , direct in vivo physiological evidence that inhibitory connections between neurons are impaired in any in vivo model of fxs is lacking . in the fruit fly antennal lobe , lateral inhibition across olfactory glomeruli is mediated by gabaergic local interneurons that can act on both olfactory receptor neuron terminals and on projection neurons [ 44 , 51 , 52 ] . to directly test the action of local interneurons on the activity of projection neurons , we performed intracellular recordings of projection neurons while optogenetically stimulating gabaergic local interneurons expressing channelrhodopsin-2 ( figure 5a ) [ 53 , 54 ] . optogenetic activation of local interneurons consistently hyperpolarized the membrane potential of wt projection neurons ( figures 5b5d ) . in contrast , dfmr1 projection neurons exhibited significantly smaller or no hyperpolarization in their membrane potential ( figures 5b5d ) . importantly , we observed that dfmr1 projection neurons exhibit a prominent excitation upon optogenetic local interneuron stimulation ( figure 5c ) , which is mediated by the gap junctions between local interneurons and projection neurons . during these recordings , we kept the antennae dry and the olfactory nerve intact , which ensures that the olfactory receptor neurons are undamaged and sustain a healthy level of background activity . as previously shown , this remaining olfactory receptor neuron background firing results in prominent subthreshold synaptic activity and spontaneous action potential firing in our recorded projection neurons ( figures 5b , 5e , and 5 g ) . the optogenetic activation of local interneurons reduced the firing rate of wt projection neurons significantly more than that of dfmr1 projection neurons ( figures 5e5h ) . in line with the remaining gap - junction - mediated lateral excitation , we observed a slight increase in projection neuron firing rates of dfmr1 flies ( figures 5e and 5f ) . the observed impaired inhibition in the projection neurons of dfmr1 flies could , in principle , be the consequence of a less effective optogenetic activation of local interneurons . to rule out this possibility , we recorded the responses to optogenetic activation in local interneurons expressing channelrhodopsin-2 , both in wt and dfmr1 flies ( figures 6a and 6b ) . our results showed that optogenetic stimulation elicited significantly larger depolarization and higher firing rates in dfmr1 local interneurons , when compared to wt local interneurons ( figures 6c6h ) . furthermore , optogenetic stimulation of local interneurons consistently inhibit the local interneurons that do not express channelrhodopsin-2 in wt flies ( figures 6i6k ) . by contrast , little or no inhibition was observed in dfmr1 local interneurons not expressing channelrhodopsin-2 ( figures 6i6k ) . these results indicate that the reduced inhibition observed in dfmr1 projection neurons ( figure 5 ) can not be due to less effective optogenetic activation of dfmr1 local interneurons . in fact , our results suggest that optogenetic stimulation is more effective in activating dfmr1 local interneurons , especially at the later phase of the stimulation , presumably , due to less effective gabaergic inhibition across dfmr1 local interneurons . in summary , these experiments revealed that deficient inhibition of dfmr1 projection neurons ( figure 5 ) is due to less effective gabaergic inhibition from local interneurons onto the whole antennal lobe circuit , at the level of both projection neurons and local interneurons . we observed that dfmr1 flies present less strongly activated glomeruli ( figures s4a s4d ) , which could be caused by reduced lateral excitation . we , therefore , recorded lateral excitatory responses in projection neurons of flies , in which the antennae were removed and , hence , did not present spontaneous activity ( figure s7a ) . optogenetic activation of local interneurons produced an excitatory response in both wt and dfmr1 projection neurons ( figures s7b and s7c ) . however , lateral excitatory responses decay faster in dfmr1 projection neurons ( figures s7b and s7c ) and were smaller in amplitude ( figure s7d ) . this observation could , in part , explain the lower incidence of strongly activated glomeruli upon odor stimulation in dfmr1 flies ( figures s4a s4d ) . our observations , indicating that inhibition is reduced in both the projection neurons ( figure 5 ) and the local interneurons ( figure 6 ) of the antennal lobe , suggest that lack of inhibition is the neurophysiological cause of the behavioral abnormalities observed in the absence of dfmrp ( figure 1 ) . we directly tested this idea by knocking down the expression of the gabaergic rdl receptor . downregulation of rdl receptors selectively in projection neurons ( figure 7a ) or in local interneurons ( figure 7b ) resulted in lower olfactory behavioral performance in fruit flies . taken together with the previously reported decreased expression of gabaa receptors in the absence of fmrp , the electrophysiological and behavioral evidence presented in this study strongly suggests that reduced inhibition of neuronal circuits contributes to the pathophysiology of fxs . since the discovery of reduced gabaa receptor subunit expression in the absence of fmrp , accumulated evidence has pointed toward alterations in gabaergic transmission as a key component in the neurophysiology of fxs [ 50 , 56 ] . in fact , intracellular recordings on acute brain slices suggested that reduced inhibitory input from interneurons onto pyramidal neurons could result in an excitation / inhibition imbalance [ 35 , 57 ] . whether this is true in vivo and we tested this using the fruit fly antennal lobe circuit and demonstrate that gabaergic connections established by local interneurons , which mediate lateral inhibition [ 44 , 51 , 58 ] , are impaired in a drosophila melanogaster model of fxs . moreover , we show that deficits in gabaergic lateral inhibition leads to increased circuit excitability , which results in reduced stimulus selectivity in projection neurons we postulate that similar deficits in lateral inhibition impair neuronal computations in other sensory modalities . in consonance with this , it has been reported that circuit hyperexcitability leads to behavioral alterations in tactile , auditory , and olfactory tasks in mouse models of fxs [ 32 , 59 , 60 ] . our results indicate that , in the absence of dfmrp , neuronal computations are impaired in the antennal lobe of drosophila melanogaster . this is in apparent contradiction with a previous study showing long - term memory defects in dfmr1 flies and no sensory deficits . as we report , responses to many odors are still elicited in projection neurons of dfmr1 flies . we suggest that this difference may be due to a more extensive and quantitative analysis of behavior and physiology in our study that revealed defects that may have not been previously detected . alternatively , the penetrance and severity of phenotypes in fmr1 mutant animals , both mice and flies , can be sensitive to genetic background . it is possible that the previous study did not account for this . at any rate , both null alleles and rnai flies analyzed using behavioral , imaging , and electrophysiological approaches revealed that dfmr1 mutants exhibit reduced odor specificity and , thus , deficient olfactory processing . lateral inhibition across drosophila olfactory glomeruli has been proposed to be important for increasing contrast among odor representations and , therefore , for discriminating odors [ 44 , 48 ] . interestingly , such a mechanism has been suggested to be relevant for other sensory modalities . in this winner - take - all model , glomeruli with most prominent odor responses would strongly activate surrounding interneurons , spreading inhibition to nearby weakly activated glomeruli . the spread of lateral inhibition , in turn , would inhibit the odor responses of weakly activated glomeruli , while strongly activated glomeruli remain as the unique encoder of the particular odor . this model also suggests that the lack of many weakly activated glomeruli , in addition to few strongly responding but very odor - specific glomeruli , enhances the separation of odor response patterns from one another . in line with this model , we observed that lack of lateral inhibition in the antennal lobe of dfmr1 flies , indeed , leads to an increase in the number of weakly activated and less odor - specific glomeruli . by contrast , wt flies present more inhibitory and less weak excitatory responses , sparing strongly responding olfactory glomeruli that are more odor specific . this is probably a consequence of reduced lateral inhibition , which is important for contrast enhancement of odor representations . additionally , the slight decrease in lateral excitation observed in dfmr1 flies could result in less strongly represented glomeruli . importantly , defects in olfactory processing have been observed in other animal models of fxs , as well as in human patients , which display hypersensitivity to smells and to other sensory modalities involving lateral inhibitory mechanisms such as tactility and audition . beyond the olfactory system , several studies have shown that cns neurons are hyperexcitable in the absence of fmrp [ 31 , 32 , 57 ] . since activation of the group 1 mglur signaling pathway results in increased neuronal excitability [ 25 , 26 ] , circuit hyperexcitability has been attributed to the constitutively enhanced group 1 mglur signaling observed in mouse fxs models . here , we provide the first direct in vivo evidence showing that defects in lateral gabaergic inhibition significantly contribute to circuit hyperexcitability . this is consistent with downregulation of proteins involved in gabaergic transmission both in fruit flies and in rodents [ 34 , 36 ] . thus , reduced inhibition could be a consequence of decreased gaba release from local interneurons , reduced expression of postsynaptic gaba receptors , or both . further studies of protein expression profiles for the specific neuron types are needed to elucidate this . it is possible that this mechanism might explain phenotypes observed in fxs patients such as hypersensitivity , hyperarousal , hyperactivity , and epilepsy , all of which reflect hyperexcitable brain states . in summary , we demonstrate that lateral inhibition within the antennal lobe is strongly affected in dfmr1 flies due to impaired inhibitory connections from local interneurons onto projection neurons and other local interneurons . the lack of this lateral inhibition on projection neurons is probably the major cause for their increased excitability and reduced odor specificity . we propose that this compromised olfactory coding consequently leads to impaired olfactory behaviors in dfmr1 flies . more generally , we provide the missing in vivo evidence that the lack of dfmrp has a direct impact on sensory processing and animal behavior through a weakening of lateral inhibitory connections , which broadens response tuning of principal neurons . this mechanism might be ubiquitously present in the brain of fxs patients . for instance , reduced gabaergic inhibition could produce hyperexcitable neuronal circuits in fxs patients , which not only explains symptoms such as hypersensitivity , hyperarousal , or epilepsy but also potentially contributes to the misprocessing of information across the brain , which would have severe effects on human behavior . finally , given the overlap between the phenotypes of fxs and those of other neurological diseases , such as autism , rett syndrome , or dravet syndrome , and their corresponding perturbations in gabaergic transmission [ 33 , 49 , 63 ] , it is possible that similar mechanisms involving reduced lateral inhibition are also present in these neurological syndromes , which are yet to be discovered . l.m.f . , b.a.h . , and e.y . conceived the study , designed the experiments , and wrote the manuscript .
summaryfragile x syndrome ( fxs ) patients present neuronal alterations that lead to severe intellectual disability , but the underlying neuronal circuit mechanisms are poorly understood . an emerging hypothesis postulates that reduced gabaergic inhibition of excitatory neurons is a key component in the pathophysiology of fxs . here , we directly test this idea in a fxs drosophila model . we show that fxs flies exhibit strongly impaired olfactory behaviors . in line with this , olfactory representations are less odor specific due to broader response tuning of excitatory projection neurons . we find that impaired inhibitory interactions underlie reduced specificity in olfactory computations . finally , we show that defective lateral inhibition across projection neurons is caused by weaker inhibition from gabaergic interneurons . we provide direct evidence that deficient inhibition impairs sensory computations and behavior in an in vivo model of fxs . together with evidence of impaired inhibition in autism and rett syndrome , these findings suggest a potentially general mechanism for intellectual disability .
Introduction Results Discussion Author Contributions
although much of fmrp activity is thought to be related to regulation of synaptic function [ 9 , 10 , 11 ] , little is known about the potential defects in neuronal function caused by the absence of fmrp , in particular how these neurophysiological alterations lead to impairment in neuronal computations and behavior in patients with fxs . in contrast to the group 1 mglur component of fxs , the potential effects of altered synaptic inhibition on neuronal circuit excitability and how these changes might impact sensory computations and animal behavior remain unexplored . specifically , we evaluated olfactory computations in the antennal lobe , a circuit constituted by excitatory projection neurons , which receive synaptic input from their cognate olfactory receptor neurons , as well as inhibitory local interneurons involved in mediating lateral inhibition . calcium imaging data show that antennal lobe projection neurons have broader odor tuning in dfmr1 flies , leading to reduced specificity in odor coding and alterations in olfactory representations . to test whether reduced olfactory performance was due to the absence of dfmrp in the antennal lobe circuit , we knocked down dfmrp expression specifically in excitatory antennal lobe projection neurons . our results reveal an impairment in olfactory coding and odor selectivity in dfmr1 flies due to broader tuning and reduced odor selectivity of antennal lobe projection neurons . the lack of any obvious morphological alterations in projection neurons , combined with our observations of increased excitatory and reduced inhibitory odor responses , suggests that defective lateral interactions among antennal lobe neurons might be responsible for the reduced specificity of olfactory representations in dfmr1 flies . altogether , these results support the idea that lateral inhibitory interactions are impaired in the antennal lobe of dfmr1 flies , which eventually results in reduced contrast across odor representations and , therefore , poorer performance in olfactory behaviors . in line with this , several components of the gabaergic transmission machinery are reported to be downregulated in mouse and fruit fly models of fxs [ 34 , 35 ] . however , direct in vivo physiological evidence that inhibitory connections between neurons are impaired in any in vivo model of fxs is lacking . in the fruit fly antennal lobe , lateral inhibition across olfactory glomeruli is mediated by gabaergic local interneurons that can act on both olfactory receptor neuron terminals and on projection neurons [ 44 , 51 , 52 ] . in summary , these experiments revealed that deficient inhibition of dfmr1 projection neurons ( figure 5 ) is due to less effective gabaergic inhibition from local interneurons onto the whole antennal lobe circuit , at the level of both projection neurons and local interneurons . taken together with the previously reported decreased expression of gabaa receptors in the absence of fmrp , the electrophysiological and behavioral evidence presented in this study strongly suggests that reduced inhibition of neuronal circuits contributes to the pathophysiology of fxs . since the discovery of reduced gabaa receptor subunit expression in the absence of fmrp , accumulated evidence has pointed toward alterations in gabaergic transmission as a key component in the neurophysiology of fxs [ 50 , 56 ] . whether this is true in vivo and we tested this using the fruit fly antennal lobe circuit and demonstrate that gabaergic connections established by local interneurons , which mediate lateral inhibition [ 44 , 51 , 58 ] , are impaired in a drosophila melanogaster model of fxs . moreover , we show that deficits in gabaergic lateral inhibition leads to increased circuit excitability , which results in reduced stimulus selectivity in projection neurons we postulate that similar deficits in lateral inhibition impair neuronal computations in other sensory modalities . in line with this model , we observed that lack of lateral inhibition in the antennal lobe of dfmr1 flies , indeed , leads to an increase in the number of weakly activated and less odor - specific glomeruli . here , we provide the first direct in vivo evidence showing that defects in lateral gabaergic inhibition significantly contribute to circuit hyperexcitability . in summary , we demonstrate that lateral inhibition within the antennal lobe is strongly affected in dfmr1 flies due to impaired inhibitory connections from local interneurons onto projection neurons and other local interneurons . more generally , we provide the missing in vivo evidence that the lack of dfmrp has a direct impact on sensory processing and animal behavior through a weakening of lateral inhibitory connections , which broadens response tuning of principal neurons . finally , given the overlap between the phenotypes of fxs and those of other neurological diseases , such as autism , rett syndrome , or dravet syndrome , and their corresponding perturbations in gabaergic transmission [ 33 , 49 , 63 ] , it is possible that similar mechanisms involving reduced lateral inhibition are also present in these neurological syndromes , which are yet to be discovered .
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leishmaniasis refers to a variety of diseases caused by more than 20 species of intracellular protozoan parasites belonging to the genus leishmania . the clinical spectrum of the disease ranges from simple self - limiting cutaneous ulcers to severe disfiguring mucocutaneous , and even to a fatal visceral disease known as kala azar . about 98 tropical and subtropical countries are known to be endemic for this disease , with 350 million people at risk and overall prevalence of 12 million worldwide ( 1 ) . cutaneous leishmaniasis ( cl ) is commonly caused by l. major and l. tropica ( 2 ) . visceral leishmaniasis ( vl ) , the most life threatening form , is caused by l. infantum and in very rare occasions by l. tropica ( 2 , 3 ) . in vl fever and hepato - splenomegaly are the main clinical signs in which leishmania parasite is dispersed to the internal viscera like spleen , liver and bone marrow ( 4 ) . based on the leishmaniasis clinical symptoms , it is evident that the host immunity factors , leishmania species , and in some cases the leishmania strain , determines the measure of pathogenecity ( 5 ) . . has about 8000 genes among only 78 genes are restricted to individual species ( 6 ) . in spite of a few species parasite genes implicated in pathogenesis and clinical presentation , the parasite gene expression rates differ greatly among species ( 6 ) . in leishmaniasis , parasites are challenged by the host immune conditions throughout their life cycle such as temperature increase of visceral tissues ( liver , spleen or bone marrow ) . such challenges causes leishmania experience biochemical changes in which post transcriptional modification are activated and may eventuate into the emergence of the leishmaniasis pathogencity ( 714 ) . analysis of proteome is most commonly performed by a combination of 2-de and mass spectrometry ( ms ) . 2-de method could separate proteins in first and second dimensions according to their isoelectric and molecular weight points . with the help of 2-de and the ms , a variable mixture of proteins is separated , visualized and then identified ( 1516 ) . in this preliminary study , we compared the proteome mapping , in three iranian isolates of leishmania species including l. tropica , l. major and l. infantum , with immobilized ph gradient stripes with linear ph 47 . moreover , liquid chromatography ( lc ) - mass spectrometry was used for identification of a number of differentially expressed proteins among the three species . ef653267 , l. major ( jn860745 ) and l. infantum ( jx289853 ) compared and were analyzed . promastigote forms recovered from the iranian leishmania parasite bank located in leishmaniasis lab , school of public heath , tehran university of medical sciences ( tums ) . the identity of these strains was already obtained by other molecular dna based methods ( 2 , 17 ) . cell culture promastigotes recovered from liquid nitrogen ( 196 c ) , were mass cultured in rpmi1640 medium ( gibco , life technologies gmbh , frankfurt , germany ) supplemented with 15% heat inactivated fetal bovine serum ( gibco , germany ) and 100u / ml penicillin and 100ug / ml streptomycin ( gibco , germany ) and incubated at 24c . parasites were harvested washed in sterile phosphate buffered saline ( pbs , ph : 7.27.4 ) and were used for protein extraction . proteomics analysis was performed on l. tropica , l. major and l. infantum , three species at the time of study . promastigotes were harvested by centrifugation at 3000rpm , 4 c and 20 minutes and washed three times in sterile pbs ( ph : 7.27.4 ) in the same condition for 10 minutes . the cells were resuspended in 5 mm tris hcl , ph 7.8 , containing 1 mm phenylmethylsulfonyl fluoride ( pmsf ( merck , germany ) ) . proteins were precipitated by 10% ( w / v ) trichloroacetic acid ( merck , germany ) in acetone ( merck ) with 0.07 % ( w / v ) dithiothreitol ( dtt ) ( merck ) for 1 hour at 20c . the samples were then centrifuged at 17500 g ( hettich , germany ) for 15 minutes at 4 c and the pellets were washed with ice - cold acetone containing 0.07% dtt , incubated at 20c for 1 h and centrifuged at 4 c . the samples were then solubilized in lysis buffer ( 9.5 m urea ( merck ) , 2% ( w / v ) chaps ( merck ) , 0.8% ( w / v ) ampholyte ( bio - rad , usa ) ph 310 , 1% ( w / v ) dtt ) ( 18 , 19 ) . the concentration of protein was measured by the bradford assay with bovine serum albumin bsa ( merck ) as the standard ( 20 ) . for analytical and preparative gels , 120 g and ief was carried out on the 18 cm immobilized ph gradient ( ipg ) strips ( ph 47 ) ( bio - rad , usa ) . ipg strips were rehydrated overnight by loading the samples diluted with rehydration buffer containing 8 m urea , 4% chaps , 2% ampholyte , 50 mm dtt , and traces of bromophenol blue ( merck ) . isoelectric focusing was conducted at 20 c with mutiphor ii and a drystrip kit ( ge healthcare , germany ) . the running condition was as follows : 300 v for 90 minute , followed by 500 v for 90 min , 1000 v for 3 h and finally 3500 v for 16 h. the focused strips were equilibrated twice in equilibration solution . the first equilibration was performed in a solution containing 6 m urea , 20% ( w / v ) glycerol , 2% ( w / v ) sds ( merck ) , 1% ( w / v ) dtt , and 50 mm tris - hcl ( merck ) buffer , ph 8.8 . the second equilibration was performed in a solution with 2.5% ( w / v ) iodoacetamide ( merck ) . separation in the second dimension was performed by sds - page in a vertical slab of acrylamide ( merck ) ( 12% total monomer , with 2.6% cross - linker ) using a protean ii multi cell ( biorad ) . the protein spots in analytical and preparative gels were visualized by silver nitrate ( merck , germany ) and coomassie brilliant blue cbb/ g-250 ( sigma , germany ) respectively ( 19 - 21 - 22 ) . gs-800 densitometer ( bio - rad ) was used for scanning of silver stain gels . gels were analyzed using the melanie 6 software ( genebio , geneva , switzerland ) . the molecular masses of protein on gels were determined by co electrophoresis of standard protein markers ( ge heathcare ) and pi of the proteins were determined by migration of the protein spots on 18 cm ipg ( ph 47 , linear ) strips . 2-de per sample ( each species ) was run for three biologically independent replicates , percent volume of each spot was estimated and analyzed by one - way analysis of variance ( anova ) sas software , and means were compared by the lsd test at p 0.01 . spots were only considered to be significantly different in abundance at least between two leishmania species when / at p 0.01 . the protein spots of interest were excised from coomassie brilliant blue ( cbb ) stained gels and analyzed using an amazon ion trab ms / ms ( bruker daltonics ) mass spectrometer . briefly , peptides were solubilized in 0.5 % formic acid and fractionated on a nano flow uhplc system ( thermo rslcnano ) before online analysis by electrospray ionisation ( esi ) mass spectrometry on an amazon ion trap ms / ms ( bruker daltonics ) . peptide separation was performed on a pepmap c18 reversed phase column ( lc packings ) , using a 5 85% v / v acetonitrile gradient ( in 0.5% v / v formic acid ) run over 45 min . at a flow rate of 0.2 mass spectrometric ( ms ) analysis was performed using a continuous duty cycle of survey ms scan followed by up to ten ms / ms analyses of the most abundant peptides , choosing the most intense multiply charged ions with dynamic exclusion for 120s . ms data was processed using data analysis software ( bruker ) and the automated matrix science mascot daemon server ( v2.1.06 ) ( 23 ) . protein identifications were assigned using the mascot search engine to interrogate in house databases of protein sequences for l. major . ef653267 , l. major ( jn860745 ) and l. infantum ( jx289853 ) compared and were analyzed . promastigote forms recovered from the iranian leishmania parasite bank located in leishmaniasis lab , school of public heath , tehran university of medical sciences ( tums ) . the identity of these strains was already obtained by other molecular dna based methods ( 2 , 17 ) . cell culture promastigotes recovered from liquid nitrogen ( 196 c ) , were mass cultured in rpmi1640 medium ( gibco , life technologies gmbh , frankfurt , germany ) supplemented with 15% heat inactivated fetal bovine serum ( gibco , germany ) and 100u / ml penicillin and 100ug / ml streptomycin ( gibco , germany ) and incubated at 24c . parasites were harvested washed in sterile phosphate buffered saline ( pbs , ph : 7.27.4 ) and were used for protein extraction . proteomics analysis was performed on l. tropica , l. major and l. infantum , three species at the time of study . promastigotes were harvested by centrifugation at 3000rpm , 4 c and 20 minutes and washed three times in sterile pbs ( ph : 7.27.4 ) in the same condition for 10 minutes . the cells were resuspended in 5 mm tris hcl , ph 7.8 , containing 1 mm phenylmethylsulfonyl fluoride ( pmsf ( merck , germany ) ) . proteins were precipitated by 10% ( w / v ) trichloroacetic acid ( merck , germany ) in acetone ( merck ) with 0.07 % ( w / v ) dithiothreitol ( dtt ) ( merck ) for 1 hour at 20c . the samples were then centrifuged at 17500 g ( hettich , germany ) for 15 minutes at 4 c and the pellets were washed with ice - cold acetone containing 0.07% dtt , incubated at 20c for 1 h and centrifuged at 4 c . the samples were then solubilized in lysis buffer ( 9.5 m urea ( merck ) , 2% ( w / v ) chaps ( merck ) , 0.8% ( w / v ) ampholyte ( bio - rad , usa ) ph 310 , 1% ( w / v ) dtt ) ( 18 , 19 ) . the concentration of protein was measured by the bradford assay with bovine serum albumin bsa ( merck ) as the standard ( 20 ) . for analytical and preparative gels , 120 g and 1.2 mg of extracted promastigotes proteins were loaded respectively . ief was carried out on the 18 cm immobilized ph gradient ( ipg ) strips ( ph 47 ) ( bio - rad , usa ) . ipg strips were rehydrated overnight by loading the samples diluted with rehydration buffer containing 8 m urea , 4% chaps , 2% ampholyte , 50 mm dtt , and traces of bromophenol blue ( merck ) . isoelectric focusing was conducted at 20 c with mutiphor ii and a drystrip kit ( ge healthcare , germany ) . the running condition was as follows : 300 v for 90 minute , followed by 500 v for 90 min , 1000 v for 3 h and finally 3500 v for 16 h. the focused strips were equilibrated twice in equilibration solution . the first equilibration was performed in a solution containing 6 m urea , 20% ( w / v ) glycerol , 2% ( w / v ) sds ( merck ) , 1% ( w / v ) dtt , and 50 mm tris - hcl ( merck ) buffer , ph 8.8 . the second equilibration was performed in a solution with 2.5% ( w / v ) iodoacetamide ( merck ) . separation in the second dimension was performed by sds - page in a vertical slab of acrylamide ( merck ) ( 12% total monomer , with 2.6% cross - linker ) using a protean ii multi cell ( biorad ) . the protein spots in analytical and preparative gels were visualized by silver nitrate ( merck , germany ) and coomassie brilliant blue cbb/ g-250 ( sigma , germany ) respectively ( 19 - 21 - 22 ) . gs-800 densitometer ( bio - rad ) was used for scanning of silver stain gels . gels were analyzed using the melanie 6 software ( genebio , geneva , switzerland ) . the molecular masses of protein on gels were determined by co electrophoresis of standard protein markers ( ge heathcare ) and pi of the proteins were determined by migration of the protein spots on 18 cm ipg ( ph 47 , linear ) strips . 2-de per sample ( each species ) was run for three biologically independent replicates , percent volume of each spot was estimated and analyzed by one - way analysis of variance ( anova ) sas software , and means were compared by the lsd test at p 0.01 . spots were only considered to be significantly different in abundance at least between two leishmania species when / at p 0.01 . the protein spots of interest were excised from coomassie brilliant blue ( cbb ) stained gels and analyzed using an amazon ion trab ms / ms ( bruker daltonics ) mass spectrometer . briefly , peptides were solubilized in 0.5 % formic acid and fractionated on a nano flow uhplc system ( thermo rslcnano ) before online analysis by electrospray ionisation ( esi ) mass spectrometry on an amazon ion trap ms / ms ( bruker daltonics ) . peptide separation was performed on a pepmap c18 reversed phase column ( lc packings ) , using a 5 85% v / v acetonitrile gradient ( in 0.5% v / v formic acid ) run over 45 min . at a flow rate of 0.2 mass spectrometric ( ms ) analysis was performed using a continuous duty cycle of survey ms scan followed by up to ten ms / ms analyses of the most abundant peptides , choosing the most intense multiply charged ions with dynamic exclusion for 120s . ms data was processed using data analysis software ( bruker ) and the automated matrix science mascot daemon server ( v2.1.06 ) ( 23 ) . protein identifications were assigned using the mascot search engine to interrogate in house databases of protein sequences for l. major . protein extracts from the three leishmania species including l. tropica , l. major and l. infantum were separated by 2-de gel electrophoresis . multiple gels from the three independent replications were run to ensure reproducibility of the protein homogenates on the 2-de gels . show a representative example of the proteins separated / detected on a 2-de gel in l. tropica , l. major and l. infantum , where a total weight of 120 g of proteins had been applied . the gel images were analyzed by melanie software , and the percent volume of the spots was estimated and compared across the gels . we succeeded in detecting 600 100 spots on the 2-de gels , from which 638 , 590 , and 546 spots were statistically analyzed across the replicates in l. tropica , l. major , and l. infantum respectively . a number of 478 spots could be paired in all of the three species ( supplementary table 1 ) . the numbering corresponds to the 2-de gel in figure 1./ accession number in swiss - prot./ experimental pi and molecular weight./ theoretical pi and molecular weight./ mascot score./ nd : not detected ( spot ) however 34 , 33 , and 4 protein spots of l. tropica , l. major , and l. infantum were identified as leishmania species - specific spots ( fig . 2 ) . altogether , 265 protein spots exhibited reproducible quantitative ( p 0.01 ) changes across the three samples in leishmania species ( supplementary table 2 ) . among them , 35 , 22 and 11 protein spots were different between the l. tropica and l. major ( presented by a ) , l. tropica and l. infantum ( presented by b ) , l. major and l. infantum ( presented by c ) respectively . seven protein spots were different between all of the three species ( presented by abc ) . within differentially expressed proteins , which were detected on the analytical gels , we could reliably detect and excite a total number of 28 protein spots on cbb - stained preparative gels . due to the lack of the protein amount , the remaining proteins could not be detected . the excised protein spots were then analyzed by lc / ms leading to the identification of 24 proteins ( table 1 ) . these proteins were classified in multiple categories according to their species , functions , and biological processes : cell motility and cytoskeleton , cell signaling and vesicular trafficking , intracellular survival / nucleotid metabolism , protein synthesis , oxidative stress defense , microtubule motor movement proteolysis , lipid metabolism , amino - acid biosynthesis , protein ubiquitination / proteolysis , transport , stress related proteins / protein folding and hypothetical proteins ( unknown ) ( table 1 ) . it is worth noting that some of these proteins are hypothetical and their functions in leishmania still remain to be elucidated . the clustering of protein expression pattern of differentially expressed proteins in l. tropica , l. major and l. infantum is presented in fig . all quantitative information is showed using a color scale in which the color ranges from green for the highest down - regulation to red for the highest up - regulation . a comparison among the 3 species revealed that the changes in expression pattern were more pronounced in l. infantum compared with l. tropica and l. major . in addition , the number of up regulated proteins was higher than that of down - regulated proteins . the functional annotation of the 3 species identified proteins in l. tropica , l. major and l. infantum classified by biological function and processes described in table 1 . the biological function pie charts of the cutaneous species ( l. tropica and l. major ) are highly similar whereas , the pie chart for the biological function of the visceral species ( l. infantum ) is different from those of cutaneous species ( fig . most of the proteins functionalities are observed in the following categories : intracellular survival / nucleotid metabolism and cell motility/ cytoskeleton ( 2022% ) . moreover , the least functionalities are observed among cell signaling/ vesicular trafficking and lipid metabolism ( 5% ) . moreover , in the l. infantum species , most functionality are observed among protein synthesis and cell motility/ cytoskeleton ( 15% ) and least of them are observed among amino - acid biosynthesis and oxidative stress defense ( 7% ) . protein extracts from the three leishmania species including l. tropica , l. major and l. infantum were separated by 2-de gel electrophoresis . multiple gels from the three independent replications were run to ensure reproducibility of the protein homogenates on the 2-de gels . show a representative example of the proteins separated / detected on a 2-de gel in l. tropica , l. major and l. infantum , where a total weight of 120 g of proteins had been applied . the gel images were analyzed by melanie software , and the percent volume of the spots was estimated and compared across the gels . we succeeded in detecting 600 100 spots on the 2-de gels , from which 638 , 590 , and 546 spots were statistically analyzed across the replicates in l. tropica , l. major , and l. infantum respectively . a number of 478 spots could be paired in all of the three species ( supplementary table 1 ) . the numbering corresponds to the 2-de gel in figure 1./ accession number in swiss - prot./ experimental pi and molecular weight./ theoretical pi and molecular weight./ mascot score./ nd : not detected ( spot ) however 34 , 33 , and 4 protein spots of l. tropica , l. major , and l. infantum were identified as leishmania species - specific spots ( fig . 2 ) . altogether , 265 protein spots exhibited reproducible quantitative ( p 0.01 ) changes across the three samples in leishmania species ( supplementary table 2 ) . among them , 35 , 22 and 11 protein spots were different between the l. tropica and l. major ( presented by a ) , l. tropica and l. infantum ( presented by b ) , l. major and l. infantum ( presented by c ) respectively . seven protein spots were different between all of the three species ( presented by abc ) . within differentially expressed proteins , which were detected on the analytical gels , we could reliably detect and excite a total number of 28 protein spots on cbb - stained preparative gels . due to the lack of the protein amount , the remaining proteins could not be detected . the excised protein spots were then analyzed by lc / ms leading to the identification of 24 proteins ( table 1 ) . these proteins were classified in multiple categories according to their species , functions , and biological processes : cell motility and cytoskeleton , cell signaling and vesicular trafficking , intracellular survival / nucleotid metabolism , protein synthesis , oxidative stress defense , microtubule motor movement proteolysis , lipid metabolism , amino - acid biosynthesis , protein ubiquitination / proteolysis , transport , stress related proteins / protein folding and hypothetical proteins ( unknown ) ( table 1 ) . it is worth noting that some of these proteins are hypothetical and their functions in leishmania still remain to be elucidated . the clustering of protein expression pattern of differentially expressed proteins in l. tropica , l. major and l. infantum is presented in fig . all quantitative information is showed using a color scale in which the color ranges from green for the highest down - regulation to red for the highest up - regulation . black color indicates no changes in expression pattern of the 3 leishmania species . a comparison among the 3 species revealed that the changes in expression pattern were more pronounced in l. infantum compared with l. tropica and l. major . in addition , the number of up regulated proteins was higher than that of down - regulated proteins . the functional annotation of the 3 species identified proteins in l. tropica , l. major and l. infantum classified by biological function and processes described in table 1 . the biological function pie charts of the cutaneous species ( l. tropica and l. major ) are highly similar whereas , the pie chart for the biological function of the visceral species ( l. infantum ) is different from those of cutaneous species ( fig . most of the proteins functionalities are observed in the following categories : intracellular survival / nucleotid metabolism and cell motility/ cytoskeleton ( 2022% ) . moreover , the least functionalities are observed among cell signaling/ vesicular trafficking and lipid metabolism ( 5% ) . moreover , in the l. infantum species , most functionality are observed among protein synthesis and cell motility/ cytoskeleton ( 15% ) and least of them are observed among amino - acid biosynthesis and oxidative stress defense ( 7% ) . the aim of this preliminary study was to apply 2-de , which is a valuable method in the proteomics arena to analyze the protein profile patterns of the three iranian cutaneous and visceral leishmania species including l. tropica , l. major and l. infantum to search for species - specific leishmania proteins . in recent years by completion of genome sequencing leishmania parasites coupled with protein separation techniques analysis has given us an insight in better understanding the mechanisms of pathogenesis of leishmania species . proteomics approaches at the protein expression level provide additional information for further analysis with a biological function . moreover , comparative proteomics has been successful in determining the virulence biomarkers ( 24 ) . overall , the proteome 2-de maps of l. tropica , l. major and l. infantum were strikingly similar in terms of protein distribution and positioning . by using 18-cm ipg strips at the pi47 range in 2-de comparative analysis , we successfully identified more than 700 protein spots for all the three species . these numbers represent about 9% of total proteins in leishmania genome projects ( 25 ) . the analysis and discussion about the detected proteins in this study could be categorized into three parts . first , we discuss comprehensively about the biological function of proteins whose expression abundances were common among each of the three species . in the second and third part , we represent the biological function of the proteins which are different and absent / present in the three different species . as it is described in table 1 and venn diagram ( fig . 2 ) , in the first part we have studied a total number of 478 proteins among which we will discuss the common ones . among the common proteins of three leishmania species , which were surveyed , we would like to mention significant proteins such as beta tubulin and adf / coflin . adf / cofilin is existent in all eukaryotic organisms and has been involved in cell motility and cytokinesis . leishmania parasites express only one isoform of adf / cofilin , which is essential for flagellar assembly and motility ( 26 ) . beta tubulin is known as one of the members of distinct microtubule networks in leishmania and is implicated in locomotion , cell shape and division ( 27 ) . within other common proteins we could name tryparedoxin , calpain - like cysteine peptidase , and calmodulin putative from the groups of oxidative stress defense , intercellular survival / proteolysis , cell signalling , and vesicular trafficking , respectively . tryparedoxins are special thiol disulfide oxidoreductases related to thioredoxins , which play a crucial role in hydroperoxide detoxification cascades of kinetoplastida ( 28 ) . it participates in calcium signaling pathways that regulate multiple critical processes such as growth and proliferation ( 29 , 30 ) . in addition , it is an actin / microtubule - binding protein which interacts with the cytoskeleton ( 3133 ) . another common protein could be calcium channel protein that is a member of the transporter group . a huge number of proteins exist under the group of hypothetical proteins whose biological mechanisms are still not well discovered . the second group of proteins , which were studied , was the ones , which were different in all three leishmania species . the differences in proteins mainly occur between l. tropica and l. major and the least differences occur between l. major and l. infantum ( fig . it is an interesting point that the proteins , which are different between l. tropica and l. infantum are less than those of l. major and l. tropica . l. tropica is the main cause of dry cutaneous leishmaniasis lesions ; whereas , l. major is the main cause of wet cl lesion and l. infantum causes visceral leishmaniasis . recently there have been reports of viscerotropic forms caused by l. tropica in either humans or dogs infected to visceral leishmaniasis in iran and different parts of the world ( 34 , 2 ) . among significant proteins which are different in l. tropica and l. major we could mention calcium channel proteins , tryparedoxin and elongation factor from the groups of oxidative stress defense and protein synthesis respectively whose mechanism have been described previously . recently , it was demonstrated that leishmania ef-1alpha acts as a virulence factor ( 35 ) . this protein could diffuse into the cytosol of infected macrophages , where it is able to activate tyrosine phosphatase-1 leading to macrophage deactivation ( 31 ) . among the proteins different in l. tropica and l. infantum we could mention proteins such as calmadolin and trypardoxin in which the former role is to transfer the material into the cells and the latter role is defending the host cell against oxidative factors and preventing its death . nevertheless , some of these proteins have a specific domain and have a different mechanism such as hypothetical protein , conserved contains nucleoside 2-deoxyribosyltransferase domain , that have a role in nucleotide metabolism . among the most significant proteins between l. major and l. infantum another group of proteins , found in this study , was too rare and introduced as absent / present . however , further studies are needed to define precise biological function for the mentioned proteins in the process of pathogenesis . moreover , the examination of these species must be repeated with other strains in order to sanction the results . in addition , such differences must be evaluated and approved by other methods such as real time pcr and western blotting with monoclonal antibodies . the analysis of proteome mapping of 3 leishmania species including , l. tropica , l. major and l. infantum by 2-de and mass spectrometry demonstrated that the vast majority of leishmania proteins are commonly expressed among 3 species . therefore , differentiation , virulence and pathogenesis may be related not only to the immunity situation of the host but also to the differentiation expression of a number of proteins like stress related proteins / protein folding and protein ubiquitination / proteolysis . it must be pointed out that further studies must be undertaken using western blotting or real time pcr in order to support the results of the current study .
background : the mechanisms of virulence and species differences of leishmania parasites are under the influence of gene expression regulations at posttranscriptional stages . in iran , l. major and l. tropica are known as principal agents of cutaneous leishmaniasis , while l. infantum causes visceral leishmaniasis.methods:as a preliminary study , we compared the proteome mapping of the above three iranian isolates of leishmania species through the 2-dimension electrophoresis ( 2-de ) , and identified the prominent proteins by liquid chromatography ( lc ) mass spectrometry.results:we reproducibly detected about 700 protein spots in each species by using the melanie software . totally , 264 proteins exhibited significant changes among 3 species . forty nine protein spots identified in both l. tropica and l. major were similar in position in the gel , whereas only 35 of l. major proteins and 10 of l. tropica proteins were matched with those of l. infantum . having identified 24 proteins in the three species , we sought to provide possible explanations for their differential expression patterns and discuss their relevance to cell biology.conclusion:the comparison of proteome profiling pattern of the 3 species identified limit up and limit down regulated or absent /present proteins . in addition , the lc - ms data analysis showed that most of the protein spots with differential abundance in the 3 species are involved in cell motility and cytoskeleton , cell signaling and vesicular trafficking , intracellular survival / proteolysis , oxidative stress defense , protein synthesis , protein ubiquitination / proteolysis , and stress related proteins . differentially proteins distributed among the species maybe implicated in host pathogenecity interactions and parasite tropism to cutaneous or visceral tissue macrophages .
Introduction Materials and Methods Leishmania isolates and cell culture Protein Extraction Two-Dimensional electrophoresis (2-DE) Gel image Analysis Peptide extraction and mass analysis Results Comparison of proteome patterns Protein identification Discussion Conclusion
in this preliminary study , we compared the proteome mapping , in three iranian isolates of leishmania species including l. tropica , l. major and l. infantum , with immobilized ph gradient stripes with linear ph 47 . moreover , liquid chromatography ( lc ) - mass spectrometry was used for identification of a number of differentially expressed proteins among the three species . the numbering corresponds to the 2-de gel in figure 1./ accession number in swiss - prot./ experimental pi and molecular weight./ theoretical pi and molecular weight./ mascot score./ nd : not detected ( spot ) however 34 , 33 , and 4 protein spots of l. tropica , l. major , and l. infantum were identified as leishmania species - specific spots ( fig . among them , 35 , 22 and 11 protein spots were different between the l. tropica and l. major ( presented by a ) , l. tropica and l. infantum ( presented by b ) , l. major and l. infantum ( presented by c ) respectively . these proteins were classified in multiple categories according to their species , functions , and biological processes : cell motility and cytoskeleton , cell signaling and vesicular trafficking , intracellular survival / nucleotid metabolism , protein synthesis , oxidative stress defense , microtubule motor movement proteolysis , lipid metabolism , amino - acid biosynthesis , protein ubiquitination / proteolysis , transport , stress related proteins / protein folding and hypothetical proteins ( unknown ) ( table 1 ) . the functional annotation of the 3 species identified proteins in l. tropica , l. major and l. infantum classified by biological function and processes described in table 1 . the biological function pie charts of the cutaneous species ( l. tropica and l. major ) are highly similar whereas , the pie chart for the biological function of the visceral species ( l. infantum ) is different from those of cutaneous species ( fig . protein extracts from the three leishmania species including l. tropica , l. major and l. infantum were separated by 2-de gel electrophoresis . the numbering corresponds to the 2-de gel in figure 1./ accession number in swiss - prot./ experimental pi and molecular weight./ theoretical pi and molecular weight./ mascot score./ nd : not detected ( spot ) however 34 , 33 , and 4 protein spots of l. tropica , l. major , and l. infantum were identified as leishmania species - specific spots ( fig . among them , 35 , 22 and 11 protein spots were different between the l. tropica and l. major ( presented by a ) , l. tropica and l. infantum ( presented by b ) , l. major and l. infantum ( presented by c ) respectively . these proteins were classified in multiple categories according to their species , functions , and biological processes : cell motility and cytoskeleton , cell signaling and vesicular trafficking , intracellular survival / nucleotid metabolism , protein synthesis , oxidative stress defense , microtubule motor movement proteolysis , lipid metabolism , amino - acid biosynthesis , protein ubiquitination / proteolysis , transport , stress related proteins / protein folding and hypothetical proteins ( unknown ) ( table 1 ) . the functional annotation of the 3 species identified proteins in l. tropica , l. major and l. infantum classified by biological function and processes described in table 1 . the biological function pie charts of the cutaneous species ( l. tropica and l. major ) are highly similar whereas , the pie chart for the biological function of the visceral species ( l. infantum ) is different from those of cutaneous species ( fig . the aim of this preliminary study was to apply 2-de , which is a valuable method in the proteomics arena to analyze the protein profile patterns of the three iranian cutaneous and visceral leishmania species including l. tropica , l. major and l. infantum to search for species - specific leishmania proteins . overall , the proteome 2-de maps of l. tropica , l. major and l. infantum were strikingly similar in terms of protein distribution and positioning . within other common proteins we could name tryparedoxin , calpain - like cysteine peptidase , and calmodulin putative from the groups of oxidative stress defense , intercellular survival / proteolysis , cell signalling , and vesicular trafficking , respectively . it is an interesting point that the proteins , which are different between l. tropica and l. infantum are less than those of l. major and l. tropica . l. tropica is the main cause of dry cutaneous leishmaniasis lesions ; whereas , l. major is the main cause of wet cl lesion and l. infantum causes visceral leishmaniasis . the analysis of proteome mapping of 3 leishmania species including , l. tropica , l. major and l. infantum by 2-de and mass spectrometry demonstrated that the vast majority of leishmania proteins are commonly expressed among 3 species .
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the online version of this article ( doi:10.1007/s00249 - 009 - 0412 - 6 ) contains supplementary material , which is available to authorized users . analytical ultracentrifugation is an analytical absolute technique applying centrifugal force to fractionate the sample and optical detection systems to detect the concentration distribution of the sample inside the ultracentrifuge cell . these experiments yield a number of important physico - chemical quantities of the sample like size , shape , density , molar mass , sedimentation and diffusion coefficient , interaction constants and stoichiometry etc . and many of them in form of distributions . however , often the available information depends on the applied optical detection system . optical systems that have been constructed are uv / vis absorption , rayleigh interference , schlieren and the lavrenko optics ( lavrenko et al . development of new optical detection systems can expand the possible use of auc for different types of samples . size distributions can be determined with turbidity optics ( mchtle 1992 ; mller 1989 ; scholtan and lange 1972 ) . fluorescence optics has expanded the use for extremely diluted labeled samples even in presence of other solutes at a much higher concentration ( macgregor et al . the only commercially available machine at the moment is the beckman xl - i which is equipped with a uv / vis absorption optics and rayleigh interference optics ( giebeler 1992 ) . recently , the uv / vis absorption detection was significantly improved by the development of a multiwavelength uv / vis absorption detector ( mwl - auc ) . by essentially replacing the monochromator with a spectrograph , the mwl - auc delivers an entire uv / vis spectrum for each radial point instead of a single wavelength reading ( bhattacharyya et al . this technology has a number of advantages over the commercial absorption optics detecting at a single wavelength with time - consuming wavelength scanning , thus excluding the study of all fast processes , obtaining full uv / vis spectra at each point in the ultracentrifuge cell rather than a radial concentration profile at a single wavelength can give much more structural information about the sample , allows for averaging and can even decrease the experimental time when modern fast ccd based spectrometers are used ( bhattacharyya et al . successful optical , mechanical , radial scan , linearity and noise tests of the mwl detector have been published recently ( strauss et al . 2008 ) . combined with the fractionating power of the auc , application of the mwl detector with its additional structural and/or compositional information on light absorbing samples can yield distributions of the individual components in complex mixtures with respect to composition and size / density related to different chromophores . this can start with relatively straightforward issues like sample homogeneity and purity but can then get increasingly complex in case of composite and/or interacting samples . especially for such complex samples mwl - auc has a huge potential , as spectral discrimination can synergistically enhance the hydrodynamic resolution ( balbo et al . this is also an important issue for any colored industrial product composed of at least two components which at least slightly differ in their uv / vis spectra . in this work , we will show the capabilities of mwl - auc for the analysis of an industrial composite sample of -carotene and gelatin . this system was investigated before with x - ray scattering , uv / vis absorption spectroscopy , foqels ( fiber - optic quasi - elastic light scattering ) , microelectrophoresis and on basis of these results , a core - shell structure was presented ( auweter et al . the core structure with 120 nm diameter consists of partially crystallized , partially amorphous -carotene as active ingredient . this hybrid structure self - assembles in a carefully tuned co - precipitation of gelatin ( from an aqueous solution ) and the active ingredient ( from a lipophilic solvent ) . such particulate formulations can transport an active ingredient that is not water soluble across an aqueous phase with high bioavailability , in this case provitamin a. these particles are not persistent , but disassemble and get digested quickly in biological media . -carotenes can precipitate as h - aggregate or j - aggregate ; the two morphologies do not interconvert and are regarded to be kinetically stable over years . the h - aggregate is observed in precipitation of dilute solutions ( 0.3 weight% ) whereas the j - aggregate is observed at higher concentration ( 1.0 weight% ) . ( 1999 ) calculated a 40 nm hypsochromic shift observed for an h - aggregate and a bathochromic shift in j - aggregates . this results in a significant color change from yellow to red of the product depending on the precipitation conditions and hybrid particle size ( figs . 1 , 2 ) . this color change is the basis for the industrial application of the -carotenes as pigments for food applications.fig . 1dashed line uv / vis spectrum of the core - shell -carotene / gelatin sample with 0.05 g / l concentration ; solid line uv / vis spectrum of gelatin at 1 g / lfig . 1999 ) , right side color change of -carotene / gelatin microparticles due to particle size and structure dashed line uv / vis spectrum of the core - shell -carotene / gelatin sample with 0.05 g / l concentration ; solid line uv / vis spectrum of gelatin at 1 g / l left side assumed structure of the -carotene microparticle system ( auweter et al . 1999 ) , right side color change of -carotene / gelatin microparticles due to particle size and structure not only the purity of the sample concerning the color characteristics ( brilliance of color due to steep absorbance profiles ) or sample homogeneity ( different species or unbound gelatin ) is of interest for the industrial application but furthermore any possible transitions between different structures . this is a problem which can be advantageously solved in a single mwl - auc experiment , which we will describe in this work . the -carotene product was obtained in powder form as a laboratory sample from basf se , ludwigshafen . an aqueous dispersion in water the uv / vis spectrum of the dispersion and of the free gelatin is shown in fig . 1 . in contrast to conventional sedimentation velocity experiments , where the sample between boundary and bottom of the cell is only diluted by radial dilution , the sample in band centrifugation is diluted additionally by fractionation in the pure solvent . the reservoir is filled with a small amount of concentrated sample . column and sample sectors are filled with d2o with a density which is higher than that of the sample solution and lower than the density of the dispersed solute . we have prepared a 20 g / l solution and deposited 15 l of the solution into the cell reservoir . after preliminary experiments , this concentration was chosen to ensure that the individual components of the mixture are detected in as many as possible scans with od s < 1.4 in the experiment without too much dilution which causes noisy data . after cell assembly , the auc was accelerated to 5,000 rpm for 3 min to transfer the sample in the reservoir via capillaries to overlay the d2o column . forty scans were taken with a time interval of 90 s and a radial step size of 50 m to observe the full sedimentation of the sample . the selected wavelength range was 250750 nm . in the prototype setup , we apply the spectrum acquired for an empty cell as a reference for the calculation of the absorption leading to a baseline offset of 0.05 od ( see fig . 3i ) after the experiment , while cleaning the cell , we saw some precipitate in the cell reservoir . thus , not all particles were transferred to the sample column , but some big particles remained in the reservoir as they already must have completely sedimented upon speeding up the rotor to 5,000 rpm.fig . 3three - dimensional plots of the raw data from a band sedimentation experiment with -carotene detected with the mwl detector . i scan 1 ( 1.5 min ) ; ii scan 10 ( 15 min ) ; iii scan 18 ( 27 min ) ; iv scan 40 ( 60 min ) three - dimensional plots of the raw data from a band sedimentation experiment with -carotene detected with the mwl detector . i scan 1 ( 1.5 min ) ; ii scan 10 ( 15 min ) ; iii scan 18 ( 27 min ) ; iv scan 40 ( 60 min ) each of the 40 scans produces a 3-dimensional graph . we have radial position as x - dimension , wavelength as y - dimension and absorbance as z - dimension . in the present contribution , we will perform a semi - quantitative evaluation based on simple model - free transformations of the data without any prior knowledge . for evaluation , we have converted the radial position ( r ) to the sedimentation coefficient s by using eq , rm is the radial position of the meniscus and t is the run time integral.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{s } } = \frac{{{\text{ln}}\,(r / r_{\text{m } } ) } } { { \omega^{2 } t } } $ $ \end{document } the 3d absorption dataset can now be projected either onto the wavelength or sedimentation coefficient axis to better visualize the spectral changes with different sedimentation coefficients or sedimentation coefficient distributions at different wavelengths . a multiwavelength auc as described in bhattacharyya et al . ( 2006 ) and strauss et al . in contrast to conventional sedimentation velocity experiments , where the sample between boundary and bottom of the cell is only diluted by radial dilution , the sample in band centrifugation is diluted additionally by fractionation in the pure solvent . the reservoir is filled with a small amount of concentrated sample . column and sample sectors are filled with d2o with a density which is higher than that of the sample solution and lower than the density of the dispersed solute . we have prepared a 20 g / l solution and deposited 15 l of the solution into the cell reservoir . after preliminary experiments , this concentration was chosen to ensure that the individual components of the mixture are detected in as many as possible scans with od s < 1.4 in the experiment without too much dilution which causes noisy data . after cell assembly , the auc was accelerated to 5,000 rpm for 3 min to transfer the sample in the reservoir via capillaries to overlay the d2o column . forty scans were taken with a time interval of 90 s and a radial step size of 50 m to observe the full sedimentation of the sample . the selected wavelength range was 250750 nm . in the prototype setup , we apply the spectrum acquired for an empty cell as a reference for the calculation of the absorption leading to a baseline offset of 0.05 od ( see fig . 3i ) after the experiment , while cleaning the cell , we saw some precipitate in the cell reservoir . thus , not all particles were transferred to the sample column , but some big particles remained in the reservoir as they already must have completely sedimented upon speeding up the rotor to 5,000 rpm.fig . 3three - dimensional plots of the raw data from a band sedimentation experiment with -carotene detected with the mwl detector . i scan 1 ( 1.5 min ) ; ii scan 10 ( 15 min ) ; iii scan 18 ( 27 min ) ; iv scan 40 ( 60 min ) three - dimensional plots of the raw data from a band sedimentation experiment with -carotene detected with the mwl detector . i scan 1 ( 1.5 min ) ; ii scan 10 ( 15 min ) ; iii scan 18 ( 27 min ) ; iv scan 40 ( 60 min ) each of the 40 scans produces a 3-dimensional graph . we have radial position as x - dimension , wavelength as y - dimension and absorbance as z - dimension . in the present contribution , we will perform a semi - quantitative evaluation based on simple model - free transformations of the data without any prior knowledge . for evaluation , we have converted the radial position ( r ) to the sedimentation coefficient s by using eq . 1 . in eq . 1 , rm is the radial position of the meniscus and t is the run time integral.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{s } } = \frac{{{\text{ln}}\,(r / r_{\text{m } } ) } } { { \omega^{2 } t } } $ $ \end{document } the 3d absorption dataset can now be projected either onto the wavelength or sedimentation coefficient axis to better visualize the spectral changes with different sedimentation coefficients or sedimentation coefficient distributions at different wavelengths . in principle , the entire dataset can be evaluated globally , and efforts are underway to incorporate such routine into the ultrascan evaluation software package ( demeler 2005 ) . however , even then we are confronted with a confounded polydispersity of both optical and colloidal / hydrodynamic properties . in the left side of fig . 2 , the assumed core - shell structure of a -carotene microparticle is shown ( auweter et al . 1999 ) . such a complex hybrid particle exhibits several levels of polydispersity , which impact the distribution of sedimentation coefficients observed in an auc . oil content , of the inner core;concentration of the adsorbed protection colloid ( gelatin);degree of swelling of the gelatin . oil content , of the inner core ; concentration of the adsorbed protection colloid ( gelatin ) ; degree of swelling of the gelatin . parameters 1 and 2 determine the optical properties and bioavailability that are decisive for the commercial application profile . for smallest particle sizes , -carotene is an h - aggregate while for the biggest particle sizes -carotene forms j - aggregates . intermediate particle sizes are assumed to integrate h- and j - aggregates in differing ratio ( auweter et al . parameters 3 and 4 determine the thickness of the protection colloid layer , which is typically 40 nm in pure water . the buoyant density of gelatin is rather high ( above 1.3 g / cm ) , and can not be matched with a non - interfering solvent such as heavy water . all parameters 14 enter into the calculation of the effective density and the hydrodynamic diameter of the hybrid particle . the frictional force under sedimentation depends on the ion concentration and ph because the gelatin may collapse or swell thus changing the effective frictional forces ( and thus changing the observable sedimentation constant ) although the chemical composition and buoyant density , which in principle could be measured in a krattky gauge or density gradient , stay the same . the swelling of the gelatin corona alone impedes an exact conversion from measured sedimentation constants to hydrodynamic diameters . considering that also parameters 1 and 2 contribute to the polydispersity in the observable distribution of sedimentation coefficients , we decided to limit ourselves to a conservative evaluation on the level of sedimentation fractions , not sizes . we now discuss the optical properties that result from the specific colloidal microstructures as discussed above . due to different preparation conditions , the morphology of the -carotene core changes . h- and j - aggregates have different uv / vis spectra , shown as visual impression on the right side of fig . 2 . figure 1 dot line curve shows the uv / vis spectrum of 0.05 g / l product without any ultracentrifugation . four peaks at 288 , 449 , 478 and 518 nm can be seen . the three peaks in the visible can be attributed to the 1ag ( s0)1bu+ ( s2 ) transition with the vibrational progression 20 , 10 , 00 of the c c stretch vibration along the alternatingly double bonded electronically conjugated backbone of the carotenoid ( polivka and sundstrom 2004).the uv peak partially can be attributed also to the carotenoid transition 1ag1ag+ , which is forbidden by symmetry , but becomes allowed in the crystalline assembly . the spectrum of the composite particle indicates the -carotene j - aggregate ( auweter et al . 1999 ) . it can be seen that gelatin only contributes to the uv region of the spectra below 280 nm . however , the contribution of gelatin is vanishing compared to the three times stronger absorption of the composite sample at 20 times lower overall concentration . another component that presumably contributes to the uv absorption is the ascorbylpalmitat dispersant that is added during the co - precipitation . if we put all these 40 scans in sequence , we can form a 3d movie of the sedimentation process . figure 3.1 shows scan 1 where particles just have been transferred from the reservoir to the sample column . the baseline offset is 0.05 ( purple ) due to the absorption calculation with an empty cell as reference . , there is an overlay of two peaks , one is the uv peak of -carotene ( see fig . 2 ) and the other is the uv signal of gelatin . after 15 min of sedimentation , fractionation of the sample is obvious and the first sedimentation fraction proceeds to the bottom of the cell . scan 10 ( 15 min ) is the last scan where the entire particle range can be seen before the first particles reach the bottom . if we compare the height of the peak in the uv and visible region at different radial positions , the ratio changes . this is the first important result , demonstrating that the sample is not homogenous . instead the observed effect can be explained by a higher content of stabilizing agent that induces smaller particle sizes . 1 ) does not exactly match the gelatin absorption and that the expected contribution of gelatin is weak at the applied concentrations , hinting at a combined action of both gelatin and the ascorbylpalmitat added during the co - precipitation in particle synthesis . the third part of fig . , the fastest particles have sedimented already . in the fourth part of fig . 3 , we see the last fraction that remained after 60 min of sedimentation , which is mainly composed of gelatin . however , some -carotene absorption is still visible , which seems to be solubilized in small amount by the excess gelatin or excess ascorbylpalmitat . we do not detect free gelatin in the analysis . in an independent experiment we measured the characteristic sedimentation behavior of gelatin with the interference optics of the beckman xli auc at 44,000 rpm . this confirms our assignment that the last fraction can not be pure gelatin . to summarize the global evaluation , fig . we can differentiate particles , observe the full uv / vis spectra of the particles and can already draw conclusions about the different components in the complex sample mixture without any further evaluation , as the y - axis shows the full uv / vis wavelength range . we can also use projections of the data onto individual axes and proceed thus to a more quantitative evaluation . in order to calculate the full s - distribution of all particles , we have selected scan 10 for all further evaluation as this scan shows fractionation of the mixture while no particles are yet lost due to complete sedimentation . more information is potentially available with a global evaluation of the entire dataset . in fig . 4 , the s - distribution of the particles is shown for 5 different representative wavelengths out of 330 ( 250750 nm with a wavelength resolution of 1.5 nm ) . we have selected the wavelengths according to the peaks of the -carotene microparticles : 260 , 280 , 450 , 480 and 520 nm in fig . 4sedimentation coefficient distributions at different wavelengths sedimentation coefficient distributions at different wavelengths the s - distribution is obviously very broad . due to the chemical heterogeneity of the particles and the resulting density distribution , however , all important sample characteristics can be discussed for the s - distributions . from fig . 4 , we conclude that there are at least three fractions in the sample : small hybrid particles with s < 25 s , a main fraction around 100 s and larger particles around 200 s. their absorption spectra ( and chemical composition ) are clearly different as can be seen in fig . 5.fig . 5top normalized uv / vis spectra of particles with different sedimentation coefficients , bottom zoom the range around 450 nm and peak positions of 10.6 s ( 448 nm ) up to 232 s ( 439 nm ) top normalized uv / vis spectra of particles with different sedimentation coefficients , bottom zoom the range around 450 nm and peak positions of 10.6 s ( 448 nm ) up to 232 s ( 439 nm ) in fig . 5 , seven representative uv / vis spectra are shown . the spectra agree well with those of pure h - aggregates ( auweter et al . however , the original sample contained j - aggregates too ( fig . 1 , dashed line ) . we believe that the j - aggregates were already precipitated before the first scan in the auc cell was taken . indeed precipitation of particulate material inside the reservoir of the vinograd cell was observed visually after cell disassembly following the experiment . however , irrespective of the actual nature of the particulate material that remained in the vinograd cell reservoir , the result stands for itself that the coloristic polydispersity ( fig . 1 ) is not due to an intra - particle but inter - particle distribution of morphologies ( fig . this result is contrary to the previous assumption that is sketched in fig . 2 , where h / j - aggregates would coexist in the particles.fig . 6ad hoc structural model of the -carotene microparticle system on basis of the presented auc results . the different color of the samples does not originate from the intraparticular coexistence of h- and j - aggregates as assumed before ( fig . 2 ) ( auweter et al . 1999 ) but instead separate particles contain pure h- or j - aggregates and the concentration ratio between these particles determines the colour of the final sample . note the difference to fig . 2 ad hoc structural model of the -carotene microparticle system on basis of the presented auc results . the different color of the samples does not originate from the intraparticular coexistence of h- and j - aggregates as assumed before ( fig . 2 ) ( auweter et al . 1999 ) but instead separate particles contain pure h- or j - aggregates and the concentration ratio between these particles determines the colour of the final sample . 2 the peak around 520 nm slightly shifts to lower wavelength with increasing sedimentation coefficient and the peak height also decreases . therefore , this excitation of -carotene microparticles decreases with increasing sedimentation coefficient . for the peak at 450 nm , only the spectral shift to lower wavelength is observed with increasing sedimentation coefficient . we suspect that the displacement of the electronic potential energy surfaces changes , such that the frank factors change for the vibrational progression , due to the changing incorporation of the chromophore into the partially crystalline assembly . as discussed above , an overlay of the signal from -carotene , gelatin and the ascorbylpalmitat a shift of the peak maximum to higher wavelength with increasing sedimentation coefficient is detected . in addition , a drastic decrease of the peak height relative to the 450 nm peak is observed with increasing sedimentation coefficient . the particles that sediment slower show stronger uv absorption , which we attribute to a higher content of ascorbylpalmitat , and hence smaller particle diameters due to the formation process in co - precipitation auweter et al . 1999 ) . to our knowledge , it is the first time that relatively small steps of spectral shift among an h - aggregate are shown for composite particles . although the spectral changes appear to be continuous , that does not exclude defined and different spectra for different particle populations as the detected raw signals are those of a sample band , which is broadened by polydispersity in size , composition and diffusional broadening . industrial -carotene gelatin composite particles are a highly heterogeneous system both in particle size and chemical composition . fractionation of this mixture in mwl - auc allows to resolve individual components in the mixture and detect compositional changes in an experiment , which takes only 1 h. this proves the power of mwl - auc as a direct technique that can differentiate particles with respect to size and uv / vis spectra . although our current analysis does not allow to unambiguously assign the spectra to defined particles as the particle density , swelling , composition and size maybe varying simultaneously , the presented multiwavelength analysis allows insights into this complex system , which were not possible before by other techniques . we do not further evaluate the directly experimentally determined sedimentation coefficients as for our sample , in addition to the above mentioned polydispersity , ph effects as well as charge interactions between the colloids also have to be taken into account . despite these restrictions , we have shown the existence of h - aggregates inside a sample that was previously known as j - aggregate and have detected spectral changes of different h - aggregate populations as well as changes in the electronic potential energy surfaces of different hybrid particles . we restricted ourselves to a semi - quantitative evaluation based on simple model - free transformations of the data of 1 out of 40 scans without any prior knowledge . clearly , even richer phenomena can potentially be discovered with a global evaluation of the entire dataset . below is the link to the electronic supplementary material . supplementary information ( avi 10.4 mb ) .
a multiwavelength uv / vis detector for the analytical ultracentrifuge ( mwl - auc ) has been developed recently . in this work , -carotene gelatin composite particles are investigated with mwl - auc . band centrifugation with a vinograd cell is used to ensure maximum sample separation . spectral changes of the system are observed in dependence of the sedimentation coefficient and are attributed to a previously unknown inhomogeneity of the -carotene chemical composition with both h- and j - aggregates coexisting in a mixture . in addition , our data suggest that pure h- and j - aggregates exist in a particle while their relative concentrations in a mixture determine the color characteristics of the sample . the unique abilities and properties of mwl - auc include sedimentation coefficient distributions for all possible wavelengths , full uv / vis spectra of each different species in the mixture and 3d movies of the sedimentation process . these properties significantly extend the scope of the analytical ultracentrifuge technique and show that complex biopolymer multicomponent mixtures can be resolved into their individual species.electronic supplementary materialthe online version of this article ( doi:10.1007/s00249 - 009 - 0412 - 6 ) contains supplementary material , which is available to authorized users .
Electronic supplementary material Introduction Materials and methods Multi-wavelength analytical ultracentrifugation (MWL-AUC) Results and discussion Conclusion Electronic supplementary material
the online version of this article ( doi:10.1007/s00249 - 009 - 0412 - 6 ) contains supplementary material , which is available to authorized users . recently , the uv / vis absorption detection was significantly improved by the development of a multiwavelength uv / vis absorption detector ( mwl - auc ) . by essentially replacing the monochromator with a spectrograph , the mwl - auc delivers an entire uv / vis spectrum for each radial point instead of a single wavelength reading ( bhattacharyya et al . this technology has a number of advantages over the commercial absorption optics detecting at a single wavelength with time - consuming wavelength scanning , thus excluding the study of all fast processes , obtaining full uv / vis spectra at each point in the ultracentrifuge cell rather than a radial concentration profile at a single wavelength can give much more structural information about the sample , allows for averaging and can even decrease the experimental time when modern fast ccd based spectrometers are used ( bhattacharyya et al . in this work , we will show the capabilities of mwl - auc for the analysis of an industrial composite sample of -carotene and gelatin . 1999 ) , right side color change of -carotene / gelatin microparticles due to particle size and structure not only the purity of the sample concerning the color characteristics ( brilliance of color due to steep absorbance profiles ) or sample homogeneity ( different species or unbound gelatin ) is of interest for the industrial application but furthermore any possible transitions between different structures . this is a problem which can be advantageously solved in a single mwl - auc experiment , which we will describe in this work . in contrast to conventional sedimentation velocity experiments , where the sample between boundary and bottom of the cell is only diluted by radial dilution , the sample in band centrifugation is diluted additionally by fractionation in the pure solvent . for evaluation , we have converted the radial position ( r ) to the sedimentation coefficient s by using eq , rm is the radial position of the meniscus and t is the run time integral.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{s } } = \frac{{{\text{ln}}\,(r / r_{\text{m } } ) } } { { \omega^{2 } t } } $ $ \end{document } the 3d absorption dataset can now be projected either onto the wavelength or sedimentation coefficient axis to better visualize the spectral changes with different sedimentation coefficients or sedimentation coefficient distributions at different wavelengths . h- and j - aggregates have different uv / vis spectra , shown as visual impression on the right side of fig . the three peaks in the visible can be attributed to the 1ag ( s0)1bu+ ( s2 ) transition with the vibrational progression 20 , 10 , 00 of the c c stretch vibration along the alternatingly double bonded electronically conjugated backbone of the carotenoid ( polivka and sundstrom 2004).the uv peak partially can be attributed also to the carotenoid transition 1ag1ag+ , which is forbidden by symmetry , but becomes allowed in the crystalline assembly . we can differentiate particles , observe the full uv / vis spectra of the particles and can already draw conclusions about the different components in the complex sample mixture without any further evaluation , as the y - axis shows the full uv / vis wavelength range . the different color of the samples does not originate from the intraparticular coexistence of h- and j - aggregates as assumed before ( fig . industrial -carotene gelatin composite particles are a highly heterogeneous system both in particle size and chemical composition . fractionation of this mixture in mwl - auc allows to resolve individual components in the mixture and detect compositional changes in an experiment , which takes only 1 h. this proves the power of mwl - auc as a direct technique that can differentiate particles with respect to size and uv / vis spectra . despite these restrictions , we have shown the existence of h - aggregates inside a sample that was previously known as j - aggregate and have detected spectral changes of different h - aggregate populations as well as changes in the electronic potential energy surfaces of different hybrid particles .
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opportunistic sampling of mammalian roadkill took place over a two - year period near the university of oklahoma campus ( norman , ok , usa ) . carcasses deemed fresh ( generally determined to have been struck by motor vehicles no more than 10 h prior to sampling ) were selected , and those with one or more intact orifices ( i.e. , mouth , nose , ear , eye , and rectum ) or gastrointestinal tracts were sampled roadside with sterile swabs . the mammalian roadkill carcasses that were sampled included ( in order of increasing frequency ) the following : skunk ( mephitis mephitis ) , armadillo ( dasypus novemcinctus ) , deer ( odocoileus virginianus ) , raccoon ( procyon lotor ) , squirrel ( sciurus carolinensis ) , and opossum ( dideiphis virginiana ) . sampling of these carcasses led to the generation of 3659 bacterial isolates ( figure 2 ) . not only were opossum carcasses the most frequently encountered , they were also a particularly rich source of morphologically unique bacteria , accounting for 39% ( 1425 ) of the roadkill - associated isolates collected ( figure 2 ) . the phylogenetic diversity of the opossum microbiome was further evaluated by sequencing 16s rrna gene libraries . swabs taken from five opossum body sites were used to generate 13 994 sequences , which clustered into 73 operational taxonomic units ( otus ) at 97% sequence similarity ( figure 3 ) . members of the gammaproteobacteria dominated the communities from the mouth , nose , and ear , constituting greater than 90% of each community . the upper gastrointestinal tract also contained a sizable population of gammaproteobacteria ( 72.4% ) and bacilli ( 21.7% ) . the rectum was notably more diverse containing large populations of fusobacteria ( 47.2% ) , clostridia ( 19.1% ) , and bacteroidia ( 10.8% ) . the gammaproteobacteria were largely represented by two abundant operational taxonomic units , one of which was unclassified and another assigned most closely to the genus pseudomonas . ( a ) breakdown of the 3659 roadkill microbiome isolates based on the source organisms from which they were derived . ( b ) distribution of the isolates based on the locations / orifices on the carcasses from which they were derived . ( c ) categorization of the isolate data illustrating the percent contribution of each body site to the overall bacterial collection prepared from the different animal species ( note that the colors and categories used to construct the slices within each of the pie charts in panel c are the same as those used for the pie chart in panel b ) . all of the bacterial isolates were individually cultured in two or more broth media , and a library of their ethyl - acetate - soluble natural products was prepared . one of the opossum ear samples showed appreciable activity in an assay designed to identify compounds that inhibited c. albicans biofilm formation . however , upon closer scrutiny , this sample was determined to be derived from a culture containing two bacteria that each produced slightly different - sized white , opaque , mucoidal colonies . when the two microorganisms were obtained in pure culture , the 16s rrna genes of both isolates were sequenced , and the bacteria were identified as members of the genera pseudomonas and serratia . representative colonies of both isolates were analyzed in situ by laser ablation electrospray ionization mass spectrometry ( laesims ) . ( figure 4 ) , which was determined to be viscosin ( 1 ) based on its ms / ms fragmentation pattern relative to an authentic standard . its assignment was later confirmed by a single - crystal x - ray diffraction experiment of the metabolite following its scale - up production and purification ( figure 5 ) . laesims analysis of the serratia sp . isolate led to the purification of serrawettin w2 ( 2 ) , as well as three co - metabolites that could not be dereplicated ( i.e. , m / z values and fragmentation patterns did not produce any reasonable matches to reported bacterial natural products ) . although the information derived from the lc - ms investigation of the compounds ( ms data , lc retention times , and uv vis pda profiles ) enabled us to postulate that the serratia sp . metabolites were analogues of 2 , we decided to pursue their scale - up purification for structure confirmation and bioactivity testing . phylogenetic diversity of mammalian microbiome bacteria from different orifices / body sites of a roadkill opossum carcass . the inset shows bacterial colonies growing on the surface of an agar plate . the plate was placed inside of the laesims chamber for mass spectrometry profiling . a subset of representative colonies was selected ( indicated by red arrows ) , and a virtual grid was laid over these colonies using the instrument s software to target where laser ablation would occur . the light blue polygons show where mass data were collected from the colonies within the range of m / z 2002000 . the presented mass data were derived from the circled colony ( average of several locations taken from the colony and subtracted from mass data obtained from a blank [ uncolonized ] portion of the plate ) , which reveals prominent single and doubly charged sodium adduct ions for viscosin ( 1 ) . ortep rendering of viscosin ( 1 ) illustrating the metabolite s absolute configuration ( determined by refinement of the flack parameter ; the water molecule identified near atom n2 has been removed from the figure for clarity ; however , the position of the water can be found in the supporting information ) . the numbering system used for this structure reflects the atom assignments used in the supporting information and in the cambridge structural database ( ccdc 1511786 ) . upon partitioning of the liquid cultures of the serratia sp . isolate , the combined ethyl acetate layer was determined to retain the putative serrawettin analogues , as well as the biofilm inhibition activity . laesims - guided fractionation of the organic layer by hp20ss vlc , as well as preparative and semipreparative c18 hplc resulted in the purification of compounds 35 , along with 2 , whose structure was subsequently confirmed by ms experiment , h and c nmr data ( tables 1 and 2 , respectively ) , and marfey s analysis . analysis of hresims data for compound 3 provided a prominent ion with m / z 740.4235 [ m + na ] that supported a molecular formula of c37h59n5o9 . this indicated that compound 3 likely differed from 2 by the loss of a ch2 unit . h tocsy data for 3 versus 2 revealed that the spin system associated with the isoleucine residue was altered . inspection of the h ( table 1 ) and h h dqfcosy nmr data for 3 showed that the amide proton ( 8.28 , d , j = 7.22 , 1 h ) coupled with the -proton ( 3.73 , t , j = 7.76 , 7.76 , 1 h ) , which coupled to a methine ( 1.95 , m , 2 h ) that in turn coupled with methyl protons ( 0.84 , m , 6 h ) . these results could be accounted for if the isoleucine residue in 2 changed to a valine residue in 3 . subsequent investigation of the metabolite by hydrolysis followed by marfey s analysis confirmed that the isoleucine residue was no longer present and instead an l - valine had been incorporated . thus , metabolite 3 was assigned the trivial name serrawettin w4 in recognition of its structural relationship to metabolite 2 . a similar structure determination strategy was applied to compound 4 after observing that the hresims data contained a prominent ion with m / z 726.4073 [ m + na ] , which corresponded to a molecular formula of c36h57n5o9 . this indicated that the metabolite was deficient for two ch2 units relative to compound 2 . h tocsy data for 4 provided strong evidence that metabolites 2 and 4 shared identical macrocycles . this was supported by hydrolysis followed by marfey s analysis , which confirmed the presence of the same amino acid residues in 2 and 4 . turning our attention to the ms result , as well as h nmr data attributable to the hydrocarbon chain , it was determined that the lipid - derived portion of 4 was missing two ch2 units relative to 2 . with the structure of metabolite 4 confirmed , the new compound was given the trivial name serrawettin w5 . an evaluation of metabolite 5 by hresims provided an ion with m / z 770.4346 [ m + na ] that enabled us to determine its molecular formula was c38h61n5o10 . this represented an increase of one oxygen atom in 5 relative to compound 2 . focusing on the h nmr data ( table 1 ) for the amino acid portion of the macrocycle in 5 , it was readily apparent that the aromatic protons associated with the phenylalanine residue in 2 were altered and that one hydrogen bonded to a carbon was missing . the new aromatic spin - coupled network appeared as an aabb system , which led us to propose that 5 incorporated a tyrosine residue in place of the phenylalanine in 2 . the assignments of the carbon and hydrogen spins for the tyrosine were subsequently confirmed by hmbc experiment , as well as hydrolysis followed by marfey s analysis , which together established the presence of an l - tyrosine in 5 . metabolite 5 was assigned the trivial name serrawettin w6 . with the stereochemical assignments of the amino acid residues completed , we focused on securing the configurations of the c-3 positions in each compound . applying biosynthetic - based logic to the problem , it appeared reasonable to presume that the stereochemistries of the metabolites c-3 positions were set upon incorporation of either 3-hydroxydecanoic acid ( 2 , 3 , and 5 ) or 3-hydroxyoctanoate ( 4 ) . a survey of the chemical literature concerning the spectroscopic properties of -hydroxy fatty acids revealed a decisive trend in their chiral - optical data ( table s1 ) . namely , in chloroform , r - configured -hydroxy fatty acids displayed consistent levorotatory activities , whereas in ethanol , these molecules exhibited dextrorotatory properties . returning to the acid hydrolysates prepared from 25 , we were able to purify milligram quantities of enantiopure -hydroxy fatty acids from each sample . specific rotation values were obtained for the products in both ethanol and chloroform , which enabled us to assign r configurations to the c-3 positions in 25 . the genome of the serratia sp . isolate was sequenced to link the identified natural products to their prospective biosynthetic gene cluster . over 5.4 million reads were generated and assembled into 35 contiguous sequences after quality control . the annotated 16s rrna gene was extracted and aligned to otus classified as gammaproteobacteria , which we had observed from the opossum ears . these showed close ( 98% ) homology to an otu classified as a member of the enterobacteriaceae . a total of eight potential secondary - metabolite - producing biosynthetic gene clusters were identified by antismash analysis , including four nrps clusters . one of the candidate clusters presented a near - perfect match to what was required to construct metabolites 25 ( si figure s69 ) . the purified compounds were evaluated for their abilities to inhibit growth and biofilm production of c. albicans . none of the metabolites were able to kill fungal cells at concentrations up to 100 m . isolate proved to be the most potent inhibitor of c. albicans biofilm formation , with an ic50 value of 4.6 1.0 m . the serratia sp . metabolites exhibited a range of biofilm inhibition capabilities , with 2 being the most potent ( ic50 = 7.7 0.7 m ) , followed by 4 ( ic50 = 13.4 0.2 m ) , 5 ( ic50 = 29.2 0.4 m ) , and 3 ( ic50 = 59.8 5.7 m ) . the abilities of these bacterial cyclic lipodepsipeptides to limit biofilm formation , while not impacting yeast cell survival , hint at the possibility of alternative chemical options for controlling c. albicans infections by means of biofilm modulation . the use of opportunistic sampling to humanely investigate the microbiomes of a broad range of animals offers an intriguing method to accelerate the exploration of this resource for new natural products . notably , the discovery pipeline presented here is an enabling tool for identifying bacterial natural products from nonhominid mammals . one potential concern centers on how well our collection strategy accurately reflects the native microbiomes of living mammals since the microbiomes of humans are known to change as the time since death increases . while our investigation did not test this point specifically , we did make efforts to sample only recently deceased mammals and took care to avoid orifices that were markedly compromised by collisions with vehicles . therefore , we remain confident that the isolates obtained in this study are representative members of mammalian microbiomes and not taxa simply derived from the immediate environment or strictly enhanced via decomposition . further studies are anticipated to provide additional microbiome - associated bacteria , their natural products , and associated biosynthetic gene clusters , which might serve as candidates for future microbiome engineering endeavors . indeed , colony profiling by laesims suggests the presence of many bacteria - derived peptide - like products that we anticipate will possess potentially useful therapeutic properties . nmr data were obtained on varian vnmr spectrometers ( agilent technologies , inc . , santa clara , ca , usa ) with broadband and triple resonance probes at 25 0.5 c unless otherwise noted . optical rotation measurements were made on a rudolph research autopol iii automatic polarimeter ( rudolph research analytical corp . uv data were measured with a hewlett - packard 8452a diode array spectrophotometer ( agilent technologies , inc . ) . ir spectra were obtained on a bruker vector 22 ft - ir spectrometer ( bruker corp . , billerica , ma , usa ) . accurate mass electrospray - ionization mass spectrometry data were collected on an agilent 6538 high - mass - resolution qtof mass spectrometer ( agilent technologies , inc . ) . hplc separations were carried out on a shimadzu system using a scl-10a vp controller ( shimadzu scientific instruments inc . , columbia , md , usa ) and gemini 5 m c18 and hexyl - phenyl columns ( 110 , 250 21.2 mm and 250 10.0 mm , respectively , phenomenex inc . , torrance , ca , usa ) with flow rates of 10 or 4 ml / min . all chemicals and solvents used in the study were of research grade quality or better . a 48 km section of oklahoma state highway 9 from the university of oklahoma campus , norman , ok , to the city of tecumseh , ok , was used for this study . this route was chosen because it traverses a state park ( lake thunderbird state park ) , a significant quantity of acreage devoted to agriculture , and forested land , which are all prime habitats for many of oklahoma s major mammalian species . additionally , the highway is heavily traveled ( a major east west thoroughfare from arkansas to texas ) with posted speed limits of 65 mph ( 105 kph ) . the juxtaposition of having an active highway that intersects such a variety of mammalian habitats made this a prime location for near daily animal vehicle collisions . a sampling permit was obtained from the state of oklahoma ( scientific collector permit # 5250 ) , and the university of oklahoma iacuc was informed of the experiments , resulting in the assignment of an internal case - tracking number ( r11 - 021 ) . when fresh roadkill was encountered ( generally less than 10 h old ) , the intact orifices , which included the mouth , ear , nose , and rectum , were sampled with sterile cotton - tipped swabs and then immediately plunged into sterile phosphate - buffered saline ( pbs ) buffer . when opportunities presented themselves , samples were also taken by swabbing the inner portions of the lower gastrointestinal tract . the cotton tips of the swabs were placed into 1.0 ml of pbs and vortexed . these suspensions were diluted 10 with pbs , and 50 l aliquots were spread onto three types of solid agar media ( 10% tryptic soy agar , 10% brain heart infusion agar , and dm7 agar ) and incubated at 30 c for 3 days . colony picking was performed using sterile toothpicks and the inoculum used to prepare streak plates on dm7 medium . single colonies were picked with toothpicks , which were then dipped into tryptic soy broth containing 15% glycerol and incubated at room temperature for 1 week . cultures of isolates were used to generate samples for cryogenic preservation at 80 c , as well as inoculate various media for further chemical and bioassay studies . for these experiments , 5 l aliquots were inoculated into the wells of three 24-well plates containing 50% tryptic soy broth , 50% brain heart infusion broth , and dm7 broth and incubated for 7 days at 30 c . these cultures were subjected to partitioning three times against equal volumes of ethyl acetate . the organic layers from each sample were combined , and the solvent was removed in vacuo . extracts were evaluated for bioactivities , and those determined to be active were examined by lcms and laesims . isolates were obtained from swabbing the ear canal of a roadkill opossum encountered on oklahoma state highway 9 near norman , ok . while the original colony selected for the study was thought to be a single isolate , subsequent cultivation of the bacteria led us to determine that the culture contained two taxa , which were separated through isolation on a solid agar medium and analyzed in pure culture . a single colony of the serratia sp . was used to inoculate 1/10th strength tryptic soy broth medium , which , after 16 h , was used to obtain genomic dna by means of an xpedition soil / fecal dna miniprep kit following the manufacturer s directions ( zymo research , irvine , ca , usa ) . the genomic dna was quantified using the qubit br assay ( life technologies , carlsbad , ca , usa ) , and approximately 1 g was sheared to a mean insert size of 500 bp and sequenced using pe250 v2 chemistry on the illumina miseq platform ( illumina , san diego , ca , usa ) . the reference strain c. albicans sc5314 was cultured in brain - heart infusion medium ( bhi , becton dickinson ) or rpmi-1640 plus mops medium [ rpmi-1640 medium ( sigma ) buffered to ph 7.0 with 0.17 m mops ( 3-(n - morpholino)propanesulfonic acid , sigma ) ] as required . sequence data used for 16s rrna gene analysis are available at the ncbi sra under accession number srx1601902 . genome is available under the accession number gca_001643155.1 , and the raw reads used to assemble the genome are available for download at the ncbi sra under the accession number srx1585056 . the opossum carcass was sampled with sterile cotton swabs , and the swabs were frozen at 20 c until dna extraction was performed . the samples were thawed and then homogenized ( biospec products , bartlesville , ok , usa ) for 30 s. community genomic dna was extracted using the mobio power biofilm dna extraction kit ( mobio laboratories , inc . , libraries of bacterial and archaeal 16s rrna gene fragments were amplified from each dna extraction by pcr with primers that spanned the v4 region between positions 519 and 802 ( e. coli numbering ) , producing a 300 bp fragment . these primers evenly amplify a broad range of both the bacteria and archaea . the forward primer ( m13l-519f : 5-gta aaa cga cgg cca gcacmg ccg cgg taa-3 ) contained the m13 forward primer ( in bold ) , followed by the 16s rrna gene - specific sequence ( underlined ) . the reverse primer ( 785r : 5-tac nvg ggt atc taa tcc-3 ) was taken directly from s - d - bact07850b - a-18 in klindworth et al . each 50 l pcr consisted of 3 u of recombinant taq polymerase ( thermo fisher scientific inc . , grand island , ny , usa ) , 1.5 mm mgcl2 , 0.1 m dntps ( thermo fisher scientific inc . ) , 0.2 m of each primer , and 4 l of a dna extraction . initial amplification was conducted using the following protocol : initial denaturation at 95 c for 2 min , followed by 30 cycles of denaturation at 95 c for 45 s , annealing at 52 c for 45 s , and extension at 72 c for 45 s , followed by a final extension at 72 c for 5 min . the amplified 16s rrna gene fragments in each library were purified using ampurexp paramagnetic beads according to the manufacturer s protocols ( beckman coulter , inc . , indianapolis , in , usa ) . a second , six - cycle pcr was used to add a unique 12 bp barcode to each amplicon library using a unique forward primer containing the barcode + m13 forward sequence ( 53 ) and the 785r primer using reaction conditions identical to those listed above . the resulting barcoded pcr products were quantified using the qubit hs assay ( life technologies ) , pooled in equimolar amounts , and concentrated to a final volume of 80 l using amicon ultra-0.5 ml 30k centrifugal filters ( merck millipore corp . , the pooled library was then submitted for sequencing on the miseq platform using pe250 v2 chemistry ( illumina ) . after sequencing , reads were merged using pear , demultiplexed in qiime , filtered by quality , and clustered into otus using uparse . the mapping file used to demultiplex the samples is present as a supplementary table . the taxonomy of each otu was assigned using uclust and the silva database ( release 123 ) . for the genomic sequence , reads were first quality trimmed , and sequence adapters were removed using peat before assembly within spades using kmer values of 21 , 33 , 55 , 77 , 99 , and 127 . after assembly , contiguous sequences less than 300 bp in length were discarded , and contaminating phix sequences were removed after visualization of the genome assembly graph in bandage . the assembled genome was annotated using the ncbi pgap annotation pipeline , and putative metabolite clusters were identified using antismash . single colonies of the serratia sp . isolate were transferred to dm7 and vortexed . aliquots of this suspension were inoculated into 40 high - aeration shake flasks ( 2 l ultra yield flask ) containing 1 l of dm7 . the flasks were shaken at 135 rpm for 7 days at room temperature on an innova 5000 shaker . the dm7 broth consisted of ( per liter ) monopotassium phosphate , 2.0 g ; ammonium chloride , 1.5 g ; magnesium sulfate heptahydrate , 0.5 g ; glycerol , 12.6 g ; myo - inositol , 0.4 g ; monosodium glutamate , 5.0 g ; sodium fluoride , 0.084 g ; iron(ii ) sulfate heptahydrate , 0.025 g ; zinc(ii ) sulfate heptahydrate , 0.01 g ; cobalt(ii ) chloride , 0.01 g ; calcium carbonate , 0.25 g ; and p - aminobenzoate , 0.001 g ; with the ph of the final solution adjusted to 7 . the broth from the liquid culture of the pseudomonas sp . was partitioned against ethyl acetate . during the partitioning process , crystals of 1 formed on the inner surface of the separatory funnel . the crystals were rinsed from the funnel under a stream of ethyl acetate , and the solvent was removed by leaving the crystals overnight in an open vial at room temperature . a colorless needle - shaped crystal of 1 of dimensions 0.26 0.04 0.04 mm was selected for structure analysis . intensity data for this compound were collected using a diffractometer with a bruker apex ccd area detector and graphite - monochromated mo k radiation ( = 1.541 78 ) . the sample was cooled to 100(2 ) k. cell parameters were determined from a nonlinear least - squares fit of 2010 peaks in the range 4.2 < < 68.2. a total of 39 420 data were measured in the range of 3.626 < < 71.353 using and oscillation frames . the data were corrected for absorption by the empirical method giving minimum and maximum transmission factors of 0.835 and 0.972 . the data were merged to form a set of 11 566 independent data with r(int ) = 0.145 and a coverage of 98.5% . the orthorhombic space group p212121 was determined by systematic absences and statistical tests and verified by subsequent refinement . the structure was solved by direct methods and refined by full - matrix least - squares methods on f. the positions of hydrogens bonded to carbons were initially determined by geometry and were refined using a riding model . hydrogens bonded to nitrogens and oxygens were located on a difference map , and their positions were refined independently with x - h restraints . a total of 767 parameters were refined against 51 restraints and 11 566 data to give wr(f ) = 0.3772 and s = 1.238 for weights of w = 1/[(f ) + ( 0.1800p ) + 30.0000p ] , where p = [ fo + 2fc]/3 . the final r(f ) was 0.1281 for the 11 016 observed , [ f > 4(f ) ] data . the largest shift / s.u . the final difference map had maxima and minima of 0.783 and 0.891 e / , respectively . the absolute configuration of the structure was determined by refinement of the flack parameter . the broth from each culture flask was pooled and partitioned three times against equal volumes of ethyl acetate . the solvent was removed in vacuo to generate a crude organic residue ( 10.3 g ) . the organic material was processed by vlc over hp20ss resin using a step gradient going from water to methanol to generate six fractions . fraction 4 was further separated by preparative c18 hplc ( gradient elution with meoh h2o , 75:25 , to 100% organic in 30 min ) to yield three subfractions ( 13 ) . subfraction 2 was further separated by semipreparative c18 hplc ( mecn h2o , 55:65 ) to provide four subfractions ( a d ) . subfraction 2c was further purified by semipreparative c18 hplc ( mecn h2o , 60:40 ) to yield 2 ( 46.7 mg , 4.53% yield ) . subfraction 2b was further purified by semipreparative c18 hplc ( mecn h2o , 65:35 ) to yield 3 ( 47.1 mg , 4.57% yield ) . subfraction 2a was further purified by semipreparative hexyl - phenyl hplc ( mecn0.1% formic acid in h2o , 65:35 ) to yield 4 ( 2.8 mg , 0.271% yield ) and 5 ( 3.7 mg , 0.359% yield ) . samples of the metabolites ( 300 g ) were incubated overnight in 6 m hcl ( 500 l ) at 110 c . the hydrosylates were dried under nitrogen gas , and 20 l of 1 m nahco3 was added to each sample to facilitate neutralization . next , 100 l of 1% 1-fluoro-2,4-dinitrophenyl 5-l - alanine amide was added to the samples , and the resulting solutions were heated at 45 c for 1 h. the reaction mixtures were neutralized with 20 l of 1 m hcl and diluted with 500 l of mecn . the samples were centrifuged and analyzed by lc - ms ( 100 , c18 kinetex 2.6 m , 75 3.0 mm column with gradient elution using mecn with 0.1% formic acid in h2o , 10:90 , to 100% organic over 15 min . ) . standards eluted as l - isoleucine at 7.96 min , d - isoleucine at 8.19 min , l - valine at 7.68 min , d - valine at 7.68 min , l - leucine at 7.78 min , d - leucine at 8.26 min , l - phenylalanine at 7.80 min , d - phenylalanine at 8.13 min , l - serine at 7.57 min , d - serine at 8.13 min , l - tyrosine at 8.57 min , d - tyrosine at 8.88 min , l - threonine at 8.53 min , l - allo - threonine at 5.73 min , and d - allo - threonine at 5.90 min . the hydrosylates were partitioned with equal portions of hexanes ( 3 ) . the hexanes were removed in vacuo , the resultant compound was suspended in 2 ml of chloroform or ethanol , and their specific rotation values were determined . the effects of compounds on the growth of c. albicans were tested using the method described in the nccls 2008 clsi m27-a3 guidelines with the following modifications . cells of c. albicans sc5314 were cultured in bhi medium ( becton dickinson co. , franklin lakes , nj , usa ) at 37 c overnight . the cells were pelleted by centrifugation , washed with sterile pbs ( ph 7.4 ) , and resuspended in rpmi-1640 plus mops medium . test compounds were prepared in dmso at stock concentrations of 2 mm before being diluted in rpmi-1640 plus mops for testing . aliquots of yeast suspension ( 100 l containing 2.5 10 cells ml ) were added to the medium containing the diluted compounds or dmso [ final concentrations did not exceed 2% ( v / v ) ] before being transferred to 96-well plates ( corning ) . after 48 h of incubation at 37 c , the viability of the yeast was measured using the xtt assay . in brief , yeast cells were treated with 0.1 mg ml xtt at 37 c for 1 h. absorbance measurements were taken at 492 nm using a microplate reader ( infinite m200 ) . the minimum inhibitor concentrations ( mic ) for growth were defined as the lowest antifungal concentrations that caused 85% reduction in metabolic activity . for measuring biofilm formation , the medium was aspirated and the wells were washed twice with sterile pbs to remove nonadherent cells . fresh medium ( 100 l of rpmi-1640 plus mops ) was then added back to each well . the 50% inhibitory concentration ( ic50 ) values for biofilm inhibition were calculated using graphpad prism 5 . white , powdery solid ; [ ]d20 = 7.3 ( c 0.164 , chcl3 ) ; uv ( meoh ) max ( log ) 206 ( 3.52 ) ; ir ( film ) max 3600 , 3516 , 3412 , 3003 , 2965 , 2926 , 1715 , 1425 , 1362 , 1221 , 1092 , 903 , 783 , 527 ; h nmr ( dmso - d6 , 600 mhz ) refer to table 1 ; c nmr ( dmso - d6 , 151 mhz ) refer to table 2 ; hresims m / z 730.4402 [ m h ] ( calcd for c38h60n5o9 , 730.4391 ) . white , powdery solid ; [ ]d20 = 7.4 ( c 0.270 , chcl3 ) ; uv ( meoh ) max ( log ) 206 ( 3.17 ) ; ir ( film ) max 3607 , 3524 , 3412 , 3003 , 2967 , 2925 , 1713 , 1707 , 1427 , 1361 , 1221 , 1094 , 903 , 783 , 527 ; h nmr ( dmso - d6 , 600 mhz ) refer to table 1 . c nmr ( dmso - d6 , 151 mhz ) refer to table 2 ; hresims m / z 740.4235 [ m + na ] ( calcd for c37h59n5o9na , 740.4210 ) . white , powdery solid ; [ ]d20 = 7.6 ( c 0.245 , chcl3 ) ; uv ( meoh ) max ( log ) 204 ( 3.05 ) ; ir ( film ) max 3603 , 3520 , 3412 , 3003 , 2968 , 2926 , 1714 , 1427 , 1362 , 1221 , 1094 , 905 , 783 , 523 ; h nmr ( dmso - d6 , 600 mhz ) refer to table 1 . c nmr ( dmso - d6 , 151 mhz ) refer to table 2 ; hresims m / z 726.4073 [ m + na ] ( calcd for c36h57n5o9na , 726.4054 ) . white , powdery solid ; [ ]d20 = 7.4 ( c 0.140 , chcl3 ) ; uv ( meoh ) max ( log ) 206 ( 3.11 ) ; ir ( film ) max 3609 , 3528 , 3412 , 3003 , 2695 , 2924 , 1746 , 1712 , 1422 , 1362 , 1222 , 1092 , 903 , 785 , 530 ; h nmr ( dmso - d6 , 600 mhz ) refer to table 1 . c nmr ( dmso - d6 , 151 mhz ) refer to table 2 ; hresims m / z 770.4346 [ m + na ] ( calcd for c38h61n5o10na , 770.4316 ) .
few secondary metabolites have been reported from mammalian microbiome bacteria despite the large numbers of diverse taxa that inhabit warm - blooded higher vertebrates . as a means to investigate natural products from these microorganisms , an opportunistic sampling protocol was developed , which focused on exploring bacteria isolated from roadkill mammals . this initiative was made possible through the establishment of a newly created discovery pipeline , which couples laser ablation electrospray ionization mass spectrometry ( laesims ) with bioassay testing , to target biologically active metabolites from microbiome - associated bacteria . to illustrate this process , this report focuses on samples obtained from the ear of a roadkill opossum ( dideiphis virginiana ) as the source of two bacterial isolates ( pseudomonas sp . and serratia sp . ) that produced several new and known cyclic lipodepsipeptides ( viscosin and serrawettins , respectively ) . these natural products inhibited biofilm formation by the human pathogenic yeast candida albicans at concentrations well below those required to inhibit yeast viability . phylogenetic analysis of 16s rrna gene sequence libraries revealed the presence of diverse microbial communities associated with different sites throughout the opossum carcass . a putative biosynthetic pathway responsible for the production of the new serrawettin analogues was identified by sequencing the genome of the serratia sp . isolate . this study provides a functional roadmap to carrying out the systematic investigation of the genomic , microbiological , and chemical parameters related to the production of natural products made by bacteria associated with non - anthropoidal mammalian microbiomes . discoveries emerging from these studies are anticipated to provide a working framework for efforts aimed at augmenting microbiomes to deliver beneficial natural products to a host .
Results and Discussion Experimental Section
the mammalian roadkill carcasses that were sampled included ( in order of increasing frequency ) the following : skunk ( mephitis mephitis ) , armadillo ( dasypus novemcinctus ) , deer ( odocoileus virginianus ) , raccoon ( procyon lotor ) , squirrel ( sciurus carolinensis ) , and opossum ( dideiphis virginiana ) . the phylogenetic diversity of the opossum microbiome was further evaluated by sequencing 16s rrna gene libraries . ( c ) categorization of the isolate data illustrating the percent contribution of each body site to the overall bacterial collection prepared from the different animal species ( note that the colors and categories used to construct the slices within each of the pie charts in panel c are the same as those used for the pie chart in panel b ) . all of the bacterial isolates were individually cultured in two or more broth media , and a library of their ethyl - acetate - soluble natural products was prepared . when the two microorganisms were obtained in pure culture , the 16s rrna genes of both isolates were sequenced , and the bacteria were identified as members of the genera pseudomonas and serratia . representative colonies of both isolates were analyzed in situ by laser ablation electrospray ionization mass spectrometry ( laesims ) . laesims analysis of the serratia sp . although the information derived from the lc - ms investigation of the compounds ( ms data , lc retention times , and uv vis pda profiles ) enabled us to postulate that the serratia sp . phylogenetic diversity of mammalian microbiome bacteria from different orifices / body sites of a roadkill opossum carcass . the presented mass data were derived from the circled colony ( average of several locations taken from the colony and subtracted from mass data obtained from a blank [ uncolonized ] portion of the plate ) , which reveals prominent single and doubly charged sodium adduct ions for viscosin ( 1 ) . laesims - guided fractionation of the organic layer by hp20ss vlc , as well as preparative and semipreparative c18 hplc resulted in the purification of compounds 35 , along with 2 , whose structure was subsequently confirmed by ms experiment , h and c nmr data ( tables 1 and 2 , respectively ) , and marfey s analysis . inspection of the h ( table 1 ) and h h dqfcosy nmr data for 3 showed that the amide proton ( 8.28 , d , j = 7.22 , 1 h ) coupled with the -proton ( 3.73 , t , j = 7.76 , 7.76 , 1 h ) , which coupled to a methine ( 1.95 , m , 2 h ) that in turn coupled with methyl protons ( 0.84 , m , 6 h ) . this was supported by hydrolysis followed by marfey s analysis , which confirmed the presence of the same amino acid residues in 2 and 4 . focusing on the h nmr data ( table 1 ) for the amino acid portion of the macrocycle in 5 , it was readily apparent that the aromatic protons associated with the phenylalanine residue in 2 were altered and that one hydrogen bonded to a carbon was missing . the assignments of the carbon and hydrogen spins for the tyrosine were subsequently confirmed by hmbc experiment , as well as hydrolysis followed by marfey s analysis , which together established the presence of an l - tyrosine in 5 . the genome of the serratia sp . the annotated 16s rrna gene was extracted and aligned to otus classified as gammaproteobacteria , which we had observed from the opossum ears . further studies are anticipated to provide additional microbiome - associated bacteria , their natural products , and associated biosynthetic gene clusters , which might serve as candidates for future microbiome engineering endeavors . a 48 km section of oklahoma state highway 9 from the university of oklahoma campus , norman , ok , to the city of tecumseh , ok , was used for this study . a sampling permit was obtained from the state of oklahoma ( scientific collector permit # 5250 ) , and the university of oklahoma iacuc was informed of the experiments , resulting in the assignment of an internal case - tracking number ( r11 - 021 ) . isolates were obtained from swabbing the ear canal of a roadkill opossum encountered on oklahoma state highway 9 near norman , ok .
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nurse practitioners ( nps ) and clinical nurse specialists ( cnss ) have practiced for over 50 years in the united states , followed closely by canada and the united kingdom , and the roles are increasingly being implemented in other countries . the quest for improved quality of care and control of healthcare costs are important drivers in the decision to implement these roles . we conducted a systematic review to assess the evidence of cost - effectiveness of np and cns roles . nps are defined as rns who have additional education in recognized programs , preferably at the graduate level . they demonstrate advanced competencies to practice autonomously and collaboratively to perform assessments , order laboratory and diagnostic tests , diagnose , prescribe medications and treatments , and perform procedures , as authorized by legislation and their regulatory scope of practice , as well as performing an advanced nursing role that includes consultation , collaboration , education , research , and leadership . cnss are registered nurses ( rns ) with a graduate degree in nursing who have expertise in a clinical specialty and perform an advanced nursing role that includes practice , consultation , collaboration , education , research , and leadership . those working in alternative roles provide similar services to those for whom they are substituting , usually physicians . those working in complementary roles provide additional services that are intended to complement or extend existing services . the intention of the alternative role is typically to reduce cost or workload or to address workforce shortages while maintaining or improving the quality of care ; in contrast , the intention of the complementary role is to improve the quality of care . during the 1970s , the first randomized controlled trials ( rcts ) of nps demonstrated their safety and effectiveness , as well as patient satisfaction with the np role [ 617 ] . nps improved resource utilization and access to care [ 14 , 1820 ] , increased primary care services in the community , and reduced costs . over the past 30 years , a number of literature reviews and systematic reviews have summarized the findings of studies evaluating nps [ 2125 ] . the reviews have consistently shown no difference in the health outcomes of patients receiving np care when compared to patients receiving physician care , but often both quality of care and patient satisfaction are higher with np care . most rcts of cns roles have been published since 1980 except one . in 1977 , pozen and colleagues found that the cns increased the knowledge of heart disease in patients with myocardial infarction resulting in an increased rate of return to work and a reduction in smoking . literature reviews and systematic reviews of cnss [ 25 , 27 ] reveal that cnss are associated with reductions in hospital length of stay , readmissions , emergency room visits , and costs , as well as improvements in staff nurse knowledge , functional performance , mood state , quality of life , and patient satisfaction . study findings are consistent that nps and cnss , either in alternative or complementary provider roles , deliver high quality patient care that results in high patient satisfaction . to address a question that often surfaces , are nps and cnss cost - effective ? , we conducted a systematic review of rcts of np and cns cost - effectiveness ( defined broadly to also include studies measuring health resource utilization ) entitled a systematic review of the cost - effectiveness of nurse practitioners and clinical nurse specialists : 1980 to july 2012 . the purpose of this paper is to report on the methodological strengths and threats to internal and external validity of these rcts . we sought rcts of np and cns cost - effectiveness between january 1980 and july 2012 . due to inconsistencies in the use of titles and lack of role clarity for these two roles , we developed specific criteria to decide if the role was an np , a cns , or an rn in an expanded role . to be deemed an np , the nurse had to have completed a formal postbaccalaureate or graduate np education program or be licensed as an np . to be deemed a cns , the nurse had to have completed a graduate degree and the role had to be reflective of the cns role definition . if necessary , we contacted the lead author and/or experts in advanced practice nursing from the country where the study was conducted to determine eligibility . the principal outcomes of interest in this review were objective measures of health system utilization . these included length of stay , rehospitalization , costs of healthcare ( e.g. , hospital , professional , and family costs ) , and health resource use ( e.g. , diagnostic tests and prescriptions ) . because it is important to examine health system utilization in the context of patient and provider outcomes , we also extracted data on all patient ( e.g. , mortality , morbidity , quality of life , and satisfaction with care ) and provider ( e.g. , quality of care and job satisfaction ) outcomes . participants were patients of any age receiving care in all types ( e.g. , teaching and nonteaching , public and private ) , sizes ( e.g. , small , medium , and large ) , and locations ( e.g. , rural and urban ) of hospitals or community settings ( e.g. , long - term care , primary care , and home care ) . substantive developments since 1980 ( e.g. , training , payment models , and scope of practice of nps ) have reduced the relevance of pre-1980 studies to modern - day policy . in consultation with a policy advisor , we chose to exclude pre-1980 studies from this review . studies were also excluded if ( 1 ) the np or cns education failed to meet our criteria or if we could not contact the author for clarification despite repeated attempts ; ( 2 ) the np or cns was part of a multicomponent or multidisciplinary intervention in which the impact of their contribution could not be isolated from other healthcare providers on the team ; ( 3 ) the study evaluated a very specific intervention ( e.g. , cognitive behavioural therapy ) that was delivered by an np or cns but could be delivered by other clinicians , such as an rn ; ( 4 ) the control group was also exposed to an np or cns during the study ; ( 5 ) a measure of health system utilization was not included ; ( 6 ) true randomization was not used ( randomization was predictable , for example , assignment by day of hospital admission and alternating assignment ) . a search was conducted to identify all relevant published and unpublished rcts reported from january 1980 to july 2012 . medical librarians conducted a comprehensive search of the literature using cinahl , embase , global health , healthstar , medline , allied and complementary medicine database ( amed ) , cochrane library database of systematic reviews and controlled trials register , database of abstracts of reviews of effects ( dare ) , health economics evaluation database ( heed ) , and web of science . relevant medical subject headings ( mesh ) keywords , inclusive suffixes , and search strings formed the search strategy ( appendix ) . in addition , the following methods were used to identify primary studies : handsearching of 16 high - yield journals , checking reference lists of all relevant papers and reviews , contacting authors of an early list of relevant studies , searching personal files , reviewing bibliographies , and searching websites of nursing research and professional organizations and national , provincial / state , and territorial governments . we uploaded all identified citations to a web - based reference management program ( refworks ) and removed duplicate entries . two - member teams independently screened titles and abstracts of these citations for relevance using prespecified criteria . translators assisted with the review of all citations in languages other than french or english . the full - text of a published paper and/or study report was obtained if it appeared to meet the inclusion criteria , if an abstract was unavailable , or if it was not possible to determine relevance from the title and abstract review . in instances where a study was reported in more than one paper , we grouped the study 's papers in a constellation and collectively reviewed them . two - member teams independently screened these full - text papers for eligibility based on the inclusion criteria . two team members ( ad and kr ) independently assessed the methodological quality of the studies for internal validity and disagreements were resolved through discussion and consensus . the internal validity of each study was assessed using a slightly modified version of the cochrane risk of bias criteria ; modifications to the criteria were three - fold . first , we did not assess for blinding of participants and personnel because the nature of np and cns interventions precludes this possibility . second , outcome assessment and completeness of outcome data were evaluated separately for objective and subjective outcomes within a study . we looked for evidence of key outcomes that would typically be measured for each study 's research question . third , if outcomes had more than 20% missing data , we judged the study to be at high risk of bias for incomplete outcome data . we assessed studies , assigning a high , low , or unclear risk of bias for each of the following eight questions : ( 1 ) to avoid selection bias , was the strategy used for random sequence generation likely to produce comparable groups ( e.g. , random number table , computer random number generator ) ? ( 2 ) to avoid selection bias , was a method used to conceal the allocation sequence so that group allocation could not be foreseen in advance ( e.g. , sequentially numbered , opaque , sealed envelopes ; central allocation office ) ? ( 3 ) to avoid detection bias , was an appropriate method / source used to collect objective ( e.g. , mortality ) measures ( e.g. , death records , blinding of outcome assessor , trained chart abstracter ) ? ( 4 ) to avoid detection bias , was an appropriate method used to collect subjective ( e.g. , quality of life ) measures ( e.g. , blinding of outcome assessor ; use of reliable , valid , established self - administered questionnaires ) ? ( 5 ) to avoid attrition bias , was outcome data complete for the objective measures ( i.e. , complete for 80% of sample ; missing data balanced between groups ; missing data imputed using appropriate methods ) ? ( 6 ) to avoid attrition bias , was outcome data complete for the subjective measures ( i.e. , complete for 80% of sample ; missing data balanced between groups ; missing data imputed using appropriate methods ) ? ( 7 ) to avoid reporting bias , were all outcomes described in the methods section of the study reported in the results and were all key outcomes reported ? ( 8) were other biases detected in the study ( e.g. , contamination bias in which the control group had exposure to the intervention ) ? we sought clarification from 40 of the 43 study authors when there were insufficient details in the paper to determine the risk of bias and we received 28 ( 70% ) responses . an overall risk of bias was assigned to each study as follows : low risk of bias ( at risk in 0 - 1 category ) , moderate risk of bias ( at risk in 2 - 3 categories ) , high risk of bias ( at risk in 46 categories ) , and very high risk of bias ( at risk in 7 - 8 categories ) . external validity refers to the generalization or applicability of the study to other circumstances . to assess external validity , two team members independently assessed the generalizability of the study population , intervention , control , and outcomes ( pico ) . historically , rcts of nps and cnss have been criticized because the number evaluated in any study has been small ( e.g. , one or two nps ) causing concern that those willing to be evaluated may be atypical in training , experience , knowledge , skills , or practice characteristics . we consulted with our policy advisor and together decided that 10 nps or cnss either within a single study or across studies combined in meta - analyses would be a reasonable minimum sample necessary to generalize results to similar np or cns roles . as reported in a separate paper , we applied the quality of health economic studies ( qhes ) instrument [ 3133 ] to evaluate the economic analyses in each study . the quality of the body of evidence for individual outcomes was evaluated using the grading of recommendations assessment , development and evaluation ( grade ) system [ 34 , 35 ] and gradepro software . a trained research assistant ( kr ) extracted data from each study into a summary table regarding general information ( i.e. , author , country , setting , language of publication , and publication status ) , characteristics of the study ( design and group allocation ) , characteristics of the participants ( number per group , sex , ages , and health conditions ) , characteristics of the intervention ( number and type of nps or cnss , education and training , specific role , and comparison intervention ) , outcomes ( health system , patient , and provider ) , length of follow - up , proportion followed to study completion , and study findings . if the findings of a single study were reported in two or more papers , they were extracted as one study . team members checked the accuracy of extractions and discrepancies were resolved through discussion and consensus . studies were categorized into the following six groupings : np - outpatient , np - transition , np - inpatient , cns - outpatient , cns - transition , and cns - inpatient . in a transition role , the np or cns could provide time - limited services designed to ensure healthcare continuity , avoid preventable poor outcomes among at - risk populations , and promote the safe and timely transfer of patients from one level of care to another or from one type of setting to another [ 37 , page 747 ] . within these groupings , studies were further categorized into alternative or complementary np or cns role function . the strengths and threats to internal and external validity of the included rcts the searches yielded 4,397 unique records of which 3,981 were excluded during title and abstract review . based on full - text review of the remaining 416 papers , 351 were excluded based on reasons listed in figure 1 . the remaining 65 papers described 43 relevant rcts ( 28 studies reported in single papers and 15 studies reported in 37 papers ) . all studies were published in english . in general , the control intervention was usual care . the distribution of the 43 rcts across groupings was np - outpatient ( n = 11 ) , np - transition ( n = 5 ) , np - inpatient ( n = 2 ) , cns - outpatient ( n = 11 ) , cns - transition ( n = 13 ) , and cns - inpatient ( n = 1 ) . we summarize the results by grouping beginning first with a brief overview of the study characteristics ( tables 1 and 2 ) followed by a description of threats to internal validity ( figures 2 and 3 ) . eleven rcts of nps in outpatient care [ 3848 ] met our inclusion criteria ( table 1 ) . they were conducted in the united states ( n = 7 ) , united kingdom ( n = 2 ) , or the netherlands ( n = 2 ) . six studies evaluated nps in alternative provider roles and five in complementary provider roles . the number of nps ranged from one to 20 in np alternative provider studies and from one to four in np complementary provider studies . some of the trials were quite large with over 1000 patients , while most of the trials examining specific patient populations tended to be much smaller . overall , six of the 11 rcts were judged to be at low risk of bias ( in other words , the methods were of high quality ) , four at moderate , and one at high risk of bias ( figure 2 ) . with regard to selection bias , nine studies used a random sequence generation process that was likely to produce comparable groups ; for two studies , we had insufficient information about the sequence generation process to permit judgement , despite contact with one of the authors . seven trials used an adequate process to conceal allocation so that participants and those enrolling participants could not foresee the group to which the next patient would be assigned . we judged one study as unclear because there was insufficient information and three at high risk of selection bias . all the rcts were judged to be at low risk of detection bias with respect to objective outcome measures ( e.g. , blood levels and medical records abstraction ) and all but two trials were assessed at low risk of detection bias for subjective measures because most used established validated self - report instruments ( e.g. , sf-12 and sf-36 ) . two studies were judged as unclear , one because they used self - reported dietary intake and physical activity which can be subject to recall and social desirability bias and the other because clinicians self - recorded the length of time they spent with each patient . seven rcts were judged to be at low risk of attrition bias for the objective measures ; one study reported a follow - up rate less than 80% for a blood cholesterol measure , and two did not report all follow - up rates . the risk of attrition bias for subjective measures was high or unclear for six studies due to failure to report follow - up rates or poor response rates for at least one self- or interviewer - administered questionnaire by last follow - up . one study was judged at high risk of reporting bias because they did not report any patient outcomes such as child 's health status , quality of life , or parent satisfaction in a study of the appropriateness of follow - up care after attendance at an emergency department . we rated one study at high risk of other bias because there was substantial baseline imbalance which was not adjusted for in the analyses . five rcts evaluated nps in a transition role [ 4953 ] ( table 1 ) . three studies were conducted in the us , one in canada , and one in the uk . one study evaluated nps in an alternative provider role and four in complementary provider roles . one or two nps were evaluated in each study . the number of patients included in the trials ranged from 54 to 750 and they were conducted at between one and 10 sites . overall , two studies were judged to be at low risk of bias and three at high risk of bias ( figure 2 ) . the trials assessed to be at low risk of selection bias used a random number generator that revealed the intervention assignment when a patient was ready for allocation and a computer generated sequence concealed in sequentially numbered , opaque , sealed envelopes . the other three studies provided insufficient information to fully judge random sequence generation and allocation concealment . all five rcts were judged to be at low risk of detection bias for objective measures as they used abstraction of hospital administrative records or blinded outcome assessment . with respect to subjective measures , two trials were at high risk of detection bias because patients self - reported their smoking cessation success and the np , who delivered the intervention , also collected baseline and outcome data from the comparison groups during a guided interview . all but one trial were at low risk of attrition bias for objective measures as they followed over 80% of participants and this was balanced across comparison groups within each study . with respect to subjective data , three trials scored high for risk of attrition bias due to poor response rates to self- or interviewer - administered questionnaires . two studies identified outcomes that they planned to measure but did not report , placing them at risk of reporting bias . two rcts of nps in inpatient settings met our inclusion criteria , both of which evaluated the np in an alternative provider role [ 54 , 55 ] ( table 1 ) . the number of nps in the trials ranged from 2.5 to 4.5 full - time equivalent nps . the number of patients included in the trials ranged from 381 to 821 and each study was conducted at one site . overall , the two studies were judged to be at low risk of bias ( figure 2 ) . both studies were at low risk of selection bias having used acceptable random sequence generation processes ( table of random numbers ; computer random number generator ) and having concealed allocation through the use of sequentially numbered , sealed , opaque envelopes . both were at low risk of detection bias as they relied on medical record and hospital database extraction of objective data such as mortality , medical complications , and length of hospital stay . in cases where study participants completed questionnaires , there were reliable , valid measures such as the sf-36 and the minnesota infant development inventory ( midi ) . both studies were judged to be at low risk of attrition bias for the objective measures but at high risk of attrition bias for the subjective measures . while many of the primary objective outcome data were available for all study participants ( e.g. , mortality , complications , and length of stay ) , subjective self - report measures often had response rates less than 80% . we judged both studies to be at low risk of reporting bias and other biases . eleven rcts [ 5666 ] addressed the cns role in delivering outpatient care ( table 2 ) . six studies were conducted in the us , two in the uk , two in the netherlands , and one in china . four trials evaluated one to six cnss in the alternative provider role , while seven trials evaluated one to nine cnss in the complementary provider role . the number of patients included in the trials ranged from 20 to 643 and the studies were conducted at between one and six sites . overall , five of the eleven studies were assessed at low risk , four at moderate risk , and two at high risk of bias ( figure 3 ) . while seven studies used valid methods to generate the random sequence and were at low risk of selection bias , we judged the remaining four to be at unclear risk of bias because the authors did not include this information in their papers and we did not receive responses to our request for further details . with respect to allocation concealment , five trials were assessed at low risk of selection bias ( e.g. , central allocation and sealed envelopes ) and three at unclear risk of bias because methods were not described . we judged one at high risk of bias because the patients were randomly assigned by the cns to one of the study groups by drawing the next allocation from an envelope ; using this method , it is possible that the drawn assignment could be returned to the envelope and redrawn if allocation was deemed unsuitable . two studies used cluster randomization and allocation concealment was not applicable as the clusters were all randomized at one time . all the studies were rated as low in risk of detection bias for objective outcome measures ( e.g. , mortality and rehospitalization ) . of the nine studies that included subjective outcomes , eight were judged at low risk of bias as they used established , validated instruments , or blinded outcome assessment and one was at high risk of bias because the cns who delivered the intervention also collected data from both groups before and after the intervention via telephone interviews . with respect to attrition bias , two of the studies judged at unclear risk of bias provided insufficient information to assess the completeness of all objective outcome measures and one , judged at high risk of bias , did not have cost data for at least 80% of the study participants . while seven trials were judged to be at low risk of reporting bias , four were judged at high risk because they did not fully report all the outcomes they collected or did not collect all patient - important outcomes that would have been expected ( e.g. , patient / parent satisfaction with care and quality of life ) . finally , one trial was judged at high risk of other bias because they did not adjust for cluster randomization . thirteen rcts [ 6779 ] evaluated the cns in the delivery of transition care in the us ( n = 12 ) and in the uk ( n = 1 ) ( table 2 ) . the number of patients included in the trials ranged from 40 to 375 and the studies were conducted at between one and six sites . overall , three of the thirteen trials were at low risk , eight at moderate risk , and two at a high risk of bias ( figure 3 ) . all but one trial used valid methods to generate the random sequence and all but one trial concealed allocation . all trials were rated at low risk of detection bias for objective measures , except one . in this study , the risk of bias was unclear because healthcare utilization outcomes were based on self - report rather than medical record review data . for subjective measures , two trials were judged to be at unclear risk of detection bias because the validity of their scales was not described , and , in one trial , treatment adherence was based on self - report rather than objective measures , such as pill counts and was assessed at high risk of bias . for objective measures , six trials had a low risk of attrition bias but , for five trials , the risk was unclear and , for two , it was high . for subjective measures , four trials had a low risk of attrition bias but , for six trials , the risk was unclear and , for three , it was high . for those studies in which it was unclear , the response rates were not specified or data were imputed ; for those at high risk of bias , the follow - up rate was less than 80% . of the 13 trials , four were at high risk of reporting bias , three of which did not report on all outcomes measured and one of which did not include a measure of health status . one study was at unclear risk of bias because it was unclear if measures reported at baseline should have been reported as outcomes . finally , seven trials were assessed at high risk of other bias because there were baseline differences between the groups for which adjustments were not made . only one study , conducted in the us in 1990 , evaluated the cns delivering inpatient care ( table 2 ) . two cnss participated in the study , which included 107 patients and was conducted at one site . overall , the risk of bias for this study was judged as moderate ( figure 3 ) . the study , however , was judged to be at high risk of attrition bias because over 20% of patients were dropped from the study after randomization as the intervention they received was changed ( e.g. , sitters discontinued and control group receiving cns consultation ) resulting in unequal distribution of patients in the two groups . the study was also at high risk of reporting bias because they did not report whether the cns and staff nurse intervention influenced patient risk behaviours as intended . contamination bias was possible because the same staff nurses who received coaching from the cns for intervention group patient management and for charting nursing observations cared for the control group and might have provided the same patient management and charting strategies for them . because the associated risk of bias was unknown , we judged this as unclear other bias . overall , we assessed that 18 of the 43 trials ( 42% ) were at low risk , 17 ( 39% ) at moderate risk , and 8 ( 19% ) at high risk of overall bias ( figures 2 and 3 ) . figures 4 and 5 summarize the studies by type of bias . with respect to the np trials , many studies were at high risk of detection bias with incomplete ( < 80% ) follow - up for subjective outcomes ( e.g. , self - administered scales ) . in cns trials , a number of studies were at high risk of reporting bias because they either did not report on all outcomes measured or did not include a key outcome that we would have expected . a number of studies ( especially smaller studies ) had baseline differences with no mention of adjusting the analyses to account for these differences . some of the potential threats to validity may not in reality be threats , but rather it may be an issue of lack of reporting . there were many instances that we rated categories as unclear risk of bias because there was insufficient information in the paper or from the author to permit judgment of low or high risk of bias . of the 43 rcts , 70% of the studies were conducted in the united states ( n = 30 ) and the remainder in four other countries : the united kingdom ( n = 6 ; 14% ) , the netherlands ( n = 4 ; 9% ) , canada ( n = 2 ; 5% ) , and china ( n = 1 ; 2% ) . given that healthcare systems and np and cns education , role implementation , and scope of practice vary internationally , applicability of study findings from one country to another may be compromised . some rcts evaluating np and cns roles were conducted across many sites which may enhance generalizability . however , many trials were conducted in single sites , which likely limits the generalizability of study findings . of the 43 rcts , 13 ( 30% ) studies were published prior to the year 2000 . given the substantive progress that has occurred in the development of np and cns roles and dynamic changes in healthcare systems internationally , the results of these studies may be less relevant to current - day policy . although we found a substantial number of eligible rcts , when broken down by grouping , we identified only one dated rct of cnss in the nontransitional care role for inpatient settings . this rct evaluated two cnss providing consultation for a small very particular population of medical - surgical patients requiring sitters due to the risk of self - harm or unpredictable behaviour . similarly , we identified only two rcts of nps in the nontransitional care role in inpatient settings , both of which were published over 10 years ago . one study evaluated nps caring for a homogeneous population of critically ill infants in a canadian hospital and the other evaluated nps caring for a heterogeneous population of adults admitted to general medical wards in a us - based hospital . given the existence of only three fairly dated rcts of nps or cnss in inpatient settings and somewhat specific populations , caution is needed in generalizing these results to nps and cnss in other inpatient settings . nine ( 21% ) trials were conducted with small numbers of patients ( n < 100 ) with specific health conditions . the larger studies with patients experiencing common conditions are more readily generalizable to the general population than smaller trials with patients experiencing a specific condition . however , one of the larger trials limited study entry to poor , non - english speaking hispanic people which may limit the generalizability of the findings to other patients seeking primary healthcare . twenty - seven ( 63% ) of the rcts evaluated one or two nps or cnss , 9 ( 21% ) evaluated three to five , four ( 9% ) evaluated six to nine , and three ( 7% ) evaluated 10 or more all of which were np - outpatient studies . the small number of nps and cnss evaluated in any study raises concern that the results may not be generalizable to colleagues in similar roles . in some cases when study outcomes were similar we were able to combine study findings which increased the number of nps or cnss evaluated for that outcome . about two - thirds of the studies ( n = 29 ; 67% ) specified that they evaluated experienced nps or cnss ( i.e. , nps or cnss who had completed their training at least one year before the evaluation and/or had graduate degrees ) . one study posed concern , as it compared novice nps who had completed a two - year advanced nursing practice graduate degree in the previous two months with general practitioners who had an average of 16 years work experience . most studies used reliable and valid outcome measures to evaluate patient - important outcomes such as health status , quality of life , and satisfaction with care which strengthens the generalizability of the findings ; however , some studies had very short - term follow - up periods ( e.g. , two weeks after the patient appointment ) which may compromise generalizability of study findings over the long term [ 39 , 48 , 60 ] . the purpose of this paper was to report on the methodological strengths and threats to internal and external validity of rcts of np and cns cost - effectiveness . based on a comprehensive search of the international literature , we identified 43 rcts , evaluating nps ( n = 18 ) and cnss ( n = 25 ) . while 43 rcts sound like a large number of evaluations of nps and cnss , categorizing the studies by np or cns role ( i.e. , alternative or complementary ) and by setting ( i.e. , outpatient , transition , or inpatient ) reveals the areas where further research is still required . for example , we found only one rct of the cns in a nontransitional role in the inpatient setting and only two rcts of the np in a nontransitional role in the inpatient settings , both of which were alternative provider roles . of the 43 rcts , 70% ( n = 30 ) were conducted in the united states with far fewer conducted in four other countries ( canada , china , the netherlands , and united kingdom ) . in 2011 , newhouse et al . they chose to restrict the review to studies conducted in the united states to enhance the applicability of study findings to the united states healthcare system . a recent systematic review that also includes studies conducted outside the united states therefore , we chose to broaden our search to include international studies in order to learn more about where np and cns role evaluations have been conducted and how the roles are being enacted globally . our assessment of the risk of bias revealed that about two - fifths ( n = 18 ; 42% ) of the 43 studies were at low risk of bias , close to the same number ( n = 17 ; 39% ) were at moderate risk of bias , and about one - fifth ( n = 8 ; 19% ) at high risk of bias . when examined by date , 31% of the 13 rcts published before the year 2000 were at high risk of bias compared to 13% of the 30 rcts published in or after the year 2000 that were at high risk of bias which may mean that study validity is improving over time . in many cases it was unclear if the authors met the risk of bias criteria because the required information was not reported in the paper consequently , we rated a large number of categories as unclear risk of bias . to permit complete and accurate assessment of risk of bias , researchers are encouraged to use a guide such as the cochrane risk of bias criteria when planning and reporting future studies . a clear brief description of the sequence generation ( e.g. , random number table ; computer random number generator ) is needed to allow the reader to determine if the process should provide comparable groups . a description of allocation concealment ( e.g. , sequentially numbered , opaque , and sealed envelope ) is important for the reader to determine if allocation to groups could be manipulated . while blinding of participants is not possible in a study incorporating nps or cnss , a description of procedures used to blind outcome assessors and/or the description of valid outcome measures is needed to assess the quality of the study . completeness of outcome data for each outcome measure and group , including the description of missing data and details of all participants excluded , lost to follow - up ( e.g. , dropped out of study or died ) , or reincluded at each stage , also needs to be reported . if researchers do not report outcomes that were measured or key outcomes that would be expected , a clear description is needed of the reasons for failing to report the outcome . a description of how any other biases were managed that threaten the quality of the study should also be reported . more detailed recommendations for reporting rcts can be found in the consolidated standards of reporting trials ( consort ) 2010 statement [ 81 , 82 ] . when authors are faced with cutting back on the number of words in a publication , a suggestion is to reduce the introductory sections to provide sufficient space to describe in detail the strategies used to prevent or minimize threats to internal validity . as others have found [ 5 , 24 ] , a challenge in conducting this systematic review was determining the fidelity of the intervention . the definition of the role and the education , training , and experience of the nps or cnss were often inadequately described or missing . when we contacted authors for this information , we found that some studies were conducted with rns who had received as little as a few weeks of training or one course and were then called nps . over half ( n = 27 ; 63% ) of the 43 studies evaluated only one or two nps or cnss and only three trials , all of nps in outpatient settings , evaluated 10 or more . approximately two - thirds of the studies ( n = 29 ; 67% ) evaluated experienced nps or cnss . researchers are encouraged to include a detailed description of the nps or cnss being evaluated in their study ( role in the context of an internationally accepted definition , education , experience in the role , and training for the specific intervention if applicable ) . furthermore , evaluations of these roles should not be initiated while the nps or cnss are still novices but rather when they have had sufficient experience in their role ( i.e. , at least 12 months ) . challenging as it is , researchers are encouraged to plan multisite studies , to increase the number of nps or cnss evaluated , to increase the number of patients enrolled in the study , and to account for variations in practice to enhance the generalizability of study findings . restricting this review to rcts may be viewed as a strength or limitation , depending on the perspective of the reader . health service settings are complex and research is confounded by multiple variables that challenge the ability to evaluate the effectiveness of an intervention , such as np and cns roles . when feasible , randomization of participants to intervention and control groups is considered the optimal design to control known and unknown complexities and confounding variables [ 8386 ] . the quality of evidence in this review demonstrates that it is feasible to conduct well - designed rcts to evaluate the effectiveness of np and cns roles in a variety of settings , remuneration mechanisms , and patient populations . strengths of our review include use of numerous strategies to identify all rcts in any language ( published or unpublished ) that met our inclusion criteria , contact with authors and international expert advisors when it was unclear whether a study met our inclusion criteria , use of current education and credentialing criteria to verify that the trial was indeed evaluating an np or cns , use of duplicate assessment by independent reviewers and a consensus process for every stage of the review , use of an internationally recognized and established tool to assess the overall risk of bias of each trial and contact with authors when additional information was required to make our assessment , use of an established tool ( quality of health economic studies ) to evaluate the health economic analysis in each study , use of grade to evaluate outcome - specific quality of evidence , consideration of external as well as internal validity , grouping of trials by type ( np or cns ) , setting ( inpatient , transition , or outpatient ) , and role ( alternative or complementary ) , and conducting meta - analyses whenever possible . in future publications , we will summarize our assessment of the quality of the economic analyses of each rct and outcome - specific quality of evidence using grade for each of the six groupings . with respect to limitations , despite our attempts to identify all relevant rcts , we may have missed some relevant studies or included some that do not meet our criteria based on author responses , advisor advice , or our interpretation of the description of the education or role . with respect to generalizability , we did not use a specific tool to assess threats to external validity but did consider the country and year of publication , number of nps or cnss in the study , the number of settings , and characteristics of the population , setting , intervention , and outcomes . we do not know how the exclusion of observational studies that investigate the effectiveness of np and cns roles may have influenced our findings . this paper builds on the body of knowledge regarding quality of rcts of np and cns cost - effectiveness ( defined broadly to also include studies measuring health resource utilization ) . we have used an international lens and inclusion criteria that meet today 's definitions of the np and cns roles . while almost half the rcts were found to be at low risk of bias , incomplete reporting of study methods and lack of details about np and cns education , experience , and roles make it difficult to fully evaluate the internal and external validity of studies of these roles . future studies that adhere to current standards for internal validity , such as cochrane risk of bias , consort [ 81 , 82 ] , and grade [ 34 , 35 ] , will contribute to a stronger body of evidence to address policy makers ' questions regarding the cost - effectiveness of np and cns roles .
background . improved quality of care and control of healthcare costs are important factors influencing decisions to implement nurse practitioner ( np ) and clinical nurse specialist ( cns ) roles . objective . to assess the quality of randomized controlled trials ( rcts ) evaluating np and cns cost - effectiveness ( defined broadly to also include studies measuring health resource utilization ) . design . systematic review of rcts of np and cns cost - effectiveness reported between 1980 and july 2012 . results . 4,397 unique records were reviewed . we included 43 rcts in six groupings , np - outpatient ( n = 11 ) , np - transition ( n = 5 ) , np - inpatient ( n = 2 ) , cns - outpatient ( n = 11 ) , cns - transition ( n = 13 ) , and cns - inpatient ( n = 1 ) . internal validity was assessed using the cochrane risk of bias tool ; 18 ( 42% ) studies were at low , 17 ( 39% ) were at moderate , and eight ( 19% ) at high risk of bias . few studies included detailed descriptions of the education , experience , or role of the nps or cnss , affecting external validity . conclusions . we identified 43 rcts evaluating the cost - effectiveness of nps and cnss using criteria that meet current definitions of the roles . almost half the rcts were at low risk of bias . incomplete reporting of study methods and lack of details about np or cns education , experience , and role create challenges in consolidating the evidence of the cost - effectiveness of these roles .
1. Introduction 2. Background 3. Methods 4. Results 5. Discussion 6. Conclusions
the quest for improved quality of care and control of healthcare costs are important drivers in the decision to implement these roles . we conducted a systematic review to assess the evidence of cost - effectiveness of np and cns roles . , we conducted a systematic review of rcts of np and cns cost - effectiveness ( defined broadly to also include studies measuring health resource utilization ) entitled a systematic review of the cost - effectiveness of nurse practitioners and clinical nurse specialists : 1980 to july 2012 . we sought rcts of np and cns cost - effectiveness between january 1980 and july 2012 . studies were categorized into the following six groupings : np - outpatient , np - transition , np - inpatient , cns - outpatient , cns - transition , and cns - inpatient . the distribution of the 43 rcts across groupings was np - outpatient ( n = 11 ) , np - transition ( n = 5 ) , np - inpatient ( n = 2 ) , cns - outpatient ( n = 11 ) , cns - transition ( n = 13 ) , and cns - inpatient ( n = 1 ) . overall , six of the 11 rcts were judged to be at low risk of bias ( in other words , the methods were of high quality ) , four at moderate , and one at high risk of bias ( figure 2 ) . overall , five of the eleven studies were assessed at low risk , four at moderate risk , and two at high risk of bias ( figure 3 ) . of the nine studies that included subjective outcomes , eight were judged at low risk of bias as they used established , validated instruments , or blinded outcome assessment and one was at high risk of bias because the cns who delivered the intervention also collected data from both groups before and after the intervention via telephone interviews . overall , three of the thirteen trials were at low risk , eight at moderate risk , and two at a high risk of bias ( figure 3 ) . overall , we assessed that 18 of the 43 trials ( 42% ) were at low risk , 17 ( 39% ) at moderate risk , and 8 ( 19% ) at high risk of overall bias ( figures 2 and 3 ) . of the 43 rcts , 70% of the studies were conducted in the united states ( n = 30 ) and the remainder in four other countries : the united kingdom ( n = 6 ; 14% ) , the netherlands ( n = 4 ; 9% ) , canada ( n = 2 ; 5% ) , and china ( n = 1 ; 2% ) . the purpose of this paper was to report on the methodological strengths and threats to internal and external validity of rcts of np and cns cost - effectiveness . our assessment of the risk of bias revealed that about two - fifths ( n = 18 ; 42% ) of the 43 studies were at low risk of bias , close to the same number ( n = 17 ; 39% ) were at moderate risk of bias , and about one - fifth ( n = 8 ; 19% ) at high risk of bias . while blinding of participants is not possible in a study incorporating nps or cnss , a description of procedures used to blind outcome assessors and/or the description of valid outcome measures is needed to assess the quality of the study . strengths of our review include use of numerous strategies to identify all rcts in any language ( published or unpublished ) that met our inclusion criteria , contact with authors and international expert advisors when it was unclear whether a study met our inclusion criteria , use of current education and credentialing criteria to verify that the trial was indeed evaluating an np or cns , use of duplicate assessment by independent reviewers and a consensus process for every stage of the review , use of an internationally recognized and established tool to assess the overall risk of bias of each trial and contact with authors when additional information was required to make our assessment , use of an established tool ( quality of health economic studies ) to evaluate the health economic analysis in each study , use of grade to evaluate outcome - specific quality of evidence , consideration of external as well as internal validity , grouping of trials by type ( np or cns ) , setting ( inpatient , transition , or outpatient ) , and role ( alternative or complementary ) , and conducting meta - analyses whenever possible . this paper builds on the body of knowledge regarding quality of rcts of np and cns cost - effectiveness ( defined broadly to also include studies measuring health resource utilization ) . while almost half the rcts were found to be at low risk of bias , incomplete reporting of study methods and lack of details about np and cns education , experience , and roles make it difficult to fully evaluate the internal and external validity of studies of these roles . future studies that adhere to current standards for internal validity , such as cochrane risk of bias , consort [ 81 , 82 ] , and grade [ 34 , 35 ] , will contribute to a stronger body of evidence to address policy makers ' questions regarding the cost - effectiveness of np and cns roles .
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realizar a traduo e a validao cultural para a lngua portuguesa falada no brasil e determinar a concordncia e a confiabilidade dos instrumentos perme intensive care unit mobility score ( designado perme escore ) e icu mobility scale ( designada escala de mobilidade em uti , emu ) . os processos de traduo e adaptao cultural seguiram as seguintes etapas : preparao , traduo , reconciliao , sntese , traduo reversa , reviso , aprovao e pr - teste . aps esses processos , as verses em portugus dos dois instrumentos foram utilizadas por dois pesquisadores na avaliao de pacientes crticos em uti . o ndice kappa ponderado e a disposio grfica de bland - altman foram utilizados para verificar a concordncia entre os instrumentos . o coeficiente alfa de cronbach foi utilizado para verificar a confiabilidade entre as respostas dos avaliadores dentro de cada domnio dos instrumentos . a correlao entre os instrumentos foi verificada pelo teste de correlao de spearman a amostra foi composta por 103 pacientes , sendo a maioria homens ( n = 56 ; 54% ) , com mdia de idade = 52 18 anos . o principal motivo de internao nas utis foi insuficincia respiratria ( em 44% ) . os dois instrumentos apresentaram excelente concordncia interobservador ( > 0,90 ) e confiabilidade ( > 0,90 ) em todos os domnios . constatou - se um baixo vis interobservador na emu e no perme escore ( 0,048 0,350 e 0,06 0,73 , respectivamente ) . os ic95% para os mesmos instrumentos variaram , respectivamente , de 0,73 a 0,64 e de 1,50 a 1,36 , respectivamente . alm disso , verificou - se alta correlao positiva entre os dois instrumentos ( r = 0,941 ; p < 0,001 ) . early mobilization is part of the rehabilitation process for icu patients and is currently considered a way to prevent icu - acquired muscle weakness and worsening of physical function . some studies have associated the practice of early mobilization with decreased duration of mechanical ventilation and reduced length of icu and hospital stays , as well as with the promotion of functional improvement in icu survivors . there are currently 26 published instruments that purport to assess function in icu patients . of those , the functional independence measure and the barthel index have been used both in clinical practice and in research . however , few of these instruments were developed and validated to assess function and/or mobility in icu patients . in fact , only 6 were developed specifically for the icu setting and have published clinimetric data . these instruments are the physical function in intensive care test scored , the chelsea critical care physical assessment tool , the perme intensive care unit mobility score , the surgical intensive care unit optimal mobilization score , the icu mobility scale , and the functional status score for the icu . however , none of them are considered " gold standard " to assist multidisciplinary teams in quantifying the patient 's degree of mobility in a rapid , easy , and objective way . in addition , there are conditions extrinsic to the patient that affect the patient 's mobility in bed , such as the presence of access ports , lines , and chest tubes , which can be interpreted as a barrier to mobility , and this presence is not scored or considered in most instruments . taking into account such limitations , perme et al . developed a specific instrument for measuring improvement in mobility status , with a view to standardizing the evaluation of icu patients - the perme intensive care unit mobility score - hereafter referred to as the perme score , which is an instrument that objectively measures the mobility status of icu patients , starting with the ability to follow commands and culminating in the distance walked in two minutes . this mobility instrument is scored from 0 to 32 and comprises 15 items grouped into 7 categories : mental status ; potential mobility barriers ; functional strength ; bed mobility ; transfers ; gait ( with or without assistive devices ) ; and endurance . in this instrument , in contrast , a low score indicates low mobility and an increased need for assistance . with similar objectives to those of the perme score , hodgson et al . designated the icu mobility scale ( ims ) , this single - domain instrument is scored from 0 to 10 , with a score of 0 meaning low mobility ( interpreted as a patient being capable of performing only passive exercises in bed ) and a score of 10 meaning high mobility ( interpreted as a patient being capable of independent ambulation , without aid ) . the clinical utility of a tool has to be determined on the basis of a logical assessment of its validation , reliability , and applicability . in order to choose the best tool that can effectively assess the functional changes that will occur in the patient during the icu stay , health care professionals and researchers should consider which tools have clinimetric data that are more robust and appropriate for the functional outcomes that they want to analyze . to date , neither the perme score nor the ims has been appropriately translated and validated for use in brazil , taking into account the language and cultural differences . therefore , the objective of the present study was to translate these two icu mobility instruments into portuguese , creating versions that are cross - culturally adapted for use in brazil , and to determine inter - rater agreement and reliability for both versions . the present study was approved by the research ethics committee and the comisso de anlises de projetos de pesquisa ( cappesq , committee for the analysis of research projects ) of the university of so paulo school of medicine hospital das clnicas ( ruling no . the translated instruments were evaluated at two clinical icus ( 10 beds ) and one surgical icu ( 20 beds ) of the university of so paulo school of medicine hospital das clnicas central institute , in the city of so paulo , brazil , between april and june of 2015 . in the phase of instrument testing , since physical therapy evaluation was part of the routine care at those icus , being performed several times a day , the health care professionals who participated in the study , scoring and comparing the instruments , gave written informed consent , rather than the patients . the methodology for translating and cross - cultural adapting and validating the instruments followed a rigorous process , in accordance with current guidelines for the translation and cross - cultural adaptation of instruments . the following steps were performed : 1 ) preparation : the author of the project contacted the authors of the original instruments and obtained the rights to use , translate , and cross - culturally validate the instruments ; 2 ) translation from english into portuguese : the instruments were independently translated into the target language by two translators who were native speakers of portuguese and fluent in english , one of whom was familiar with the instruments and was aware of the objective of the present study and the other of whom was not familiar with the instruments ; 3 ) reconciliation and synthesis : the two initial portuguese - language versions were compared , item by item , with the original english - language versions by two physical therapists who were familiar with the instruments . any existing discrepancies were analyzed and discussed by three researchers , leading to the production of a second portuguese - language version for each of the two instruments ; 4 ) back - translation : the second portuguese - language version of each of the two instruments was sent to two translators who were native speakers of english and fluent in portuguese , neither of whom had contact with the original english - language versions , for back - translation ; 5 ) review and harmonization of the back - translation : the back - translated versions of the instruments were compared with their original english - language versions by a review committee comprising three researchers , in order to identify potential discrepancies and make the necessary adjustments , item by item , thereby producing the final back - translated version of each of the two instruments ; 6 ) approval from the authors of the original instruments : the final back - translated versions were sent to the authors of the original instruments for evaluation and comments on their consistency . an expert committee comprising three physical therapists analyzed the evaluations and comments of the authors of the original instruments , incorporating their suggestions , and thereby produced the final portuguese - language version of each of the two instruments ; and 7 ) pre - test : raters were trained on the administration and scoring of the final portuguese - language version of each of the two instruments . after training , a pilot study involving 40 patients was conducted in which two raters administered the two instruments following the methodology described in the original articles ; during these evaluations , the raters could discuss the scores and the difficulties in administering each instrument . data were collected by two raters , one of whom was a senior ( > 5 years of experience ) physical therapist ( rater 1 ) and one of whom was a junior ( < 5 years of experience ) physical therapist ( rater 2 ) . the two raters performed the scoring according to the rules of the two mobility instruments , which were administered after an initial evaluation made by the icu physical therapist . while one of the raters evaluated the patient , the other one only observed the procedure , without having any physical contact with the patient . each rater was responsible for 50% of the evaluations , and the functions of rater and observer were swapped every two patients . both raters completed the scoring sheet of the perme score and of the ims according to the highest activity level . in an attempt to avoid biases , the scoring sheets were completely separate and there was no communication between the raters . data on age , gender , reason for icu admission , mechanical ventilation use , vasoactive drug use , and score on the simplified acute physiology score 3 were collected to determine the clinical characteristics of the population . the sample size was calculated with a level of significance of 5% and a power of 80% , taking into account that the instruments could be equal ( 50% ) or not ( 50% ) . this is possible through the use of the bernoulli probability distribution ; in addition , we considered a delta of 10% , that is , the probability of equality could range from 40% to 60% , and found that a sample size of 100 individuals was required . statistical analysis was performed with the statistical package for the social sciences , version 17 ( spss inc . , the clinical characteristics of the patients were descriptively expressed as mean and standard deviation , median and interquartile range , or proportion , depending on data type and normality of distribution . the level of inter - rater agreement in the scoring of each instrument was determined using weighted kappa statistics and 95% ci . inter - rater reliability ( internal consistency ) in scoring was determined using cronbach 's alpha coefficient . for the perme score , inter - rater agreement and reliability were assessed individually for each domain ( items 1 to 15 ) . in addition , bland - altman plots were used to determine inter - rater agreement in total score ( sum of all domains ) both for the perme score and the ims . the proportions of evaluations with minimum scores ( floor effect ) and maximum scores ( ceiling effect ) were also calculated . finally , the kolmogorov - smirnov test and levene 's test were used to determine normality and homoscedasticity , respectively . since these principles were not met , spearman 's correlation coefficient was used to test the correlation between the two instruments . for this correlation analysis table 1 shows the clinical characteristics of the patients evaluated in the present study . slightly more than half ( 54% ; n = 56 ) of our sample was male and 67% ( n = 69 ) of the patients were admitted for clinical reasons , the most prevalent being respiratory disorders ( n = 45 ) . mechanical ventilation was present in 36% ( n = 37 ) of the cases , and vasoactive drug use occurred in 51% ( n = 53 ) . appendices show the portuguese - language versions of the ims and the perme score , both of which were translated from the original instruments . they are available online at http://www.jornaldepneumologia.com.br/detalhe_anexo.asp?id=47 table 1characteristics of the patients ( n = 103 ) . characteristicresultage , years 52 18male gender56 ( 54)saps3 66 reason for icu admission clinical69 ( 67)respiratory45 ( 44)renal9 ( 9)neurological8 ( 8)rheumatological5 ( 5)hepatic2 ( 2)surgical26 ( 25)gastroenterological11 ( 11)hepatic9 ( 9)cardiac3 ( 3)neurological2 ( 2)respiratory1 ( 1)trauma8 ( 8)vasoactive drug use53 ( 51)mechanical ventilation37 ( 36)duration of mechanical ventilation , days 4 tracheostomy9 ( 9)length of icu stay at the time of evaluation , days 5.5 saps3 : simplified acute physiology score 3 . table 2 shows the inter - rater agreement ( kappa statistics and 95% ci ) and reliability ( internal consistency , cronbach 's alpha coefficient ) for the ims and for each domain of the perme score . in addition , the inter - rater agreement for each item of the perme score ranged from 78% to 100% , and the inter - rater reliability ( cronbach 's alpha coefficient ) ranged from 88% to 100% , meaning that there was excellent inter - rater agreement and reliability for all items . figure 1 presents the bland - altman plots for the ims and for the total score on the perme score . inter - rater bias was low both for the ims ( 0.048 0.35 ) and the perme score ( 0.06 0.73 ) . the 95% cis ranged from 0.73 to 0.64 for the ims and from 1.50 to 1.36 for the perme score . table 2inter - rater reliability and agreement for the icu mobility scale ( ims ) and the perme icu mobility score.instrumentrater 1rater 2reliabilityagreement median [ min - max]median [ min - max](cronbach 's alpha coefficient)(95% ci)ims1 [ 0 - 10]1 [ 0 - 10]0.990.99 ( 0.98 - 0.99)perme icu mobility score mental status : item 12 [ 0 - 2]2 [ 0 - 2]0.970.94 ( 0.92 - 0.96)mental status : item 21 [ 0 - 1]1 [ 0 - 1]1.001.00potential barriers : item 31 [ 0 - 1]1 [ 0 - 1]1.001.00potential barriers : item 40 [ 0 - 1]0 [ 0 - 1]0.960.92 ( 0.88 - 0.94)potential barriers : item 50 [ 0 - 1]0 [ 0 - 1]0.970.95 ( 0.93 - 0.96)potential barriers : item 60 [ 0 - 1]0 [ 0 - 1]0.880.78 ( 0.70 - 0.85)functional strength : item 7 ( left leg)1 [ 0 - 1]1 [ 0 - 1]0.990.98 ( 0.97 - 0.98)functional strength : item 7 ( right leg)1 [ 0 - 1]1 [ 0 - 1]1.001.00functional strength : item 8 ( right arm)0 [ 0 - 1]0 [ 0 - 1]0.990.98 ( 0.97 - 0.98)functional strength : item 8 ( left arm)1 [ 0 - 1]1 [ 0 - 1]0.990.98 ( 0.97 - 0.98)bed mobility : item 90 [ 0 - 3]0 [ 0 - 3]0.980.97 ( 0.96 - 0.98)bed mobility : item 100 [ 0 - 3]0 [ 0 - 3]0.990.99 ( 0.99 - 0.99)transfers : item 110 [ 0 - 3]0 [ 0 - 3]0.980.97 ( 0.95 - 0.98)transfers : item 120 [ 0 - 3]0 [ 0 - 3]0.990.99 ( 0.99 - 0.99)transfers : item 130 [ 0 - 3]0 [ 0 - 3]0.990.99 ( 0.99 - 0.99)gait item 140 [ 0 - 3]0 [ 0 - 3]1.001.00endurance : item 150 [ 0 - 3]0 [ 0 - 3]0.990.99 ( 0.98 - 0.99)perme icu mobility score ( total ) 8 [ 0 - 32]8 [ 0 - 32 ] figure 1bland - altman plots of inter - rater score differences and mean scores for the icu mobility scale ( in a ) and the perme icu mobility score ( in b ) . ula : upper 95% limit of agreement ; and lla : lower 95% limit of agreement . the floor effect for the ims and the perme score was found to be 36% and 20% , respectively . the ceiling effect for the ims and the perme score was found to be 6% and 3% , respectively . the mean completion time for the scoring sheets was two minutes for the perme score and less than one minute for the ims . there was also a strong positive correlation between the use of the two instruments in the evaluation of the patients ( r = 0.941 ; p < 0.001 ) . for the present study , two instruments for evaluating mobility in icu patients were carefully translated into portuguese and validated for use in brazil , with technical and semantic equivalence having been achieved between the original versions and the portuguese - language versions . our results show that , in their versions adapted for use in brazil , both instruments showed high inter - rater agreement and reliability after a brief period of familiarization and training with them . in addition , there was a strong positive correlation between the two instruments . performing physical therapy in critically ill patients is currently in the spotlight , with numerous publications commenting on its prevalence and benefits . in this context , some instruments for evaluating function and mobility have been developed specifically for this population in order to improve physical therapy care in terms of performing and progressing the exercises in icu patients according to the mobility milestones that each individual can reach . to date , as mentioned above , no instrument for evaluating mobility in icu patients has been cross - culturally adapted for use in brazil . the careful translation and cross - cultural validation of such an instrument makes it possible for health care professionals nationwide to have access to a tool that can improve the quality of care to critically ill patients in the icu , as well as allowing the comparison of results across studies conducted in different countries . in their versions adapted for use in brazil , both the ims and the perme score showed excellent inter - rater agreement and reliability ( > 0.9 and > 0.9 for most domains ) . although the group who developed the perme score reported moderate to high reliability , our study reported an even higher level of inter - rater reliability . one possible explanation for this finding it that , in our study , the sample size was larger than those of the two previous studies . in view of the ease of learning and ease of use of the perme score , any disagreements in score had a lesser impact in our study than in the validation studies for the original english - language version of the instrument . our study also reported higher inter - rater reliability for the portuguese - language version of the ims than that reported in the validation study for the original english - language version of the instrument . in this case , the raters performed the scoring simultaneously but in an independent fashion , each being blinded to the scoring by the other rater , whereas in the study by hodgson et al . interestingly , the ims showed excellent inter - rater reliability and agreement , although its single domain is scored from 0 to 10 , an 11-point range . although it is a greater range than that in each domain of the perme score , the ims comprises mobility milestones that are clear and can easily be evaluated by the rater . item 6 in the perme score , " potential mobility barriers - continuous intravenous infusion " , was the one showing the lowest inter - rater reliability and agreement in our study . although some patients had venous access for administration of saline or drugs , in some cases , the infusion was not being administered at the time of evaluation , which may have confused the raters in scoring this item . however , we emphasize that , even with this likely difference , the reported inter - rater reliability and agreement were higher than 75 - 80% . the bland - altman plots showed high inter - rater score agreement and low inter - rater score variability for the ims and for the sum of all domains of the perme score . since the evaluation of each domain had shown excellent inter - rater agreement and reliability , the sum of all domains did not change this behavior . on the basis of the 95% cis , the maximum inter - rater difference was 2 points for the perme score and less than 1 point for the ims . it was expected that the scores on the two instruments would be highly correlated , given that both instruments measure the same property and therefore should show a similar behavior . finally , instrument floor and ceiling effects of 15% or less are considered acceptable . in our study , floor effects were found to be higher for the two instruments ( 20% and 36% for the perme escore and the ims , respectively ) . knowing that these instruments purport to assess functioning , floor effects were expected to be higher than normal , given the high incidence of sedated or unconscious patients in icus . although data collection was performed at three different icus in order to try to minimize this drawback , in 35% of the evaluations , the patients were unconscious or had lethargic responses , which made it impossible to perform more functional tasks or mobilizations at the time of evaluation . the lower floor effect of the perme score as compared with that of the ims can be explained by the scoring of the different domains of the former , such as patient cooperation , presence of pain , and presence of barriers to mobilization . although they are not mobilization aspects per se , they end up affecting the ease or difficulty of mobilization first , no clinimetric analyses were performed other than inter - rater reliability and agreement testing and inter - instrument correlation analysis . however , the primary objective of the present study was the cross - cultural validation of the instruments for use in brazil . we recognize that the applicability of the instruments and their predictive and concurrent validity have yet to be tested . although the clinimetric properties of the ims were tested against those of the physical function in intensive care test scored , the same was not true for the perme score . second , in our study , the two participating raters who were tested for inter - rater reliability and agreement were physical therapists . knowing that such health care professionals are directly related to the process of functional evaluation and early mobilization of critically ill patients , analysis of inter - rater reliability and agreement involving such professionals was fundamental . however , we can not state that the characteristics reported in the present study can be obtained by the other health care professionals who comprise multidisciplinary icu teams , such as nurses and physicians , and will eventually use the instruments . finally , the evaluations took place concurrently . therefore , aspects such as tone of voice , personal approach , and instruction of patients , all of which may differ from one health care professional to another , were not fully evaluated in our study . however , each rater was responsible for half of the evaluations , which to some extent minimized this effect . therefore , we conclude that the brazilian portuguese - language versions of the ims and the perme score were appropriately translated and cross - culturally validated , following strict guidelines , and can be used in brazil .
abstractobjective : to translate the perme intensive care unit mobility score and the icu mobility scale ( ims ) into portuguese , creating versions that are cross - culturally adapted for use in brazil , and to determine the interobserver agreement and reliability for both versions . methods : the processes of translation and cross - cultural validation consisted in the following : preparation , translation , reconciliation , synthesis , back - translation , review , approval , and pre - test . the portuguese - language versions of both instruments were then used by two researchers to evaluate critically ill icu patients . weighted kappa statistics and bland - altman plots were used in order to verify interobserver agreement for the two instruments . in each of the domains of the instruments , interobserver reliability was evaluated with cronbach 's alpha coefficient . the correlation between the instruments was assessed by spearman 's correlation test . results : the study sample comprised 103 patients-56 ( 54% ) of whom were male - with a mean age of 52 18 years . the main reason for icu admission ( in 44% ) was respiratory failure . both instruments showed excellent interobserver agreement ( > 0.90 ) and reliability ( > 0.90 ) in all domains . interobserver bias was low for the ims and the perme score ( 0.048 0.350 and 0.06 0.73 , respectively ) . the 95% cis for the same instruments ranged from 0.73 to 0.64 and 1.50 to 1.36 , respectively . there was also a strong positive correlation between the two instruments ( r = 0.941 ; p < 0.001 ) . conclusions : in their versions adapted for use in brazil , both instruments showed high interobserver agreement and reliability .
Objetivo: Mtodos: Resultados: Concluses: INTRODUCTION METHODS RESULTS DISCUSSION
therefore , the objective of the present study was to translate these two icu mobility instruments into portuguese , creating versions that are cross - culturally adapted for use in brazil , and to determine inter - rater agreement and reliability for both versions . any existing discrepancies were analyzed and discussed by three researchers , leading to the production of a second portuguese - language version for each of the two instruments ; 4 ) back - translation : the second portuguese - language version of each of the two instruments was sent to two translators who were native speakers of english and fluent in portuguese , neither of whom had contact with the original english - language versions , for back - translation ; 5 ) review and harmonization of the back - translation : the back - translated versions of the instruments were compared with their original english - language versions by a review committee comprising three researchers , in order to identify potential discrepancies and make the necessary adjustments , item by item , thereby producing the final back - translated version of each of the two instruments ; 6 ) approval from the authors of the original instruments : the final back - translated versions were sent to the authors of the original instruments for evaluation and comments on their consistency . the 95% cis ranged from 0.73 to 0.64 for the ims and from 1.50 to 1.36 for the perme score . table 2inter - rater reliability and agreement for the icu mobility scale ( ims ) and the perme icu mobility score.instrumentrater 1rater 2reliabilityagreement median [ min - max]median [ min - max](cronbach 's alpha coefficient)(95% ci)ims1 [ 0 - 10]1 [ 0 - 10]0.990.99 ( 0.98 - 0.99)perme icu mobility score mental status : item 12 [ 0 - 2]2 [ 0 - 2]0.970.94 ( 0.92 - 0.96)mental status : item 21 [ 0 - 1]1 [ 0 - 1]1.001.00potential barriers : item 31 [ 0 - 1]1 [ 0 - 1]1.001.00potential barriers : item 40 [ 0 - 1]0 [ 0 - 1]0.960.92 ( 0.88 - 0.94)potential barriers : item 50 [ 0 - 1]0 [ 0 - 1]0.970.95 ( 0.93 - 0.96)potential barriers : item 60 [ 0 - 1]0 [ 0 - 1]0.880.78 ( 0.70 - 0.85)functional strength : item 7 ( left leg)1 [ 0 - 1]1 [ 0 - 1]0.990.98 ( 0.97 - 0.98)functional strength : item 7 ( right leg)1 [ 0 - 1]1 [ 0 - 1]1.001.00functional strength : item 8 ( right arm)0 [ 0 - 1]0 [ 0 - 1]0.990.98 ( 0.97 - 0.98)functional strength : item 8 ( left arm)1 [ 0 - 1]1 [ 0 - 1]0.990.98 ( 0.97 - 0.98)bed mobility : item 90 [ 0 - 3]0 [ 0 - 3]0.980.97 ( 0.96 - 0.98)bed mobility : item 100 [ 0 - 3]0 [ 0 - 3]0.990.99 ( 0.99 - 0.99)transfers : item 110 [ 0 - 3]0 [ 0 - 3]0.980.97 ( 0.95 - 0.98)transfers : item 120 [ 0 - 3]0 [ 0 - 3]0.990.99 ( 0.99 - 0.99)transfers : item 130 [ 0 - 3]0 [ 0 - 3]0.990.99 ( 0.99 - 0.99)gait item 140 [ 0 - 3]0 [ 0 - 3]1.001.00endurance : item 150 [ 0 - 3]0 [ 0 - 3]0.990.99 ( 0.98 - 0.99)perme icu mobility score ( total ) 8 [ 0 - 32]8 [ 0 - 32 ] figure 1bland - altman plots of inter - rater score differences and mean scores for the icu mobility scale ( in a ) and the perme icu mobility score ( in b ) . there was also a strong positive correlation between the use of the two instruments in the evaluation of the patients ( r = 0.941 ; p < 0.001 ) . in their versions adapted for use in brazil , both the ims and the perme score showed excellent inter - rater agreement and reliability ( > 0.9 and > 0.9 for most domains ) .
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readers of this review should consider that many age - related processing speed findings reported in the literature are cross - sectional in nature , including the primary findings reviewed here . this is important because there are developmental ( laasonen et al . , 2002 ; deary et al . , 2010 ) and sampling factors that could affect the outcome of cross - sectional studies on processing speed in addition , the neural systems that are critical for understanding individual variation in cognition may differ across the lifespan ( zimmerman et al . , 2006 ) . there may be qualitative neurobiological changes that distinguish older adults from the oldest adults . our approach has been to include adults across the lifespan because cognitive aging appears to begin when people reach 2030 years of age ( salthouse , 2009 ) . for this reason , we consider many of the findings summarized in this review to reflect effects of chronological age . importantly , however , longitudinal findings of processing speed change have supported the robust cross - sectional findings of processing speed declines on cognitive function ( finkel et al . , 2009 ) . we anticipate that neural systems exhibiting cross - sectional processing speed changes will also exhibit robust within subject changes with processing speed . one early neurobiological explanation for age - related changes in processing speed focused on cortical declines in neuropil ( morris and mcmanus , 1991 ) . age - related differences in total gray matter volume have been related to differences in processing speed ( chee et al . , 2009 ) , which theoretically could result in degraded or noisy representations that would slow the recognition of stimuli and decision making . for example , primary auditory cortex neurons in the aged rat exhibit elevated excitation that has been related to a loss of gabaergic function ( caspary et al . , 2008 ) . these changes likely stem from reduced inhibitory interneuron input onto dendritic arbors that are diminished in the aged rat ( vaughan , 1977 ) and in older adult human auditory association cortex ( jacobs and scheibel , 1993 ) . similar changes could occur across perceptual , planning , and motor systems that support speeded task performance . thus , age - related changes in cognition are likely to be driven by changes in the connectivity of neural systems ( dickstein et al . , 2007 ) . another prominent explanation for processing speed declines is that a loss of myelination could slow conduction rates and therefore slow processing speed ( morris and mcmanus , 1991 ; fjell and walhovd , 2010 ) . age - related differences in whole brain white matter fractional anisotropy have been associated with processing speed ( coffey et al . , 2001 ; charlton et al . , 2006 ; there is a myelin - specific explanation that could account for age - related changes in processing speed . the number of oligodendrocytes does not decline with age in the rhesus monkey and have been observed to re - myelinate axons whose oligodendrocytes have died ( peters , 2009 ) . the consequence , however , is ( 1 ) a shortening in the lengths of the new myelin sheaths , ( 2 ) additional nodes of ranvier that could alter conductance speed , and ( 3 ) the potential for altered neural firing patterns that could further diminish the quality of neural signals affected by axonal and neuropil loss ( peters , 2009 ) . the early hypotheses for processing speed decline focused on global changes in gray and white matter , due in part to the available evidence . over the last 20 years there has been mounting evidence for regional specificity in the severity of age - related change in gray matter volume . 2009 ) , anterior insula , inferior frontal , superior frontal , medial frontal , and cerebellar regions exhibit some of the most pronounced changes with age when measured with automated ( good et al . , 2001 ; ; tisserand et al . , 2004 ; lemaitre et al . , 2005 ; chee et al . , 2009 ) and manual measures ( raz et al . , 1997 , 2005 , 2010 ; , 2005 ; raz and rodrigue , 2006 ) . given the substantial changes in frontal white matter that also occur with age ( bendlin et al . 2010 ) , these results suggest that frontal cortex is undergoing de - afferentation and subsequent decline with increasing age . we return to this premise below with respect to vascular explanations for changes in processing speed . one important point is that these findings could explain why frontal cortex exhibits fewer functional connections with other cortical and sub - cortical regions in older compared to younger adults ( meunier et al . , 2009 ) , with reduced control of sensory and perceptual processing . similarly , the cerebellar hemispheres could be considered as having domain general roles in the support of cognitive function , where the substantial aging effects on morphology ( raz et al . , because of their widespread influence on behavior , frontal and cerebellar regions are consistently related to a variety of speeded or timed cognitive tasks . frontal cortex has been a primary target for the study of age - related processing speed changes because of the relatively pronounced aging effects on frontal cortex morphology and interest in prominent hypotheses for cognitive aging such as the frontal aging hypothesis ( west , 1996 ) and the inhibition deficit hypothesis ( hasher and zacks , 1988 ; hasher et al . , 1999 ) . volumetric , voxel - based , and sulcal morphometry examinations of frontal gray matter have demonstrated associations with performance across a wide variety of cognitive tasks . importantly , the frontal gray matter findings appear to be dependent on the susceptibility of frontal white matter to age - related decline . regions of frontal gray matter that exhibit cross - sectional change with age and predict processing speed are presented in figure 1 from a sample of 42 adults that ranged in age from 19 to 79 years ( eckert et al . younger adults with elevated medial frontal and lateral frontal gray matter volume demonstrated faster processing speed compared to older adults with lower gray matter volume in these regions . this pattern of frontal results is consistent with volumetric and voxel - based gray matter associations with processing speed that were observed in a sample of 248 subjects ranging in age from 55 to 86 years ( chee et al . , 2009 ) . similarly , the volume of prefrontal cortex has been observed to predict a drawing speed ( kennedy and raz , 2005 ) , an estimate of perceptual comparison speed ( schretlen et al . , 2000 ) , and a measure of fluid intelligence in older adults ( raz et al . , 2008 ; kennedy et al . , 2009 ) frontal sulcal span , an indirect measure of gray matter declines in frontal cortex , has also been associated with processing speed ( kochunov et al . , 2010 ) . voxel - based gray matter variation ( modulated ) predicts connections simple processing speed performance ( a ) . these results are not significant at the p < 0.001 ( uncorrected ) level after controlling for age , thereby demonstrating the dependence of the results on age . posterior cingulate , cuneus , cerebellar , and medial temporal regions exhibited weak associations with processing speed ( p < 0.05 , uncorrected ) after controlling for age , suggesting that age may exaggerate normal variation in brain morphology and processing speed . sbm ( please see figure 3 for an sbm summary ) demonstrates unique patterns of frontal and cerebellar gray matter variance ( b , c ) that each accounted for different distributions of the processing speed results [ ( d ) : p < 0.001 after controlling for variance representing either the frontal or the cerebellar independent components ] . importantly , by controlling for either gray matter component we can see that there are at least two sources that contribute to the association between gray matter and processing speed in this sample . in a separate analysis , these component specific effects were not significant using a p < 0.001 threshold when age was covaried . there were weak p < 0.05 associations with processing speed in cingulate and cerebellar regions after controlling for either component and age , again suggesting the influence of additive developmental factors ( deary et al . , 2010 ) . individual variation in prefrontal gray matter volume among older adults is strongly related to frontal white matter volume ( raz et al . , 2003 ) . perhaps then it is not surprising that multiple measures of frontal white matter morphology or integrity have been associated with processing speed , including transverse relaxation rate ( bartzokis et al . , 2010 ) , midline genu area of the corpus callosum ( jokinen et al . , 2007 ) , and fractional anisotropy and/or mean diffusivity ( deary et al , 2007 ; bucur et al . , 2008 ; kennedy and raz , 2009a ; schiavone et al . , 2009 ; bendlin et al . , 2010 ; an example of the robust association between fractional anisotropy in frontal lobe white matter and processing speed is presented in figure 2 . individual variation in frontal lobe fractional anisotropy has been related to processing speed in healthy young adults ( turken et al . , 2008 ) , suggesting the interesting possibility that aging exaggerates normal structure function associations ( harris et al . , 2009 ) . voxel - based analysis of fractional anisotropy data demonstrates a right frontal association with connections simple processing speed ( p < 0.01 , family - wise discovery rate corrected ) in 36 of the 42 subjects whose data were included in the figure 1 analyses . the graph shows the association between frontal fractional anisotropy and the frontal gray matter component from figure 1 , the presence of white matter hyperintensities among people with low fractional anisotropy and low frontal gray matter ( circle color : no hyperintensities blue ; some hyperintensities orange ; pronounced hyperintensities green ) , and that ps is related to each of these variables ( larger circle size reflects faster processing speed ) . ( the tspoon approach designed to control for smoothing kernel effects , lee et al . , 2009 , was used to process the 2 mm 2 mm in plane resolution images that were collected on a philips 3 t with 32 directions using an 8-channel phased array head coil . the b0 and fa data were co - registered to each subject 's t1 image and then normalized into study - specific space using the dartel parameters obtained for the t1 images . ) a large body of literature implicates white matter pathology in the cognitive slowing of older adults ( gunning - dixon and raz , 2000 ; kennedy and raz , 2009b ; vernooij et al . , 2009 ; kochunov et al . , 2010 ; vidal et al . , 2010 ; please see gunning - dixon et al . white matter hyperintensities , a common age - related finding in periventricular regions ( almkvist et al . , 1992 ; soderlund et al . , 2003 ; cook et al . , 2004 ) , have a gradual impact on cognition over time ( silbert et al . , 2009 ) and appear to affect regional gray matter morphology even before overt cognitive changes are observable ( ota et al . , 2010 ) . they are generally considered to be a sign of cerebral small vessel disease and the severity of the hyperintensities has been related to hypertension ( brickman et al . , 2010 ) , blood pressure ( kochunov et al . , 2010 ; kuo et al . , 2010 ) , coronary artery calcification ( vidal et al . , 2010 ) , and endothelial dependent vasodilation ( hoth et al . , 2007 ) . white matter pathology has been observed independently of effects related to systolic blood pressure and cholesterol ( chowdhury et al . age - related changes in pericytes , cells that contribute to the blood brain barrier , may provide an explanation for both hypoxia related damage and neurotoxic damage stemming from microvessel leakage . pericyte deficient mice exhibit progressive loss of cerebral perfusion and a loss of dendritic spines that has been attributed to the leakage of damaging proteins through the blood brain barrier and subsequent neuronal loss ( bell et al . , 2010 ) . both conditions would produce an inflammatory response and could explain why variation in interleukin-6 has been associated with changes in macaque frontal white matter ( willette et al . , 2010 ) . regardless of the mechanism , damage to periventricular white matter appears to disrupt pathways projecting to and from frontal lobe regions that support performance on processing speed tasks . cerebellar morphology also undergoes robust age - related structural change but has not received the same experimental attention as frontal cortex with respect to age - related changes in processing speed . this is due , in part , to image processing choices that exclude the cerebellum and because the prominent theories of cognitive aging ( the frontal aging hypothesis and the inhibition deficit hypothesis ) focus on frontal cortex . while there may be a publication bias related to negative findings , relations between cerebellar morphology and processing speed have been observed and provide support for a fronto - cerebellar aging hypothesis ( hogan , 2004 ) . hogan ( 2004 ) proposed that changes in feedback and feedforward cerebro - cerebellar interaction result in greater within subject variation in task performance , reduced behavioral automaticity , and increased demands on cognitive control , as well as reduced working memory capacity . this hypothesis is based on normative and patient evidence linking the cerebellum to cognitive and perceptual functions ( e.g. , eckert et al . , 2003 ; chen and desmond , 2005 ; desmond et al . , 2005 ; steinlin , 2007 ) and its distinct patterns of structural and functional connectivity with motor , sensory , and attention - related cortical regions ( e.g. , kelly and strick , 2003 ; habas et al . , 2009 ; krienen and buckner , 2009 ; schmahmann , 2010 ) . in particular , limits on the cerebellum 's role in modifying motor function in response to sensory and perceptual feedback ( ivry , 1997 ) could affect performance on perceptual and motor processing speed tasks . cross - sectional age - related changes in cerebellar morphology have been associated with processing speed ( maclullich et al . , 2004 ; paul et al . , 2009 ; eckert et al . , 2010 ) and a measure of general intelligence among older adults ( hogan et al . , 2011 ) . in particular , we observed that processing speed was predicted by regional cerebellar gray matter volume ( figure 1 ) . this voxel - based effect was present throughout the vermis and cerebellar hemispheres ( superior to the horizontal fissure ) . the vermis finding is spatially consistent with a finding that the midline surface area of vermis ( declive , folium , tuber region ) predicted age - related digit - symbol substitution test performance , which was significant even after controlling for an estimate of intracranial volume ( maclullich et al . , 2004 ) . in addition , a volumetric measure of the same vermis region and total cerebellar gray matter volume were significantly related to age - related changes in trail making test ( alternating condition ) performance ( paul et al . , 2009 ) . in the paul et al . ( 2009 ) study , however , the vermis measure did not significantly predict trail making task performance after accounting for a measure of prefrontal volume ( brodmann areas 9 and 46 ) . the specificity of these frontal and cerebellar findings is addressed below . as suggested by hogan ( 2004 ) , the processing speed and cerebellar findings could be explained by problems with sensory motor integration . for example , a history of falling and slow processing speed has been associated with delayed horizontal saccades during the initiation of a footlift for a stepping task ( greany and di fabio , 2008 ) . in addition , left cerebellar volume has been related to gait speed in older adults ( rosano et al . , 2007 ; although see rosano et al . , 2008 ) and right cerebellar hemisphere volume has been associated with balance difficulty among older adults ( rosano et al . , 2007 ) . a sensory motor integration explanation could also explain why cerebellar volumes have been associated with perceptual motor skill as measured by total time on target for a pursuit rotor task ( raz et al . , 2000 ) , although not for a timed mirror tracing task ( kennedy and raz , 2005 ) . age - related differences in cerebellar volume has also been significantly correlated with associative learning as measured by eye - blink conditioning ( woodruff - pak et al . , 2000 , 2001 ) . together , these findings suggest that the behavioral effects of cerebellar aging are variable across subjects , perhaps because of individual variability in developmental factors ( deary et al . , 2010 ) , and may have widespread effects on a variety of cerebellar functions . based on the perceptual motor skill associations with cerebellar morphology , we asked whether performance on the connections processing speed test could be influenced by increasing performance across the two trials of each sub - test [ numbers only , letters only , numbers letters ( starting with numbers ) , letters numbers ( starting with numbers ) ] . there was no evidence indicating that people with elevated cerebellar gray matter exhibited the greatest improvement in performance across the trials . people with low cerebellar gray matter volume were more likely to improve across the first to second trials of the alternating letters numbers condition compared to people with elevated cerebellar gray matter volume ( p < 0.05 uncorrected ; no significant relations were seen for the other connections conditions ) . this finding suggests that the processing speed and cerebellar relation was not due to a specific learning effect , however it is possible that we could not detect evidence of procedural learning because of ( 1 ) the balanced order in which the sub - tests are administered or ( 2 ) ceiling effects on the first trial of the connections sub - tests in people with the greatest cerebellar gray matter volume . importantly , however , these results are consistent with evidence that variation in cerebellar morphology is related to the initial stages of skill acquisition ( raz , 2000 ) . returning to the hogan ( 2004 ) cerebellar hypothesis for cognitive aging , we also wondered whether variation in performance across the connections tests could be related to age and cerebellar morphology . a measure of each subject 's variance in processing speed was obtained from the sd of subject z scores from the connections sub - tests , where the z score was based on the mean performance of the sample for each sub - test . rather than greater variability among the oldest subjects , an inverted u function was observed where by middle - aged subjects exhibited the most variable connections performance ( age connections variance ; quadratic r = 0.41 , p < 0.05 ; linear r = 0.05 , ns ) . there were not clear linear or non - linear relations with cerebellar morphology and the connections variance measure , but it is intriguing to consider that elevated variance in performance among middle - aged subjects may be an early behavioral marker for neurobiological declines that slow processing speed and cognition . finally , there are questions about the etiological factors that drive age - related cerebellar changes and that would contribute to slowed processing speed . we observed that cerebellar associations with processing speed occurred independently of periventricular white matter hyperintensities , suggesting that cerebral small vessel disease was not a primary factor for changes in cerebellar morphology in our sample . similarly , white matter hyperintensities did not account for the association between cerebellar volume and gait speed observed in the rosano et al . the results of other studies , however , have indicated that primary declines in prefrontal cortex contribute to cerebellar changes in morphology ( bugalho et al . , 2007 ) there is the strong likelihood that cerebellar and frontal regions vary in susceptibility to different age - related risk factors . the high metabolic demands of cerebellar purkinje cells , which demonstrate a substantial loss in number with age ( hall et al . , 1975 ; andersen et al . , 2003 ) , may increase the risk for cell death due to oxidative stress . indeed , oxidative stress is one factor contributing to cerebellar aging in mice ( lee et al . , 2000 ) . the results of human imaging studies indicate that leptin may serve to protect against oxidative stress based on evidence that plasma concentration of leptin was associated with elevated cerebellar and hippocampal gray matter in middle - aged and older adults ( narita et al . , 2009 ) and evidence that leptin treatment increases cerebellar gray matter in leptin deficient patients ( matochik et al . , 2005 ) . these findings , together with developmental findings in c57 mice showing that leptin promotes cerebellar purkinje cell survival and neurite outgrowth ( oldreive et al . , 2008 ) , suggest the interesting possibility that leptin limits the impact of oxidative stress on cerebellar purkinje cells whose high metabolic rate places them at risk for age - related declines as it does for in vitro hippocampal purkinje cells ( guo et al . , 2008 ) . this leptin explanation for cerebellar health is just one example for what is likely a multifactorial aging process in the cerebellum . one early neurobiological explanation for age - related changes in processing speed focused on cortical declines in neuropil ( morris and mcmanus , 1991 ) . age - related differences in total gray matter volume have been related to differences in processing speed ( chee et al . , 2009 ) , which theoretically could result in degraded or noisy representations that would slow the recognition of stimuli and decision making . for example , primary auditory cortex neurons in the aged rat exhibit elevated excitation that has been related to a loss of gabaergic function ( caspary et al . , 2008 ) . these changes likely stem from reduced inhibitory interneuron input onto dendritic arbors that are diminished in the aged rat ( vaughan , 1977 ) and in older adult human auditory association cortex ( jacobs and scheibel , 1993 ) . similar changes could occur across perceptual , planning , and motor systems that support speeded task performance . thus , age - related changes in cognition are likely to be driven by changes in the connectivity of neural systems ( dickstein et al . , 2007 ) . another prominent explanation for processing speed declines is that a loss of myelination could slow conduction rates and therefore slow processing speed ( morris and mcmanus , 1991 ; fjell and walhovd , 2010 ) . age - related differences in whole brain white matter fractional anisotropy have been associated with processing speed ( coffey et al . , 2001 ; charlton et al . , 2006 ; vernooij et al . , 2008 ; schiavone et al . , 2009 ) , although this variation likely reflect a loss of myelinated axons . there is a myelin - specific explanation that could account for age - related changes in processing speed . the number of oligodendrocytes does not decline with age in the rhesus monkey and have been observed to re - myelinate axons whose oligodendrocytes have died ( peters , 2009 ) . the consequence , however , is ( 1 ) a shortening in the lengths of the new myelin sheaths , ( 2 ) additional nodes of ranvier that could alter conductance speed , and ( 3 ) the potential for altered neural firing patterns that could further diminish the quality of neural signals affected by axonal and neuropil loss ( peters , 2009 ) . the early hypotheses for processing speed decline focused on global changes in gray and white matter , due in part to the available evidence . over the last 20 years there has been mounting evidence for regional specificity in the severity of age - related change in gray matter volume . 2009 ) , anterior insula , inferior frontal , superior frontal , medial frontal , and cerebellar regions exhibit some of the most pronounced changes with age when measured with automated ( good et al . , 2001 ; , 2004 ; tisserand et al . , 2004 ; lemaitre et al . , 2005 ; chee et al . , 2009 ) and manual measures ( raz et al . , 1997 , 2005 , 2010 ; jernigan et al . , 2001 ; tisserand et al . , 2002 ; decarli et al . , 2005 ; raz and rodrigue , 2006 ) . given the substantial changes in frontal white matter that also occur with age ( bendlin et al . 2010 ) , these results suggest that frontal cortex is undergoing de - afferentation and subsequent decline with increasing age . we return to this premise below with respect to vascular explanations for changes in processing speed . one important point is that these findings could explain why frontal cortex exhibits fewer functional connections with other cortical and sub - cortical regions in older compared to younger adults ( meunier et al . , 2009 ) , with reduced control of sensory and perceptual processing . similarly , the cerebellar hemispheres could be considered as having domain general roles in the support of cognitive function , where the substantial aging effects on morphology ( raz et al . , 2010 ) could affect perceptual and cognitive task performance . perhaps because of their widespread influence on behavior , frontal and cerebellar regions are consistently related to a variety of speeded or timed cognitive tasks . frontal cortex has been a primary target for the study of age - related processing speed changes because of the relatively pronounced aging effects on frontal cortex morphology and interest in prominent hypotheses for cognitive aging such as the frontal aging hypothesis ( west , 1996 ) and the inhibition deficit hypothesis ( hasher and zacks , 1988 ; hasher et al . , 1999 ) . volumetric , voxel - based , and sulcal morphometry examinations of frontal gray matter have demonstrated associations with performance across a wide variety of cognitive tasks . importantly , the frontal gray matter findings appear to be dependent on the susceptibility of frontal white matter to age - related decline . regions of frontal gray matter that exhibit cross - sectional change with age and predict processing speed are presented in figure 1 from a sample of 42 adults that ranged in age from 19 to 79 years ( eckert et al . , younger adults with elevated medial frontal and lateral frontal gray matter volume demonstrated faster processing speed compared to older adults with lower gray matter volume in these regions . this pattern of frontal results is consistent with volumetric and voxel - based gray matter associations with processing speed that were observed in a sample of 248 subjects ranging in age from 55 to 86 years ( chee et al . , 2009 ) . similarly , the volume of prefrontal cortex has been observed to predict a drawing speed ( kennedy and raz , 2005 ) , an estimate of perceptual comparison speed ( schretlen et al . , 2000 ) , and a measure of fluid intelligence in older adults ( raz et al . , 2008 ; kennedy et al . , frontal sulcal span , an indirect measure of gray matter declines in frontal cortex , has also been associated with processing speed ( kochunov et al . , 2010 ) . voxel - based gray matter variation ( modulated ) predicts connections simple processing speed performance ( a ) . these results are not significant at the p < 0.001 ( uncorrected ) level after controlling for age , thereby demonstrating the dependence of the results on age . posterior cingulate , cuneus , cerebellar , and medial temporal regions exhibited weak associations with processing speed ( p < 0.05 , uncorrected ) after controlling for age , suggesting that age may exaggerate normal variation in brain morphology and processing speed . sbm ( please see figure 3 for an sbm summary ) demonstrates unique patterns of frontal and cerebellar gray matter variance ( b , c ) that each accounted for different distributions of the processing speed results [ ( d ) : p < 0.001 after controlling for variance representing either the frontal or the cerebellar independent components ] . importantly , by controlling for either gray matter component we can see that there are at least two sources that contribute to the association between gray matter and processing speed in this sample . in a separate analysis , these component specific effects were not significant using a p < 0.001 threshold when age was covaried . there were weak p < 0.05 associations with processing speed in cingulate and cerebellar regions after controlling for either component and age , again suggesting the influence of additive developmental factors ( deary et al . , 2010 ) . individual variation in prefrontal gray matter volume among older adults is strongly related to frontal white matter volume ( raz et al . , 2003 ) . perhaps then it is not surprising that multiple measures of frontal white matter morphology or integrity have been associated with processing speed , including transverse relaxation rate ( bartzokis et al . , 2010 ) , midline genu area of the corpus callosum ( jokinen et al . , 2007 ) , and fractional anisotropy and/or mean diffusivity ( deary et al kennedy and raz , 2009a ; schiavone et al . , 2009 ; bendlin et al . an example of the robust association between fractional anisotropy in frontal lobe white matter and processing speed is presented in figure 2 . individual variation in frontal lobe fractional anisotropy has been related to processing speed in healthy young adults ( turken et al . , 2008 ) , suggesting the interesting possibility that aging exaggerates normal structure function associations ( harris et al . , 2009 ) . voxel - based analysis of fractional anisotropy data demonstrates a right frontal association with connections simple processing speed ( p < 0.01 , family - wise discovery rate corrected ) in 36 of the 42 subjects whose data were included in the figure 1 analyses . the graph shows the association between frontal fractional anisotropy and the frontal gray matter component from figure 1 , the presence of white matter hyperintensities among people with low fractional anisotropy and low frontal gray matter ( circle color : no hyperintensities blue ; some hyperintensities orange ; pronounced hyperintensities green ) , and that ps is related to each of these variables ( larger circle size reflects faster processing speed ) . ( the tspoon approach designed to control for smoothing kernel effects , lee et al . , 2009 , was used to process the 2 mm 2 mm in plane resolution images that were collected on a philips 3 t with 32 directions using an 8-channel phased array head coil . the b0 and fa data were co - registered to each subject 's t1 image and then normalized into study - specific space using the dartel parameters obtained for the t1 images . ) a large body of literature implicates white matter pathology in the cognitive slowing of older adults ( gunning - dixon and raz , 2000 ; kennedy and raz , 2009b ; vernooij et al . , 2009 ; kochunov et al . , 2010 ; vidal et al . , 2010 ; please see gunning - dixon et al . , 2009 for additional review ) . white matter hyperintensities , a common age - related finding in periventricular regions ( almkvist et al . , 1992 ; soderlund et al . , 2003 ; cook et al . , 2004 ) , have a gradual impact on cognition over time ( silbert et al . , 2009 ) and appear to affect regional gray matter morphology even before overt cognitive changes are observable ( ota et al . , 2010 ) . they are generally considered to be a sign of cerebral small vessel disease and the severity of the hyperintensities has been related to hypertension ( brickman et al . , 2010 ) , blood pressure ( kochunov et al . , 2010 ; kuo et al . , 2010 ) , coronary artery calcification ( vidal et al . , 2010 ) , and endothelial dependent vasodilation ( hoth et al . , 2007 ) . white matter pathology has been observed independently of effects related to systolic blood pressure and cholesterol ( chowdhury et al . age - related changes in pericytes , cells that contribute to the blood brain barrier , may provide an explanation for both hypoxia related damage and neurotoxic damage stemming from microvessel leakage . pericyte deficient mice exhibit progressive loss of cerebral perfusion and a loss of dendritic spines that has been attributed to the leakage of damaging proteins through the blood brain barrier and subsequent neuronal loss ( bell et al . , 2010 ) . both conditions would produce an inflammatory response and could explain why variation in interleukin-6 has been associated with changes in macaque frontal white matter ( willette et al . , 2010 ) . regardless of the mechanism , damage to periventricular white matter appears to disrupt pathways projecting to and from frontal lobe regions that support performance on processing speed tasks . cerebellar morphology also undergoes robust age - related structural change but has not received the same experimental attention as frontal cortex with respect to age - related changes in processing speed . this is due , in part , to image processing choices that exclude the cerebellum and because the prominent theories of cognitive aging ( the frontal aging hypothesis and the inhibition deficit hypothesis ) focus on frontal cortex . while there may be a publication bias related to negative findings , relations between cerebellar morphology and processing speed have been observed and provide support for a fronto - cerebellar aging hypothesis ( hogan , 2004 ) . hogan ( 2004 ) proposed that changes in feedback and feedforward cerebro - cerebellar interaction result in greater within subject variation in task performance , reduced behavioral automaticity , and increased demands on cognitive control , as well as reduced working memory capacity . this hypothesis is based on normative and patient evidence linking the cerebellum to cognitive and perceptual functions ( e.g. , eckert et al . , 2003 ; chen and desmond , 2005 ; desmond et al . , 2005 ; steinlin , 2007 ) and its distinct patterns of structural and functional connectivity with motor , sensory , and attention - related cortical regions ( e.g. , kelly and strick , 2003 ; habas et al . , 2009 ; krienen and buckner , 2009 ; schmahmann , 2010 ) . in particular , limits on the cerebellum 's role in modifying motor function in response to sensory and perceptual feedback ( ivry , 1997 ) could affect performance on perceptual and motor processing speed tasks . cross - sectional age - related changes in cerebellar morphology have been associated with processing speed ( maclullich et al . , 2004 ; paul et al . , 2009 ; eckert et al . , 2010 ) and a measure of general intelligence among older adults ( hogan et al . , 2011 ) . in particular , we observed that processing speed was predicted by regional cerebellar gray matter volume ( figure 1 ) . this voxel - based effect was present throughout the vermis and cerebellar hemispheres ( superior to the horizontal fissure ) . the vermis finding is spatially consistent with a finding that the midline surface area of vermis ( declive , folium , tuber region ) predicted age - related digit - symbol substitution test performance , which was significant even after controlling for an estimate of intracranial volume ( maclullich et al . , 2004 ) . in addition , a volumetric measure of the same vermis region and total cerebellar gray matter volume were significantly related to age - related changes in trail making test ( alternating condition ) performance ( paul et al . , 2009 ) . ( 2009 ) study , however , the vermis measure did not significantly predict trail making task performance after accounting for a measure of prefrontal volume ( brodmann areas 9 and 46 ) . the specificity of these frontal and cerebellar findings is addressed below . as suggested by hogan ( 2004 ) , the processing speed and cerebellar findings could be explained by problems with sensory motor integration . for example , a history of falling and slow processing speed has been associated with delayed horizontal saccades during the initiation of a footlift for a stepping task ( greany and di fabio , 2008 ) . in addition , left cerebellar volume has been related to gait speed in older adults ( rosano et al . , 2007 ; although see rosano et al . , 2008 ) and right cerebellar hemisphere volume has been associated with balance difficulty among older adults ( rosano et al . , 2007 ) . a sensory motor integration explanation could also explain why cerebellar volumes have been associated with perceptual motor skill as measured by total time on target for a pursuit rotor task ( raz et al . , 2000 ) , although not for a timed mirror tracing task ( kennedy and raz , 2005 ) . age - related differences in cerebellar volume has also been significantly correlated with associative learning as measured by eye - blink conditioning ( woodruff - pak et al . together , these findings suggest that the behavioral effects of cerebellar aging are variable across subjects , perhaps because of individual variability in developmental factors ( deary et al . , 2010 ) , and may have widespread effects on a variety of cerebellar functions . based on the perceptual motor skill associations with cerebellar morphology , we asked whether performance on the connections processing speed test could be influenced by increasing performance across the two trials of each sub - test [ numbers only , letters only , numbers letters ( starting with numbers ) , letters numbers ( starting with numbers ) ] . there was no evidence indicating that people with elevated cerebellar gray matter exhibited the greatest improvement in performance across the trials . people with low cerebellar gray matter volume were more likely to improve across the first to second trials of the alternating letters numbers condition compared to people with elevated cerebellar gray matter volume ( p < 0.05 uncorrected ; no significant relations were seen for the other connections conditions ) . this finding suggests that the processing speed and cerebellar relation was not due to a specific learning effect , however it is possible that we could not detect evidence of procedural learning because of ( 1 ) the balanced order in which the sub - tests are administered or ( 2 ) ceiling effects on the first trial of the connections sub - tests in people with the greatest cerebellar gray matter volume . importantly , however , these results are consistent with evidence that variation in cerebellar morphology is related to the initial stages of skill acquisition ( raz , 2000 ) . returning to the hogan ( 2004 ) cerebellar hypothesis for cognitive aging , we also wondered whether variation in performance across the connections tests could be related to age and cerebellar morphology . a measure of each subject 's variance in processing speed was obtained from the sd of subject z scores from the connections sub - tests , where the z score was based on the mean performance of the sample for each sub - test . rather than greater variability among the oldest subjects , an inverted u function was observed where by middle - aged subjects exhibited the most variable connections performance ( age connections variance ; quadratic r = 0.41 , p < 0.05 ; linear r = 0.05 , ns ) . there were not clear linear or non - linear relations with cerebellar morphology and the connections variance measure , but it is intriguing to consider that elevated variance in performance among middle - aged subjects may be an early behavioral marker for neurobiological declines that slow processing speed and cognition . finally , there are questions about the etiological factors that drive age - related cerebellar changes and that would contribute to slowed processing speed . we observed that cerebellar associations with processing speed occurred independently of periventricular white matter hyperintensities , suggesting that cerebral small vessel disease was not a primary factor for changes in cerebellar morphology in our sample . similarly , white matter hyperintensities did not account for the association between cerebellar volume and gait speed observed in the rosano et al . the results of other studies , however , have indicated that primary declines in prefrontal cortex contribute to cerebellar changes in morphology ( bugalho et al . , 2007 ) there is the strong likelihood that cerebellar and frontal regions vary in susceptibility to different age - related risk factors . the high metabolic demands of cerebellar purkinje cells , which demonstrate a substantial loss in number with age ( hall et al . , 1975 ; andersen et al . , 2003 ) , may increase the risk for cell death due to oxidative stress . indeed , oxidative stress is one factor contributing to cerebellar aging in mice ( lee et al . , 2000 ) . the results of human imaging studies indicate that leptin may serve to protect against oxidative stress based on evidence that plasma concentration of leptin was associated with elevated cerebellar and hippocampal gray matter in middle - aged and older adults ( narita et al . , 2009 ) and evidence that leptin treatment increases cerebellar gray matter in leptin deficient patients ( matochik et al . , 2005 ) . these findings , together with developmental findings in c57 mice showing that leptin promotes cerebellar purkinje cell survival and neurite outgrowth ( oldreive et al . , 2008 ) , suggest the interesting possibility that leptin limits the impact of oxidative stress on cerebellar purkinje cells whose high metabolic rate places them at risk for age - related declines as it does for in vitro hippocampal purkinje cells ( guo et al . , this leptin explanation for cerebellar health is just one example for what is likely a multifactorial aging process in the cerebellum . the review above suggests that age - related changes across multiple systems could affect processing speed . the frontal findings , in particular , could explain why changes in auditory gap detection ( harris et al . , 2010 ) and tactile temporal order thresholds ( craig et al . , 2010 ) it is difficult to determine , however , from the results presented in figure 1a whether widespread anatomical effects stem from a common mechanism or whether unique neural systems are affected . this is a particularly significant problem for cross - sectional chronological aging studies where disentangling neurobiological patterns of aging can be difficult . our approach to addressing this challenge has been to use ica or source based morphometry to identify unique patterns of structural covariance in the data that uniquely predict age - related differences in processing speed and that might provide insight into the affected neural systems . independent component analysis of functional imaging data is helping to identify consistent patterns of neural activity across experiments that are thought to reflect distinct neural systems for focused and scanning attention , motor function , or auditory and visual processing ( beckmann et al . , 2005 ; eckert et al . , 2008a ) . indeed , this type of analysis may help to identify atypical coherence in neural systems that underlie changes in processing speed . for example , the ability to switch between functional networks that are thought to represent focused attention to a task ( dorsal attention network ; sridharan et al . , 2007 ) and self - referential thinking ( default mode network ; andrews - hanna et al . , 2010 ) has been related to within subject variability in children 's task performance ( kelly et al . , 2008 ) , appears to be impaired in older adults ( grady et al . , 2006 ) , and may also relate to variable processing speed performance in middle - aged adults . independent component analysis of structural mri data has been described as source based morphometry because unique patterns of variation are thought to have common underlying influences or sources ( xu et al . , 2009 ; specific regions of gray matter may covary across subjects because of variation in fiber pathways connecting distant areas , the amount of neuropil , vascular support , or image artifact . for example , this technique identified periventricular white matter regions where white matter hyperintensities were observed ( figure 6 in eckert et al . , 2010 ) . in addition , the component included regions where there was less gray matter in frontal cortex ( ic7 , figure 3 ) . this result suggested that a common source , presumably cerebral small vessel disease , was affecting gray and white matter segmentation in people with reduced frontal gray matter and lower fractional anisotropy in frontal white matter . source based morphometry or ica of gray matter probability images across 42 subjects . each subject 's t1-weighted image is segmented to generate a gray matter probability image that is normalized to a common coordinate space and smoothed . the degree to which each independent component ( ic ) or unique pattern of variance can be compared to the other ics with a variety of metrics , including an estimate of similarity space . the brain regions that contribute most to each component can be identified by displaying each component with scaled intensity values ( z score = 13 above ) . each ic also has an inverse component or regions that are negatively correlated with regions in the ic . an example is presented for ic7 where white matter hyperintensity related segmentation error ( yellow arrow ) is identified by ica and is inversely correlated with decreased frontal gray matter ( e.g. , anterior cingulate , anterior insula , and superior frontal sulcus regions represented by hot signal intensities above ) . this is important because these results suggest there are independent age - based sources that affect gray matter variation in cerebellar ( ic4 ) and frontal ( ic7 ) regions that are associated with processing speed . the source based morphometry analysis also identified six other independent spatial patterns of gray matter covariance ( figure 3 ) . we identified multiple patterns of gray matter variation that were related to age but only some were related to processing speed . one component coincided spatially with the default mode network ( ic1 ) , which was nearly perfectly correlated with total brain volume ( r = 0.90 , p < 0.001 ) , related to age ( r = 0.34 , p < 0.05 ) , but not uniquely predictive of processing speed . the frontal component ( ic7 ) related to vessel disease and a cerebellar component ( ic4 ) were each associated with age and uniquely predicted processing speed ( figures 1 and 3 ) . importantly , unique frontal - cerebellar predictors of processing speed were identified with this analysis . a nice feature of the source based morphometry approach is that each component can be covaried in a whole brain analysis to identify regions uniquely associated a variable of interest . this type of analysis is presented in figure 1 and demonstrates : ( 1 ) that the frontal and cerebellar regions were uniquely predictive of processing speed ; ( 2 ) the spatial extent of these effects ; and ( 3 ) that some brain regions were significantly correlated with processing speed after controlling for either component . the latter is a particularly exciting demonstration of the potential for source based morphometry as an analytical tool because some of these regions , the cingulate for example , had voxels that were influenced by both the source of the frontal component and the source of the cerebellar component . in other words , this is like height being influenced by iodine deficiency and parental height , but in this case the gray matter volume of a specific brain region appears to be influenced by multiple additive aging risk factors . figure 4 provides a summary of these results , presented in the context of the multifactorial nature cognitive aging . summary of potential factors affecting frontal and cerebellar morphology and processing speed . on the left side of the figure , aging affects brain morphology through the damaging effects of oxidative stress and inflammation ( small vessel disease is in red because of consistent evidence linking cerebral small vessel disease to structural declines and slowed processing speed ) . these detrimental effects are likely buffered by neuroprotective factors such as ( 1 ) leptin and growth factor levels that limit the impact of oxidative stress , ( 2 ) norepinephrine or blueberry limits on inflammatory responses in animal models ( heneka et al . , 2010 ; willis et al . , 2009 ) , and ( 4 ) positive lifestyle behaviors such as aerobic exercise ( rosano et al . , 2010 ; voss et al . , 2010 ) . the gray arrow indicates that oxidative stress and inflammation likely affects cerebellar morphology , in comparison to the black arrows for which there is empirical evidence of significant associations . the right side of the figure is designed to emphasize that early development has a significant influence on the degree to which older adults will demonstrate atypical morphology and impaired processing speed ( deary et al . , 2006 , 2010 ) , but could also influence the expression of neuroprotective factors and the development of lifestyle patterns of behavior and modulate risk for age - related neural declines . while this summary figure is general in nature , it is designed to emphasize the multifactorial and interactive effects of aging neural systems on processing speed . the structural components that we identified with source based morphometry included patterns of variation that have been observed in functional imaging studies using ica ( beckmann et al . , 2005 ; damoiseaux et al . , 2006 ; de luca et al . , 2006 ; habas et al . , these results suggested that source based morphometry could be used to identify structural variation that corresponds to functionally defined neural systems . although source based morphometry results will be dependent on variation within a sample , further support for the potential of the source based morphometry approach to identify neural systems was the observation that individual variation in a cerebellar component was related to individual variation in a white matter component that included the middle cerebellar peduncles , cerebral peduncles , and posterior limb of the internal capsule ( figure 5 in eckert et al . , 2010 ) . the possibility that source based morphometry captures variance reflecting neural systems may therefore provide some insight into the neural systems that influence processing speed . our results and the existing literature suggest that a dorsal attention ( sweeney et al . , 2001 ) or prefrontal - mediated system for maintaining focused attention , inhibitory control , and working memory is significantly affected by microvascular damage . this interpretation is consistent with evidence that prefrontal gray matter volume has been related to perseveration on the wisconsin card sorting task ( raz et al . , 2003 , 2008 ) . it is important , however , to note that our processing speed results were most pronounced for the connections simple test compared to the connections complex task that required inhibiting a previous response . older adults may become particularly dependent on prefrontal cortex to maintain normal performance when age - related declines in sensory systems make relatively easy tasks more challenging because of degraded stimulus representations ( eckert et al . indeed , elevated frontal activity is a common finding in functional imaging studies of aging ( cabeza et al . , 2002 ; grady et al . , 2005 ) and appears to reflect compensatory engagement of frontal cortex in some studies ( townsend et al . , 2006 ) . performance across a variety of tasks may slow or exhibit impairment with subsequent declines in a dorsal attention system , particularly for tasks that require sustained attention or the inhibition of distracting information . for this reason , changes in a dorsal attention system could account for slowed processing speed across sensory domains . interestingly , the dorsal attention system appears to include regions of the cerebellar hemispheres ( krienen and buckner , 2009 ) . ( 2010 ) did exhibit some spatial overlap even though they each predicted additive variance in those overlapping regions . it is possible that primary aging effects are occurring across the dorsal attention system , but are differentially concentrated across people . this could explain why cerebellar measures might be uniquely predictive of processing speed in some studies and not others . the same neural system may be affected , but there may be regional specificity in the degree to which it is affected . changes in a dorsal attention or executive attention system could also explain why older adults exhibit difficulty inhibiting attention , as measured by eye - movements , to distracting or irrelevant visual targets ( kramer et al . this would have the downstream effect of slowing performance because of difficulty making a saccade to a new target , a function that is critical for many processing speed tasks . it is also possible that direct effects on an eye - movement system could influence processing speed , which is suggested by our findings that processing speed covaries with gray matter variation in the frontal eye - fields , cingulate , dorsolateral prefrontal cortex , and vermis ( fujikado and noda , 1987 ; hayakawa et al . older adults have been observed to exhibit impaired horizontal saccades ( carter et al . , 1983 ; sharpe and zackon , 1987 ) , but not in every study ( pratt et al . , 2006 ) . understanding the degree to which frontal and cerebellar declines relate to horizontal saccade performance may provide insight into the statistical linkage between neuroanatomical variation in these regions and processing speed . cerebellar morphology associations with processing speed most likely reflect a change in the ability of the cerebellum to integrate sensory and motor information to help guide smooth and quick movements . impaired integration of visual and proprioceptive information because of granule cell loss and/or impaired output of this information because of purkinje cell loss could slow behavior and increase movement errors . this prediction is supported by evidence that older adults have difficulty stopping arm movements smoothly and make more corrective movements during reaching than younger adults ( brown , 1996 ) . it is unclear , however , whether specific cerebellar zones and related function are affected because of the widespread cerebellar vermis and hemisphere effects observed in mri studies ( figure 1 ) . in summary , the existing literature and our sbm findings strongly implicate a dorsal attention system in the processing speed declines of older adults . impaired function of a domain general system would account for slowed task performance across such a wide variety of behavioral tasks . questions remain how best to enhance healthy aging of this system , although exercise appears to have positive benefits ( colcombe et al . in addition , the degree to which cerebellar declines reflect impairments in specific cerebellar systems and/or reflect changes in a dorsal attention system is unclear . addressing these questions and identifying the mechanisms for declines in neural systems that support processing speed has the potential to enhance healthy cognitive aging . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
processing speed , or the rate at which tasks can be performed , is a robust predictor of age - related cognitive decline and an indicator of independence among older adults . this review examines evidence for neurobiological predictors of age - related changes in processing speed , which is guided in part by our source based morphometry findings that unique patterns of frontal and cerebellar gray matter predict age - related variation in processing speed . these results , together with the extant literature on morphological predictors of age - related changes in processing speed , suggest that specific neural systems undergo declines and as a result slow processing speed . future studies of processing speed dependent neural systems will be important for identifying the etiologies for processing speed change and the development of interventions that mitigate gradual age - related declines in cognitive functioning and enhance healthy cognitive aging .
Neurobiological Predictors of Processing Speed A brief history and global cortical declines Frontal cortex and processing speed Cerebro-cerebellar and processing speed declines Identifying Unique Patterns of Cerebral Aging Toward a Systems Understanding of Age-Related Changes in Processing Speed Conflict of Interest Statement
one early neurobiological explanation for age - related changes in processing speed focused on cortical declines in neuropil ( morris and mcmanus , 1991 ) . age - related differences in total gray matter volume have been related to differences in processing speed ( chee et al . , 2006 ; there is a myelin - specific explanation that could account for age - related changes in processing speed . frontal cortex has been a primary target for the study of age - related processing speed changes because of the relatively pronounced aging effects on frontal cortex morphology and interest in prominent hypotheses for cognitive aging such as the frontal aging hypothesis ( west , 1996 ) and the inhibition deficit hypothesis ( hasher and zacks , 1988 ; hasher et al . sbm ( please see figure 3 for an sbm summary ) demonstrates unique patterns of frontal and cerebellar gray matter variance ( b , c ) that each accounted for different distributions of the processing speed results [ ( d ) : p < 0.001 after controlling for variance representing either the frontal or the cerebellar independent components ] . cerebellar morphology also undergoes robust age - related structural change but has not received the same experimental attention as frontal cortex with respect to age - related changes in processing speed . cross - sectional age - related changes in cerebellar morphology have been associated with processing speed ( maclullich et al . in addition , a volumetric measure of the same vermis region and total cerebellar gray matter volume were significantly related to age - related changes in trail making test ( alternating condition ) performance ( paul et al . this finding suggests that the processing speed and cerebellar relation was not due to a specific learning effect , however it is possible that we could not detect evidence of procedural learning because of ( 1 ) the balanced order in which the sub - tests are administered or ( 2 ) ceiling effects on the first trial of the connections sub - tests in people with the greatest cerebellar gray matter volume . one early neurobiological explanation for age - related changes in processing speed focused on cortical declines in neuropil ( morris and mcmanus , 1991 ) . there is a myelin - specific explanation that could account for age - related changes in processing speed . over the last 20 years there has been mounting evidence for regional specificity in the severity of age - related change in gray matter volume . frontal cortex has been a primary target for the study of age - related processing speed changes because of the relatively pronounced aging effects on frontal cortex morphology and interest in prominent hypotheses for cognitive aging such as the frontal aging hypothesis ( west , 1996 ) and the inhibition deficit hypothesis ( hasher and zacks , 1988 ; hasher et al . sbm ( please see figure 3 for an sbm summary ) demonstrates unique patterns of frontal and cerebellar gray matter variance ( b , c ) that each accounted for different distributions of the processing speed results [ ( d ) : p < 0.001 after controlling for variance representing either the frontal or the cerebellar independent components ] . cerebellar morphology also undergoes robust age - related structural change but has not received the same experimental attention as frontal cortex with respect to age - related changes in processing speed . in addition , a volumetric measure of the same vermis region and total cerebellar gray matter volume were significantly related to age - related changes in trail making test ( alternating condition ) performance ( paul et al . this finding suggests that the processing speed and cerebellar relation was not due to a specific learning effect , however it is possible that we could not detect evidence of procedural learning because of ( 1 ) the balanced order in which the sub - tests are administered or ( 2 ) ceiling effects on the first trial of the connections sub - tests in people with the greatest cerebellar gray matter volume . our approach to addressing this challenge has been to use ica or source based morphometry to identify unique patterns of structural covariance in the data that uniquely predict age - related differences in processing speed and that might provide insight into the affected neural systems . this is important because these results suggest there are independent age - based sources that affect gray matter variation in cerebellar ( ic4 ) and frontal ( ic7 ) regions that are associated with processing speed . , 2006 , 2010 ) , but could also influence the expression of neuroprotective factors and the development of lifestyle patterns of behavior and modulate risk for age - related neural declines . older adults may become particularly dependent on prefrontal cortex to maintain normal performance when age - related declines in sensory systems make relatively easy tasks more challenging because of degraded stimulus representations ( eckert et al . it is also possible that direct effects on an eye - movement system could influence processing speed , which is suggested by our findings that processing speed covaries with gray matter variation in the frontal eye - fields , cingulate , dorsolateral prefrontal cortex , and vermis ( fujikado and noda , 1987 ; hayakawa et al . addressing these questions and identifying the mechanisms for declines in neural systems that support processing speed has the potential to enhance healthy cognitive aging .
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increasing age and cancer are well - established risk factors for venous thromboembolism ( vte).16 given that older persons bear most of the cancer burden , as the population ages the incidence of cancer - associated vtes will increase as well.7,8 however , the epidemiology of vte in older cancer patients has not been well described . prior efforts have largely focused on either hospitalized patients or were not able to collect longitudinal data on a sufficient number of patients.1,9,10 as a result , we lack information about the degree to which vte increases the risk of death or anticoagulation - associated complications such as hemorrhage at the population level . prior work has suggested that cancer patients with a vte have a two- or threefold greater risk of death.3,11 if a vte diagnosis truly does independently increase the risk of death threefold , it would have profound implications for prognostication , management , and prophylaxis of vte in cancer patients . it is also possible that the impact of vte on mortality may be less pronounced than prior studies have suggested because vtes are associated with more aggressive tumor characteristics and later stage at diagnosis.10 prior studies have not fully accounted for patient and tumor factors and it is unclear to what degree vtes are truly a risk factor for mortality or if they are simply a marker of more aggressive tumors.11 furthermore , given that cancers vary in the degree to which they are associated with thrombosis , it could be expected that the impact of vte on subsequent survival could vary across cancer types.12 information about whether vte is associated with increased risk of death for different types of cancer could inform decision - makers about the intensity and duration of vte prophylaxis and treatment . although some work has suggested that patients with a vte could benefit from prolonged or even indefinite anticoagulation , the hemorrhage risks for patients with vte are uncertain.13,14 while the frequency of major hemorrhage reported in clinical trials was quite low , recent analyses of anticoagulation treatment of both cancer and noncancer patients in the community setting have suggested that major bleeding rates were up to tenfold higher.4,15,16 moreover , subgroup analyses suggested that patients with vte in the setting of cancer may have a higher hemorrhage risk than patients with vte alone.4,15,16 in addition , patients with some types of malignancies may be more prone to major hemorrhage than others because of anatomic location or disordered hemostasis associated with the underlying malignancy.17 as a result of these areas of uncertainty , there has been a call for population - based data documenting the rate of hemorrhage among cancer patients with vte.18 to address these knowledge gaps , we conducted a longitudinal population - based study of older cancer patients to determine how frequently vte is diagnosed concomitantly with cancer and the degree to which vtes increase the risk of death or major hemorrhage for different types of cancer . we obtained data on patients diagnosed with cancer during 1995 through 1999 from the linked national cancer institute ( nci ) surveillance , epidemiology , and end results ( seer)medicare database . all incident cancer patients who reported to the seer registries are cross - matched with a master file of medicare enrollment.19,20 patient level information available includes sociodemographic characteristics , cancer type , grade , site , and stage.21 we identified all patients who were diagnosed with 1 of 10 common cancer types , selected on the basis of their high overall incidence rates as well as prior studies demonstrating a relationship with vte.11,12 to allow for a 2-year ascertainment period for previous vte as well as other comorbid conditions before their cancer diagnosis ( see below ) , we only included patients who were 67 years of age and older at the time of their cancer diagnosis . table 1 demonstrates the construction of the study sample . of a total of 410,315 patients in the database , we included only the 247,785 individuals who were 67 years of age or older at the time of diagnosis , had a malignant primary lesion diagnosed during 1995 through 1999 , and had a known cancer type and month of diagnosis . exclusions included date of death before cancer diagnosis ( 23 patients ) , ineligible for medicare part a or part b ( 17,477 ) , and enrollment in a managed care plan during a 2-year period before cancer diagnosis ( 60,771 ) . these latter groups of patients were excluded because claims from these beneficiaries were not included in medicare claim files . patients who dropped coverage and/or enrolled in a managed care plan after their cancer diagnosis were censored at the month the fee - for - service coverage terminated . finally , we restricted our study sample to patients who had no diagnosis of vte ( defined as the absence of any icd-9 codes ) between 6 and 24 months before their cancer diagnosis to increase the likelihood that patients defined as having a concomitant vte did not have a prior vte . table 1construction of study sample number of patientspercentagetotal patients410,315100.0exclusions not malignant primary30,4507.6 first cancer diagnosis outside of 1995199940,20710.0 age < 67111,14227.7 cancer type not of interest12,2413.1 unknown month of cancer diagnosis2,0510.5eligible247,78561.7additional exclusions date of death before cancer diagnosis230.0 not eligible for part a or part b during 2-year period before cancer diagnosis17,4777.1 hmo enrollment during 2-year period before cancer diagnosis60,77124.5 prior claim for vte3,6320.9study sample167,38540.8vte = venous thromboembolism . a vte was defined as the diagnosis of either deep venous thrombosis ( dvt ) or pulmonary embolism ( pe ) . cases were ascertained through hospital admissions associated with specific international classification of diseases , 9th revision , clinical modification ( icd-9-cm ) codes . the relevant icd-9-cm codes used for the diagnosis of dvt or pe were 451.1 , 451.11 , 451.19 , 453.2 , 451.81 , 453.2 , 453.3 , 453.8 , 453.9 , 415.1 , 415.11 , and 415.19 . we defined a vte as presenting concomitantly with a cancer diagnosis if the vte diagnosis was made between 6 months prior and 1 month after the initial cancer diagnosis . the 6-month prediagnosis window was selected because many cancer - related vtes may be diagnosed before recognition of the underlying malignancy.3,22,23 we defined major hemorrhage as an intracranial or gastrointestinal hemorrhage requiring hospital admission . codes 430 , 431 , or 432were derived from validated work using administrative claims data.24 similarly , icd-9-cm codes for gastrointestinal hemorrhage ( 456.0x , 530.7 , 530.82 , 5315 , 537.83 , 562.0203 , 562.1213 , 569.3 , 569.85 , and 578.xx ) were also derived from previously published approaches.25 cancer stage was determined using seer american joint committee on cancer ( ajcc ) stage or historical stage ( coded as local , regional , or distant ) for cancer types in which ajcc was not available ( lymphoma , pancreatic , renal , and prostate cancers ) . we used median income according to patient zip code of residence as a proxy for socioeconomic status ( ses ) . dichotomous cancer treatment variables were created using seer data and medicare claims to identify patients who had received cancer - specific surgery , chemotherapy , and radiation therapy.26,27 pertinent comorbidity diagnosis codes during the period before each patient s cancer diagnosis were obtained from the inpatient and outpatient claims to identify conditions that comprise the charlson comorbidity index.28 we only included conditions that appeared on either one inpatient or two outpatient claims.28 to enable us to capture additional conditions that could alter the risk of developing a vte , we also identified patients with a diagnosis code for atrial fibrillation ( icd-9 code 427.3 ) prosthetic heart valve ( icd-9 code 35.20 , 35.22 , 35.24 , 35.26 , or 3528 ) , obesity ( 278.x ) , hip fracture ( icd-9 code 352.0 , 352.2 , 352.4 , 352.6 , or 352.8 ) , or insertion of a central venous catheter ( cpt code 36533 ) during the 2-year period before their cancer diagnosis . to understand the degree to which the relation between vte and mortality is mediated by cancer - related and other factors the initial cox proportional hazards model incorporated only age ( by 5-year age group because of the nonlinear relation between age and mortality ) , race , and gender as covariates , with vte as the independent variable and death as the outcome . we then added cancer characteristics and therapy to the model , including stage at diagnosis , histological grade , chemotherapy , radiation , and cancer - specific surgery . because comorbid conditions could affect the probability of receiving treatment or of developing a vte , comorbidity was then added to the model . while ses would not be expected to alter the physiologic impact of a vte , we considered lower ses as a potential mediator of stage and comorbidity . we calculated the proportion of patients who had a major hemorrhage during the first year after cancer diagnosis for each cancer type according to vte status . the excess hemorrhage risk in vte patients was estimated by deriving the absolute difference in hemorrhage rates between the vte and non - vte groups . finally , we explored the potential role of hemorrhage in mediating the relationship between vte and mortality by adding hemorrhage as a covariate to the final multivariate model . we obtained data on patients diagnosed with cancer during 1995 through 1999 from the linked national cancer institute ( nci ) surveillance , epidemiology , and end results ( seer)medicare database . all incident cancer patients who reported to the seer registries are cross - matched with a master file of medicare enrollment.19,20 patient level information available includes sociodemographic characteristics , cancer type , grade , site , and stage.21 we identified all patients who were diagnosed with 1 of 10 common cancer types , selected on the basis of their high overall incidence rates as well as prior studies demonstrating a relationship with vte.11,12 to allow for a 2-year ascertainment period for previous vte as well as other comorbid conditions before their cancer diagnosis ( see below ) , we only included patients who were 67 years of age and older at the time of their cancer diagnosis . table 1 demonstrates the construction of the study sample . of a total of 410,315 patients in the database , we included only the 247,785 individuals who were 67 years of age or older at the time of diagnosis , had a malignant primary lesion diagnosed during 1995 through 1999 , and had a known cancer type and month of diagnosis . exclusions included date of death before cancer diagnosis ( 23 patients ) , ineligible for medicare part a or part b ( 17,477 ) , and enrollment in a managed care plan during a 2-year period before cancer diagnosis ( 60,771 ) . these latter groups of patients were excluded because claims from these beneficiaries were not included in medicare claim files . patients who dropped coverage and/or enrolled in a managed care plan after their cancer diagnosis were censored at the month the fee - for - service coverage terminated . finally , we restricted our study sample to patients who had no diagnosis of vte ( defined as the absence of any icd-9 codes ) between 6 and 24 months before their cancer diagnosis to increase the likelihood that patients defined as having a concomitant vte did not have a prior vte . table 1construction of study sample number of patientspercentagetotal patients410,315100.0exclusions not malignant primary30,4507.6 first cancer diagnosis outside of 1995199940,20710.0 age < 67111,14227.7 cancer type not of interest12,2413.1 unknown month of cancer diagnosis2,0510.5eligible247,78561.7additional exclusions date of death before cancer diagnosis230.0 not eligible for part a or part b during 2-year period before cancer diagnosis17,4777.1 hmo enrollment during 2-year period before cancer diagnosis60,77124.5 prior claim for vte3,6320.9study sample167,38540.8vte = venous thromboembolism . a vte was defined as the diagnosis of either deep venous thrombosis ( dvt ) or pulmonary embolism ( pe ) . cases were ascertained through hospital admissions associated with specific international classification of diseases , 9th revision , clinical modification ( icd-9-cm ) codes . the relevant icd-9-cm codes used for the diagnosis of dvt or pe were 451.1 , 451.11 , 451.19 , 453.2 , 451.81 , 453.2 , 453.3 , 453.8 , 453.9 , 415.1 , 415.11 , and 415.19 . we defined a vte as presenting concomitantly with a cancer diagnosis if the vte diagnosis was made between 6 months prior and 1 month after the initial cancer diagnosis . the 6-month prediagnosis window was selected because many cancer - related vtes may be diagnosed before recognition of the underlying malignancy.3,22,23 we defined major hemorrhage as an intracranial or gastrointestinal hemorrhage requiring hospital admission . the icd-9-cm codes for intracranial hemorrhage codes 430 , 431 , or 432were derived from validated work using administrative claims data.24 similarly , icd-9-cm codes for gastrointestinal hemorrhage ( 456.0x , 530.7 , 530.82 , 5315 , 537.83 , 562.0203 , 562.1213 , 569.3 , 569.85 , and 578.xx ) were also derived from previously published approaches.25 cancer stage was determined using seer american joint committee on cancer ( ajcc ) stage or historical stage ( coded as local , regional , or distant ) for cancer types in which ajcc was not available ( lymphoma , pancreatic , renal , and prostate cancers ) . we used median income according to patient zip code of residence as a proxy for socioeconomic status ( ses ) . dichotomous cancer treatment variables were created using seer data and medicare claims to identify patients who had received cancer - specific surgery , chemotherapy , and radiation therapy.26,27 pertinent comorbidity diagnosis codes during the period before each patient s cancer diagnosis were obtained from the inpatient and outpatient claims to identify conditions that comprise the charlson comorbidity index.28 we only included conditions that appeared on either one inpatient or two outpatient claims.28 to enable us to capture additional conditions that could alter the risk of developing a vte , we also identified patients with a diagnosis code for atrial fibrillation ( icd-9 code 427.3 ) prosthetic heart valve ( icd-9 code 35.20 , 35.22 , 35.24 , 35.26 , or 3528 ) , obesity ( 278.x ) , hip fracture ( icd-9 code 352.0 , 352.2 , 352.4 , 352.6 , or 352.8 ) , or insertion of a central venous catheter ( cpt code 36533 ) during the 2-year period before their cancer diagnosis . to understand the degree to which the relation between vte and mortality is mediated by cancer - related and other factors , we constructed a series of sequential models for each cancer type . the initial cox proportional hazards model incorporated only age ( by 5-year age group because of the nonlinear relation between age and mortality ) , race , and gender as covariates , with vte as the independent variable and death as the outcome . we then added cancer characteristics and therapy to the model , including stage at diagnosis , histological grade , chemotherapy , radiation , and cancer - specific surgery . because comorbid conditions could affect the probability of receiving treatment or of developing a vte , comorbidity was then added to the model . while ses would not be expected to alter the physiologic impact of a vte , we considered lower ses as a potential mediator of stage and comorbidity . we calculated the proportion of patients who had a major hemorrhage during the first year after cancer diagnosis for each cancer type according to vte status . the excess hemorrhage risk in vte patients was estimated by deriving the absolute difference in hemorrhage rates between the vte and non - vte groups . finally , we explored the potential role of hemorrhage in mediating the relationship between vte and mortality by adding hemorrhage as a covariate to the final multivariate model . the median age of patients in the study sample was 75 years ( interquartile range 71 , 81 years ) . overall , about 1% of the patients was diagnosed with a vte concomitantly with their cancer diagnosis ( table 2 ) . the cancer types that were most frequently associated with vte were ovarian , kidney , and pancreatic ( 2.7% , 2.5% , and 2.2% , respectively ) . conversely , breast ( 0.4% ) and prostate ( 0.4% ) cancers were rarely associated with concomitant vte . in the bivariate ( unadjusted ) analysis , concomitant vte was strongly associated with mortality for patients with all types of cancer ( table 3 ) . the hazard ratio ( hr ) for the risk of mortality in patients with a concomitant vte compared with those without a vte ranged from 1.30 ( 95% confidence interval [ ci ] 1.181.44 ) among patients with lung cancer to a high of 3.06 for patients with cancer of the uterus ( 95% ci 2.324.04 ) . table 2concomitant venous thromboembolism ( vte ) according to cancer typecancer typenumber of patientsconcomitant vte ( occurring 6 months before to 1 month after cancer diagnosis)n%prostate40,7101520.37breast26,5631020.38bladder11,063790.71uterus5,685711.25lung32,3484491.39colorectal29,1014531.56lymphoma8,0221451.81pancreas6,3931392.17kidney4,1411032.49ovary3,359922.74total167,3851,7851.07table 3concomitant venous thromboembolism ( vte ) and risk of death according to cancer typecancer typehazard of death associated with vte ( vs no vte)model a : unadjustedmodel b : adjusted for age , sex , racemodel c : adjusted for factors in model b + cancer characteristics and treatmentmodel d : adjusted for factors in model c + comorbiditymodel e : adjusted for factors in model d + socioeconomic statushazard ratio95% cihazard ratio95% cihazard ratio95% cihazard ratio95% cihazard ratio95% ciprostate2.241.782.811.811.442.271.441.141.811.200.951.511.210.961.52breast2.301.763.011.951.482.551.110.841.451.010.771.321.010.771.33bladder2.802.173.622.842.203.661.571.212.031.541.192.001.431.131.80uterus3.062.324.043.472.634.592.081.572.751.981.482.641.961.472.62lung1.301.181.441.321.201.451.201.0911.11.151.041.271.161.051.27colorectal1.331.181.491.321.171.481.241.101.391.191.061.331.191.061.34lymphoma1.951.622.341.821.522.191.781.482.141.621.351.961.631.351.97pancreas1.311.101.551.351.141.601.281.081.521.261.061.491.261.061.49kidney1.491.191.881.611.282.021.491.181.881.411.121.781.431.131.80ovary1.271.001.611.160.911.471.351.071.721.321.041.681.321.031.68each row represents a unique model , as patients with each type of cancer were analyzed separately . hazard ratio represents the hazard of death for patients with a concomitant vte compared to patients with the same cancer type , but without a concomitant vte . concomitant venous thromboembolism ( vte ) according to cancer type concomitant venous thromboembolism ( vte ) and risk of death according to cancer type each row represents a unique model , as patients with each type of cancer were analyzed separately . hazard ratio represents the hazard of death for patients with a concomitant vte compared to patients with the same cancer type , but without a concomitant vte . mortality after accounting for demographic factors , cancer characteristics , and comorbidity , the relation between concomitant vte and mortality was attenuated substantially for patients with cancer of the breast or prostate . for example , among patients with breast cancer , the unadjusted hr associated with vte was 2.30 ( 95% ci 1.763.01 ) . after adjusting for age and gender , the hr decreased to 1.95 ( 95% ci 1.482.55 ) ( table 3 ) . after accounting for cancer characteristics and anticancer treatment received , the hr decreased to 1.11 and was no longer statistically significant ( 95% ci 0.841.45 ) . finally , after accounting for ses and comorbidity , the hr was 1.01 ( 95% ci 0.771.33 ) . a similar trend was noted among patients with prostate cancer , in that in the unadjusted hr associated with vte was 2.24 ( 95% ci 1.782.81 ) ; the hr decreased sequentially after adjusting for demographics ( hr 1.81 , 95% ci 1.442.27 ) , cancer characteristics and treatment ( hr 1.44 , 95% ci 1.141.81 ) , and finally ses and comorbidity ( hr 1.21 , 95% ci 0.961.52).unlike breast and prostate cancers , concomitant vte was significantly associated with mortality for patients with the other eight cancer types , even after adjusting for patient and cancer factors ( table 3 ) . the hrs associated with vte ranged from 1.16 ( 95% ci 1.051.27 ) for patients with lung cancer to 1.96 ( 95% ci 1.472.62 ) for patients with cancer of the uterus . when we added hemorrhage as a covariate to the analysis , there was little change in the hrs associated with vte for any of the cancer types ( data not shown ) . hemorrhage approximately 16.8% ( 95% ci 14.918.8 ) of patients with a concomitant vte were admitted to the hospital with a major hemorrhage during the year after their cancer diagnosis . in contrast , 7.9% ( 95% ci 7.78.0 ) of patients without a vte experienced a hemorrhage - related admission . the excess risk of hemorrhage in vte patients was 8.9% ( 95% ci 7.0 , 10.8 ; p value for difference < .001 ) . a concomitant vte was associated with a significantly higher hemorrhage risk for patients with 8 of the 10 cancer types studied ( table 4 ) . there was substantial variation across cancer types with regard to both baseline hemorrhage risk ( i.e. , proportion of patients with no vte who had a hemorrhage ) and the excess hemorrhage rate associated with vte . table 4concomitant venous thromboembolism ( vte ) and major hemorrhage during the first year after cancer diagnosis according to cancer typecancer typeconcomitant vteno concomitant vteexcess hemorrhage rate in vte patients ( % ) p value*n% hemorrhagen% hemorrhageprostate1298.538,3713.64.90.007breast8010.024,8223.16.90.0035bladder6813.210,3275.37.90.001uterus541.95,2484.42.50.36lung38212.626,8978.14.50.003colorectal38825.825,83617.97.9<0.001lymphoma13122.16,93610.611.5<0.001pancreas11524.34,83616.47.90.020kidney9510.53,6128.42.10.45ovary8214.62,7738.06.60.033total1,52416.8149,6587.98.9<0.001concomitant vte : vte diagnosed between 6 months before and 1 month after cancer diagnosis . major hemorrhage defined as intracranial or gastrointestinal bleeding requiring hospitalization.*p value with two - sided fisher s exact test for difference in hemorrhage rate between patients with vte versus without vte for each cancer type.the excess risk of hemorrhage associated with concomitant vte ( and the related anticoagulation therapy ) was approximately 68% for most cancer types . among patients with bladder cancer , for instance , approximately 13.2% of those who had a concomitant vte suffered a major hemorrhage , in comparison to only 5.3% of patients without a concomitant vte , yielding an excess hemorrhage rate of 7.9% . the attributable risk for hemorrhage associated with vte ranged from no significant excess ( kidney and uterine cancer ) hemorrhage risk to 11.5% ( lymphoma ) . concomitant venous thromboembolism ( vte ) and major hemorrhage during the first year after cancer diagnosis according to cancer type concomitant vte : vte diagnosed between 6 months before and 1 month after cancer diagnosis . major hemorrhage defined as intracranial or gastrointestinal bleeding requiring hospitalization . * p value with two - sided fisher s exact test for difference in hemorrhage rate between patients with vte versus without vte for each cancer type . in a population - based cohort of older patients with cancer , we found concomitant vtes were associated with increased risk of death as well as major hemorrhage for patients with most types of cancer . the 9% absolute increase in hemorrhage rate associated with vte suggests that treatment of vte is associated with substantial risk . the risk of death associated with vte did not change substantively after adding hemorrhage to the multivariate model , suggesting that hemorrhage is not the mechanism through which vte increases the risk of death . a major hemorrhage leading to hospitalization is costly , frightening , and can be associated with substantial morbidity among patients with a relatively short life expectancy . our analysis builds upon these studies by providing quantitative , population - based estimates of hemorrhage risk in the older cancer population . in one analysis of 181 cancer patients with vte , the 12-month cumulative incidence of major bleeding was 12.4% ( 95% ci 6.518.2).4 of note , the same analysis also investigated the hemorrhage rate in vte patients without cancer and found that it was only 4.9% ( 95% ci 2.57.4).4 similarly , a review of more than 2,000 patients with vte , with and without cancer , demonstrated a bleeding - related hospitalization rate of 11.4 to 14.9 per 100 patient - years , and that patients with cancer had a higher hemorrhage risk.15 these hemorrhage rates are substantially higher than the rate ( 1.1 events/100 person - years ) reported in a recent meta - analysis of anticoagulation trials , which included patients with and without cancer , across a spectrum of age groups.16 this discrepancy between trial and community outcomes is likely because of differences in study populations , monitoring of therapy , and the fact that many of the trials have included patients with and without cancer.16,29 a previously validated tool for assessing hemorrhage risk is the outpatient bleeding risk index , which includes risk factors such as age greater than 65 years , prior gastrointestinal hemorrhage , and specific comorbid conditions including atrial fibrillation stroke , anemia , prior myocardial infarction , or renal insufficiency.18,30 in the context of prior work showing that cancer increases the risk of hemorrhage , and our finding that hemorrhage risk can vary substantially across cancer types , future work should seek to identify bleeding prediction tools for validation in cancer patients . our findings suggest that prior estimates of the relation between vte and mortality may have been inaccurate because of confounding by tumor characteristics.1 in one case control study of cancer patients that accounted for age , gender , and cancer type , patients with a vte had a hr for mortality of 2.20 ( 95% ci 2.052.45).3 although patients with a vte were significantly more likely to have metastatic disease than patients without a vte , cancer stage was not accounted for in this analysis . in contrast , we found that the impact of vte on mortality was attenuated after accounting for these factors in patients with breast or prostate cancer . this suggests that a substantial portion of the increased mortality risk previously attributed to vte for patients with these cancer types may actually be attributed to underlying tumor characteristics and stage at presentation . because they are common malignancies and are frequently diagnosed at an early stage , inadequate adjustment for cancer type and stage can lead to biased assessments of vte outcomes . although other authors have used icd-9 codes to identify patients with diagnosed vte , and some studies have reported that this is a relatively accurate approach , many cases of vte may not be clinically recognized , and some clinically recognized cases may not be appropriately coded.1,23,3133 we focused on vtes that were diagnosed with an associated hospital admission to increase the likelihood that the vtes were acute . however , because it is unclear whether administrative data can reliably capture the clinical diagnosis of vte , future work should use alternate data sources to assess outcomes associated with vte . similarly , while there has been a trend toward increased treatment of vtes in the outpatient setting , low molecular weight heparin was not approved for outpatient vte treatment until december 31 , 1998near the end of our study period.34 our study included only older persons , so it is unclear whether our findings would generalize to a younger cancer patient population . finally , because pharmacologic and laboratory data were not available , we were unable to identify anticoagulation management patterns for patients with vte . further work should identify the degree to which the substantial hemorrhage rate noted in our sample is attributable to appropriateness of therapeutic monitoring , or the interaction of anticoagulation risks with specific cancer characteristics or other comorbid illnesses . finally , we repeated the analysis after excluding patients who were diagnosed in 1999 , as low molecular weight heparin was available during this time and outpatient treatment would have affected the analysis . we found no substantive change in the adjusted hazard of death associated with vte after excluding patients diagnosed in 1999 . our findings shed new light on the scope and impact of vte on older cancer patients . the substantial risk of hospitalization with a major hemorrhage in the first year after diagnosis of a vte emphasizes the importance of identifying opportunities for risk reduction in this population . we also found that although vte is associated with increased risk of death , this risk varied across cancer types and was , in many cases , attenuated by adjusting for underlying tumor characteristics . with the number of newly diagnosed cancer patients and cancer survivors increasing dramatically , it is imperative to further clarify the optimal approach to older cancer patients who are at risk for vte and those who have already experienced a vte
backgroundamong older cancer patients , there is uncertainty about the degree to which venous thromboembolism ( vte ) and its treatment increase the risk of death or major hemorrhage.objectiveto determine the prevalence of vte in a cohort of older cancer patients , as well as the degree to which vte increased the risk of death or major hemorrhage.methodswe conducted a retrospective cohort study of linked surveillance , epidemiology , and end results cancer registry and medicare administrative claims data . patients with any of ten invasive cancers diagnosed during 1995 through 1999 were included ; the independent variable was vte diagnosed concomitantly with cancer diagnosis . outcomes included major hemorrhage during the first year after cancer diagnosis and all - cause mortality;resultsoverall , about 1% of patients who were diagnosed with cancer also had a vte diagnosed concomitantly . after adjusting for sociodemographic factors and cancer stage and grade , concomitant vte was associated with a relative increase in the risk of death for 8 of the 10 cancer types ; the increase in risk tended to range 2040% across most cancer types . approximately 16.8% ( 95% confidence interval [ ci ] 14.918.8% ) of patients with a concomitant vte and 7.9% ( 95% ci 7.78.0% ) of patients without a vte experienced a major hemorrhage during the year after cancer diagnosis ( p value < .001 ) . the excess risk of hemorrhage associated with vte varied substantially across cancer types , ranging from no significant excess ( kidney and uterine cancer ) to 11.5% ( lymphoma).conclusionconcomitant vte is not only a marker and potential mediator of increased risk of death among older cancer patients , but patients with a vte have a marked increased risk of major hemorrhage .
BACKGROUND METHODS Data Sources and Study Sample Construction of Variables Statistical Analysis RESULTS DISCUSSION
it is also possible that the impact of vte on mortality may be less pronounced than prior studies have suggested because vtes are associated with more aggressive tumor characteristics and later stage at diagnosis.10 prior studies have not fully accounted for patient and tumor factors and it is unclear to what degree vtes are truly a risk factor for mortality or if they are simply a marker of more aggressive tumors.11 furthermore , given that cancers vary in the degree to which they are associated with thrombosis , it could be expected that the impact of vte on subsequent survival could vary across cancer types.12 information about whether vte is associated with increased risk of death for different types of cancer could inform decision - makers about the intensity and duration of vte prophylaxis and treatment . although some work has suggested that patients with a vte could benefit from prolonged or even indefinite anticoagulation , the hemorrhage risks for patients with vte are uncertain.13,14 while the frequency of major hemorrhage reported in clinical trials was quite low , recent analyses of anticoagulation treatment of both cancer and noncancer patients in the community setting have suggested that major bleeding rates were up to tenfold higher.4,15,16 moreover , subgroup analyses suggested that patients with vte in the setting of cancer may have a higher hemorrhage risk than patients with vte alone.4,15,16 in addition , patients with some types of malignancies may be more prone to major hemorrhage than others because of anatomic location or disordered hemostasis associated with the underlying malignancy.17 as a result of these areas of uncertainty , there has been a call for population - based data documenting the rate of hemorrhage among cancer patients with vte.18 to address these knowledge gaps , we conducted a longitudinal population - based study of older cancer patients to determine how frequently vte is diagnosed concomitantly with cancer and the degree to which vtes increase the risk of death or major hemorrhage for different types of cancer . the hazard ratio ( hr ) for the risk of mortality in patients with a concomitant vte compared with those without a vte ranged from 1.30 ( 95% confidence interval [ ci ] 1.181.44 ) among patients with lung cancer to a high of 3.06 for patients with cancer of the uterus ( 95% ci 2.324.04 ) . a similar trend was noted among patients with prostate cancer , in that in the unadjusted hr associated with vte was 2.24 ( 95% ci 1.782.81 ) ; the hr decreased sequentially after adjusting for demographics ( hr 1.81 , 95% ci 1.442.27 ) , cancer characteristics and treatment ( hr 1.44 , 95% ci 1.141.81 ) , and finally ses and comorbidity ( hr 1.21 , 95% ci 0.961.52).unlike breast and prostate cancers , concomitant vte was significantly associated with mortality for patients with the other eight cancer types , even after adjusting for patient and cancer factors ( table 3 ) . hemorrhage approximately 16.8% ( 95% ci 14.918.8 ) of patients with a concomitant vte were admitted to the hospital with a major hemorrhage during the year after their cancer diagnosis . in one analysis of 181 cancer patients with vte , the 12-month cumulative incidence of major bleeding was 12.4% ( 95% ci 6.518.2).4 of note , the same analysis also investigated the hemorrhage rate in vte patients without cancer and found that it was only 4.9% ( 95% ci 2.57.4).4 similarly , a review of more than 2,000 patients with vte , with and without cancer , demonstrated a bleeding - related hospitalization rate of 11.4 to 14.9 per 100 patient - years , and that patients with cancer had a higher hemorrhage risk.15 these hemorrhage rates are substantially higher than the rate ( 1.1 events/100 person - years ) reported in a recent meta - analysis of anticoagulation trials , which included patients with and without cancer , across a spectrum of age groups.16 this discrepancy between trial and community outcomes is likely because of differences in study populations , monitoring of therapy , and the fact that many of the trials have included patients with and without cancer.16,29 a previously validated tool for assessing hemorrhage risk is the outpatient bleeding risk index , which includes risk factors such as age greater than 65 years , prior gastrointestinal hemorrhage , and specific comorbid conditions including atrial fibrillation stroke , anemia , prior myocardial infarction , or renal insufficiency.18,30 in the context of prior work showing that cancer increases the risk of hemorrhage , and our finding that hemorrhage risk can vary substantially across cancer types , future work should seek to identify bleeding prediction tools for validation in cancer patients .
[ 0, 0, 0, 1, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 0, 0, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 ]
hepatic stem - like ( hsl ) cells have a potential of self - renewal and an ability to differentiate to hepatocytes and biliary duct cells ( nagai et al . ( 2005 ) described that hsl cells converted to pancreatic neuroendocrine cells when incubated with sodium butyrate ( nab ) and betacellulin . while many tissue stem cells are cultured in vitro in the medium with some growth factors , cytokines or extracellular matrix , hsl cells proliferate in dulbecco s modified eagle s medium ( dmem ) with 10% fetal calf serum ( fcs ) without differentiation or tumorigenesis . if the mechanisms underlying continuous proliferation of hsl cells without differentiation become clear , many tissue stem cells will be applicable to cell transplantation therapy for many types of diseases . a histone deacetylase inhibitor , nab , acts as a differentiation - promoting agent for a wide variety of cell types . in mice , nab induces differentiation of immature hepatocytes into mature parenchymal cells and reduces their proliferation ( iwai et al . recently , it was shown that human and murine embryonic stem cells were able to differentiate into hepatocyte - like cells by treatment with nab ( zhou et al . when hsl cells were cultured with nab , they converted to flattened cells with large cytoplasm ( yamada et al . there are many studies about nab induced differentiation , while there is little information about anti - proliferative effect of nab on hsl cells . to reveal the molecular mechanisms that sustain proliferative activity of hsl cells with pluripotency , it is necessary to identify the molecular event induced by nab treatment . in this study , we performed two - dimensional electrophoresis ( 2-de ) to search for proteins statistically more abundant in undifferentiated hsl cells than in nab - treated cells , and identified those proteins by matrix - assisted laser desorption / ionization - time of flight mass spectrometry ( maldi - tof - ms ) analysis . these proteins might be participated in the molecular mechanism that causes the vigorous proliferative activity of the hsl cells . hsl cells were cultured in dmem ( asahi technoglass co. , tokyo , japan ) with 10% fcs ( biofluids , rockville , md , usa ) at 37 c in a 5% co2 atmosphere . an hsl pellet about 70 mg in weight ( 2 dishes of 100 mm culture dish , nearly confluent ) were lysed in 350 l of extraction solution ( 8 m urea , 1 m thiourea , 0.5% 2-mercaptoethanol , 3% nonidet p-40 ) with 50 l of protease inhibitor cocktail ( complete mini edta - free ; roche diagnostics gmbh , mannheim , germany ) by microhomogenizer ( ina optica , osaka , japan ) . the homogenate was centrifuged at 15,000 g for 20 min , 4 c and the clear supernatant was subjected to the zoom strip isoelectric focusing ( ief ) gel . total protein concentration of the supernatant was determined with coomassie protein assay kit ( pierce , rockford , il , usa ) according to the bradford method ( bradford 1976 ) . the ief gel strips ( zoom strip ph 3 - 10nl , invitrogen corp . , carlsbad , ca , usa ) were rehydrated at room temperature for 1 h with ca . 30 g of protein for analytical assay or 150 g of protein for in - gel digestion by trypsin . after rehydration , ief was carried out at 175 v for 20 min , followed by voltage ramping to 2,000 v for 45 min , focusing for 30 min , with total focusing for 1,800 vh . following ief , the ipg strips were thawed and incubated for 15 min in 5 ml lithium dodesyl sulfate ( lds ) reducing solution containing 1.25 ml nupage lds sample buffer and 0.5 ml nupage sample reducing agent followed by incubation with s - alkylation solution containing 116 mg iodoacetamide and 1.25 ml nupage lds sample buffer for 15 min at room temperature . finally , the ipg strips and precision plus unstained protein standards ( bio - rad laboratories , hercules , ca , usa ) were positioned on top of nupage 4 - 12% bis tris zoom gel ( invitrogen corp . , gels were run in the xcell surelock mini - cell system ( invitrogen corp . , carlsbad , ca , usa ) , at 200 v for 50 min . following electrophoresis , the gels were stained using the deep purple total protein stain ( amersham biosciences corp . , piscataway , gel images were acquired using the typhoon 9200 variable image analyzer ( amersham biosciences corp . , the density of protein spots were calculated using image quant software ( amersham biosciences corp . , the spots were selected randomly with the goal of detecting as many proteins as possible . the electrophoresis was repeated 10 times , the densities of 198 spots on each gel were compared using student s t - test , and differentially expressed spots ( p preparative electrophoresed gel for in - gel tryptic digestion was fixed in 45% methanol and 10% acetic acid overnight and stained by coomassie brilliant blue r-250 . the proteins were washed , dehydrated and digested in situ within the gel with 1 l of 5 ng trypsin in 50 mm ammonium bicarbonate overnight at 37 c . the resulting peptides were extracted from the gel matrix with 3 l of 0.1% trifluoroacetic acid/50% acetonitrile . the gel pieces were extracted again with 3 l of 0.1% trifluoroacetic acid/50% acetonitrile . the supernatants were combined with the first extraction and the pooled digests were lyophilized and resuspended in 3 l of 0.1% trifluoroacetic acid . following peptide extraction , the samples were prepared for maldi - tof - ms analysis with a ziptip c18 ( millipore , billerica , ma , usa ) according to manufacturer s instructions . the peptides were eluted into 3 l of 5% acetonitrile in water with 0.1% trifluoroacetic acid and spotted on the prespotted anchorchip ( bruker daltonics inc . , the peptide mass spectra analyzed by auto flex maldi - tof - ms system ( bruker daltonics inc . , billerica , ma , usa ) were searched against the national center for biotechnology information ( ncbi ) nonredundant database using mascot search engine ( perkins et al . 1999 ) . cultured hsl cells were washed three times with sterile ice cold phosphate buffered saline ( pbs : 137 mm nacl , 2.7 mm kcl , 10 mm na2hpo4 12h2o , 2 mm kh2po4 ) . total rna was prepared from scraped hsl cells ( 10 cells ) using micro - to - midi total rna purification system ( invitrogen corp . , hsl cell cdna was prepared from total rna 5 g by revertra ace ( toyobo co. ltd . , , 1 l of cdna solution was added by 5 l 2 gotaq green master mix ( promega co. , madison , wi , usa ) , 1 l of 10 m forward and reverse primers and 2 l nuclease free water . pcr was made as below : pre - denaturation 95 c , 2 min , 30 cycles of denaturation 95 c , 30 s , annealing 60 c , 30 s , elongation 72 c , 3 min , and post - elongation 72 c , 10 min . the primers used were : ezrin forward ; 5-cagagagtcatggaccagca-3 , ezrin reverse ; 5-cggggtcaacttgtcatctt-3 , prohibitin forward ; 5-ggcatgcctgagtagaccttg-3 , prohibitin reverse ; 5-tcacggttaagagggaatgg-3 , vimentin forward ; 5-agatcgatgtggacgtttcc-3 , vimentin reverse ; 5-cacctgtctccggtattcgt-3 , annexin a3 forward ; 5-ttgatgccaagcaactgaag-3 , annexin a3 reverse ; 5-caggctttcgtctcttccac-3 , glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) forward ; 5-ccatcaccatcttccaggag-3 , gapdh reverse ; 5-cctgctcaccaccttcttg-3. the primer sequence was selected using amplified dnas were separated on 1% agarose gel electrophoresis and stained with 0.5 g / ml ethidium bromide . hsl cells were cultured in dmem ( asahi technoglass co. , tokyo , japan ) with 10% fcs ( biofluids , rockville , md , usa ) at 37 c in a 5% co2 atmosphere . an hsl pellet about 70 mg in weight ( 2 dishes of 100 mm culture dish , nearly confluent ) were lysed in 350 l of extraction solution ( 8 m urea , 1 m thiourea , 0.5% 2-mercaptoethanol , 3% nonidet p-40 ) with 50 l of protease inhibitor cocktail ( complete mini edta - free ; roche diagnostics gmbh , mannheim , germany ) by microhomogenizer ( ina optica , osaka , japan ) . the homogenate was centrifuged at 15,000 g for 20 min , 4 c and the clear supernatant was subjected to the zoom strip isoelectric focusing ( ief ) gel . total protein concentration of the supernatant was determined with coomassie protein assay kit ( pierce , rockford , il , usa ) according to the bradford method ( bradford 1976 ) . the ief gel strips ( zoom strip ph 3 - 10nl , invitrogen corp . , carlsbad , ca , usa ) were rehydrated at room temperature for 1 h with ca . 30 g of protein for analytical assay or 150 g of protein for in - gel digestion by trypsin . after rehydration , ief was carried out at 175 v for 20 min , followed by voltage ramping to 2,000 v for 45 min , focusing for 30 min , with total focusing for 1,800 vh . following ief , the ipg strips were thawed and incubated for 15 min in 5 ml lithium dodesyl sulfate ( lds ) reducing solution containing 1.25 ml nupage lds sample buffer and 0.5 ml nupage sample reducing agent followed by incubation with s - alkylation solution containing 116 mg iodoacetamide and 1.25 ml nupage lds sample buffer for 15 min at room temperature . finally , the ipg strips and precision plus unstained protein standards ( bio - rad laboratories , hercules , ca , usa ) were positioned on top of nupage 4 - 12% bis tris zoom gel ( invitrogen corp . , carlsbad , ca , usa ) . gels were run in the xcell surelock mini - cell system ( invitrogen corp . , carlsbad , ca , usa ) , at 200 v for 50 min . following electrophoresis , the gels were stained using the deep purple total protein stain ( amersham biosciences corp . , piscataway , gel images were acquired using the typhoon 9200 variable image analyzer ( amersham biosciences corp . , the density of protein spots were calculated using image quant software ( amersham biosciences corp . , the spots were selected randomly with the goal of detecting as many proteins as possible . the electrophoresis was repeated 10 times , the densities of 198 spots on each gel were compared using student s t - test , and differentially expressed spots ( p < 0.05 ) were identified . preparative electrophoresed gel for in - gel tryptic digestion was fixed in 45% methanol and 10% acetic acid overnight and stained by coomassie brilliant blue r-250 . the proteins were washed , dehydrated and digested in situ within the gel with 1 l of 5 ng trypsin in 50 mm ammonium bicarbonate overnight at 37 c . the resulting peptides were extracted from the gel matrix with 3 l of 0.1% trifluoroacetic acid/50% acetonitrile . the gel pieces were extracted again with 3 l of 0.1% trifluoroacetic acid/50% acetonitrile . the supernatants were combined with the first extraction and the pooled digests were lyophilized and resuspended in 3 l of 0.1% trifluoroacetic acid . following peptide extraction , the samples were prepared for maldi - tof - ms analysis with a ziptip c18 ( millipore , billerica , ma , usa ) according to manufacturer s instructions . the peptides were eluted into 3 l of 5% acetonitrile in water with 0.1% trifluoroacetic acid and spotted on the prespotted anchorchip ( bruker daltonics inc . , the peptide mass spectra analyzed by auto flex maldi - tof - ms system ( bruker daltonics inc . , billerica , ma , usa ) were searched against the national center for biotechnology information ( ncbi ) nonredundant database using mascot search engine ( perkins et al . cultured hsl cells were washed three times with sterile ice cold phosphate buffered saline ( pbs : 137 mm nacl , 2.7 mm kcl , 10 mm na2hpo4 12h2o , 2 mm kh2po4 ) . total rna was prepared from scraped hsl cells ( 10 cells ) using micro - to - midi total rna purification system ( invitrogen corp . , hsl cell cdna was prepared from total rna 5 g by revertra ace ( toyobo co. ltd . , osaka , japan ) with oligo ( dt)20 ( toyobo co. ltd . , , 1 l of cdna solution was added by 5 l 2 gotaq green master mix ( promega co. , madison , wi , usa ) , 1 l of 10 m forward and reverse primers and 2 l nuclease free water . pcr was made as below : pre - denaturation 95 c , 2 min , 30 cycles of denaturation 95 c , 30 s , annealing 60 c , 30 s , elongation 72 c , 3 min , and post - elongation 72 c , 10 min . the primers used were : ezrin forward ; 5-cagagagtcatggaccagca-3 , ezrin reverse ; 5-cggggtcaacttgtcatctt-3 , prohibitin forward ; 5-ggcatgcctgagtagaccttg-3 , prohibitin reverse ; 5-tcacggttaagagggaatgg-3 , vimentin forward ; 5-agatcgatgtggacgtttcc-3 , vimentin reverse ; 5-cacctgtctccggtattcgt-3 , annexin a3 forward ; 5-ttgatgccaagcaactgaag-3 , annexin a3 reverse ; 5-caggctttcgtctcttccac-3 , glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) forward ; 5-ccatcaccatcttccaggag-3 , gapdh reverse ; 5-cctgctcaccaccttcttg-3. the primer sequence was selected using primer3 software ( rozen and skaletsky 2000 ) . gapdh was used as internal control as described before ( miura et al . amplified dnas were separated on 1% agarose gel electrophoresis and stained with 0.5 g / ml ethidium bromide . hsl cells proliferated with a cobblestone appearance ( fig . 1 ) and did not show any changes in shape spontaneously even in long - term culture ( nagai et al . 2002 ) . in this study , morphological changes occurred in response to 5 mm nab in some population of cells on dishes , and 20 mm nab was more effective ( fig . 1 ) . in the following study , hsl cells were incubated with 20 mm nab for 18 h. to clarify the proteins involved in proliferation of hsl cells , we compared the proteins expressed in hsl cells before and after nab treatment by 2-de . at the first glance , quite similar expression patterns with little difference were observed in both samples ( fig . 2 ) . we selected 198 spots on the gel , and the spot concentrations were calculated by image quant software . one was the ratio of the concentration of one spot to the total spot intensity of selected 198 spots ( fig . the other was the ratio of the concentration of one spot to the randomly selected spot intensity as standard . the numbers of selected spots for standard were 21 , 46 , 57 , 77 and 116 ( fig . 3 ) . we performed statistical analysis by using student s t - test and the spots with statistically different intensity ( p < 0.05 ) using all 6 data sets , were selected . we found 5 proteins up - regulated after nab treatment and 14 proteins down - regulated after nab treatment . the down - regulated proteins were the candidate proteins which might have some roles in the hsl cell proliferation . then those proteins were in - gel digested by trypsin and analyzed by maldi - tof - ms . mascot search identified 6 proteins , namely prohibitin , vimentin , ezrin , annexin a3 , acidic ribosomal phosphoprotein p0 and grp75 ( fig . we compared the protein spots on 2-de gels stained by deep purple fluorescence dye between control hsl cells extract and nab treated hsl cells extract , whereas the protein spots on the 2-de gels for maldi - tof - ms analysis were stained by coomassie brilliant blue . the amounts of the 8 proteins were too low even under deep purple staining to see under coomassie brilliant blue staining or detect by mass spectrometer . to subconfluent hsl cells cultured in dmem , final 2.5 , 5 , 10 or 20 mm nab were added and incubated for 0 , 12 , 15 , 18 , 21 and 24 h. cells were observed by phase - contrast microscope . significant morphological changes were observed 18 h after 20 mm nab treatment hsl cellsfig . cell extracts prepared from hsl cells ( a ) or nab treated hsl cells ( b ) were separated first by isoelectric focusing on ph gradient between 3 and 10 , and then on 412% gradient polyacrylamide gel electrophoresis . proteins were stained by fluorescent dye , deep purple , and detected by laser scanner typhoon 9200 as described in experimental procedure . the expression patterns were almost the same between control and nab - treated hsl cells . to obtain statistically reliable results , 3statistically conferred protein spots . to confer expression intensity between control and nab treated cells statistically , 198 spots were selected randomly . the spots , those intensity were used as standard , were shown as the number . the proteins down - regulated by nab treatment were identified by mascot searchtable 1the proteins identified by mascot searchspot no.giidentified proteinscoremasspicover ( % ) matched peptidetotal peptide2962664759prohibitin6027,7575.44439283551980303annexin a314736,5695.966321383611693176acidic ribosomal phosphoprotein p06134,3655.914810324414389299vimentin16053,7575.06643553501000439grp758573,9845.873619357917902245ezrin6254,2546.16391938gi : accession number , mass : calculated molecular weight , pi : calculated iso - electric point , total peptide : number of the peptide used to search the data base , matched peptide : number of the peptide contained the hit protein , cover : sequence cover ratio with detected peptide on complete amino acid sequence , score : highest hit score of several mascot searches morphological changes in hsl cells treated with nab . to subconfluent hsl cells cultured in dmem , final 2.5 , 5 , 10 or 20 mm nab were added and incubated for 0 , 12 , 15 , 18 , 21 and 24 h. cells were observed by phase - contrast microscope . significant morphological changes were observed 18 h after 20 mm nab treatment hsl cells two - dimensional electrophoresis . cell extracts prepared from hsl cells ( a ) or nab treated hsl cells ( b ) were separated first by isoelectric focusing on ph gradient between 3 and 10 , and then on 412% gradient polyacrylamide gel electrophoresis . proteins were stained by fluorescent dye , deep purple , and detected by laser scanner typhoon 9200 as described in experimental procedure . the expression patterns were almost the same between control and nab - treated hsl cells . to obtain statistically reliable results , 2-de was executed 10 times for each sample statistically conferred protein spots . to confer expression intensity between control and nab treated cells statistically the spots , those intensity were used as standard , were shown as the number . the proteins down - regulated by nab treatment were identified by mascot search the proteins identified by mascot search gi : accession number , mass : calculated molecular weight , pi : calculated iso - electric point , total peptide : number of the peptide used to search the data base , matched peptide : number of the peptide contained the hit protein , cover : sequence cover ratio with detected peptide on complete amino acid sequence , score : highest hit score of several mascot searches prohibitin is expressed in wide variety of tissues such as heart , liver and neurons . it has been shown that prohibitin localizes in mitochondrial inner membrane and may play a role in regulating mictochondrial respiration activity and in aging ( rajalingam and rudol 2005 ; mishra et al . a recent study indicated that prohibitin is required for raf activation induced by ras , a downstream signal activated by epidermal growth factor ( rajalingam et al . 2005 ) . it is expected that prohibitin has some role in activating classical mitogen - activated protein ( map ) kinase cascade in intact hsl cells to continuous proliferation . vimentin is the cell matrix proteins which constructs cell structures ( clarke and allan 2002 ; wangi and stamenovic 2002 ) . vimentin is well known as an intermediate filament found in various nonepithelial cells , especially mesenchymal cells . it was also reported that vimentin interacted with a phosphorylated erk and protected it from dephosphorylation ( perlson et al . ( chen et al . 2007 ) , in hsl cells , nab down regulates prohibitin and vimentin expression . ezrin is thought to be involved in connections of major cytoskeletal structure of the plasma membrane ( bretscher et al . the expression levels increase in the fetal rat gut epithelium between day 15 and day 20 of gestation and during the first week after birth ( baril et al . ezrin also serves as a substrate for protein tyrosine kinases and mediates adhesion - mediated events ( srivastava et al . it is reasonable to suppose that those cell matrix proteins are involved in the alteration of morphology of hsl cells after 18 h culture with nab ( fig . 1 ) . annexin a3 is one of the annexin family proteins that is described as an inhibitor of phospholipase a2 , also possesses anti - coagulant properties . however , the biological role of annexin a3 is not totally clear . in the liver , annexin a3 rna interference study suggested that annexin a3 has a role in dna synthesis ( niimi et al . nab is known as an inhibitor of histone deacetylase that modulates the expression of genes by causing in histone acetylation ( heinz et al . 2002 ) . to confirm whether prohibitin , vimentin , ezrin and annexin a3 expression was regulated transcriptionally , 4 , the mrna expression levels of the proteins selected were not changed 18 h after nab treatment . because a slight difference in protein expression level was observed between in the control hsl cells and in the nab treated hsl cells , mrna transcription activity did not change dramatically . other possible mechanisms were those , the protein degradation was stimulated , the expression level of the protein was regulated by translation or the protein was modified by some post - translational modifications . total rnas were extracted from control hsl cells ( c ) or nab treated hsl cells ( n ) . the sizes of the amplified bands of prohibitin , vimentin , ezrin and annexin a3 were about 200 bp as expected . no significant differences were observed between control and nab treated hsl cells expression of mrna for prohibitin , vimentin , ezrin and annexin a3 . total rnas were extracted from control hsl cells ( c ) or nab treated hsl cells ( n ) . the sizes of the amplified bands of prohibitin , vimentin , ezrin and annexin a3 were about 200 bp as expected . no significant differences were observed between control and nab treated hsl cells grp75 is known as mitochondrial stress-70 protein belonging to the heat shock protein 70 family ( wadhwa et al . high level expression of grp75 in hsl cells suggested that hsl proliferation is controlled not only under growth signal but also under protection from aging . acidic ribosomal phosphoprotein p0 forms a pentameric complex by interaction with dimers of p1 and p2 , and constructs ribosomal large subunit . , our study demonstrates that the proteins regulating the map kinase cascade , cytoskeletons , chaperon and protein synthesis system , have some role to continue proliferation of hsl cells without differentiation . these results suggest that the map kinase cascade and related pathways are activated continuously in proliferating hsl cells . further study is required to elucidate how these proteins regulate continuous proliferation of hsl cells .
to understand the molecular mechanism underlying vigorous proliferative activity of hepatic stem - like ( hsl ) cells , we performed two - dimensional electrophoresis to identify the proteins statistically more abundant in rapidly growing undifferentiated hsl cells than in sodium butyrate - treated differentiated hsl cells . matrix - assisted laser desorption / ionization - time of flight mass spectrometry and mascot search identified 6 proteins including prohibitin , vimentin , ezrin , annexin a3 , acidic ribosomal phosphoprotein p0 and grp75 . prohibitin and vimentin control the mitogen - activated protein ( map ) kinase pathway . ezrin is phosphorylated by various protein - tyrosine kinases and modulates interactions between cytoskeletal and membrane proteins . annexin a3 has a role in dna synthesis . acidic ribosomal phosphoprotein p0 and grp75 play in protein synthesis . these results suggest that the proteins related to the map kinase cascade had some role in continuous proliferation of hsl cells without differentiation .
Introduction Experimental procedure Cell culture Two-dimensional gel electrophoresis MALDI-TOF-MS analysis Reverse transcription-polymerase chain reaction (RT-PCR) Results and discussion
hepatic stem - like ( hsl ) cells have a potential of self - renewal and an ability to differentiate to hepatocytes and biliary duct cells ( nagai et al . if the mechanisms underlying continuous proliferation of hsl cells without differentiation become clear , many tissue stem cells will be applicable to cell transplantation therapy for many types of diseases . to reveal the molecular mechanisms that sustain proliferative activity of hsl cells with pluripotency , it is necessary to identify the molecular event induced by nab treatment . in this study , we performed two - dimensional electrophoresis ( 2-de ) to search for proteins statistically more abundant in undifferentiated hsl cells than in nab - treated cells , and identified those proteins by matrix - assisted laser desorption / ionization - time of flight mass spectrometry ( maldi - tof - ms ) analysis . these proteins might be participated in the molecular mechanism that causes the vigorous proliferative activity of the hsl cells . the primers used were : ezrin forward ; 5-cagagagtcatggaccagca-3 , ezrin reverse ; 5-cggggtcaacttgtcatctt-3 , prohibitin forward ; 5-ggcatgcctgagtagaccttg-3 , prohibitin reverse ; 5-tcacggttaagagggaatgg-3 , vimentin forward ; 5-agatcgatgtggacgtttcc-3 , vimentin reverse ; 5-cacctgtctccggtattcgt-3 , annexin a3 forward ; 5-ttgatgccaagcaactgaag-3 , annexin a3 reverse ; 5-caggctttcgtctcttccac-3 , glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) forward ; 5-ccatcaccatcttccaggag-3 , gapdh reverse ; 5-cctgctcaccaccttcttg-3. the primers used were : ezrin forward ; 5-cagagagtcatggaccagca-3 , ezrin reverse ; 5-cggggtcaacttgtcatctt-3 , prohibitin forward ; 5-ggcatgcctgagtagaccttg-3 , prohibitin reverse ; 5-tcacggttaagagggaatgg-3 , vimentin forward ; 5-agatcgatgtggacgtttcc-3 , vimentin reverse ; 5-cacctgtctccggtattcgt-3 , annexin a3 forward ; 5-ttgatgccaagcaactgaag-3 , annexin a3 reverse ; 5-caggctttcgtctcttccac-3 , glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) forward ; 5-ccatcaccatcttccaggag-3 , gapdh reverse ; 5-cctgctcaccaccttcttg-3. in the following study , hsl cells were incubated with 20 mm nab for 18 h. to clarify the proteins involved in proliferation of hsl cells , we compared the proteins expressed in hsl cells before and after nab treatment by 2-de . mascot search identified 6 proteins , namely prohibitin , vimentin , ezrin , annexin a3 , acidic ribosomal phosphoprotein p0 and grp75 ( fig . significant morphological changes were observed 18 h after 20 mm nab treatment hsl cells two - dimensional electrophoresis . it is expected that prohibitin has some role in activating classical mitogen - activated protein ( map ) kinase cascade in intact hsl cells to continuous proliferation . 2007 ) , in hsl cells , nab down regulates prohibitin and vimentin expression . in the liver , annexin a3 rna interference study suggested that annexin a3 has a role in dna synthesis ( niimi et al . to confirm whether prohibitin , vimentin , ezrin and annexin a3 expression was regulated transcriptionally , 4 , the mrna expression levels of the proteins selected were not changed 18 h after nab treatment . the sizes of the amplified bands of prohibitin , vimentin , ezrin and annexin a3 were about 200 bp as expected . no significant differences were observed between control and nab treated hsl cells expression of mrna for prohibitin , vimentin , ezrin and annexin a3 . the sizes of the amplified bands of prohibitin , vimentin , ezrin and annexin a3 were about 200 bp as expected . , our study demonstrates that the proteins regulating the map kinase cascade , cytoskeletons , chaperon and protein synthesis system , have some role to continue proliferation of hsl cells without differentiation . these results suggest that the map kinase cascade and related pathways are activated continuously in proliferating hsl cells .
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there is evidence that sufficient and restoring sleep is associated with a wide variety of increased cognitive performance1,2 and emotional processing,3,4 and also with personality traits5 such as optimism6 and mental toughness.7,8 in contrast , dysfunctional cognitive emotional processes such as symptoms of depression,9,10 anxiety,9 and perceived stress1113 are associated with poor sleep quality . as regards the personality traits of perfectionism , numerous studies indicate that perfectionism and poor sleep are associated both cross - sectionally14,15 and longitudinally.16,17 perfectionism is understood as a set of cognitive emotional attitudes such as unrealistically high and rigid standards for performance , fear of failure , excessive self - criticism , and an inability to derive satisfaction from achieved goals.1820 in a search for the developmental origins of perfectionism , hibbart and walton21 conducted semistructured interviews and observed that unlike nonperfectionist undergraduate students , perfectionists reported feeling pressure from their families to succeed . moreover , perfectionists also reported that their parents were overly critical of their mistakes when they were growing up . hibbart and walton21 concluded that perfectionism was a personality trait that seemed to develop over childhood and adolescence as a result of parents ( dysfunctional ) expectancies and pressure . as regards the definition of the personality trait of perfectionisms , there is agreement that perfectionism consists of a set of dysfunctional cognitive emotional processes.1821 this definition is important because numerous studies indicate that dysfunctional cognitive emotional processes cause sleep disturbances . to illustrate , riemann et al13 proposed a cognitive behavioral and neurobiological model of insomnia to explain this link : briefly , psychological stress and inadequate problem - solving , along with worrying and rumination , lead to sleep disruptions ( ie , delayed sleep onset latency , more awakenings after sleep onset , early awakening in the morning ) . in parallel to these cognitive emotional processes and sleep disruptions , physiological processes such as an increased secretion of cortisol and orexin , and a decreased secretion of serotonin , lead in the longer term to acute cortical arousal and a deterioration in homeostasis . riemann et al13 thus explain sleep disturbances as the result of cognitive emotional processes in conjunction with underlying neurophysiological processes . in addition , harvey22 claims that individuals who suffer from insomnia tend to be overly worried about their sleep and about the daytime consequences of not getting enough sleep . put simply , people suffering from insomnia maintain sleep disturbances via further dysfunctional cognitive emotional processes . however , only a few studies have focused so far on the relationship between perfectionism and sleep , and this relationship deserves particular attention . first , de azevedo et al14 observed , among a sample of 1,163 undergraduate students aged between 17 and 25 years , that socially prescribed perfectionism was associated with sleep disturbance . second , azevedo et al16 showed that socially prescribed perfectionism predicted poor sleep 1 and 2 years later , suggesting therefore that perfectionism traits causally predicted sleep disturbances among young adults . the authors observed that relative to non - insomniacs , insomniacs were more likely to report doubts about action , frequent parental criticism , and concerns about action . however , focusing on sleep continuity variables , only the perception of increased parental criticism was associated with delayed sleep onset , whereas all other perfectionism traits ( concerns about action , doubts about action ) were unrelated to sleep continuity variables . additionally , fourth , spiegelhalder et al23 investigated punctuality behavior ( here understood as a sign of perfectionist behavior ) among patients admitted to the sleep lab and observed that arriving at the sleep lab earlier than arranged was not associated with scores for insomnia , but a strong relationship was observed between polysomnographic sleep parameters and punctuality ; short sleep duration was significantly associated with early arrival times at the sleep laboratory , suggesting therefore , that dimensions of perfectionist traits ( here , punctuality ) may causally be linked to poor sleep . whereas the pattern of results of the abovementioned studies suggest a bidirectional relationship between poor sleep and perfectionist traits , to our knowledge possible confounders and latent factors have not thus far been taken into account . this might be surprising given that , among dysfunctional cognitive emotional processes , perceived stress or poor emotion regulation , for example , might contribute to an unknown degree to the explained variance between perfectionism traits and sleep . fifth , in these respects , and again to our knowledge , jansson - frjmark and linton17 were the first to investigate the predictive value of perfectionism traits for preexisting and future sleep disturbances , while introducing emotional distress as a control . second , when introducing emotional distress as a confounder , none of the perfectionism subscales contributed significantly to the explanation of preexisting or future insomnia . importantly , we note that the approach of jansson - frjmark and linton17 corresponds to a mediational model of coping , emotional regulation as well as perceived stress in the relationship of trait perfectionism on current sleep disturbance.2428 collectively , only five scientific studies focused on the relationship between perfectionism and sleep more thoroughly , and only one study17 introduced possible mediating or confounding factors . we took these observations into account and investigated the associations between perfectionism traits and sleep pattern , while concomitantly assessing perceived stress , coping strategies , and dimensions of emotion regulation . we believe that the results may be potentially of practical importance in that , by definition , cognitive behavioral therapies focus on modifying dysfunctional cognitive emotional processes such as perceived stress , coping , and emotion regulation , while , on the flip side , modifying personality traits , which are products of long - term developmental processes , requires more psychotherapeutic effort , and with uncertain results ; accordingly , treating personality - related sleep disturbances would turn out much more difficult . or simply put , if it turns out that perceived stress , coping , and dysfunctional emotion regulation are responsible for the association between personality traits of perfectionism and sleep , then a cognitive behavioral approach to treat these dysfunctional coping strategies and emotion regulation underlying perfectionism traits and sleep disturbances might be more successful . the following two hypotheses were formulated : first , following azevedo et al,16 de azevedo et al14 and vincent and walker15 we expected a direct association between perfectionism traits and poor sleep . second , following jansson - frjmark and linton17 we expected that the significant association between perfectionism and sleep would disappear when introducing perceived stress , coping , and emotion regulation as mediating factors , that is , following others2428 we expected that the association between perfectionism and sleep disturbances would be mediated via coping , emotional regulation , and perceived stress . a total of 346 young adults ( m=23.87 years ; sd [ standard deviation ] = 1.93 ; 54.6% females ) took part in the study . they were recruited from among students of the faculties of medicine and psychology at the university of basel ( switzerland ) . all participants were informed of the purpose of the study and the voluntary basis of their participation . afterwards , they completed a booklet consisting of questionnaires related to perfectionism , sleep , stress , emotion regulation , and mental toughness , as described in more detail in the tools section . after completion , participants received a voucher of chf15.00 ( about 12/usd 15.00 ) for a lunch at the university canteen . the study was approved by the local ethical committee of basel , switzerland ( ethic commission beider basel , basel , switzerland ) and performed in accordance with the ethical standards laid down in the declaration of helsinki . a total of 346 young adults ( m=23.87 years ; sd [ standard deviation ] = 1.93 ; 54.6% females ) took part in the study . they were recruited from among students of the faculties of medicine and psychology at the university of basel ( switzerland ) . all participants were informed of the purpose of the study and the voluntary basis of their participation . afterwards , they completed a booklet consisting of questionnaires related to perfectionism , sleep , stress , emotion regulation , and mental toughness , as described in more detail in the tools section . after completion , participants received a voucher of chf15.00 ( about 12/usd 15.00 ) for a lunch at the university canteen . the study was approved by the local ethical committee of basel , switzerland ( ethic commission beider basel , basel , switzerland ) and performed in accordance with the ethical standards laid down in the declaration of helsinki . perfectionism was assessed with the german version ( fmps - d)19 of the frost multidimensional perfectionism scale ( fmps).18 this self - report measure consists of 35 items which , following frost et al18 are divided between the following dimensions : concern over mistakes , doubts about actions ; parental expectations ; parental criticism , and personal standards . here , however , for the german version , we followed stber19 and scored the questionnaire for the following dimensions : a composite combining items for concerns over mistakes ( i should be upset if i make a mistake . ) and doubts about action ( it takes me a long time to do something right ) , labeled concerns and doubts.a composite combining items for parental expectations ( eg , my parents have expected excellence from me . ) and parental criticism ( eg , ) , labeled parental expectations and criticisms.personal standards ( eg , i set higher goals than most people . ) . a composite combining items for concerns over mistakes ( i should be upset if i make a mistake . ) and doubts about action ( it takes me a long time to do something right ) , labeled concerns and doubts . a composite combining items for parental expectations ( eg , my parents have expected excellence from me . ) and parental criticism ( eg , answers were given on 5-point likert scales ranging from 0 (= not at all true ) to 4 (= definitively true ) , with higher scores reflecting greater perfectionism ( cronbach s =0.90 ) . the seven items , answered on five - point rating scales ( 1= not at all , 5= very much ) , refer to difficulty in falling asleep , difficulties maintaining sleep , increased daytime fatigue , and worrying about sleep . the higher the overall score , the more the respondent is assumed to suffer from insomnia ( cronbach s =0.89 ) . to assess sleep schedules and sleep - related psychological functioning , we administered a brief questionnaire based on the pittsburgh sleep quality index ( psqi).30 this asks about sleep duration ( hours ) , sleep onset latency ( minutes ) , and number of awakenings after sleep onset for the last five working days . additionally , participants reported , on eight - point likert - scales , for the mornings : sleep quality ( 8= very good sleep quality ) , feeling of being restored ( 8= completely restored ) , mood in the morning ( 8= very good mood ) ; during the day : tiredness during the day ( 8= very tired ; higher scores reflect greater tiredness ) , and concentration during the day ( 8= very good concentration ) ; for the evenings : mood in the evening ( 8= very good mood ) ; ( cronbach s =0.84 ) . the perceived stress scale31 consists of ten items and was used to determine perceived overall stress that occurred over the previous month . answers were given on five - point rating scales ranging from 1 ( never ) to 5 ( very often ) , with higher scores reflecting greater perceived stress ( cronbach s =0.89 ) . the questionnaire consists of 18 items and assesses positive and negative coping strategies.32 positive coping strategies are those that reduce tension in both the short- and long - term , including minimizing the situation , controlling the situation , and self - instruction . negative coping strategies are those that reduce tension in the short - term but increase stress in the long - term , including social withdrawal , rumination , and resignation . answers were given on five - point rating scales ranging from 1 ( very unlikely ) to 5 ( very likely ) . the higher the score , the more pronounced is the coping strategy ( cronbach s =0.87 ) . two composite mean scores were computed reflecting positive and negative coping strategies , respectively . to further calculate an overall score for coping strategies , the ratio between positive and negative coping was calculated , with higher ratios reflecting a greater reliance on positive as opposed to negative coping strategies . to assess emotion regulation , we used the emotionale- kompetenz - fragebogen ( emotional competencies inventory).33 the inventory consists of 62 items and assesses the following dimensions : perception and acknowledgement of own emotions ( pae : sometimes i feel sad without knowing why ) ; regulation and control of own emotions ( rce : when i feel myself getting angry , i know how to cool down again ) ; emotional expressivity ( ee : i can express my feelings very well ) ; perception of others emotions ( poe : i can perceive and describe my friend s emotions very well ) . answers are given on five - point - likert scales with the anchor points 1 (= never / not at all true ) to 5 (= practically always / definitively true ) . participants were asked to fill in the 18-item mental toughness questionnaire , the short form of the mtq48.34 the short form provides a general score for mental toughness . answers on the mtq18 are given on five - point likert - type scales ranging from 1 (= strongly disagree ) to 5 (= strongly agree ) . items were summed , with higher scores reflecting greater mt ( cronbach s =0.91 ) . next , to calculate the relation between perfectionism traits and sleep parameters ( sleep disturbance , sleep duration , sleep onset latency , awakenings after sleep onset , daytime sleepiness ) , perceived stress , coping , mental toughness , and emotion regulation , a series of pearson s correlations was calculated . finally , to calculate both the direct and indirect relations of perfectionism with sleep disturbance via perceived stress , coping , mental toughness , and emotion regulation , a structural equation model ( sem ) was computed , using amos ( ibm corporation , armonk , ny , usa ) . , all statistical analyses were calculated with spss 20.0 ( imb corporation , armonk , ny , usa ) for apple mcintosh . perfectionism was assessed with the german version ( fmps - d)19 of the frost multidimensional perfectionism scale ( fmps).18 this self - report measure consists of 35 items which , following frost et al18 are divided between the following dimensions : concern over mistakes , doubts about actions ; parental expectations ; parental criticism , and personal standards . here , however , for the german version , we followed stber19 and scored the questionnaire for the following dimensions : a composite combining items for concerns over mistakes ( i should be upset if i make a mistake . ) and doubts about action ( it takes me a long time to do something right ) , labeled concerns and doubts.a composite combining items for parental expectations ( eg , my parents have expected excellence from me . ) and parental criticism ( eg , ) , labeled parental expectations and criticisms.personal standards ( eg , i set higher goals than most people . ) . a composite combining items for concerns over mistakes ( i should be upset if i make a mistake . ) and doubts about action ( it takes me a long time to do something right ) , labeled concerns and doubts . a composite combining items for parental expectations ( eg , my parents have expected excellence from me . ) and parental criticism ( eg , answers were given on 5-point likert scales ranging from 0 (= not at all true ) to 4 (= definitively true ) , with higher scores reflecting greater perfectionism ( cronbach s =0.90 ) . the seven items , answered on five - point rating scales ( 1= not at all , 5= very much ) , refer to difficulty in falling asleep , difficulties maintaining sleep , increased daytime fatigue , and worrying about sleep . the higher the overall score , the more the respondent is assumed to suffer from insomnia ( cronbach s =0.89 ) . to assess sleep schedules and sleep - related psychological functioning , we administered a brief questionnaire based on the pittsburgh sleep quality index ( psqi).30 this asks about sleep duration ( hours ) , sleep onset latency ( minutes ) , and number of awakenings after sleep onset for the last five working days . additionally , participants reported , on eight - point likert - scales , for the mornings : sleep quality ( 8= very good sleep quality ) , feeling of being restored ( 8= completely restored ) , mood in the morning ( 8= very good mood ) ; during the day : tiredness during the day ( 8= very tired ; higher scores reflect greater tiredness ) , and concentration during the day ( 8= very good concentration ) ; for the evenings : mood in the evening ( 8= very good mood ) ; ( cronbach s =0.84 ) . the perceived stress scale31 consists of ten items and was used to determine perceived overall stress that occurred over the previous month . answers were given on five - point rating scales ranging from 1 ( never ) to 5 ( very often ) , with higher scores reflecting greater perceived stress ( cronbach s =0.89 ) . the questionnaire consists of 18 items and assesses positive and negative coping strategies.32 positive coping strategies are those that reduce tension in both the short- and long - term , including minimizing the situation , controlling the situation , and self - instruction . negative coping strategies are those that reduce tension in the short - term but increase stress in the long - term , including social withdrawal , rumination , and resignation . answers were given on five - point rating scales ranging from 1 ( very unlikely ) to 5 ( very likely ) . the higher the score , the more pronounced is the coping strategy ( cronbach s =0.87 ) . two composite mean scores were computed reflecting positive and negative coping strategies , respectively . to further calculate an overall score for coping strategies , the ratio between positive and negative coping was calculated , with higher ratios reflecting a greater reliance on positive as opposed to negative coping strategies . to assess emotion regulation , we used the emotionale- kompetenz - fragebogen ( emotional competencies inventory).33 the inventory consists of 62 items and assesses the following dimensions : perception and acknowledgement of own emotions ( pae : sometimes i feel sad without knowing why ) ; regulation and control of own emotions ( rce : when i feel myself getting angry , i know how to cool down again ) ; emotional expressivity ( ee : i can express my feelings very well ) ; perception of others emotions ( poe : i can perceive and describe my friend s emotions very well ) . answers are given on five - point - likert scales with the anchor points 1 (= never / not at all true ) to 5 (= practically always / definitively true ) . participants were asked to fill in the 18-item mental toughness questionnaire , the short form of the mtq48.34 the short form provides a general score for mental toughness . answers on the mtq18 are given on five - point likert - type scales ranging from 1 (= strongly disagree ) to 5 (= strongly agree ) . items were summed , with higher scores reflecting greater mt ( cronbach s =0.91 ) . next , to calculate the relation between perfectionism traits and sleep parameters ( sleep disturbance , sleep duration , sleep onset latency , awakenings after sleep onset , daytime sleepiness ) , perceived stress , coping , mental toughness , and emotion regulation , a series of pearson s correlations was calculated . finally , to calculate both the direct and indirect relations of perfectionism with sleep disturbance via perceived stress , coping , mental toughness , and emotion regulation , a structural equation model ( sem ) was computed , using amos ( ibm corporation , armonk , ny , usa ) . , all statistical analyses were calculated with spss 20.0 ( imb corporation , armonk , ny , usa ) for apple mcintosh . perfectionism traits were associated with sleep disturbances ( isi ) , more awakenings after sleep onset , lower mood in the morning and in the evening , feeling less restored in the morning , poorer concentration during the day , and greater tiredness during the day . no significant correlations were found for sleep quality , sleep duration , and for sleep onset latency ( with one exception ) . perfectionism traits were significantly associated with greater perceived stress , more reliance on negative coping strategies , and less reliance on positive coping strategies . perfectionisms traits were significantly associated with lower scores for perception and acknowledgement of own emotion , and the regulation and control of own emotions . no significant associations were found for emotional expressivity ( with one exception ) or for the perception of others emotions . two perfectionism traits ( concerns and doubts ; personal standards ) were associated with lower mental toughness . to calculate both the direct and indirect effects of perfectionism on sleep disturbance via perceived stress , coping , mental toughness , and emotion regulation , a sem was computed . with respect to the goodness of fit criteria as proposed by hu and bentler35 and mcdonald and ho,36 the model represented a satisfying fit ( goodness of fit in square brackets ) : /df=22.36 , p<0.05 [ < df ; here , 4 ] , agfi ( adjusted goodness of fit index ) = 0.91 [ 0.95 ] , pclose = 0.78 [ > 0.50 ] , cfi ( confirmatory fit index ) = 0.1791 [ > 0.90 ] , rmr ( root mean squared residual ) = 0.018 [ < 0.08 ] and rmsea ( root mean square error of approximation ) = 0.025 [ 0.05 ] . the statistically significant and direct association between perfectionism and sleep disturbances ( isi ) was =0.187 ( p<001 ) . when perceived stress , coping , emotion regulation , and mental toughness were entered in the equation , the direct effect of perfectionism on sleep disturbance ( isi ) decreased to 0 ; sleep disturbance was predicted by greater perceived stress , poor coping , low emotion regulation , and low mental toughness ( figure 1 ) . ( sobel z = 2.01 , p<0.05 ; 95% asymmetric ci [ confidence interval ] = 0.010.05 , p<0.05 ) . to calculate both the direct and indirect effects of perfectionism on sleep disturbance via perceived stress , coping , mental toughness , and emotion regulation , a sem was computed . with respect to the goodness of fit criteria as proposed by hu and bentler35 and mcdonald and ho,36 the model represented a satisfying fit ( goodness of fit in square brackets ) : /df=22.36 , p<0.05 [ < df ; here , 4 ] , agfi ( adjusted goodness of fit index ) = 0.91 [ 0.95 ] , pclose = 0.78 [ > 0.50 ] , cfi ( confirmatory fit index ) = 0.1791 [ > 0.90 ] , rmr ( root mean squared residual ) = 0.018 [ < 0.08 ] and rmsea ( root mean square error of approximation ) = 0.025 [ 0.05 ] . the statistically significant and direct association between perfectionism and sleep disturbances ( isi ) was =0.187 ( p<001 ) . when perceived stress , coping , emotion regulation , and mental toughness were entered in the equation , the direct effect of perfectionism on sleep disturbance ( isi ) decreased to 0 ; sleep disturbance was predicted by greater perceived stress , poor coping , low emotion regulation , and low mental toughness ( figure 1 ) . ( sobel z = 2.01 , p<0.05 ; 95% asymmetric ci [ confidence interval ] = 0.010.05 , p<0.05 ) . the key findings of the present study are that perfectionism traits were associated with poorer sleep , though if perceived stress , coping , emotion regulation , and mental toughness were entered into the equation , the importance of perfectionism traits dramatically decreased . therefore , the pattern of results suggests that the underlying psychological mechanisms of perfectionism seem to be greater perceived stress , poor coping , low emotion regulation , and low mental toughness , which are in turn associated with poorer sleep . our first hypothesis was that there would be a significant association between perfectionism traits and poor sleep , and this hypothesis was supported . in this respect , the present pattern of results concurs with previous research,1416,23 showing that perfectionism , understood as a dysfunctional set of attitudes toward achieving high performance as a function of one s own standards , concerns about mistakes and doubts , but above all as a function of parents expectations and criticism , is associated with poor sleep . the present study expands upon previous work in that we showed that in a sample of young adults perfectionism traits were also negatively associated with a broad range of psychological processes such as greater perceived stress , poor coping , poor emotion regulation , and low mental toughness , along with increased tiredness and decreased concentration during the day , and lower mood in the morning and in the evening . our second hypothesis , however , was that psychological factors such as stress and emotion regulation would mediate the relation between sleep and perfectionism . this hypothesis was derived from the findings of morin et al 24 leblanc et al25,26 and fernandez - mendoza et al27,28 who claimed the importance of coping and perceived stress as an important factor to explain the emergence and maintenance of sleep disturbances , and from the study by jansson - frjmark and linton.17 they reported two main findings : first , concerns over mistakes were significantly related to preexisting and future insomnia . second , when introducing emotional distress as a control , none of the perfectionism subscales contributed significantly to explaining preexisting and future insomnia . we took this pattern of results into account and confirmed the second hypothesis , that is jansson - frjmark and lintons17 observation : when perceived stress , poor coping , low emotion regulation , and low mental toughness were entered in the equation , perfectionism traits were no longer associated with poor sleep , that is to say , they were mediated by coping and perceived stress . therefore , we conclude that dysfunctional cognitive emotional processes such as stress perception and coping , and poor emotion regulation underlying perfectionist behavior explain most of the association between perfectionism traits as such and poor sleep . most importantly , the present pattern of results fits well within the large body of research showing that it is not personality traits per se , but the underlying coping strategies and emotion regulation processes , which are predisposing and perpetuating factors of sleep disturbance.2428 the present data do not shed any further light on the reasons for the association between perfectionism and stress or poor emotion regulation , though three overlapping theoretical frameworks support the following possibility . both riemann et al13 and harvey22 claim that sleep disturbances are caused and maintained by dysfunctional cognitive emotional processes ; more specifically , riemann et al13 propose that psychological stress and inadequate problem - solving , along with worrying and rumination , lead to sleep disruptions ( ie , delayed sleep onset latency , more awakenings after sleep onset , early awakening in the morning ) . importantly , spiegelhalder et al23 showed that hyper - punctuality among patients admitted to the sleep lab arriving earlier than agreed at the lab ( interpreted here as a form of perfectionist behavior ) was significantly associated with poor polysomnographic sleep parameters . short sleep duration was significantly associated with early arrival times at the sleep laboratory , suggesting therefore , that aspects of perfectionism ( here , punctuality ) may be causally linked to poor sleep . likewise , harvey22 claims that individuals who suffer from insomnia tend to be overly worried about their sleep and about the daytime consequences of not getting enough sleep or , put simply , people suffering from insomnia maintain sleep disturbances via further dysfunctional cognitive emotional processes . last , morin et al24 leblanc et al25,26 and fernandez- mendoza et al27,28 showed that it is not personality traits per se , but the underlying coping strategies and emotion regulation processes , which are predisposing and perpetuating factors of sleep disturbance . taken together , the three theoretical frameworks help to explain why it is not perfectionism per se , but rather the underlying psychological mechanisms that best explain the association between perfectionism and poor sleep . as regards the association between mental toughness ( a construct consisting of a high degree of control , commitment , challenge , and confidence ; clough et al34 ) and sleep , we observed that the present pattern of results fits well within previous literature showing that mental toughness was associated with increased subjective7,37 and objective sleep8 among adolescents . in this respect , the present findings expand upon earlier results in that the positive association between higher mental toughness and favorable sleep also holds among young adults . despite the intriguing results , several limitations warrant against overgeneralization of the results , and findings should be interpreted cautiously . first , participants were recruited from among university students and therefore the sample is not representative of young adults as a whole . more specifically , it remains unanswered to what extent the present sample is representative for other samples of adults such as nonacademic people , older adults , or people suffering from severe insomnia . however , we claim that the present pattern of results is a useful step in differentiating cognitive processes that may impact sleep , which could act as a catalyst for future research in clinical samples . second , the data consists entirely of self - reports ; experts ratings such as a brief psychiatric interview could have provided further insight into possible psychiatric or personality disorders . third , and most importantly , sleep was not objectively assessed , leaving it uncertain to what extent participants with poor sleep were also inclined to report poor psychological functioning . or simply put : there is the risk , that self - reported poor sleep quality was associated with inadequate and negative self - reported psychological functioning . future studies should therefore also include objective sleep measurements ( actigraphy , sleep - eeg [ electroencephalogram ] ) , though we also note that for large samples questionnaire - based measures remain the gold standard.38 fourth , no biological markers were assessed , though , specifically , there is evidence that , for instance , cortisol secretion is associated with sleep and stress regulation . further , bdnf as indicator of neuronal plasticity plays a role in insomnia and coping with stress.39 fifth , the present pattern of results might have emerged due to further latent but unassessed variables . sixth , no cognitive academic performance was assessed , which is a global concern for students ; accordingly , a wealth of studies shows the association between cognitive abilities , academic performance , and sleep.1,40 last , the cross - sectional design of the study precludes any conclusion as to the direction of influence between perfectionism traits , perceived stress , coping , mental toughness , and sleep , and haario et al41 showed the bidirectional association between sleep and psychological functioning . longitudinal studies such as the research of jansson - frjmark and linton17 and azevedo et al16 allow for a more detailed picture as to the cause the pattern of results suggests that among a sample of young adults , perfectionism traits are related to poor sleep , though cognitive emotional processes such as stress , coping , and emotion regulation underlying perfectionism traits better explain variance in sleep than perfectionism traits per se . in our opinion , these results should allow more focused and successful psychotherapeutic treatment of perfectionist patients suffering from poor sleep . however , to further prove these observations , the present pattern of results should be replicated among clinical samples .
backgroundperfectionism is understood as a set of personality traits such as unrealistically high and rigid standards for performance , fear of failure , and excessive self - criticism . previous studies showed a direct association between increased perfectionism and poor sleep , though without taking into account possible mediating factors . here , we tested the hypothesis that perfectionism was directly associated with poor sleep , and that this association collapsed , if mediating factors such as stress and poor emotion regulation were taken into account.methodsthree hundred and forty six young adult students ( m=23.87 years ) completed questionnaires relating to perfectionism traits , sleep , and psychological functioning such as stress perception , coping with stress , emotion regulation , and mental toughness.resultsperfectionism was directly associated with poor sleep and poor psychological functioning . when stress , poor coping , and poor emotion regulation were entered in the equation , perfectionism traits no longer contributed substantively to the explanation of poor sleep.conclusionthough perfectionism traits seem associated with poor sleep , the direct role of such traits seemed small , when mediating factors such as stress perception and emotion regulation were taken into account .
Introduction Methods Sample Tools Perfectionism Subjective sleep Sleep schedules and sleep-related psychological functioning Perceived stress Coping with stress Emotion regulation Mental toughness Statistical analysis Results Direct and indirect associations between perfectionism and sleep Discussion Conclusions
as regards the personality traits of perfectionism , numerous studies indicate that perfectionism and poor sleep are associated both cross - sectionally14,15 and longitudinally.16,17 perfectionism is understood as a set of cognitive emotional attitudes such as unrealistically high and rigid standards for performance , fear of failure , excessive self - criticism , and an inability to derive satisfaction from achieved goals.1820 in a search for the developmental origins of perfectionism , hibbart and walton21 conducted semistructured interviews and observed that unlike nonperfectionist undergraduate students , perfectionists reported feeling pressure from their families to succeed . we believe that the results may be potentially of practical importance in that , by definition , cognitive behavioral therapies focus on modifying dysfunctional cognitive emotional processes such as perceived stress , coping , and emotion regulation , while , on the flip side , modifying personality traits , which are products of long - term developmental processes , requires more psychotherapeutic effort , and with uncertain results ; accordingly , treating personality - related sleep disturbances would turn out much more difficult . or simply put , if it turns out that perceived stress , coping , and dysfunctional emotion regulation are responsible for the association between personality traits of perfectionism and sleep , then a cognitive behavioral approach to treat these dysfunctional coping strategies and emotion regulation underlying perfectionism traits and sleep disturbances might be more successful . second , following jansson - frjmark and linton17 we expected that the significant association between perfectionism and sleep would disappear when introducing perceived stress , coping , and emotion regulation as mediating factors , that is , following others2428 we expected that the association between perfectionism and sleep disturbances would be mediated via coping , emotional regulation , and perceived stress . afterwards , they completed a booklet consisting of questionnaires related to perfectionism , sleep , stress , emotion regulation , and mental toughness , as described in more detail in the tools section . afterwards , they completed a booklet consisting of questionnaires related to perfectionism , sleep , stress , emotion regulation , and mental toughness , as described in more detail in the tools section . next , to calculate the relation between perfectionism traits and sleep parameters ( sleep disturbance , sleep duration , sleep onset latency , awakenings after sleep onset , daytime sleepiness ) , perceived stress , coping , mental toughness , and emotion regulation , a series of pearson s correlations was calculated . when perceived stress , coping , emotion regulation , and mental toughness were entered in the equation , the direct effect of perfectionism on sleep disturbance ( isi ) decreased to 0 ; sleep disturbance was predicted by greater perceived stress , poor coping , low emotion regulation , and low mental toughness ( figure 1 ) . when perceived stress , coping , emotion regulation , and mental toughness were entered in the equation , the direct effect of perfectionism on sleep disturbance ( isi ) decreased to 0 ; sleep disturbance was predicted by greater perceived stress , poor coping , low emotion regulation , and low mental toughness ( figure 1 ) . the key findings of the present study are that perfectionism traits were associated with poorer sleep , though if perceived stress , coping , emotion regulation , and mental toughness were entered into the equation , the importance of perfectionism traits dramatically decreased . therefore , the pattern of results suggests that the underlying psychological mechanisms of perfectionism seem to be greater perceived stress , poor coping , low emotion regulation , and low mental toughness , which are in turn associated with poorer sleep . the present study expands upon previous work in that we showed that in a sample of young adults perfectionism traits were also negatively associated with a broad range of psychological processes such as greater perceived stress , poor coping , poor emotion regulation , and low mental toughness , along with increased tiredness and decreased concentration during the day , and lower mood in the morning and in the evening . we took this pattern of results into account and confirmed the second hypothesis , that is jansson - frjmark and lintons17 observation : when perceived stress , poor coping , low emotion regulation , and low mental toughness were entered in the equation , perfectionism traits were no longer associated with poor sleep , that is to say , they were mediated by coping and perceived stress . therefore , we conclude that dysfunctional cognitive emotional processes such as stress perception and coping , and poor emotion regulation underlying perfectionist behavior explain most of the association between perfectionism traits as such and poor sleep . sixth , no cognitive academic performance was assessed , which is a global concern for students ; accordingly , a wealth of studies shows the association between cognitive abilities , academic performance , and sleep.1,40 last , the cross - sectional design of the study precludes any conclusion as to the direction of influence between perfectionism traits , perceived stress , coping , mental toughness , and sleep , and haario et al41 showed the bidirectional association between sleep and psychological functioning . longitudinal studies such as the research of jansson - frjmark and linton17 and azevedo et al16 allow for a more detailed picture as to the cause the pattern of results suggests that among a sample of young adults , perfectionism traits are related to poor sleep , though cognitive emotional processes such as stress , coping , and emotion regulation underlying perfectionism traits better explain variance in sleep than perfectionism traits per se .
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our understanding of the mechanisms that regulate the organization of developing tissues is based on the idea that cells gain information determining their fates by monitoring the levels of morphogens released by discrete signaling centers , or organizers , in their vicinity ( rogers and schier , 2011 ) . one possibility is that feedback from transcription factors whose expression is regulated by morphogens contributes to the control of the organizers and their morphogen production . the identification of such mechanisms is particularly interesting because they are likely to play a major role in enhancing the precision , stability , and robustness of gene expression patterns in the developing embryo ( sokolowski et al . , 2012 ) . here , we tested whether feedback via the transcription factor pax6 regulates the size and function of a forebrain organizer , the zona limitans intrathalamica ( zli ) . the diencephalon is the caudalmost component of the forebrain and contains the thalamus . during development , interactions between genes expressed around and within the thalamic anlage establish regions with different identities and fates along the embryonic rostral - caudal axis . the transcription factors fezf1 and fezf2 specify a rostral diencephalic domain ( the future prethalamus ; hirata et al . , 2006 ; jeong et al . , 2007 ) , whereas the transcription factors otx2 and irx1 specify a caudal diencephalic domain ( the future thalamus ; hirata et al . , 2006 ) . the zli forms as a thin strip of tissue in the progenitor cell layer at the interface between these domains . it contributes to the organization of the regions around it mainly through its expression of the diffusible morphogen shh ( hashimoto - torii et al . , 2003 ; jeong et al . , 2011 ; kiecker and lumsden , 2004 ; scholpp et al . , 2006 ; scholpp and lumsden , 2010 ; zhou et al . , 2004 ; robertshaw et al . , it appears as a thin spike of shh - expressing tissue extending from basal plate through alar plate toward roof plate ( shimamura et al . , 1995 ) . immediately caudal to it , a small rostral area ( called pth - r ; figure 1b ) , which comprises thalamic progenitors exposed to relatively high levels of shh , expresses ascl1 and nkx2.2 and generates mostly gabaergic neurons that contribute to the ventral lateral geniculate ( vlg ) nucleus ( inamura et al . , 2011 ; suzuki - hirano et al . , 2011 ; vue et al . , 2007 ; robertshaw et al . , . a larger region of thalamic progenitors caudal to pth - r , called pth - c ( figure 1b ) , expresses ngn2 and olig3 rather than ascl1 and nkx2.2 and generates glutamatergic neurons that innervate cortex ( vue et al . , 2007 ; it starts in the neural plate and is initially throughout the entire alar forebrain neuroepithelium ( mastick et al . pax6 is repressed in the zli by developing shh expression ( ericson et al . , 1997 ; macdonald et al . , 1995 ; robertshaw et al . , pax6 is retained by prethalamic progenitors and postmitotic cells and by thalamic progenitors ; the latter express it in a gradient , with pax6 levels increasing with distance from the zli . mutant mice lacking pax6 show progressive defects of diencephalic size and patterning ( grindley et al . , it has been considered that pax6 functions downstream of shh , which represses pax6 ( ericson et al . , 1997 ; 2013 ) , but it has also been reported that loss of pax6 increases the size of the shh - producing zli ( grindley et al . , 1997 ; pratt et al . , 2000 ; chatterjee et al . , 2014 ) , suggesting that pax6 might somehow regulate shh . we examined the influence of pax6 on diencephalic patterning and identified an important cell - autonomous action of pax6 on the expression of shh . our findings indicate a crucial role for mutual repression between pax6 and shh in diencephalic cell specification . in normal e12.5 mice , pax6 expression is highest in the progenitor and postmitotic layers of the prethalamus , absent along the thalamic / prethalamic border , and distributed in a gradient through the progenitor layer of the thalamus ( lowest near to the zli ) ( figure 1a ) . we examined the consequences of pax6 absence for patterning in each of these regions ( figure 1b ) . a marker of prethalamic progenitors , gsx2 , was undetectable in pax6 diencephalon ( figures 1c and 1d ) , and numbers of islet1 + cells were greatly reduced in prethalamic postmitotic cells ( asterisk in figure 1d ) . the absence of gsx2-expressing lineages was shown using a cre recombinase transgene controlled by the gsx2 promoter ( kessaris et al . , 2006 ) with a floxed - stop - gfp reporter ( miyoshi et al . , 2010 ) . in controls , most prethalamic cells expressed gfp ( figure 1e ) , consistent with their descent from gsx2 + prethalamic progenitors , but no gfp+ cells were detected in an equivalent region of pax6 mutants ( asterisk in figure 1f ) . to test whether pax6 is required cell autonomously for expression of gsx2 by prethalamic cells the contributions of pax6 cells to each chimera varied ( nine chimeras were analyzed ) , allowing us to analyze situations in which mutant cells were surrounded by much larger numbers of wild - type cells and those where the opposite was the case . in all , pax6 cells contributed to the prethalamus . in chimeras with a high contribution of mutant cells , none of these cells expressed gsx2 , whereas even small clusters of wild - type prethalamic progenitors embedded among them retained gsx2 expression ( figure 1h ) . similarly , in chimeras with a low contribution of mutant cells , all pax6 progenitors were gsx2 , even those in very small isolated groups ( figures 1i1 m ) , whereas all wild - type progenitors , even individually isolated ones , were gsx2 + ( figures 1l and 1 m ) . in low - contribution chimeras , islet1 was expressed by most pax6 cells ( as it was by most wild - type cells ) exiting the progenitor layer ( figure 1 m ) , but in high - contribution chimeras , it was expressed by a smaller proportion ( about 50% ) of differentiating cells ( figure 1 g ) . these results indicate that cells in the prethalamus have an absolute cell - autonomous requirement for pax6 in order to express gsx2 . pax6 is not required for prethalamic islet1 expression , although the observation that proportions of islet1 + cells are reduced when large proportions of prethalamic cells are pax6 suggests a non - cell - autonomous effect of extensive pax6 loss on prethalamic islet1 expression . in normal diencephalic development , nkx2.2 expression marks a thin strip of cells around the prethalamic / thalamic border , including progenitors around the zli that coexpress olig3 ( figure 2a ) and postmitotic cells extending through the neuroepithelial wall to the developing vlg ( figures 1e and 2a ; kitamura et al . , 1997 ; scholpp and lumsden , 2010 ; vue et al . , 2007 ) . in pax6 diencephalon , this domain was greatly expanded ( double arrow in figure 1f ; figure 2b ) ; it still reached a collection of superficially located nkx2.2 + cells in the same relative position as the vlg of normal embryos ( arrowheads in figure 2b ) . the domains of lhx1 and lhx5 expression in postmitotic cells around the prethalamic / thalamic border were also greatly enlarged in pax6 embryos ( figures 2c2f , s1a , and s1b ) . we tested whether the mechanism of expansion of these domains of expression involved incorrect cellular specification , causing an abnormally broad swathe of cells , many of which would normally have adopted distinct thalamic or prethalamic identities , to take on the identities of cells normally located very close to the prethalamic / thalamic border . ngn2 + lineage cells , which populate the thalamus of wild - type and pax6 embryos ( figures 2i and 2j ) , were labeled using a transgene expressing a tamoxifen - inducible cre recombinase , creer , under the control of the ngn2 promoter ( zirlinger et al . , 2002 ) and a floxed - stop - gfp reporter allele ( miyoshi et al . , 2010 ) . pregnant females received tamoxifen at e10.5 so as to induce reporter activation by e12.5 ( kessaris et al . , 2006 ) , and embryos were collected at e14.5 . in e14.5 controls , gfp+ cells were present throughout the body of the thalamus , and their axons could be seen exiting through the prethalamus ; ngn2 lineage cells close to the thalamic / prethalamic border did not express lhx1/lhx5 ( figures 2i and 2k ) . in e14.5 pax6 embryos , about 75% of the gfp+ cells located in the rostral part of the thalamus were double labeled for lhx1/lhx5 ( figures 2j and 2l ; cells were counted in three sections from each of three embryos ) , providing evidence for the misspecification of many postmitotic cells in this region . examination of the thalamus in pax6 embryos showed an overall reduction in its size ( as marked by sox2 , gbx2 , and ngn2 expression : figures 1e , 1f , 2c , 2d , and s1c s1f ) but an expansion of the part of its progenitor domain closest to the zli at e12.3e13.5 ( figures 2e2h , 2 m , 2n , s1 g , s1h , and s2 ) . this region , known as pth - r ( inamura et al . , 2011 ; suzuki - hirano et al . , 2011 ; 2011 ) , expresses ascl1 , gsx1 , and nkx2.2 but only low levels of ngn2 , and its expansion in pax6 embryos coincides with a reduction in the size of the pth - c , the caudal region of thalamic progenitors , which expresses ngn2 . previous studies showed that pth - r contributes gabaergic neurons ( vue et al . , 2007 ) . our analysis of postmitotic gabaergic neurons ( vgat and gad67 ) and glutamatergic neurons ( mglur1 ) confirmed shrinkage of the glutamatergic population in caudal thalamus and expansion of the gabaergic population in rostral thalamus ( figures s3a s3i ) . expression of six3 in a small group of very rostral postmitotic thalamic cells was also expanded in pax6 mutants ( figures s3j s3l , arrows ) . these findings indicate that the absence of pax6 results in expansion of both pth - r and the gabaergic population of cells that it generates at the expense of the glutamatergic pth - c - derived population . the fact that pax6 is expressed more strongly in pth - c than pth - r progenitors and that rostral territory expands if pax6 is lost suggests that pax6 in pth - c normally suppresses the molecular phenotype associated with the rostral thalamus . to begin addressing how pax6 suppresses the rostral thalamic phenotype , we tested whether its requirement is cell autonomous by examining the expression of rostral thalamic markers in pax6 pax6 chimeras . figure 3 shows examples of chimeras containing high or low proportions of pax6 ( gfp+ ) cells . regardless of the balance of wild - type and mutant cells , the boundary region between the prethalamus and thalamus was always identifiable . its progenitor layer contained cells that were both olig3 + and nkx2.2 + ( figures 3a , 3b , 3d , and 3e ) , and the overlying mantle zone contained nkx2.2 + cells ( figures 3a and 3d ) and lhx1/lhx5 + cells ( figure 3f ) , whose expression overlapped slightly with that of thalamic sox2 expression as occurs in wild - type embryos ( figures 3 g and 3h ) . both wild - type and pax6 cells contributed to this boundary region , which was broader in those chimeras that contained a high proportion of mutant cells ( marked by double - headed arrows in figure 3a ) . nkx2.2 , lhx1/lhx5 , and ascl1 are not normally expressed through the body of the thalamus , but they were expressed by some thalamic pax6 cells in chimeras . examples are shown in figures 3a , 3c , 3f , 3i , 3l , and 3n3q . interestingly , the mutant thalamic cells that showed abnormal expression of nkx2.2 , lhx1/lhx5 , and ascl1 , which their surrounding wild - type neighbors did not , were all located relatively close to the thalamic / prethalamic border ( within about 100200 m ) in clusters of 100200 cells or more ( figures 3a , 3c , 3f , 3i , 3l , and 3n3q ) . even large clusters of pax6 cells more distant from the border expressed the same markers as their wild - type neighbors ( figures 3a , 3f , 3i , 3l , and 3n ) . smaller clusters of pax6 cells always showed the same expression as their wild - type neighbors ( figures 3d , 3j , 3k , and 3r3v ) . these findings indicate that absence of pax6 does not cause a straightforward cell - autonomous upregulation of the rostral thalamic phenotype ( nkx2.2 + , lhx1/lhx5 + , and ascl1 + ) . although mutant cells do upregulate rostral thalamic markers in some circumstances , they need to be in a critical mass relatively close to the thalamic / prethalamic border . another revealing result was that in chimeras containing a high contribution of mutant cells , we observed expanded ascl1 domains immediately caudal to the expanded pax6 zli ( i.e. , corresponding to expanded pth - r ) that comprised wild - type cells , providing clear evidence for a non - cell - autonomous upregulation of ascl1 and loss of ngn2 around the expanded mutant zli ( figures 3l and 3 m ) . in summary , our findings strongly suggest that a major underlying cause of abnormal thalamic patterning in the absence of pax6 is a position - dependent defect of local intercellular signaling . this contrasts with the absolute cell - autonomous requirement for pax6 for gsx2 expression in prethalamus described above . position - dependent defects of signaling among cells around the thalamic / prethalamic border might be caused by defects of the zli . we compared the expression of markers of the zli in control and pax6 embryos using in situ hybridization and immunofluorescence at e12.5e13.5 . the wild - type e12.5 zli is a thin shh - expressing wedge - shaped structure in the alar plate ( figure 4a ) . close to its tip , it appears as a single line of cells expressing both shh and ngn2 ( vue et al . , 2007 ) . we found , however , that as it widens toward its base ( e.g. , at the level of the broken line in figure 4a ) , only its caudal part expresses both ngn2 and shh ( figure 4c ; solid double - headed arrow in figure 4e ) . its rostral part expresses dbx1 ( broken double - headed arrow in figure 4 g ) , and a central domain is both dbx1 + and ngn2 + ( arrow in figure 4i ; summarized in figure 4k ) . in e12.5 pax6 mutants , there was a large rostrocaudal expansion of shh expression in the alar plate in a location equivalent to that of the zli ( figures 4b , 4d , 4f , 4h , 4j , and 4l ) . this was divisible into an enlarged caudal domain expressing both shh and ngn2 ( solid double - headed arrow in figure 4f ) and an enlarged rostral domain expressing both shh and dbx1 ( broken double - headed arrow in figure 4h ) with an enlarged central domain expressing dbx1 and ngn2 + ( double - headed arrow in figure 4j ) . these results indicate that , in the absence of pax6 , the zli and its subdomains , identified by expression / coexpression of shh , ngn2 , and dbx1 , are enlarged , as summarized in figures 4k and 4l . the pitx2-expressing mantle zone immediately superficial to the zli is also expanded in pax6 mutants ( figures s3m s3o ) . we then tested whether the expansion of the zli in pax6 mutants results from the misspecification of cells flanking the normal position of the zli ( e.g. , rather than an overproliferation of correctly specified zli progenitors ) . the normal zli develops at about e9.5e10.5 in the mouse at the interface between a rostral diencephalic fezf1 + domain ( figure 4 m ) and a caudal diencephalic irx1 + domain ( figure 4o ) ( hirata et al . , 2006 ) . it comprises shh+ cells sandwiched between pax6 + domains rostral and caudal to it ( figures 4q and 4u ) . we tested whether expansion of the zli in pax6 embryos involves the erroneous activation of shh by flanking cells expressing the pax6 gene . we used two approaches to identify the pax6 + cells in pax6 mutants . in the first , they were labeled by gfp produced from a pax6-yeast artificial chromosome - reporter transgene that expresses gfp in pax6-expressing cells regardless of whether they are pax6 or not ( pax6-gfp ; tyas et al . , in e10.5 pax6 embryos , the boundary between fezf1 and irx1 expression was as clear as in controls ( arrows in figures 4m4p ) , indicating that pax6 is not required for this early patterning and allowing us to identify the position of zli formation . in complete contrast to controls ( figures 4q and 4u ) , we observed a relatively large block of tissue whose cells coexpressed high levels of pax6-gfp and shh ( figures 4r and 4v ) . this tissue was largely caudal to the position marked for zli development by the interface of fezf1 + and irx1 + territories ( compare figures 4n and 4p with figures 4r and 4v ) . normally , the difference in pax6 expression between prethalamus ( high ) and thalamus ( low ) becomes greater by e12.5 , as shown by expression of pax6-gfp in figures 4s and 4w . as at e10.5 , in e12.5 pax6 embryos ( figures 4 t and 4x ) , the caudal part of the abnormally broad shh+ domain extended abnormally far into the low pax6-gfp - expressing thalamic progenitor layer ( double - headed arrow in figure 4x ) . the rostral part of the abnormally broad shh+ domain overlapped the region of high pax6-gfp prethalamic expression ( broken double - headed arrow in figure 4x ) , with cells in this region being double positive for shh and pax6-gfp expression . at e12.5 , we also observed abnormal coexpression of pax6 and ngn2 mrnas in this rostrally extended part of the expanded pax6 zli ( figures 4y4bb ) . whereas ngn2 and pax6 expression is normally complementary at e12.5 ( with ngn2 in the zli and high pax6 rostral to it ; figures 4y and 4aa ) , in pax6 embryos , many ngn2 + cells in the expanded zli coexpress high levels of pax6 ( figures 4z and 4bb ) . these findings indicate that pax6 progenitor cells rostral and caudal to the position of normal zli formation are misspecified . the failure of cells around the thalamic / prethalamic border to repress shh expression in pax6 embryos might provide an explanation for aspects of the mis - patterning of this region in terms of its abnormal marker - gene expression in pax6 embryos and chimeras . to test this , we first examined chimeras to discover whether the abnormal upregulation of shh in pax6 cells around the thalamic / prethalamic border is cell autonomous . we focused on chimeric embryos in which small minorities of pax6 cells were surrounded by vast majorities of wild - type cells , in which influences from outside pax6 cell groups should be relatively normal . in chimeras , small clusters of pax6 ( gfp+ ) cells throughout the diencephalon all expressed shh regardless of their size and whether they were in the zli , thalamus , or prethalamus ( figures 5a5e ) . cells embedded in the zli expressed shh at levels that were indistinguishable from those of their shh - expressing wild - type neighbors ( figures 5a5c ) . outside the zli , pax6 cells achieved levels of shh expression that , although raised above those of their wild - type neighbors , were lower than those of cells in the zli ( e.g. , horizontal arrow in figure 5b , and double - headed arrows in figures 5d and 5e ) . we measured the relative intensities of shh labeling across the diencephalon of wild - type and pax6 embryos and in the clusters of pax6 cells in chimeras . the relationship between intensity and distance from the zli was very similar in pax6 mutants and in pax6 cells in chimeras ( figure 5f ) . in control chimeras comprising a mixture of pax6 and pax6 gfp - expressing cells , the gfp+ cells were not clustered but were scattered throughout the diencephalon , the in situ hybridization staining for shh appeared normal , and there was no evidence that levels of shh depended on whether cells were gfp+ or not ( figures s4a s4c ) . these findings indicate that pax6 diencephalic cells activate shh cell autonomously because activation occurred even when mutant cells were embedded in an environment comprising a large majority of wild - type cells . moreover , the results suggest that the mechanism that sets the level of activation with reference to distance from the zli in the absence of pax6 acts cell autonomously because the relationship between the locations of the pax6 patches and their levels of shh expression reflected the relationship in pax6 embryos . these results have the potential to explain the observed mis - patterning of those pax6 cells close to the thalamic / prethalamic border in pax6 mutants and chimeras . they might also explain the expanded ascl1 domain affecting wild - type cells close to the border ( figures 3l and 3 m ) and the lack of repatterning among wild - type cells further from the border . we do not exclude the possibility that factors other than levels of shh expression might also be important in region - specific repatterning . previous studies have shown that expression of ptch1 and gli1 can be upregulated by shh signaling and is normally high in cells adjacent to the zli ( figures 5 g , 5i , and 5k ; bai et al . , 2002 , 2004 ; , 1997 ; goodrich et al . , 1996 ; kiecker and lumsden , 2004 ; lee et al . , 1997 ; marigo and tabin , 1996 ; wijgerde et al . , 2002 ; vue et al . , we tested the prediction that if the abnormal shh expression of pax6 cells has functional consequences for those cells , then they should upregulate ptch1 and gli1 . in situ hybridizations on e10.5 pax6 whole mounts showed that staining for ptch1 spread more broadly than normal through surrounding diencephalic tissue ( arrowheads in figures 5 g and 5h ) . coronal sections of e12.5e13.5 pax6 embryos showed expansion of the ptch1 + areas and abnormally strong gli1 expression in the thalamus and prethalamus ( figures 5i5l ) . outside the zli , clusters of pax6 ( gfp+ ) cells were ptch1 + ( figures 5m5o ) , indicating a response to the shh that they produce . wild - type cells surrounding the zli were ptch1 + , as in normal embryos ( figures 5q and 5r ) . interestingly , ptch1 and gli1 are normally downregulated within areas of high shh expression in the zli and basal plate ( figures 5 g , 5i , and 5k ; marigo and tabin , 1996 ) . ptch1 and gli1 are also downregulated in the expanded zli of pax6 embryos ( figures 5j and 5l ) and by pax6 cells embedded in the zli in chimeras ( figures 5p5r ) . to test whether this might also be caused by exposure to high levels of shh , we administered vismodegib ( gdc-0449 ) , a selective inhibitor of the shh receptor smo ( expressed throughout control and pax6 diencephalon ; figures s5a and s5b ) , to pax6 embryos . this treatment caused a dose - dependent overall reduction of the area of ptch1 expression but an upregulation of ptch1 at the expanded shh+ pax6 zli ( figures s5c s5f ) , in agreement with the hypothesis above . in the forebrain , pax6 is also strongly expressed in the cerebral cortex , and a previous microarray - based analysis found small but significant upregulation of both shh and ptch1 in cortical cells from pax6 embryos ( shh : + 1.38-fold , p = 0.038 ; ptch1 : + 1.36-fold , p = 0.043 ; mi et al . , 2013 ) . in situ hybridization on pax6 embryos did not show obvious upregulation of shh or ptch1 ( figures s4d s4 g ) , but in chimeras , where mutant and wild - type cells are together in the same sections , we did detect slightly stronger staining for shh and ptch1 in pax6 cells ( figures s4h s4k ) . these findings indicate that pax6 also has a cell - autonomous repressive effect on shh in cortex , although its magnitude appears less than in diencephalon . we then tested whether abnormalities of diencephalic patterning in pax6 embryos result from abnormally high shh signaling around the zli , by administering vismodegib ( gdc-0449 ) to inhibit shh signaling in pax6 embryos . we examined the effects of shh blockade on markers whose expression is altered at or around the zli and the thalamic / prethalamic border in pax6 mutants ( dbx1 , ngn2 , lhx5 , nkx2.2 , and ascl1 ) . in wild - types , dbx1 is expressed in the zli and in a gradient through the thalamus ( figures 4 g , 4i , 4k , and 6a ) , whereas in pax6 mutants , its expression is expanded at the zli and greatly reduced in the thalamus ( figures 4h , 4j , 4l , and 6d ) . its expression at the zli was abolished by administration of vismodegib , both in wild - types and pax6 embryos ( figures 6b , 6c , 6e , and 6f ) , indicating that its expression / expanded expression in this region is dependent on shh . in wild - types , the intensity of staining for lhx5 expression around the thalamic / prethalamic border declined with increasing doses of vismodegib , but otherwise , the pattern of expression appeared unaffected ( figures 6g6i ) . in pax6 mutants , the expanded domain of intense lhx5 expression around the thalamic / prethalamic border ( whose position was recognized by shh expression in adjacent sections ) was reduced by increasing doses of vismodegib ( figures 6j6n ) , indicating the importance of shh signaling for this aspect of the mutant phenotype . the pattern of ngn2 expression in wild - types appeared unaffected by treatment with vismodegib , showing high expression in the thalamus and zli with reduced expression in pth - r ( figures 6o and 6p ) . in pax6 mutants , however , the region of low ngn2 expression at the pth - r was no longer visible after vismodegib treatment ( figures 6q6s ) . ascl1 has a complementary expression pattern to that of ngn2 : wild - types show high expression in pth - r and prethalamus and low expression in the zli and thalamus ( figures 2 g and s6a ) , whereas pax6 mutants show expanded expression at the pth - r ( figures 2h and s6b ) . vismodegib had no obvious effect on the ascl1 expression pattern in wild - types ( figure s6c ) but caused a marked loss of expression at the pth - r of pax6 mutants ( figure s6d ) , reducing both the area containing ascl1 + cells and their density ( from 20.5 1.9 sem , n = 3 , to 9.78 1.8 , n = 3 , cells per 0.005 mm ; p < 0.05 , student s t test ) . vismodegib had little effect on nkx2.2 expression in wild - types ( figures 6 t , 6u , and 2a ) , but it reduced substantially the expanded nkx2.2 expression around the zli in pax6 mutants ( figures 2b and 6v6x ) . these findings indicate that altered patterns of ngn2 , ascl1 , and nkx2.2 expression around the thalamic / prethalamic border of pax6 mutants are dependent on shh signaling . our finding that the normal expression of these three genes is not shh dependent indicates that other factors can maintain their normal patterns . they suggest that enhanced shh signaling is a major contributor to many diencephalic patterning defects in pax6 mutants . we examined the potential functional consequence of the slight increase in shh signaling in the pax6 cortex but could not detect any effects of even high doses of vismodegib on the abnormal patterning in this region . for example , loss of pax6 causes upregulation of ascl1 expression in the cortex ( figures s6e and s6f ) , but vismodegib did not reverse this defect ( figure s6 g ) . this indicates that the small upregulation of shh expression in pax6 cortex is not a significant cause of patterning defects in this region , highlighting regional differences in pax6 s mode of action . we carried out luciferase assays using a 775 bp sequence corresponding to the shh promoter ( table s1 ; mutoh et al . , 2010 ) inclusion of the shh promoter into the firefly luciferase reporter construct caused a large increase in firefly luciferase over renilla luciferase activity ; this increase was significantly reduced when cells were cotransfected with a pax6-expressing construct ( mi et al . chromatin immunoprecipitation ( chip ) experiments ( figure 7b ) showed significant enrichment of fragments containing shh promoter sequence ( table s1 ) compared to that of fragments containing syt8 promoter ( a pax6 nonbound control ; mi et al . , 2013 ) , demonstrating that pax6 binds to the shh promoter in vivo . interestingly , bioinformatics using position weight matrices based on previously reported pax6 binding sites did not identify the likely positions of pax6 binding within the shh promoter , indicating that pax6 binds this region through one or more previously unrecognized , noncanonical sites . we also tested whether an indirect mechanism might contribute to the upregulation of shh in pax6 diencephalic cells . it was possible that pax6 loss might increase wnt signaling around the zli , and this might cause upregulation of shh because early wnt expression in the zli permits the induction of shh ( martinez - ferre et al . , 2013 ) . we used a -galactosidase transgenic reporter ( bat - gal ) ( maretto et al . , 2003 ) to show strong wnt/-catenin signaling around the zli whose domain expands in pax6 mutants ( figures s7a s7f ) . in chimeras , however , we found no evidence that clusters of pax6 cells expressed increased levels of axin2 and lef1 ( figures s7g s7l ) , which are readouts of levels of wnt signaling ( hsu et al . , 1998 ; jho et al . , 2002 ) , indicating that increased wnt signaling is unlikely to be a cause of changes in shh expression by pax6 diencephalic cells . previous studies showed that loss of pax6 results in diencephalic patterning defects including expansion of the zli ( grindley et al . , 1997 ; pratt et al . , 2000 ; chatterjee et al . , we find that a combination of cell - autonomous and non - cell - autonomous effects is responsible . pax6 is required cell autonomously to repress diencephalic shh expression . some of the major patterning defects that occur around the zli when pax6 is absent are caused by enhanced shh activity . we show that expansion of the zli and territories around the thalamic / prethalamic border is caused by cells acquiring abnormal molecular identities . theoretical studies by others have indicated that the involvement in control systems of elements that mutually repress each other , such as shh and pax6 , can enhance the system s robustness by buffering it against stochastic , interindividual fluctuations or temporal changes in morphogen levels and can generate bistability ( sokolowski et al . , 2012 ) . these properties are exactly those required within the diencephalon , which is a relatively small but intricately patterned structure , to maintain its cells in either a zli or a non - zli state with a sharp , reproducibly positioned transition between them . we propose that direct repression of shh by pax6 creates a feedback loop that is critical for the precision of normal diencephalic patterning by ensuring extremely tight , robust control of the size and influence of the zli . several aspects of our findings highlight the importance of the context within which interactions between regulatory molecules occur for their outcomes . although previous studies have shown that shh can repress pax6 expression , including at the zli ( ericson et al . , 1997 ; kiecker and lumsden , 2004 ; macdonald et al . , 1995 ) , we found no evidence for repression of the pax6 gene expression in diencephalic regions surrounding the normal position of the zli in pax6 mutants . in these areas , expanded shh expression caused an anticipated increase in the expression of its targets ptch1 and gli1 , but it did not prevent pax6 transcription , as shown by the abnormal double labeling of cells around the zli for both shh and the pax6 reporter or pax6 mrna . this suggests that , whereas shh may normally contribute to the absence of pax6 at the zli , elsewhere , the potential repressive effects of shh on pax6 expression are likely moderated by other factors . another example of context dependency is seen in the prethalamus , where pax6 is required for gsx2 expression , whereas in the telencephalon , loss of pax6 results in upregulation of gsx2 in cortex , where it would not normally be expressed ( rallu et al . , 2002 ; toresson et al . , 2000 ; yun et al . , the way in which a transcription factor affects its target genes probably depends mainly on the nature of the cofactors that are available for it to interact with . many studies have shown that shh can drive expression of ptch1 ( bai et al . , 2002 , 2004 ; balaskas et al . , 2012 ; goodrich et al . , 1996 ; this explains the expression of ptch1 by cells adjacent to the zli in normal embryos and by pax6 cells more widely throughout the diencephalon in mutants and chimeras , but it leaves unexplained the paradoxical observation that ptch1 is not expressed within the zli itself . ptch1 and gli1 are downregulated in regions expressing the highest levels of shh not only at the zli but also along the neural tube ( marigo and tabin , 1996 ) , where cells become progressively refractory to shh ( ribes et al . , this suggests a biphasic response of ptch1 and gli1 to shh , with very high levels of shh repressing their expression ( figure 7c ) . they indicate that administration of shh inhibitor to mutant embryos caused ptch1 upregulation at the expanded zli , where shh levels were blocking ptch1 production , while simultaneously reducing ptch1 expression in cells outside the expanded zli , where lower levels of shh were activating ptch1 . in our model , shh activates dbx1 in the zli s ventricular zone and enhances lhx5 expression in the overlying differentiating zone . because the shh receptor ptch1 is not expressed at the zli , it is likely that this activation and enhancement involve double repression , i.e. , ptch1 receptor activation likely represses dbx1 and lhx5 , and so loss of ptch1 in the ventricular zone at the zli would allow expression of dbx1 by progenitors and lhx5 by their progeny . other factors must also be involved in fine - tuning the expression of dbx1 because dbx1 is not expressed throughout all of the shh - rich zli , but only in its rostral part . lineage analysis has shown that progeny from the zli are incorporated into the rostral vlg , a nucleus that comprises gabaergic neurons whose axons do not project to the cortex ( suzuki - hirano et al . , 2011 ; vue et al . it is possible that there are subtle differences in the vlg neurons derived from the different types of zli progenitor . in summary , we found that mutual antagonism between pax6 and shh , involving cell - autonomous repression of shh by pax6 , constrains the development of the zli , its production of shh , and its influence on surrounding diencephalon . pax6 is likely to have similar effects on shh in other regions of the cns , including the cortex ( present findings and mi et al . , 2013 ) and the spinal cord , where its misexpression has been shown to reduce shh expression ( lek et al . , 2010 ) . the functional importance of this repression outside the diencephalon is currently unclear ; we show here that inhibition of shh signaling in pax6 cortex does not prevent abnormal cortical patterning . in the diencephalon , however , we conclude that pax6 plays many of its essential roles in patterning by cell autonomously regulating shh expression at the zli . animals were bred in - house following home office ( uk ) regulations . the pax6 null allele used was pax6 . pax6 and wild - type embryonic stem ( es ) cells were stably transfected with tau - gfp expression construct ptp6 ( pratt et al . , 2000 ) . chimeric embryos were produced by injection of pax6 tau - gfp or pax6 tau - gfp es cells into blastocysts . antibodies used were mouse anti - islet1 ( 39.4d5 ) , mouse anti - lhx1/lhx5 ( 4f2 ) , and mouse anti - nkx2.2 ( 74.5a5 ) obtained from the developmental studies hybridoma bank ( university of iowa ) , mouse anti - ascl1 ( bd biosciences ) , mouse anti - ngn2 ( clone 7g4 ; lo et al . , 2002 ) , rabbit anti - olig3 ( chemicon ) , rabbit anti - sox2 ( ab5603 ; chemicon ) , goat and rabbit anti - gfp ( abcam ) , and mouse anti - gfp ( roche ) . probes were labeled with digoxigenin , fluorescein , or dinitrophenol ( dnp ) . for fluorescence double in situ hybridization , the probes were detected sequentially , and the slides were incubated in 10 mm hcl before detection of the second probe . suspensions of gdc-0449 powder were prepared in methylcellulose - tween vehicle ( mct ) ( lipinski et al . , 2010 ) . dna - protein complexes were precipitated with anti - pax6 antibody ( covance ) or with anti - immunoglobulin g ( igg ) antibody ( abcam ) ( sansom et al . , 2009 ) . the amount of qpcr product obtained with anti - pax6 antibody was expressed relative to that obtained with anti - igg antibody . shh promoter sequence ( table s1 ) was cloned into pgla4.10 promoterless firefly luciferase reporter vector ( promega ) . pax6 was expressed using the pcmv - pax6 construct ( mi et al . , 2013 ) . human embryonic kidney 293 cells were transfected using lipofectamine 2000 ( invitrogen ) , harvested 48 hr after transfection , and analyzed with the dual - luciferase reporter assay system using the glomax luminometer ( both promega ) . did most of the experiments in collaboration with m.m . and helped write the manuscript .
summaryduring development , region - specific patterns of regulatory gene expression are controlled by signaling centers that release morphogens providing positional information to surrounding cells . regulation of signaling centers themselves is therefore critical . the size and the influence of a shh - producing forebrain organizer , the zona limitans intrathalamica ( zli ) , are limited by pax6 . by studying mouse chimeras , we find that pax6 acts cell autonomously to block shh expression in cells around the zli . immunoprecipitation and luciferase assays indicate that pax6 can bind the shh promoter and repress its function . an analysis of chimeras suggests that many of the regional gene expression pattern defects that occur in pax6/ diencephalic cells result from a non - cell - autonomous position - dependent defect of local intercellular signaling . blocking shh signaling in pax6/ mutants reverses major diencephalic patterning defects . we conclude that pax6 s cell - autonomous repression of shh expression around the zli is critical for many aspects of normal diencephalic patterning .
Introduction Results Discussion Experimental Procedures Author Contributions
here , we tested whether feedback via the transcription factor pax6 regulates the size and function of a forebrain organizer , the zona limitans intrathalamica ( zli ) . , 1997 ; 2013 ) , but it has also been reported that loss of pax6 increases the size of the shh - producing zli ( grindley et al . we examined the influence of pax6 on diencephalic patterning and identified an important cell - autonomous action of pax6 on the expression of shh . pax6 is not required for prethalamic islet1 expression , although the observation that proportions of islet1 + cells are reduced when large proportions of prethalamic cells are pax6 suggests a non - cell - autonomous effect of extensive pax6 loss on prethalamic islet1 expression . , corresponding to expanded pth - r ) that comprised wild - type cells , providing clear evidence for a non - cell - autonomous upregulation of ascl1 and loss of ngn2 around the expanded mutant zli ( figures 3l and 3 m ) . in summary , our findings strongly suggest that a major underlying cause of abnormal thalamic patterning in the absence of pax6 is a position - dependent defect of local intercellular signaling . position - dependent defects of signaling among cells around the thalamic / prethalamic border might be caused by defects of the zli . these results indicate that , in the absence of pax6 , the zli and its subdomains , identified by expression / coexpression of shh , ngn2 , and dbx1 , are enlarged , as summarized in figures 4k and 4l . the failure of cells around the thalamic / prethalamic border to repress shh expression in pax6 embryos might provide an explanation for aspects of the mis - patterning of this region in terms of its abnormal marker - gene expression in pax6 embryos and chimeras . moreover , the results suggest that the mechanism that sets the level of activation with reference to distance from the zli in the absence of pax6 acts cell autonomously because the relationship between the locations of the pax6 patches and their levels of shh expression reflected the relationship in pax6 embryos . to test whether this might also be caused by exposure to high levels of shh , we administered vismodegib ( gdc-0449 ) , a selective inhibitor of the shh receptor smo ( expressed throughout control and pax6 diencephalon ; figures s5a and s5b ) , to pax6 embryos . we then tested whether abnormalities of diencephalic patterning in pax6 embryos result from abnormally high shh signaling around the zli , by administering vismodegib ( gdc-0449 ) to inhibit shh signaling in pax6 embryos . in pax6 mutants , the expanded domain of intense lhx5 expression around the thalamic / prethalamic border ( whose position was recognized by shh expression in adjacent sections ) was reduced by increasing doses of vismodegib ( figures 6j6n ) , indicating the importance of shh signaling for this aspect of the mutant phenotype . this indicates that the small upregulation of shh expression in pax6 cortex is not a significant cause of patterning defects in this region , highlighting regional differences in pax6 s mode of action . , we find that a combination of cell - autonomous and non - cell - autonomous effects is responsible . some of the major patterning defects that occur around the zli when pax6 is absent are caused by enhanced shh activity . we propose that direct repression of shh by pax6 creates a feedback loop that is critical for the precision of normal diencephalic patterning by ensuring extremely tight , robust control of the size and influence of the zli . , 1995 ) , we found no evidence for repression of the pax6 gene expression in diencephalic regions surrounding the normal position of the zli in pax6 mutants . in these areas , expanded shh expression caused an anticipated increase in the expression of its targets ptch1 and gli1 , but it did not prevent pax6 transcription , as shown by the abnormal double labeling of cells around the zli for both shh and the pax6 reporter or pax6 mrna . this suggests that , whereas shh may normally contribute to the absence of pax6 at the zli , elsewhere , the potential repressive effects of shh on pax6 expression are likely moderated by other factors . in summary , we found that mutual antagonism between pax6 and shh , involving cell - autonomous repression of shh by pax6 , constrains the development of the zli , its production of shh , and its influence on surrounding diencephalon . in the diencephalon , however , we conclude that pax6 plays many of its essential roles in patterning by cell autonomously regulating shh expression at the zli .
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increasing prevalence of type 2 diabetes mellitus ( t2 dm ) has stimulated research focused on the pathogenesis and treatment of t2 dm and its complications . initial studies examining fracture as a possible complication of t2 dm indicated that type 2 diabetics were not at risk of fracture based on bone mineral density ( bmd ) , the clinical standard for screening [ 13 ] . however , data analyzed from clinical trials with fracture as an outcome variable instead of bmd revealed that both men and women with t2 dm experience an increase in fracture ( i.e. , 1.53-fold ) beginning 510 years after diagnosis [ 48 ] . collectively , the clinical evidence indicates that , independent of bmd , type 2 diabetics are at increased risk of fracture that is exacerbated over time . rodent models have enabled investigators to study the molecular mechanisms involved in altering bone quality in t2 dm . one of the most commonly utilized models has been the c57bl/6 young growing mouse fed a diet high in total and saturated fat ( hf ) , which exhibits an increase in adiposity , impaired glucose tolerance , and dyslipidemia , similar to prediabetes in humans [ 1012 ] . c57bl/6 mice have been reported to exhibit decreased trabecular bone and either increased , decreased , or no change in cortical bone in response to long - term intake of a hf diet [ 1317 ] . when alterations in bone microarchitecture occur , they are usually accompanied by impaired bone quality as evidenced by compromised biomechanical properties [ 15 , 1721 ] . factors that may contribute to some of the discrepancies in the literature describing the skeletal response to a hf diet could be due to the composition of the diets , the age of the mice , the duration of the study , and , importantly , differences in the c57bl/6 mouse substrain 's response ( e.g. , c57bl/6j or c57bl/6n ) [ 1316 , 21 ] . a review of published reports revealed that studies utilizing different c57bl/6 substrains ( e.g. , c57bl/6j and c57bl/6n ) are often treated interchangeably without mention of genetic variations that could have important implications on the results and their interpretation . for example , the c57bl/6j mouse has a missense mutation in the gene encoding nicotinamide nucleotide transhydrogenase ( nnt ) that alters rna splicing and leads to the deletion of exons 711 [ 2224 ] . when c57bl/6j mice are fed a high fat diet , they exhibit impaired glucose tolerance that appears to result from suppressed insulin secretion by pancreatic -cells . thus , genetic differences in the c57bl/6j mouse could contribute to some of the discrepancies in the literature in regard to metabolic and skeletal responses reported in the diet - induced obesity model of t2 dm . in contrast to the c57bl/6 mouse , the c3h / hej mouse model has been used in mechanistic studies because of its blunted metabolic response to a high fat diet [ 26 , 27 ] . c3h / hej mice have a nonfunctional toll - like receptor ( tlr ) 4 due to a point mutation in the toll - interleukin 1 receptor domain [ 2628 ] . tlr-4 is expressed on bone cells ( i.e. , both the osteoblasts and osteoclasts ) [ 29 , 30 ] . our lab and others [ 3134 ] have shown that tlr-4 ligands ( e.g. , lipopolysaccharide or lps and saturated free fatty acids or sffas ) as well as downstream inflammatory mediators have the potential to uncouple bone turnover . because of interest in sffas and gut - derived lps in the pathophysiology of t2 dm and its complications , the c3h / hej strain has become an important research tool to examine the role of tlr-4 in these metabolic responses . to date , a direct comparison of the long - term metabolic and skeletal responses of the c57bl/6j and c57bl/6n substrains to a hf diet has not been reported in the literature . if , as we hypothesized , the metabolic response to a hf diet in these two substrains differs due to genetic variations , this may alter the inflammatory response , hormones , and adipokines , subsequently affecting the bone . such differences would be important relative to the interpretation of results and could assist investigators in selecting the most appropriate model . furthermore , because of our laboratory 's interest in tlr-4 and bone , in this study c3h / hej mice were used as a negative control for comparative purposes [ 26 , 27 ] . eight - week - old male mice , c57bl/6n from charles river ( wilmington , ma ) and c57bl/6j and c3h / hej mice from jackson labs ( bar harbor , me ) , were obtained ( n = 30 mice / strain ) for these studies . animals were acclimated for 7 days and then randomly assigned to a control ain-93 m ( 10% kcals from fat ) or a hf ( 45% kcals from fat ; harlan teklad , td.06415 ) diet for 24 wk . body weight and food intake were recorded throughout the study . total feed efficiency was calculated by determining the gain in body weight ( mg ) per energy unit consumed ( kcal ) . venous tail blood was collected following a 6 hr fast for evaluation of glucose and insulin at 4 wk intervals . after 24 wk , mice were anesthetized ( ketamine / xylazine cocktail 70 and 30 mg / kg body weight , resp . ) as previously reported and whole body dxa ( lunar pixi , ge medical systems , madison , wi ) scans were performed . an aliquot of blood was collected for total white blood cell ( wbc ) counts and the remainder processed for plasma in edta coated tubes and stored at 80c . all procedures were approved by the institutional animal care and use committee of oklahoma state university . one week prior to the end of the study ( 23rd wk ) , mice were fasted for 6 hrs and an intraperitoneal ( ip ) glucose tolerance test ( igtt ) was performed . an ip glucose solution ( 2 g glucose / kg bodyweight ) was administered , followed by blood glucose monitoring at 15 , 30 , 60 , 90 , and 120 min . area under the curve ( auc ) was determined by calculating the sum of rectangular area between each time point . plasma insulin was assessed at 4 wk intervals , whereas plasma leptin , adiponectin , and osteocalcin ( ocn ) , both total ocn ( gla - ocn ) and undercarboxylated ocn ( glu - ocn ) , were determined only at the final time point . all assays were performed using commercially available elisa kits including insulin ( crystal chem , downers grove , il ) , leptin and adiponectin ( emd millipore , billerica , ma ) , and gla - ocn and glu - ocn ( clontech takara bio , mountain view , ca ) , following the manufacturer 's protocol . gla - ocn is reported as an indicator of bone turnover and given the importance of the carboxylation status of ocn relative to total ocn on glucose metabolism , the ratio of [ glu - ocn]/[gla - ocn ] was calculated to provide insight into the relationship between the skeletal and metabolic response to treatment . whole body dxa scans were performed to determine body composition , bone mineral area ( bma ) , content ( bmc ) , and bmd . all scans were analyzed using piximus series software version 1.4x ( ge lunar pixi , madison , wi ) . micro - ct ( micro - ct 40 , scanco medical , switzerland ) was used to evaluate bone microarchitecture at the proximal tibia metaphysis , tibia middiaphysis , and 4th lumbar vertebral body . analysis of trabecular bone was performed at the proximal tibia metaphysis on high resolution scans ( 2048 2048 pixels ) and the volume of interest ( voi ) included 750 m of secondary spongiosa . the voi was analyzed using a threshold of 300 , a sigma of 0.7 , and support of 1.0 . trabecular bone of the vertebra was assessed on images 80 m from the dorsal and caudal growth plates at medium resolution ( 1024 1024 pixels ) and included only secondary spongiosa . images generated from the scans of the vertebrae were analyzed at a threshold of 340 and a sigma and support of 1.2 and 2.0 , respectively . trabecular parameters evaluated included trabecular bone volume expressed as a percentage of total volume ( bv / tv ) , trabecular number ( tb.n . ) , trabecular thickness ( tb.th . ) , trabecular separation ( tb.sp . ) cortical bone was evaluated by analyzing a 120 m section at the mid - diaphysis of the tibia . assessment of cortical bone parameters included cortical porosity , thickness , area , and medullary area of the tibial middiaphysis . the acquired images were analyzed at a threshold of 300 , a sigma of 0.7 , and support of 1.0 . tibiae were cleaned of soft - adhering tissue and stored in phosphate buffered saline ( pbs ) at 4c until analyses were performed . reference point indentation ( rpi ) was applied laterally at the tibia - fibula junction using a biodent ( active life scientific , inc . , santa barbara , ca ) , and the first cycle indentation distance and touchdown distance were recorded . each tibia was subjected to a testing protocol of 2 n force , 2 hz , and 10 cycles . fixed ( 10% neutral buffered formalin ) liver samples were processed and sectioned ( 5 m ) for staining with hematoxylin and eosin to assess histological changes associated with nonalcoholic fatty liver disease ( nafld ) that occurs in obesity and/or diabetes . steatosis and fibrosis were scored on a scale from 0 to 4 , with 0 indicating the absence of hepatic lipid droplets or fibrosis , whereas 4 indicated pronounced steatosis or fibrosis . lobular and portal inflammation was scored using a range of 03 , with 0 indicating the absence of macrophage infiltration and 3 corresponding to severe inflammation . balloon degeneration was scored using a 02 system , with 0 defined as the lack of degeneration and 2 indicating modest presence of parenchymal cell death . total rna was isolated from the liver and bone marrow using trizol reagent ( invitrogen , grand island , ny ) as previously described [ 36 , 37 ] . cdna was synthesized following a standardized laboratory protocol and qpcr was performed using sybr green chemistry ( 7900ht fast real - time , applied biosystems , foster city , ca ) . hepatic genes of interest included fatty acid synthase ( fasn ) , sterol regulatory element - binding protein ( srebp1c ) , glucose transporter 2 or solute carrier family ( slc2a2 ) , peroxisome proliferative - activator ( ppara ) , and glutathione peroxidase ( gpx1 ) and in the bone marrow fasn , ppara , and gpx1 ( table s1 available online at http://dx.doi.org/10.1155/2015/758080 ) . all qpcr results were evaluated by the comparative cycle number at threshold ( cq ) method ( user manual # 2 , applied biosystems ) using peptidylprolyl isomerase b or cyclophilin b ( ppib ) as the invariant control . statistical analyses were performed using statistical analysis software version 9.3 ( sas institute , nc ) . the primary objective was to determine the difference in response to a hf diet of a given strain and , therefore , student 's paired t - test was used unless stated otherwise . however , to further assess differences in the responsiveness between the two c57bl/6 substrains , if a statistical difference ( p < 0.05 ) was observed for a given parameter between con and hf within a given strain , the magnitude of response ( i.e. , percent change of hf compared to con ) was compared between strains using one - way anova . when the f value was < 0.05 , post hoc analyses were performed with fischer 's least square means separation test . all data are presented as mean standard error ( se ) and a p < 0.05 was considered statistically significant . at baseline , body weight between strains differed ( c3h / hej > c57bl/6n > c57bl/6j ) ; however , no differences existed within a given strain between the two dietary treatment groups ( i.e. , con versus hf ; data not shown ) . after 5 wk on the hf diet , the c57bl/6j exhibited a significant increase in body weight compared to the c57bl/6j con , whereas the c57bl/6n on the hf diet had a higher ( p < 0.05 ) body weight after only 3 wk ( figure 1 ) . the c3h / hej mice on the hf diet also exhibited a more rapid increase in body weight after only 1 wk compared to their respective controls ( figure 1 ) . analysis of body composition revealed the increase in body weight was due to a significant increase in both lean and fat mass for the two c57bl/6 substrains as well as the c3h / hej mice ( table 1 ) . the amount of food consumed was less for the mice on the hf diet in each strain ( table 1 ) . however , on a kcal basis the c57bl/6n mice on the hf diet consumed + 2.1 kcal / day and the c57bl/6j and c3h / hej on the hf diet consumed + 1.2 kcal / day compared to their respective controls ( data not shown ) . overall , feed efficiency was higher in the c57bl/6n mice than in the c57bl/6j mice on the hf diet ( table 1 ) , further demonstrating the differences in metabolic responsiveness between these two substrains . after 24 wk on a hf diet the c57bl/6n mice exhibited splenomegaly , thymic hypertrophy , and decreased wbc , but the c57bl/6j mice failed to demonstrate these immunological changes ( table 1 ) . c3h / hej mice had a similar response to the hf diet in terms of tissue weights ( i.e. , spleen and thymus ) and total wbcs compared to the c57bl/6n mice ( table 1 ) . c57bl/6n mice on the hf diet were the only strain that had elevated fasting blood glucose ( figure 2(a ) ) and plasma insulin ( figure 2(b ) ) after 4 , 8 , 12 , 16 , 20 , and 24 wk of treatment compared to their con counterparts . the c57bl/6j substrain on the hf diet was hyperglycemic at 16 and 20 wk ( figure 2(a ) ) and hyperinsulinemic at 24 wk ( figure 2(b ) ) . importantly , neither substrain achieved a fasting blood glucose consistent with frank diabetes ( i.e. , > 250 mg / dl ) that is associated with polyuria and polydipsia . similar to the c57bl/6j mice , the c3h / hej strain on the hf diet exhibited delayed - onset of hyperglycemia ( figure 2(a ) ) , while their plasma insulin was increased at 12 , 20 , and 24 wk ( figure 2(b ) ) . at the end of the study , igtt showed that the c57bl/6j and c57bl/6n as well as the c3h / hej mice on the hf diet exhibited impaired glucose intolerance ( figures 3(a ) and 3(b ) ) . the percent change in auc to the hf diet demonstrated that the magnitude of response of the two c57bl/6 substrains was similar ( data not shown ) . the c3h / hej mice also exhibited impaired glucose intolerance after 24 wk on a hf diet ( figure 3 ) . it should be noted that , despite elevated auc , the c3h / hej mice on the hf diet maintained the ability to restore blood glucose by the final igtt time point . both the c57bl/6j and c57bl/6n substrains had elevated plasma leptin after 24 wk on a hf diet ( table 1 ) , but there was no significant difference in the magnitude of the response between the two substrains . similarly , the c3h / hej mice on the hf diet also had higher plasma leptin ( table 1 ) . interestingly , at 24 wk the c57bl/6j mice , but not the c57bl/6n substrain , exhibited a decrease in plasma adiponectin in response to a hf diet ( table 1 ) . after 24 wk on a hf diet , there were no differences in gla - ocn as an indicator of bone turnover due to diet in the c57bl/6 substrains or c3h / hej mice ( data not show ) . the carboxylation status of ocn ( i.e. , glu / gla - ocn ratio ) , which has been shown to influence insulin sensitivity and systemic energy metabolism , was reduced only in the c57bl/6n mice after 24 wk on a hf diet ( figure 4 ) . representative micrographs of liver sections from each group show that the c57bl/6j and c57bl/6n strains as well as the c3h / hej strain experienced some degree of hepatic steatosis in response to the hf diet ( figure 5 ) . the c57bl/6n mice on the hf diet had a significantly higher lobular and portal inflammation mean score compared to the con ( table 2 ) . although the c57bl/6j mice on the hf diet had more lobular inflammation than their respective controls ( p = 0.0038 ) , the frequency of the inflammatory response was markedly lower in this substrain compared to the c57bl/6n ( i.e. , lobular inflammation in 92% c57bl/6n versus 54% c57bl/6j and portal inflammation in 77% c57bl/6n versus 23% c57bl/6j ) ( table 2 ) . while none of the c57bl/6j mice on the hf diet exhibited liver fibrosis , 23% of the treated c57bl/6n mice had fibrotic changes ( table 2 ) . balloon degeneration was also more severe in the c57bl/6n mice on the hf diet compared to the c57bl/6j ( table 2 ) . despite a lack of lobular and portal inflammation and fibrosis in the c3h / hej mice , balloon degeneration was severe in this strain ( table 2 ) . both the c57bl/6j and c57bl/6n mice demonstrated a decrease in whole body bmc and bma , but no change in whole body bmd in response to the hf diet after 24 wk ( table 3 ) . when bmd was expressed relative to body weight , differences due to diet were observed suggesting that the bone density did not increase relative to the increase in body weight ( table 3 ) . micro - ct analyses of the lumbar vertebra revealed significant loss of trabecular bone or bv / tv with the hf diet in c57bl/6n , while the skeletal response of the c57bl/6j mice did not reach the level of statistical significance ( p < 0.0579 ) ( figure 6(a ) ) . as expected , the c3h / hej mice were protected from vertebral bone loss ( figure 6 ) or nonmorphometric parameters with hf diet ( table 3 ) . both the c57bl/6j and c57bl/6n mice on the hf diet had a higher smi indicative of a weaker , more rod - like trabecular bone in the vertebra ( table 3 ) . in contrast to the vertebra , no changes were observed in trabecular or cortical parameters analyzed at the proximal tibial metaphysis or the tibial middiaphysis in the c57bl/6j or the c57bl/6n mice . the c3h / hej mice failed to demonstrate alterations in trabecular bone of the proximal tibia but did exhibit an increase in the medullary area at the middiaphysis ( table 3 ) . based on reference point indentation testing on cortical bone at the tibia - fibula junction , no changes were observed in first cycle indentation distance or touchdown distance in any strain following 24 wk on a hf diet when compared to their respective con ( table 3 ) . determination of genes involved in hepatic metabolism and inflammation revealed that the c57bl/6n mice on the hf diet had altered metabolic processes , including the upregulation of glucose uptake ( slc2a2 ) , triglyceride storage ( fasn and srebp1c ) and adipogenesis ( ppara ) , as well as antioxidant capacity ( gpx1 ) ( table 4 ) . interestingly , none of these alterations in gene expression were observed in the c57bl/6j mice after 24 wk on the hf diet . to determine the degree to which oxidative stress and adipogenesis contributed to bone loss with the hf diet model , gpx1 and pparg mrna abundance similar to the hepatic tissue , the abundance of gpx1 mrna was increased in the c57bl/6n mice on the hf diet , suggesting an increase in antioxidant capacity ( table 4 ) . in contrast , the c57bl/6j mice on the hf diet demonstrated a decrease in the relative abundance of gpx1 ( table 4 ) . additionally , no alterations were observed in the transcriptional regulator of adipogenesis , pparg , in any strain after 24 wk ( table 4 ) . the findings of this study show that the c57bl/6j and the c57bl/6n mouse differ in their metabolic response to a hf diet over a 24 wk study period . discrepancies in the metabolic response between the two strains may be attributed in part to the missense mutation ( m35 t ) in exon 1 and a multiexon deletion of nnt in the c57bl/6j mice [ 39 , 40 ] . this mutation in nnt has been reported to uncouple -cell mitochondrial metabolism leading to less atp production in pancreatic islets , enhanced katp channel activity , and , consequently , impaired glucose - stimulated insulin secretion [ 23 , 39 , 41 ] . only fasting insulin was assessed in the current study ; however , early onset of hyperinsulinemia with hf diet was only observed in the c57bl/6n mice with an intact , functional nnt . the coincident lower feed efficiency in the c57bl/6j compared to the c57bl/6n mice resulted in a delay in the development of hyperglycemia and shorter duration of exposure to conditions associated with impaired glucose tolerance . the c57bl/6j and c57bl/6n mice had a markedly different hepatic response to the hf diet after 24 wk . increased mrna abundance of fasn and a modest increase in srebpc1 in the presence of severe liver steatosis in the c57bl/6n mice on the hf diet suggest an increase in hepatic triglyceride synthesis and storage . conversely , the c57bl/6j substrain , which has lower glucokinase activity and thus impaired glucose sensing , may explain the lack of transcriptional regulation of fasn and srebp1c . furthermore , c57bl/6n mice on the hf diet demonstrated an increase in slc2a2 gene expression , which encodes the non - insulin - sensitive glucose transporter 2 and has been reported to be upregulated in response to a hf diet . histological evaluation suggests that the c57bl/6n mice on the hf diet also experienced the most pronounced hepatic inflammation , compared to the c57bl/6j mice . interestingly , the hf - induced nafld that develops in the c57bl/6j was previously attributed , in part , to the spontaneous mutation in the nnt [ 39 , 43 ] ; however , the data demonstrate that the c57bl/6n mice , with a functional nnt , develop more severe nafld in response to hf diet feeding . given the more pronounced metabolic phenotype in the c57bl/6n mice on the hf diet , it was counterintuitive that only the c57bl/6j mice demonstrated the anticipated decrease in plasma adiponectin following a hf diet . although the mechanism for reduced adiponectin during obesity and t2 dm has been attributed , in part , to an increase in local tnf- in adipose tissue , strain - related variability of adiponectin gene expression and plasma has been previously documented . as mice continue to be used for models of impaired glucose homeostasis , further investigation is warranted to fully understand these strain differences relative to metabolic handling . the findings of this study also demonstrate that the c3h / hej mice may not be completely resistant to diet - induced obesity and the subsequent metabolic changes . differences in the c3h / hej strain 's response to a high fat diet compared to previous reports may be attributed to the difference in the control strain used [ 26 , 28 ] . specifically , previous studies have compared the c3h / h3j response to hf diet to c3h / heouj or c3h / hen , both of which have a functional tlr-4 [ 26 , 28 ] . however , in these studies , no comparisons were made with c3h / hej mice on a control diet . therefore , it is not possible to determine if the differences in metabolic response to a hf diet are a result of tlr-4 or genetic variability in the control substrain background . furthermore , the c3h / hej strain has recently been shown to have a genetic variation in the leptin receptor gene ( lepr ) . this mutation could account for the impaired metabolic response observed in the c3h / hej strain on a hf diet due to the central role leptin has on regulating energy intake and expenditure . however , in the present study , food intake in the c3h / hej strain was not significantly altered and the absence of a leptin - mediated effect on food or energy intake does not rule out implications of the lepr defect on the skeletal response [ 18 , 19 , 47 ] . in conjunction with the metabolic comparisons , the other primary objective of this study was to compare the skeletal response to a hf diet in two commonly used c57bl/6 substrains . the c57bl/6j mice on the hf diet experienced a 13.6% reduction in trabecular bone of the vertebra , although not statistically significant . c57bl/6n was the only strain that exhibited significant trabecular bone loss which occurred only in the vertebra . in the absence of alterations in tibia trabecular and cortical bone microarchitecture , it is conceivable that the absence of alterations in the tibia could result from site - specific changes associated with increased adiposity and greater weight - bearing , which could offset some of the negative effects of glucose intolerance on bone [ 48 , 49 ] . based on reports in the literature that bone biomechanical properties are changed in t2 dm independent of alterations in bone mass [ 8 , 50 , 51 ] , the tibia was subjected to rpi testing . no detectable alterations in cortical bone strength were observed after 6 months in the absence of structural changes . the c57bl/6n mice had more prolonged exposure to hyperglycemia and hyperinsulinemia in response to the hf diet compared to the c57bl/6j substrain , and c57bl/6n were the only substrain to lose significant trabecular bone in the spine . while there have been conflicting reports on how a hf diet impacts bone in c57bl/6 mice [ 12 , 1417 ] , the results of this study indicate the skeletal response may be linked to the duration of disrupted insulin signaling and glucose intolerance . this idea is further supported by the response of the c3h / hej mice in which case an attenuated glucose , leptin , and insulin response to the hf diet failed to induce bone loss . several reports have shown that a high fat diet uncouples bone turnover by increasing bone resorption and decreasing bone formation in various rodent models [ 16 , 17 , 21 ] . in this study , osteocalcin which is considered a marker of bone turnover was the only bone marker assessed and it was not altered at the end of the study . although this does not rule out alterations in bone metabolism occurring earlier , future studies are needed to investigate the mechanism involved in the site - specific loss of bone observed in this animal model . based on recent literature describing the hormone ocn as a regulator of systemic energy metabolism [ 5254 ] , the role of ocn on both metabolic and skeletal changes induced by hf was investigated . after 24 wk , the c57bl/6n mice on the hf diet had a lower ratio of plasma glu - ocn / gla - ocn . because circulating undercarboxylated ( glu - ocn ) can act directly on pancreatic -cells to stimulate insulin secretion [ 20 , 5557 ] , it would be expected that a reduction in glu - ocn would lead to a decrease in insulin secretion . instead , fasting plasma insulin was elevated in the c57bl/6n mice on the hf diet compared to their respective controls . alternatively , these results could indicate the direct effects impaired glucose tolerance has on osteoclastogenesis and bone resorption . in this regard , the acidic milieu of the osteoclast - resorption lacunae is capable of decarboxylating ocn , resulting in its release from the bone matrix and its subsequent circulation in the blood . the complexity of ocn 's role on bone and energy metabolism during glucose intolerance , as well as these implications in various mouse strains , warrants further investigation . additional studies are also needed to determine how genes involved in the gamma carboxylation of ocn ( i.e. , ggcx and esp1 ) by osteoblasts as well as the role of decarboxylation of ocn by osteoclast are regulated in response to changes energy homeostasis [ 55 , 59 ] . to date , this is the first study to directly compare the c57bl/6j and c57bl/6n substrains ' response to a hf diet from a metabolic and skeletal perspective . although neither substrain developed frank t2 dm , the data presented here show that c57bl/6n mice exhibit an earlier metabolic response consistent with impaired glucose tolerance or prediabetes to the hf diet compared to the c57bl/6j mice . moreover , the skeletal response followed that of the metabolic changes ; this was demonstrated by the fact that significant trabecular bone loss occurred in the c57bl/6n mice , which demonstrated the robust metabolic alterations associated with clinical t2 dm . given the observed differences in feed efficiency between the c57bl/6 substrains , further research is also warranted to identify the mechanisms underlying altered energy utilization . in contrast to the c57bl/6 mice , the c3h / hej strain was protected from the metabolic and skeletal changes induced by a hf diet . while a number of questions remain including how bone metabolism is being altered in response to a high fat diet on a molecular level , this study highlights the need to consider not only the most appropriate strain but also the most appropriate substrain of mouse when designing experiments . other important factors to consider when studying the relationship between glucose intolerance and bone include the site - specific skeletal response and the study duration . these decisions could significantly impact data interpretation and the translational implications as they relate to understanding how bone metabolism is altered in the context of t2 dm .
type 2 diabetes mellitus ( t2 dm ) represents a complex clinical scenario of altered energy metabolism and increased fracture incidence . the c57bl/6 mouse model of diet - induced obesity has been used to study the mechanisms by which altered glucose homeostasis affects bone mass and quality , but genetic variations in substrains of c57bl/6 may have confounded data interpretation . this study investigated the long - term metabolic and skeletal consequences of two commonly used c57bl/6 substrains to a high fat ( hf ) diet . male c57bl/6j , c57bl/6n , and the negative control strain , c3h / hej , mice were fed a control or hf diet for 24 wks . c57bl/6n mice on a hf diet demonstrated an increase in plasma insulin and blood glucose as early as 4 wk , whereas these responses were delayed in the c57bl/6j mice . the c57bl/6n mice exhibited more severe hepatic steatosis and inflammation . only the c57bl/6n mice lost significant trabecular bone in response to the high fat diet . the c3h / hej mice were protected from bone loss . the data show that c57bl/6j and c57bl/6n mice differ in their metabolic and skeletal response when fed a hf diet . these substrain differences should be considered when designing experiments and are likely to have implications on data interpretation and reproducibility .
1. Introduction 2. Methods 3. Results 4. Conclusions
one of the most commonly utilized models has been the c57bl/6 young growing mouse fed a diet high in total and saturated fat ( hf ) , which exhibits an increase in adiposity , impaired glucose tolerance , and dyslipidemia , similar to prediabetes in humans [ 1012 ] . factors that may contribute to some of the discrepancies in the literature describing the skeletal response to a hf diet could be due to the composition of the diets , the age of the mice , the duration of the study , and , importantly , differences in the c57bl/6 mouse substrain 's response ( e.g. in contrast to the c57bl/6 mouse , the c3h / hej mouse model has been used in mechanistic studies because of its blunted metabolic response to a high fat diet [ 26 , 27 ] . to date , a direct comparison of the long - term metabolic and skeletal responses of the c57bl/6j and c57bl/6n substrains to a hf diet has not been reported in the literature . however , on a kcal basis the c57bl/6n mice on the hf diet consumed + 2.1 kcal / day and the c57bl/6j and c3h / hej on the hf diet consumed + 1.2 kcal / day compared to their respective controls ( data not shown ) . after 24 wk on a hf diet the c57bl/6n mice exhibited splenomegaly , thymic hypertrophy , and decreased wbc , but the c57bl/6j mice failed to demonstrate these immunological changes ( table 1 ) . interestingly , at 24 wk the c57bl/6j mice , but not the c57bl/6n substrain , exhibited a decrease in plasma adiponectin in response to a hf diet ( table 1 ) . after 24 wk on a hf diet , there were no differences in gla - ocn as an indicator of bone turnover due to diet in the c57bl/6 substrains or c3h / hej mice ( data not show ) . representative micrographs of liver sections from each group show that the c57bl/6j and c57bl/6n strains as well as the c3h / hej strain experienced some degree of hepatic steatosis in response to the hf diet ( figure 5 ) . both the c57bl/6j and c57bl/6n mice demonstrated a decrease in whole body bmc and bma , but no change in whole body bmd in response to the hf diet after 24 wk ( table 3 ) . the findings of this study show that the c57bl/6j and the c57bl/6n mouse differ in their metabolic response to a hf diet over a 24 wk study period . furthermore , c57bl/6n mice on the hf diet demonstrated an increase in slc2a2 gene expression , which encodes the non - insulin - sensitive glucose transporter 2 and has been reported to be upregulated in response to a hf diet . interestingly , the hf - induced nafld that develops in the c57bl/6j was previously attributed , in part , to the spontaneous mutation in the nnt [ 39 , 43 ] ; however , the data demonstrate that the c57bl/6n mice , with a functional nnt , develop more severe nafld in response to hf diet feeding . given the more pronounced metabolic phenotype in the c57bl/6n mice on the hf diet , it was counterintuitive that only the c57bl/6j mice demonstrated the anticipated decrease in plasma adiponectin following a hf diet . differences in the c3h / hej strain 's response to a high fat diet compared to previous reports may be attributed to the difference in the control strain used [ 26 , 28 ] . in conjunction with the metabolic comparisons , the other primary objective of this study was to compare the skeletal response to a hf diet in two commonly used c57bl/6 substrains . the c57bl/6n mice had more prolonged exposure to hyperglycemia and hyperinsulinemia in response to the hf diet compared to the c57bl/6j substrain , and c57bl/6n were the only substrain to lose significant trabecular bone in the spine . to date , this is the first study to directly compare the c57bl/6j and c57bl/6n substrains ' response to a hf diet from a metabolic and skeletal perspective . in contrast to the c57bl/6 mice , the c3h / hej strain was protected from the metabolic and skeletal changes induced by a hf diet . while a number of questions remain including how bone metabolism is being altered in response to a high fat diet on a molecular level , this study highlights the need to consider not only the most appropriate strain but also the most appropriate substrain of mouse when designing experiments .
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appropriate management of farm waste such as manure is critical in controlling the spread of pathogens such as e. coli o157:h7 to vegetable crops . in most management schemes , fumigants are used for the control of plant pathogens , nematodes , and weeds before high - value cash crops such as strawberry and tomato are planted . outbreaks of e. coli o157:h7 infections historically have been associated with consumption of undercooked ground beef ; however , many recent outbreaks have resulted from consumption of contaminated raw vegetables , including lettuce [ 13 ] . although many pathogens have been associated with fresh produce , e. coli o157:h7 is of particular concern because ingestion of relatively few cells can cause illness . e. coli o157:h7 can survive for 60 to 120 days in water and in soil , and under dry and acidic conditions . the steps in the production chain that have the greatest potential for pathogen contamination are soil preparation ( use of uncomposted manures ) and planting and growing ( use of contaminated irrigation water and animal manures and manure from animals grazing locally or nearby ) [ 68 ] . prevention of preharvest contamination of fresh produce is an essential part of systems approach focused on interventions designed to achieve delivery of microbiological safe produce to consumers . suppression of human pathogens in agricultural soils and the subsequent prevention of spread into the food chain by contamination of produce must be realized by the adoption of best management practices . in the absence of known phytopathogens , many crops have exhibited an increased growth response when planted into soil that had been fumigated with mebr at the recommended application rate . one of the likely reasons for this observation may be that fumigation altered the microbial composition of the soil , either enhancing beneficial colonizers or reducing populations of deleterious rhizosphere colonizers . fumigation is to control plant pathogens such as nematodes , soil - borne diseases , and weeds . the immediate impact of fumigation may be the reduction of certain bacterial species in the soil and the development of new communities after a few weeks [ 11 , 12 ] . some fumigants may be toxic to some microbes , and this may enhance the selection of microbes that may be beneficial to plants [ 11 , 12 ] . due to the increased focus on food safety related to fresh produce , there are more studies using real - time pcr to quantify pathogens such as e. coli o157:h7 in the environment [ 13 , 14 ] . the main objectives of this study were to determine the effects of soil microbial diversity and fumigation on the survival of e. coli o157:h7 in soils contaminated with the pathogen . to accomplish these objectives , a preliminary study was conducted to determine the survival of the pathogen in both autoclaved and nonautoclaved soils at different concentrations of the pathogen . for our main objectives , both plate count and real - time pcr approaches were used to determine the survival of e. coli o157:h7 in the two soils . this strain produces shiga - like toxin stx1 and stx2 and the pgfp expressing green fluorescent protein ( gfp ) and ampicilin resistance . e. coli o157:h7 was cultured at 37c overnight in modified tryptic soy broth ( mtsb)(difco laboratories inc . , cockeysville , md ) supplemented with 100 g of ampicillin ml ( sigma , st louis , mo ) . cells were harvested by centrifugation at 3500 x g for 10 min and resuspended in phosphate buffered saline ( pbs ) ( fisher scientific , pittsburgh , pa ) to a concentration of ~10 cfu ml . bacterial strains ( except strain 72 ) used to determine the specificity and sensitivity of pcr assays were obtained from the national animal disease center ( ames , ia ) and were cultured on luria - bertani ( lb ) broth agar and sorbitol macconkey ( smac ) agar plates at 37c . clay soil ( willows silty clay , saline - alkaline ) and sandy soil ( dello loamy sand ) were collected from mystic lake dry bed and the santa ana river bed , respectively , and treated as described by ibekwe and grieve . the clay soil had a bulk density of 1.51 mg m with 3.7% sand , 49.1% silt , and 47.2% clay . the sandy soil has a bulk density of 1.67 mg m with 99.1% sand , 0.20% silt , and 0.70% clay . the moisture content of the clay soil was 4.02 % and that of sandy soil was 5.32% , before both were increased to about 12% at the start of the experiment . the ph of sand was 6.85 and that of clay was 7.45 . each soil ( 100 g dry wt ) was placed in 150 ml beakers ( 18 beakers of each soil type ) and the soil was autoclaved for 1 h , cooled for 24 h , and autoclaved again before use for the study . inoculums were made in pbs and added to the appropriate soil beaker by spraying and mixing 10 ml of the culture mixture with a spray bottle ( sprayco , detroit , mich . ; sanitized by soaking in 70% ethanol ) on the surface of 100 g of soil to obtain the following inoculums concentrations in triplicate : 10 , 10 , 10 , 10 , 10 , and 0 for both autoclaved and unautoclaved soils.serial dilution was made with 10 g portion of each soil for the enumeration of bacteria . soils were mixed for 5 min with sterile specula to homogeneously distribute the e. coli o157:h7 , and covered with foil and incubated at 20 c in the growth chamber for the duration of the experiment . e. coli o157:h7 population was determined by plating on tryptic soy agar ( tsa ; becton dickinson ) plates containing 100 g of ampicillin ml ( tsa - a ) at days 0 ( inoculation ) , 1 , 3 , 5 , 10 , 20 , 30 , 40 , 50 , and 60 . the fumigant methyl iodide ( mei , iodomethane , > 99% purity ) was purchased from chem service ( west chester , pa ) and methyl bromide ( mebr > 99% purity ) was obtained from great lakes chemical company ( west lafayette , in ) . plastic trays ( 58.2 43.2 18.5 cm ) were filled with approximately 40 kg of soil . bacteria were inoculated into the irrigation lines with a cole - parmer hplc pump ( cole - parmer , chicago , illinois ) and delivered through pvc pipes to each tray with five surface drip lines . soil samples were collected on the day of inoculation for community analysis , e. coli o157:h7(pgfp ) concentration and heterotrophic plate counts . after the initial sample collection , trays were manually covered with a virtually impermeable plastic film ; 0.038 mm hytibar film ( klerk plastics , belgium ) and fumigants were applied . fumigant rates and application methods were selected according to the recommended field application rate for each chemical by california department of pesticide regulation ( http://www.cdpr.ca.gov/ ) . to avoid the emission of fumigants to the growth chamber , syringes were used to inject fumigant ( mebr - gas and mei - liquid ) into the trays ; injection ports covered immediately with duct tape and left in the growth chamber for 10 days . trays remained outside in an area covered with barb wires , opened and aerated for 2 days before they were moved back to the growth chamber for the continuation of the experiment . at this point , a total of 14 days has elapsed since fumigation , and soil samples were collected for e. coli o157:h7(pgfp ) concentration , bacterial diversity , and heterotrophic plate counts . the sandy soil received the above nutrient solution twice daily , because this was a very poor river bank soil with poor nutrients for plant growth . soil samples were collected weekly for 5 weeks for e. coli o157:h7 , heterotrophic plate counts and for total bacterial dna extraction . soil was transferred to ziplock bags and 10 g sample was used for serial dilution . e. coli / pgfp colonies were enumerated under a hand - held spectroline ultraviolet lamp ( spectronics corporation , westbury , n.y ) . community dna was extracted from 0.5 g soil with the ultra clean soil dna kit ( mobio laboratories , solana beach , ca ) according to the manufacturer 's protocol and stored at 20 c. a 236-bp dna fragment in the v3 region of the small subunit ribosomal rna genes of eubacteria was amplified by using primer set prba338f and prun518r . ready - to - go pcr beads ( amersham pharmacia biotech , piscataway , nj ) and 5 pmol of primers in a total volume of 25 ml were used in the pcr reaction . pcr amplifications were done under the following conditions : 92c for 2 min ; 30 cycles of 92c for 1 min , 55c for 30 s , 77c for 1 min followed by a final extension at 72c for 6 min . dgge was performed with 8% ( wt / vol ) acrylamide gels containing a linear chemical gradient ranging from 30% to 70% denaturant with 100% defined as 7 m urea and 40% formamide . gels were run for 3 h at 200 v with the dcode universal mutation system ( bio - rad laboratories , hercules , ca ) . dna was visualized after ethidium bromide staining by uv transillumination and photographed with a polaroid camera . dna fingerprints obtained from the 16s rrna banding patterns on the dgge gels were photographed and digitized using imagemaster labscan ( amersham - pharmacia biotech , uppsala , sweden ) and analyzed . the comparison of diversity was done by using a one - way analysis of variance , and tukey hsd test for post hoc analysis . diversity was calculated by using the shannon index of diversity ( h ) to compare changes in diversity of microbial communities within all treatments at each time by using the following function : ( 1)h = pi log pi , when pi = ni / n , ni is the height of peak , and n is the sum of all peak heights in the curve . genomic dna was isolated from pure culture of e. coli o157:h7 , grown for 12 h at 37c and extracted with the qiagen tissue kit ( qiaamp dna mini kit ; valencia , ca ) . dna extracted from o157:h7 was used for the construction of standard curve and for the determination of detection limits of the e. coli by real - time pcr . total bacterial dna was extracted from soil with the ultra clean soil dna kit ( mobio laboratories , solana beach , ca ) as stated above and stored at 20c . primers and probes used for the detection and quantification of the stx1 , stx2 , and the eae gene in e. coli o157:h7 were as described [ 21 , 22 ] . real - time , quantitative pcr was performed with the icycler iq ( bio - rad , hercules , ca ) as described by ibekwe et al . . briefly , pcr was performed in a total volume of 50 l volume containing 200 m of dntps , 2 l of genomic dna from each concentration , 2.5 u of amplitaq gold polymerase , 5 l of 10x taqman buffer ( pe applied biosystems , foster city , ca ) , 0.3 m of each primer , 0.1 m of probe , and 3.5 mm of mgcl2 . genomic dna purified from e. coli o157:h7 was used as a template for the positive control and no template for negative control . pcr was performed using the following cycle conditions : denaturation at 95c for 10 min , 50 cycles of 94c for 20 s , 55c for 30 s , 72c for 40 s , followed by a 5 min extension at 72c and a hold at 4c . standard curves generated from plotting the threshold cycle ( ct ) versus log10 of starting dna quantities ( pg ) were used for determining the detection limit of the assay . optimization of the multiplex assay was done as previously discussed [ 21 , 22 ] . amplification efficiency ( e ) was estimated by using the slope of the standard curve and the formula : e = ( 10)1 . reaction with 100% efficiency generated a slope of 3.32 . statistical analyses were done with the general linear model ( glm ) procedure of the statistical analysis system . the population data were log transformed to obtain a normal distribution of the data . comparisons between pairs of treatment means at any date were accomplished with the tukey 's test . the log - transform data of e. coli o157:h7 population size of all individual samples were plotted over time after inoculation , and analyzed by regression analysis . plate counts and real time pcr data were transformed to log10 values and survival curves were obtained by plotting the logarithm of survivors against the treatment time . the survival data were fitted to a biphasic model as proposed by coroller et al . [ 23 , 24 ] with the geeraerd and van impe inactivation model - fitting tool ( ginafit ) as shown in ( 2 ) and ( 3 ) and as described by franz et al . : ( 2)n(t)=n01 + 10[10(t/1)p++10(t/2)p],(3)=log 10 ( f1f ) , where n is the number of survivors , n0 is the inoculums size ; t is the time ; p is the shape parameter , when p > 1 a convex curve is observed ; when p < 1 a concave curve is observed , when p = 1 a linear curve is observed . f , varying from 0 to 1 , is the fraction of subpopulation 1 in the population . another parameter , , varying from negative infinity to positive infinity , is obtained by logit transformation of f as shown in equation 2 . the strong correlation between the scale ( ) and the shape ( p ) parameters makes it possible for the double weibull model to fit most of the shapes of deactivation curves . additionally , when 1 = 2 , the double weibull model can be simplified into a single weibull model , and the survival curve can be described by only three parameters . a very important and useful parameter , time to detection limit ( td ) can also be calculated when using ginafit to fit the experimental survival data . the effects of inoculum density on the survival of e. coli o157:h7 in sandy and clay soils was first determined in the two soils used for this study . this was done to determine the influence of indigenous microorganism on the survival of e. coli o157:h7 in autoclaved and unautoclaved soils . data from the survival study showed that within the first 7 days e. coli o157:h7 populations decreased by ca . 0.24 log10cfu g in the 10 dilution and by 0.67 log10cfu g in the 10 dilution for the unautoclaved soil ( figures 1(a ) and 1(b ) ) . the reverse was the case with autoclaved soil where there was an increased in population by 2.13 log10 cfu g in the 10 dilution and an increased of 1.68 log10 cfu g in the 10 dilution . survival curves showed a concave curvature in autoclave sandy soil and a convex curvature in the unautoclaved sandy soil ( figure 1(a ) ) . in the clay soil , the shape parameter was different from sandy soil with soils with inoculums density of 10 showing the convex shape whereas soils with cells at 10 showed either concave or linear shape . modeling parameters ( alpha ( ) , delta ( ) , and the shape parameter - p ) were calculated from equation ( 2 ) and ( 3 ) used to explain the inactivation kinetics . more variations in values were observed from different soils ( figures 1(c ) and 1(d ) ) . when the strain was characterized in sandy and clay soils , distinct 1 and 2 were observed indicating that the two subpopulations behaved differently in both soils , thus the survival data in both soils might not be simplified into the single weibull model that can be described by only three parameters , , and p. the initial sharp decrease in cell numbers in sandy soil ( concave shape ) might largely be attributed to the faster decline of subpopulation as shown with smaller 1 ( figure 1(c ) ) . however , with the time going , the subpopulation with greater 2 ( i.e. , the more resistant ) dominated the cell population , leading to a slower and steadily decline of the cell concentration as the curves showed little or no decline . after the first 10 days , e. coli o157:h7 populations in unautoclaved soil declined considerably more rapidly than in autoclaved soil below the detection limits of 10 cfug soil . after 60 days , the concentration of e. coli o157:h7 in the 10 dilution was undetectable by plate count , and there was a 6.18 log10cfu g reduction for the 10 dilution . survival of pathogen was greater in the sandy soil ( p = .05 ) than clay unautoclaved soil within the first 7 days ( figure 1 ) . e. coli o157:h7 at 10 cfu g dilutions survived for more than 60 days in both unautoclaved and autoclaved soils used in this study . before the enumeration of e. coli o157:h7 in the different matrices , background concentrations of heterotrophic bacterial the initial heterotrophic plate count in soil was 2.1 10 cfu g. after storage at 20c in the growth chamber , the total aerobic plate counts decreased steadily from ca . there were no differences in the levels of heterotrophic plate count in the two soils during the study period ( data not shown ) . mean comparison by days and methods were used to determine the impact of fumigants on the survival of e. coli o157:h7 in the two soils after fumigation ( table 1 ) . since one of our objectives was to determine the effects of fumigants on e. coli o157:h7 on a weekly basis , direct comparison of the two fumigants and the control was done using plate count and real - time pcr to quantify the concentrations of e. coli o157:h7 . in the growth chamber soil , ten days after fumigation , e. coli o157:h7 was significantly lower ( p = .0001 ) in fumigated soils than the control clay soil at the recommended application rate . during the rest of the study , there were no significant differences on the effect of the two fumigants on the pathogen , except on day 36 ( p = .046 ) where the effects varied . real - time pcr analysis showed that 10 days after fumigation , e. coli o157:h7 concentration in non - fumigated soils was significantly higher ( p = .002 ) in sandy soil than clay soil . there were no significant differences ( p = .56 ) in pathogen concentration during day 23 when real - time pcr was used for the analysis . the same effect was observed during day 36 and 50 ( data not shown ) . direct comparison between mebr and mei within each soil showed that neither had significant greater impact on e. coli o157:h7 . the majority of the survival curves ( figure 2 ) showed a concave shape , with a relatively fast initial decline followed by a slower decline phase . survival reached the detection limit faster in clay soil than in sandy soil without fumigation using plate counts ( figures 2(a ) and 2(b ) ) . when the pathogen was exposed to fumigants ( mei ) , inactivation was faster than in control , especially with plate count ( figures 2(c ) and 2(d ) ) . the same pattern was observed with mebr ( figures 2(e ) and 2(f ) ) . however , for both control treatments the population size did not reach the detection limit ( ttd ) of 10 cfu g during the experiment due to earlier onset of tailing at about 35 days using real - time pcr . also , both soils showed that it took less than a day to inactivate the first log10 of microbial population in most of the fumigated samples . effects of soil types on the survival e. coli o157:h7 in clay and sandy soils after fumigation was model by fitting the experimental data into the survival functions ( figure 3 ) . similar modeling parameters ( , , and p ) were calculated when they were inoculated into the same soil ( figures 3(a)3(f ) ) . however , there more little variations in these parameters from the two soils , except the values . when the pathogen was characterized in sandy and clay soils , distinct 1 and 2 were observed indicating that the two subpopulations behave differently in both soils . the goodness - of - fit statistics ( r ) did not differ significantly between survival curves in sandy soil irrespective of fumigants or no fumigant . the same effect was observed in clay soil , indicating that the model is suitable to fit survival curves of e. coli o157:h7 in an array of different soils . dgge analysis of 16s rrna fragments was used to examine the effects of mebr and mei on soil microbial communities during week 1 to7 after fumigation . the most drastic effect occurred on the first week of the experiment where there was a significant effect ( p .05 ) of fumigants as determined by the shannon - weaver index of diversity between clay and sandy soil ( table 2 ) . during this period , microbial diversity in the mebr fumigated treatments was significantly lower ( p = .0003 ) in sandy soil than in clay soil at the recommended application rate . the same effect was observed with mei fumigated soil . bacterial communities were not different at week three ( p = .13 ) , week four ( p = .06 ) , and week five ( p = .11 ) . however , at week seven there was a significant ( p = .0001 ) shift in microbial community structure as determined by diversity index with all the treatments ( table 2 ) , and the effect was greater in sandy soil than clay soil . this resulted in the initial higher survival rate of e. coli o157:h7 in sandy soil compared to clay soil . microbial diversity ( expressed as shannon weaver index of diversity h ) was positively correlated with survival of e. coli o157:h7 in sandy soils ( figure 4(a ) ; r = 0.56 , p = .015 ) and in clay soil using the plate count method ( figure 4b ; r = 0.47 ; p = .019 ) . using data from real - time pcr analysis , survival of e. coli o157:h7 were positively correlated with microbial diversity in both clay and sandy soils ( data not shown ) . before the start of this experiment , a preliminary study was conducted in autoclaved and unautoclaved soil to determine the influence of indigenous soil microorganisms on the survival and growth of e. coli o157:h7 ( figures 1(a ) and 1(b ) ) . the antagonistic effect of indigenous soil microorganisms was likely a factor in killing e. coli o157:h7 cells in unautoclaved soils used in this study . e. coli o157:h7 was inactivated more rapidly in unautoclaved soil than in autoclaved soil in all the different dilutions of e. coli o157:h7 used as inoculums ( figures 1(a ) and 1(b ) for 10 and 10 cfu g ) . our study is in agreement with jiang et al . who showed that small numbers of e. coli o157:h7 from an unautoclaved soil were only detected by enrichment culture , and survived for longer period of time at 15c than 21c . this suggests that there was a small population of cells that have survived in the soil under different environmental stress . the long - term survival of this pathogen in the environment has been reported by many authors [ 6 , 2731 ] , but very little has been done on the survival in fumigated soil . we have shown from this study that e. coli o157:h7 can survive in fumigated soils for over 60 days due to long - term persistence of a small percent of the population . our study showed that e. coli o157:h7 can survive longer in sandy soil than in clay soil during a short term experiment . however , populations persisted longer in clay than in sandy soil during a long - term study . our results showed that e. coli o157:h7 survived longer than 60 days in both soils . others have reported survival of more than 54 days in manure amended soil [ 28 , 3234 ] and 34 days or more in sandy loam soil amended with cow manure [ 26 , 35 ] , and over 90 days in clay soils ( 8 , 40 ) . others reported longer e. coli o157:h7 survival times of between 154 and 217 d in soils amended with inoculated compost . this study with longer survival period agrees with our study because both studies relied on inoculating the substrate with relatively high densities of the pathogen ( > 10 cfu g ) . also , during our preliminary experiment with e. coli o157:h7 with population of lesser than 10 cfu g , survival of the pathogen was less than seven days this is in agreement with franz et al . that monitored the fate of the pathogens in manure - amended soil after they declined to below detection limit within a short period with low and more realistic levels of pathogens inoculated into manure ( approximately 10 cfu g ) . therefore , the survival of e. coli o157:h7 in the environment may depend on the initial concentration at the beginning of the experiment . in this study , we used the double weibull model , which is the cumulative form of the underlying distribution of individual inactivation kinetics , and it was a suitable model for describing the decline of e. coli o157:h7 . the survival curves generated from our study in most cases showed a convex fitting , indicating changes in biological stress over time . the model is sufficiently flexible to account for different survival patterns and has been previously used to model thermal inactivation of listeria monocytogenes in sucrose solutions of various water activities and the survival of e. coli o157:h7 in manure amended soil . franz et al . ; van boekel , ; peleg , discussed the different processes and mechnisms responsible for the different shape parameters during inactivation of e. coli o157:h7 in soil . they pointed out that even though the weibull model is an empirical model , it can be linked to physiological properties at population level and that the population is heterogeneous with respect to the stress encountered in the soil . these authors noted that a convex curve would mean that the remaining cells become increasingly susceptible to stress , and the cells are therefore subjected to more damages with time . a linear survival curve means that inactivation does not depend on time or other biological activities and the concave survival curve means that sensitive members of the population are rapidly eliminated and that the sturdier survivors remain . longer persistence of e. coli o157:h7 in clay soil may be influenced by interaction between soil particles in the clay particle sizes that provided niches for the pathogen and moisture / nutrients within the niches . other factors that contributed to the survival of pathogen in soil were soil microbial diversity . recently , the effects of e. coli o157:h7 was assessed in a loamy sand soil obtained from species - rich grassland , in which the microbial community composition had been modified by progressively enhanced fumigation depths . the authors showed that e. coli o157:h7 in the soils with modified community structures due to fumigation was clearly consistent with the hypothesis that within the single selected habitat ( soil ) , which was relatively unaffected with respect to abiotic conditions like ph , moisture and soil chemical conditions , microbial community structure was the main determinant of the survival of the pathogen . with the present study we found that the values of the log reduction time and the shape parameter of the double weibull model were higher for clay soils compared to sandy soils . this means that with sandy soils the initial rate of decline of e. coli o157:h7 was faster than in clay soil . e. coli o157:h7 was more vulnerable to mortality during the first few weeks in the sandy soil than in clay soil . finer - textured ( clayey ) soils result in prolonged survival of introduced bacteria compared with coarser - textured ( sandy ) soils because of higher availability of protective pore spaces against feeding by soil fauna like protozoa . this could explain the faster initial decrease in e. coli o157:h7 numbers in the sandy soils compared with the loamy soils , but survivors are increasingly more sturdy compared with survival in the clay soils . the implication of long - term survival of this pathogen in the environment may involve the recontamination of the environment after the initial contamination event from few surviving strains . e. coli o157:h7 was significantly lower in fumigated soils than the control at the normal application rate during the first 2 weeks of the experiment . however , survival was significantly affected by soil type , with survival greater in sandy soil in the short run than clay soil . however , long - term persistence occurred in clay soil than sandy soil due to properties of clay soil . none of the fumigants showed significant higher toxicity effects on the pathogen at the normal application on the long run , and toxicity was higher in fumigated soil than nonfumigated soils during the first two weeks of the study . therefore , mebr and mei have identical toxicity effects on e. coli o157:h7 at the normal application rate .
persistence of escherichia coli ( e. coli ) o157:h7 in the environment is a major concern to vegetable and fruit growers where farms and livestock production are in close proximity . the objectives were to determine the effects of preplant fumigation treatment on the survival of e. coli o157:h7 in two soils and the effects of indigenous bacterial populations on the survival of this pathogen . real - time pcr and plate counts were used to quantify the survival of e. coli o157:h7 in two contrasting soils after fumigation with methyl bromide ( mebr ) and methyl iodide ( mei ) . ten days after fumigation , e. coli o157:h7 counts were significantly lower ( p = .0001 ) in fumigated soils than in the non - fumigated . direct comparison between mebr and mei within each soil indicated that these two fumigants showed similar impacts on e. coli o157:h7 survival . microbial species diversity as determined by dgge was significantly higher in clay soil than sandy soil and this resulted in higher initial decline in population in clay soil than in sandy soil . this study shows that if soil is contaminated with e. coli o157:h7 , fumigation alone may not eliminate the pathogen , but may cause decrease in microbial diversity which may enhance the survival of the pathogen .
1. Introduction 2. Materials and Methods 3. Results 4. Discussion
due to the increased focus on food safety related to fresh produce , there are more studies using real - time pcr to quantify pathogens such as e. coli o157:h7 in the environment [ 13 , 14 ] . the main objectives of this study were to determine the effects of soil microbial diversity and fumigation on the survival of e. coli o157:h7 in soils contaminated with the pathogen . for our main objectives , both plate count and real - time pcr approaches were used to determine the survival of e. coli o157:h7 in the two soils . the effects of inoculum density on the survival of e. coli o157:h7 in sandy and clay soils was first determined in the two soils used for this study . this was done to determine the influence of indigenous microorganism on the survival of e. coli o157:h7 in autoclaved and unautoclaved soils . mean comparison by days and methods were used to determine the impact of fumigants on the survival of e. coli o157:h7 in the two soils after fumigation ( table 1 ) . since one of our objectives was to determine the effects of fumigants on e. coli o157:h7 on a weekly basis , direct comparison of the two fumigants and the control was done using plate count and real - time pcr to quantify the concentrations of e. coli o157:h7 . in the growth chamber soil , ten days after fumigation , e. coli o157:h7 was significantly lower ( p = .0001 ) in fumigated soils than the control clay soil at the recommended application rate . during the rest of the study , there were no significant differences on the effect of the two fumigants on the pathogen , except on day 36 ( p = .046 ) where the effects varied . real - time pcr analysis showed that 10 days after fumigation , e. coli o157:h7 concentration in non - fumigated soils was significantly higher ( p = .002 ) in sandy soil than clay soil . direct comparison between mebr and mei within each soil showed that neither had significant greater impact on e. coli o157:h7 . survival reached the detection limit faster in clay soil than in sandy soil without fumigation using plate counts ( figures 2(a ) and 2(b ) ) . effects of soil types on the survival e. coli o157:h7 in clay and sandy soils after fumigation was model by fitting the experimental data into the survival functions ( figure 3 ) . during this period , microbial diversity in the mebr fumigated treatments was significantly lower ( p = .0003 ) in sandy soil than in clay soil at the recommended application rate . however , at week seven there was a significant ( p = .0001 ) shift in microbial community structure as determined by diversity index with all the treatments ( table 2 ) , and the effect was greater in sandy soil than clay soil . this resulted in the initial higher survival rate of e. coli o157:h7 in sandy soil compared to clay soil . microbial diversity ( expressed as shannon weaver index of diversity h ) was positively correlated with survival of e. coli o157:h7 in sandy soils ( figure 4(a ) ; r = 0.56 , p = .015 ) and in clay soil using the plate count method ( figure 4b ; r = 0.47 ; p = .019 ) . before the start of this experiment , a preliminary study was conducted in autoclaved and unautoclaved soil to determine the influence of indigenous soil microorganisms on the survival and growth of e. coli o157:h7 ( figures 1(a ) and 1(b ) ) . the long - term survival of this pathogen in the environment has been reported by many authors [ 6 , 2731 ] , but very little has been done on the survival in fumigated soil . our study showed that e. coli o157:h7 can survive longer in sandy soil than in clay soil during a short term experiment . therefore , the survival of e. coli o157:h7 in the environment may depend on the initial concentration at the beginning of the experiment . longer persistence of e. coli o157:h7 in clay soil may be influenced by interaction between soil particles in the clay particle sizes that provided niches for the pathogen and moisture / nutrients within the niches . the authors showed that e. coli o157:h7 in the soils with modified community structures due to fumigation was clearly consistent with the hypothesis that within the single selected habitat ( soil ) , which was relatively unaffected with respect to abiotic conditions like ph , moisture and soil chemical conditions , microbial community structure was the main determinant of the survival of the pathogen . e. coli o157:h7 was more vulnerable to mortality during the first few weeks in the sandy soil than in clay soil .
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the dna in eukaryotic cells is wrapped around histones which can be post - translationally modified to regulate transcription . large genomes , such as the mouse and human genomes , are replete with repetitive sequences including tandem repeats at centromeres and telomeres , and interspersed repeats ( e.g. lines , sines , alu repeats and ervs ) . in order to prevent aberrant transcription , these genomic repeats are packaged into constitutive heterochromatin , a condensed chromatin structure which restricts accessibility to transcriptional machinery . aberrant transcription of these repeats can compromise genome integrity and maintaining the heterochromatic architecture at these sites is critical to genome stability ( 1 ) . the passage of polymerases during dna replication or transcription poses a threat to chromatin and the timely restoration of nucleosomes is important for maintaining epigenetic memory ( 2 ) . the replication of dna during s - phase is accompanied by the deposition of newly synthesised histones as chromatin is rapidly reassembled behind the replication fork ( 3 ) . the canonical histones , h3.1/h3.2 , are specifically expressed during this period and incorporated into chromatin in a replication - coupled manner . in contrast , the histone variant h3.3 is expressed throughout the cell cycle and deposition of this variant is replication - independent ( 46 ) . in addition to the distinct expression patterns , h3.3 differs from the canonical h3.1/h3.2 counterparts at five amino acid residues . these substitutions in h3.3 mediate interactions with chaperone complexes which are unique to h3.3 ( 7,8 ) and facilitate the replication - independent deposition of this variant ( 4 ) . two major h3.3-specific chaperones have been identified ; the hira complex which deposits h3.3 at genic regions ( 5,6 ) and the atrx / daxx complex which is responsible for h3.3 deposition at repetitive regions of the genome ( 911 ) . the association of h3.3 with heterochromatin was first reported at the telomeres of mouse es cells ( 12 ) . this was closely followed by the identification of a chaperone complex comprised of atrx and daxx ( 9,10 ) . atrx is a chromatin remodeller which is thought to localise to telomeres via interactions with g - quadruplex secondary structures ( 13,14 ) while daxx is an h3.3-specific chaperone ( 8) . both components of the atrx / daxx complex were found to be essential for the deposition of h3.3 at telomeres ( 9,10 ) and pericentric heterochromatin ( pch ; 11 ) though the functional significance of this pathway was unclear . more recent studies have now demonstrated that atrx / daxx mediated deposition of h3.3 is not limited to telomeric and pericentric repeats but occurs at heterochromatin distributed throughout the genome of mouse es cells ( 1518 ) , including endogenous retroviral repeats ( ervs ; 1618 ) , imprinted differentially methylated regions ( dmrs ) and selected intragenic methylated cpg islands ( cgis ; 15 ) . enrichment at these sites coincides with the h3k9me3 heterochromatin modification and disruption of atrx , daxx or h3.3 led to a loss of h3k9me3 at these regions ( 15,16,1820 ) . this review will consolidate the major findings of these studies and outline the evidence to support a model where atrx / daxx deposits h3.3 to maintain h3k9me3 heterochromatin in the genome . the interaction between the atrx / daxx complex and histone h3.3 these highly repetitive regions of the genome are archetypal constitutive heterochromatin , and well documented as being enriched for h3k9me3 , h4k20me3 and dna methylation . more recent chip - seq studies have demonstrated that atrx binding sites across the genome are generally associated with heterochromatic modifications ( h3k9me3 , h4k20me3 , dna methylation ; 15 ) . further supporting the idea that atrx localises to heterochromatin , a number of studies detected atrx ( 1518 ) and daxx ( 16,18 ) enrichment at erv repeats , particularly at iap repeats ( intracisternal a particle ; 16,18 ) . ervs are transposable elements which are known to be modified as silent heterochromatin , as aberrant expression results in genome instability ( 1 ) . consistent with this , atrx / daxx binding at ervs coincided with enrichment of h3k9me3 and the co - repressor complex kap1 ( aka trim28 ) and setdb1 ( aka eset ; 16,17 ) , a lysine methyltransferase which catalyses h3k9me3 . it is likely that the heterochromatin modifications at these regions direct atrx binding as the add domain of atrx has been demonstrated to specifically recognise h3k9me3 ( 2123 ) . this interaction has been confirmed at the cellular level where atrx localisation to pch was demonstrated to be dependent on h3k9me3 catalysed by suv39h ( 23 ) . furthermore the monoallelic localisation of atrx to the methylated , heterochromatic allele of imprinted dmrs ( 15 ) demonstrates that chromatin modifications play an important role in directing atrx localisation . the atrx / daxx complex is known to be important for deposition of h3.3 at telomeres and pch . chip - seq of endogenous h3.3 in wt , atrx ko ( 15,16 ) and daxx ko ( 16 ) cells demonstrated that this was also true for the methylated allele of imprinted genes ( 15 ) as well as certain tandem repeats and intragenic cgis ( 15 ) . h3.3 was found to be generally enriched at atrx binding sites in wt cells and this enrichment was lost in atrx ko ( 15,16 ) and daxx ko cells ( 16 ) . h3.3 was also found to be enriched at iap / erv retrotransposons ( 1517 ) although there is some disagreement about the requirement of atrx / daxx for h3.3 deposition at these sites . two studies reported that atrx / daxx ko results in loss of h3.3 at iap / ervs ( 15,16 ) while one study reported increased h3.3 at iap / ervs in atrx ko cells ( 17 ) . the primary difference between these studies is that chip was performed either against endogenous h3.3 ( 15,16 ) or an yfp - tagged h3.3 ( 9 ) . we have eliminated bioinformatics analysis as a possible variable by using a single method to re - align all three data sets . ( 17 ) , when we mapped the yfp - tagged h3.3 data set ( 9 ) to iapez repeats ( figure 1 ) , we detected a gain of yfp - h3.3 at this erv in atrx ko cells . however , the two h3.3 data sets generated using an antibody against endogenous h3.3 ( 15,16 ) did not recapitulate this result and h3.3 enrichment at iapez ervs was consistently lost in atrx ko cells ( figure 1 ) . h3.3 chip - seq reads from all three data sets were also mapped to the telomeres as a control . atrx ko led to decreased h3.3 incorporation at telomeres in all three data sets although telomere enrichment was clearer for endogenous h3.3 , compared to the yfp - tagged h3.3 , in normal cells ( figure 1 ) . the relative enrichment profile of h3.3 at ervs compared to telomeres in the endogenous h3.3 chip data sets are in agreement with immunofluorescence analyses ( 10,12 ) . in addition , upregulated iap expression was detected in both atrx ko ( 17 ) and h3.3 ko cells ( 16 ; see below ) , supporting a role for atrx / daxx - dependent deposition of h3.3 at ervs , in agreement with the endogenous h3.3 chip - seq data . raw read files of yfp - h3.3 chip ( 9 ) , endogenous h3.3 chip ( 15,16 ) and matched input sequencing from wt and atrx ko cells were downloaded from geo ( 35,36 ) and mapped to repeats with repeat enrichment estimator ( 37 ) . samples were normalised for total read counts by dividing mapped reads against total mappable reads . results show normalised reads counts of h3.3 chip and matched input samples for each data set at ( a ) iapez repeats and ( b ) telomere repeats . yfp - h3.3 was increased at iapez repeats in atrx ko relative to wt cells . all three data sets showed decreased h3.3/yfp - h3.3 at telomeres in atrx ko relative to wt cells . it is possible that the discrepancy between the endogenous h3.3 and yfp - h3.3 has arisen due to the introduction of the yfp tag . for example , h3.3 turnover has been demonstrated to be important for regulation ( 24 ) and the yfp tag may interfere with this process . however , it is clear that further experiments are required to reconcile the discrepancies between these data sets . future analysis using a different epitope tag , a different antibody or h3.3 k9 mutant cells would be useful for clarifying the differences between these studies . nonetheless , when taken together , these results suggest that atrx / daxx localises to selected heterochromatic regions in the genome and deposits h3.3 . this pathway has now been extended from telomeres and pch to also include methylated imprinted dmrs , ervs / iaps and selected short tandem repeats . in addition to the loss of h3.3 , disruption of the atrx / daxx complex in mouse es cells also led to the loss of h3k9me3 at some genomic regions . atrx ko led to a reduction in h3k9me3 at methylated imprinted dmrs , intragenic cgis ( 15 ) and iap repeats ( 16 ) as detected by chip - qpcr and chip - seq . similarly , a reduction in h3k9me3 was also detected at iaps ( 16 ) and telomeres ( 18 ) in daxx ko cells . in addition , direct knockout of h3.3 also led to decreased h3k9me3 at iaps ( 16 ) and telomeres ( 19,20 ) . chip - rechip assays of h3.3 and h3k9me3 on a genome - wide basis ( 16 ) and specifically at telomeres ( 19 ) demonstrated that h3.3 at these heterochromatic sites were directly modified with k9me3 ( h3.3 k9me3 ) . overall , these data support a model where atrx / daxx mediates deposition of h3.3 which can be modified with k9me3 to facilitate the maintenance of heterochromatin . the h3k9me3 modification is catalysed by suv39h1/2 at telomeres and pch ( 25,26 ) while setdb1 acts in a complex with kap1 to catalyse h3k9me3 at ervs ( 27,28 ) and methylated imprinted dmrs ( 28,29 ) . the kap1/setdb1 complex was found to co - localise with atrx at ervs ( 17 ) and generally with atrx / daxx / h3.3/h3k9me3 across the genome ( 16 ) . consistent with previous studies , depletion of setdb1 led to reduced h3k9me3 at iaps / ervs ( 16,17 ) and also at telomeres ( 19 ) , albeit to a lesser degree . a direct interaction between daxx and kap1 has been identified by co - immunoprecipitation ( 16 ) and suggests that daxx contributes to kap1 binding at some genomic sites . although krab - znfs , such as zfp809 at ervs ( 30 ) and zfp57 at imprinted genes ( 29,31 ) , are known to be the main determinant for kap1 localisation , it is possible that daxx may help to further stabilise these interactions . overall , these studies suggest that the kap1/setdb1 complex is able to catalyse h3.3k9me3 at these sites . in addition , daxx ( 18 ) and h3.3 ( 19 ) were also found to interact with suv39h and depletion of suv39h led to a loss in h3.3 k9me3 at telomeres ( 19 ) . combined , these results demonstrate that both setdb1 and suv39h are able to catalyse the trimethylation of lysine 9 on h3.3 . previous studies have shown that atrx ko leads to increased terra transcription at telomeres ( 9 ) and this was verified by sirna depletion of atrx and daxx ( 18 ) . knockout of histone h3.3 similarly led to increased terra transcription ( 19 ) confirming that the atrx / daxx deposition of h3.3 is important for suppressing telomere transcription . this has now been extended to other genomic loci ; atrx ko cells failed to silence a mini - iap transgene ( 17 ) and resulted in aberrant allelic expression of imprinted genes ( 15 ) while h3.3 ko led to increased erv transcription in mouse es cells ( 16 ) . it should be noted that multiple mechanisms may mediate transcriptional silencing at these genomic regions and the atrx / daxx / h3.3 complex is one of many heterochromatin factors which maintain silencing . the degree of transcriptional deregulation varies depending on which heterochromatin pathway is disrupted , however , aberrant transcription is a reliable indicator of alterations in the underlying chromatin structure . the importance of atrx / h3.3 in heterochromatin maintenance is further demonstrated by the decrease in other silent chromatin modifications including h4k20me3 at telomeres ( 19 ) and dna methylation at dmrs of imprinted genes ( 15 ) . taken together , these studies demonstrate that atrx / daxx deposition of h3.3 k9me3 is important for maintaining silent heterochromatin at a number of genomic sites . the interaction between the atrx / daxx complex and histone h3.3 was first described at telomeres and pch ( 911 ) . these highly repetitive regions of the genome are archetypal constitutive heterochromatin , and well documented as being enriched for h3k9me3 , h4k20me3 and dna methylation . more recent chip - seq studies have demonstrated that atrx binding sites across the genome are generally associated with heterochromatic modifications ( h3k9me3 , h4k20me3 , dna methylation ; 15 ) . further supporting the idea that atrx localises to heterochromatin , a number of studies detected atrx ( 1518 ) and daxx ( 16,18 ) enrichment at erv repeats , particularly at iap repeats ( intracisternal a particle ; 16,18 ) . ervs are transposable elements which are known to be modified as silent heterochromatin , as aberrant expression results in genome instability ( 1 ) . consistent with this , atrx / daxx binding at ervs coincided with enrichment of h3k9me3 and the co - repressor complex kap1 ( aka trim28 ) and setdb1 ( aka eset ; 16,17 ) , a lysine methyltransferase which catalyses h3k9me3 . it is likely that the heterochromatin modifications at these regions direct atrx binding as the add domain of atrx has been demonstrated to specifically recognise h3k9me3 ( 2123 ) . this interaction has been confirmed at the cellular level where atrx localisation to pch was demonstrated to be dependent on h3k9me3 catalysed by suv39h ( 23 ) . furthermore the monoallelic localisation of atrx to the methylated , heterochromatic allele of imprinted dmrs ( 15 ) demonstrates that chromatin modifications play an important role in directing atrx localisation . the atrx / daxx complex is known to be important for deposition of h3.3 at telomeres and pch . chip - seq of endogenous h3.3 in wt , atrx ko ( 15,16 ) and daxx ko ( 16 ) cells demonstrated that this was also true for the methylated allele of imprinted genes ( 15 ) as well as certain tandem repeats and intragenic cgis ( 15 ) . h3.3 was found to be generally enriched at atrx binding sites in wt cells and this enrichment was lost in atrx ko ( 15,16 ) and daxx ko cells ( 16 ) . h3.3 was also found to be enriched at iap / erv retrotransposons ( 1517 ) although there is some disagreement about the requirement of atrx / daxx for h3.3 deposition at these sites . two studies reported that atrx / daxx ko results in loss of h3.3 at iap / ervs ( 15,16 ) while one study reported increased h3.3 at iap / ervs in atrx ko cells ( 17 ) . the primary difference between these studies is that chip was performed either against endogenous h3.3 ( 15,16 ) or an yfp - tagged h3.3 ( 9 ) . we have eliminated bioinformatics analysis as a possible variable by using a single method to re - align all three data sets . ( 17 ) , when we mapped the yfp - tagged h3.3 data set ( 9 ) to iapez repeats ( figure 1 ) , we detected a gain of yfp - h3.3 at this erv in atrx ko cells . however , the two h3.3 data sets generated using an antibody against endogenous h3.3 ( 15,16 ) did not recapitulate this result and h3.3 enrichment at iapez ervs was consistently lost in atrx ko cells ( figure 1 ) . h3.3 chip - seq reads from all three data sets were also mapped to the telomeres as a control . atrx ko led to decreased h3.3 incorporation at telomeres in all three data sets although telomere enrichment was clearer for endogenous h3.3 , compared to the yfp - tagged h3.3 , in normal cells ( figure 1 ) . the relative enrichment profile of h3.3 at ervs compared to telomeres in the endogenous h3.3 chip data sets are in agreement with immunofluorescence analyses ( 10,12 ) . in addition , upregulated iap expression was detected in both atrx ko ( 17 ) and h3.3 ko cells ( 16 ; see below ) , supporting a role for atrx / daxx - dependent deposition of h3.3 at ervs , in agreement with the endogenous h3.3 chip - seq data . raw read files of yfp - h3.3 chip ( 9 ) , endogenous h3.3 chip ( 15,16 ) and matched input sequencing from wt and atrx ko cells were downloaded from geo ( 35,36 ) and mapped to repeats with repeat enrichment estimator ( 37 ) . samples were normalised for total read counts by dividing mapped reads against total mappable reads . results show normalised reads counts of h3.3 chip and matched input samples for each data set at ( a ) iapez repeats and ( b ) telomere repeats . yfp - h3.3 was increased at iapez repeats in atrx ko relative to wt cells . all three data sets showed decreased h3.3/yfp - h3.3 at telomeres in atrx ko relative to wt cells . it is possible that the discrepancy between the endogenous h3.3 and yfp - h3.3 has arisen due to the introduction of the yfp tag . for example , h3.3 turnover has been demonstrated to be important for regulation ( 24 ) and the yfp tag may interfere with this process . however , it is clear that further experiments are required to reconcile the discrepancies between these data sets . future analysis using a different epitope tag , a different antibody or h3.3 k9 mutant cells would be useful for clarifying the differences between these studies . nonetheless , when taken together , these results suggest that atrx / daxx localises to selected heterochromatic regions in the genome and deposits h3.3 . this pathway has now been extended from telomeres and pch to also include methylated imprinted dmrs , ervs / iaps and selected short tandem repeats . in addition to the loss of h3.3 , disruption of the atrx / daxx complex in mouse es cells also led to the loss of h3k9me3 at some genomic regions . atrx ko led to a reduction in h3k9me3 at methylated imprinted dmrs , intragenic cgis ( 15 ) and iap repeats ( 16 ) as detected by chip - qpcr and chip - seq . similarly , a reduction in h3k9me3 was also detected at iaps ( 16 ) and telomeres ( 18 ) in daxx ko cells . in addition , direct knockout of h3.3 also led to decreased h3k9me3 at iaps ( 16 ) and telomeres ( 19,20 ) . chip - rechip assays of h3.3 and h3k9me3 on a genome - wide basis ( 16 ) and specifically at telomeres ( 19 ) demonstrated that h3.3 at these heterochromatic sites were directly modified with k9me3 ( h3.3 k9me3 ) . overall , these data support a model where atrx / daxx mediates deposition of h3.3 which can be modified with k9me3 to facilitate the maintenance of heterochromatin . the h3k9me3 modification is catalysed by suv39h1/2 at telomeres and pch ( 25,26 ) while setdb1 acts in a complex with kap1 to catalyse h3k9me3 at ervs ( 27,28 ) and methylated imprinted dmrs ( 28,29 ) . the kap1/setdb1 complex was found to co - localise with atrx at ervs ( 17 ) and generally with atrx / daxx / h3.3/h3k9me3 across the genome ( 16 ) . consistent with previous studies , depletion of setdb1 led to reduced h3k9me3 at iaps / ervs ( 16,17 ) and also at telomeres ( 19 ) , albeit to a lesser degree . a direct interaction between daxx and kap1 has been identified by co - immunoprecipitation ( 16 ) and suggests that daxx contributes to kap1 binding at some genomic sites . although krab - znfs , such as zfp809 at ervs ( 30 ) and zfp57 at imprinted genes ( 29,31 ) , are known to be the main determinant for kap1 localisation , it is possible that daxx may help to further stabilise these interactions . overall , these studies suggest that the kap1/setdb1 complex is able to catalyse h3.3k9me3 at these sites . in addition , daxx ( 18 ) and h3.3 ( 19 ) were also found to interact with suv39h and depletion of suv39h led to a loss in h3.3 k9me3 at telomeres ( 19 ) . combined , these results demonstrate that both setdb1 and suv39h are able to catalyse the trimethylation of lysine 9 on h3.3 . previous studies have shown that atrx ko leads to increased terra transcription at telomeres ( 9 ) and this was verified by sirna depletion of atrx and daxx ( 18 ) . knockout of histone h3.3 similarly led to increased terra transcription ( 19 ) confirming that the atrx / daxx deposition of h3.3 is important for suppressing telomere transcription . this has now been extended to other genomic loci ; atrx ko cells failed to silence a mini - iap transgene ( 17 ) and resulted in aberrant allelic expression of imprinted genes ( 15 ) while h3.3 ko led to increased erv transcription in mouse es cells ( 16 ) . it should be noted that multiple mechanisms may mediate transcriptional silencing at these genomic regions and the atrx / daxx / h3.3 complex is one of many heterochromatin factors which maintain silencing . the degree of transcriptional deregulation varies depending on which heterochromatin pathway is disrupted , however , aberrant transcription is a reliable indicator of alterations in the underlying chromatin structure . the importance of atrx / h3.3 in heterochromatin maintenance is further demonstrated by the decrease in other silent chromatin modifications including h4k20me3 at telomeres ( 19 ) and dna methylation at dmrs of imprinted genes ( 15 ) . taken together , these studies demonstrate that atrx / daxx deposition of h3.3 k9me3 is important for maintaining silent heterochromatin at a number of genomic sites . a substantial proportion of mammalian genomes is comprised of repetitive dna which includes telomeres , centromeres and ervs . aberrant transcription of these repeats is deleterious to the organism and maintaining heterochromatic silencing at these repeats is critical for protecting genomic integrity . chromatin modifications , such as dna methylation , h3k9me3 and h4k20me3 , are known to be important for maintaining silencing . in addition , a recent set of studies have now also identified the histone variant h3.3 as an important component of heterochromatin in mouse es cells . the complex which deposits h3.3 at heterochromatin is comprised of daxx , an h3.3 specific chaperone , and a chromatin remodeller , atrx . this chaperone complex both targets h3.3 to specific heterochromatic sites and interacts with histone modifiers to maintain the k9me3 heterochromatin mark . the add domain of atrx preferentially interacts with h3k9me3 which likely drives the localisation of atrx / daxx / h3.3 to heterochromatin modified regions including telomeres , ervs and the methylated imprinted dmrs . daxx is then able to interact with either the kap1/setdb1 co - repressor complex or suv39h , both of which catalyse h3k9me3 . atrx / daxx is therefore able to target heterochromatin for deposition of h3.3 and recruit methyltransferases to mediate direct modification of k9me3 on h3.3 . this represents a self - reinforcing pathway which is able to protect heterochromatin integrity throughout the cell cycle ( figure 2 ) . ( a ) heterochromatic regions such as telomeres , iap ltrs and methylated imprinted dmrs are distributed throughout the genome and enriched for h3k9me3 . ( b ) atrx recognises h3k9me3 and acts with daxx to deposit h3.3 to replace histones which are lost . atrx / daxx / h3.3 are able to act continuously through the cell cycle to ensure constant maintenance of h3k9me3 heterochromatin . these studies have identified the atrx / daxx / h3.3 complex as an important contributor to heterochromatin silencing in mouse es cells however , a number of outstanding questions remain . one possible clue could lie in the replication - independent expression and incorporation of the h3.3 variant . this would suggest that atrx / daxx / h3.3 are particularly important outside of s - phase when the canonical histones are unavailable . future analyses with highly quantitative time - lapse imaging experiments would provide useful information of the dynamics of h3.3 , including turnover and deposition of newly synthesised h3.3 , and recruitment of other chromatin repressors at these sites . furthermore , while it is evident that depletion of atrx / daxx / h3.3 leads to loss of heterochromatin , the overall phenotypic consequences remain unclear . this is particularly pertinent to alt cancers where atrx mutations have been strongly linked to increased recombination at telomeres ( 32,33 ) . several studies have demonstrated that disruption of heterochromatin facilitates aberrant telomere recombination ( 34 ) . detailed analyses of how heterochromatin structure , including h4k20me3 and dna methylation , is disrupted in atrx / daxx / h3.3 deficient cells would provide insights into how this pathway might promote genome instability in cancer . australia research council ( arc ) future fellowship award from the arc , australia [ to l.h.w . ] .
a number of studies have demonstrated that various components of the atrx / daxx / histone h3.3 complex are important for heterochromatin silencing at multiple genomic regions . we provide an overview of the individual components ( atrx , daxx and/or h3.3 ) tested in each study and propose a model where the atrx / daxx chaperone complex deposits h3.3 to maintain the h3k9me3 modification at heterochromatin throughout the genome .
INTRODUCTION The ATRX/DAXX complex localises to heterochromatin ATRX/DAXX is required for H3.3 deposition at heterochromatin ATRX/DAXX mediated deposition of H3.3 is required for maintaining H3K9me3 SUV39H and SETDB1 catalyse K9me3 on H3.3 Disruption of ATRX/DAXX/H3.3K9me3 leads to aberrant transcription CONCLUSIONS FUNDING
two major h3.3-specific chaperones have been identified ; the hira complex which deposits h3.3 at genic regions ( 5,6 ) and the atrx / daxx complex which is responsible for h3.3 deposition at repetitive regions of the genome ( 911 ) . both components of the atrx / daxx complex were found to be essential for the deposition of h3.3 at telomeres ( 9,10 ) and pericentric heterochromatin ( pch ; 11 ) though the functional significance of this pathway was unclear . more recent studies have now demonstrated that atrx / daxx mediated deposition of h3.3 is not limited to telomeric and pericentric repeats but occurs at heterochromatin distributed throughout the genome of mouse es cells ( 1518 ) , including endogenous retroviral repeats ( ervs ; 1618 ) , imprinted differentially methylated regions ( dmrs ) and selected intragenic methylated cpg islands ( cgis ; 15 ) . this review will consolidate the major findings of these studies and outline the evidence to support a model where atrx / daxx deposits h3.3 to maintain h3k9me3 heterochromatin in the genome . the interaction between the atrx / daxx complex and histone h3.3 these highly repetitive regions of the genome are archetypal constitutive heterochromatin , and well documented as being enriched for h3k9me3 , h4k20me3 and dna methylation . the atrx / daxx complex is known to be important for deposition of h3.3 at telomeres and pch . nonetheless , when taken together , these results suggest that atrx / daxx localises to selected heterochromatic regions in the genome and deposits h3.3 . in addition to the loss of h3.3 , disruption of the atrx / daxx complex in mouse es cells also led to the loss of h3k9me3 at some genomic regions . overall , these data support a model where atrx / daxx mediates deposition of h3.3 which can be modified with k9me3 to facilitate the maintenance of heterochromatin . knockout of histone h3.3 similarly led to increased terra transcription ( 19 ) confirming that the atrx / daxx deposition of h3.3 is important for suppressing telomere transcription . it should be noted that multiple mechanisms may mediate transcriptional silencing at these genomic regions and the atrx / daxx / h3.3 complex is one of many heterochromatin factors which maintain silencing . taken together , these studies demonstrate that atrx / daxx deposition of h3.3 k9me3 is important for maintaining silent heterochromatin at a number of genomic sites . the interaction between the atrx / daxx complex and histone h3.3 was first described at telomeres and pch ( 911 ) . the atrx / daxx complex is known to be important for deposition of h3.3 at telomeres and pch . nonetheless , when taken together , these results suggest that atrx / daxx localises to selected heterochromatic regions in the genome and deposits h3.3 . in addition to the loss of h3.3 , disruption of the atrx / daxx complex in mouse es cells also led to the loss of h3k9me3 at some genomic regions . overall , these data support a model where atrx / daxx mediates deposition of h3.3 which can be modified with k9me3 to facilitate the maintenance of heterochromatin . knockout of histone h3.3 similarly led to increased terra transcription ( 19 ) confirming that the atrx / daxx deposition of h3.3 is important for suppressing telomere transcription . it should be noted that multiple mechanisms may mediate transcriptional silencing at these genomic regions and the atrx / daxx / h3.3 complex is one of many heterochromatin factors which maintain silencing . taken together , these studies demonstrate that atrx / daxx deposition of h3.3 k9me3 is important for maintaining silent heterochromatin at a number of genomic sites . this chaperone complex both targets h3.3 to specific heterochromatic sites and interacts with histone modifiers to maintain the k9me3 heterochromatin mark . the add domain of atrx preferentially interacts with h3k9me3 which likely drives the localisation of atrx / daxx / h3.3 to heterochromatin modified regions including telomeres , ervs and the methylated imprinted dmrs . these studies have identified the atrx / daxx / h3.3 complex as an important contributor to heterochromatin silencing in mouse es cells however , a number of outstanding questions remain .
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lower urinary tract symptoms ( luts ) are common , especially in older men , and are most often caused by benign prostatic hyperplasia ( bph ) which results in benign prostatic enlargement ( bpe ) and bladder outlet obstruction ( boo ) . patients with severe luts unresponsive to pharmacological management , and those with bph complications are candidates for surgical therapy . transurethral resection of the prostate ( turp ) has been considered the surgical gold standard in terms of efficacy and re treatment rate . however , recent scientific and technological advances have challenged the traditional surgical approach to bph . the morbidity and late complications of turp were the major impetus driving the development of new techniques such as bipolar turp and laser enucleation or vaporization . open prostatectomy , recommended by the european association of urology for patients with prostate volumes > 80 ml , is associated with a prolonged hospital stay and serious complications such as severe blood loss and infection . additionally , patients may require subsequent surgical revision . despite the wide acceptance of open prostatectomy ; blunt dissection from the capsule , particularly in the apical area , may be technically challenging for the less experienced surgeons and is frequently associated with complications . laparoscopic , and more recently robotic techniques , have provided a minimally invasive alternative to open prostatectomy , with equal efficacy , faster recovery , and shorter hospital stay . there is less pain and improved cosmetic results , although operative time ( ot ) and estimated blood loss are dependent on surgical expertise . these endoscopic procedures allow improved visualization and reduced morbidity and are well established in the management of prostate carcinoma , providing a rationale for their use in the treatment of bph [ 35 ] . articles for this review were identified through pubmed searches from january 2004 through december 2013 . various algorithms were used including : benign prostatic hyperplasia , adenomectomy , simple prostatectomy , laparoscopic adenomectomy , and robotic assisted simple prostatectomy . the final reference list was approved by authors rs and tb based on originality and relevance to the scope of this review article . prior to endoscopic procedures , patients underwent a detailed history assessment , a thorough physical examination , and an appropriate laboratory assessment . the severity of luts was evaluated by most authors using the international prostate symptom score ( ipss ) questionnaire and the maximal flow rate ( qmax ) . for precise postoperative functional evaluation , some authors used the sexual health inventory for men ( shim ) , typically 3 , 6 , and 12 months postoperatively [ 6 , 7 ] . prostate biopsy was performed as a standard procedure in some studies , but in other studies it was only performed in selected cases [ 8 , 9 ] . peri and postoperative assessment included ot , blood loss , transfusion requirements , catheterization time , and duration of hospital stay . complications were evaluated based on their severity , usually according to clavien et al . . the longest follow up period was several years in patients after la , and up to 10 months in patients after robotic assisted adenomectomy [ 11 , 12 ] . in 2002 , mariano et al . described the first la in a 71year old man with an ultrasonography estimated 173ml prostate and a 26.6 ng / ml prostate specific antigen ( psa ) level . the procedure was performed with five intraperitoneal trocars , with anterior prostate capsulotomy and placement of hemostatic sutures at the 5 and 7 o'clock positions after adenoma removal . van velthoven et al . described their technique of la , which included hemostatic control of the lateral venous vesicoprostatic pedicles , transverse anterior incision of the prostatic capsule , adenoma enucleation using the harmonic scalpel , and reconstruction of the posterior bladder neck and prostatic capsule . the authors reported very good functional outcomes ; mean qmax was increased by 4.3 ml / s postoperatively . in 2005 , sotelo et al . stepwise , the technique included transverse cystotomy just proximal to the prostatovesical junction , subcapsular plane development , prostatic adenomectomy , prostatic fossa trigonization , and prostatic capsule suture repair . the mean ot was 156 min and blood loss was 516 ml ( 1002500 ml ) . a 7 ml / s increase in qmax was noted postoperatively . since these initial reports , laparoscopic simple prostatectomy has been more widely adopted , offering surgeons an extra or intraperitoneal approach , following a transcapsular or transvesical route . in selected patients , finger assistance has been used for rapid enucleation of large adenomas [ 2 , 16 , 17 , 8 ] . one technique involves an incision under the umbilicus with carbon dioxide insufflation into the extraperitoneal space via a veress needle to 12 mmhg . a second technique involves making a 2cm vertical midline incision above the pubic arch followed by blunt dissection of the preperitoneal and retzius space with an index finger and a 700ml self dilating balloon . in a third technique the retroperitoneal space is then bluntly dissected with an 8001200ml infusion of sterile saline solution into the balloon . the operation can also begin with the primary insertion of a 10mm infraumbilical port and laparoscope . then , dissection of the preperitoneal space is completed with the aid of the laparoscope and insufflation . usually , 4 trocars , 5 mm or 12 mm , are inserted in a fan shape , to introduce a needle for suturing , as in extraperitoneal radical prostatectomy . a single 10mm port is inserted infraumbilically as the camera port ( figure 1 ) . the dorsal vein complex is assessed and then carefully coagulated using bipolar forceps cranially , keeping an appropriate distance from the puboprostatic ligaments . in a fourth technique , two hemostatic sutures are applied to these vessels . using the bladder catheter or a special metal guide inserted into the urethra as a reference point , the interface between the bladder neck and prostate base is identified . if necessary , two cross stitch hemostatic sutures are placed on the lateral surface of the prostate at the level of the bilateral vesicoprostatic vessels . the prostatic capsule is opened 34 cm transversally and 1 cm distal to the bladder neck . the capsular incision is carried to a depth that first reveals the off white tissue of the adenoma . monopolar scissors and the suction irrigation cannula are used to develop the plane between the prostatic adenoma and the capsule ( figure 2 ) . the anterior plane is then developed , followed by lateral and posterior dissection . in some cases , where improved exposure is required , the incision is extended in the shape of an inverted t on the prostatic capsule . to enucleate the lateral lobes , a harmonic scalpel is used to develop the surgical avascular capsular plane in the distal projection towards the apex , the lateral projection to the posterior plane , and the cranial projection to the bladder neck , in a fashion similar to that used in open surgery . in a modified version of the procedure , two lateral stay sutures are used between the cut prostatic capsule and the cooper ligament , providing a clear visualization of the fossa and the cleavage plan . the adenoma is excised , and then one or two corresponding specimens are placed outside the capsule in the lateral prostatic fossa , e.g. , in close proximity to the obturator fossa , to be removed later . hemostasis is obtained with stitches for transcapsular arteries and bipolar or monopolar electrocoagulation for minor vessels . the prostatic fossa is inspected for any remaining adenoma nodules . to facilitate the re epithelialization of the prostatic fossa and to achieve more effective control of hemostasis , the prostatic fossa is trigonized using 24 stitches between the sacral lip of the bladder neck and posterior surgical capsule . an interrupted or running suture is used for prostatic capsule reconstruction , followed by the introduction of a bag via the lateral 10mm port for collection of the fragmented adenoma . when the adenoma is too large to be removed as a whole , morcellation is performed before extraction . a drain ( redon type ) is inserted via the port , the infraumbilical incision is enlarged to enable intact retrieval , and the bag with the specimen is removed . the prostatic capsule can also be opened through a longitudinal opening in the anterior aspect of the bladder and extended to the anterior aspect of the prostatic capsule using bipolar diathermy , scissors , or a harmonic scalpel . stay sutures are placed between the edges of the open bladder on each side of the cooper ligament . most published series describe the extraperitoneal technique for la , but a transperitoneal approach has also been described . with the patient in a steep trendelenburg position , the pneumoperitoneum is created , and five intraperitoneal trocars are placed in a w fashion . after the peritoneum is incised and the retzius space is dissected , the bladder and prostate are taken down . a midline incision covering the anterior aspect of the prostatic capsule and bladder neck exposes the adenoma . retrigonization of the mucosa and hemostatic sutures of the bladder neck are performed with intracorporeal sutures . some authors described another modification with an extraperitoneal transvesical approach [ 17 , 23 ] . in this case , a transverse cystotomy incision is made proximal to the junction of the bladder and prostate . in this manner , next , a circular incision is made on the vesicle mucosa overlying the prostate lobes , and made deeper to reach the prostatic adenoma . the procedure ends with closure of the transverse cystotomy to ensure water tightness . in some patients , when the layer for surgical exposure of a large adenoma is unclear , or if the surgeon is inexperienced , the adenoma can be dissected using the finger assisted technique [ 2 , 7 , 16 , 18 , 22 ] . after opening the prostatic capsule and developing the plane of cleavage between the prostatic adenoma and capsule , gas flow is stopped and the index finger is introduced through a 23 cm suprapubic incision into this developed plane . this can be performed easily and is assisted by digital rectal examination ( dre ) [ 18 , 19 , 23 ] . the specimen is removed via the incision , and insufflation is restarted after closure of the suprapubic incision [ 2 , 22 ] . increased prostate weight , and hence prostate volume , have been correlated with increased ot . excellent visualization and bloodless resection of the adenoma limits complications and decreases ot , especially with larger lesions . the use of vascular control , bladder neck and capsular incisions , and ultrasonic scissors as a sharp and blunt dissection instrument allow easier enucleation of larger adenomas . there have been no reports of conversion to open surgery due to unexpected difficulties corresponding to prostate size . in a recently published series , after excluding the first 10 cases of la , univariate analysis of the next 78 patients with a large prostate ( > 90 ml ) showed a correlation of prostate volume and ot with complications . even with a large prostate ( > 90 ml ) , la provides excellent operative and perioperative results and patient satisfaction ( table 1 ) . series ( with > 15 cases ) on laparoscopic simple prostatectomy n number of patients ; t transperitoneal approach ; e extraperitoneal approach ; catheter catheterization time after the procedure ; i pss international prostate symptom score ; qmax the maximal flow rate ; nr not received a major benefit of la includes improved control of bleeding , possibly because of improved visualization and vascular compression from insufflated gas . morbidity and pain are reduced compared to those in the open , procedure as incisions are smaller and there is no need for retraction . la has better esthetic results , reduced need for analgesics , fewer wound infections , shorter hospital stay , and an earlier return to normal activities compared with the open procedure ( table 1 ) . intraoperative complications , mostly bleeding , are rare ( < 2.5% ) with no effect on clinical outcome and a decreased need for transfusion [ 4 , 22 ] . intraoperative blood loss does not usually correlate with the amount of enucleated tissue ( table 1 ) . decreased blood loss is achieved via gas compression of the venous system , aiding hemostasis , and allowing more precise dissection and coagulation of the adenomatous cleavage plane [ 11 , 13 ] . furthermore , better visualization improves hemostasis . the early complication rate of 14% is acceptable , particularly since most are less than clavien grade ii . long term complications rarely occur ( 2.5% at the 12month follow up , < 5% at the 30month follow up ) , and mainly include new obstructive urinary symptoms including urinary tract infections such as pyelonephritis , prostatitis , or epididymitis that are usually treated with conservative methods , and short presphincteric urethral stenosis usually treated with endoscopic urethrotomy . most researchers observed a significant increase in the qmax in comparison to preoperative values ( mean increased qmax 14.4 ml / s ) and a marked decrease in ipss ( mean decrease 17.2 ) postoperatively . ipss , ipss in the quality of life domain , and the expanded prostate cancer index composite ( epic ) questionnaire score remained stable at 3 , 6 , and > 12 months postoperatively . erectile function , evaluated with iief5 or other tools , did not change significantly as a result of la . persistent retrograde ejaculation was a consequence of the surgery , but had no significant impact on sexual function . psa levels were significantly different preoperatively and postoperatively , and later psa levels remained stable during follow up . most investigators use the extraperitoneal technique based on the millin technique employed in open procedures [ 7 , 11 , 14 , 15 , 1719 ] . with this approach , the risk of bladder tamponade by clots is avoided along with the additional anesthesiology risks associated with the steep trendelenburg position that is necessary in the transperitoneal approach . the transperitoneal technique was used by some investigators [ 11 , 13 , 17 ] , but is associated with a risk of ileus , peritonitis , and bowel injuries along with urine leakage and possible urine peritonitis from the bladder suture . no studies have compared extra and transperitoneal laparoscopic adenomectomy in the way laparoscopic or robotic radical prostatectomy has been compared [ 2426 ] . however , in la , these two approaches give comparable results ( table 1 ) . la has a relatively short learning curve in comparison with radical laparoscopic prostatectomy , and has been estimated at 510 surgeries . if conversion is required during a procedure using the preperitoneal access , no new access is needed because the surgery can be continued within the same space . as the number of surgeries performed and laparoscopic skills increase , there is a clear reduction in ot [ 11 , 15 , 21 ] . studies comparing la and open surgery have shown that both procedures offer the same benefits in terms of functional results , but that la performed by experienced surgeons provided greater perioperative benefits such as reduced bleeding and transfusions , shorter irrigation and catheterization times , shorter hospital stays , lower analgesic requirements , shorter recovery times , and improved cosmetic results [ 4 , 20 , 21 , 27 ] ( table 2 ) . comparative series between open and laparoscopy adenomectomy catheterization time after the procedure the introduction of the da vinci robotic surgical system [ intuitive surgical , inc . , sunnyvale , ca , usa ] for urological procedures , including radical prostatectomy , has been a major step towards a minimally invasive approach . in 2008 , sotelo et al . used a newly developed technique for rasp in 7 patients with a mean prostate volume of 77.66 ml . the functional outcomes were very good with reduction in the ipss by 14.5 points and an increase in the qmax by 37.75 ml / min the authors concluded that robotic simple prostatectomy is a feasible and reproducible procedure for symptomatic bph . in the same year , yuh reported rasp using a technique similar to conventional millin surgery . many clinical studies and case series using rasp have been published , but all have a small sample size and non comparative designs . port placement and surgical access in rasp are similar to those used in robotic assisted radical prostatectomy . after exposure of the retropubic space , the endopelvic fascia is opened bilaterally to expose the puboprostatic ligaments . the dorsal venous complex is ligated and access to the adenoma is achieved through the transvesical [ 2831 ] or prevesical approach [ 9 , 12 ] . in the prevesical approach , the plane between the adenoma and the prostatic capsule is identified and dissected , and the prostatic urethra is carefully transected to prevent external sphincter damage . finally , the adenoma is removed and retrigonization is achieved by suturing the posterior edge of the bladder neck mucosa to the posterior edge of the urethra . in another technique , a horizontal cystotomy of the prostatic capsule is made . after removing the adenoma , some authors suggest a modification of retrigonization by folding the posterior prostatic capsule , suturing the anterior prostatic capsule to the anterior bladder wall , and performing a modified van velthoven continuous vesicourethral anastomosis . patients treated with rasp show significant increases in qmax and reductions in ipss after surgery . ot is usually slightly longer or comparable to that in la , although in some reports , ot was > 3 h. blood loss has also been comparable to that reported in la , and in most cases no blood transfusions were necessary [ 30 , 32 ] . hospital stays were short , and most patients were discharged 12 days after surgery ( range 13.2 days ) [ 6 , 9 , 30 , 32 ] . reported follow up periods have been short and it is difficult to precisely define the long term outcome and complications . the laparoendoscopic single site ( less ) procedure is also used to remove prostatic adenomas [ 33 , 34 ] . the initial procedures used classical laparoscopic instruments with modifications , including the use of a port device for pre or transvesical access to the prostate [ 3538 ] . to avoid the basic limitations of less , collision of the robot 's arms and small operative space , single site instruments designed for the da vinci surgical system were used to perform single port transvesical enucleation of the prostate ( step ) [ 36 , 37 ] . in recent years , several clinical studies and case reports have been published on less [ 35 , 3742 ] . preliminary functional outcomes are encouraging , but the procedure is associated with a high risk of complications , and its role has yet to be determined . prior to endoscopic procedures , patients underwent a detailed history assessment , a thorough physical examination , and an appropriate laboratory assessment . the severity of luts was evaluated by most authors using the international prostate symptom score ( ipss ) questionnaire and the maximal flow rate ( qmax ) . for precise postoperative functional evaluation , some authors used the sexual health inventory for men ( shim ) , typically 3 , 6 , and 12 months postoperatively [ 6 , 7 ] . prostate biopsy was performed as a standard procedure in some studies , but in other studies it was only performed in selected cases [ 8 , 9 ] . peri and postoperative assessment included ot , blood loss , transfusion requirements , catheterization time , and duration of hospital stay . complications were evaluated based on their severity , usually according to clavien et al . . the longest follow up period was several years in patients after la , and up to 10 months in patients after robotic assisted adenomectomy [ 11 , 12 ] . in 2002 , mariano et al . described the first la in a 71year old man with an ultrasonography estimated 173ml prostate and a 26.6 ng / ml prostate specific antigen ( psa ) level . the procedure was performed with five intraperitoneal trocars , with anterior prostate capsulotomy and placement of hemostatic sutures at the 5 and 7 o'clock positions after adenoma removal . van velthoven et al . described their technique of la , which included hemostatic control of the lateral venous vesicoprostatic pedicles , transverse anterior incision of the prostatic capsule , adenoma enucleation using the harmonic scalpel , and reconstruction of the posterior bladder neck and prostatic capsule . the authors reported very good functional outcomes ; mean qmax was increased by 4.3 ml / s postoperatively . in 2005 , sotelo et al . stepwise , the technique included transverse cystotomy just proximal to the prostatovesical junction , subcapsular plane development , prostatic adenomectomy , prostatic fossa trigonization , and prostatic capsule suture repair . the mean ot was 156 min and blood loss was 516 ml ( 1002500 ml ) . a 7 ml / s increase in qmax was noted postoperatively . since these initial reports , laparoscopic simple prostatectomy has been more widely adopted , offering surgeons an extra or intraperitoneal approach , following a transcapsular or transvesical route . in selected patients , finger assistance has been used for rapid enucleation of large adenomas [ 2 , 16 , 17 , 8 ] . one technique involves an incision under the umbilicus with carbon dioxide insufflation into the extraperitoneal space via a veress needle to 12 mmhg . a second technique involves making a 2cm vertical midline incision above the pubic arch followed by blunt dissection of the preperitoneal and retzius space with an index finger and a 700ml self dilating balloon . in a third technique the retroperitoneal space is then bluntly dissected with an 8001200ml infusion of sterile saline solution into the balloon . the operation can also begin with the primary insertion of a 10mm infraumbilical port and laparoscope . then , dissection of the preperitoneal space is completed with the aid of the laparoscope and insufflation . usually , 4 trocars , 5 mm or 12 mm , are inserted in a fan shape , to introduce a needle for suturing , as in extraperitoneal radical prostatectomy . a single 10mm port is inserted infraumbilically as the camera port ( figure 1 ) . the dorsal vein complex is assessed and then carefully coagulated using bipolar forceps cranially , keeping an appropriate distance from the puboprostatic ligaments . in a fourth technique , a special metal guide inserted into the urethra as a reference point , the interface between the bladder neck and prostate base is identified . if necessary , two cross stitch hemostatic sutures are placed on the lateral surface of the prostate at the level of the bilateral vesicoprostatic vessels . the prostatic capsule is opened 34 cm transversally and 1 cm distal to the bladder neck . the capsular incision is carried to a depth that first reveals the off white tissue of the adenoma . monopolar scissors and the suction irrigation cannula are used to develop the plane between the prostatic adenoma and the capsule ( figure 2 ) . the anterior plane is then developed , followed by lateral and posterior dissection . in some cases , where improved exposure is required , the incision is extended in the shape of an inverted t on the prostatic capsule . to enucleate the lateral lobes , a harmonic scalpel is used to develop the surgical avascular capsular plane in the distal projection towards the apex , the lateral projection to the posterior plane , and the cranial projection to the bladder neck , in a fashion similar to that used in open surgery . in a modified version of the procedure , two lateral stay sutures are used between the cut prostatic capsule and the cooper ligament , providing a clear visualization of the fossa and the cleavage plan . any bladder stones are removed during the intervention through a capsular incision . the adenoma is excised , and then one or two corresponding specimens are placed outside the capsule in the lateral prostatic fossa , e.g. , in close proximity to the obturator fossa , to be removed later . hemostasis is obtained with stitches for transcapsular arteries and bipolar or monopolar electrocoagulation for minor vessels . the prostatic fossa is inspected for any remaining adenoma nodules . to facilitate the re epithelialization of the prostatic fossa and to achieve more effective control of hemostasis , the prostatic fossa is trigonized using 24 stitches between the sacral lip of the bladder neck and posterior surgical capsule . an interrupted or running suture is used for prostatic capsule reconstruction , followed by the introduction of a bag via the lateral 10mm port for collection of the fragmented adenoma . when the adenoma is too large to be removed as a whole , morcellation is performed before extraction . a drain ( redon type ) is inserted via the port , the infraumbilical incision is enlarged to enable intact retrieval , and the bag with the specimen is removed . the prostatic capsule can also be opened through a longitudinal opening in the anterior aspect of the bladder and extended to the anterior aspect of the prostatic capsule using bipolar diathermy , scissors , or a harmonic scalpel . stay sutures are placed between the edges of the open bladder on each side of the cooper ligament . most published series describe the extraperitoneal technique for la , but a transperitoneal approach has also been described . with the patient in a steep trendelenburg position , the pneumoperitoneum is created , and five intraperitoneal trocars are placed in a w fashion . after the peritoneum is incised and the retzius space is dissected , the bladder and prostate are taken down . a midline incision covering the anterior aspect of the prostatic capsule and bladder neck exposes the adenoma . retrigonization of the mucosa and hemostatic sutures of the bladder neck are performed with intracorporeal sutures . some authors described another modification with an extraperitoneal transvesical approach [ 17 , 23 ] . in this case , a transverse cystotomy incision is made proximal to the junction of the bladder and prostate . in this manner , next , a circular incision is made on the vesicle mucosa overlying the prostate lobes , and made deeper to reach the prostatic adenoma . the procedure ends with closure of the transverse cystotomy to ensure water tightness . in some patients , when the layer for surgical exposure of a large adenoma is unclear , or if the surgeon is inexperienced , the adenoma can be dissected using the finger assisted technique [ 2 , 7 , 16 , 18 , 22 ] . after opening the prostatic capsule and developing the plane of cleavage between the prostatic adenoma and capsule , gas flow is stopped and the index finger is introduced through a 23 cm suprapubic incision into this developed plane . this can be performed easily and is assisted by digital rectal examination ( dre ) [ 18 , 19 , 23 ] . the specimen is removed via the incision , and insufflation is restarted after closure of the suprapubic incision [ 2 , 22 ] . increased prostate weight , and hence prostate volume , have been correlated with increased ot . excellent visualization and bloodless resection of the adenoma limits complications and decreases ot , especially with larger lesions . the use of vascular control , bladder neck and capsular incisions , and ultrasonic scissors as a sharp and blunt dissection instrument allow easier enucleation of larger adenomas . there have been no reports of conversion to open surgery due to unexpected difficulties corresponding to prostate size . in a recently published series , after excluding the first 10 cases of la , univariate analysis of the next 78 patients with a large prostate ( > 90 ml ) showed a correlation of prostate volume and ot with complications . even with a large prostate ( > 90 ml ) , la provides excellent operative and perioperative results and patient satisfaction ( table 1 ) . series ( with > 15 cases ) on laparoscopic simple prostatectomy n number of patients ; t transperitoneal approach ; e extraperitoneal approach ; catheter catheterization time after the procedure ; i pss international prostate symptom score ; qmax the maximal flow rate ; nr not received a major benefit of la includes improved control of bleeding , possibly because of improved visualization and vascular compression from insufflated gas . morbidity and pain are reduced compared to those in the open , procedure as incisions are smaller and there is no need for retraction . la has better esthetic results , reduced need for analgesics , fewer wound infections , shorter hospital stay , and an earlier return to normal activities compared with the open procedure ( table 1 ) . intraoperative complications , mostly bleeding , are rare ( < 2.5% ) with no effect on clinical outcome and a decreased need for transfusion [ 4 , 22 ] . intraoperative blood loss does not usually correlate with the amount of enucleated tissue ( table 1 ) . decreased blood loss is achieved via gas compression of the venous system , aiding hemostasis , and allowing more precise dissection and coagulation of the adenomatous cleavage plane [ 11 , 13 ] . furthermore , better visualization improves hemostasis . the early complication rate of 14% is acceptable , particularly since most are less than clavien grade ii . long term complications rarely occur ( 2.5% at the 12month follow up , < 5% at the 30month follow up ) , and mainly include new obstructive urinary symptoms including urinary tract infections such as pyelonephritis , prostatitis , or epididymitis that are usually treated with conservative methods , and short presphincteric urethral stenosis usually treated with endoscopic urethrotomy . most researchers observed a significant increase in the qmax in comparison to preoperative values ( mean increased qmax 14.4 ml / s ) and a marked decrease in ipss ( mean decrease 17.2 ) postoperatively . ipss , ipss in the quality of life domain , and the expanded prostate cancer index composite ( epic ) questionnaire score remained stable at 3 , 6 , and > 12 months postoperatively . erectile function , evaluated with iief5 or other tools , did not change significantly as a result of la . persistent retrograde ejaculation was a consequence of the surgery , but had no significant impact on sexual function . psa levels were significantly different preoperatively and postoperatively , and later psa levels remained stable during follow up . most investigators use the extraperitoneal technique based on the millin technique employed in open procedures [ 7 , 11 , 14 , 15 , 1719 ] . with this approach , the risk of bladder tamponade by clots is avoided along with the additional anesthesiology risks associated with the steep trendelenburg position that is necessary in the transperitoneal approach . the transperitoneal technique was used by some investigators [ 11 , 13 , 17 ] , but is associated with a risk of ileus , peritonitis , and bowel injuries along with urine leakage and possible urine peritonitis from the bladder suture . no studies have compared extra and transperitoneal laparoscopic adenomectomy in the way laparoscopic or robotic radical prostatectomy has been compared [ 2426 ] . however , in la , these two approaches give comparable results ( table 1 ) . la has a relatively short learning curve in comparison with radical laparoscopic prostatectomy , and has been estimated at 510 surgeries . if conversion is required during a procedure using the preperitoneal access , no new access is needed because the surgery can be continued within the same space . as the number of surgeries performed and laparoscopic skills increase , there is a clear reduction in ot [ 11 , 15 , 21 ] . studies comparing la and open surgery have shown that both procedures offer the same benefits in terms of functional results , but that la performed by experienced surgeons provided greater perioperative benefits such as reduced bleeding and transfusions , shorter irrigation and catheterization times , shorter hospital stays , lower analgesic requirements , shorter recovery times , and improved cosmetic results [ 4 , 20 , 21 , 27 ] ( table 2 ) . comparative series between open and laparoscopy adenomectomy from published series the introduction of the da vinci robotic surgical system [ intuitive surgical , inc . , sunnyvale , ca , usa ] for urological procedures , including radical prostatectomy , has been a major step towards a minimally invasive approach . in 2008 , sotelo et al . used a newly developed technique for rasp in 7 patients with a mean prostate volume of 77.66 ml . the functional outcomes were very good with reduction in the ipss by 14.5 points and an increase in the qmax by 37.75 ml / min the authors concluded that robotic simple prostatectomy is a feasible and reproducible procedure for symptomatic bph . in the same year , yuh reported rasp using a technique similar to conventional millin surgery . many clinical studies and case series using rasp have been published , but all have a small sample size and non comparative designs . port placement and surgical access in rasp are similar to those used in robotic assisted radical prostatectomy . after exposure of the retropubic space , the endopelvic fascia is opened bilaterally to expose the puboprostatic ligaments . the dorsal venous complex is ligated and access to the adenoma is achieved through the transvesical [ 2831 ] or prevesical approach [ 9 , 12 ] . in the prevesical approach , the plane between the adenoma and the prostatic capsule is identified and dissected , and the prostatic urethra is carefully transected to prevent external sphincter damage . finally , the adenoma is removed and retrigonization is achieved by suturing the posterior edge of the bladder neck mucosa to the posterior edge of the urethra . in another technique , a horizontal cystotomy of the prostatic capsule is made . after removing the adenoma , some authors suggest a modification of retrigonization by folding the posterior prostatic capsule , suturing the anterior prostatic capsule to the anterior bladder wall , and performing a modified van velthoven continuous vesicourethral anastomosis . patients treated with rasp show significant increases in qmax and reductions in ipss after surgery . ot is usually slightly longer or comparable to that in la , although in some reports , ot was > 3 h. blood loss has also been comparable to that reported in la , and in most cases no blood transfusions were necessary [ 30 , 32 ] . hospital stays were short , and most patients were discharged 12 days after surgery ( range 13.2 days ) [ 6 , 9 , 30 , 32 ] . reported follow up periods have been short and it is difficult to precisely define the long term outcome and complications . the laparoendoscopic single site ( less ) procedure is also used to remove prostatic adenomas [ 33 , 34 ] . the initial procedures used classical laparoscopic instruments with modifications , including the use of a port device for pre or transvesical access to the prostate [ 3538 ] . to avoid the basic limitations of less , collision of the robot 's arms and small operative space , single site instruments designed for the da vinci surgical system were used to perform single port transvesical enucleation of the prostate ( step ) [ 36 , 37 ] . in recent years , several clinical studies and case reports have been published on less [ 35 , 3742 ] . preliminary functional outcomes are encouraging , but the procedure is associated with a high risk of complications , and its role has yet to be determined . despite its recent development , la has become a well established option for the surgical treatment of bph patients . la is standardized and reproducible and offers good functional results and a minimal complication rate along with other benefits of minimally invasive surgery . rasp offers the benefits of robotic surgery , and a shorter hospital stay , faster recovery , and quicker return to work than la . additionally , robotic surgery offers a number of benefits including stereoscopic vision and 6 degrees of freedom . longer terms studies of the functional outcomes , complications , and cost analysis of rasp will further define this procedure 's place in the urological surgeon 's armamentarium .
introductionmany options exist for the surgical treatment of lower urinary tract symptoms ( luts ) due to benign prostatic hyperplasia ( bph ) , including transurethral resection of the prostate ( turp ) , laser surgery , and open adenomectomy . recently , endoscopic techniques have been used in the treatment of bph.material and methodswe reviewed clinical studies in pubmed describing minimally invasive endoscopic procedures for the treatment of bph.resultslaparoscopic adenomectomy ( la ) and robotic assisted simple prostatectomy ( rasp ) were introduced in the early 2000s . these operative techniques have been standardized and reproducible , with some individual modifications . studies analyzing the outcomes of la and rasp have reported significant improvements in urinary flow and decreases in patient international prostate symptom score ( ipss ) . these minimally invasive approaches have resulted in a lower rate of complications , shorter hospital stays , smaller scars , faster recoveries , and an earlier return to work.conclusionsminimally invasive techniques such as la and rasp for the treatment bph are safe , efficacious , and allow faster recovery . these procedures have a short learning curve and offer new options for the surgeon treating bph .
INTRODUCTION METHODS RESULTS Pre and postoperative evaluation Laparoscopic adenomectomy (LA): Initial studies LA procedure Comparison studies between LA and open prostatectomy Roboticassisted simple prostatectomy (RASP) CONCLUSIONS
lower urinary tract symptoms ( luts ) are common , especially in older men , and are most often caused by benign prostatic hyperplasia ( bph ) which results in benign prostatic enlargement ( bpe ) and bladder outlet obstruction ( boo ) . transurethral resection of the prostate ( turp ) has been considered the surgical gold standard in terms of efficacy and re treatment rate . laparoscopic , and more recently robotic techniques , have provided a minimally invasive alternative to open prostatectomy , with equal efficacy , faster recovery , and shorter hospital stay . these endoscopic procedures allow improved visualization and reduced morbidity and are well established in the management of prostate carcinoma , providing a rationale for their use in the treatment of bph [ 35 ] . various algorithms were used including : benign prostatic hyperplasia , adenomectomy , simple prostatectomy , laparoscopic adenomectomy , and robotic assisted simple prostatectomy . the severity of luts was evaluated by most authors using the international prostate symptom score ( ipss ) questionnaire and the maximal flow rate ( qmax ) . to enucleate the lateral lobes , a harmonic scalpel is used to develop the surgical avascular capsular plane in the distal projection towards the apex , the lateral projection to the posterior plane , and the cranial projection to the bladder neck , in a fashion similar to that used in open surgery . series ( with > 15 cases ) on laparoscopic simple prostatectomy n number of patients ; t transperitoneal approach ; e extraperitoneal approach ; catheter catheterization time after the procedure ; i pss international prostate symptom score ; qmax the maximal flow rate ; nr not received a major benefit of la includes improved control of bleeding , possibly because of improved visualization and vascular compression from insufflated gas . la has better esthetic results , reduced need for analgesics , fewer wound infections , shorter hospital stay , and an earlier return to normal activities compared with the open procedure ( table 1 ) . studies comparing la and open surgery have shown that both procedures offer the same benefits in terms of functional results , but that la performed by experienced surgeons provided greater perioperative benefits such as reduced bleeding and transfusions , shorter irrigation and catheterization times , shorter hospital stays , lower analgesic requirements , shorter recovery times , and improved cosmetic results [ 4 , 20 , 21 , 27 ] ( table 2 ) . many clinical studies and case series using rasp have been published , but all have a small sample size and non comparative designs . to avoid the basic limitations of less , collision of the robot 's arms and small operative space , single site instruments designed for the da vinci surgical system were used to perform single port transvesical enucleation of the prostate ( step ) [ 36 , 37 ] . the severity of luts was evaluated by most authors using the international prostate symptom score ( ipss ) questionnaire and the maximal flow rate ( qmax ) . to enucleate the lateral lobes , a harmonic scalpel is used to develop the surgical avascular capsular plane in the distal projection towards the apex , the lateral projection to the posterior plane , and the cranial projection to the bladder neck , in a fashion similar to that used in open surgery . series ( with > 15 cases ) on laparoscopic simple prostatectomy n number of patients ; t transperitoneal approach ; e extraperitoneal approach ; catheter catheterization time after the procedure ; i pss international prostate symptom score ; qmax the maximal flow rate ; nr not received a major benefit of la includes improved control of bleeding , possibly because of improved visualization and vascular compression from insufflated gas . la has better esthetic results , reduced need for analgesics , fewer wound infections , shorter hospital stay , and an earlier return to normal activities compared with the open procedure ( table 1 ) . studies comparing la and open surgery have shown that both procedures offer the same benefits in terms of functional results , but that la performed by experienced surgeons provided greater perioperative benefits such as reduced bleeding and transfusions , shorter irrigation and catheterization times , shorter hospital stays , lower analgesic requirements , shorter recovery times , and improved cosmetic results [ 4 , 20 , 21 , 27 ] ( table 2 ) . the functional outcomes were very good with reduction in the ipss by 14.5 points and an increase in the qmax by 37.75 ml / min the authors concluded that robotic simple prostatectomy is a feasible and reproducible procedure for symptomatic bph . many clinical studies and case series using rasp have been published , but all have a small sample size and non comparative designs . despite its recent development , la has become a well established option for the surgical treatment of bph patients . rasp offers the benefits of robotic surgery , and a shorter hospital stay , faster recovery , and quicker return to work than la .
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generation of 3-mb - pp13-mb - pp1 was synthesized using a previously described route with slightly modified conditions ( 18 ) . p116 , a zap-70-deficient jurkat - derived t cell line , was obtained from r. abraham ( the burnham institute , la jolla , ca ) . 293 cells , a kidney epithelial cell line , were obtained from the american type culture collections . , 20 10 cells were transfected with 5 g of expression construct and 25 g of empty vector . stable p116 clones expressing zap-70 constructs were selected with blasticidin ( 10 g / ml ; invitrogen ) . transient transfections of 293 cells were carried out in 24-well plates using lipofectamine plus reagent ( invitrogen ) according to the manufacturer 's instructions . plasmids a quikchange site - directed mutagenesis kit ( stratagene ) and standard pcr techniques were used to prepare zap-70 mutations m414a ( zap-70 ) and m414a / c405v ( zap-70 ) in the plasmid pbluescript ( invitrogen ) . the mutated versions and wild - type human zap-70 were then subcloned into expression vector pef6.a ( invitrogen ) via ecori digest . for transfections , the following previously described plasmids were used : lck ( 19 ) , flag - tagged lat ( 20 ) , cd8- ( 21 ) , hemagglutinin - tagged rat plc1 ( 22 ) , and hemagglutinin - tagged murine tec kinase ( 22 ) . the diacylglycerol kinase ( dgk ) construct was a gift from gary koretzky . an enhanced green fluorescent protein plasmid from invitrogen was used in cotransfection experiments . antibodies ascites of c305 , an anti - jurkat tcr -chain monoclonal antibody was used for tcr stimulations ( 23 ) . for stimulation via cd28 the following antibodies were used for western blotting : plc1-py783 , lat - py132 ( biosource ) , zap-70-py319 , thr / tyr for phospho - p44/42 mapk ( cell signaling ) , lck ( 1f6 from j. b. bolen ) , anti - phosphotyrosine ( 4g10 ; upstate biotechnology ) , -tubulin ( sigma ) , lat ( abcam ) , slp-76 ( santa cruz biotechnology ) , active p38 , active jnk ( promega ) , plc1 mixed monoclonal antibodies , and tec antibody ( upstate biotechnology ) . the following antibodies have been described previously : 2f3.2 ( anti - zap-70 ) ( 24 ) and 6b10.2 ( anti - tcr- ) ( 25 ) . flow cytometry assays for cd69 experiments , 6 h after transfection , cells were washed in rpmi and resuspended at 1 10 cell / ml . cells were incubated with dmso ( vehicle ) or 3-mb - pp1 and stimulated with anti - tcr antibody ( 1:1000 c305 ) or phorbol 12-myristate 13-acetate ( pma ) ( 25 ng / ml ) . cells were left overnight at 37 c with 5% co2 and then stained with allophycocyanin - conjugated cd69 ( bd biosciences ) . cells were then fixed with bd cytofix ( bd biosciences ) , washed twice with facs buffer , and then permeabilized in caltag fix / perm medium b and stained for intracellular zap-70 ( caltag ) . for intracellular flow cytometry analysis of perk , p116 stable lines were incubated with anti - tcr antibody ( 1:2000 c305 ) for a 30-min time course , with vehicle ( dmso ) or 3-mb - pp1 , in a 96-well round bottom plate . cells were fixed by adding bd cytofix , washed twice with facs buffer , and then permeabilized with 100% ice - cold methanol . cells were then washed three times , stained with primary perk antibody , washed twice , and then stained with allophycocyanin - conjugated goat anti - rabbit secondary ( jackson immunoresearch ) . cell were stimulated with anti - tcr antibody ( 1:2000 c305 ) in the presence of dmso , 3-mb - pp1 , or pp2 luciferase assays stable cell lines were transfected with 20 g of nfat / ap-1 luciferase and 20 g of vector only . approximately 16 h after transfection , cells were stimulated with anti - tcr antibody ( 1:2000 c305 ) or pma ( 50 ng / ml ) and ionomycin ( 1 m ) . six hours later , the cells were harvested , lysed , and assayed for luciferase activity using a mithras lb 940 ( berthold technologies ) . superantigen stimulation and interleukin-2 ( il-2 ) measurement superantigen - loaded antigen - presenting cells were prepared by incubating raji b cells with 100 ng / ml staphylococcus enterotoxin ( toxin technology ) . 10 raji cells ( staphylococcus enterotoxin ) were then incubated with an equal number of zap-70 or zap-70 cells per well in a 96-well plate in a total volume of 0.2 ml of medium ( rpmi with 5% fetal calf serum supplemented with penicillin , streptomycin , and glutamine ) treated with the indicated concentrations of 3-mb - pp1 . after 18 h at 37 c with 5% co2 , the il-2 concentration was determined by using human il-2 elisa ready - set - go ! cells were treated with pma ( 20 ng / ml ) plus ionomycin ( 1 m ) as a control . stimulations and immunoblot analysis for 293 cell experiments , inhibitor or dmso control was added at the time of transfection , and cells were incubated for 24 h. cells were then harvested , spun down , and lysed in 2 concentrated sds sample buffer . lysates were cleared by ultracentrifugation at 440,000 g for 30 min at 24 c . supernatants were then collected , and dithiothreitol was added to a final concentration of 1% . p116 stable lines were stimulated ( 25 10/ml ) in rpmi with anti - tcr antibody ( 1:2000 c305 ) for the indicated time . for most experiments , cells were lysed as noted in 2 concentrated sds sample buffer and subjected to ultracentrifugation as described above . for immunoprecipitation experiments , 10 10 cells were stimulated and lysed in ice - cold lysis buffer ( 10 mm tris , ph 7.6 , 150 mm nacl , 1% nonidet p-40 , and a mixture of protease and phosphatase inhibitors ) . postnuclear supernatant was used for immunoprecipitation with antibody bound to protein a or protein g beads ( amersham biosciences ) . samples were analyzed by sds - page , and immunoblotting was performed using primary and horseradish peroxidase - conjugated secondary antibodies . proteins were detected by chemiluminescence ( western lightning ) using a kodak image station ( kodak ) . generation of analog - sensitive zap-70 allele we generated an analog - sensitive zap-70 allele by mutating the gatekeeper methionine to alanine to create greater access in the atp pocket of the kinase domain . the resulting m414a mutant is referred to as analog - sensitive allele 1 ( as1 ) ( fig . , we introduced a secondary mutation , c405v , in conjunction with m414a , in an attempt to restore stability to the -sheet in the n - terminal kinase domain , which could potentially be compromised by the parental m414a mutation ( 26 ) . 1b ) . as seen in fig . 1a , the mutation of the gatekeeper methionine to the smaller alanine residue generates more space in the catalytic domain , allowing room for binding of bulky pp1 analogs . in these studies , we chose to use the pp1 analog 3-mb - pp1 over other pp1 analogs , based upon its potent inhibition of zap-70 and the lack of effect on wild - type cells in a screen for t cell activation ( data not shown ) . 3-mb - pp1 contains an extra methylene bridge to the phenyl substituent of pp1 and a 3-methyl substituent that has been shown to be important for reducing binding to wild - type kinases ( fig . 1c ) ( 27 ) . to test the cellular activity of the zap-70 mutants and their sensitivity to 3-mb - pp1 , we transiently transfected 293 cells with zap-70 , or either one of the zap-70 constructs , along with lck to activate zap-70 and the transmembrane adaptor lat , which serves as a zap-70-specific substrate . as shown in fig 2a , zap-70 was insensitive to the addition of 3 or 6 m 3-mb - pp1 . in contrast , both zap-70 and zap-70 were inhibited by 3-mb - pp1 in a dose - dependent manner as measured by decreased lat phosphorylation . importantly , the 293 transfection data also showed that the analog - sensitive zap-70 exhibited reduced catalytic activity when compared with the wild - type ; however , both alleles were able to phosphorylate lat in the absence of inhibitor . we estimate the analog - sensitive zap-70 mutant has an average 2.1-fold reduction in cellular activity relative to wild - type , based upon quantification of the intensity of the total level of zap-70 relative to that of the phosphorylated lat in a series of immunoblots ( data not shown ) . to further characterize the sensitivity of zap-70 to 3-mb - pp1 , we transiently transfected the zap-70 constructs into the zap-70-deficient jurkat - derived t cell line , p116 . p116 fails to effectively initiate signaling events downstream of zap-70 , including protein tyrosine phosphorylation , ca mobilization , ras / mapk activation , nfat - directed transcription , and expression of a variety of downstream genes such as cd69 ( 28 , 29 ) . we tested the transiently transfected cells for surface cd69 expression , mediated via the ras / mapk pathway , after overnight stimulation with an anti - tcr antibody ( c305 ) in the presence or absence of 10 m 3-mb - pp1 . both zap-70- and zap-70-expressing cells efficiently up - regulated cd69 following tcr stimulation in the presence of vehicle ( dmso ) . however , the activation of cells expressing zap-70 was markedly impaired in the presence of 3-mb - pp1 ( fig . no 3-mb - pp1-mediated effect was seen with either of the zap-70 alleles after pma stimulation , which bypasses proximal tcr signaling . thus , the zap-70 is functional and is inhibited by 3-mb - pp1 in a t cell system . moreover , although lck and many downstream kinases are required for cd69 induction , only the cells expressing the zap-70 alleles are sensitive to inhibition by 3-mb - pp1 . a , ribbon structure of a portion of the kinase domain of zap-70 ( left ) ( protein data bank code 2ozo ( 35 ) ) and zap-70 ( m414a was manually introduced in pymol ) ( right ) . zap-70 generation . a , ribbon structure of a portion of the kinase domain of zap-70 ( left ) ( protein data bank code 2ozo ( 35 ) ) and one of the principal challenges to developing inhibitor systems is avoiding off - target effects . therefore , we further tested the specificity of 3-mb - pp1 inhibition by analyzing its effect on other relatively upstream kinase / substrate pairs implicated in tcr signaling in 293 transient transfection assays ( fig . lck activity was monitored by cotransfecting a cd8- chimera , which contains the cytoplasmic domain of and the extracellular domain of cd8 , as a substrate . both lck and tec kinase activity were only minimally affected even after incubation with high doses ( 9 - 10 m ) of 3-mb - pp1 . zap-70 is required to both initiate and maintain tcr - mediated increases in cytoplasmic free calcium ( [ ca]i)zap-70-deficient p116 cells fail to increase [ ca]i after tcr stimulation . therefore , we wanted to test the effect of 3-mb - pp1 on [ ca]i . we first generated p116 clones that stably expressed zap-70 , zap-70 , or zap-70 to reconstitute tcr signaling . both of the zap-70 clones expressed more zap-70 than the parental jurkat line ( supplemental fig . 1 ) . therefore , we selected zap-70 and zap-70 , which express comparable amounts of zap-70 to the wild - type clones . in addition , we utilized zap-70 because it expresses zap-70 equivalently to parental jurkat cells and , therefore , allowed us to rule out any potential artifact of zap-70 overexpression . to test the requirement of zap-70 for initiating [ ca]i increases , cells were pretreated with a 6 m dose of 3-mb - pp1 or vehicle for 75 s and then stimulated with anti - tcr antibody . in both zap-70 and zap-70 stably transfected cells , 3-mb - pp1 treatment blocked tcr - mediated signaling , and there was only a negligible effect on zap-70 cells ( data not shown ; fig . , src family kinases such as lck in jurkat t cells are also required for inducing [ ca]i increases because of their ability to mediate itam phosphorylation and zap-70 activation . treatment with the src family kinase inhibitor pp2 had a comparable effect to that of inhibiting zap-70 in both wild - type and mutant stables , whereas only zap-70 was sensitive to blockade by 3-mb - pp1 , suggesting a high degree of specificity . there was also no effect of 3-mb - pp1 treatment on ionomycin - dependent calcium release in zap-70 or zap-70 cells ( data not shown ) . this block in ca signaling was interesting but was anticipated from studies of the parental p116 cells . however , we were more interested in determining the requirement of the zap-70 catalytic function for maintaining [ ca]i elevation . the inhibitor system provides a powerful tool for answering such questions because 3-mb - pp1 can be added post - stimulation . to test this idea directly , we treated the stably transfected cells with 3-mb - pp1 after the maximal ca response was achieved ( 75 s after stimulation ) . interestingly , under these conditions , 3-mb - pp1 treatment completely abrogated tcr - mediated [ ca]i increase in analog - sensitive stables , returning [ ca]i to base line within 20 s ( fig . this suggests an ongoing requirement for zap-70 catalytic function beyond signal initiation by the tcr . the striking inhibition of calcium flux was also noted in a dose - response analysis to 3-mb - pp1 . as shown in fig . 4c , itis clear that the calcium flux of zap-70 cells is sensitive to even very low doses ( including 0.5 and 1.0 m ) of 3-mb - pp1 . a , 293 transient transfection of zap-70 constructs in the presence or absence of 3-mb - pp1 . cells were lysed in 2 concentrated sds - page sample buffer and analyzed by immunoblotting with antibody against phosphotyrosine ( top panel ) . total zap-70 , lck , and lat levels were determined by blotting with specific antibodies . unless noted otherwise , all experiments shown in figs . 2 , 3 , 4 , 5 , 6 , 7 and 8 are representative of at least three independent experiments . b , facs analysis of zap-70 and cd69 expression after stimulation of p116 cells transiently transfected with either zap-70 or zap-70 . six h after transfection , cells were left unstimulated or stimulated with the anti - tcr antibody c305 or with pma ( 25 ng / ml ) for 16 h in the presence of vehicle ( dmso ) or 3-mb - pp1 . cells were stained for surface cd69 and then fixed , permeabilized , and stained for intracellular zap-70 . the numbers represent the percentage of zap-70cd69 ( upper right ) and zap-70cd69 ( lower right ) . although zap-70 staining was used in the experiment shown here , future experiments confirmed this result using cotransfected green fluorescent protein as a surrogate marker of zap-70 . zap-70 cellular activity is selectively inhibited by 3-mb - pp1 . a , 293 transient transfection of zap-70 constructs in the presence or absence of 3-mb - pp1 . cells were lysed in 2 concentrated sds - page sample buffer and analyzed by immunoblotting with antibody against phosphotyrosine ( top panel ) . total zap-70 , lck , and lat levels were determined by blotting with specific antibodies . unless noted otherwise , all experiments shown in figs . 2 , 3 , 4 , 5 , 6 , 7 and 8 are representative of at least three independent experiments . b , facs analysis of zap-70 and cd69 expression after stimulation of p116 cells transiently transfected with either zap-70 or zap-70 . six h after transfection , cells were left unstimulated or stimulated with the anti - tcr antibody c305 or with pma ( 25 ng / ml ) for 16 h in the presence of vehicle ( dmso ) or 3-mb - pp1 . cells were stained for surface cd69 and then fixed , permeabilized , and stained for intracellular zap-70 . the numbers represent the percentage of zap-70cd69 ( upper right ) and zap-70cd69 ( lower right ) . although zap-70 staining was used in the experiment shown here , future experiments confirmed this result using cotransfected green fluorescent protein as a surrogate marker of zap-70 . a , 293 transient transfection of lck and cd8- , serving as the kinase substrate , in the presence or absence of 3-mb - pp1 . total lck , cd8- , tec , and plc1 were determined by blotting with specific antibodies . a , 293 transient transfection of lck and cd8- , serving as the kinase substrate , in the presence or absence of 3-mb - pp1 . total lck , cd8- , tec , and plc1 were determined by blotting with specific antibodies . the requirement of zap-70 for [ ca]i elevation was striking , but these measurements were made during short time intervals following the initiation of tcr signaling . therefore , it was important to monitor nfat transcriptional events in order to determine the long - term effect of this failure to mobilize intracellular calcium . we transfected a nfat / ap-1 luciferase reporter into our stable lines and monitored the activity after 6 h of tcr stimulation . as expected from the calcium data , nfat activity was severely and equivalently diminished by 3-mb - pp1 treatment in both the zap-70 and zap-70 lines after anti - tcr antibody stimulation ( fig . in addition , the response was dose - dependent . the highest dose at 6 m , which effectively blocked calcium responses , inhibited almost all nfat - driven luciferase activity ( 5% remaining activity compared with vehicle treated ) . zap-70 inhibition suppresses superantigen - mediated il-2 induction the inhibition of calcium increases and nfat - transcription in 3-mb - pp1-treated cells demonstrated the utility of this analog approach and exposed the requirement of zap-70 to both initiate and maintain the calcium response after tcr antibody stimulation . however , we also wanted to test the system using a more physiological approach . therefore , we decided to measure il-2 production following stimulation with staphylococcus enterotoxin e superantigen - loaded antigen - presenting cells . this stimulation method is more physiological than antibody treatment because it requires antigen - presenting cells and also because humans t cells respond to superantigens in pathologic situations such as toxic shock syndrome or food poisoning . as shown in fig . 5b thus , our findings show that this analog approach works in a physiologic context and that zap-70 is required to mediate a superantigen response to an enterotoxin . interestingly , the zap-70 cells were less sensitive to 3-mb - pp1 than in the nfat - transcriptional assay shown in fig . 5a . il-2 production is a more integrated response and therefore may be less sensitive to lower levels of zap-70 inhibition . cells were loaded with indo-1 dye and stimulated with anti - tcr antibody and either pretreated ( a ) or treated post - maximal ca flux ( b ) with 6 m 3-mb - pp1 or vehicle ( v ; dmso ) . in a , pp2 was added at a final concentration of 20 m . the experiment in a is representative of multiple independent experiments , but pp2 was added as a control in two experiments . c , zap-70 cells were pretreated with a range of 3-mb - pp1 concentrations as indicated . cells were loaded with indo-1 dye and stimulated with anti - tcr antibody and either pretreated ( a ) or treated post - maximal ca flux ( b ) with 6 m 3-mb - pp1 or vehicle ( v ; dmso ) . in a the experiment in a is representative of multiple independent experiments , but pp2 was added as a control in two experiments . c , zap-70 cells were pretreated with a range of 3-mb - pp1 concentrations as indicated . the cells were then left unstimulated , stimulated with anti - tcr antibody , or stimulated with 50 ng / ml pma plus 1 m ionomycin for 6 h at 37 c and then assayed for luciferase activity . the nfat response was first calculated as a percentage of the maximum as determined by pma plus ionomycin treatment . then the activity was determined for each 3-mb - pp1 concentration relative to untreated ( no 3-mb - pp1 or 0 m 3-mb - pp1 ) . b , zap-70 and zap-70 cells were incubated with staphy - lococcus enterotoxin e - loaded antigen - presenting cells for 18 h in the presence of a range of 3-mb - pp1 concentrations before il-2 concentration was determined by enzyme - linked immunosorbent assay . the cells were then left unstimulated , stimulated with anti - tcr antibody , or stimulated with 50 ng / ml pma plus 1 m ionomycin for 6 h at 37 c and then assayed for luciferase activity . the nfat response was first calculated as a percentage of the maximum as determined by pma plus ionomycin treatment . then the activity was determined for each 3-mb - pp1 concentration relative to untreated ( no 3-mb - pp1 or 0 m 3-mb - pp1 ) . b , zap-70 and zap-70 cells were incubated with staphy - lococcus enterotoxin e - loaded antigen - presenting cells for 18 h in the presence of a range of 3-mb - pp1 concentrations before il-2 concentration was determined by enzyme - linked immunosorbent assay . data are representative of two experiments . decreased phosphorylation of the lat - slp-76-plc1 signalosome upon zap-70 inhibition zap-70 has been thought to have at least two direct downstream targets , the adaptor proteins lat and slp-76 . both lat and slp-76 are key signaling adaptors , and cells that lack expression of either protein fail to propagate many of the downstream tcr signals . in zap-70-deficient p116 , neither lat nor slp-76 is efficiently phosphorylated ; and , the same cells overexpressing a kinase - inactive form of zap-70 also fail to exhibit these phosphorylated substrates ( 1 , 28 ) . lat and slp-76 interact indirectly and are responsible for forming a complex of signaling molecules downstream of the tcr . slp-76 then recruits a number of important molecules including plc1 , guanine nucleotide exchange factor vav , and the tec family kinase , interleukin-2-inducible t cell kinase ( itk ) , the latter two of which appear to require slp-76 phosphorylation ( 31 ) . in addition to its indirect interaction with slp-76 via gads , lat also interacts with numerous signaling molecules through its phosphorylated tyrosines , including the adaptor grb2 ( growth factor receptor - bound protein 2 ) as well as plc1 . the formation of a lat- and slp-76-containing signalosome serves as a nucleation point for tcr signaling events . phosphorylation of these adaptors , mediated by zap-70 , plays a critical role in the formation and functional activity of this signaling complex . because phosphorylation of lat and slp-76 by zap-70 is so critical for initiation of many of the downstream signaling events , we wanted to examine the phosphorylation status of lat and slp-76 after treatment with 3-mb - pp1 in the analog - sensitive clones . however , to first control for more proximal events that are not zap-70-dependent , we monitored lck - dependent tcr phosphorylation in zap-70 and zap-70 cells . 6a , tcr phosphorylation was not significantly altered after incubation with the inhibitor in either cell line , thereby reinforcing the idea that 3-mb - pp1 specifically targets zap-70-dependent events . it is important to note that the increase in total tcr immunoprecipitated from 3-mb - pp1-treated zap-70 cells was not reproducible . we then examined lat tyr phosphorylation , because plc1 has been shown to bind to this tyrosine when phosphorylated ( 32 ) . once activated , plc1 cleaves phosphatidylinositol 4,5-bisphosphate ( pip2 ) into diacylglycerol and inositol 1,4,5-trisphosphate ( ip3 ) , where ip3 binds receptors on the endoplasmic reticulum that lead to release of ca stores . mutation of tyr to phenylalanine has been shown to decrease plc1 phosphorylation and its binding to lat , in addition to diminishing the overall [ ca]i increase ( 30 , 32 ) . 6b , induced phosphorylation of lat tyr is markedly inhibited after cotreatment with anti - tcr antibody and 5 - 10 m 3-mb - pp1 . in addition to lat phosphorylation , total slp-76 phosphorylation was reduced over a 2-min time course ( fig . vehicle - treated zap-70 mutant cell samples often exhibited a somewhat decreased phosphorylation relative to zap-70 cells , which we hypothesized was due to the decreased catalytic activity of the analog - sensitive mutant . despite the decreased magnitude of phosphorylation in the untreated zap-70 cells , moreover , in very preliminary studies , we have been able to reconstitute t cell development of zap-70 null mice with the zap-70 clone expressed as a transgene , suggesting that the reduced catalytic activity of the mutant zap-70 is not substantially functionally impaired . p116 stable lines were incubated with vehicle or 5 m 3-mb - pp1 ( unless indicated otherwise ) and treated with either anti - tcr antibody or left unstimulated . a , after stimulation , 10 10 cells were lysed in 1% nonidet p-40 lysis buffer , and tcr was immunoprecipitated . immunoprecipitated lysates were immunoblotted for total phosphotyrosine and then stripped and blotted for total tcr. b , cells were lysed in 2 concentrated sds - page sample buffer and analyzed by immunoblotting with phosphotyrosine - specific lat ptyr and total zap-70 antibodies . approximately 0.4 10 cell equivalents were loaded onto a sds - polyacrylamide gel . c , after stimulation , slp-76 was immunoprecipitated as described in a. immunoprecipitated lysates were immunoblotted for total phosphotyrosine and slp-76 . results are representative of three independent experiments , which were carried out using either zap-70 or zap-70 . d , lysates were prepared as described in b and immunoblotted with antibodies specific for plc1 ptyr , zap-70 ptyr , and tubulin . p116 stable lines were incubated with vehicle or 5 m 3-mb - pp1 ( unless indicated otherwise ) and treated with either anti - tcr antibody or left unstimulated . a , after stimulation , 10 10 cells were lysed in 1% nonidet p-40 lysis buffer , and tcr was immunoprecipitated . immunoprecipitated lysates were immunoblotted for total phosphotyrosine and then stripped and blotted for total tcr. b , cells were lysed in 2 concentrated sds - page sample buffer and analyzed by immunoblotting with phosphotyrosine - specific lat ptyr and total zap-70 antibodies . approximately 0.4 10 cell equivalents were loaded onto a sds - polyacrylamide gel . c , after stimulation , slp-76 was immunoprecipitated as described in a. immunoprecipitated lysates were immunoblotted for total phosphotyrosine and slp-76 . results are representative of three independent experiments , which were carried out using either zap-70 or zap-70 . d , lysates were prepared as described in b and immunoblotted with antibodies specific for plc1 ptyr , zap-70 ptyr , and tubulin . in particular , phosphorylation of tyr has been shown to facilitate the interaction between tyr and the c - terminal sh2 domain of plc1 , which leads to enzyme activation ( 33 ) . recently , it was demonstrated that slp-76-bound itk phosphorylates plc1 on this key residue ( 34 ) . as shown in fig . 6d , phosphorylation of tyr on plc1 was markedly reduced in 3-mb - pp1-treated zap-70 cells . the specificity of the 3-mb - pp1 toward zap-70 was further emphasized in this experiment by the finding that zap-70 tyr phosphorylation , mediated by lck , was not affected by the addition of the inhibitor ( fig . recent crystallographic studies have shown that the tyr residue , which is phosphorylated by lck , is involved in the autoinhibition of zap-70 ( 35 ) . together , the loss of lat , slp-76 , and plc1 phosphorylation can account for the marked inhibition we observed in calcium mobilization . we noticed only a modest decrease in total itk phosphorylation in 3-mb - pp1-treated zap-70 and zap-70 cells ( data not shown ) . this is in agreement with the fact that src kinases regulate tec kinase phosphorylation ( 36 ) ; however , itk phosphorylation has recently been shown to be defective in slp-76-deficient cells ( 34 ) . the remaining slp-76 phosphorylation in our 3-mb - pp1 cells may be sufficient to support itk phosphorylation . thus , these studies indicate that phosphorylation of two of the most important downstream zap-70 substrates is impaired upon 3-mb - ppl treatment of zap-70-expressing cells but that other substrates such as the tcr chain , itk , and zap-70 itself , which are all phosphorylated by lck , are not substantially affected . maximal and persistent ras / mapk phosphorylation requires zap-70 catalytic activity given the substantial 3-mb - pp1-mediated inhibition of calcium and plc1 phosphorylation in zap-70-expressing cells , we wanted to examine other plc1-mediated signaling events , particularly activation of the ras / mapk pathway via the generation of diacylglycerol . we had already demonstrated during the initial screening of 3-mb - pp1 that inhibitor treatment diminishes cd69 up - regulation , a downstream transcriptional target of ras activation , in cells transiently transfected with zap-70 ( fig . , all zap-70 lines failed to maximally up - regulate cd69 after 16 h of stimulation with anti - tcr antibody . however , there was no effect on pma - mediated activation , which bypasses proximal tcr signaling ( fig . the failure to express surface cd69 in 3-mb - pp1-treated analog - sensitive cells was expected to correlate with the lack of activation of the mapk , erk . however , we repeatedly noticed that we had substantial , albeit diminished and delayed , erk phosphorylation at 2 min in 3-mb - pp1-treated zap-70 cells . it became clear , both by western blotting and intracellular staining of perk using flow cytometry , that erk phosphorylation was delayed and not maintainable in the inhibitor - treated zap-70 cells ( fig . analysis of erk phosphorylation by the use of a phosphospecific erk antibody on whole cell lysates examined by sds - page showed clear induction of erk phosphorylation by 1 min in untreated zap-70 cells and zap-70 cells , but there was no detectable perk phosphorylation above basal levels in inhibitor - treated zap-70 cells until 1.5 - 2 min after anti - tcr antibody stimulation ( fig . this pattern of erk phosphorylation held true for other mapk members , p38 and jnk , albeit basal levels of pp38 were also reduced . we were especially interested in p38 induction given the direct role that zap-70 is thought to play in activating p38 in t cells ( 37 ) . analysis of perk by intracellular flow cytometry showed similar results at the early time points , with only a small percentage of the cells containing phosphorylated erk 5 min post - stimulation ( fig . this minimal phosphorylation was not sustained . by 30 min , erk phosphorylation in the analog - sensitive cells returned to base line , whereas untreated cells were still substantially positive for perk ( fig . there was no difference in erk phosphorylation between zap-70 and zap-70 cells treated with vehicle . a , facs analysis of cd69 expression after stimulating stable lines for 16 h with or without anti - tcr antibody or pma ( 25 ng / ml ) in the presence or absence of 10 m 3-mb - pp1 . b , p116 stable lines were incubated for a 2-min time course with either vehicle or 5 m 3-mb - pp1 and treated with anti - tcr antibody or left unstimulated . cells were lysed in 2 concentrated sds sample buffer and blotted for perk , pjnk , pp38 , and tubulin . c , p116 stable lines were incubated with anti - tcr antibody over a 30-min time course with vehicle ( filled histogram ) or 5 m 3-mb - pp1 ( open histogram ) . at the appropriate time points , d , same as in c except cells were treated with either vehicle ( filled ) , 5 m 3-mb - pp1 ( solid line ) , or 10 m 3-mb - pp1 ( dotted line ) . e , zap-70 was transiently transfected with 20 g of green fluorescent protein vector and either empty vector ( left ) or 20 g of dgk ( right ) and stimulated and stained as described in c. transfected cells were identified by gating on gfp cells . the histograms are labeled as follows : unstimulated ( dotted line ) ; 5 min c305 + dmso ( filled ) ; 5 min c305 + 5 m 3-mb - pp1 ( solid line ) . zap-70 catalytic activity is required for complete and persistent ras / mapk phosphorylation . a , facs analysis of cd69 expression after stimulating stable lines for 16 h with or without anti - tcr antibody or pma ( 25 ng / ml ) in the presence or absence of 10 m 3-mb - pp1 . b , p116 stable lines were incubated for a 2-min time course with either vehicle or 5 m 3-mb - pp1 and treated with anti - tcr antibody or left unstimulated . cells were lysed in 2 concentrated sds sample buffer and blotted for perk , pjnk , pp38 , and tubulin . c , p116 stable lines were incubated with anti - tcr antibody over a 30-min time course with vehicle ( filled histogram ) or 5 m 3-mb - pp1 ( open histogram ) . at the appropriate time points , d , same as in c except cells were treated with either vehicle ( filled ) , 5 m 3-mb - pp1 ( solid line ) , or 10 m 3-mb - pp1 ( dotted line ) . e , zap-70 was transiently transfected with 20 g of green fluorescent protein vector and either empty vector ( left ) or 20 g of dgk ( right ) and stimulated and stained as described in c. transfected cells were identified by gating on gfp cells . the histograms are labeled as follows : unstimulated ( dotted line ) ; 5 min c305 + dmso ( filled ) ; 5 min c305 + 5 m 3-mb - pp1 ( solid line ) . although we were able effectively to eliminate increases in [ ca]i with 3-mb - pp1 , treatment with the same inhibitor still generated a substantial , yet abbreviated , pattern of erk phosphorylation . this discrepancy between the ca data and the erk phosphorylation was interesting because it either suggested that the level of plc1 activity post - treatment was sufficient for some erk phosphorylation but not ca mobilization or that the residual erk phosphorylation was plc1-independent . such a plc1-independent pathway could be mediated by grb2-sos recruitment to the phosphorylated tcr -chain , as had been suggested previously ( 38 ) , although grb2-sos is also known to be recruited to phosphorylated lat ( 39 , 40 ) . interestingly , the existence of a zap-70-independent pathway leading to erk phosphorylation has been reported . in these studies , p116 cells repeatedly exhibited delayed and transient erk phosphorylation ; however , this required the use of high levels of cd3 cross - linking ( 40 , 41 ) . we used two approaches to test whether residual plc1 activity was the mediator of the erk activation . first , we increased the concentration of 3-mb - pp1 to 10 m and found that this eliminated the remaining erk phosphorylation ( fig . second , we transiently overexpressed diacylglycerol kinase ( dgk ) , which converts diacylglycerol into phosphatidic acid by phosphorylation of the free hydroxyl group . we hypothesized that if the remaining erk phosphorylation were plc1-dependent , then overexpression of dgk would eliminate the remaining erk in the 3-mb - pp1-treated zap-70 cells . overexpression of dgk did , in fact , eliminate the remaining erk phosphorylation at 5 min post - stimulation in the presence of 3-mb - pp1 , thus providing additional evidence that the erk phosphorylation appears to be plc1-dependent ( fig . therefore , although [ ca]i increases were severely inhibited by 3-mb - pp1 treatment , similar doses of the inhibitor still allowed for enough plc1 activity to initiate the mapk cascade to some extent . however , this level of residual erk phosphorylation was not sufficient to maintain long - term phosphorylation or promote maximal transcriptional changes . these data are consistent with the notion that the mechanisms to amplify or produce positive feedback on erk activation are more sensitive to low levels of plc1 activity than the mechanisms that regulate the calcium pathway . superagonist cd28 antibody is zap-70-dependent one of the benefits of utilizing a small molecule inhibitor system is to be able to test the role of the given protein in a variety of pathways where its function is controversial or not easily determined . for instance , zap-70 is well known for its critical role in proximal tcr signaling , but its function in cd28 superagonist signaling has been unclear . conventional anti - cd28 antibodies require tcr coengagement to induce proliferation , but there is a class of anti - cd28 antibodies , termed stimulating or superagonists , that can stimulate t cell il-2 production and proliferation in the absence of tcr antibodies . one human cd28 superagonist was recently utilized in a clinical trial because it had been shown to induce development of regulatory t cells , but when administered to the healthy volunteers in a phase i clinical trial , the superagonist induced an unanticipated cytokine storm followed by multiorgan failure in volunteer subjects participating in this trial ( 42 ) . thus , understanding the biochemical mechanisms by which cd28 superagonist antibodies function might help to prevent such unforeseen , disastrous complications with immunologically active agents in the future . although the use of stimulating anti - cd28 antibody has been shown to activate some common events downstream of the tcr , including slp-76 and vav phosphorylation , all tcr - mediated pathways are not induced ( 43 ) . moreover , initially , it was assumed that zap-70 did not function in superagonist stimulation because zap-70 did not appear to be phosphorylated after stimulation in primary rat cells ( 44 ) . however , although a more recent study also did not observe zap-70 phosphorylation , the same group found that overexpression of a dominant negative zap-70 construct resulted in inhibition of the anti - cd28-induced il-2 production , thus suggesting an important yet unappreciated role for zap-70 in this signaling process ( 43 ) . therefore , given the controversial role for zap-70 signaling in this signaling cascade and the importance of understanding how superagonists function , we decided to utilize our inhibitor system to dissect the role of this kinase in superagonist stimulation . previous researchers had not observed zap-70 phosphorylation following cd28 superagonist stimulation , but the stimulating cd28 an28.1/5d10 antibody induced detectable zap-70 tyr phosphorylation after a 1-min stimulation of jurkat t cells , albeit at a substantially lower level than anti - tcr - stimulated cells ( fig . interestingly , as has been reported previously , the same cells induced substantial plc1 tyr phosphorylation , with delayed kinetics when compared with tcr stimulation . therefore , the presence of zap-70 phosphorylation in these cells suggests that zap-70 could play a role in the signaling cascade . to explore this idea further , we first used p116 to see if the cells could be activated by anc28.1 stimulation . 8a demonstrates that superagonist stimulation induces perk , so we decided to monitor cd69 as a readout for anc28.1-mediated ras activation , which has been reported previously to be induced in superagonist - treated cells ( 44 ) . although zap-70 cells clearly up - regulated cd69 after overnight stimulation with soluble anc28.1 at 1 g / ml , p116 failed to induce surface cd69 expression ( fig . a , jurkat cells were stimulated for 1 or 5 min with either anti - tcr antibody or a final concentration of 1 , 5 , or 10 g / ml anc28.1/5d10 antibody at 37 c . cells were lysed in 2 concentrated sds sample buffer and analyzed by immunoblotting for plc1 ptyr , zap-70 ptyr , perk , and tubulin . b , facs analysis of cd69 expression after stimulating zap-70 or p116 for 16 h with anc28.1 antibody ( 1 g / ml ) or pma ( 25 ng / ml ) . the histograms are labeled as follows : unstimulated ( dotted line ) ; anc28.1 ( solid line ) ; pma ( filled ) . c , cd69 analysis of zap-70 and zap-70 after anc28.1 antibody ( 1 g / ml ) stimulation with or without 5 m 3-mb - pp1 . the histograms are labeled as follows : unstimulated ( dotted line ) ; anc28.1 + vehicle ( filled ) ; anc28.1 + 3-mb - pp1 ( solid line ) . 7c , except cells were stimulated with anc28.1 antibody ( 1 g / ml ) for 5 min with or without 5 m 3-mb - pp1 . a , jurkat cells were stimulated for 1 or 5 min with either anti - tcr antibody or a final concentration of 1 , 5 , or 10 g / ml anc28.1/5d10 antibody at 37 c . cells were lysed in 2 concentrated sds sample buffer and analyzed by immunoblotting for plc1 ptyr , zap-70 ptyr , perk , and tubulin . b , facs analysis of cd69 expression after stimulating zap-70 or p116 for 16 h with anc28.1 antibody ( 1 g / ml ) or pma ( 25 ng / ml ) . the histograms are labeled as follows : unstimulated ( dotted line ) ; anc28.1 ( solid line ) ; pma ( filled ) . c , cd69 analysis of zap-70 and zap-70 after anc28.1 antibody ( 1 g / ml ) stimulation with or without 5 m 3-mb - pp1 . the histograms are labeled as follows : unstimulated ( dotted line ) ; anc28.1 + vehicle ( filled ) ; anc28.1 + 3-mb - pp1 ( solid line ) . 7c , except cells were stimulated with anc28.1 antibody ( 1 g / ml ) for 5 min with or without 5 m 3-mb - pp1 . the induction of zap-70 phosphorylation in jurkat and the failure of p116 to be activated by anc28.1 clearly suggest that stimulating cd28 antibody treatment requires zap-70 . however , previous research by dennehy et al . ( 43 ) suggests that only tonic signaling via zap-70 might be required to prime the system . this hypothesis can not be tested in p116 cells , which do not express zap-70 and thus would not exhibit zap-70-dependent tonic signals . the best way to test this hypothesis is with a small molecule inhibitor ; therefore , we treated our zap-70 cells with 3-mb - pp1 concurrently with 1 g / ml anc28.1 antibody . this treatment inhibited cd69 up - regulation and perk phosphorylation in two analog - sensitive lines but had no effect on zap-70 cells ( fig . thus , the use of 3-mb - pp1 and the zap-70-expressing cells demonstrates the requirement of zap-70 in cd28 superagonist - mediated signaling as well as the utility of having an inhibitor system to study the role of zap-70 . we successfully generated two zap-70 alleles that retain kinase activity but are sensitive to inhibition by the pp1 analog 3-mb - pp1 . in both 293 and p116 cells , zap-70 reconstitutes zap-70 functions but is inhibitable by the addition of 3-mb - pp1 in a dose - dependent fashion . failure to initiate or maintain calcium mobilization was associated with and a likely consequence of the inhibition of phosphorylation of the key zap-70 adaptor targets , lat and slp-76 . without the phosphorylation of those important adaptor molecules , plc1 can not be recruited to the membrane , phosphorylated , or activated properly to generate the ip3 required for [ ca]i increase . the physiological relevance of this approach was , in part , verified by the ability of 3-mb - pp1 to inhibit il-2 production in superantigen - stimulated zap-70 cells . interestingly , although il-2 production was inhibited , it was relatively insensitive to lower concentrations of 3-mb - pp1 as compared with other readouts such as calcium flux . this differential sensitivity in the various assays might be due to the difference in stimulations but also may be the result of the former being a more integrated response . overall , we found that the half - maximal inhibitory concentration ( ic50 ) varied among different readouts used throughout this study , although it was consistently within the range of 1 to 10 m . such differences in the ic50 between different functional readouts have been reported for other kinases ( 45 ) . although phosphorylation of tyr on plc1 was not readily inducible nor were elevations of [ ca]i detectable following 3-mb - pp1 treatment of zap-70 cells , we consistently observed evidence of erk phosphorylation in a percentage of these cells . the level of erk phosphorylation was delayed , reduced in magnitude , and not sustained when compared with zap-70 or untreated zap-70 cells . nonetheless , this diminished response was observed reproducibly . it had been reported previously that zap-70-deficient cells , p116 , are capable of activating erk , thus providing support for a zap-70-independent mechanism ( 40 , 41 ) . however , such zap-70-independent activation required high levels of tcr stimulation . it was also plausible to consider a possible plc1-independent mechanism , as grb2-sos have been reported to interact with the phosphorylated tcr chain ( 38 ) and also with phosphorylated lat ( 40 ) . thus , there could be separate zap-70- and plc1-independent mechanisms leading to the activation of the ras / mapk pathway . however , when we increased the dose of 3-mb - pp1 or , more importantly , transiently transfected dgk to convert the remaining diacylglycerol into phosphatidic acid , perk was no longer induced . it remains unclear why 3-mb - pp1 completely inhibited calcium mobilization but not erk phosphorylation . the experiments were performed under different conditions , with the cells being suspended in a larger volume in the ca assay than in the perk experiment . this larger volume increases the ratio of drug to cell even though similar concentrations of 3-mb - pp1 were used in the two different experiments . in addition , given the delay in erk phosphorylation in 3-mb - pp1-treated zap-70 cells , we hypothesized that a positive feedback loop over erk is strong enough to support transient erk phosphorylation in these cells . this positive feedback could involve the functional amplification loop of the two guanine nucleotide exchange factors , rasgrp and sos , that we have described previously ( 46 ) . thus , our studies herein support the notion that grb2-sos plays a less important role than rasgrp in tcr signaling leading to ras activation . zap-70 not only has kinase function but also has been shown to serve as an adaptor for many proteins , including lck , vav , c - cbl , and crk , via its phosphorylated tyrosine residues . one of the outstanding questions has been : what downstream tcr signaling functions is zap-70 able to fulfill as an adaptor in the absence of its catalytic function ? following the addition of 3-mb - pp1 to zap-70 stable cell lines , we did not observe that any substantial tcr signaling functions were maintained . however , we did not determine whether the association of zap-70 with the other proteins was still inducible . our laboratory had previously reported that the deletion of the region between the second sh2 domain and kinase domain of zap-70 , termed interdomain b , which contains the key tyrosines that are known to interact with other proteins when phosphorylated , does not eliminate kinase activity ( 47 ) . in addition , point mutations of tyrosines 315 and 319 to alanine or glutamic acid do not ablate kinase activity ( 1 ) . the recently solved crystal structure of zap-70 supports a role for the interdomain b tyrosines in maintaining zap-70 in an inactive and closed conformation when unphosphorylated ( 35 ) . upon phosphorylation of zap-70 by lck thus , although biochemical evidence has shown that the zap-70 tyrosines promote binding with other important signaling molecules , these events are clearly not sufficient for substantial downstream signaling in the absence of zap-70 catalytic activity . overall , these findings allow for the possibility that the role of zap-70 as an adapter might be significant only in the context of a catalytically active protein kinase . one of the benefits of using a small molecule inhibitor is to be able to study an enzymatic role in a process where having a genetically deficient system is undesirable . although previous researchers did not observe zap-70 phosphorylation upon superagonist stimulation , we were able to detect induction of zap-70 phosphorylation , albeit weaker than after tcr antibody stimulation . recently , a role for zap-70 has been implicated in superagonist stimulation , but it was hypothesized that only tonic signaling is required ( 43 ) . our data using 3-mb - pp1 and analog - sensitive zap-70 alleles suggest that zap-70 is actively required in this signaling cascade . although the level of zap-70 phosphorylation is lower following stimulation with the cd28 superagonist , the induced signaling events appear to be zap-70-dependent , suggesting the presence of an amplification loop downstream of zap-70 . ( 43 , 44 ) did not observe it in either rat primary t cells or jurkat cells . the stimulating cd28 antibody used in our current experiment was also different than the one used in those previous studies . overall , these data support the general utility of this zap-70 inhibitor system and the general benefit of using multiple biological methods to dissect protein function . , broad expression of many of these enzymes makes them unappealing targets because inhibition of the kinase , when outside of the disease context , can lead to undesirable side effects . zap-70 is an attractive target because it is expressed predominantly in t cells . in situations where overactive t cells are a substantial component of disease , such as in autoimmune diseases or transplantation , targeting zap-70 could provide a means to target the t cells while avoiding adverse effects on other cells . the system that we have generated for analyzing the effect of zap-70 inhibition will allow us to identify the utility of a zap-70 inhibitor in preclinical models in mice that we have recently reconstituted with the analogsensitive allele of zap-70 . importantly , in preliminary studies , we have confirmed that peripheral t cells isolated ex vivo from the analog - sensitive transgenic animals are uniquely susceptible to inhibition by 3-mb - pp1 , thus validating an in vivo system for our future studies . these studies together with crystal structures of the intact autoinhibited zap-70 protein and the activated isolated kinase domain may help in the development of a clinical zap-70 inhibitor ( 35 , 48 ) .
zap-70 is a cytoplasmic protein tyrosine kinase that is required for t cell antigen receptor ( tcr ) signaling . both mice and humans deficient in zap-70 fail to develop functional t cells , thus demonstrating its necessity for t cell development and function . there is currently no highly specific , cell - permeable , small molecule inhibitor for zap-70 ; therefore , we generated a mutant zap-70 allele that retains kinase activity but is sensitive to inhibition by a mutant - specific inhibitor . we validated the chemical genetic inhibitor system in jurkat t cell lines , where the inhibitor blocked zap-70-dependent tcr signaling in cells expressing the analog - sensitive allele . interestingly , the inhibitor also ablated cd28 superagonist signaling , thereby demonstrating the utility of this system in dissecting the requirement for zap-70 in alternative mechanisms of t cell activation . thus , we have developed the first specific chemical means of inhibiting zap-70 in cells , which serves as a valuable tool for studying the function of zap-70 in t cells .
EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material
in these studies , we chose to use the pp1 analog 3-mb - pp1 over other pp1 analogs , based upon its potent inhibition of zap-70 and the lack of effect on wild - type cells in a screen for t cell activation ( data not shown ) . to test the cellular activity of the zap-70 mutants and their sensitivity to 3-mb - pp1 , we transiently transfected 293 cells with zap-70 , or either one of the zap-70 constructs , along with lck to activate zap-70 and the transmembrane adaptor lat , which serves as a zap-70-specific substrate . thus , the zap-70 is functional and is inhibited by 3-mb - pp1 in a t cell system . moreover , although lck and many downstream kinases are required for cd69 induction , only the cells expressing the zap-70 alleles are sensitive to inhibition by 3-mb - pp1 . therefore , we further tested the specificity of 3-mb - pp1 inhibition by analyzing its effect on other relatively upstream kinase / substrate pairs implicated in tcr signaling in 293 transient transfection assays ( fig . therefore , we selected zap-70 and zap-70 , which express comparable amounts of zap-70 to the wild - type clones . , src family kinases such as lck in jurkat t cells are also required for inducing [ ca]i increases because of their ability to mediate itam phosphorylation and zap-70 activation . treatment with the src family kinase inhibitor pp2 had a comparable effect to that of inhibiting zap-70 in both wild - type and mutant stables , whereas only zap-70 was sensitive to blockade by 3-mb - pp1 , suggesting a high degree of specificity . zap-70 inhibition suppresses superantigen - mediated il-2 induction the inhibition of calcium increases and nfat - transcription in 3-mb - pp1-treated cells demonstrated the utility of this analog approach and exposed the requirement of zap-70 to both initiate and maintain the calcium response after tcr antibody stimulation . because phosphorylation of lat and slp-76 by zap-70 is so critical for initiation of many of the downstream signaling events , we wanted to examine the phosphorylation status of lat and slp-76 after treatment with 3-mb - pp1 in the analog - sensitive clones . vehicle - treated zap-70 mutant cell samples often exhibited a somewhat decreased phosphorylation relative to zap-70 cells , which we hypothesized was due to the decreased catalytic activity of the analog - sensitive mutant . despite the decreased magnitude of phosphorylation in the untreated zap-70 cells , moreover , in very preliminary studies , we have been able to reconstitute t cell development of zap-70 null mice with the zap-70 clone expressed as a transgene , suggesting that the reduced catalytic activity of the mutant zap-70 is not substantially functionally impaired . for instance , zap-70 is well known for its critical role in proximal tcr signaling , but its function in cd28 superagonist signaling has been unclear . one human cd28 superagonist was recently utilized in a clinical trial because it had been shown to induce development of regulatory t cells , but when administered to the healthy volunteers in a phase i clinical trial , the superagonist induced an unanticipated cytokine storm followed by multiorgan failure in volunteer subjects participating in this trial ( 42 ) . therefore , given the controversial role for zap-70 signaling in this signaling cascade and the importance of understanding how superagonists function , we decided to utilize our inhibitor system to dissect the role of this kinase in superagonist stimulation . the best way to test this hypothesis is with a small molecule inhibitor ; therefore , we treated our zap-70 cells with 3-mb - pp1 concurrently with 1 g / ml anc28.1 antibody . thus , the use of 3-mb - pp1 and the zap-70-expressing cells demonstrates the requirement of zap-70 in cd28 superagonist - mediated signaling as well as the utility of having an inhibitor system to study the role of zap-70 . we successfully generated two zap-70 alleles that retain kinase activity but are sensitive to inhibition by the pp1 analog 3-mb - pp1 . following the addition of 3-mb - pp1 to zap-70 stable cell lines , we did not observe that any substantial tcr signaling functions were maintained . the system that we have generated for analyzing the effect of zap-70 inhibition will allow us to identify the utility of a zap-70 inhibitor in preclinical models in mice that we have recently reconstituted with the analogsensitive allele of zap-70 . importantly , in preliminary studies , we have confirmed that peripheral t cells isolated ex vivo from the analog - sensitive transgenic animals are uniquely susceptible to inhibition by 3-mb - pp1 , thus validating an in vivo system for our future studies .
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mental , emotional , and autonomic functions of the brain arise from interactions between the nearly 100 billion neurons that comprise this organ in humans . on average , synapses are asymmetric , complex , and highly dynamic [ 1 , 2 ] . the plasticity of synapses and dendritic spines , the morphological structures that are the loci of most excitatory synapses in the central nervous system ( cns ) , are widely believed to underlie learning and memory and are frequently altered in neurodevelopmental and neurodegenerative diseases [ 3 , 4 ] . thus , understanding the development , dynamics , and elimination of synapses is crucial for human health . a dizzying array of signals coordinates these processes , and thus receptors are an integral component of the synaptic regulatory machinery [ 14 ] . receptors also represent the most accessible point at which to manipulate these processes pharmacologically . g - protein coupled receptors ( gpcrs ) comprise a superfamily of approximately 800 members in humans , including many important drug targets . they exhibit a characteristic seven - transmembrane ( 7tm ) core structure by which gpcrs interact with and activate a variety of heterotrimeric g - proteins , which in turn activate or repress intracellular signaling cascades . adhesion - gpcrs are a gpcr subfamily with 33 members in humans that are characterized by an extended n - terminal extracellular segment connected to the core gpcr structure by a distinctive gpcr autoproteolysis - inducing ( gain ) domain , which is present in all adhesion - gpcrs except gpr123 [ 8 , 9 ] . the n - terminal segments of most adhesion - gpcrs contain multiple domains capable of binding to other cells or the extracellular matrix [ 810 ] . these include at least 16 different types of domain , with multiple types frequently occurring within the same protein ; domains include cadherin - like repeats , thrombospondin - like repeats , rhamnose - binding lectin domains , and calnexin domains . adhesion - gpcrs can be divided into 9 subfamilies based on phylogenetic analysis of the gpcr moiety ; members of the different subfamilies generally also have related complements of n - terminal adhesive domains [ 9 , 10 ] . gain domains mediate autoproteolytic cleavage of adhesion - gpcrs during translation in the er at a site within the gain domain called the gpcr proteolysis site ( gps ) [ 11 , 12 ] . after cleavage , the n- and c - terminal fragments ( ntfs , ctfs ) of most adhesion - gpcrs remain noncovalently associated [ 9 , 10 ] . however , this scenario is complicated . some adhesion - gpcrs do not undergo autoproteolysis , and some that do may even swap ntfs with other adhesion - gpcrs resulting in hybrid adhesion - gpcrs [ 9 , 13 , 14 ] . it has been widely believed that the ntfs may repress the signaling mediated by ctfs , and that ligand binding relieves this inhibition , possibly by causing dissociation of the ntf from the ctf [ 8 , 15 ] . recently , a peptide agonist sequence named stachel was identified on the c - terminal side of the gps of adhesion - gpcrs . this sequence , which is specific for a given adhesion - gpcr , can activate g - protein dependent signaling through the adhesion - gpcr when it is unmasked by removal of the ntf or conformational changes in the protein ( either of which is presumably ligand - induced ) . identification of the gain domain and stachel sequence are both recent findings , illustrating a rapid advance in the knowledge of adhesion - gpcr biology after years lagging behind other gpcrs . adhesion - gpcrs function in various tissues throughout organisms [ 8 , 9 ] , but an important driving force of recent rapid advances in adhesion - gpcr biology has been the discovery that adhesion - gpcrs regulate the development and function of many aspects of the nervous system . these include migration of neuronal precursors , axon guidance , myelination of axons , vascularization of the brain , and synapse formation and function [ 8 , 9 ] . in this brief review , we highlight the roles of the brain - specific angiogenesis inhibitor ( bai ) subfamily of adhesion - gpcrs at neuronal synapses . adhesion - gpcr nomenclature arose over a long period of time and in a nonsystemic manner . thus , the members of the bai subfamily , bai13 , would now be named adgrb13 . this new nomenclature is not yet in standard use , and we will use the traditional names for adhesion - gpcrs , noting the new designations of adhesion - gpcrs we discuss . for general information on adhesion - gpcr function bai1 , bai2 , and bai3 ( adgrb13 ) comprise a subfamily of adhesion - gpcrs that are highly expressed in the brain [ 9 , 17 ] . bais are large proteins , approximately 200 kda in size , with each possessing a long n - terminal region containing multiple adhesive thrombospondin type 1 repeats ( tsrs ) , a hormone - binding domain , and the autoproteolysis - inducing gain domain ( figure 1 ) . bais also contain an extended intracellular region c - terminal to the conserved 7tm gpcr domain that terminates in a pdz - binding motif , qtev ( gln - thr - glu - val ) . bai1 contains an additional tsr ( five in total ) , an integrin - binding rgd ( arg - gly - asp ) motif , and a c - terminal proline - rich region not present in the other two bai family members ( figure 1 ) . bais are widely expressed in postnatal and adult brain , with bai1 and bai2 mrna levels peaking at postnatal day 10 ( p10 ) , while the level of bai3 mrna is highest 1 day after birth . bai1 protein is present in neurons , glia , and macrophages , with particularly high expression in cortical and hippocampal pyramidal neurons [ 2226 ] . less is known about the cellular distribution of bai2 and bai3 proteins , although bai3 is abundant in cerebellar purkinje cells [ 2729 ] . in neurons , bai1 and bai3 are both enriched in the postsynaptic density ( psd ) , suggesting a role for these proteins in synapse development and/or function [ 25 , 30 , 31 ] . like most adhesion - gpcrs , bais possess a gain domain , but their ability to undergo autoproteolytic cleavage appears to be cell - type specific and not required for proper surface trafficking . for instance , while bai1 is cleaved at the gps site in mouse brain and human malignant glioma cells [ 11 , 3234 ] , uncleaved full - length bai1 is also clearly present in hippocampal and cortical neurons . cleavage of the bai1 gain domain generates a secreted 120 kda fragment called vasculostatin-120 ( vstat120 ) , which is capable of inhibiting angiogenesis and tumor formation [ 32 , 33 ] . bai1 is also cleaved at a second site n - terminal to the gain domain by matrix metalloproteinase 14 ( mmp-14 ) . this cleavage event generates a 40 kda fragment called vasculostatin-40 ( vstat40 ) , which also has antiangiogenic activity . the antiangiogenic effects of vstat120 and vstat40 are primarily mediated by the tsrs , which bind to the scavenger receptor cd36 and induce proapoptotic signaling . while proteolytic cleavage of bai proteins is thought to both modulate the function of the full - length receptors and release their ntfs , which can exert their own physiological effects , more work needs to be done to understand how cleavage is regulated and what precise consequences it has on bai function . research in the last decade has revealed a number of important roles for bai family members in diverse cellular processes [ 17 , 36 ] . as indicated above , bai proteins can function as potent inhibitors of angiogenesis and tumor progression . bai1 expressed in macrophages has also been shown to bind to phosphatidylserine ( ps ) and lipopolysaccharide ( lps ) and mediate the engulfment of apoptotic cells and gram - negative bacteria , respectively [ 24 , 37 ] . bai1 promotes engulfment in response to ps or lps binding by activating the associated elmo / dock180 signaling module , which in turn activates the small gtpase rac1 and induces rac1-dependent actin cytoskeletal remodeling required for internalization of apoptotic cells or bacteria [ 24 , 37 ] . the ability to bai1 to bind to ps is also important for myoblast fusion , and loss of bai1 results in a reduction in myofiber size and impaired muscle regeneration in mice . the tsrs on the n - terminus of bai family members are essential for their capacity to regulate these diverse cellular processes , and therefore proteolysis of the bai extracellular domain may dramatically alter bai function . despite the recent advances in our understanding of bai function , until recently , little was known about the roles of bai adhesion - gpcrs in neurons . over the last few years , bais have emerged as important regulators of synaptogenesis and synaptic plasticity . below , we consider the synaptic functions of each of the bai family members in turn . bai1 is enriched in , though not exclusively localized to , the psd in dendritic spines in hippocampal neurons ; this has been shown by biochemical fractionation and immunocytochemistry in rat hippocampal neurons and mouse brains [ 25 , 31 ] . this enrichment indicated that bai1 might play a role in synaptic formation or function , and this problem was attacked in two different ways . in both cases , our approach was to acutely knock down bai1 both in vitro using cultured rat hippocampal neurons and in vivo using in utero electroporation of shrnas directed against bai1 . in both systems , we found that bai1 plays a key role in dendritic spine formation . knockdown of bai1 in cultured primary hippocampal neurons resulted in a loss of spine and synapse density with a shift of remaining spines to an immature elongated morphology . in vivo knockdown also resulted in a dramatic loss of spine density and a shift toward less mature spines in the somatosensory and the cingulate cortices . bai1 's prospinogenic and prosynaptogenic activities are mediated through its interactions with the cell polarity complex tiam1/par3 through its c - terminal pdz - binding motif ( figure 2 ) . tiam1 is an activator of the small gtpase rac1 , which directs the actin cytoskeletal remodeling that drives spine and synapse development . tiam1 couples rac1-dependent spine and synapse formation to extracellular signals , including glutamate ( via nmda receptors ) , ephrin - b ( via ephb receptors ) , and bdnf ( via trkb receptors ) . bai1 anchors the tiam1/par3 complex to dendritic spines where localized rac1 activation promotes the formation of dendritic spines and subsequent excitatory synaptogenesis . of note , although other rac1 activators such as elmo / dock180 bind to bai1 , rac1 activation leading to spinogenesis requires only tiam1 , as bai1 mutants lacking the tiam1/par3-interacting motif can not rescue the knockdown phenotype , whereas mutants that do not interact with elmo / dock180 can . consistent with these results , knockout mouse studies recently revealed a requirement for bai1 in spatial learning and synaptic plasticity . bai1-null mice have severe deficits in both hippocampus - dependent spatial learning and memory along with enhanced long - term potentiation ( ltp ) and impaired long - term depression ( ltd ) . an interesting result arising from this study was the discovery that bai1 contributes to proper synapse formation through its ability to stabilize the expression of the postsynaptic scaffold protein psd95 . it was determined that bai1 binds to and inhibits the e3 ubiquitin ligase mdm2 , thereby preventing the psd95 degradation that was responsible for the spatial learning and plasticity phenotypes observed in bai1-null mice ( figure 2 ) . although both of these studies agreed that bai1 plays a role in synapse function , there were important differences in the results . our results using shrnas against bai1 led to stark and obvious loss of spines , while the results with the bai1-null mice showed no difference in spine density . there are obvious differences in the techniques used that could have given rise to these differences , and we will return to this issue below . bai1 's c - terminal pdz - binding motif also interacts with a variety of other synaptic molecules . proteomic analysis reveals that the c - terminal segment of bai1 can bind to pdz - domain - containing proteins such as sap97 ( dlg1 ) , densin-180 , magi-1/bap1 , magi-2 , and magi-3 . however , the exact functions of the majority of these interactions are not well understood . one potentially interesting bai1-binding protein is the insulin receptor substrate 53 ( irsp53 ) , which binds to a proline - rich region in bai1 's intracellular c - terminal segment and is also enriched in the psd [ 43 , 44 ] . since irsp53 is itself a downstream effector of rac1 and cdc42 and a regulator of dendrite spine morphogenesis , future studies that explore the effects of irsp53-bai1 interactions could elucidate key mechanisms of spinogenesis and synaptogenesis . irsp53 's potential role in autism spectrum disorder ( asd ) makes this an even more interesting interaction to investigate . like bai1 roles for bai2 in neurogenesis and synaptogenesis have been suggested but not well established experimentally . bai2-deficient mice were found to display increased resistance to social defeat stress and reduced immobility in the tail suspension test , two behavioral assays that assess depressive behavior in rodents . bai2-deficient mice were also shown to exhibit increased neurogenesis in the dentate gyrus of the hippocampus , where bai2 is highly expressed [ 47 , 48 ] . these two observations are likely related since enhanced adult neurogenesis has been shown to positively correlate with resistance to depression . it is also consistent with reports that bai2 suppresses the expression of vascular endothelial growth factor ( vegf ) , as vegf stimulates adult neurogenesis in the dentate gyrus . loss of bai2 could therefore increase vegf levels , resulting in enhanced neurogenesis and increased resistance to stress . this idea will need to be further investigated . furthermore , since stress and depression are known to induce synapse loss , while antidepressants promote synaptogenesis , in future studies it will be interesting to investigate the possible roles of bai2 at synapses . biochemical fractionation studies have revealed that like bai1 , bai3 localizes to excitatory synapses in the brain [ 30 , 53 ] . furthermore , overexpression studies examining the localization of bai3 in transfected hippocampal neurons have shown that it is highly enriched in spines where it colocalizes with the postsynaptic marker psd95 . indeed , recently bai3 was shown to regulate excitatory synapse connectivity and formation in the mouse cerebellum [ 28 , 29 ] ( figure 2 ) . knockdown of bai3 using lentivirus - delivered shrna in p7 pups induced clear deficits in connectivity between cerebellar climbing fibers and their target purkinje cells and between parallel fibers and purkinje cells by p21 . dendritic spine density and vglut1-positive synaptic contacts were both decreased in purkinje cells with reduced bai3 levels . similarly , mice lacking bai3 specifically in purkinje cells show a significant decrease in the number of vglut2-positive puncta in the cerebellum . bai3 's role at climbing fiber synapses is mediated through its interactions with a class of secreted complement proteins known as the c1q - like complement ( c1ql ) family . c1ql proteins are broadly expressed in the brain with different spatial and temporal expression patterns shown by family members c1ql14 . in particular , c1ql1 is highly expressed during the first 2 postnatal weeks in various neuronal populations , particularly in the hippocampus , cerebral cortex , and cerebellum . transient c1ql1 secretion in the cerebellum promotes purkinje cell spinogenesis , and the effect of modulating c1ql1 expression on purkinje cell spinogenesis depends on the expression levels of bai3 . critically , the c1ql1-bai3 interaction promotes developmental synapse refinement and triggers elimination of surplus climbing fiber synapses , helping to select and maintain a single winning climbing fiber . bai3 expression in purkinje cells is required for this process , and the climbing fiber is the source of c1ql . moreover , continued expression of bai3 is necessary for maintenance of climbing fiber synapses , and adult mice lacking c1ql , which possess excess climbing fiber synapses per purkinje cell , eliminate these extra synapses when c1ql is introduced into the animals . c1ql1 interacts with bai3 through the n - terminal cub domain , which is unique to bai3 . bai3 also interacts with another c1ql family member , c1ql3 , through its tsrs . incubating cultured hippocampal neurons with c1ql3 was shown to decrease excitatory synaptic density , and this effect was reversed by adding the isolated tsrs of bai3 to the culture . this result suggests a role for bai3/c1ql3 in hippocampal synapse development akin to the bai3/c1ql1-mediated pruning function in the cerebellum described above . it is not known if bai3 also plays an earlier role in promoting synapse formation in the hippocampus . further , since the tsrs in bai3 are present in all bais , it is possible that c1ql3 also interacts with bai1 and bai2 , but this remains to be investigated . bai3 's role in synapse elimination during cerebellar development could shed some light on the differences observed in the shrna - transfected versus bai1-null mice described above . if proper spine formation requires a competition to sort out the winning synapse , expression profiles of relevant proteins in participating neurons might contribute to the resolution of this competition . in the neurons in which bai1 was removed via shrna , only the transfected cells had a deficit in bai1 , and they represented a small fraction ( < 5% ) of the total population . if they were in competition with bai1-expressing neurons for the establishment of synapses , and bai1 promotes winning the competition , then the bai1 knockdown neurons would be at a decided disadvantage relative to the vast majority of neurons expressing normal levels of bai1 . this state of affairs would hold for both the cultured neurons and the in vivo preparations . on the other hand , the neurons examined in the bai1 null mice existed on a background of bai1 null neurons . therefore , the unmarked neurons would not have an advantage in preserving synapses and this may explain why no loss of dendritic spines was observed . such argument by analogy can only go so far , and compensation by other bai family members could also be a factor , but this hypothesis warrants further investigation . in addition to the roles that bais play in synaptogenesis and synaptic function , there is evidence that additional adhesion - gpcrs function in these roles . latrophilins are an adhesion - gpcr subfamily comprised of 3 members latrophilins 13 ( lphn13 or adgrl13 ) and eltd1 ( adgrl4 ) in humans and represent one of only two subfamilies conserved in invertebrates . latrophilins were identified as receptors for the black widow spider toxin -latrotoxin , which causes a massive ca - mediated exocytosis of neurotransmitter - containing vesicles from the presynaptic side of the synapses . lphn1 and lphn3 are largely restricted to the brain , while lphn2 is expressed in many tissues . in addition to their gain domains , lphns contain a hormone receptor motif , an olfactomedin - like domain , and a rhamnose - binding lectin domain in their ntfs . lphn1 is thought to mediate its effects on synapse formation via interactions with teneurin-2/lasso [ 57 , 58 ] , neurexin-1/2 , and fibronectin leucine - rich transmembrane proteins ( flrts ) . presynaptic lphn1 binds to teneurin-2 via its lectin domain with nanomolar affinity in a manner regulated by alternate splicing of lphn1 [ 57 , 58 ] . the lphn1/teneurin interaction leads to presynaptic ca increases , and disruption of the interaction using the teneurin - binding segment of the lphn1 ntf decreases both excitatory and inhibitory synapse density in rat hippocampal neurons . lphn1 's interaction with neurexins also has nanomolar affinity and is regulated by alternate splicing of neurexins but is largely mediated by lphn1 's olfactomedin domain . this interaction is especially intriguing because neurexins and their canonical binding partners , neuroligins , form trans - synaptic complexes and are strongly implicated in asd . it is not yet known what function the lphn1/neurexin interaction serves at synapses , but given the known roles of both proteins , it is likely to be of high interest . lphn3 has received increased attention of late due to a strong emerging correlation with attention deficit / hyperactivity disorder ( adhd ) in humans [ 61 , 62 ] . lphn3 binds to flrt-3 via its olfactomedin domain and to teneurin-1 via its olfactomedin and lectin domains [ 63 , 64 ] . presynaptic lphn3 interacts with postsynaptic flrt-3 to promote synapse formation in hippocampal neurons and in cortical synapses from layers 2/3 to layer 5 [ 63 , 64 ] . interestingly , flrt-3 and teneurins vary in their distributions throughout the layered structure of the cortex , suggesting that lphn3 could serve different functions in different regions of the brain by interacting with distinct ligands . in short , lphns are implicated in both presynaptic function and in directing synapse formation by forming complexes with transmembrane ligands in neuronal membranes . the celsr adhesion - gpcr subfamily is characterized by the presence of atypical cadherin repeats , calcium - binding egf - like domains , laminin g domains , and a hormone receptor motif in their ntfs in addition to the gain domain [ 9 , 10 ] . like latrophilins , this subfamily is conserved in invertebrates , with flamingo in drosophila melanogaster , fmi-1/2 in caenorhabditis elegans , and celsr13 ( adgrc13 ) in humans [ 9 , 10 ] . adhesion - gpcrs of the flamingo / celsr subfamily function in many aspects of nervous system development , including neural tube closure , axon guidance , and the formation of dendritic arbors [ 9 , 6568 ] . these effects are mediated through the now classical interaction of these proteins with the cellular planar cell polarity ( pcp ) machinery , as well as camp- and ca - dependent mechanisms [ 9 , 65 , 66 , 68 ] . synaptic defects are observed when expression of celsr - subfamily adhesion - gpcrs is altered or repressed , but it is difficult to determine whether these are direct effects on synaptic formation and/or maintenance , or whether they arise secondarily from malformation of axons and dendrites . loss of flamingo leads to formation of ectopic neuromuscular junctions , or synapses between axons and muscle , in drosophila . it also leads to malformed en passant synapses in this system , though these synapses are functional . further , aging animals lacking flamingo exhibit a decrease in neuromuscular junctions , though this appears to be an effect of axonal degeneration . numerous questions remain to be answered in order to determine the specific roles of celsr subfamily adhesion - gpcrs in synaptic formation and function . finally , very large gpcr 1 ( vlgr1 or adgrv1 ) has been implicated in the formation of cochlear synapses , though its specific role remains unclear . identification of adhesion - gpcrs involved in synaptic formation and function as well as elucidation of the mechanisms and signals that underlie these roles are important challenges for both adhesion - gpcr and synaptic biology . given the important roles that bai adhesion - gpcrs play in promoting synapse development and plasticity and inhibiting angiogenesis and tumor formation , it is not surprising that they have been implicated in a number of human diseases . for instance , single nucleotide polymorphisms ( snps ) and copy number variations in the human bai3 gene have been associated with schizophrenia [ 7173 ] , bipolar disorder , and drug addiction , brain disorders characterized by synapse abnormalities . furthermore , bai3 expression is affected by lithium treatment , which is often used to treat patients with bipolar disorder and schizophrenia [ 74 , 76 ] . the human bai1 gene is also located in a hot spot for de novo germline mutations in patients with autism , and bai1 expression is upregulated in mouse models of rett and mecp2 duplication syndromes . conversely , bai1 expression is downregulated in glioblastoma and is inversely correlated with neovascularization in colorectal and lung cancers . the growing evidence that bais play critical roles in human disease suggests that they may make good therapeutic targets in the future . gpcrs are generally considered to be the most successful therapeutic targets for a broad spectrum of diseases . indeed , greater than 50% of the current therapeutic agents on the market target these proteins [ 79 , 80 ] . greater insight into the regulation and function of bais could therefore facilitate the development of novel therapies for the treatment of brain disorders and cancer . after years of relative obscurity , there have been rapid recent advances in understanding the biology of bais and other adhesion - gpcrs . these molecules are intriguing because they tend to have multiple ligand binding domains that suggest that they are signal integrators , recognize large , complex substrates , and/or detect coincidences . the complexities added by ntf swapping , signaling by both gpcr - dependent and -independent modes , splice variants , and potential formation of higher level complexes are only beginning to be understood in a functional context . these complexities lend themselves to neuronal and synaptic function , given the role that these cells and structures play in storing and processing information . bais in particular are demonstrating key roles in synaptic function , though they play other roles in and out of the brain as well . a full appreciation of bai function will require the identification of all bai ligands , complete elucidation of bai expression patterns and localization , identification of all binding partners and modes of signaling , and dynamic measurements of these properties . these are exciting challenges that hold great promise for increasing our understanding of synaptic function , as well as treating synaptic dysfunction .
synapses mediate communication between neurons and enable the brain to change in response to experience , which is essential for learning and memory . the sites of most excitatory synapses in the brain , dendritic spines , undergo rapid remodeling that is important for neural circuit formation and synaptic plasticity . abnormalities in synapse and spine formation and plasticity are associated with a broad range of brain disorders , including intellectual disabilities , autism spectrum disorders ( asd ) , and schizophrenia . thus , elucidating the mechanisms that regulate these neuronal processes is critical for understanding brain function and disease . the brain - specific angiogenesis inhibitor ( bai ) subfamily of adhesion g - protein - coupled receptors ( adhesion - gpcrs ) has recently emerged as central regulators of synapse development and plasticity . in this review , we will summarize the current knowledge regarding the roles of bais at synapses , highlighting their regulation , downstream signaling , and physiological functions , while noting the roles of other adhesion - gpcrs at synapses . we will also discuss the relevance of bais in various neurological and psychiatric disorders and consider their potential importance as pharmacological targets in the treatment of these diseases .
1. Introduction 2. Adhesion-GPCRs 3. The BAI Subfamily of Adhesion-GPCRs 4. Roles of BAIs at Synapses 5. Other Adhesion-GPCRs Involved in Synapses 6. BAIs' Disease Relevance and Potential as Therapeutic Targets 7. Conclusions
the plasticity of synapses and dendritic spines , the morphological structures that are the loci of most excitatory synapses in the central nervous system ( cns ) , are widely believed to underlie learning and memory and are frequently altered in neurodevelopmental and neurodegenerative diseases [ 3 , 4 ] . g - protein coupled receptors ( gpcrs ) comprise a superfamily of approximately 800 members in humans , including many important drug targets . adhesion - gpcrs are a gpcr subfamily with 33 members in humans that are characterized by an extended n - terminal extracellular segment connected to the core gpcr structure by a distinctive gpcr autoproteolysis - inducing ( gain ) domain , which is present in all adhesion - gpcrs except gpr123 [ 8 , 9 ] . gain domains mediate autoproteolytic cleavage of adhesion - gpcrs during translation in the er at a site within the gain domain called the gpcr proteolysis site ( gps ) [ 11 , 12 ] . after cleavage , the n- and c - terminal fragments ( ntfs , ctfs ) of most adhesion - gpcrs remain noncovalently associated [ 9 , 10 ] . some adhesion - gpcrs do not undergo autoproteolysis , and some that do may even swap ntfs with other adhesion - gpcrs resulting in hybrid adhesion - gpcrs [ 9 , 13 , 14 ] . this sequence , which is specific for a given adhesion - gpcr , can activate g - protein dependent signaling through the adhesion - gpcr when it is unmasked by removal of the ntf or conformational changes in the protein ( either of which is presumably ligand - induced ) . adhesion - gpcrs function in various tissues throughout organisms [ 8 , 9 ] , but an important driving force of recent rapid advances in adhesion - gpcr biology has been the discovery that adhesion - gpcrs regulate the development and function of many aspects of the nervous system . these include migration of neuronal precursors , axon guidance , myelination of axons , vascularization of the brain , and synapse formation and function [ 8 , 9 ] . in this brief review , we highlight the roles of the brain - specific angiogenesis inhibitor ( bai ) subfamily of adhesion - gpcrs at neuronal synapses . this new nomenclature is not yet in standard use , and we will use the traditional names for adhesion - gpcrs , noting the new designations of adhesion - gpcrs we discuss . for general information on adhesion - gpcr function bai1 , bai2 , and bai3 ( adgrb13 ) comprise a subfamily of adhesion - gpcrs that are highly expressed in the brain [ 9 , 17 ] . in neurons , bai1 and bai3 are both enriched in the postsynaptic density ( psd ) , suggesting a role for these proteins in synapse development and/or function [ 25 , 30 , 31 ] . despite the recent advances in our understanding of bai function , until recently , little was known about the roles of bai adhesion - gpcrs in neurons . over the last few years , bais have emerged as important regulators of synaptogenesis and synaptic plasticity . biochemical fractionation studies have revealed that like bai1 , bai3 localizes to excitatory synapses in the brain [ 30 , 53 ] . in addition to the roles that bais play in synaptogenesis and synaptic function , there is evidence that additional adhesion - gpcrs function in these roles . adhesion - gpcrs of the flamingo / celsr subfamily function in many aspects of nervous system development , including neural tube closure , axon guidance , and the formation of dendritic arbors [ 9 , 6568 ] . numerous questions remain to be answered in order to determine the specific roles of celsr subfamily adhesion - gpcrs in synaptic formation and function . identification of adhesion - gpcrs involved in synaptic formation and function as well as elucidation of the mechanisms and signals that underlie these roles are important challenges for both adhesion - gpcr and synaptic biology . given the important roles that bai adhesion - gpcrs play in promoting synapse development and plasticity and inhibiting angiogenesis and tumor formation , it is not surprising that they have been implicated in a number of human diseases . for instance , single nucleotide polymorphisms ( snps ) and copy number variations in the human bai3 gene have been associated with schizophrenia [ 7173 ] , bipolar disorder , and drug addiction , brain disorders characterized by synapse abnormalities . greater insight into the regulation and function of bais could therefore facilitate the development of novel therapies for the treatment of brain disorders and cancer . after years of relative obscurity , there have been rapid recent advances in understanding the biology of bais and other adhesion - gpcrs .
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cancer is a huge burden of our societies with an overall worldwide incidence of 182,3 cases per 100.000 inhabitants and an overall mortality of 102,4/100.000 according to the international agency for research on cancer of the world health organization ( estimated age - standardized incidence and mortality rates ( asr ) for both sexes ) . highest incidence rates are reported for breast , colorectal and cervical cancer in women and lung and prostate cancer in men . current treatment options comprise of surgery , chemotherapy or radiation plus more recently introduced targeted therapies . targeted therapies aim to specifically address malignantly transformed cells while sparing healthy tissues . thus , receptors , which are important during embryonic development and readopted by cancer cells , belong to the most promising targets . one of the most prominent molecules of that kind is the human epidermal growth factor receptor-2 ( her-2 ) . her-2 is a receptor tyrosine kinase , mediating signals for cell proliferation , cell mobility and survival . in the absence of a known ligand her-2 is very important during embryonic development , e.g. it plays a role in ductal morphogenesis of the mammary gland , but it is almost not expressed on adult tissue , except the heart . on the contrary , her-2 is overexpressed in breast , ovarian , gastric , colorectal , pancreatic , and endometrial cancers . another closely related receptor tyrosine - kinase is the epidermal growth factor receptor ( egfr ) . its overexpression is associated with head and neck squamous cell carcinoma ( hnscc ) , non - small - cell lung cancer ( nsclc ) , colorectal cancer ( crc ) , breast and pancreatic cancer , but also with certain types of brain cancer . in contrast to her-2 , egfr senses the epidermal growth factor ( egf ) and other important growth signals , such as transforming growth factor- ( tgf- ) or amphiregulin [ 11 - 14 ] . egfr is physiologically required for promoting cell proliferation and dna repair , but can also lead to tumor growth , progression , and evasion of apoptosis via the activation of plc--pkc , ras - raf - mek , pi-3k - akt - mtor and jak2-stat3 pathways . overall , egfr and her-2 together with her-3 and her-4 belong to the erbb - family , which derives its name from the homology to the erythroblastic leukemia viral oncogene protein ( v - erb - b , ) . currently two forms of targeted therapies against egfr and her-2 are in clinical use : i ) blocking the intracellular receptor tyrosine kinase with small molecules and ii ) attacking the extracellular domains of the receptor with monoclonal antibodies . small molecules targeting egfr comprise erlotinib ( tarceva , roche ) and gefitinib ( iressa , astrazeneca ) plus the dual kinase inhibitors lapatinib ( tykerb , glaxosmithkline ) and afatinib ( gilotrif , bhringer ingelheim ) , the latter inhibiting her-2 as well ( , see table 1 ) . especially the reversible inhibitors gefitinib , being fda - approved in may 2003 and erlotinib , with fda - approval in november 2004 , are successfully applied in non - small - cell lung cancer . although gefitinib was recalled from that indication in the us , it is still widely used in japan , where patients display a higher rate of egfr - mutations in nsclc , and also received marketing authorization in the european union in 2009 . moreover , erlotinib is approved for the treatment of advanced pancreatic cancer and several next generation irreversible egfr - tyrosine kinase inhibitors , like canertinib , are under investigation for their efficacy in breast , colorectal , lung , pancreatic , renal , head and neck , gynecologic and prostate cancer . the most prominent tyrosine kinase inhibitor ( tki ) for her-2 is lapatinib ( tykerb , glaxosmithkline ) , the above mentioned reversible dual inhibitor of her-2 and egfr , which was fda - approved in march 2007 for the treatment of advanced breast cancer . also in this case , irreversible inhibitors , like neratinib or again canertinib are widely investigated . in contrast to small molecules that intracellularly interfere signaling via blocking the kinase activity , monoclonal antibodies directed against egfr and her-2 aim to extracellularly inhibit ligand binding or dimerization of these receptors , respectively . for targeting egfr , two monoclonal antibodies are currently in clinical use , cetuximab ( erbitux , merck kgaa ) , which was fda - approved in february 2004 and panitumumab ( vectibix , amgen ) , which received fda - approval in september 2006 ( , table 1 ) . in particular cetuximab , a human - murine chimeric igg1 antibody has become an indispensable cornerstone in the treatment of advanced - stage metastatic crc and advanced hnscc . cetuximab finds its epitope within the ligand - binding site of egfr ( extracellular domain iii ) and can thus block binding of growth signals . panitumumab works mechanistically similar ; it can also prevent egf - binding via sterical hindrance , but on a different epitope on domain iii , though partially overlapping . panitumumab is successfully applied in the treatment of metastatic colorectal cancer . for all above mentioned therapeutics , wild type ( wt ) kirsten rat sarcoma viral oncogene homolog ( kras)-status of the patient is of uttermost importance , as it could be demonstrated , that acquired kras mutations lead to resistance against egfr targeting . as kras is a downstream effector - protein in the egfr - signaling pathway , mutations that lead to constitutive activation of kras counteract growth signal inhibition of all egfr targeting drugs . therefore kras - status is meanwhile routinely determined for every human patient before any egfr - specific treatment is initiated . also in case of her-2 targeting , two monoclonal antibody therapies are fda - approved : trastuzumab ( herceptin , roche ) and pertuzumab ( perjeta , genentech ) . especially trastuzumab , being fda approved in september 1998 , proved to be highly successful for the treatment of metastatic breast cancer and has later received importance also for treatment of metastatic gastric cancer and tumors of the gastroesophageal junction ( gej , ) . trastuzumab has been so successful in breast cancer therapy , that very recently , in february 2013 , also a drug - conjugated trastuzumab derivate , trastuzumabemtansine ( t - dm1 , kadcyla , roche ) was approved by the fda for treatment of advanced breast cancer , fuelling the emerging field of antibody - drug conjugates . the other her-2 targeting antibody , pertuzumab received fda - approval in june 2012 and is also applied for treatment of metastatic breast cancer , as specified in table 1 . trastuzumab and pertuzumab target different epitopes on her-2 : trastuzumab binds to subdomain iv on the extracellular domain ( ecd ) of her-2 , whereas pertuzumab targets subdomain ii . as her-2 has no endogenous ligand , the mechanisms of action of trastuzumab and pertuzumab differ from those of the mentioned egfr - targeting immunoglobulins . trastuzumab sterically prevents the formation of her-2 homodimers or highly active heterodimers with other erbb - family members , mainly her-3 and egfr ( . moreover , upon trastuzumab binding , her-2 gets endocytosed and shedding of the receptor is inhibited , which otherwise would lead to an actively - signaling p95-remnant on the cancer cell surface . pertuzumab , on the other hand , binds more distant from the cell membrane , is more efficient in preventing heterodimer formation and in contrast to trastuzumab , which inhibits ligand - independent dimerization , pertuzumab especially inhibits ligand - induced her-2 heterodimers . this different mode of action prompted researchers to investigate a combination therapy of both antibodies in preclinical models , resulting in more complete her-2 blockade , and efficacy in cases where cancer had progressed after trastuzumab monotherapy . also in clinical settings , this combination therapy proved to be highly effective and peaked in june 2012 in the first fda approval of a dual her-2 targeting regimen for metastatic breast cancer . the mechanism of action of monoclonal antibodies , however , can not be confined to their growth signal inhibitory capacity only ; as all mentioned monoclonal antibodies feature fully functional human constant regions , thus they are also able to attract immune effector cells to the site of the tumor and trigger immune cell mediated cancer cell death . among the attracted immune cells are monocytes and macrophages dominant , which are known for their tumoricidic potential in mediating antibody - dependent cell - mediated cytotoxicity ( adcc ) via insertion of granzyme b and caspase enzymes . moreover , our group could demonstrate that monocytes are also able to mediate high levels of antibody - dependent cell - mediated phagocytosis ( adcp ) upon trastuzumab treatment . other attracted cell types expressing fc - receptors for antibody binding , are nk - cells and neutrophilic granulocytes , which have been shown to bear high tumoricidic potential with regard to adcc . moreover , professional antigen - presenting cells , that also express fcreceptors such as dendritic or langerhans cells can be attracted and can induce activation of tumor - reactive t - cells upon facilitated antigen uptake and presentation . this mechanism has been demonstrated to lead to tumor regression in a xenograft model of cetuximab treatment in concert with reconstituted immune cells . upon a closer look , all mentioned egfr or her-2 targeting antibodies belong to the igg class of immunoglobulins , with cetuximab , trastuzumab and pertuzumab being igg1 antibodies and panitumumab , being an igg2 . in fact , all currently fda - approved monoclonal antibodies for therapy of cancer are gamma - immunoglobulins . this is indeed astonishing as the human immunoglobulin repertoire consists of 5 different classes : iga , igd , ige , igg and igm . based on their different constant domains , all of them bear distinct physiological properties with respect to distribution , tissue penetration and function , such as complement - activation or adcc and adcp mediation : igm , the primary antibody response against pathogens is highly active in opsonization , which leads to pathogen clearance by phagocytic cells . iga protects body surfaces , such as the respiratory , gastrointestinal or genitourinary tract and is abundantly found in secrets like tear fluid or saliva , where it exerts neutralizing functions . the function of iga in mother s milk is of special interest as it protects the infant against pathogens in the mother s environment , which are also of high risk to the child . igg is the most abundant antibody isotype in the bloodstream , as it constitutes 70% of all serum immunoglobulins . igg antibodies , which can be further subdivided in igg1 - 4 in humans , exert systemic immune - protection by binding to bacteria , viruses and fungi with high affinity . in general , igg1 and igg3 antibodies are mainly produced in response to protein antigens , whereas igg2 and igg4 antibodies react against pathogens with polysaccharide capsules , like streptococcus pneumoniae . the biological role of igd , however , remains still enigmatic ; discovered late ( in 1965 ) in the serum of a myeloma patient , it was long neglected , because it is only cleaved in minor amounts . membrane - bound igd on the surface of b - cells though , was found to regulate b - cell activation . despite thorough investigation in recent years , the physiological and pathophysiological role of igd is still unclear ; however , an interesting aspect is , that igd was found to bind to certain bacterial proteins with relatively high affinity , but not via its antigen binding - site , as rather through sugar residues on its constant region . moreover , it has recently been shown , that circulating igd can activate antimicrobial , proinflammatory and b cell - stimulating effects in basophilic granulocytes . ige , finally , is the most recently discovered immunoglobulin subclass , as it was only first described in 1966 by teruko and kimishige ishizaka as a novel immunoglobulin in the serum of an atopic individual . at the same time , bennich and johannsson could purify a paraprotein from serum of a myeloma patient , which they termed ignd . they could soon link this novel ignd to asthma and it turned out to be the same protein as the group from japan had found . on a meeting in lausanne in february 1968 , finally , under the guidance of the who immunoglobuline reference laboratory , it was decided to designate this novel immunoglobuline ige . ige plays an important role in defense against parasites and is a key molecule in the pathophysiology of allergic diseases such as atopic dermatitis , asthma , food allergy or anaphylaxis . upon crosslinking of ige - antibodies , bound to fcri - receptors on the surface of mast cells or basophils , ige is mostly known for its detrimental role in allergy , but several studies have for long pointed towards a natural tumor surveillance function of this antibody isotype . interestingly , large epidemiologic studies could reveal an inverse association between the history of atopic diseases and cancer . in 2005 , turner et al . published a study enrolling 1.1 million us - american adults with self - reported , physician - diagnosed asthma or hay fever , who had no cancer at baseline and were followed up for 18 years . in this population , a relative - risk reduction for all cancer mortality could be observed [ relative risk ( rr ) = 0.88 ; 95% confidence interval ( ci ) 0.83 - 0.93 ] . however , in a separate analysis of never - smokers , this effect still persisted , but was not significant anymore . in a following literature analysis of studies from the medline database from 1966 to august 2005 , the same group described strong inverse associations for pancreatic cancer and glioma , whereas lung cancer was positively associated with asthma . however , methodical issues to these historical studies with regard to exposure assessment , confounding and bias were addressed by the authors . the most recent study investigating a possible association between ige and cancer was published in 2010 : van hemelrijck et al . reviewed 27 studies from pubmed and embase and surveyed a swedish cohort of 24.820 people , who underwent ige measurements . here , the authors could show a weak inverse association in their cohort , and a pattern by cancer type in the meta - analysis of the historical studies . another interesting observation was made when the anti - ige antibody omalizumab ( xolair , novartis ) underwent clinical trials and pooled phase i to iii data was evaluated . omalizumab , which removes ige from the circulation , is currently approved for therapy of severe persistent asthma . in those mentioned phase i to iii trials with allergics undergoing omalizumab treatment , a slightly higher number of malignant neoplasms was observed in the anti - ige - treated group ( 20 of 4127=0.5% compared to 5 of 2236=0.2% in the control group ) . malignancies that occurred in the treatment group comprised of breast , non - melanoma skin , prostate , melanoma and parotid cancer . subsequently , busse et al . analyzed 67 phase i to iv trials of omalizumab and could not confirm any possible association between omalizumab treatment and cancer in this extended study . summarizing all epidemiologic observations one can only state , that a possible association between ige and cancer remains still unclear due to the lack of big prospective studies . they could isolate ige antibodies which were not only specific for a 50 kda pancreatic cancer antigen but were indeed able to mediate adcc of pancreatic cancer cells in vitro , pointing towards a benefitial role of ige - antibodies in defense against cancer . pioneer studies with igg and ige antibodies of the same epitope specificity tested head - to - head revealed a higher potential of the ige in terms of cytotoxicity . the very first studies were performed with mov18igg and ige , antibodies that target the folate receptor ( fr)-. fr- ( also known as folate - binding protein , lk26 trophoblastic antigen or gp38 ) is a glycosylphosphatidylinositol ( gpi)-anchored membrane protein that binds folic acid and is regarded as a tumor - associated - antigen ( taa ) in gynecologic malignancies , due to its overexpression in more than 90% of epithelial ovarian cancers and in a subpopulation of uterine carcinomas . as folate is a necessary micronutrient of replicating cells , overexpression of fr- facilitates enhanced growth of cancer cells . for targeting of this receptor , gould et al could demonstrate in an ovarian cancer model , that mov18ige was able to mediate adcc of fr- expressing tumor cells in vitro and in vivo . in a mouse model using xenografted human fr- overexpressing cells , mice that received mov18ige treatment developed in the presence of human peripheral blood mononuclear cells ( pbmcs ) significantly smaller tumors than those treated with mov18igg . in a follow - up study , it could further be demonstrated , that also cytotoxic killing by monocytes can be efficiently triggered with ige . in a subsequent nude mouse study , where again fr- overexpressing tumors were grafted and pbmcs were reconstituted , the ige - treated group had shown monocytic infiltration of the tumor xenografts , which was still persistent after 3 weeks and led to significantly longer survival . moreover , upon a closer look in an in vitro flow cytometric model , specific adcc of tumor cells , executed by monocytes upon mov18ige stimulation could be displayed . finally phagocytosis of fr- positive tumor cells by monocytes armed with fr- specific ige could be displayed by fluorescence microscopy . subsequently , we could demonstrate similar results for the her-2 system in close collaboration with prof . karagiannis : upon generation of a recombinant trastuzumab - like ige , constituted of the same variable regions as original trastuzumab ( being an igg1 ) , it was shown in a flow cytometric assay , that the ige antibody is highly effective in mediating adcc of monocytes against her-2 overexpressing cells . interestingly , in this model , the ige antibody mediated high levels of adcc but only background adcp , whereas the picture was completely opposite for the igg , which mediated killing of tumor cells almost exclusively via adcp . this could be a first hint towards distinct mechanisms of tumor cell killing mediated by different immunoglobulin classes ; however , this still has to be confirmed in more extensive experiments and has to be investigated also for other cancer types . apart from a possibly higher potential for mediating adcc , ige - based immunotherapies of cancer could have other beneficial effects : first , ige antibodies have a uniquely high affinity to their receptors on immune cells ( ka~ 1010/m for fcri and ka~ 108 - 109/m for the cd23 trimer complex ) , which significantly exceeds the affinities of igg1 - 4 to their high - affinity receptor fcri . thus , due to its rapid binding to fc-receptors on cells , ige is quickly removed from the circulation , which is advantageous in terms of side - effects because of the short duration of the compound in the bloodstream . moreover , potential ige - immunotherapies would be effectively distributed to tumor tissues , as ige antibodies bound to fc-receptors on e.g. mast cells can use those cells as shuttle systems to penetrate malignancies and as mast cells are tissue - resident immune cells , this transport would be highly efficient . consistently , we would like to quote the review problems of delivery of monoclonal antibodies by reilly et al . , who wrote that the clinical success of monoclonal antibody - based cancer diagnosis and therapy depends , however , on solving a number of pharmacokinetic delivery problems . these include : ( i ) slow elimination of monoclonal antibodies from the blood and poor vascular permeability ; ( ii ) low and heterogeneous tumour uptake , and state that those two substantial challenges of anti - tumor immunotherapy could be simply addressed by using ige . other possible advantages include the high sensitivity of ige - effector cells to activation by antigens and the speed and amplitude of the response , which can most impressively be seen during allergic and anaphylactic reactions , typically beginning within minutes upon allergen exposure . that is at the same time also the biggest concern of using ige - based immunotherapies against cancer : recombinant ige , applied intravenously , always bears the risk of anaphylactic reactions ; therefore , careful selection of the target epitope is of uttermost importance in this regard . during an anaphylactic reaction , preformed ige , that is bound to fc-receptors on the surface of mast cells or basophilic granulocytes is cross - linked by allergens , which induces release of stored granules , containing vasoactive amines ( e.g. : histamine ) or lipid mediators ( e.g. prostaglandin d2 , platelet - activating factor , or leukotrienes ) . this rapid release can lead within minutes to fatal symptoms like asphyxiation from laryngeal swelling , circulatory collapse from hypotensive shock , cardiac arrest , or respiratory failure because of bronchoconstriction . in order to prevent such effects , the target structure for designing passive immunotherapies with ige - antibodies should not be expected to be cross - linking , which means , that the epitope should be monovalent andit should not circulate in the blood , or if , it should only circulate in a monomeric form . it should not circulate in the blood , or if , it should only circulate in a monomeric form . these requirements are fulfilled for the mentioned anti - fr- antibody mov18ige , but also for the anti - her-2 antibody trastuzumab - like ige . for both antibodies it could be demonstrated , that monomeric target molecules do not trigger mediator release of mast cells , which were preloaded with their specific ige - antibodies , respectively . furthermore , rudman et al . could demonstrate , that although serum levels of fr- were increased in ovarian cancer patients ( up to 40 ng / ml ) compared to healthy controls ( mean=1,73 ; sd=3,45 ) , basophilic granulocytes loaded with mov18ige were not significantly activated upon incubation with fr- even at a concentration of 300 ng / ml . on the other hand , both antibodies were shown to mediate mast cell degranulation upon incubation with tumor cells , displaying high numbers of target molecules in a repetitive manner on their surface . here , cross - linking of fc-receptors is highly efficient , and therefore local anaphylaxis at the tumor site could be expected , which would again be beneficial , as it results in initiation of a strong immune response . as mast cells also store tumorinhibiting agents in their granules , e.g. tumor necrosis factor ( tnf)- , this degranulation could also result in direct tumor cell killing . moreover , also other cells involved in anaphylactic reactions , such as eosinophils , have been shown to execute tumoricidic functions , e.g. via secretion of granzyme a or eosinophilic peroxidase . another big challenge of current immunotherapies with igg antibodies is that not all human fc-receptors are immune - activating , but one among them , fcriib is -inhibiting . therefore , the tumoricidic effects of igg - based immunotherapies also depend on the net ratio of binding to activating and inhibiting receptors . as it has recently been shown for igg4 , a subclass that shows relatively high binding affinity to fcriib , this antibody is not able to trigger immune cell - mediated tumor cell killing in vitro , despite being taa - specific . moreover it was demonstrated , that igg4 antibodies significantly impaired the killing potential of igg1 antibodies of the same specificity in vitro and in vivo . strategies to overcome this limitation include modification of the posttranslational glycosylation of the igg - constant regions heavy chains , as these sugar residues have been identified to be of high relevance for distinct binding affinities to different fc - receptors . for ige on the other hand , there are no inhibitory receptors , so again this isotype could contribute to overcome a current challenge of immunotherapies of cancer . however the fc-receptor - biology differs considerably between humans and mice , as the high affinity ige receptor fcri is only expressed on mouse basophils and mast cells , whereas it has been described in humans on mast cells , basophils , eosinophils , monocytes , langerhans and dendritic cells . this is a huge limitation of current mouse models in displaying all mentioned in vivo benefits and risks of ige - based immunotherapy of cancer and the great benefit of using a comparative oncology approach . although human and veterinary medicine share the same goals and aims , namely to treat patients and promote health , currently both of them are distinct sciences with distinct studies taught on separate universities . research is presently going on in one or the other , but there is little crosstalk between the two specialties . the concept of comparative medicine , however , aims to study naturally occurring diseases across species to improve both human and veterinary medicine . there is in fact no explicit reason for a strict separation of studies for humans or other mammals , as many pathophysiological processes have been shown to be similar and highly comparable , or as the german pathologist rudolf virchow stated : between animal and human medicine the object is different but the experience obtained constitutes the basis of all medicine . this view , which is also in line with the wider concepts of one medicine , or one health , bringing together aspects of health of humans , animals and also their environment is not really novel . browsing through the history of medicine , this approach appears at many points , starting with hippocrates and plato in ancient greece . even further back in time , physicians in ancient india were trained to treat humans and animals , especially cattle , elephants and horses and in ancient china , medical treatment for horses was highly elaborated as well . also in europe and north america human and veterinary medicine were closely interconnected for a long period of time , peaking in the 18th , 19th and early 20th century . many important findings were made by comparative observation and experimentation during that time , for instance edward jenner ( 1749 - 1823 ) noted that milkers who had been in contact with cowpox - infected cows did not develop smallpox ( variola minor ) but only the milder cowpox . he also observed that this protection could be mediated by inoculation of a small amount of pus from cowpox blisters , a method he called vaccination because of the latin word for cow , vacca . similarly , louis pasteur ( 1822 - 1895 ) worked on cholera in humans and chicken , robert koch ( 1843 - 1910 ) researched on human and bovine tuberculosis , and the mentioned rudolf virchow ( 1821 - 1902 ) worked on trichinella infections in humans and pigs . besides virchow , the most important proponent of comparative medicine was sir william osler , a canadian physician ( 1849 - 1919 ) , who studied , worked and taught in london , berlin , vienna , toronto and montreal , and was one of the four founding professors of johns hopkins hospital in baltimore . osler lectured at mcgill university for medical students as well as for students from the veterinary medical college with emphasis on comparative topics . his research in this field concentrated on infectious diseases in dogs , pigs and cattle and his deep interest is documented in many editorials of the journal of comparative medicine and surgery . recent developments are no longer dependent on distinguished individuals , but comprise the establishment of special departments for comparative medicine within universities , such as at stanford , yale or at the university of california , davis . whereas comparative medicine focused mainly on infectious diseases in previous centuries , modern approaches tend to tackle another big burden of our societies , cancer , via the principle of comparative oncology . the comparative oncology approach aims to speed up drug development simultaneously for human and animal cancer patients via clinical trials in pet patients , primarily cats and dogs . although murine models have been proven to be highly effective with regards for understanding basic principles of malignant transformation , cancer signal transduction pathways or drug resistance formation , these models often poorly mimic human cancer for drug testing . for many compounds , the translation of a safe and efficacious agent in mice into an actual drug fails , due to the poor presentation of key features of human cancer in murine tumor models , such as genomic instability , long latency periods or the lack of intra - tumor heterogeneity . moreover , the concept of toxicity studies , which are conducted in healthy animals , followed immediately by phase i and phase ii trials in humans , often leaves many important questions unanswered , before treating a relatively high number of human patients . on the other hand , also more and more extensive studies in animals can not be the solution , as it would increase the ethical dilemma that potential benefits for humans stand against the costs sustained by animals . thus , legislators and regulatory bodies state in their directives on drug development that the 3rs should be applied on animal experiments , which are : replacement , reduction , and refinement [ 143,144 ] . in line with these concepts , clinical studies in animal patients , which suffer from spontaneously developed tumors , would allow the investigation of drug effects on malignancies that developed naturally within intact immune systems , in the context of their original tumor microenvironment in pet animals that share similar environmental factors as their owners , for instance pollution . such trials in veterinary patients could replace many preclinical experiments , refine our models and ultimately reduce animal experiments . the information obtained from these studies would be highly relevant and valuable , while the treated veterinary patients would be provided with cutting edge research simultaneously . studies in this field of comparative oncology with treated dog patients , led to the advancement of surgical techniques , like limb sparing for sarcoma patients , elucidating hyperthermia or evaluating novel delivery strategies , like inhalation of cytokines or chemotherapies . ( cotc ) , a multicenter initiative of twenty comparative oncology centers throughout the usa . founded and centrally managed by the national cancer institute of the national institutes of health ( nih ) , 11 clinical trials have been conducted so far , ranging from all fields of cancer therapy attempts like evaluation of rgd targeted delivery of phage expressing tnf - alpha to tumor bearing dogs ( cotc001 , closed trial ) via preclinical comparison of three indenoisoquinolines candidates in tumor bearing dogs ( cotc007b , open trial ) to evaluation of the mtor inhibitor rapamycin in dogs with metastatic osteosarcoma one of the most successful example of a recent clinical comparative oncology trial was published in 2009 , in which the tyrosine kinase inhibitor toceranib phosphate ( palladia , su11654 , pfizer ) , targeting kit , vascular endothelial growth factor receptor 2 ( vegfr2 ) and platelet derived growth factor receptor - beta ( pdgfr ) was tested in dog cancer patients with recurrent mast cell tumors . this large clinical phase iii trial could demonstrate significant effects of toceranib phosphate with regard to overall response rates , median duration of objective responses and time to tumor progression , which has finally led to the approval of toceranib phosphate for mast cell tumors in dogs and to the approval of sunitinib ( sutent , su11248 , pfizer ) , a similar compound , for therapy of human renal cell cancer and gastrointestinal stromal tumors ( gist , ) . also for evaluation of ige - based immunotherapies of cancer , trials in dog cancer patients would be highly valuable , due to the fact that dogs , in contrast to mice , share important principles of the ige - biology with humans , underlined by the clinical observation that dogs also suffer from ige - mediated diseases , such as atopic dermatitis or food allergies . thus , the introduction of passive immunotherapy in veterinary clinical oncology would be highly valuable to elucidate the full potential of ige - based immunotherapy of cancer . we contributed to this strategy by our recent molecular characterization of canine egfr and the generation of a recombinant canine ige of the exact cetuximab specificity [ 155 , 156 ] .
antibody - based immunotherapies are important therapy options in human oncology . although human humoral specific immunity is constituted of five different immunoglobulin classes , currently only igg - based immunotherapies have proceeded to clinical application.this review , however , discusses the benefits and difficulties of ige - based immunotherapy of cancer , with special emphasis on how to translate promising preclinical results into clinical studies . pursuing the comparative oncology approach , novel drug candidates are investigated in clinical trials with veterinary cancer patients , most often dogs . by this strategy drug development could be speeded up , animal experiments could be reduced and novel therapy options could be introduced benefitting humans as well as man s best friend .
Targeted Therapies of Cancer Immunological Effects of Antibodies IgE and Cancer IgE-based Immunotherapies of Cancer - Pioneer Studies Possible Advantages and Pitfalls of IgE-based Cancer Immunotherapies Comparative Medicine Comparative Oncology
the mechanism of action of monoclonal antibodies , however , can not be confined to their growth signal inhibitory capacity only ; as all mentioned monoclonal antibodies feature fully functional human constant regions , thus they are also able to attract immune effector cells to the site of the tumor and trigger immune cell mediated cancer cell death . upon crosslinking of ige - antibodies , bound to fcri - receptors on the surface of mast cells or basophils , ige is mostly known for its detrimental role in allergy , but several studies have for long pointed towards a natural tumor surveillance function of this antibody isotype . in those mentioned phase i to iii trials with allergics undergoing omalizumab treatment , a slightly higher number of malignant neoplasms was observed in the anti - ige - treated group ( 20 of 4127=0.5% compared to 5 of 2236=0.2% in the control group ) . this could be a first hint towards distinct mechanisms of tumor cell killing mediated by different immunoglobulin classes ; however , this still has to be confirmed in more extensive experiments and has to be investigated also for other cancer types . apart from a possibly higher potential for mediating adcc , ige - based immunotherapies of cancer could have other beneficial effects : first , ige antibodies have a uniquely high affinity to their receptors on immune cells ( ka~ 1010/m for fcri and ka~ 108 - 109/m for the cd23 trimer complex ) , which significantly exceeds the affinities of igg1 - 4 to their high - affinity receptor fcri . , who wrote that the clinical success of monoclonal antibody - based cancer diagnosis and therapy depends , however , on solving a number of pharmacokinetic delivery problems . that is at the same time also the biggest concern of using ige - based immunotherapies against cancer : recombinant ige , applied intravenously , always bears the risk of anaphylactic reactions ; therefore , careful selection of the target epitope is of uttermost importance in this regard . therefore , the tumoricidic effects of igg - based immunotherapies also depend on the net ratio of binding to activating and inhibiting receptors . this is a huge limitation of current mouse models in displaying all mentioned in vivo benefits and risks of ige - based immunotherapy of cancer and the great benefit of using a comparative oncology approach . osler lectured at mcgill university for medical students as well as for students from the veterinary medical college with emphasis on comparative topics . whereas comparative medicine focused mainly on infectious diseases in previous centuries , modern approaches tend to tackle another big burden of our societies , cancer , via the principle of comparative oncology . the comparative oncology approach aims to speed up drug development simultaneously for human and animal cancer patients via clinical trials in pet patients , primarily cats and dogs . thus , legislators and regulatory bodies state in their directives on drug development that the 3rs should be applied on animal experiments , which are : replacement , reduction , and refinement [ 143,144 ] . in line with these concepts , clinical studies in animal patients , which suffer from spontaneously developed tumors , would allow the investigation of drug effects on malignancies that developed naturally within intact immune systems , in the context of their original tumor microenvironment in pet animals that share similar environmental factors as their owners , for instance pollution . founded and centrally managed by the national cancer institute of the national institutes of health ( nih ) , 11 clinical trials have been conducted so far , ranging from all fields of cancer therapy attempts like evaluation of rgd targeted delivery of phage expressing tnf - alpha to tumor bearing dogs ( cotc001 , closed trial ) via preclinical comparison of three indenoisoquinolines candidates in tumor bearing dogs ( cotc007b , open trial ) to evaluation of the mtor inhibitor rapamycin in dogs with metastatic osteosarcoma one of the most successful example of a recent clinical comparative oncology trial was published in 2009 , in which the tyrosine kinase inhibitor toceranib phosphate ( palladia , su11654 , pfizer ) , targeting kit , vascular endothelial growth factor receptor 2 ( vegfr2 ) and platelet derived growth factor receptor - beta ( pdgfr ) was tested in dog cancer patients with recurrent mast cell tumors . also for evaluation of ige - based immunotherapies of cancer , trials in dog cancer patients would be highly valuable , due to the fact that dogs , in contrast to mice , share important principles of the ige - biology with humans , underlined by the clinical observation that dogs also suffer from ige - mediated diseases , such as atopic dermatitis or food allergies . thus , the introduction of passive immunotherapy in veterinary clinical oncology would be highly valuable to elucidate the full potential of ige - based immunotherapy of cancer .
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increases in the concentration of intracellular ca ( [ ca]i ) control diverse functions in virtually all cell types . these ca signals differ spatially , temporally and in magnitude and are mediated by numerous channels , transporters and ca - sensing proteins . opening of ca channels in either the plasma membrane or the membrane of intracellular ca stores directly elevates [ ca]i . ca entry from extracellular space and ca release from endoplasmic reticulum ( er ) operate independently or together to regulate different ca - dependent processes , such as exocytosis of neurotransmitters , contraction of skeletal muscle , activation of t lymphocytes or contraction of smooth muscle cells . signaling via many cell surface receptors involves the activation of ca release from er via inositol-1,4,5-triphosphate ( ip3 ) . because a second phase of ip3-mediated ca mobilization was frequently observed and identified as ca influx from extracellular space , the hypothesis of capacitative ca entry was formulated by putney in 1986 . he proposed a receptor - regulated pathway for ca that enters the depleted ca pool directly via plasma and er membranes and is controlled by ip3 and the ca content of the er . the capacitative or store - operated ca entry ( soce ) , has been recognized as a an essential route for ca uptake in a wide variety of cell types to replenish intracellular ca stores and to regulate secretion , gene transcription and cell cycle progression . several candidate molecules and ion channels mediating soce were discussed in recent years . in particular , some members of the transient receptor potential ( trp ) cation channel family were found to generate receptor - activated ca entry . however , electrophysiological characterization of soce in mast cells revealed a ca - release activated ca ( crac ) current , whose properties can be clearly distinguished from those of trp channels . in 2005 and 2006 , different groups identified stromal interaction molecules ( stim ) and orai proteins ( named after the keepers of heaven 's gate in greek mythology ) as essential components of soce . stromal interaction molecule 1 ( stim1 ) was originally identified as a potential tumor suppressor gene coding for a transmembrane protein . in vertebrates , one additional stim1-related gene , stim2 , both stim homologues are ubiquitously expressed in different cell types with higher stim1 levels in most tissues but a predominant expression of stim2 in brain . stim1 and stim2 each contain highly conserved domains , including a single transmembrane segment , an ef - hand ca - binding domain , a sterile -motif ( sam ) domain and coiled - coil regions ( for review see refs . stim1 and stim2 are primarily located to the er ; stim1 was also detected in the plasma membrane . the ef - hand ca - binding domains ( together with the sam domains ) of stims are responsible for sensing [ ca ] in the lumen of the er . a decrease in er luminal ca concentration results in dissociation of ca from the ef - hand domain which , in turn , triggers oligomerization and activation of stim1 , a process that is reversed when luminal [ ca ] returns to resting level ( that is , 250600 m ) . the luminal ef - hand domain of stim1 binds ca with an apparent dissociation constant ( kd ) of 250 m , whereas the kd of stim2 for ca is 500 m . hence , stim2 is more sensitive to small changes in luminal [ ca ] and partially active at resting er ca levels . stim2 shows a slower oligomerization kinetics and is less effective than stim1 in orai1 binding and activation , which might be critical for limiting excessive soce . orai1 and its homologues orai2 and orai3 were discovered by genome wide rnai screens for soce inhibition and by positional cloning in patients with immune deficiency and crac channel dysfunction . co - expression with stim1 showed that orai1 is the pore - forming subunit of the crac channel . orai1 channels ( therefore , also named crac modulator 1 ; cracm1 ) are activated upon ca store depletion , translocation of activated stim1 to er - plasma membrane junctions and interaction of cytosolic stim1 domains with the c - terminal domains of orai1 tetramers . heterologously expressed orai2 and orai3 channels ( together with stim1 ) conduct crac - like currents but exhibit distinct inactivation and permeability properties . store - depletion signaling via stim2 also activates all 3 orai channels . over - expression of orai1 or orai2 ( but not orai3 ) alone although trp channels do not account for crac currents , a contribution of some trp family members to stim1-induced and store - dependent ca entry has been shown . in this context , a soce - signaling complex consisting of trpc1 , orai1 , and stim1 has been proposed . the principal components stim1 and orai1 essentially or nearly exclusively contribute to soce in different cell types . the function of the innate and adaptive immune system requires stim1/orai1-dependent soce . in mouse t lymphocytes , orai2 and/or orai3 appear to have redundant functions in mouse t cells , whereas human t cells lacking functional orai1 failed to proliferate in vitro and to produce cytokines . stim1 and orai1 regulate mast cell activation , and stim1 is essential for fc receptor - dependent ca signaling and phagocytosis in macrophages and neutrophils . roles of both stim1 and orai1 in other cell types include growth and contractility of skeletal muscle , proliferation of vascular smooth muscle cells , and aggregation of platelets . soce in the nervous system has been reviewed with respect to ca signaling and homeostasis in neurons and neuroglia . the present review is mostly based on studies regarding expression analyses or using gene silencing or knockout of stims and orais and gives a current overview on the putative functions of these soce components in both neurons and glial cells , i.e. , astrocytes and microglia . stim and orai isoforms are broadly expressed in murine and human tissues . at the rna level , stim1 was consistently detected in skeletal muscle and brain , whereas stim2 was preferentially found in brain and heart from both species . both stim proteins were detected in murine and human brain . within the murine brain , stim1 and stim2 are distributed in the cerebral cortex and can be assigned to hippocampal and cerebellar structures . whereas stim1 is most prominent in the cerebellum , stim2 dominates in hippocampus and cortex . in human brain , stim1 protein expression is high in cerebellum , medium in cerebral cortex , and low in hippocampus . stim2 protein levels are high in hippocampus and cerebral cortex , and medium in cerebellum , indicating that the differential distribution of stim1 and stim2 in cerebrum and cerebellum is similar in human and murine brain . expression of stim mrnas at the cellular level was analyzed using cell isolation or separation by laser capture , cell soma harvest and cell culture . in primary hippocampal and cortical neurons , stim2 is the predominant stim isoform . in hippocampal cultures , stim2 was detected in both neuronal soma and dendrites , whereas stim1 protein is restricted to the soma . furthermore , the expression of stim1 and stim2 increases during development of hippocampal neurons in vitro . in purkinje neurons , the principal neurons of the cerebellar cortex , stim1 levels were higher than those of stim2 . the second most important neuron within the cerebellum is the cerebellar granule cell , which is located in the nuclear layer and whose parallel fibers project to purkinje neurons in the upper molecular layer . interestingly , orai1 mrna levels in both species appeared to be lower than those of orai3 . a high abundance of orai2 was found in hippocampal neurons , cerebellar tissue and purkinje neurons . the origin of the strong orai3 expression , particularly in human brain , is presently unclear and might come from glial cells . in mouse cerebellum , the role of orai2 in brain as well as in other tissues is still unclear . store - operated ca entry has been implicated in neuronal ca signaling even before the discovery of stim and orai . ca imaging experiments revealed soce in cultured and freshly dissociated cortical and hippocampal neurons . a role for soce in spontaneous synaptic activity and in synaptic plasticity of hippocampal neurons was suggested from studies using the common soce inhibitors 2-aminoethoxydiphenyl borate ( 2-apb ) , skf96365 and la . first molecular evidence for neuronal soce provided a study using stim and orai knockout mice . have shown that stim2-deficient mice were protected from cerebral damage after ischemic stroke . the reduced infarct size and the improved neurological outcome of stim2 animals were independent of functional changes within the haematopoietic system . in cultured cortical neurons , soce was significantly decreased in the absence of stim2 but not of stim1 or orai1 . hypoxia and hypoglycemia induced an increase in [ ca]i which was markedly reduced in stim2 neurons . hypoxia / hypoglycemia inhibits atp - dependent ca transport into the er and might , therefore , trigger persistent stim2 activity and soce - induced accumulation of cytosolic ca ( fig . glutamate - induced excitotoxicity is a process triggered by and contributing to neuronal ca accumulation during ischemia . cholesterol , a major component of the plasma membrane , plays a key role in regulating neuronal functions . glutamate - induced cholesterol loss required high intracellular [ ca ] and functional stim2 in hippocampal neurons . apart from the role of stim2 in ischemic stroke , behavioral tests revealed an impairment of hippocampus - dependent spatial learning in stim2-deficient mice . ( a ) proposed model for the involvement of stim2 in ischemic ca accumulation in hippocampal and cortical neurons . a disturbed refilling of intracellular ca stores during ischemia may be induced by inhibition of sarco - endoplasmic reticulum ca pumps . the reduced [ ca ] in the endoplasmic reticulum ( er ) , [ ca]er , leads to the activation of stim2 and , possibly , of orai2 channels in the plasma membrane . opening of orai channels results in soce , which contributes to deleterious ca accumulation in the cytosol . ( b ) role of stim2 in maintenance of postsynaptic mushroom spines in hippocampal neurons . activation of stim2 due to reduced [ ca]er induces continuous soce via orai ( supposedly , orai2 ) channels . increased cytosolic [ ca ] supports constant levels of ca / calmodulin - dependent protein kinase ii ( camkii ) and long - term stability of mushroom spines . activation of metabotropic glutamate receptor type 1 ( mglur1 ) induces ca release from er , a decrease in [ ca]er , and the activation of stim1 . soce results in ca store refilling and supports ca - dependent activation of the transient receptor potential channel trpc3 . trpc3 mediates slow excitatory postsynaptic currents ( epsc ) which are important for purkinje neuron function and cerebellar motor behavior . ( a ) proposed model for the involvement of stim2 in ischemic ca accumulation in hippocampal and cortical neurons . a disturbed refilling of intracellular ca stores during ischemia may be induced by inhibition of sarco - endoplasmic reticulum ca pumps . the reduced [ ca ] in the endoplasmic reticulum ( er ) , [ ca]er , leads to the activation of stim2 and , possibly , of orai2 channels in the plasma membrane . opening of orai channels results in soce , which contributes to deleterious ca accumulation in the cytosol . ( b ) role of stim2 in maintenance of postsynaptic mushroom spines in hippocampal neurons . activation of stim2 due to reduced [ ca]er induces continuous soce via orai ( supposedly , orai2 ) channels . increased cytosolic [ ca ] supports constant levels of ca / calmodulin - dependent protein kinase ii ( camkii ) and long - term stability of mushroom spines . activation of metabotropic glutamate receptor type 1 ( mglur1 ) induces ca release from er , a decrease in [ ca]er , and the activation of stim1 . soce results in ca store refilling and supports ca - dependent activation of the transient receptor potential channel trpc3 . trpc3 mediates slow excitatory postsynaptic currents ( epsc ) which are important for purkinje neuron function and cerebellar motor behavior . whereas no obvious abnormalities were reported for brain structures of stim2 mice , sun et al . showed that conditional knockout of the stim2 gene in the hippocampus of 26 month old mice induced a massive neuronal loss in this brain region . deletion of stim2 in hippocampal neurons caused a moderate reduction of somatic soce but a dramatic decrease of soce in dendritic spines . the absence of stim2 also reduced the number of spines and changed their morphology by reducing the fraction of mushroom spines which play an important role in the storage of memories . a loss of mushroom spines , concurrent with the decrease in synaptic soce and the downregulation of stim2 was observed in hippocampal neurons from the presenilin-1 m146v knockin mouse model of alzheimer 's disease . overexpression of stim2 ( but not of stim1 ) rescued synaptic soce and increased the expression of phosphorylated ca / calmodulin - dependent protein kinase ii ( camkii ) suggesting that stim2-dependent soce is required for camkii activity and stabilization of mushroom spines in healthy neurons ( fig . it is not clear why stim2 but not stim1 regulates synaptic soce in hippocampal neurons . stim2 has a higher kd for ca than stim1 and induces soce near resting er ca levels . thus , stim2 stabilizes basal [ ca]i even at incomplete store depletion and , in turn , maintains steady - state camkii activity and integrity of mushroom spines . another recent study supports the role of stim2 in regulating dendritic spine density and morphology in hippocampal neurons . showed that stim2 preferentially localizes to large dendritic spines and is enriched in the postsynaptic density . stim2 is required for regular synaptic activity and mediates camp - dependent phosphorylation and trafficking of the ampa receptor subunit glua1 to plasma membrane - er junctions . analogous to stim interaction with orai1 , the authors suggest that stim2 binds via its cytosolic domain to glua1 and couples glua1 to camp - dependent protein kinase ( pka ) . signaling of stim proteins through other pathways than soce has also been shown for the interaction of stim1 with the voltage - gated ca channel ( vgcc ) subtype cav1.2 . stim1 inhibits vgcc activation by binding to cav1.2 via the cytosolic stim - orai activating domain . it has been suggested that stim1-mediated suppression of vgccs is involved in the differentiation of neurons from embryonic stem cells . knockdown of stim1 or stim2 reduced soce and inhibited entry of mouse embryonic stem cells into neural lineage . stim1 knockdown induced an increase in voltage - dependent ca entry and treatment of cells with the vgcc blocker nifedipine facilitated neural differentiation . found a markedly reduced soce after knockdown / knockout of stim1 or orai1 in embryonic and neonatal neural precursor cells . suppression of stim1 or orai1 diminished proliferation of neural precursors and inhibited activation of the transcription factor nfat ( nuclear factor of activated t cells ) . whereas stim2 regulates soce in hippocampal and cortical neurons , stim1 appears to be the primary stim isoform in cerebellar granule cells and purkinje neurons . lalonde et al . have shown that changes in the extracellular k concentration rapidly induce redistribution of overexpressed stim1 together with overexpressed orai1 and orai2 in cerebellar granule neurons . because a switch of extracellular [ k ] from a high ( 2565 mm ) to a low level ( 5 mm ; near resting extracellular [ k ] ) induces plasma membrane hyperpolarization ( repolarization ) , the authors suggest that changes in the membrane potential induce soce in granule neurons . furthermore , ryanodine receptor - dependent ca stores were depleted by k - induced hyperpolarization and a sustained soce signal was observed at resting extracellular [ k ] . knockdown of stim1 in a neuroblastoma cell line and pharmacological inhibition of soce in granule neurons inhibited hyperpolarization - induced degradation of the neuron - specific transcription factor sp4 . sp4 has been implicated in dendrite patterning in cerebellar and hippocampal neurons as well as in memory and synaptic plasticity . the activation of soce at resting ( hyperpolarized ) membrane potential suggests a homeostatic function of stims in neurons , i.e. the regulation of ca - dependent gene transcription by controlling resting intracellular ca levels . reported that stim1 controls glutamate receptor - dependent synaptic transmission in purkinje neurons and motor learning in mice . the metabotropic glutamate receptor type 1 ( mglur1 ) , that is highly expressed in purkinje neurons , plays an important role in cerebellar functions like motor coordination . signal transduction downstream of mglur1 involves the ip3-dependent ca release from er and the activation of a slow excitatory postsynaptic current which is mediated by the trp channel trpc3 . in the absence of stim1 , the ip3-dependent ca release from dendritic er ca stores and the trpc3-mediated currents were abolished . interestingly , stim1 was only required for ca store refilling and slow synaptic currents when purkinje neurons were held at resting membrane potential . depolarization - induced ca entry via vgccs , however , induced a stim1-independent recovery of er ca release and trpc3 activity . the latter observation leads to the conclusion that activation of trpc3 channels depends on intracellular [ ca ] but not on interaction with stim1 or the mglur1-induced activation of phospholipase c ( fig . . the essential role of stim1 in resting purkinje neurons suggests that neuronal soce replenishes intracellular and er ca levels when vgcc activity is low . astrocytes are glial cells which are derived , like neurons and oligodendrocytes , from neuroepithelial progenitors . they perform a variety of homeostatic functions within the nervous system and communicate with neighboring glial cells and neurons by generating ( intercellular ) ca signals and by releasing and binding of transmitter molecules . cultured astrocytes and tumor cells of astroglial origin , i.e. , glioblastoma cells , exhibit soce in response to metabotropic receptor agonists like atp , histamine , and glutamate . stim1 and stim2 proteins are expressed in cultured astrocytes while stim1 appeared to be the dominant isoform . reported that knockdown of stim1 and orai1 or of orai1 alone diminishes thrombin - induced ca signals and soce in cultured rat astrocytes . . showed a reduction of soce and of crac currents by silencing of stim1 or orai1 in human glioblastoma cells . knockdown of stim1 and orai1 slightly affected proliferation but clearly inhibited invasive glioblastoma cell migration . surprisingly , the authors were unable to detect orai1 but found a dominant expression of orai3 which was 6-fold more abundant than orai2 . furthermore , previous studies suggest a role of trp channels in astrocytic soce and , therefore , challenge a sole contribution of orai channels . golovina reported that antisense oligonucleotides targeted to the trp channel gene trpc1 inhibited soce in murine astrocytes . however , in a later study the same group showed a profound inhibition of astrocytic soce by knockdown of orai1 . reported that an antibody against trpc1 reduces soce and abolishes atp - mediated ca entry in cultured rat astrocytes . astrocytes also express further trp channel subtypes , including trpc4 and trpc5 , which can form heteromeric channels with trpc1 . a concordant regulation of trpc1 , trpc4 , orai3 , and of receptor - induced ca entry has been observed in astrocytes treated with different pro - inflammatory agents and amyloid- protein . thrombin treatment of astrocytes induced the up - regulation of a further trp homolog , trpc3 , and an increase in soce . in contrast to crac channels , which are highly selective for ca , trpc3 is permeable for some other divalent cations , including sr . have shown that store depletion induces only a negligible sr entry in astrocytes suggesting a minor role of trpc3 in astrocytic soce . microglia are derived from myeloid precursors and constitute up to 20% of the total glia population . microglia sense disturbances or loss of brain homeostasis and undergo morphological and functional changes in response to brain injury and infection . this microglial activation includes shape changes toward an ameboid appearance , directed movement , proliferation , phagocytosis , and release of cytokines . microglia are equipped with a variety of surface receptors for neurotransmitters , signaling molecules and pathogens . extracellular nucleotides , i.e. atp , adp and udp , accumulate during brain injury , activate purinergic p2y receptors and stimulate migration and phagocytosis in microglia . showed that crac / orai channels rather than trp channels mediate soce in cultured rat microglia . soce was inhibited by a high concentration of 2-apb ( 50 m ) and by exchange of extracellular ca by sr or ba . from the high expression of orai1 and orai3 in microglia and the agonistic action of 50 m 2-apb on orai3 , the authors suggested a mayor role of orai1 in microglial soce . the same group detected high levels of orai1 and stim1 in microglial podosomes , actin - rich structures involved in cell motility and invasion . treatment of cultured rat microglia with soce inhibitors strongly reduced migration , i.e. , transmigration across filters with defined pores , and invasion , i.e. transmigration through pores coated with extracellular matrix molecules . confirmed the expression of all orai isoforms and of stim1 in purified cultured mouse microglia and showed that downregulation of orai1 and stim1 reduces soce and udp - induced ca signals . silencing of stim1 inhibited lipopolysaccharide - induced activation of nfat1 and production of interleukin 6 ( il-6 ) but did not affect activation of nf-b . stim1 knockdown slightly decreased release of tnf- and reduced the udp - stimulated phagocytosis of bacterial particles . the p2y6 receptor agonist udp was previously shown to induce activation of nfat1 and nfat2 as well as expression of the chemokines ccl2 and ccl3 in microglia . we investigated the role of stim1 , stim2 , and orai1 in microglia . to estimate the relative contribution of stim1 and stim2 to microglial responses and to evaluate the role of orai1 purity of microglial cultures was tested by staining with isolectin b4 and by using transgenic cx3cr1 mice , where gfp is exclusively expressed in microglia . in our primary cultures , which showed a purity of 99.5% , we analyzed the mrna levels of stim and orai isoforms by quantitative rt - pcr . calcium imaging revealed a graded suppression of soce in the absence of stim1 , stim2 , or orai1 . soce evoked by blockage of sarco - endoplasmic reticulum atpase ( serca ) was nearly absent in stim1 microglia ( 91% inhibition ) , whereas stim2 cells showed a less pronounced but significant inhibition ( by 30% ) . purinergic stimulation with the p2y6 receptor agonist udp or the p2y12 receptor agonist 2-methylthio - adp ( 2-mesadp ) caused soce amplitudes which were significantly smaller than those evoked by serca inhibition . the p2y12 receptor - mediated soce was blocked by 40% in stim2 microglia and reduced by 77% in stim1 cells , suggesting a decreased effect of stim1 and an increased impact of stim2 on soce of lower magnitude . a role for stim2 in modulating the threshold for ca entry showed that stim2 exclusively contributed to ca signals evoked by tyrosine kinase receptors but not by g - protein coupled receptors in leukemia cells . found a stim2-dependent soce signal after mild store depletion which was completely replaced by a stim1-dependent soce upon strong store depletion . ong et al . proposed that stim2 triggers soce by recruiting stim1 at low stimulus intensities when er ca stores are mildly depleted . together , these data suggest that stim2 increases the sensitivity of intracellular ca signals to extracellular agonists . to evaluate the role of soce in microglial migration and phagocytosis we used stim and orai knockout mice , the soce blockers 2-apb , la , and the trp channel blocker n-(p - amylcinnamoyl)anthranilic acid ( aca ) . we found that aca effectively blocks soce in microglia with an ic50 of 0.4 m . transmigration of cultured microglia stimulated with atp was largely reduced by aca and soce inhibitors . uptake of zymosan particles by udp - stimulated microglia was nearly abolished in the presence of 2-apb . p2y receptor - induced migration ( induced by 2-mesadp , atp , and udp ) was inhibited in stim1 , stim2 , and orai1 microglia . p2y6 receptor - dependent phagocytosis was nearly abolished in stim1 cells and largely decreased in stim2 and orai1 microglia . basal migration and phagocytosis in the absence of purinergic stimuli were not affected by stim or orai knockout . from these data we concluded that stim1 and orai1 are the mayor constituents of microglial soce and that stim2 is an important component of this signaling pathway . consequently , soce is essential for p2y receptor - dependent ca entry , migration , and phagocytosis in microglia ( fig . 2 ) . possible downstream effectors of microglial soce involve protein kinase c ( pkc ) and the ca / calmodulin - activated myosin light chain kinase ( mlck ) which both activate phagocytosis in microglia . the ca - dependent phosphorylation of protein kinase b ( akt ) is required for adp - induced chemotaxis of microglia . the g protein - coupled receptors p2y6 and p2y12 are activated by udp and adp / atp , respectively . activation of p2y receptors induces ca release from endoplasmic reticulum ( er ) and reduces [ ca]er . in turn , the resulting soce promotes activation of different ca - dependent signaling molecules , including ca / calmodulin - activated myosin light chain kinase ( mlck ) , protein kinase c ( pkc ) , the serin / threonine specific kinase akt , and the transcription factor nfat . remodeling of actin - myosin skeleton is probably involved in mlck / pkc - dependent phagocytosis and in akt - dependent cell migration . the g protein - coupled receptors p2y6 and p2y12 are activated by udp and adp / atp , respectively . activation of p2y receptors induces ca release from endoplasmic reticulum ( er ) and reduces [ ca]er . in turn , the resulting soce promotes activation of different ca - dependent signaling molecules , including ca / calmodulin - activated myosin light chain kinase ( mlck ) , protein kinase c ( pkc ) , the serin / threonine specific kinase akt , and the transcription factor nfat . remodeling of actin - myosin skeleton is probably involved in mlck / pkc - dependent phagocytosis and in akt - dependent cell migration . stim and orai proteins have been implicated in normal brain function but also in neurological disorders . stim1 , preferentially expressed in cerebellar neurons , and stim2 , more abundant in hippocampal and cortical neurons , trigger neuronal soce . the versatile role of stims and , consequently , of soce in neuronal signaling was rather unanticipated because synaptic activity was thought to provide a sufficient supply for intracellular ca . however , recent studies suggest that soce is a mayor source for intracellular ca in resting neurons . stim1 is required for slow glutamate receptor signals in purkinje neurons and for ca - dependent gene transcription in cerebellar granule neurons . stim2 controls steady - state activity of camkii and thereby stabilizes mushroom spines in hippocampal neurons . decreased stim2 activity and loss of mushroom spines may be responsible for memory loss in aging neurons and in alzheimer 's disease . stim2 is also a potential target for the treatment of glutamate - induced excitotoxicity during epilepsy , brain trauma and cerebral infarction . the role of orai proteins in neuronal ca signaling is still unresolved . in particular , consequences of the dominant orai2 expression , orai2-mediated currents show a smaller amplitude but undergo a decreased inactivation at higher [ ca]i . in analogy to functional differences between stim1 and stim2 , orai2 might , therefore , support a persistent and moderate less is known about the role of stims and orais in astrocytes . here , a further orai isoform , orai3 , might play a central role . orai3 is , in contrast to orai1 and orai2 , inhibited by reactive oxygen species ( ros ) , and is required for store - independent crac channels activated by arachidonic acid , a phospholipase a2 ( pla2 ) metabolite . both , pla2 activity and ros production were implicated in neurodegenerative diseases , such as cerebral ischemia and alzheimer 's disease , while astrocytes play diverse supportive roles in maintaining neuronal health . in a mouse model of alzheimer 's disease , a deregulation of astrocytic ca homeostasis and signaling , including increased basal [ ca]i and spontaneous network activity , was observed . since soce is upregulated in astrocytes treated with amyloid- , future work on the role of stim and orai proteins might help to understand the pathophysiological regulation of astrocytic ca signaling and function . in microglia , stim1 , stim2 , and orai1 while stim1 is the primary er ca sensor , stim2 contributes to soce , particularly upon weak ca store depletion . several microglial functions , i.e. , release of cytokines , chemotaxis , and stimulated phagocytosis , are impaired in the absence or after downregulation of these soce components . dysfunction and dystrophy of microglia has been recognized as a potential cause of neurodegeneration and impaired neuronal development . for example , engulfment of dysfunctional synapses during development and phagocytosis of cellular debris ( such as amyloid- protein ) are possibly regulated by intracellular ca release and soce in microglia . therefore , stim1 , stim2 , and orai1 represent possible targets for the treatment of microglial dysfunction that may underlie neurodegenerative and neurodevelopmental disorders . the author was supported by the deutsche forschungsgemeinschaft ( dfg ; kr3408/2 - 1 ) .
stromal interaction molecules ( stim ) 1 and 2 are sensors of the calcium concentration in the endoplasmic reticulum . depletion of endoplasmic reticulum calcium stores activates stim proteins which , in turn , bind and open calcium channels in the plasma membrane formed by the proteins orai1 , orai2 , and orai3 . the resulting store - operated calcium entry ( soce ) , mostly controlled by the principal components stim1 and orai1 , has been particularly characterized in immune cells . in the nervous system , all stim and orai homologs are expressed . this review summarizes current knowledge on distribution and function of stim and orai proteins in central neurons and glial cells , i.e. astrocytes and microglia . stim2 is required for soce in hippocampal synapses and cortical neurons , whereas stim1 controls calcium store replenishment in cerebellar purkinje neurons . in microglia , stim1 , stim2 , and orai1 regulate migration and phagocytosis . the isoforms orai2 and orai3 are candidates for soce channels in neurons and astrocytes , respectively . due to the role of soce in neuronal and glial calcium homeostasis , dysfunction of stim and orai proteins may have consequences for the development of neurodegenerative disorders , such as alzheimer 's disease .
STIM and ORAI Proteins Mediate Store-Operated Ca Expression of STIMs and ORAIs in Brain Neurons Astrocytes Microglia Conclusions and Implications for Neurological Disorders Funding Disclosure of Potential Conflicts of Interest
the capacitative or store - operated ca entry ( soce ) , has been recognized as a an essential route for ca uptake in a wide variety of cell types to replenish intracellular ca stores and to regulate secretion , gene transcription and cell cycle progression . in 2005 and 2006 , different groups identified stromal interaction molecules ( stim ) and orai proteins ( named after the keepers of heaven 's gate in greek mythology ) as essential components of soce . a decrease in er luminal ca concentration results in dissociation of ca from the ef - hand domain which , in turn , triggers oligomerization and activation of stim1 , a process that is reversed when luminal [ ca ] returns to resting level ( that is , 250600 m ) . the principal components stim1 and orai1 essentially or nearly exclusively contribute to soce in different cell types . soce in the nervous system has been reviewed with respect to ca signaling and homeostasis in neurons and neuroglia . the present review is mostly based on studies regarding expression analyses or using gene silencing or knockout of stims and orais and gives a current overview on the putative functions of these soce components in both neurons and glial cells , i.e. in primary hippocampal and cortical neurons , stim2 is the predominant stim isoform . in hippocampal cultures , stim2 was detected in both neuronal soma and dendrites , whereas stim1 protein is restricted to the soma . store - operated ca entry has been implicated in neuronal ca signaling even before the discovery of stim and orai . the reduced [ ca ] in the endoplasmic reticulum ( er ) , [ ca]er , leads to the activation of stim2 and , possibly , of orai2 channels in the plasma membrane . the reduced [ ca ] in the endoplasmic reticulum ( er ) , [ ca]er , leads to the activation of stim2 and , possibly , of orai2 channels in the plasma membrane . stim2 is required for regular synaptic activity and mediates camp - dependent phosphorylation and trafficking of the ampa receptor subunit glua1 to plasma membrane - er junctions . whereas stim2 regulates soce in hippocampal and cortical neurons , stim1 appears to be the primary stim isoform in cerebellar granule cells and purkinje neurons . the activation of soce at resting ( hyperpolarized ) membrane potential suggests a homeostatic function of stims in neurons , i.e. we investigated the role of stim1 , stim2 , and orai1 in microglia . calcium imaging revealed a graded suppression of soce in the absence of stim1 , stim2 , or orai1 . to evaluate the role of soce in microglial migration and phagocytosis we used stim and orai knockout mice , the soce blockers 2-apb , la , and the trp channel blocker n-(p - amylcinnamoyl)anthranilic acid ( aca ) . p2y receptor - induced migration ( induced by 2-mesadp , atp , and udp ) was inhibited in stim1 , stim2 , and orai1 microglia . in turn , the resulting soce promotes activation of different ca - dependent signaling molecules , including ca / calmodulin - activated myosin light chain kinase ( mlck ) , protein kinase c ( pkc ) , the serin / threonine specific kinase akt , and the transcription factor nfat . stim1 , preferentially expressed in cerebellar neurons , and stim2 , more abundant in hippocampal and cortical neurons , trigger neuronal soce . stim1 is required for slow glutamate receptor signals in purkinje neurons and for ca - dependent gene transcription in cerebellar granule neurons . in analogy to functional differences between stim1 and stim2 , orai2 might , therefore , support a persistent and moderate less is known about the role of stims and orais in astrocytes . orai3 is , in contrast to orai1 and orai2 , inhibited by reactive oxygen species ( ros ) , and is required for store - independent crac channels activated by arachidonic acid , a phospholipase a2 ( pla2 ) metabolite . since soce is upregulated in astrocytes treated with amyloid- , future work on the role of stim and orai proteins might help to understand the pathophysiological regulation of astrocytic ca signaling and function . in microglia , stim1 , stim2 , and orai1 while stim1 is the primary er ca sensor , stim2 contributes to soce , particularly upon weak ca store depletion . therefore , stim1 , stim2 , and orai1 represent possible targets for the treatment of microglial dysfunction that may underlie neurodegenerative and neurodevelopmental disorders .
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acidic foods are consumed globally [ 1 , 2 ] but universally assumed to be innocent as to their effects on the mouth . acidic food and beverages can affect natural teeth , and chronic exposure often leads to the development of dental frangibles ( attrition , erosion , abrasion , and decay ) . not only is acid derived directly from colas , but also after imbibing the cola , acid is produced from biofilm bacteria metabolizing fermentable sugars in the drinks . human saliva acts as a neutralizing and/or buffering solution on imbibed acid beverages [ 4 , 5 ] . intraoral ph ( ph 6.8 ) decreases from , after drinking , an acidulated drink to below ph5 within 2 to 3 minutes . oral acidogenic bacterial action on fermentable carbohydrates ( monosaccharides like glucose and fructose ; disaccharides like maltose and sucrose ) also aggravates the ph reduction to below ph4 . intraoral ph takes about 25 minutes to change the acid environment , as further stimulated saliva neutralises any residual acid . the critical ph , at which hydroxyapatite dissolves , is ph5.5 , and because teeth are composed of calcium - deficient carbonated hydroxyapatite , they are vulnerable to decalcification in acid media . tooth material in saliva as a saturated solution of calcium and phosphate will show dissolution or remineralisation , depending on incumbent acidity of the surrounding saliva . this is illustrated by the following formula : ( 1)ca10(po4)6 ( oh)210ca2++6po43 + 2oh. should the ph fall ( increased h concentration and accordingly acidity ) , the 6po4 ions convert to h2po4 or h2po , and the oh ions are neutralized to water , driving the reaction to the right , that is , dissolution of calcium . if the h concentration diminishes ( i.e. , ph rises and acid activity falls ) , the reaction is driven to the left producing remineralization [ 5 , 6 ] . repeated exposure to acid drinks changes the intraoral ph to below the critical ph ( ph5.5 ) , when chemical dissolution of calcium from dental hydroxyapatite occurs . this sustained low ph , < ph5.5 , allows mordant development of frangibles starting and deriving from decalcification . besides ph and buffering , calcium , phosphate , and to a lesser extent fluoride chronic frequency of imbibing , method of drinking , timing of consumption , or type and quantity of beverage are known to influence the realization of frangibles . the formulae and contents of pop - cola beverages are closely guarded industrial secrets , and sparse data about acidity is provided on their consumer packages . in light of knowing the critical ph for hydroxyapatite , and also that acid drinks require a lot of alkaline salivary flow to neutralize dietary acid , it is most important to know the ph of pop - cola drinks , the strength of their acids , particularly their buffering capacities as to how much alkali is needed to change their ph . various methods are used to assess ultramicroscopic effects of beverages on teeth ; these include surface hardness measurements , surface profilometry , iodide permeability tests , chemical analysis of dissolved minerals , microradiography , confocal scanning microscopy , quantitative light - induced fluorescence , atomic force microscopy , element analysis of solid samples , nano - indentation , ultrasonic measurements , and scanning electron microscopy ( sem ) with environmental sem ( esem ) . with esem , minimal sample preparation is required , is economical , and minimizes risks for artefacts . also esem and sem permit progressive assessment of the identical test - tooth material without carbon or metal coating of native surfaces . recently ( 2010 ) , variable pressure scanning electron microscope ( vp - sem ) has refined this technique further . aimthis study aims to ( i ) assess ph , and buffering capacities of six pop - cola beverages ( initial rise of one ph unit to the critical ph and to neutral ph ) ; ( ii ) measure calcium and phosphorous content of cola after rinsing from mouths with ( dentate ) and without teeth ( edentulous ) ; ( iii ) assess scanning electron microscopic ( vp - sem ) erosive effects on native dental hard tissues after exposure to pop colas ; ( iv ) describe a clinical case of pop - cola erosion . this study aims to ( i ) assess ph , and buffering capacities of six pop - cola beverages ( initial rise of one ph unit to the critical ph and to neutral ph ) ; ( ii ) measure calcium and phosphorous content of cola after rinsing from mouths with ( dentate ) and without teeth ( edentulous ) ; ( iii ) assess scanning electron microscopic ( vp - sem ) erosive effects on native dental hard tissues after exposure to pop colas ; ( iv ) describe a clinical case of pop - cola erosion . six pop colas , namely , pepsi , diet pepsi , coca cola , diet coca cola , and selection cola and diet selection cola were analyzed . standard aliquots of cola liquid were tested for ph and buffering capacity ( batches of potable over - the - counter colas were purchased by chance at local retail outlets ) . the acidity as ph and buffering capacity was measured with an automated mettler dl25 titrator ( at ) . buffering capacities were assessed with 0.5 m naoh solution and titrated to raise the measured cola ph one ph unit , then to ph 5.5 , and to ph 7 . two fresh 60mls aliquots were collected directly from each of 12 cans and placed in separate polystyrene falcon test tubes for measurements . titration measures were repeated for each sample from different cans in the same batch . for each of the 6 colas , automatic titrator ( at ) the at was used to determine the ph and buffering capacities of the various pop - cola liquids . for this analysis , the at was used according to described methods by larsen and nyvad 1998 method and measured initial ph 's ( figure 1 ) and buffering capacities at the three different stated levels : ( i ) ph change of one unit from the incipient ph ( orange bars , figure 2 ) , ( ii ) ph change up to the critical level ph 5.5 ( red bars , figure 2 ) , and ( iii ) ph change up to the neutral level ph 7.0 ( green bars , figure 2 ) . all measures were assessed at room temperature of 23c . to ensure measurement accuracy and establish baseline controls , the at was calibrated each time before use , at ph 2 , 4 , 7 , and 10 , using standard buffer solutions for all levels . a 60 ml volume of the tested sample was then titrated using a 0.5 m naoh solution . the at was used to determine the ph and buffering capacities of the various pop - cola liquids . for this analysis , the at was used according to described methods by larsen and nyvad 1998 method and measured initial ph 's ( figure 1 ) and buffering capacities at the three different stated levels : ( i ) ph change of one unit from the incipient ph ( orange bars , figure 2 ) , ( ii ) ph change up to the critical level ph 5.5 ( red bars , figure 2 ) , and ( iii ) ph change up to the neutral level ph 7.0 ( green bars , figure 2 ) . all measures were assessed at room temperature of 23c . to ensure measurement accuracy and establish baseline controls , the at was calibrated each time before use , at ph 2 , 4 , 7 , and 10 , using standard buffer solutions for all levels . a 60 ml volume of the tested sample was then titrated using a 0.5 m naoh solution . the six different pop - colas mentioned ( coca cola , pepsi cola , selection cola , diet coke , diet pepsi , and diet selection cola ) were tested . two volunteer cohorts , one fully dentate ( mean age 22 , m : f 4 : 2 , n = 6 ) the second edentulous ( mean age 52 , m : f 3 : 3 , n = 6 ) , were used to swish with 20 ml aliquots of deionized aquafina water as baseline and subsequently a 20 ml aliquots cola for 60 seconds for each volunteer . every cola was sampled and analyzed twice , giving 12 analyses for swishes , using standardized analytical chemistry methods . the colas from source and postswish expectorates were analyzed for calcium , and phosphorous contents using inductively coupled plasma with optical emission spectroscopy ( icp - oes ) and ion chromatography ( ic ) were used to determine concentrations of anions ( fluoride ) present in the six colas direct from their cans . ( see appendices a.2 and a.3 ) for our analysis , concentrations of calcium and the major ions of interest as phosphate and fluoride are reported here . to eliminate inter- and intraoperator bias , blindly by third - party technicians unaware of the source of procured samples . results analysis reveals consistent significant ( p < .01 student - t ) increases in calcium leeched from dentate subjects after swishing with all the pop - colas tested . the icp - oes and ic measures were compared and there were no significant differences ( p > .99 ) with regard to results obtained for phosphorous . variable amounts of phosphorous were found in the various pop - colas tested , and this was reflected in the swish findings of phosphorous present after rinsing with these pop - colas . hard tissue erosion , as chemical dissolution of calcium , may differ between colas , but is produced by all the colas tested . in none of the pop - colas is hard tissue erosive action absent from postswishing exposure , and calcium content in the swish expectorates with teeth all showed significant ( p < .01 student - t ) increases in dissolved calcium when compared to swishing with water , or swishing without teeth ( see figure 3 ) . < .01 student - t ) increases in calcium leeched from dentate subjects after swishing with all the pop - colas tested . the icp - oes and ic measures were compared and there were no significant differences ( p > .99 ) with regard to results obtained for phosphorous . variable amounts of phosphorous were found in the various pop - colas tested , and this was reflected in the swish findings of phosphorous present after rinsing with these pop - colas . hard tissue erosion , as chemical dissolution of calcium , may differ between colas , but is produced by all the colas tested . in none of the pop - colas is hard tissue erosive action absent from postswishing exposure , and calcium content in the swish expectorates with teeth all showed significant ( p < .01 student - t ) increases in dissolved calcium when compared to swishing with water , or swishing without teeth ( see figure 3 ) . healthy teeth condemned for extraction ( orthodontics or impactions ) were used . immediately after extraction , teeth were placed in deionized water refrigerated at 4c for a maximum of two hours . each cola was tested at three selected sites on the same tooth ; this was repeated on three teeth from the same set of wisdom teeth , which provided fresh native material for assessment . for enamel , flat tooth surfaces were selected ; for dentine , samples were obtained by cutting horizontally through the middle of the crown , and the enamel - cemental junction on each tooth used was also tested and examined directly . after samples were cut , each specimen was rinsed with deionized water and air stream dried . all areas of focused interest were examined using scanning electron microscopy with vp - sem . resolution was at 700x , with accelerating voltage 2025 kv , to assess fracture patterns and morphology . immediately after scanning controls , each sample was immersed in a cola for 30 s , and subsequently the specimens were immersed for a further 30 s ( for a total of 60 s ) , and the same location was scanned and recorded . image analysis of morphology , properties and characteristics ( cracks , openings , and changes of identifiable shapes ) were done with microscopic scales and by micrometer measures using the same microscope magnification of the identical areas tested . after 60 seconds of cola exposure , the specimens were brushed with a standardized rotary tooth brush for 5 seconds under 60 grams pressure . results ultrascaled measures ( 700x ) reveal significant losses of hard tissue from erosion ( p < .01 , student 's t - test paired ) . initial erosion is seen after exposure to the colas , as the chemical loss of calcium widens cracks and causes surface crenellations to develop . after 60 s cola exposure , soft surface crenellations develop . after brushing , ultrascaled measures ( 700x ) reveal significant losses of hard tissue from erosion ( p < .01 , student 's t - test paired ) . initial erosion is seen after exposure to the colas , as the chemical loss of calcium widens cracks and causes surface crenellations to develop . after 60 s cola exposure , soft surface crenellations develop . after brushing , evidence of tooth - brush abrasion enamel , ecj and dentin at 60 seconds exposure at 700 show effects after brushing . an 18-year - old male presented complaining of temperature sensitive teeth and that his teeth were getting smaller . his medical history revealed nothing significant to indicate that he may suffer from gord ( gastro - oesophageal reflux disorder ) . the aetiology of gord is variable , but when the gastric contents flow from the stomach to the mouth , it is called gastro - oesophageal reflux disorder ( gord ) . common symptoms are heart burn along the oesophageal pathway ; epigastric pain localised over the stomach ; regurgitation into the mouth ; dysphagia with or without pain ; noncardiac retrosternal pain ; chronic coughing and sore throat from laryngitis ; vocal hoarseness ; a throat globus [ 16 , 17 ] . he attended his general dentist regularly and brushed and flossed his teeth regularly , morning and evening every day . he used fluoridated toothpaste and used a new soft nylon tufted tooth brush every month . his diet was derived from foods in all food groups ( meat / fish , dairy , grain / cereals , and vegetables ) , but he specifically stressed that he did not drink fresh fruit juices and rarely ate fresh fruit because they hurt his teeth . however , he reported drinking copious amounts of cola solidly from my last two years of in primary school till now , the time which was calculated to be about ten years . he drank all brands of conventionally ( carbohydrate ) sweetened colas , until three years prior to presenting , the point from which he changed to drinking exclusively synthetically sweetened diet - colas . a fortnight of dietary analysis confirmed these predilections and showed that he consumed between at least one litre to one and a half litres of cola daily , from cans , or bottles depending on availability . this daily cola intake was a habitual part of his diet , and he would swish the cola over his teeth to reduce the gas and enhance the flavour . on presentation ( figure 12(a)12(k ) ) , his teeth appeared reduced in size apparently by erosion with reduced enamel and dentin exposure . the upper anteriors were more affected than the rest of the teeth , along with molar and premolar cuspal reduction and incisal attrition . subgingival probing revealed no periodontal disease ( no sulcus depth exceeding 3 mm ) , and further subgingival exploration showed only healthy tooth surfaces and no caries . all the standard cola drinks show a progressive increased requirement of base to neutral ph 7 , as does all the diet cola beverages . the ph and buffering do not fully explain the erosive potential of colas , as the mineral content , concentrations of organic acids ( phosphoric and citric ) , fluoride , and the ability of the mix to remove calcium from the mineral surface are all relevant . however , ph - expressing acid content , buffering ability , and acidic ions available for the overall general mordant effect of acid - cola beverages are more important in producing erosion , as without an acid environment the other stated ions are not active . figure 1 shows how low the ph of the selected group of pop cola drinks is . all the cola drinks register ph values well below the critical ph 5.5 , at which tooth decalcification occurs ( figure 1 ) . among the tested drinks , pepsi cola is the most acidic ( ph 2.53 ) while diet selection cola is the least ( ph 3.4 ) . from the results presented in figure 2 , selection cola and diet pepsi - cola have the largest buffering capacities for a ph change of one unit . as for the ph change up to its critical value ph 5.5 ( figure 2 ) , pepsi cola requires the largest amount of sodium hydroxide ; diet pepsi cola ( figure 2 ) finally appears to be the most resistant drink to a ph change up to neutralization ( ph 7.0 ) . these results are noteworthy as the low ph values reflect that the pop - cola drinks are extremely acidic and consequently could all contribute to the decalcification of teeth ( see figures 611 ) . it is also important to say that an average of 5.86 ml of base is required to bring the ph back to the neutral level of ph 7.0 ( figure 2 ) . this indicates that not only are the cola drinks highly acidic when they are first exposed to teeth , but they would also require a large amount of alkaline - stimulated saliva to be neutralized . whole saliva as a complex buffering solution may provide a definite protective buffering and neutralizing effect on acid dietary drinks [ 1921 ] . but with acid drinks , including pop colas , this buffering / neutralizing effect may be overwhelmed . the colas tested are not all chemically identical ; yet they all leech calcium from teeth in vivo after contact for 60 seconds ( figure 3 ) . the phosphorous content varies ( figure 4 ) because the phosphorous content of the colas from the cans varies . the dental and salivary calcium salts absorb some of the phosphorous from the cola ; also the acidity from each cola varies as does the buffering , and reflex stimuli may vary in intensity of reaction . other intraoral phosphate sources may derive from gingivo - crevicular fluid and calculus ( tricalcium phosphate , octa - calcium phosphate , dicalcium diphosphate ) . because the phosphorous composition of the colas is variable , the amount of phosphates produced after swishing is also variable ( figure 4 ) . although the chemical composition of the colas tested varied , they all had high buffering capacities and acid activity ( ph ) well below the critical ph 5.5 . but even when accounting for any extra calcium released in stimulated saliva , the calcium found in swished expectorates from all these colas is consistently and significantly ( p < .01 student - t ) higher when compared to the calcium content in the can . tooth erosion , as chemical dissolution of calcium , is derived from an intraoral source and is reflected by an increased content of calcium in all the swishes from the colas tested . the increase in calcium shown in the swishing experiment without teeth ( figure 3 ) after rinsing with the colas may be derived from calcium ions secreted in stimulated saliva . other sources of calcium may derive in miniscule aliquots from minor salivary glands and circulating oral glycoprotein . the calcium content in the expectorates varies , but the only confounding factor , as a variable that explains the increase of calcium in the swish expectorates , is the presence of teeth ; this is particularly highlighted by comparisons in each cola of calcium content in the expectorate swish with teeth that shows significant ( p < .01 , student - t ) increases in dissolved calcium when compared to swishing with water , or swishing without teeth . some phosphates may derive from stimulated saliva , and some from the teeth or other intraoral phosphate sources , but the amounts of calcium contents measured in the swishes can not be explained in this experiment , from sources other than from the natural teeth in vivo . considering that the average rate of resting - saliva secretion is 0.78 ml / min , this would also mean that a long period of time is needed for the mouth to return to neutral ph 7 [ 4 , 11 ] . knowing that acids are the most potent stimuli for reflexive stimulated - salivary flow , the rate of stimulated secretion can increase to reach a maximum limit of 8ml saliva per minute . even if this maximum rate is reached when consuming cola drinks , neutralisation to physiological stable oral ph levels would still need a long period of time to be achieved , and the calcium content from saliva is too low to account for calcium increases after rinsing . typically after one test bolus in the mouth mg / l , f fails to reduce erosion of teeth in vitro , even with the mean fluoride content from the cans , it was 3.5 mg / l ( ranging from 2 to 6 mg / l ) ; this concentration does not stop calcium dissolution after swishing ( see figure 5 ) . increasing the fluoride content is feasible , but higher concentrations would be undesirable , as it would be unacceptably toxic and also negatively affect organoleptic taste properties . the dietary acids from the colas over power any protective effect from saliva and the same applies to the fluoride content of the colas . some other important biological factors may slightly affect decalcification and tooth erosion , such as the saliva flow rate , its composition , buffering capacity and stimulation capacity , and the acquired pellicle , which has diffusion - limiting properties by its composition , maturation , and thickness . the type of dental substrate and its density of composition also can effect erosion , as does the dental anatomy and occlusion influencing the flow of liquids over the tooth surfaces ; and besides the anatomy and histology , the vigorous function of oral soft tissues in relationship to the teeth affects the development of erosion . none of these are as important as the acid composition and ph of the pop - cola drink in producing erosion . keratosis on the tongue seems to act as a rasper that removes surface tooth material softened by decalcification . decalcification caused by regurgitated gastric contents in bulimia often manifests first as palatal erosion because of tongue thrusting , removing softened tooth material [ 15 , 23 , 24 ] . the acidulated colas also act as a stimulus for stimulated saliva to flow which contains calcium . but the calcium content of saliva is negligible ( mean 5.7 mg/100 ml ; range : 210 mg/100 ml ) , and even with maximum secretion rate of saliva with ranges of 7 to 8 ml in one minute , comparisons between swish expectorates ( with and without teeth ) indicate that it is impossible for stimulated saliva to secrete calcium in amounts recorded after 60 seconds of swishing . also it is inordinately difficult to procure age - matched ( edentulous ) controls without teeth below 35 ( dentate controls with teeth had a mean age of 22 years old ) , or to find people aged 52 years ( the mean age of the test edentulous group ) without any dental restorative work . the recent increase of consumption of pop drinks is reflected in increased reporting of dental erosion [ 3 , 27 ] . data reported here shows that damage occurs at a microscopic level and corroborates information gleaned from epidemiology and marketing . some consider a 30- or 60-second exposure unreasonably long ; this is not valid criticism , as most reported data from other investigators uses target times in excess of 5 minutes , even hours or days of immersion , and 30 seconds and 60 seconds could well approximate the total time a 350 ml can of cola may be exposed to teeth , when people swallow a 60ml bolus and swish it for 5 to 10 seconds . dental ravages from cola drinks have a high prevalence in children and young adults [ 7 , 29 ] . evidence presented here in this electron microscopic study re - enforces and confirms this theory . this study also demonstrates this interaction results in surface etching ( figure 6 ) and cracks ( figures 8 and 10 ) . this sem evidence confirms observations about smear layer removal , opening of dentine tubules , and increasing of tubules diameter based on randomly selected sites [ 30 , 31 ] . this is probably due to disruption of fluid dynamics in the tubules as well as by mechanical loss of tooth material [ 30 , 31 ] . the results also indicate that with light brushing after 60 s exposure to pop colas , abrasion will be obtained . tooth wear results for three main processes erosion , attrition , and abrasion , with chemical , physical , and physiological forces interacting to produce a clinical case of dental frangibles . therapy ranges from eschewing acid drinks , avoiding brushing immediately after drinking pop - drinks , reducing frequency of drinking to fissure sealants and coating , occlusal build - up with overlays , or comprehensive oral rehabilitation with full - coverage crowns . ( i ) pop - cola drinks tested are acid and may decalcify tooth material . ( ii)this study provides evidence that all these six common colas ( pepsi cola , diet pepsi cola , coca cola , diet coke , selection cola and diet selection cola ) , leech calcium out of teeth , after rinsing with pop - colas . ( iii ) this sem experiment shows that acid pop - cola ignores the smear layer , softens and erodes dental hard tissues , and facilitates abrasion . ( iv ) the clinical case report shows erosion from chronic imbibing of acid pop colas . these data collectively provide more evidence as a proof that chemical dissolution by tooth decalcification is caused by drinking pop colas . this study demonstrates clear visual evidence of dental erosion with altered enamel , and dentine morphology changes due to short exposure to pop colas . the automatic titrator instrument was standardized with certified reference buffers at ph 2 , 4 , 7 , and 10 . the icp - oes was used to detect the concentrations of calcium , phosphorus ( and sodium , potassium , boron , tin , arsenic , iron , nickel , lead , copper , magnesium , zinc , sulphur , chrome , cobalt , and manganese not reported ) present in the cola drinks being tested . in order to obtain accurate and precise results , twelve replicates for every drink being tested along with a deionized aquafina water blank were prepared with due diligence . initial precleaned polypropylene digitubes ( 50 ml ) were clearly labeled , and 20 ml samples were dispensed to each tube with a pipette . because there is a high sugar content in the regular drinks , a matrix effect is formed , preventing the icp - oes from determining accurate concentrations of those elements being tested . to eliminate any potential matrix , 5 ml of trace metal grade nitric acid was added to each sample , and the samples were digested to 95c for 120 minutes using a 24-position digiprep equipped with a digiprobe . the probe was placed in a sample in order to maintain and monitor the digestion procedure . once digestion was finished ( 1 ) the samples were cooled to room temperature , ( 2 ) volumes of twelve replicates and a blank were completed to 50 ml with the deionized water . a calibration curve was prepared from 0.5 to 50 mg / l with certified icp - oes standards diluted in a solution of 2% nitric acid . a total of four points including a calibration blank ( consisting of 2% trace metal grade nitric acid ) were prepared . furthermore , two quality control standards were prepared from a different lot of certified icp - oes standards . they were prepared at a concentration of 5 and 25 mg / l . the ic is used to determine the concentration of different anions present in the six cola drinks being tested . for the purpose of our analysis , the concentrations of the major anions , which are fluoride , acetate , formate , chloride , phosphate , nitrate , and sulphate were studied . other ions were also assessed but not reported here . a fixed volume ( 5 ml ) of the cola is accurately delivered in a properly labeled digitubes ( 50 ml ) . the cola samples were then filtered using a very small filter ( diameter of pores = 0.45 m ) to remove all sediments and impurities present that would cause microbial alterations . a volume of 4 ml of every filtered sample the calibration curve covered a range from 0.01 to 10 mg / l for each anion . furthermore , two quality control standards were used at concentration of 0.02 and 5 mg / l . the concentration of each ion can then be obtained by evaluating the area under each peak . the concentration of the anions in the cola drinks was determined by plotting calibration curves .
introduction . manufactured colas are consumed universally as soft drinks . evidence about the acid contents of cola - beverages and its effects on teeth is rare . aim . to assess ( i ) cola acidity and buffering capacity in vitro , ( ii ) tooth erosion after swishing with colas in vivo ( iii ) scanning electron microscopic effects on teeth of colas , and tooth - brush abrasion , and ( iv ) report a clinical case of erosion from cola consumption . materials and methods . ( i ) we measured six commercially available pop cola beverages , ph , and buffering capacities using a ph - mettler automatic titrator , with weak solution of sodium hydroxide ( ii ) two cohorts , one with teeth , the second without teeth rinsed with aliquots of cola for 60 seconds . swished cola samples tested for calcium and phosphorus contents using standardized chemical analytical methods ( iii ) enamel , dentine , and the enamel - cemental junction from unerupted extracted wisdom teeth were examined with a scanning electron microscope after exposure to colas , and tested for tooth - brush abrasion ; ( iv ) a clinical case of pop cola erosion presentation , are all described . results . comparisons among pop colas tested in vitro reveal high acidity with very low ph . buffering capacities in millilitres of 0.5 m naoh needed to increase one ph unit , to ph 5.5 and ph 7 are reported . rinsing in vivo with pop cola causes leeching of calcium from teeth ; sem shows dental erosion , and pop - cola consumption induces advanced dental erosion and facilitates abrasion . conclusions . ( i ) pop - cola acid activity is below the critical ph 5.5 for tooth dissolution , with high buffering capacities countering neutralization effects of saliva ; ( ii ) calcium is leeched out of teeth after rinsing with pop colas ; ( iii ) sem evidence explains why chronic exposure to acid pop colas causes dental frangibles ; ( iv ) a clinical case of pop - cola erosion confirms this .
1. Introduction 2. Methodology 3. Discussion 4. Concluding Remarks A. Descriptions for Technique Details of ICP-OES and IC
repeated exposure to acid drinks changes the intraoral ph to below the critical ph ( ph5.5 ) , when chemical dissolution of calcium from dental hydroxyapatite occurs . in light of knowing the critical ph for hydroxyapatite , and also that acid drinks require a lot of alkaline salivary flow to neutralize dietary acid , it is most important to know the ph of pop - cola drinks , the strength of their acids , particularly their buffering capacities as to how much alkali is needed to change their ph . aimthis study aims to ( i ) assess ph , and buffering capacities of six pop - cola beverages ( initial rise of one ph unit to the critical ph and to neutral ph ) ; ( ii ) measure calcium and phosphorous content of cola after rinsing from mouths with ( dentate ) and without teeth ( edentulous ) ; ( iii ) assess scanning electron microscopic ( vp - sem ) erosive effects on native dental hard tissues after exposure to pop colas ; ( iv ) describe a clinical case of pop - cola erosion . this study aims to ( i ) assess ph , and buffering capacities of six pop - cola beverages ( initial rise of one ph unit to the critical ph and to neutral ph ) ; ( ii ) measure calcium and phosphorous content of cola after rinsing from mouths with ( dentate ) and without teeth ( edentulous ) ; ( iii ) assess scanning electron microscopic ( vp - sem ) erosive effects on native dental hard tissues after exposure to pop colas ; ( iv ) describe a clinical case of pop - cola erosion . buffering capacities were assessed with 0.5 m naoh solution and titrated to raise the measured cola ph one ph unit , then to ph 5.5 , and to ph 7 . for this analysis , the at was used according to described methods by larsen and nyvad 1998 method and measured initial ph 's ( figure 1 ) and buffering capacities at the three different stated levels : ( i ) ph change of one unit from the incipient ph ( orange bars , figure 2 ) , ( ii ) ph change up to the critical level ph 5.5 ( red bars , figure 2 ) , and ( iii ) ph change up to the neutral level ph 7.0 ( green bars , figure 2 ) . for this analysis , the at was used according to described methods by larsen and nyvad 1998 method and measured initial ph 's ( figure 1 ) and buffering capacities at the three different stated levels : ( i ) ph change of one unit from the incipient ph ( orange bars , figure 2 ) , ( ii ) ph change up to the critical level ph 5.5 ( red bars , figure 2 ) , and ( iii ) ph change up to the neutral level ph 7.0 ( green bars , figure 2 ) . two volunteer cohorts , one fully dentate ( mean age 22 , m : f 4 : 2 , n = 6 ) the second edentulous ( mean age 52 , m : f 3 : 3 , n = 6 ) , were used to swish with 20 ml aliquots of deionized aquafina water as baseline and subsequently a 20 ml aliquots cola for 60 seconds for each volunteer . for enamel , flat tooth surfaces were selected ; for dentine , samples were obtained by cutting horizontally through the middle of the crown , and the enamel - cemental junction on each tooth used was also tested and examined directly . the ph and buffering do not fully explain the erosive potential of colas , as the mineral content , concentrations of organic acids ( phosphoric and citric ) , fluoride , and the ability of the mix to remove calcium from the mineral surface are all relevant . although the chemical composition of the colas tested varied , they all had high buffering capacities and acid activity ( ph ) well below the critical ph 5.5 . the calcium content in the expectorates varies , but the only confounding factor , as a variable that explains the increase of calcium in the swish expectorates , is the presence of teeth ; this is particularly highlighted by comparisons in each cola of calcium content in the expectorate swish with teeth that shows significant ( p < .01 , student - t ) increases in dissolved calcium when compared to swishing with water , or swishing without teeth . tooth wear results for three main processes erosion , attrition , and abrasion , with chemical , physical , and physiological forces interacting to produce a clinical case of dental frangibles . ( ii)this study provides evidence that all these six common colas ( pepsi cola , diet pepsi cola , coca cola , diet coke , selection cola and diet selection cola ) , leech calcium out of teeth , after rinsing with pop - colas . ( iii ) this sem experiment shows that acid pop - cola ignores the smear layer , softens and erodes dental hard tissues , and facilitates abrasion .
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the fourth diabetes atlas , published by the international diabetes federation ( idf ) , revealed that there were an estimated 50.8 million cases of diabetes in india in 2010 . data on hypertension , available from several regional studies , indicate an increase of about 30 times among urban indians and 10 times among rural indians , within a span of three and six decades , respectively . to date , no nation - wide epidemiological study aimed at determining the prevalence of hypertension has been reported . hypertension is 1.52 times more prevalent in diabetic as in non - diabetic individuals and about 3035% of hypertensives are detected to have diabetes . the combination of diabetes and hypertension imparts an additive risk and accelerates progression of both microvascular and macrovascular complications . hence , these two conditions contribute significantly to the growing load of cardiovascular diseases and lead to a high economic burden that accounts for 520% of the total indian healthcare expenditure . this rapid increase in diabetes and hypertension in india is most likely to result in a twin epidemic . the screening india 's twin epidemic ( site ) study was aimed at determining the number of diagnosed and undiagnosed cases of diabetes and hypertension in india and reporting the measures employed to manage and control these diseases in an outpatient setting . the study was conducted in eight states maharashtra , delhi , tamil nadu , west bengal , karnataka , andhra pradesh , madhya pradesh , and gujarat . although site is not a population - based study , it is one of the largest studies of its kind . also , in lieu of poor existing information , site serves as a good proxy for providing valuable insights on the national prevalence and management of both diabetes and hypertension . we present herewith the site study design , methodologies and highlight the amendments made as the study progressed . site was a cross - sectional , national , multicentric , non - randomized , observational study . the primary objective of the study was to estimate the prevalence of diagnosed and undiagnosed diabetes and hypertension in outpatient settings in major indian states . the secondary objectives included estimation of the prevalence of other cardiovascular risk factors such as dyslipidemia , obesity [ body mass index ( bmi ) , waist circumference ( wc ) , and waist - hip ratio ( whr ) ] , metabolic syndrome , urine microalbuminuria , arrhythmia , diet , smoking and alcohol consumption ; estimation of the prevalence of abdominal obesity in sub - groups of patients ( hypertension , diabetes , dyslipidemia , smokers and alcoholics ) ; and understanding the management and the level of control of hypertension and diabetes . the study was conducted in accordance with the guidelines for good epidemiology practices . the protocol complied with recommendations of the 18 world health congress ( helsinki , 1964 ) and the local laws and regulations of india . the sample size depended on the expected prevalence of diabetes / hypertension ( whichever was lower ) . based on the conservative expected prevalence of 15% for diabetes , it was determined that a sample size of 1225 patients was required per state . assuming a dropout rate of 39% , it was estimated that 2000 patients were required to ensure 95% confidence such that the prevalence of diabetes is between 13% and 17% , that is , 15% 2% in each state . it was initially planned to conduct the study in 10 states across five zones , north , south , east , west and central , in waves one state at a time and recruit 2000 patients from 100 centers per wave . physician selection : the study was conducted in outpatient settings and the data were collected from general practitioners rather than specialists . also , the source of any potential bias was much reduced , as the number of people presenting the disease would evidently be higher at a specialist clinic rather than a general practitioner 's clinic . patient selection : patients 18 years of age and those ready to sign the data release consent form along with an acceptance to take the screening tests were included in the study . pregnant women are predisposed to gestational diabetes and weight gain , and their exclusion decreased eventual confounding effects on the results . the first 10 patients visiting the physicians clinic on two consecutive days , irrespective of the purpose of the visit , and satisfying the inclusion criteria , were selected for the study . during 20092010 , site was conducted in 8 states including 7 of the top 10 most populous indian states and the national capital territory [ figure 1 ] . for the remaining two planned states , on the basis of the fact that the sample size was calculated per state and that a low dropout rate was observed among the patients enrolled , the national coordinators deemed the data adequate to pan out substantial results . so , by the end of the 8 wave , the recruitment was declared completed . because of a national multicentric approach of the study , the data obtained were representative of diverse demographic populations within india . the states in which the screening india 's twin epidemic study was conducted the study was sponsored by sanofi - aventis ( india ) . site management organization ( smo ) and data management services were provided by max neeman ltd . the sponsor ensured that the central laboratory , smo , and data management personnel dedicated to study , as well as the participating physicians , received adequate training on various aspects of the study . thus , the complete study team was well versed with the data collection form ( dcf ) and the study procedures were executed uniformly . the data collection process was carried out in two steps [ figure 2 ] . data collection process of the screening india 's twin epidemic study the dcf used was designed in line with the study objectives . it captured details on the following aspects : demographics ( age and gender ) , anthropometrics ( height , weight , waist and hip circumference ) , behavioral ( smoking and alcohol use ) , medical history ( diabetes and its treatment , hypertension and its treatment , and cardiovascular diseases like ischemic heart disease , myocardial infarction and stroke ) , type of diet ( vegetarian / non - vegetarian ) , and family history of diabetes and hypertension . details on the patient 's socioeconomic , educational , psychosocial , and physical activity status were not collected . anthropometric measurements : height was measured without shoes , in centimeters , using a standard stadiometer . weight was measured with light clothes and without shoes , in kilograms , using a professional calibrated weighing scale , and rounded off to the nearest number . since no standard weighing machine was employed , inter - instrument variations can not be ruled out . body mass index ( bmi ) was calculated using the formula : weight ( kg)/height ( m ) . the hip and waist measurements were taken using a standard non - stretchable anthropometric measure tape across all study sites . the hip circumference ( hc ) was measured over light clothing . the patient was made to stand erect with arms at the sides , feet placed together with weight equally distributed on each leg , and the gluteal muscles relaxed . the tape was placed horizontally around the point of the maximum posterior extension of the buttocks . the patient 's measured hc was calculated as the mean of the two observations or the mean of the two closest measurements . the patient was made to stand erect with arms at the sides and feet placed about 2530 cm apart with weight equally distributed on each leg . the wc was measured at the part of the trunk located midway between the lower costal margin and the iliac crest . the measurer stood beside the patient and made the tape fit snugly , without compressing any underlying soft tissues . the circumference was measured in centimeters to the nearest 0.5 cm at the end of a normal expiration . the method and angles of measurement were adequately detailed to ensure limited variance in the technique . whr was calculated by dividing the wc ( cm ) by the hc ( cm ) . blood pressure ( bp ) measurement : a mercury sphygmomanometer was used to measure the bp , preferably from the right arm of the patient . an average of two readings , recorded at 5-minutes interval , was taken to ensure accuracy . at the end of visit 1 , patients received a coupon of identification and were required to visit any of the nearest metropolis clinical diagnostic centers , within 2 - 5 days . fasting lipid profile , microalbuminuria , fasting plasma glucose ( fpg ) , and hba1c were determined for each patient presenting a valid coupon . hba1c was measured by the bio - rad d10 high - pressure liquid chromatography method . urine microalbumin was measured by a turbidimetric inhibition immunoassay technique and enzymatic reactions were applied to quantify total cholesterol ( cholesterol esterase ) , high - density lipoproteins ( hdl ; cholesterol oxidase , esterase , and peroxidase ) , and triglycerides ( tg ; lipoprotein lipase , glycerol kinase , glycerol-3-phosphate oxidase , and peroxidase ) . low - density lipoprotein ( ldl ) was also calculated by using friedewald 's equation for tg < 300 mg / dl . diabetes was diagnosed according to the american diabetes association ( ada ) guidelines , and hypertension was classified according to the recommendations of the seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ( jnc 7 ) . metabolic syndrome was diagnosed on the basis of national cholesterol education program ( ncep ) . bmi was categorized into normal ( < 23 kg / m ) , overweight ( 2325 kg / m ) and obese ( > 25 kg / m ) , according to the guidelines jointly suggested by the indian health ministry , diabetes foundation of india , all india institute of medical sciences , indian council of medical research , and national institute of nutrition and 20 other institutions . normal and truncal obesity was categorized according to the whr , as suggested by the ncep . fasting lipid profile revealed the total cholesterol , tg , hdl - cholesterol , and ldl - cholesterol levels . of these , elevated tg ( 150 mg / dl ) and concomitant low hdl - cholesterol ( < 40 mg / dl ) were used to identify dyslipidemia . the criteria used are ( 1 ) primarily attributed to atherogenic dyslipidemia , ( 2 ) linked to insulin resistance , ( 3 ) measurable during the initial phase of development of metabolic syndrome , ( 4 ) individually extremely inheritable , ( 5 ) simple to measure , and ( 6 ) determinants of the atherogenic index of plasma ( aip ) , a key prognostic marker of cardiovascular disease , calculated as log ( tg / hdl - c ) . urine microalbumin was defined according to the albumin creatinine ratio ( acr ) as normoalbuminuria ( acr < 30 g / mg ) , microalbuminuria ( 30300 g / mg ) , and macroalbuminuria ( > 300 g / mg ) . monitoring visits were randomly conducted for at least 20% of the active sites ( enrolled at least one patient ) and the reports documented . phlebotomy services , testing , processing , shipment of blood and urine samples , and preparation and distribution of reports were undertaken by the central laboratory ( metropolis healthcare ltd . ) . data entry , verification , and validation were carried out using pheedit database system , version 3.1 ( sas institute inc . , cary , nc , usa ) . a double - entry method was used to ensure that the data ( except comments ) were transferred accurately from the dcfs to the database . moreover , every modification in the database could be traced using an audit trail . a data checking plan was established to define all automatic validation checks , as well as the supplemental manual checks . all discrepancies were researched until resolved . additionally , a national steering committee comprising india 's leading endocrinologists and cardiologists was established to provide scientific direction for the study . the total sample number , mean , standard deviation , and range ( minimum and maximum ) were calculated for all continuous variables ( age , height , wc , hc , and lipid values ) , and frequency and percentage were calculated for all categorical variables ( gender , presence of hypertension and diabetes and their treatment , smoking status , and urine microalbuminuria ) . student 's t - test and chi - square test were used to make comparisons between groups , with p value < 0.05 considered statistically significant . a multivariate analysis was performed to estimate the association of hypertension and diabetes with dyslipidemia , obesity , metabolic syndrome , microalbuminuria , arrhythmia , smoking , alcohol consumption , and diet . a simple linear regression analysis was performed to estimate the contribution of individual variables to systolic and diastolic bp . relative risk for developing diabetes and hypertension was calculated by assessing the following variables : age , gender , bmi , whr , family history of diabetes and hypertension , and smoking status . site was a cross - sectional , national , multicentric , non - randomized , observational study . the primary objective of the study was to estimate the prevalence of diagnosed and undiagnosed diabetes and hypertension in outpatient settings in major indian states . the secondary objectives included estimation of the prevalence of other cardiovascular risk factors such as dyslipidemia , obesity [ body mass index ( bmi ) , waist circumference ( wc ) , and waist - hip ratio ( whr ) ] , metabolic syndrome , urine microalbuminuria , arrhythmia , diet , smoking and alcohol consumption ; estimation of the prevalence of abdominal obesity in sub - groups of patients ( hypertension , diabetes , dyslipidemia , smokers and alcoholics ) ; and understanding the management and the level of control of hypertension and diabetes . the study was conducted in accordance with the guidelines for good epidemiology practices . the protocol complied with recommendations of the 18 world health congress ( helsinki , 1964 ) and the local laws and regulations of india . the sample size depended on the expected prevalence of diabetes / hypertension ( whichever was lower ) . based on the conservative expected prevalence of 15% for diabetes , it was determined that a sample size of 1225 patients was required per state . assuming a dropout rate of 39% , it was estimated that 2000 patients were required to ensure 95% confidence such that the prevalence of diabetes is between 13% and 17% , that is , 15% 2% in each state . it was initially planned to conduct the study in 10 states across five zones , north , south , east , west and central , in waves one state at a time and recruit 2000 patients from 100 centers per wave . physician selection : the study was conducted in outpatient settings and the data were collected from general practitioners rather than specialists . also , the source of any potential bias was much reduced , as the number of people presenting the disease would evidently be higher at a specialist clinic rather than a general practitioner 's clinic . patient selection : patients 18 years of age and those ready to sign the data release consent form along with an acceptance to take the screening tests were included in the study . pregnant women are predisposed to gestational diabetes and weight gain , and their exclusion decreased eventual confounding effects on the results . the first 10 patients visiting the physicians clinic on two consecutive days , irrespective of the purpose of the visit , and satisfying the inclusion criteria , were selected for the study . during 20092010 , site was conducted in 8 states including 7 of the top 10 most populous indian states and the national capital territory [ figure 1 ] . for the remaining two planned states , on the basis of the fact that the sample size was calculated per state and that a low dropout rate was observed among the patients enrolled , the national coordinators deemed the data adequate to pan out substantial results . so , by the end of the 8 wave , the recruitment was declared completed . because of a national multicentric approach of the study , the data obtained were representative of diverse demographic populations within india . site management organization ( smo ) and data management services were provided by max neeman ltd . the sponsor ensured that the central laboratory , smo , and data management personnel dedicated to study , as well as the participating physicians , received adequate training on various aspects of the study . thus , the complete study team was well versed with the data collection form ( dcf ) and the study procedures were executed uniformly . the data collection process was carried out in two steps [ figure 2 ] . data collection process of the screening india 's twin epidemic study the dcf used was designed in line with the study objectives . it captured details on the following aspects : demographics ( age and gender ) , anthropometrics ( height , weight , waist and hip circumference ) , behavioral ( smoking and alcohol use ) , medical history ( diabetes and its treatment , hypertension and its treatment , and cardiovascular diseases like ischemic heart disease , myocardial infarction and stroke ) , type of diet ( vegetarian / non - vegetarian ) , and family history of diabetes and hypertension . details on the patient 's socioeconomic , educational , psychosocial , and physical activity status were not collected . anthropometric measurements : height was measured without shoes , in centimeters , using a standard stadiometer . weight was measured with light clothes and without shoes , in kilograms , using a professional calibrated weighing scale , and rounded off to the nearest number . since no standard weighing machine was employed , inter - instrument variations can not be ruled out . body mass index ( bmi ) was calculated using the formula : weight ( kg)/height ( m ) . the hip and waist measurements were taken using a standard non - stretchable anthropometric measure tape across all study sites . the hip circumference ( hc ) was measured over light clothing . the patient was made to stand erect with arms at the sides , feet placed together with weight equally distributed on each leg , and the gluteal muscles relaxed . the tape was placed horizontally around the point of the maximum posterior extension of the buttocks . the patient 's measured hc was calculated as the mean of the two observations or the mean of the two closest measurements . the patient was made to stand erect with arms at the sides and feet placed about 2530 cm apart with weight equally distributed on each leg . the wc was measured at the part of the trunk located midway between the lower costal margin and the iliac crest . the measurer stood beside the patient and made the tape fit snugly , without compressing any underlying soft tissues . the circumference was measured in centimeters to the nearest 0.5 cm at the end of a normal expiration . the method and angles of measurement were adequately detailed to ensure limited variance in the technique . whr was calculated by dividing the wc ( cm ) by the hc ( cm ) . blood pressure ( bp ) measurement : a mercury sphygmomanometer was used to measure the bp , preferably from the right arm of the patient . an average of two readings , recorded at 5-minutes interval , was taken to ensure accuracy . at the end of visit 1 , patients received a coupon of identification and were required to visit any of the nearest metropolis clinical diagnostic centers , within 2 - 5 days . fasting lipid profile , microalbuminuria , fasting plasma glucose ( fpg ) , and hba1c were determined for each patient presenting a valid coupon . hba1c was measured by the bio - rad d10 high - pressure liquid chromatography method . urine microalbumin was measured by a turbidimetric inhibition immunoassay technique and enzymatic reactions were applied to quantify total cholesterol ( cholesterol esterase ) , high - density lipoproteins ( hdl ; cholesterol oxidase , esterase , and peroxidase ) , and triglycerides ( tg ; lipoprotein lipase , glycerol kinase , glycerol-3-phosphate oxidase , and peroxidase ) . low - density lipoprotein ( ldl ) was also calculated by using friedewald 's equation for tg < 300 mg / dl . it captured details on the following aspects : demographics ( age and gender ) , anthropometrics ( height , weight , waist and hip circumference ) , behavioral ( smoking and alcohol use ) , medical history ( diabetes and its treatment , hypertension and its treatment , and cardiovascular diseases like ischemic heart disease , myocardial infarction and stroke ) , type of diet ( vegetarian / non - vegetarian ) , and family history of diabetes and hypertension . details on the patient 's socioeconomic , educational , psychosocial , and physical activity status were not collected . anthropometric measurements : height was measured without shoes , in centimeters , using a standard stadiometer . weight was measured with light clothes and without shoes , in kilograms , using a professional calibrated weighing scale , and rounded off to the nearest number . since no standard weighing machine was employed , inter - instrument variations can not be ruled out . body mass index ( bmi ) was calculated using the formula : weight ( kg)/height ( m ) . the hip and waist measurements were taken using a standard non - stretchable anthropometric measure tape across all study sites . the hip circumference ( hc ) the patient was made to stand erect with arms at the sides , feet placed together with weight equally distributed on each leg , and the gluteal muscles relaxed . the tape was placed horizontally around the point of the maximum posterior extension of the buttocks . the patient 's measured hc was calculated as the mean of the two observations or the mean of the two closest measurements . the patient was made to stand erect with arms at the sides and feet placed about 2530 cm apart with weight equally distributed on each leg . the wc was measured at the part of the trunk located midway between the lower costal margin and the iliac crest . the measurer stood beside the patient and made the tape fit snugly , without compressing any underlying soft tissues . the circumference was measured in centimeters to the nearest 0.5 cm at the end of a normal expiration . the method and angles of measurement were adequately detailed to ensure limited variance in the technique . whr was calculated by dividing the wc ( cm ) by the hc ( cm ) . blood pressure ( bp ) measurement : a mercury sphygmomanometer was used to measure the bp , preferably from the right arm of the patient . an average of two readings , recorded at 5-minutes interval , was taken to ensure accuracy . at the end of visit 1 , patients received a coupon of identification and were required to visit any of the nearest metropolis clinical diagnostic centers , within 2 - 5 days . fasting lipid profile , microalbuminuria , fasting plasma glucose ( fpg ) , and hba1c were determined for each patient presenting a valid coupon . hba1c was measured by the bio - rad d10 high - pressure liquid chromatography method . urine microalbumin was measured by a turbidimetric inhibition immunoassay technique and enzymatic reactions were applied to quantify total cholesterol ( cholesterol esterase ) , high - density lipoproteins ( hdl ; cholesterol oxidase , esterase , and peroxidase ) , and triglycerides ( tg ; lipoprotein lipase , glycerol kinase , glycerol-3-phosphate oxidase , and peroxidase ) . low - density lipoprotein ( ldl ) was also calculated by using friedewald 's equation for tg < 300 mg / dl . diabetes was diagnosed according to the american diabetes association ( ada ) guidelines , and hypertension was classified according to the recommendations of the seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ( jnc 7 ) . metabolic syndrome was diagnosed on the basis of national cholesterol education program ( ncep ) . bmi was categorized into normal ( < 23 kg / m ) , overweight ( 2325 kg / m ) and obese ( > 25 kg / m ) , according to the guidelines jointly suggested by the indian health ministry , diabetes foundation of india , all india institute of medical sciences , indian council of medical research , and national institute of nutrition and 20 other institutions . normal and truncal obesity was categorized according to the whr , as suggested by the ncep . fasting lipid profile revealed the total cholesterol , tg , hdl - cholesterol , and ldl - cholesterol levels . of these , elevated tg ( 150 mg / dl ) and concomitant low hdl - cholesterol ( < 40 mg / dl ) were used to identify dyslipidemia . the criteria used are ( 1 ) primarily attributed to atherogenic dyslipidemia , ( 2 ) linked to insulin resistance , ( 3 ) measurable during the initial phase of development of metabolic syndrome , ( 4 ) individually extremely inheritable , ( 5 ) simple to measure , and ( 6 ) determinants of the atherogenic index of plasma ( aip ) , a key prognostic marker of cardiovascular disease , calculated as log ( tg / hdl - c ) . urine microalbumin was defined according to the albumin creatinine ratio ( acr ) as normoalbuminuria ( acr < 30 g / mg ) , microalbuminuria ( 30300 g / mg ) , and macroalbuminuria ( > 300 g / mg ) . monitoring visits were randomly conducted for at least 20% of the active sites ( enrolled at least one patient ) and the reports documented . phlebotomy services , testing , processing , shipment of blood and urine samples , and preparation and distribution of reports were undertaken by the central laboratory ( metropolis healthcare ltd . ) . data entry , verification , and validation were carried out using pheedit database system , version 3.1 ( sas institute inc . , a double - entry method was used to ensure that the data ( except comments ) were transferred accurately from the dcfs to the database . moreover a data checking plan was established to define all automatic validation checks , as well as the supplemental manual checks . all discrepancies were researched until resolved . additionally , a national steering committee comprising india 's leading endocrinologists and cardiologists was established to provide scientific direction for the study . the total sample number , mean , standard deviation , and range ( minimum and maximum ) were calculated for all continuous variables ( age , height , wc , hc , and lipid values ) , and frequency and percentage were calculated for all categorical variables ( gender , presence of hypertension and diabetes and their treatment , smoking status , and urine microalbuminuria ) . student 's t - test and chi - square test were used to make comparisons between groups , with p value < 0.05 considered statistically significant . a multivariate analysis was performed to estimate the association of hypertension and diabetes with dyslipidemia , obesity , metabolic syndrome , microalbuminuria , arrhythmia , smoking , alcohol consumption , and diet . a simple linear regression analysis was performed to estimate the contribution of individual variables to systolic and diastolic bp . relative risk for developing diabetes and hypertension was calculated by assessing the following variables : age , gender , bmi , whr , family history of diabetes and hypertension , and smoking status . screening studies play a very important role in determining the trends of disease prevalence ; such trends can provide valuable insight regarding necessary corrective measures that can be implemented in a given population . india has witnessed a constant increase in the prevalence of diseases such as diabetes and hypertension . however , most previous reports on the prevalence of both diabetes and hypertension are either regional or vary in methodologies and sampling frames . site aims not only to indicate the most accurate prevalence data of diabetes and hypertension in an outpatient setting in india at the state , regional , and national levels , but also to raise awareness on the need for early diagnosis and on the management of these diseases to reduce complications . after wave-1 and wave-2 , the protocol was refined and the laboratory investigations simplified and then employed for all other subsequent waves . during the first wave conducted in maharashtra , the investigations included : random blood sugar ( rbs ; in all patients ) , electrocardiogram ( ecg ; in patients 40 years of age ) , and hba1c ( in known cases of diabetes ) at visit-1 ; and the oral glucose tolerance test ( ogtt ; in patients with rbs 140 mg / dl ) and microalbuminuria test ( in patients with rbs 140 mg / dl and in patients with hypertension ) at visit-2 . the ogtt involved a lengthy procedure that was logistically difficult to perform in general clinics . going forward , this attrition could have potentially introduced a bias in the study results . thus , based on the experiences of the pilot study in maharashtra , certain protocol improvements were suggested for the second wave conducted in the national capital territory , delhi . the ada recommends either ogtt or fpg and disease - related symptoms ( polydipsia , polyuria , and unexplained weight loss ) for the confirmation of diabetes . during wave-2 , the ogtt was not performed ; fpg was measured for all unknown cases of diabetes , as opposed to rbs in the pilot study . though signs and symptoms of diabetes were recorded in this amended protocol , they did not play a decisive role in diagnosing new cases of diabetes . additionally , specifications with regard to alcohol consumption and diet of patients were included in the dcf . alcohol consumption and diet are established high - risk factors leading to the development and progression of diabetes and hypertension . since significant regional differences exist in the indian diet , it would be interesting to assess if a correlation exists between the regional prevalence of these diseases and diet . to establish a connection between lifestyle factors ( diet and alcohol consumption ) and prevalence of diabetes and hypertension this inclusion also broadened the scope of secondary analyses to be performed with the results of this study . during wave-2 , difficulties in maintaining uniformity in the ecg recordings and in dealing with logistical hurdles of organizing the ecg test at the sites surfaced . the inability to standardize ecg testing in a study of this magnitude would lead to inaccurate data generation . additionally , as an effort to further simplify the protocol , the fpg and hba1c testing was conducted in all patients from wave-3 onward . however , by employing hba1c as a screening tool as per the newly proposed guidelines released in july 2009 , our approach to diagnosing diabetes was further simplified . additionally , the correlation between fpg and hba1c for the diagnosis of diabetes could also be studied . an important limitation of this study is the reliance on a single - day fpg and bp measurement to detect new cases of diabetes and hypertension , respectively . also , owing to the cross - sectional nature of our study , the possibility of residual confounding can not be ruled out . nonetheless , site is one of the largest prevalence studies on diabetes and hypertension conducted in outpatient settings in india . due to the national multicentric approach of the site study , the amendments to the site protocol were efforts made to maintain consistency and accuracy of the data collected as the study progressed through each subsequent wave . the wave-3 protocol ( conducted in tamil nadu ) reflected the most adequate data collection process for this study and was employed for all other waves , permitting robust analyses . in conclusion , site will provide a real - world national perspective on diabetes and hypertension and their contributing risk factors . it will identify practice variations across states and evaluate compliance to international guidelines for the management of diabetes and hypertension . the standardization of the data collection process and the data analyses will justify various comparisons . the study will raise awareness on the need for early diagnosis and management of disease to reduce complications . site data will be useful for national recommendations , policies and guidelines , and will also support future exploratory researches .
objectives : the recent years have seen a surge in the prevalence of both diabetes and hypertension . significant demographic variations reported on the prevalence patterns of diabetes and hypertension in india establish a clear need for a nation - wide surveillance study . the screening india 's twin epidemic ( site ) study aimed at collecting information on the prevalence of diagnosed and undiagnosed diabetes and hypertension cases in outpatient settings in major indian states to better understand disease management , as well as to estimate the extent of underlying risk factors.materials and methods : during 20092010 , site was conducted in eight states , in waves one state at a time . it was planned to recruit about 2000 patients from 100 centers per wave . each center enrolled the first 10 eligible patients ( 18 years of age , not pregnant , signed data release consent form , and ready to undergo screening tests ) per day on two consecutive days . patient demographics , medical history , and laboratory investigation results were collected and statistically interpreted . the protocol defined diabetes and hypertension as per the american diabetes association ( ada ) and seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ( jnc 7 ) recommendations , respectively.results:after the first two pilot phases in maharashtra and delhi , the protocol was refined and the laboratory investigations were simplified to be further employed for all other states , namely , tamil nadu , west bengal , karnataka , andhra pradesh , madhya pradesh , and gujarat.conclusion:site's nation - wide approach will provide a real - world perspective on diabetes and hypertension and its contributing risk factors . results from the study will raise awareness on the need for early diagnosis and management of these diseases to reduce complications .
I M Study design Sample size calculation Sampling design Study waves Training Methods At visit 1 At visit 2 Definitions and diagnostic criteria Quality control Statistical considerations R
the screening india 's twin epidemic ( site ) study was aimed at determining the number of diagnosed and undiagnosed cases of diabetes and hypertension in india and reporting the measures employed to manage and control these diseases in an outpatient setting . the study was conducted in eight states maharashtra , delhi , tamil nadu , west bengal , karnataka , andhra pradesh , madhya pradesh , and gujarat . also , in lieu of poor existing information , site serves as a good proxy for providing valuable insights on the national prevalence and management of both diabetes and hypertension . the primary objective of the study was to estimate the prevalence of diagnosed and undiagnosed diabetes and hypertension in outpatient settings in major indian states . it was initially planned to conduct the study in 10 states across five zones , north , south , east , west and central , in waves one state at a time and recruit 2000 patients from 100 centers per wave . patient selection : patients 18 years of age and those ready to sign the data release consent form along with an acceptance to take the screening tests were included in the study . during 20092010 , site was conducted in 8 states including 7 of the top 10 most populous indian states and the national capital territory [ figure 1 ] . it captured details on the following aspects : demographics ( age and gender ) , anthropometrics ( height , weight , waist and hip circumference ) , behavioral ( smoking and alcohol use ) , medical history ( diabetes and its treatment , hypertension and its treatment , and cardiovascular diseases like ischemic heart disease , myocardial infarction and stroke ) , type of diet ( vegetarian / non - vegetarian ) , and family history of diabetes and hypertension . diabetes was diagnosed according to the american diabetes association ( ada ) guidelines , and hypertension was classified according to the recommendations of the seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ( jnc 7 ) . the primary objective of the study was to estimate the prevalence of diagnosed and undiagnosed diabetes and hypertension in outpatient settings in major indian states . it was initially planned to conduct the study in 10 states across five zones , north , south , east , west and central , in waves one state at a time and recruit 2000 patients from 100 centers per wave . patient selection : patients 18 years of age and those ready to sign the data release consent form along with an acceptance to take the screening tests were included in the study . during 20092010 , site was conducted in 8 states including 7 of the top 10 most populous indian states and the national capital territory [ figure 1 ] . it captured details on the following aspects : demographics ( age and gender ) , anthropometrics ( height , weight , waist and hip circumference ) , behavioral ( smoking and alcohol use ) , medical history ( diabetes and its treatment , hypertension and its treatment , and cardiovascular diseases like ischemic heart disease , myocardial infarction and stroke ) , type of diet ( vegetarian / non - vegetarian ) , and family history of diabetes and hypertension . it captured details on the following aspects : demographics ( age and gender ) , anthropometrics ( height , weight , waist and hip circumference ) , behavioral ( smoking and alcohol use ) , medical history ( diabetes and its treatment , hypertension and its treatment , and cardiovascular diseases like ischemic heart disease , myocardial infarction and stroke ) , type of diet ( vegetarian / non - vegetarian ) , and family history of diabetes and hypertension . diabetes was diagnosed according to the american diabetes association ( ada ) guidelines , and hypertension was classified according to the recommendations of the seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ( jnc 7 ) . site aims not only to indicate the most accurate prevalence data of diabetes and hypertension in an outpatient setting in india at the state , regional , and national levels , but also to raise awareness on the need for early diagnosis and on the management of these diseases to reduce complications . after wave-1 and wave-2 , the protocol was refined and the laboratory investigations simplified and then employed for all other subsequent waves . during the first wave conducted in maharashtra , the investigations included : random blood sugar ( rbs ; in all patients ) , electrocardiogram ( ecg ; in patients 40 years of age ) , and hba1c ( in known cases of diabetes ) at visit-1 ; and the oral glucose tolerance test ( ogtt ; in patients with rbs 140 mg / dl ) and microalbuminuria test ( in patients with rbs 140 mg / dl and in patients with hypertension ) at visit-2 . nonetheless , site is one of the largest prevalence studies on diabetes and hypertension conducted in outpatient settings in india . in conclusion , site will provide a real - world national perspective on diabetes and hypertension and their contributing risk factors . the study will raise awareness on the need for early diagnosis and management of disease to reduce complications .
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asthma control is characterised by the management of symptoms and reduction of predicted future risk , particularly exacerbations . some asthma exacerbations can be caused by a range of risk factors and irritants , including exercise , allergens , pollutants and occupational exposures , viral infections , medications and other factors such as emotional stress or co - existing medical conditions . triggers. ongoing exposure to asthma triggers is often a factor in poorly controlled asthma [ 1,37 ] . asthma management guidelines and educational materials recommend that healthcare professionals educate patients to identify those triggers that worsen asthma symptoms and lead to loss of control , and take steps to avoid these triggers [ 1,811 ] . however , there is mixed evidence whether the monitoring and provision of advice , and discussion of environmental control strategies with asthma patients is adequate or suboptimal . the reasons why education on trigger identification and avoidance may be suboptimal are unclear but may relate to a perception that little can be done about asthma triggers or that consultations focus on direct clinical management . another potential reason could be a lack of understanding of what an individual patient considers to be a trigger and the total trigger burden for that patient . therefore , there is a clear need to improve understanding of the types of triggers that affect asthma patients , how often triggers are encountered , and how the overall burden of asthma triggers impacts disease control . the current epidemiological study was conducted to identify the types , frequency and impact of asthma triggers , and to investigate the relationship between asthma trigger burden ( number and frequency ) and asthma control among adults with asthma across five european countries . this was a questionnaire- and diary - based study designed to investigate the type , frequency and impact of asthma triggers among adults with asthma . the study was conducted in five european countries ( france , germany , italy , spain and the uk ) between november and december 2010 . members of the panel were sent an invitation to take part in the survey via an email containing a link to the online questionnaire . the questionnaire contained a series of screening questions and only eligible participants completed the main questionnaire ( approximately 25 min in duration ) . a random subset of participants who completed the questionnaire and who reported that specific events or activities triggered a worsening of their asthma were then asked to complete an online diary at least every 2 d over 34 weeks . eligible participants were adults with a physician diagnosis of asthma ( self - reported ) and currently receiving maintenance asthma treatment and self - reported exposure to known asthma triggers . eligibility was determined using a series of online screening questions and only those meeting the inclusion criteria proceeded to the online questionnaire . patients were advised in the email invitation that accessing the online questionnaire provided their consent to partake in the study . the survey was developed based on qualitative research and expert opinion and was designed to describe and quantify common triggers for patients with asthma , understand and quantify coping behaviours in the presence of asthma triggers , and to understand and quantify how patients modify their lifestyles in response to triggers , and the impact this lifestyle modification has on their everyday quality of life . participants were also asked about the frequency of severe asthma attacks , asthma worsening and use of reliever medication . participants were then asked to indicate how well controlled they perceived their asthma to be on a scale of 010 ( 0 = very poorly controlled and 10 = completely controlled ) . the previously defined cut - off score of 20 was used to identify participants as having well controlled ( act score 20 ) or uncontrolled asthma ( act score < 20 ) . asthma control was also evaluated in relation to the global initiative for asthma ( gina ) guidelines . the gina guidelines recommend evaluation of asthma control in relation to five characteristics : daytime symptoms , limitation of activities , nocturnal symptoms / awakenings , need for reliever / rescue medication and lung function . participants were asked to report on the occurrence of day- and night - time asthma symptoms and the frequency with which these affected their planned activities as well as the frequency of rescue medication use in the previous week . lung function data was not available so the remaining four characteristics were used as a proxy to define the level of asthma control as follows : ( 1 ) controlled : daytime symptoms no more than twice a week , no night - time symptoms , no limitation to activities in the previous four weeks and rescue medication use no more than once a week ; ( 2 ) poorly controlled : daytime symptoms more than twice a week or night - time symptoms or limitation to activities in the previous four weeks or rescue medication use more than once a week ; and ( 3 ) uncontrolled : three or more features of poorly controlled asthma . participants were presented with a list of 36 potential asthma triggers and asked to indicate which had ever resulted in a worsening of their asthma . the list of 36 potential asthma triggers was developed based on qualitative interviews conducted with patients with asthma . the perceived impact of asthma on the daily lives of participants was derived from the question overall , how much of an impact do you think asthma has had on your life and the things you are able to do?. participants were asked to respond on a likert - type scale of 1 ( very mild ) to 10 ( very severe ) . the purpose of the diary was to understand the impact of asthma triggers over a period of time . a random subset of participants were recruited after completing the main online survey . at the end of the survey participants were asked if they wanted to take part in the diary part of the research and an additional online link was then sent to them . the diaries specifically elicited information on daily exposure to asthma triggers , the impact of exposure to asthma triggers on asthma symptoms , coping strategies and behaviours in relation to exposure to asthma triggers , as well as the impact of these strategies and behaviours on work and home activities . to investigate the relationship between asthma trigger burden ( number and frequency ) and asthma control an asthma trigger frequency index ( ranging from 0 to 1 ) was calculated using the annual self - reported trigger exposure for all asthma triggers for each individual participant . a cut off of 0.5 was used to stratify patients into high trigger frequency > 0.5 and low trigger frequency ( 0.5 ) . participants were also stratified according to the level of asthma control ( act or gina ) and their self - reported asthma trigger burden ( number of triggers identified by the patient as causing asthma symptoms ) : very low ( 15 ) , low ( 610 ) , medium ( 1115 ) and high ( 16 ) . statistically significant differences between groups in relation to trigger burden , level of asthma control and behavioural changes in relation to asthma triggers were determined using chi - square testing and analysis of variance ( kruskal wallis ) . descriptive statistics and multivariate regression analyses are presented on risk factors associated with differences in the mean number of severe attacks in a lifetime , the annual number of times individuals reported hospitalisation in the last year and the annual number of days missed at work or study in the last year due to asthma . a post hoc , investigational regression analysis was also undertaken in order to examine the impact of a variety of potential covariates on socioeconomic outcomes ( poisson regression ) , impact of daily life and activities ( linear regression ) and the number of behavioural changes patients made in order to manage their exposure to known asthma triggers ( logistic regression ) . the following covariates of interest were included in all analyses : trigger burden : very low ( reference ) , low , medium , highage group : 1830 years ( reference ) , 3144 years and > 45 yearsdwelling : rural ( reference ) , urbancountry : germany ( reference ) , uk , spain , france , italygender : male ( reference ) , femaleself - reported physician - defined asthma severity : mild ( reference ) , moderate , severe , not informed trigger burden : very low ( reference ) , low , medium , high age group : 1830 years ( reference ) , 3144 years and > 45 years dwelling : rural ( reference ) , urban country : germany ( reference ) , uk , spain , france , italy gender : male ( reference ) , female self - reported physician - defined asthma severity : mild ( reference ) , moderate , severe , not informed results are reported with 95% confidence intervals . this was a questionnaire- and diary - based study designed to investigate the type , frequency and impact of asthma triggers among adults with asthma . the study was conducted in five european countries ( france , germany , italy , spain and the uk ) between november and december 2010 . members of the panel were sent an invitation to take part in the survey via an email containing a link to the online questionnaire . the questionnaire contained a series of screening questions and only eligible participants completed the main questionnaire ( approximately 25 min in duration ) . a random subset of participants who completed the questionnaire and who reported that specific events or activities triggered a worsening of their asthma were then asked to complete an online diary at least every 2 d over 34 weeks . eligible participants were adults with a physician diagnosis of asthma ( self - reported ) and currently receiving maintenance asthma treatment and self - reported exposure to known asthma triggers . eligibility was determined using a series of online screening questions and only those meeting the inclusion criteria proceeded to the online questionnaire . patients were advised in the email invitation that accessing the online questionnaire provided their consent to partake in the study . the survey was developed based on qualitative research and expert opinion and was designed to describe and quantify common triggers for patients with asthma , understand and quantify coping behaviours in the presence of asthma triggers , and to understand and quantify how patients modify their lifestyles in response to triggers , and the impact this lifestyle modification has on their everyday quality of life . participants were also asked about the frequency of severe asthma attacks , asthma worsening and use of reliever medication . participants were then asked to indicate how well controlled they perceived their asthma to be on a scale of 010 ( 0 = very poorly controlled and 10 = completely controlled ) . as part of the survey participants the previously defined cut - off score of 20 was used to identify participants as having well controlled ( act score 20 ) or uncontrolled asthma ( act score < 20 ) . asthma control was also evaluated in relation to the global initiative for asthma ( gina ) guidelines . the gina guidelines recommend evaluation of asthma control in relation to five characteristics : daytime symptoms , limitation of activities , nocturnal symptoms / awakenings , need for reliever / rescue medication and lung function . participants were asked to report on the occurrence of day- and night - time asthma symptoms and the frequency with which these affected their planned activities as well as the frequency of rescue medication use in the previous week . lung function data was not available so the remaining four characteristics were used as a proxy to define the level of asthma control as follows : ( 1 ) controlled : daytime symptoms no more than twice a week , no night - time symptoms , no limitation to activities in the previous four weeks and rescue medication use no more than once a week ; ( 2 ) poorly controlled : daytime symptoms more than twice a week or night - time symptoms or limitation to activities in the previous four weeks or rescue medication use more than once a week ; and ( 3 ) uncontrolled : three or more features of poorly controlled asthma . participants were presented with a list of 36 potential asthma triggers and asked to indicate which had ever resulted in a worsening of their asthma . the list of 36 potential asthma triggers was developed based on qualitative interviews conducted with patients with asthma . the perceived impact of asthma on the daily lives of participants was derived from the question overall , how much of an impact do you think asthma has had on your life and the things you are able to do?. participants were asked to respond on a likert - type scale of 1 ( very mild ) to 10 ( very severe ) . the purpose of the diary was to understand the impact of asthma triggers over a period of time . a random subset of participants were recruited after completing the main online survey . at the end of the survey participants were asked if they wanted to take part in the diary part of the research and an additional online link was then sent to them . the diaries specifically elicited information on daily exposure to asthma triggers , the impact of exposure to asthma triggers on asthma symptoms , coping strategies and behaviours in relation to exposure to asthma triggers , as well as the impact of these strategies and behaviours on work and home activities . to investigate the relationship between asthma trigger burden ( number and frequency ) and asthma control an asthma trigger frequency index ( ranging from 0 to 1 ) was calculated using the annual self - reported trigger exposure for all asthma triggers for each individual participant . a cut off of 0.5 was used to stratify patients into high trigger frequency > 0.5 and low trigger frequency ( 0.5 ) . participants were also stratified according to the level of asthma control ( act or gina ) and their self - reported asthma trigger burden ( number of triggers identified by the patient as causing asthma symptoms ) : very low ( 15 ) , low ( 610 ) , medium ( 1115 ) and high ( 16 ) . statistically significant differences between groups in relation to trigger burden , level of asthma control and behavioural changes in relation to asthma triggers were determined using chi - square testing and analysis of variance ( kruskal wallis ) . descriptive statistics and multivariate regression analyses are presented on risk factors associated with differences in the mean number of severe attacks in a lifetime , the annual number of times individuals reported hospitalisation in the last year and the annual number of days missed at work or study in the last year due to asthma . a post hoc , investigational regression analysis was also undertaken in order to examine the impact of a variety of potential covariates on socioeconomic outcomes ( poisson regression ) , impact of daily life and activities ( linear regression ) and the number of behavioural changes patients made in order to manage their exposure to known asthma triggers ( logistic regression ) . the following covariates of interest were included in all analyses : trigger burden : very low ( reference ) , low , medium , highage group : 1830 years ( reference ) , 3144 years and > 45 yearsdwelling : rural ( reference ) , urbancountry : germany ( reference ) , uk , spain , france , italygender : male ( reference ) , femaleself - reported physician - defined asthma severity : mild ( reference ) , moderate , severe , not informed trigger burden : very low ( reference ) , low , medium , high age group : 1830 years ( reference ) , 3144 years and > 45 years dwelling : rural ( reference ) , urban country : germany ( reference ) , uk , spain , france , italy gender : male ( reference ) , female self - reported physician - defined asthma severity : mild ( reference ) , moderate , severe , not informed results are reported with 95% confidence intervals . a total of 1202 adults with asthma were considered eligible to participate in this study and completed the online survey ( table 1 ) . of these , 177 also completed the online diary and provided data on a total of 1947 d.table 1.demographics and disease characteristics.characteristicproportion of participants , % ( n = 1202)gender male49 female51age range 1830 years35 3144 years37 4555 years27place of residence large town or city53 small town or suburbs31 village / hamlet16working status employed full time55 not currently working25 employed part - time10 self - employed10do not currently smoke72age at diagnosis 25 years of age68 2640 years24 > 40 years of age8duration with asthma 5 years25 620 years45 > 20 years31asthma control : self - reported daytime symptoms in the last 7 d85 night - time symptoms in the last 7 d79 experienced a worsening of symptoms at least once a week53uncontrolled asthma ( act score < 20 points)76partly controlled or uncontrolled asthma ( gina guidelines ) 92 proxy measure for gina definition : daytime symptoms experienced more than twice a week , night - time symptoms , had to limit activities in the last four weeks , had to use rescue medication more than once a week . proxy measure for gina definition : daytime symptoms experienced more than twice a week , night - time symptoms , had to limit activities in the last four weeks , had to use rescue medication more than once a week . most ( 85% ) reported experiencing daytime symptoms and 79% reported experiencing night - time symptoms at least once in the past 7 d. in all , 53% of participants indicated that they experienced a worsening of their asthma symptoms at least once a week . the majority of participants ( 76% ) were classed as uncontrolled using the act score , while 92% of participants were described as partly controlled or uncontrolled according to the gina guidelines . the number of self - reported asthma triggers varied considerably between participants , ranging from 1 to 5 ( 13% of participants ) to 16 ( 35% ) ; 52% of participants reported exposure to between 6 and 15 triggers . the most common asthma triggers as reported by participants were dust or dusting ( 72% ) , colds , flu , infections or sinusitis ( 69% ) , coughing ( 68% ) , tobacco smoking ( 60% ) and smog or air pollution ( 58% ) ( table 2).table 2.self-reported asthma triggers as ranked by the proportion of participants who have ever experienced the trigger and by how frequently the trigger was reported.trigger% who have ever experienced n = 1202triggerfrequency ( weeks / year ) n = 1202dust or dusting72dust or dusting18.6cold , flu , infections , sinusitis69smoking ( e.g. cigarettes or cigars)14.2coughing68coughing11.3smoking ( e.g. cigarettes or cigars)60exercise11.1smoke59vacuum cleaning10.1smog , air pollution58perfumes , hairspray or air fresheners10.0exercise54animals9.6strong odours54smog , air pollution9.5weather changes51smoke9.0mould and mould spores51emotions8.2animals50cleaning products8.2damp places48damp places8.1humidity48weather changes8.0perfumes , hairspray or air fresheners48humidity7.9grass , mowing the lawn , weeds47strong odours7.6cold air45mould and mould spores7.4emotions43cold air7.2cleaning products42grass , mowing the lawn , weeds6.9vacuum cleaning41lying flat6.8feathers40cold , flu , infections , sinusitis6.7air conditioning37air conditioning5.8lying flat29feathers5.4your work environment25your work environment4.8rain19rain3.4food , drinks or food colourings19food , drinks or food colourings3.1alcohol18indigestion or heart burn2.8other medications18alcohol2.4indigestion or heart burn17aspirin2.1eating out at particular restaurants15other medications2.0aspirin15eating out at particular restaurants1.8hormonal changes14hormonal changes1.8paracetamol12paracetamol1.6 self - reported asthma triggers as ranked by the proportion of participants who have ever experienced the trigger and by how frequently the trigger was reported . dust and dusting were the most commonly reported triggers and the most frequently experienced ( table 2 ) . colds / flu were commonly reported asthma triggers but were experienced less frequently during the year . a high asthma trigger burden was associated with a lack of asthma control , as measured by patient - reported control ( 5.4 for patients with very low and low trigger burdens , 5.8 for patients with medium trigger burden , and 6.5 for patients with high trigger burden ) or using the act score ( figure 1 ) . patients whose asthma was not well controlled ( act score ) made more behavioural modifications than those with controlled asthma . there were statistically significant differences between control groups in terms of the amount and type of housework undertaken , the amount of exercise taken and the choice to remain indoors due to weather conditions ( p < 0.05 across groups ; chi - square test ) ( figure 2).figure 1.relationship between trigger burden ( exposure and frequency ) and asthma control as measured by the act score ( total participants = 1202 ) . panel a depicts participants ( n = 696 [ 58% ] ) with a low to medium asthma trigger burden ( < 16 self - reported known asthma triggers ) and a low total frequency burden ( annual frequency index < 0.5 ) . panel b depicts participants ( n = 107 [ 9% ] ) with a low to medium asthma trigger burden ( < 16 self - reported known asthma triggers ) and a high total frequency burden ( annual frequency index 0.5 ) . panel c depicts participants ( n = 278 [ 23% ] ) with a high asthma trigger burden ( 16 self - reported known asthma triggers ) and a low total frequency burden ( annual frequency index < 0.5 ) . panel d depicts participants ( n = 121 [ 10% ] ) with a high asthma trigger burden ( 16 self - reported known asthma triggers ) and a high total frequency burden ( annual frequency index 0.5 ) . the shaded area on each panel depicts well controlled asthma ( act score > 20 ) . figure 2.asthma control ( act score ) and behavioural changes . this figure depicts the frequency with which participants stratified according to asthma control ( act score ) reported making behavioural changes to avoid or minimise exposure to known asthma triggers . relationship between trigger burden ( exposure and frequency ) and asthma control as measured by the act score ( total participants = 1202 ) . panel a depicts participants ( n = 696 [ 58% ] ) with a low to medium asthma trigger burden ( < 16 self - reported known asthma triggers ) and a low total frequency burden ( annual frequency index < 0.5 ) . panel b depicts participants ( n = 107 [ 9% ] ) with a low to medium asthma trigger burden ( < 16 self - reported known asthma triggers ) and a high total frequency burden ( annual frequency index 0.5 ) . panel c depicts participants ( n = 278 [ 23% ] ) with a high asthma trigger burden ( 16 self - reported known asthma triggers ) and a low total frequency burden ( annual frequency index < 0.5 ) . panel d depicts participants ( n = 121 [ 10% ] ) with a high asthma trigger burden ( 16 self - reported known asthma triggers ) and a high total frequency burden ( annual frequency index 0.5 ) . the shaded area on each panel depicts well controlled asthma ( act score > 20 ) . asthma control ( act score ) and behavioural changes . this figure depicts the frequency with which participants stratified according to asthma control ( act score ) reported making behavioural changes to avoid or minimise exposure to known asthma triggers . an analysis of the number of triggers to which participants reported being exposed to did not necessarily correspond to the frequency of asthma worsening , with a minority of participants ( 9% ) experiencing few asthma triggers but frequent asthma worsening ( figure 1 ) . the majority of participants ( 99% ) believed that exposure to known asthma triggers would have a long - term effect on their asthma . one in five participants reported that they made no modifications to their behaviour in response to known asthma triggers until prompted to do so by worsening asthma symptoms . participants were asked to rate the severity of asthma worsening in relation to the most commonly reported asthma triggers ; all scored 6 or above on a scale of 0 ( very mild ) to 10 ( very severe ) . the perceived severity of asthma worsening increased as the number of triggers experienced increased . as the number of known asthma triggers increased , participants reported statistically significantly more severe asthma attacks during their lifetime , more hospitalisations in the previous year , more days missed at work or study in the last year and more symptomatic days and nights per week ( table 3).table 3.burden of asthma triggers.patient groups by trigger numbers total n n = 1202very few ( 15 ) n = 156low ( 610 ) n = 293medium ( 1115 ) n = 335high ( 16 + ) n = 418kruskal wallismean number of severe attacks in lifetime3.2 ( median 2 , range 099)2.0 ( median 1 , range 015)2.7 ( median 2 , range 099)2.6 ( median 2 , range 020)4.6 ( median 2 , range 099)<0.001mean number of times hospitalised in the last year0.7 ( median 0 , range 025)0.5 ( median 0 , range 07)0.5 ( median 0 , range 06)0.8 ( median 0 , range 025)0.8 ( median 0 , range 020)0.038mean days missed at work or study in the last year because of asthma7.9 ( median 2 , range 0365)2.9 ( median 1 , range 080)6.1 ( median 2 , range 0288)6.3 ( median 2 , range 0265)12.2 ( median 2 , range 0365)<0.001 burden of asthma triggers . a number of covariates were associated with an increase in the socioeconomic burden of asthma ( table 4 ) . any increase in the level of known asthma triggers above very low was positively associated with a statistically significant increase in mean number of severe attacks in a lifetime ( p 0.01 ) , the number of times individuals were hospitalised in the last year ( p < 0.001 ) and the number of days missed at work or study in the last year ( p < 0.001 ) due to asthma . female gender was associated with an increased number of severe attacks ( p < 0.001 ) , hospitalisation ( p < 0.001 ) and missing work ( p < older age ( > 31 years ) was associated with an increased likelihood of hospitalisations and days lost to work or study in the previous year compared with age < 30 years . moderate or severe asthma severity ( physician informed and reported by patient ) was associated with increased rates of all the three above outcomes ( p < 0.001 ) . patients who reported they had severe asthma had three times the mean number of hospitalisations and four times more days off work than patients who reported they had mild asthma.table 4.covariates associated with an increase in the socioeconomic burden of asthma ( poisson regression analysis).mean severe attacks in a lifetime hospitalisations in the previous 12 months number of days of work or study missed in the previous 12 months variableestimate ( confidence interval ) p valueestimate ( confidence interval ) p valueestimate ( confidence interval ) p valuetrigger burden ( vs very low ) low1.38 ( 1.141.6)<0.011.13 ( 0.851.51)0.402.15 ( 1.942.39)<0.01 medium1.20 ( 1.051.37)0.011.90 ( 1.472.49)<0.011.93 ( 1.742.14)<0.01 high2.07 ( 1.832.34)<0.011.95 ( 1.512.55)<0.013.45 ( 3.133.81)<0.01age ( vs < 30 years ) 3144 years0.92 ( 0.861.00)0.040.63 ( 0.540.73)<0.010.99 ( 0.941.04)0.73 > 45 years1.06 ( 0.98115)0.170.47 ( 0.390.57)<0.011.48 ( 1.401.56)<0.01dwelling ( vs rural ) urban1.03 ( 0.971.1)0.360.74 ( 0.640.86)<0.010.56 ( 0.530.58)<0.01country ( vs germany ) uk1.24 ( 1.121.37)<0.010.87 ( 0.691.09)0.221.05 ( 1.001.11)0.07 spain1.21 ( 1.091.34)<0.011.02 ( 0.831.25)0.870.59 ( 0.550.63)<0.01 france1.22 ( 1.101.35)<0.010.75 ( 0.600.94)0.010.44 ( 0.410.47)<0.01 italy0.87 ( 0.780.98)0.020.81 ( 0.641.01)0.060.62 ( 0.590.66)<0.01gender ( vs male ) female0.83 ( 0.780.89)<0.010.60 ( 0.510.69)<0.011.05 ( 1.011.09)0.02asthma severity ( vs mild ) moderate1.43 ( 1.271.62)<0.012.04 ( 1.522.80)<0.012.00 ( 1.832.19)<0.01 severe2.44 ( 2.132.80)<0.013.41 ( 2.474.79)<0.014.09 ( 3.724.51)<0.01 not told1.82 ( 1.552.15)<0.010.87 ( 0.501.47)0.610.75 ( 0.650.87)<0.01 covariates associated with an increase in the socioeconomic burden of asthma ( poisson regression analysis ) . participants reporting a high number of known asthma triggers ( 16 ) reported that their asthma had a significantly greater impact on their overall daily life and job choice compared with those reporting very few , low or medium number of triggers . for overall daily life , asthma impact score was 5.9 compared with 4.5 , 4.7 and 5.3 , respectively ( p < 0.0001 across groups ; kruskal - wallis test ) . for job choice , asthma symptom score was 5.2 compared with 3.9 , 3.9 and 4.2 , respectively ( p = 0.002 across groups ; kruskal wallis test ) . linear regression analyses indicated that participants reporting a medium or high asthma trigger burden were significantly more likely to report an adverse impact on their overall daily life , as were participants in italy ( versus those in germany ; asthma impact score 0.61 points higher , p < 0.001 ) , females ( versus males ; 0.42 points lower , p < 0.001 ) and those with moderate or severe asthma ( versus mild ; asthma impact score 1.33 and 2.36 points , respectively , p < 0.001 for both ) . participants who reported that their asthma had impacted their daily life over the previous four weeks also reported making considerable behaviour changes to manage their asthma symptoms , the frequency of which increased as the number of triggers to which participants were exposed increased . the proportion of patients making any behaviour changes ranged from 67% for those with very few triggers to 89% for those with a high trigger burden ( p < 0.001 across groups ; chi - square test ) . a logistic regression analysis showed a significant positive association between the trigger burden and the number of times behavioural changes were made in order to manage exposure to known asthma triggers in the previous four weeks . the higher the burden , the greater the number of changes , with high trigger patients being 10 times more likely to change behaviour up to five times in a week than those with very low number of triggers . most ( 85% ) reported experiencing daytime symptoms and 79% reported experiencing night - time symptoms at least once in the past 7 d. in all , 53% of participants indicated that they experienced a worsening of their asthma symptoms at least once a week . the majority of participants ( 76% ) were classed as uncontrolled using the act score , while 92% of participants were described as partly controlled or uncontrolled according to the gina guidelines . the number of self - reported asthma triggers varied considerably between participants , ranging from 1 to 5 ( 13% of participants ) to 16 ( 35% ) ; 52% of participants reported exposure to between 6 and 15 triggers . the most common asthma triggers as reported by participants were dust or dusting ( 72% ) , colds , flu , infections or sinusitis ( 69% ) , coughing ( 68% ) , tobacco smoking ( 60% ) and smog or air pollution ( 58% ) ( table 2).table 2.self-reported asthma triggers as ranked by the proportion of participants who have ever experienced the trigger and by how frequently the trigger was reported.trigger% who have ever experienced n = 1202triggerfrequency ( weeks / year ) n = 1202dust or dusting72dust or dusting18.6cold , flu , infections , sinusitis69smoking ( e.g. cigarettes or cigars)14.2coughing68coughing11.3smoking ( e.g. cigarettes or cigars)60exercise11.1smoke59vacuum cleaning10.1smog , air pollution58perfumes , hairspray or air fresheners10.0exercise54animals9.6strong odours54smog , air pollution9.5weather changes51smoke9.0mould and mould spores51emotions8.2animals50cleaning products8.2damp places48damp places8.1humidity48weather changes8.0perfumes , hairspray or air fresheners48humidity7.9grass , mowing the lawn , weeds47strong odours7.6cold air45mould and mould spores7.4emotions43cold air7.2cleaning products42grass , mowing the lawn , weeds6.9vacuum cleaning41lying flat6.8feathers40cold , flu , infections , sinusitis6.7air conditioning37air conditioning5.8lying flat29feathers5.4your work environment25your work environment4.8rain19rain3.4food , drinks or food colourings19food , drinks or food colourings3.1alcohol18indigestion or heart burn2.8other medications18alcohol2.4indigestion or heart burn17aspirin2.1eating out at particular restaurants15other medications2.0aspirin15eating out at particular restaurants1.8hormonal changes14hormonal changes1.8paracetamol12paracetamol1.6 self - reported asthma triggers as ranked by the proportion of participants who have ever experienced the trigger and by how frequently the trigger was reported . dust and dusting were the most commonly reported triggers and the most frequently experienced ( table 2 ) . colds / flu were commonly reported asthma triggers but were experienced less frequently during the year . a high asthma trigger burden was associated with a lack of asthma control , as measured by patient - reported control ( 5.4 for patients with very low and low trigger burdens , 5.8 for patients with medium trigger burden , and 6.5 for patients with high trigger burden ) or using the act score ( figure 1 ) . patients whose asthma was not well controlled ( act score ) made more behavioural modifications than those with controlled asthma . there were statistically significant differences between control groups in terms of the amount and type of housework undertaken , the amount of exercise taken and the choice to remain indoors due to weather conditions ( p < 0.05 across groups ; chi - square test ) ( figure 2).figure 1.relationship between trigger burden ( exposure and frequency ) and asthma control as measured by the act score ( total participants = 1202 ) . panel a depicts participants ( n = 696 [ 58% ] ) with a low to medium asthma trigger burden ( < 16 self - reported known asthma triggers ) and a low total frequency burden ( annual frequency index < 0.5 ) . panel b depicts participants ( n = 107 [ 9% ] ) with a low to medium asthma trigger burden ( < 16 self - reported known asthma triggers ) and a high total frequency burden ( annual frequency index 0.5 ) . panel c depicts participants ( n = 278 [ 23% ] ) with a high asthma trigger burden ( 16 self - reported known asthma triggers ) and a low total frequency burden ( annual frequency index < 0.5 ) . panel d depicts participants ( n = 121 [ 10% ] ) with a high asthma trigger burden ( 16 self - reported known asthma triggers ) and a high total frequency burden ( annual frequency index 0.5 ) . the shaded area on each panel depicts well controlled asthma ( act score > 20 ) . figure 2.asthma control ( act score ) and behavioural changes . this figure depicts the frequency with which participants stratified according to asthma control ( act score ) reported making behavioural changes to avoid or minimise exposure to known asthma triggers . relationship between trigger burden ( exposure and frequency ) and asthma control as measured by the act score ( total participants = 1202 ) . panel a depicts participants ( n = 696 [ 58% ] ) with a low to medium asthma trigger burden ( < 16 self - reported known asthma triggers ) and a low total frequency burden ( annual frequency index < 0.5 ) . panel b depicts participants ( n = 107 [ 9% ] ) with a low to medium asthma trigger burden ( < 16 self - reported known asthma triggers ) and a high total frequency burden ( annual frequency index 0.5 ) . panel c depicts participants ( n = 278 [ 23% ] ) with a high asthma trigger burden ( 16 self - reported known asthma triggers ) and a low total frequency burden ( annual frequency index < 0.5 ) . panel d depicts participants ( n = 121 [ 10% ] ) with a high asthma trigger burden ( 16 self - reported known asthma triggers ) and a high total frequency burden ( annual frequency index 0.5 ) . the shaded area on each panel depicts well controlled asthma ( act score > 20 ) . asthma control ( act score ) and behavioural changes . this figure depicts the frequency with which participants stratified according to asthma control ( act score ) reported making behavioural changes to avoid or minimise exposure to known asthma triggers . an analysis of the number of triggers to which participants reported being exposed to did not necessarily correspond to the frequency of asthma worsening , with a minority of participants ( 9% ) experiencing few asthma triggers but frequent asthma worsening ( figure 1 ) . the majority of participants ( 99% ) believed that exposure to known asthma triggers would have a long - term effect on their asthma . one in five participants reported that they made no modifications to their behaviour in response to known asthma triggers until prompted to do so by worsening asthma symptoms . participants were asked to rate the severity of asthma worsening in relation to the most commonly reported asthma triggers ; all scored 6 or above on a scale of 0 ( very mild ) to 10 ( very severe ) . the perceived severity of asthma worsening increased as the number of triggers experienced increased . as the number of known asthma triggers increased , participants reported statistically significantly more severe asthma attacks during their lifetime , more hospitalisations in the previous year , more days missed at work or study in the last year and more symptomatic days and nights per week ( table 3).table 3.burden of asthma triggers.patient groups by trigger numbers total n n = 1202very few ( 15 ) n = 156low ( 610 ) n = 293medium ( 1115 ) n = 335high ( 16 + ) n = 418kruskal wallismean number of severe attacks in lifetime3.2 ( median 2 , range 099)2.0 ( median 1 , range 015)2.7 ( median 2 , range 099)2.6 ( median 2 , range 020)4.6 ( median 2 , range 099)<0.001mean number of times hospitalised in the last year0.7 ( median 0 , range 025)0.5 ( median 0 , range 07)0.5 ( median 0 , range 06)0.8 ( median 0 , range 025)0.8 ( median 0 , range 020)0.038mean days missed at work or study in the last year because of asthma7.9 ( median 2 , range 0365)2.9 ( median 1 , range 080)6.1 ( median 2 , range 0288)6.3 ( median 2 , range 0265)12.2 ( median 2 , range 0365)<0.001 burden of asthma triggers . a number of covariates were associated with an increase in the socioeconomic burden of asthma ( table 4 ) . any increase in the level of known asthma triggers above very low was positively associated with a statistically significant increase in mean number of severe attacks in a lifetime ( p 0.01 ) , the number of times individuals were hospitalised in the last year ( p < 0.001 ) and the number of days missed at work or study in the last year ( p < 0.001 ) due to asthma . female gender was associated with an increased number of severe attacks ( p < 0.001 ) , hospitalisation ( p < 0.001 ) and missing work ( p < older age ( > 31 years ) was associated with an increased likelihood of hospitalisations and days lost to work or study in the previous year compared with age < 30 years . moderate or severe asthma severity ( physician informed and reported by patient ) was associated with increased rates of all the three above outcomes ( p < 0.001 ) . patients who reported they had severe asthma had three times the mean number of hospitalisations and four times more days off work than patients who reported they had mild asthma.table 4.covariates associated with an increase in the socioeconomic burden of asthma ( poisson regression analysis).mean severe attacks in a lifetime hospitalisations in the previous 12 months number of days of work or study missed in the previous 12 months variableestimate ( confidence interval ) p valueestimate ( confidence interval ) p valueestimate ( confidence interval ) p valuetrigger burden ( vs very low ) low1.38 ( 1.141.6)<0.011.13 ( 0.851.51)0.402.15 ( 1.942.39)<0.01 medium1.20 ( 1.051.37)0.011.90 ( 1.472.49)<0.011.93 ( 1.742.14)<0.01 high2.07 ( 1.832.34)<0.011.95 ( 1.512.55)<0.013.45 ( 3.133.81)<0.01age ( vs < 30 years ) 3144 years0.92 ( 0.861.00)0.040.63 ( 0.540.73)<0.010.99 ( 0.941.04)0.73 > 45 years1.06 ( 0.98115)0.170.47 ( 0.390.57)<0.011.48 ( 1.401.56)<0.01dwelling ( vs rural ) urban1.03 ( 0.971.1)0.360.74 ( 0.640.86)<0.010.56 ( 0.530.58)<0.01country ( vs germany ) uk1.24 ( 1.121.37)<0.010.87 ( 0.691.09)0.221.05 ( 1.001.11)0.07 spain1.21 ( 1.091.34)<0.011.02 ( 0.831.25)0.870.59 ( 0.550.63)<0.01 france1.22 ( 1.101.35)<0.010.75 ( 0.600.94)0.010.44 ( 0.410.47)<0.01 italy0.87 ( 0.780.98)0.020.81 ( 0.641.01)0.060.62 ( 0.590.66)<0.01gender ( vs male ) female0.83 ( 0.780.89)<0.010.60 ( 0.510.69)<0.011.05 ( 1.011.09)0.02asthma severity ( vs mild ) moderate1.43 ( 1.271.62)<0.012.04 ( 1.522.80)<0.012.00 ( 1.832.19)<0.01 severe2.44 ( 2.132.80)<0.013.41 ( 2.474.79)<0.014.09 ( 3.724.51)<0.01 not told1.82 ( 1.552.15)<0.010.87 ( 0.501.47)0.610.75 ( 0.650.87)<0.01 covariates associated with an increase in the socioeconomic burden of asthma ( poisson regression analysis ) . participants reporting a high number of known asthma triggers ( 16 ) reported that their asthma had a significantly greater impact on their overall daily life and job choice compared with those reporting very few , low or medium number of triggers . for overall daily life , asthma impact score was 5.9 compared with 4.5 , 4.7 and 5.3 , respectively ( p < 0.0001 across groups ; kruskal - wallis test ) . for job choice , asthma symptom score was 5.2 compared with 3.9 , 3.9 and 4.2 , respectively ( p = 0.002 across groups ; kruskal wallis test ) . linear regression analyses indicated that participants reporting a medium or high asthma trigger burden were significantly more likely to report an adverse impact on their overall daily life , as were participants in italy ( versus those in germany ; asthma impact score 0.61 points higher , p < 0.001 ) , females ( versus males ; 0.42 points lower , p < 0.001 ) and those with moderate or severe asthma ( versus mild ; asthma impact score 1.33 and 2.36 points , respectively , p < 0.001 for both ) . participants who reported that their asthma had impacted their daily life over the previous four weeks also reported making considerable behaviour changes to manage their asthma symptoms , the frequency of which increased as the number of triggers to which participants were exposed increased . the proportion of patients making any behaviour changes ranged from 67% for those with very few triggers to 89% for those with a high trigger burden ( p < 0.001 across groups ; chi - square test ) . a logistic regression analysis showed a significant positive association between the trigger burden and the number of times behavioural changes were made in order to manage exposure to known asthma triggers in the previous four weeks . the higher the burden , the greater the number of changes , with high trigger patients being 10 times more likely to change behaviour up to five times in a week than those with very low number of triggers . the aim of this large - scale epidemiological questionnaire and diary - based study conducted among adults with asthma across five european countries was to identify the types , frequency and impact of asthma triggers , and to investigate the relationship between trigger burden and asthma control . the majority of participants in the study had uncontrolled asthma and reported 615 known asthma triggers . dust and dusting , colds / flu , sinusitis and coughing were the most common known asthma triggers with dust and dusting , smoking and exercise being the most frequently experienced triggers . an increase in trigger burden was associated with an increased likelihood of uncontrolled asthma , previous severe asthma attacks during a lifetime , more hospitalisations , more missed days at work / study , and behavioural changes to manage trigger exposure . the number of known asthma triggers reported by participants in this survey varied considerably from just one to more than 16 . many of these triggers have been reported in asthma guidelines , by a systematic literature review and documented in the literature [ 1524 ] , but the identification of such triggers has typically been through studies focusing on single triggers , not through population - based approaches . however , studies that more fully characterise the wide range of triggers experienced by individual asthma patients , and how frequently they are encountered , have been limited . one such approach was the development of the asthma trigger inventory , a questionnaire designed to measure the main categories of triggers self - reported by asthma patients , with a specific focus on psychosocial triggers . the most common categories of triggers were climate , physical and air pollution ( experienced by > 50% of patients ) , followed by infection , house dust , animal allergy , pollen allergy and psychology ( > 20 to < 50% of patients ) , then respiration , food , sleep , alcohol , mould and medication ( all < 20% of patients ) . with the exception of mould , which was a much more common trigger in the present survey than in the asthma trigger inventory ( 51% versus 2% ) , some of the triggers perceived by participants in this survey are not considered to be classic asthma triggers , for example coughing , strong odours and lying flat . although coughing was the third most common trigger ( reported by 68% of participants ) , it is a well - established symptom of asthma and is not listed as an asthma trigger by the gina guidelines . there is literature to suggest that cough can be a precursor to an asthma exacerbation , but this association could be secondary to the worsening of asthma via another trigger ( leading to more symptoms , including coughing ) , rather than coughing being a true trigger . strong smells are not listed as an asthma risk factor by gina , but a small - scale study showed that perfume can cause exacerbations and airway obstruction , particularly in patients with severe asthma . there are clearly discrepancies between what patients consider to be triggers and what physicians and guidelines class as triggers . possible explanations for such discrepancies include the lack of a standardised definition of the term asthma trigger , a lack of distinction between a trigger and an irritant , and documented gaps between patient perception and probable actual role of a trigger . irritants can make the patient feel worse but may not necessarily lead to an exacerbation , while triggers are often thought to cause exacerbations . the present research suggests that patients do not discriminate between an irritant and a trigger and they will categorise any factor that increases symptoms , leads to loss of control , or causes an exacerbation as an asthma trigger . this has important implications for how healthcare professionals listen to and communicate with their patients , and emphasises the need for healthcare professionals to seriously consider all complaints about triggers , irrespective of whether they are real or perceived . the frequency with which participants reported exposure to triggers varied considerably in this survey , with self - reported exposures ranging from every few months to every few days . these findings are important as there is little information in the literature about how frequently specific asthma triggers are encountered , and they help to characterise the full extent of trigger burden experienced by patients . although the authors are not aware of comparable reports that assessed the frequency of exposure to a wide range of triggers , those triggers that were experienced most commonly by patients concord with other investigations that aimed to identify common trigger types . those triggers experienced by patients for 8 weeks of the year ( see table 2 ) generally fall into the most important trigger categories reported by a systematic literature review ( allergic , physical , environmental ) or during the development of the asthma trigger inventory ( climate , physical , air pollution , infection , house dust , animal allergy ) . the key exception was coughing , which was not reported as a trigger in either the systematic literature review or asthma trigger inventory . frequent exposure to a large number of triggers can worsen symptoms , cause exacerbations and lead to loss of asthma control [ 1,37 ] , therefore reducing a patient s exposure to triggers forms part of the overall recommended therapeutic approach to asthma management . however , the relationship between trigger burden and asthma control has not been well documented extensively in the literature . in the present study , a higher trigger burden was associated with more severe attacks in a lifetime , more hospitalisations in the last year , more days absent from work / study in the last year , and larger proportions of patients with uncontrolled asthma , as defined using the act ( see table 3 and figure 1 ) . these findings align with those of a previous study in which patients were asked at the time of hospitalisation for an exacerbation what their usual triggers were and what trigger they felt caused their exacerbation . these patients reported a high trigger burden ( mean of 12 triggers ) and there were statistically significant correlations between more triggers and more exacerbations , worse quality of life , more hospitalisation due to asthma , and oral corticosteroid use . these collective findings strongly suggest that a greater trigger burden leads to adverse asthma outcomes and worse asthma control , and highlights the importance of patient education to eliminate or mitigate trigger exposure . asthma guidelines and authoritative groups recommend patients with asthma work to identify triggers that worsen their symptoms and try to reduce exposure to those triggers with the aim of improving asthma control and reducing medication needs . however , it is recognised that a response to triggers is a marker of poorly controlled disease , therefore trigger avoidance should not be used in isolation but rather as part of the holistic approach required to improve disease control overall . in the current study the majority of participants did not report any behavioural avoidance strategies for known environmental triggers , while some did but only after being prompted to do so by worsening asthma symptoms . further , there was no apparent consistent approach to how patients managed trigger exposure and a wide range of behavioural strategies were reported . patient education of trigger avoidance and advice on trigger management strategies appear to be logical steps to reduce the impact of triggers , but there are conflicting reports whether these steps are routinely taken . tools for use in the primary care setting that aim to capture specific information on trigger exposure and other reasons for poor asthma control , such as the asthma apgar and the active life with asthma tool , may help to improve the dialogue between healthcare professionals and patients with respect to triggers . nonetheless , although some asthma patients do make temporary or permanent lifestyle changes to manage trigger exposure , literature reviews and task force recommendations have not yielded consistent and conclusive evidence that interventions and trigger avoidance strategies improve symptoms , reduce exacerbations , and increase quality of life and productivity , at least in adults . although better understanding and identification of asthma triggers , more consistent provision of advice on asthma triggers , and the development of more effective trigger avoidance strategies appear to be needed , it should also be considered whether overall asthma control can be improved to the point where the avoidance of triggers is not necessary . the strengths of the study lie in the relatively large cohort of adults with asthma who took part ( n = 1202 ) and in the validity of the act scale as a measure of asthma control both as a written and , as used in the current study , as an online questionnaire . the knowledge gained from the comprehensive characterisation of asthma triggers in this survey can be applied to broader epidemiological assessments of asthma triggers . the results should be considered within the limitations expected of the survey nature of the study design . the results apply to a large population of asthma patients from five european countries who were currently receiving maintenance treatment and who self - reported exposure to known asthma triggers ; however , generalising the findings to other populations of asthma patients is not without its limitations . furthermore , patient assessment of asthma control and trigger burden were based on self reporting with recall periods of up to one year . finally , verification of self - reported asthma worsening or healthcare service utilisation was beyond the scope of the current study . the results of the current study support those of previous surveys that suggest asthma control among adults in europe is suboptimal . individuals with asthma identify a wide range of environmental and behavioural triggers and often employ avoidance strategies or behavioural changes to manage their exposure . these behavioural changes often have a significant impact on daily life , social life and employment performance and choices . individuals who perceive that their asthma is worsened on exposure to multiple triggers may be at particular risk for poorly controlled asthma . physicians should seek to routinely assess the trigger burden for individual patients and offer education and support to enable individuals to manage their exposure while minimising the effect of behavioural changes on their daily lives .
objective : to identify the types , frequency and impact of asthma triggers and the relationship to asthma control among adults with asthma in europe.methods:adults with self - reported physician - diagnosed asthma receiving maintenance asthma treatment and self - reported exposure to known asthma triggers completed an online questionnaire ; a subset completed a diary over 34 weeks . information on asthma control ( asthma control test [ act ] ) , asthma triggers , frequency of exposure and behaviours in response or to avoid asthma triggers and the perceived impact on daily life was captured . a post - hoc analysis evaluated the impact of high trigger burden on the frequency of severe asthma exacerbations , hospitalisations and days lost at work / study.results : a total of 1202 adults participated and 177 completed the diary . asthma was uncontrolled for the majority ( 76% ) of participants and most ( 52% ) reported exposure to 615 asthma triggers . as trigger burden increased , behavioural changes to manage trigger exposure had a significantly increased impact on daily life ( p < 0.0001 ) and job choice ( p = 0.002 ) . participants reporting a high trigger burden ( > 16 ) were more likely to report uncontrolled asthma than those with a low trigger burden ( 15 ) . participants with a high trigger burden had previously experienced on average two more severe asthma attacks during a lifetime ( p < 0.001 ) , two more hospitalisations ( p < 0.001 ) and 3.5 more missed days at work or study in the last year due to their asthma ( p < 0.001 ) than those with a low trigger burden.conclusions:adults with asthma reporting a high trigger burden ( > 16 different triggers ) experience more severe asthma attacks than those reporting lower trigger burdens .
Introduction Methods Study design Participants Online survey Participant diary Statistical analysis Results Asthma control Incidence and frequency of asthma triggers Burden of asthma triggers Behavioural changes in response to asthma trigger Discussion Conclusions
the current epidemiological study was conducted to identify the types , frequency and impact of asthma triggers , and to investigate the relationship between asthma trigger burden ( number and frequency ) and asthma control among adults with asthma across five european countries . as the number of known asthma triggers increased , participants reported statistically significantly more severe asthma attacks during their lifetime , more hospitalisations in the previous year , more days missed at work or study in the last year and more symptomatic days and nights per week ( table 3).table 3.burden of asthma triggers.patient groups by trigger numbers total n n = 1202very few ( 15 ) n = 156low ( 610 ) n = 293medium ( 1115 ) n = 335high ( 16 + ) n = 418kruskal wallismean number of severe attacks in lifetime3.2 ( median 2 , range 099)2.0 ( median 1 , range 015)2.7 ( median 2 , range 099)2.6 ( median 2 , range 020)4.6 ( median 2 , range 099)<0.001mean number of times hospitalised in the last year0.7 ( median 0 , range 025)0.5 ( median 0 , range 07)0.5 ( median 0 , range 06)0.8 ( median 0 , range 025)0.8 ( median 0 , range 020)0.038mean days missed at work or study in the last year because of asthma7.9 ( median 2 , range 0365)2.9 ( median 1 , range 080)6.1 ( median 2 , range 0288)6.3 ( median 2 , range 0265)12.2 ( median 2 , range 0365)<0.001 burden of asthma triggers . any increase in the level of known asthma triggers above very low was positively associated with a statistically significant increase in mean number of severe attacks in a lifetime ( p 0.01 ) , the number of times individuals were hospitalised in the last year ( p < 0.001 ) and the number of days missed at work or study in the last year ( p < 0.001 ) due to asthma . as the number of known asthma triggers increased , participants reported statistically significantly more severe asthma attacks during their lifetime , more hospitalisations in the previous year , more days missed at work or study in the last year and more symptomatic days and nights per week ( table 3).table 3.burden of asthma triggers.patient groups by trigger numbers total n n = 1202very few ( 15 ) n = 156low ( 610 ) n = 293medium ( 1115 ) n = 335high ( 16 + ) n = 418kruskal wallismean number of severe attacks in lifetime3.2 ( median 2 , range 099)2.0 ( median 1 , range 015)2.7 ( median 2 , range 099)2.6 ( median 2 , range 020)4.6 ( median 2 , range 099)<0.001mean number of times hospitalised in the last year0.7 ( median 0 , range 025)0.5 ( median 0 , range 07)0.5 ( median 0 , range 06)0.8 ( median 0 , range 025)0.8 ( median 0 , range 020)0.038mean days missed at work or study in the last year because of asthma7.9 ( median 2 , range 0365)2.9 ( median 1 , range 080)6.1 ( median 2 , range 0288)6.3 ( median 2 , range 0265)12.2 ( median 2 , range 0365)<0.001 burden of asthma triggers . any increase in the level of known asthma triggers above very low was positively associated with a statistically significant increase in mean number of severe attacks in a lifetime ( p 0.01 ) , the number of times individuals were hospitalised in the last year ( p < 0.001 ) and the number of days missed at work or study in the last year ( p < 0.001 ) due to asthma . linear regression analyses indicated that participants reporting a medium or high asthma trigger burden were significantly more likely to report an adverse impact on their overall daily life , as were participants in italy ( versus those in germany ; asthma impact score 0.61 points higher , p < 0.001 ) , females ( versus males ; 0.42 points lower , p < 0.001 ) and those with moderate or severe asthma ( versus mild ; asthma impact score 1.33 and 2.36 points , respectively , p < 0.001 for both ) . an increase in trigger burden was associated with an increased likelihood of uncontrolled asthma , previous severe asthma attacks during a lifetime , more hospitalisations , more missed days at work / study , and behavioural changes to manage trigger exposure .
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patients presenting a carotid stenosis and contralateral occlusion ( co ) have been historically considered at high risk for carotid endarterectomy ( cea ) , since results from randomized controlled trials ( rcts ) on carotid surgery have reported morbidity and mortality rates significantly higher than in the general population affected by carotid stenosis [ 15 ] . hence , that group of patients has been frequently excluded from surgical treatment in some prospective rcts comparing results in cea and carotid artery stenting ( cas ) in the following years , as in the sapphire trial . on the other hand , some single - center reports have highlighted a nondissimilar rate of complications in patients presenting with a carotid stenosis with or without contralateral occlusion [ 610 ] . in those studies perioperative complications are reported in 0.7 to 6.9% of patients presenting with carotid stenosis and contralateral occlusion , thus reaching percentages consistent with international recommendations for cea postoperative complications in symptomatic patients , but surely exceeding those requested for cea in asymptomatic ones [ 11 , 12 ] . however , within the population of patients affected by carotid stenosis and contralateral occlusion , different risk categories could be identified with respect to general medical conditions and involvement of other vascular districts in the atherosclerotic process , as well as with respect to presence of previous brain infarct on neuroimaging . it has to be noted that frequently patients affected by co have been included in the high - surgical risk category mixed with patients presenting general medical conditions at high risk for surgery , thus generating confusion with regard to the real risk addressed by those patients [ 5 , 7 , 8 , 10 ] . we retrospectively reviewed our database on carotid endarterectomy to analyse the incidence of postoperative neurological complications in patients affected by carotid stenosis and chronic contralateral occlusion , to analyse factors associated with the development of complications in this group of patients , and to compare their results with those obtained in the group of patients without co submitted to cea at our vascular surgery division . from january 1997 to december 2012 , 1639 patients underwent primary cea at our vascular surgery division . data on demographics , risk factors , preoperative neurological evaluation and imaging , intervention , and 30-day outcomes were prospectively collected in our institutional database . from the database 136 out of 1639 ( 8.3% ) patients presenting a stenosis of internal carotid artery with a contralateral internal carotid occlusion ( co ) submitted to carotid endarterectomy in the stenotic side were identified . a carotid stenosis 70% in asymptomatic patients or 50% ( nascet stenosis evaluation criteria ) in symptomatic patients was considered indication for intervention . patients were considered symptomatic if they presented carotid - related neurological symptoms in the previous six months before operation . patients submitted to carotid endarterectomy in urgency ( within 2 weeks from last neurological symptom ipsilateral to the carotid stenosis or occlusion ) were not considered for analysis in the present series because they represent a subset of patients at higher surgical risk . all patients preoperatively underwent a complete medical examination , assessment of preoperative neurological examination by the neurologist by use of national institute of health stroke scale ( nihss ) or rankin scale evaluation , blood test , electrocardiogram , and carotid duplex ultrasound imaging to assess the degree of stenosis . brain computed tomography ( ct ) or magnetic resonance imaging ( mri ) was performed in all co patients and in all control sample patients with symptomatic or ulcerated / irregular carotid plaque detected at duplex ultrasound examination . before admission , all patients classified as having a carotid occlusion had been previously submitted to at least two different imaging modalities to confirm the obstruction ( duplex us , contrast - enhanced supra - aorticvessels ct scans and mri ) . patients were operated on on cervical block anaesthesia or , alternatively , general anaesthesia whenever a local anaesthesia was not feasible for patient - related causes . patients under local cervical anaesthesia were assessed for neurological deficit and pain throughout the entire procedure by hand - grip test . during operations under general anaesthesia neurological monitoring was performed by transcranial doppler ( tcd ) whenever possible and adjunctive quantitative electroencephalogram ( qeeg ) . cerebral protection by sundt shunting was selectively used when mean velocity in the middle cerebral artery at tcd monitoring decreased to 15 cm / sec or a significant alteration in qeeg recording was detected . patients were maintained under their scheduled asa ( 100 mg ) or ticlopidine ( 250 mg ) medication and were postoperatively reevaluated by a neurologist . follow - up was conducted at our vascular ultrasound laboratory by carotid dus and clinical examination at 1 , 6 , and 12 months and then annually . when a postoperative complication had occurred a neurological assessment outcome measures for analysis were perioperative ( 30-day ) major stroke ( assessed as a neurological deficit lasting more than 24 hours and scored as nihss 4 ) , minor stroke ( assessed as a neurological deficit lasting more than 24 hours and scored as nihss 3 ) , and stroke - related or neurological death . water - shed infarction ( cerebral border - zone infarctions ) and hyperperfusion syndrome ( defined as occurrence of severe unilateral headache , acute changes in mental status , vomiting , seizures , focal neurologic deficits , and , ultimately , intracranial hemorrhage ) occurrence were also recorded . neurological morbidity ( major + minor stroke ) and mortality were analyzed according to clinical preoperative demographics and presentation and intraoperative details and compared between patients with or without carotid occlusion contralateral to the treated carotid stenosis . univariate predictors that were significant at p < 0.05 were then entered into a multivariate model using logistic regression which was used to generate odds ratios ( ors ) and 95% confidence intervals ( cis ) for 30-day neurological outcomes , in order to identify a subset of patients at high risk for neurological complications . the demographic characteristics of the two groups ( co and control patients ) are shown in table 1 . between groups analysis disclosed that patients with carotid stenosis and contralateral occlusion ( co group ) were significantly younger ( 67.02 7.9 versus 69.72 8.13 ; p < 0.0001 ) , were more frequently males ( 114 versus 1054 ; 83.8% versus 68.3% ; p = 0.001 ) and more frequently smokers ( 103 versus 828 ; 75.7% versus 53.7% ; p < 0.0001 ) , and presented a significantly higher incidence of peripheral arterial disease ( 45 versus 248 ; 33.1% versus 16.1% ; p < 0.0001 ) than patients without contralateral occlusion ( control group ) . co patients referred previous neurological symptoms ipsilateral to the stenosis in 18 cases out of 86 , and contralateral , or ipsilateral to the carotid occlusion , in 68 cases . analysis of neurological presentation before operation disclosed that co patients were more frequently symptomatic ( 86 co patients presenting preoperative neurological symptoms versus 665 control patients ; 63.2% versus 46.4% ; p < 0.0001 ) and presented more frequently cerebral infarcts on preoperative brain imaging ( 65 co patients versus 379 control patients ; 47.8% versus 25.2% ; p < 0.0001 ) . one vertebral artery was occluded in 9 patients in the co group and in 25 patients in the control group . co patients were more frequently submitted to cea under local anaesthesia ( 93 versus 873 ; 68.4% versus 58.1% ; p = 0.02 ) and had more frequently a shunt implantation after clamping test ( 40 versus 121 ; 29.4% versus 8.1% ; p < 0.0001 ) . shunt use rate was not statistically different in patients submitted to cea under local or general anaesthesia in both groups . mean clamping time was 23.3 13.5 versus 22.9 12.5 minutes in co versus control group ( p = 0.28 ) . perioperative ( 30-day ) complications analysis disclosed no significant difference in neurological mortality rates between the two groups ( p = 0.28 ) . incidence of postoperative minor stroke was not significantly different in the two groups ( 0.7% in co group and 0.5% in control group , resp . ) , while major stroke incidence analysis disclosed a statistically significant difference ( 4.4% in co and 1.2% in control group , p = 0.009 ) , accounting for higher overall neurological morbidity and overall neurological complication rates in co group with respect to control group ( 5.1% versus 1.7% , p = 0.01 , and 6.6% versus 2.1% , p = 0.003 , resp . ) . in co patients 5 out of 9 perioperative neurological complications were related to technical defects ( carotid early thrombosis , 55.5% ) while in control cases carotid early thrombosis was responsible for neurological events in 24 out of 26 cases ( 92.3% ) . in 4 cases in co group and 2 cases in control group neurological complications were related to hypoperfusion or reperfusion injuries ( 44.5% versus 7.7% ) . no complication occurred in co patients presenting one vertebral artery occluded and 1 major stroke ( carotid thrombosis in the first hours following intervention ) occurred in one patient with an occluded vertebral artery in the control group . analysis of preoperative clinical factors and neurological outcome in co patients disclosed that neurological complications occurred more frequently in elderly patients ( patients with complications versus patients without complications mean age 73.55 4.24 versus 66.55 7.9 ; 95% ci : 1.7612.27 ; p < 0.01 ; patients with complications versus patients without complications and age > 74 years p = 0.008 ) and in patients with cerebral infarct on preoperative neuroimaging ( p = 0.036 ) . use of shunt did not show to be related to an increased risk of neurological complications ( 4 patients in the shunt group and 5 patients in the nonshunt group presented overall perioperative neurological complications combined endpoint ) . all other variables analysed did not show any statistical difference between favourable and unfavourable outcome . the abovementioned risk factors ( > 74 years derived from the intragroup continuous data analysis of age , and preoperative brain damage ) were subsequently analyzed for their role in the development of postoperative neurological complications in the whole sample . odds ratio analysis disclosed that the association of ipsilateral carotid stenosis and contralateral occlusion with bilateral or contralateral preoperative brain ischemic lesion exposed the cea patient to a higher surgical risk of postoperative neurological complications ( or 8.1 , 95% ci 1.7238.41 , p = 0.001 ; and or 3.2 , 95% ci 0.9510.96 , p = 0.04 , resp . , table 3 ) . similarly , in cea patients the association of ipsilateral stenosis and contralateral occlusion with age > 74 years increased significantly this risk ( or 11.5 , 95% ci 4.0832.62 , p < 0.0001 ) . further , in co cea patients the association of age > 74 years with ipsi / bilateral or contralateral preoperative brain damage showed an augmented surgical risk of brain lesions ( or 19.9 ; 95% ci 1.77224.56 , p = 0.0006 ; or 40.9 , 95% ci 5.61298.05 , p < 0.0001 , resp . , table 4 ) . among 11 co patients presenting both age > 74 years and a preoperative brain infarct , 5 ( 45.5% ) presented postoperative neurological complications . analysis of the causes of postoperative neurological complications in those 5 patients disclosed that one patient suffered from hemorrhagic transformation of a preoperative brain infarct and died ; one patient suffered from acute postoperative carotid thrombosis with immediate neurological deterioration and slow recovery in the following months after cea ; one patient with a very small internal carotid artery ( maximal external diameter 4 mm ) who did not tolerate carotid clamping , in which a 3 4 mm sundt shunt was employed , presented an intraoperative cerebral hypoperfusion with prompt recovery of the postoperative neurological deficit ; and two patients presented a postoperative neurological deficit related to the contralateral carotid occlusion ( watershed infarct ) with a residual small deficit in the postoperative period . in the present series cea patients affected by carotid stenosis and contralateral occlusion presented significantly higher perioperative major stroke rate ( p = 0.009 ) , overall neurological morbidity ( p = 0.01 ) , and overall neurological complication rates ( p = 0.003 ) compared to control group of patients . from our experience the co sample , as a whole , represents a subset of patients at higher surgical risk for cea when compared to the general patients affected by carotid stenosis requiring intervention , in accordance with previous reports [ 1 , 3 , 8 , 1416 ] . nevertheless , when dealing with co patients , some peculiarities must be taken into account and some ensuing considerations could be made . first , in our series co patients demographics analysis showed a higher frequency of smoking history , other vascular district involvements in the atherosclerotic process ( i.e. , peripheral arterial disease ) , neurological symptoms history , and , of course , presence of previous brain infarction on neuroimaging . those data are in accordance with previous studies by julia et al . and rockman this seems to define the picture of a population at high risk for any surgical procedure , given the extensive involvement of the blood vessels in the atherosclerotic process [ 1823 ] . this peculiarity is underlined by the presence of a preoperative neurological symptomatology in 64% of patients in our series , since the population analyzed presented more frequently carotid - related symptoms and more frequently a brain infraction on neuroimaging contralateral to the side affected by the stenosis . so the brain in those patients is somewhat more fragile and less prone to tolerate carotid clamping during cea or also eventual small and clinically silent ischemic brain lesions ( eventually accompanied by perilesional oedema ) caused by microembolic plaque particles dislodged during cea . ultimately , it could be speculated that the brain in co patients could have less cerebral functional reserve . have recently reported a significant reduction in cerebrovascular reactivity in anterior circulation of the brain hemisphere ipsilateral to a carotid stenoocclusive disease , thus implying that unilateral carotid stenosis affects the vascular reserve of both sides of the brain , so not only the hemisphere ipsilateral to an occlusion , but also the contralateral one . to further support the theory of a hemodynamic impairment in patients with stenosis and contralateral occlusion oka et al . demonstrated increased cerebral blood flow and cerebrovascular reactivity in both hemispheres 36 months after carotid stenosis treatment . in our series in co patients immediate neurological complications were related to carotid embolism in 5 cases ( 55% ) and to hypo- or reperfusion in 4 cases ( 45% ) , thus accounting for a frailer brain in those patients . these data demonstrate relevant differences concerning causes of complications in comparison with the control sample where hypo- or reperfusion were responsible for postoperative complications in 2/36 cases ( 5.5% ) , strengthening the assumption that co patients present with an increased perfusion instability . such susceptibility of the brain with respect to any type of ischemic insult in those patients is sustained also by the increased need for shunt implantation during cea in co , in accordance with previous reports [ 6 , 9 , 26 ] . some conflicting data are reported in the literature concerning the need for shunt implantation in co patients : while some authors advocate shunt implantation in all patients with co , others recommend selective shunting or no shunting at all [ 29 , 30 ] . to add to the matter , a recent study by goodney et al . showed that shunt use for co patients during cea is associated with fewer complications , but only if the surgeon used a shunt as part of his or her routine practice in cea . a careful intragroup analysis of co population has highlighted that some additional preoperative risk factors can enhance the neurological risk in cea . in our experience presence of brain infarct on preoperative neuroimaging in co patients decreases the capability of tolerating any kind of ischemic insult , as demonstrated by a 4.4-fold neurological risk increase in this subset of patients ( table 3 ) . from our analysis patients with bilateral or contralateral brain lesions unfortunately , no information was available in our database concerning the size of brain ischemic lesions but we can speculate that contralateral damage in our series , namely , ipsilateral to a carotid occlusion , might be quite wide and so it can reinforce the persuasion that those patients ' brains are less prone to tolerate any kind of hemodynamic or embolic ischemic insult . on the other hand , an incomplete willis circle might justify the higher incidence of old brain infarcts and liability to new ischemic events . moreover , advanced age , defined in our experience by the presence of more than 74 years , further increases the neurological risk by 11.5-fold ( table 4 ) . the elderly seem at higher risk of brain hemodynamic impairment during carotid clamping per se . when advanced age and preoperative brain infarct are combined , the risk of postoperative neurological complications reached prohibitive values in our series ( or 28.6 in co population , table 4 ) . analysis of specific complications developed in 5 out of 11 co patients presenting both age > 74 years and a preoperative brain infarct has confirmed the higher fragility of the brain in those patients . except for one case of carotid thrombosis , probably related to a technical defect in cea , in two patients the postoperative neurological deficit was related to a global brain hypoperfusion not clinically evident during cea and in another one to a hypoperfusion due to a very small carotid artery in which a small shunt had been implanted , thus causing a damage in the hemisphere ipsilateral to the occluded carotid artery . in one last case a preoperative brain lesion underwent a hemorrhagic transformation thus underlining the weakness produced in the blood - brain barrier by a previous damage . even if a higher neurological surgical risk in patients affected by carotid stenosis and contralateral occlusion has been reported in numerous series [ 8 , 14 , 15 ] , some single - center experiences have derived different conclusions [ 27 , 33 ] . this is why nowadays no clear indications for carotid treatment are recognized for this group of patients . historical data on cea patients have shown co to be a significant risk factor for perioperative development of neurological complications [ 13 , 15 ] . a post hoc analysis of patients enrolled in nascet and in the ontario carotid endarterectomy registry reported a significantly higher incidence of 30-day adverse neurological events in patients with co. more recently two meta - analyses of patients undergoing revascularization when presenting a contralateral carotid occlusion were independently published in 2013 [ 16 , 35 ] . antoniou et al . performed a systematic review of electronic information sources to identify studies comparing perioperative and early outcomes of cea in patients with occluded and patent contralateral carotid arteries . thirty articles were included for meta - analysis , comprising 27265 patients having undergone 28846 ceas between 1961 and 2009 . among them the incidence of stroke was 3.3% in the occluded contralateral carotid group and 1.9% in the patent contralateral carotid group ( or 1.65 ; 95% ci 1.302.09 ; p < 0.001 ) . no significant heterogeneity among the studies was identified and no statistically significant association between the time of publication of studies and the likelihood of developing perioperative neurological complications was found . analysis of complications related to routine or selective shunting was not performed given the lack of significant heterogeneity of outcome in the selected studies . patients undergoing cea in the presence of an occluded contralateral carotid artery had increased perioperative and early postoperative risk , but they also pointed out a wide variety among studies of degree of stenosis , indication for treatment , exclusion of recurrent stenosis , carotid disease diagnostic methods , selection criteria for patients enrolment , examination of the status of collateral vertebrobasilar circulation , combination of results in symptomatic and asymptomatic patients , anesthetic methods , surgical techniques , and shunt use . performed a systematic review of papers published up to march 2012 reporting results on patients with carotid stenosis and contralateral occlusion submitted to either cea or cas . the authors reported co to be considered a significant risk factor for stroke and death in cea ( or 1.82 ; 95% ci 1.572.11 ; p < 0.00001 ) but not in cas ( or 1.22 ; 95% ci 0.602.49 ; p < 0.58 ) . in cea studies the authors performed a subgroup analysis reporting separate results for symptomatic patients ( or 2.43 ; 95% ci 1.075.50 ; p = 0.03 ) , asymptomatic patients ( or 1.83 ; 95% ci 1.252.68 ; p = 0.002 ) , and patients included in studies with higher statistical power ( or 1.75 ; 95% ci 1.442.12 ; p < 0.00001 ) , thus concluding that in all analysis co represents a significant risk factor for adverse neurological events . furthermore , the analysis performed with respect to shunt use revealed that in studies reporting both selective and routine shunting an increased risk of stroke and death can be encountered in co patients ( resp . , or 1.83 ; 95% ci 1.342.52 ; p < 0.0002 ; or 2.14 ; 95% ci 1.283.32 ; p < 0.0007 ) , while in studies reporting no shunt use that risk was increased but not significantly ( or 2.61 ; 95% ci 0.917.46 ; p < 0.07 ) . no cas studies were deemed appropriate for the subgroup meta - analysis . nevertheless , based on historical data derived from rcts , showing an increased risk of neurological complications in co patients , a cas treatment has been proposed and performed in the majority of those patients for many years . retrospectively reviewed 417 cas procedures performed between may 2001 and july 2010 and concluded that a preexisting co does not seem to adversely impact cas outcomes . on the contrary , a report by brewster et al . has pointed out that although cea and cas can both be performed with satisfactory perioperative results , the observed outcomes do not support the presence of contralateral carotid occlusion as a selection criteria for cas over cea in the absence of other indications . more recently , more papers have fuelled the debate on co patients [ 3840 ] . yang et al . have found that co has no adverse impact on the development of postprocedural stroke after either cea or cas , reporting a stroke rate of 2.3% in 698 cea patients routinely submitted to shunt implantation and 4% in 455 cas patients . have derived quite different conclusions , showing that in cea patients co significantly affects perioperative stroke rates ( 3.1% versus 1.1% in patients without co , p < 0.0001 ) , while in cas patients co does not affect periprocedural stroke rates ( 2.1% versus 2.3% in patients without co , p = 0.82 ) . kang et al . reported again co to be among predictors of any stroke at 30 days after cea and also of long - term ipsilateral stroke ( hr 2.06 ; p = 0.025 ) . on the other hand , alternative strategies , such as medical therapy alone , appeared to be of low efficacy in patients affected by co in historical studies [ 15 , 41 ] . in those papers reporting on patients suffering from internal carotid artery stenosis and contralateral occlusion alarmingly high rates of recurrent stroke , ranging from 20% within 3 years to 34% within 51 months of follow - up in medically - treated patients , data from nascet reported a 14.3% risk of stroke in co patients submitted to cea , but a 69.4% 2-year risk of stroke in those patients treated by medical therapy alone , thus leaving very short room to the latter , when considering the whole population of patients affected by co. it must be acknowledged that optimal medical treatment has significantly improved since those cited studies were conducted , making it possible that medical therapy alone in asymptomatic patients with co , as well as the so - called high - risk categories , should be considered the best treatment in the near future . hence , it is of outmost importance to identify those co patients with additional risk factors for carotid surgery who can better be treated by optimized medical therapy and strict surveillance in absence of recent neurological symptoms . in our experience cea patients with co present with a heavier burden of diffuse atherosclerotic disease when compared to control sample of cea patients . moreover , they are at higher risk for postoperative neurological complications because of a higher brain susceptibility mainly encountered in a small subset of patients presenting association of two most significant risk factors for developing complications ( advanced age , i.e. , > 74 years , and preoperative brain infarction on preoperative ct scans ) . in those cases the risk of surgery seems to be excessive , and exclusion from surgery with an alternative interventional or conservative approach could be worthwhile . future studies evaluating the role of medical therapy over carotid intervention in this subgroup of patients are mandatory .
objective . to report on the incidence and factors associated with the development of perioperative neurological complications following cea in patients affected by carotid stenosis with contralateral occlusion ( co ) and to compare results between those patients and the whole group of patients submitted to cea at our vascular division from 1997 to 2012 . methods . our nonrandomized prospective experience including 1639 patients consecutively submitted to cea was retrospectively reviewed . 136 patients presented a co contralateral to the treated carotid stenosis . outcomes considered for analysis were perioperative neurological death rates , major and minor stroke rates , and a combined endpoint of all neurological complications . results . co patients more frequently were male , smokers , younger , and symptomatic ( p < 0.001 ) , presented with a preoperative brain infarct and associated peripheral arterial disease ( p < 0.0001 ) , and presented with higher perioperative major stroke rate than patients without co ( 4.4% versus 1.2% , resp . , p = 0.009 ) . factors associated with the highest neurological risk in co patients were age > 74 years and preoperative brain infarct ( p = 0.03 ) . the combination of the abovementioned factors significantly increased complication rates in co patients submitted to cea . conclusions . in our experience co patients were at high risk for postoperative neurological complications particularly when presenting association of advanced age and preoperative brain infarction .
1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions
we retrospectively reviewed our database on carotid endarterectomy to analyse the incidence of postoperative neurological complications in patients affected by carotid stenosis and chronic contralateral occlusion , to analyse factors associated with the development of complications in this group of patients , and to compare their results with those obtained in the group of patients without co submitted to cea at our vascular surgery division . between groups analysis disclosed that patients with carotid stenosis and contralateral occlusion ( co group ) were significantly younger ( 67.02 7.9 versus 69.72 8.13 ; p < 0.0001 ) , were more frequently males ( 114 versus 1054 ; 83.8% versus 68.3% ; p = 0.001 ) and more frequently smokers ( 103 versus 828 ; 75.7% versus 53.7% ; p < 0.0001 ) , and presented a significantly higher incidence of peripheral arterial disease ( 45 versus 248 ; 33.1% versus 16.1% ; p < 0.0001 ) than patients without contralateral occlusion ( control group ) . co patients were more frequently submitted to cea under local anaesthesia ( 93 versus 873 ; 68.4% versus 58.1% ; p = 0.02 ) and had more frequently a shunt implantation after clamping test ( 40 versus 121 ; 29.4% versus 8.1% ; p < 0.0001 ) . , while major stroke incidence analysis disclosed a statistically significant difference ( 4.4% in co and 1.2% in control group , p = 0.009 ) , accounting for higher overall neurological morbidity and overall neurological complication rates in co group with respect to control group ( 5.1% versus 1.7% , p = 0.01 , and 6.6% versus 2.1% , p = 0.003 , resp . ) analysis of preoperative clinical factors and neurological outcome in co patients disclosed that neurological complications occurred more frequently in elderly patients ( patients with complications versus patients without complications mean age 73.55 4.24 versus 66.55 7.9 ; 95% ci : 1.7612.27 ; p < 0.01 ; patients with complications versus patients without complications and age > 74 years p = 0.008 ) and in patients with cerebral infarct on preoperative neuroimaging ( p = 0.036 ) . further , in co cea patients the association of age > 74 years with ipsi / bilateral or contralateral preoperative brain damage showed an augmented surgical risk of brain lesions ( or 19.9 ; 95% ci 1.77224.56 , p = 0.0006 ; or 40.9 , 95% ci 5.61298.05 , p < 0.0001 , resp . analysis of the causes of postoperative neurological complications in those 5 patients disclosed that one patient suffered from hemorrhagic transformation of a preoperative brain infarct and died ; one patient suffered from acute postoperative carotid thrombosis with immediate neurological deterioration and slow recovery in the following months after cea ; one patient with a very small internal carotid artery ( maximal external diameter 4 mm ) who did not tolerate carotid clamping , in which a 3 4 mm sundt shunt was employed , presented an intraoperative cerebral hypoperfusion with prompt recovery of the postoperative neurological deficit ; and two patients presented a postoperative neurological deficit related to the contralateral carotid occlusion ( watershed infarct ) with a residual small deficit in the postoperative period . in the present series cea patients affected by carotid stenosis and contralateral occlusion presented significantly higher perioperative major stroke rate ( p = 0.009 ) , overall neurological morbidity ( p = 0.01 ) , and overall neurological complication rates ( p = 0.003 ) compared to control group of patients . have derived quite different conclusions , showing that in cea patients co significantly affects perioperative stroke rates ( 3.1% versus 1.1% in patients without co , p < 0.0001 ) , while in cas patients co does not affect periprocedural stroke rates ( 2.1% versus 2.3% in patients without co , p = 0.82 ) . in those papers reporting on patients suffering from internal carotid artery stenosis and contralateral occlusion alarmingly high rates of recurrent stroke , ranging from 20% within 3 years to 34% within 51 months of follow - up in medically - treated patients , data from nascet reported a 14.3% risk of stroke in co patients submitted to cea , but a 69.4% 2-year risk of stroke in those patients treated by medical therapy alone , thus leaving very short room to the latter , when considering the whole population of patients affected by co. it must be acknowledged that optimal medical treatment has significantly improved since those cited studies were conducted , making it possible that medical therapy alone in asymptomatic patients with co , as well as the so - called high - risk categories , should be considered the best treatment in the near future .
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population growth has increased the demand for food ; rising prosperity has increased the demand for quality food . at the same time , consumers demand convenience foods , since they are becoming increasingly health conscious ; therefore , there is a need to diversify food products . wheat is being used as a staple food for most part of the world , because of its special dough characteristic like cohesiveness and thus being used in the preparation of bread and other wide ranges of products like noodles , soups , pasta , and other foods like biscuits , cookies , cakes , and breakfast cereal . pastas include noodles in various lengths , widths and shapes and varieties that are filled with other ingredients like ravioli and tortellini . pasta is an excellent source of complex carbohydrates , which provide a slow release of energy . unlike simple sugars that offer a quick , yet fleeting boost of energy enriched varieties provide a good source of several essential nutrients , including iron and several b - vitamins . in recent years increase in popularity of pasta products and their increased consumption make it very important for increase in availability of raw materials . durum wheat , being the hardest of wheat 's is used at large scale for pasta production . being commercially expensive to produce due to the limited availability makes it a case of study . the endosperm made up of durum is completely different from other wheat species because the mineral was distributed throughout endosperm and has more carotenoids contents , low protein efficiency ratio , and excellent rheological characteristics which are desirable for making pasta products . however , over the years , numerous studies on alternate methods for production of pasta from different raw materials have been conducted , where alternates for raw materials were used for production focusing on reduced cost and to match similar parameters and to improve nutritional value . limited availability of durum wheat is noticed due to the increase in consumption of pasta products , making it commercially expensive for procurement of raw material . on the other hand however , the availability of durum wheat for production of pasta products is very limited . alternates are thus researched upon , where it is found that the common wheat widely available throughout the world is similar in comparison with durum wheat . the percentages of starch , protein , minerals , lipids , and amino acids are roughly equivalent . common bread wheat occupying 90% of the world total production makes it a cheap and readily available alternate for pasta production , thus saving money and creating ample opportunities to improve nutritional quality . the objective of the present study is to find suitability of t. aestivum milled products ( refined wheat flour , semolina , and whole wheat flour ) in the place of durum semolina for preparation of pasta , thereby reducing the cost of production , maintaining or improving the quality of the product , and then study in detail the rheological properties of the pasta dough , chemical composition , nutritional profile , and quality of developed pasta products . freshly prepared refined wheat flour , whole wheat flour , and semolina were obtained from narasu 's roller flour mills , salem , tamil nadu , india . all reagents and chemicals used are of analytical grade ( ar ) unless otherwise specified . raw materials were analyzed for particle size ( aacc 55 - 30.01 ) , moisture content ( aacc-44 - 15a ) , ash content ( aacc-80 - 01 ) , gluten content ( aacc-38 - 10 ) , and micro - kjeldahl method was used to determine nitrogen contents of pasta samples ( aacc , 2000 ) , sedimentation value ( aacc-56 - 70 ) , farinograph ( aacc-54 - 21 ) , mixograph ( aacc 54 - 40.02 ) , and alveograph characteristics ( aacc-54 - 30a ) using the mentioned standard methodologies . raw materials and water were premixed in a spar mixer at speed 1 ( 60 rpm ) for 10 min to facilitate uniform distribution of water . the premixed dough ( 500 g ) was transferred to a laboratory pasta machine ( la monferrina , model dolly , asti , italy ) . the dough was then extruded through the brass die for pasta type shells in the required size and was dried in sakar drier ( shirsat , mumbai ) at 75c for 4 h. the pasta samples were then allowed to cool at room temperature and then packed in polyethylene covers for storage . cooking loss was determined according to the bureau of indian standards ( bis 1976 ) . twenty - five grams of pasta sample was weighed and put in the 250 ml of boiling water . start timer count is and stirs well to make sure that the pieces are separated . check the piece of pasta after every 30 sec intervals for its hydration and cooking by squeezing the sample . firmness of cooked pasta was measured according to method adopted by krishnan and prabhasankar using a universal texture measuring system ( lloyds instruments , lr-5 k , hampshire , uk ) . a panel consisting of 25 panelists ( n = 25 ) , who were regular eaters of pasta , was employed for the sensory evaluation of pasta samples . briefly , panelists evaluated the randomly coded pasta samples for their colour , appearance , aroma , texture , taste , and overall acceptability . assessors were instructed to cleanse their palate with cold , filtered tap water before tasting each sample . the overall sensory attributes were measured using hedonic scale of 19 where 9 = like extremely , 8 = like very much , 7 = like moderately , 6 = like slightly , 5 = neither like nor dislike , 4 = dislike slightly , 3 = dislike moderately , 2 = dislike very much , and 1 = dislike extremely . all the parameters were carried out in quadruplicates and the means values were reported . the values of surface colour ( l , a and b ) of raw pasta in terms of lightness ( l ) and colour ( + a : red a : green ; + b : yellow ; b : blue ) and e were measured using hunter lab colour measuring system ( colour measuring labscan xe system , usa ) . the cooked pasta samples were freeze - dried using heto freeze dryer ( dw3 , allerod , denmark ) . surface and cross section of freeze - dried samples were mounted on the specimen holder and sputter - coated with gold ( 2 min , 2 mbar ) . finally , each sample was transferred to the microscope where it was observed at 15 kv and a vacuum of 9.75 10 torr . a scanning electron microscope ( leo 435 vp , leo electronic systems , cambridge , uk ) was used to scan the images . the method was followed from aoac method 32.1.17 . in vitro digestibility of starch freeze dried and ground sample ( 50 mg ) was dispersed in 4 ml of sodium acetate buffer ( ph 4.6 , 0.4 m ) containing amyloglucosidase and was incubated in water bath for 30 min at 60c . then , the enzyme was inactivated by placing the tubes in boiling water bath ( 100c ) for 15 min . the tubes were cooled to room temperature and then centrifuged at 5000 rpm for 10 min . supernatant was measured for its glucose content using a glucose oxidase - peroxidase ( god - pod ) kit ( autospan , span diagnostics limited , india ) . absorption was measured at 505 nm , and the glucose concentration was converted into starch content using a 0.9 factor . the particle size distributions of the flour samples were determined with a series of standard sieves , and the results were expressed as a percentage of the sample weight ( table 2 ) . ideally , the majority of semolina particles should fall within a narrow range of particle size range so that pasta dough water uptake will be homogenous . it was observed that the refined wheat flour ( refined wheat flour ) is much finer than the control ( semolina ) . the nonuniformity in the particle size can be attributed to the grinding of the semolina particles , which was carried out by an external minimill grinder . however , the results obtained were good within the requirements for good pasta making quality . whole wheat was ground to a granulation similar to that of the semolina ( table 2 ) . incomplete hydration of semolina or ground whole wheat would result in white specks in the spaghetti . thus , white specks would affect the appearance , mechanical strength , and cooking quality of the spaghetti . proximate analysis of all the raw materials ( refined wheat flour , whole wheat flour , semolina ) used were shown in the table 3 . it is noticed that comb1 ( t. aestivum wheat flour & semolina with additives ) with the presence of additives has moisture of 10.87% is very much higher compared to the control ( 9.8% ) . the other two blends ( comb2 & comb3 ) had acceptable levels of moisture compared to the control as they have the presence of semolina . the proximate composition of all the samples was found to be within limits of pfa and isi standards . similar results of semolina were also reported by where moisture of indian durum varieties varied from 9.0 to 11.5% , ash content varied from 0.79 to 0.86% , and the protein content varied between 12.1 and 15.9% . ash , an index of the mineral content of the flour , is of much relevance , and in that it gives the indication of the grade or the extraction of the flour . this is because of the low level of mineral content present in the endosperm when compared to the outer bran content . the bran layer is rich in ash and protein , so removing bran during milling would lower the protein and ash content . it is noticed that all the comb samples have lower ash content when compared to the control , where comb3 ( semolina ( t. aestivum ) and semolina ( t. durum ) ) ( table 3 ) has 0.596% . comb2 & comb1 samples also have lower ash values , thus improving the quality of the flour . a significant increase in protein content is noticed with the comb samples ( table 2 ) , which could be due to the addition of additives . this is similar to the results reported by prabhasankar et al . for increase in nutritional attributes of pasta samples with the addition of additives . the maximum increase is noticed in comb2 ( refined wheat flour and semolina ( t. durum ) ) which is 15.40% ( table 3 ) when compared to the control which has 12.8% ( table 3 ) of protein . increase is also noted in comb1 at a level of 13.70% , thus imparting better nutritional value . the mixograph is a primary physical dough testing procedure in the us durum wheat - breeding program . figure 1 details the results of the monograms obtained from the pasta samples . comparing the two raw materials used , dough strength is greatly deferred between the samples where figures 1(b ) and 1(c ) were comparatively stronger than the others . this is because of the presence of refined wheat flour , which is generally high in strength compared to semolina . regardless of samples , refined wheat flour had higher peak heights as shown in figures 1(b ) and 1(c ) , but semolina had higher peak width as shown in figures 1(a ) , 1(d ) , and 1(e ) . higher peak proves that the flour is very resistant to extensibility and also has greater mixing ability making it more stable as seen in control ( figure 1(b ) ) . but semolina generally is seen to have a lower peak height ( figure 1(a ) ) , thereby reducing the extensibility of the flour making it better for pasta making . mixogram dough development time ( the time required for the mixogram curve to reach maximum height ) was greater for comb1 had the highest ( 4.26 ) which was made up of refined wheat flour . based on the mixograms obtained , refined wheat flour had higher dough stability when compared to semolina as evidenced by the rapid decline of the mixogram curves after 3.5 min . dough stability indicates the time during which the dough resists mechanical action without undergoing a change in consistency . lack of dough stability or tolerance to over mixing could be related to the dilution of semolina with bran or germ as fewer storage proteins are available to form a gluten matrix . other researchers have reported that dough stability decreases with increase in bran concentration . in spite of all these issues , all the parameters were in accordance was the norms and were able to produce good pasta quality . it is seen from ( figure 1(d ) ) , that the addition of refined wheat flour along with semolina has given the flour additional properties showing a higher peak height when compared to control ( figure 1(a ) ) , thus aiding in better pasta making ability . the results obtained along with the amount of water absorption required to centre the farinogram curve on the 500 bu ( brabender units ) line varied as shown in table 4 . it is well noticed from the results that the samples with a higher concentration of refined wheat flour exhibit stronger dough characteristics ( increased water absorption , dough development time and dough stability ) , that is , refined wheat flour & comb1 in contrast to weak dough development . the whole wheat flour showed higher water absorption 63.9% due to high damages starch , whereas the dough development time and dough stability were lower than those in the whole - wheat flour . the addition of gluten as indicated in samples combs 1 , 2 , and 3 , increased water absorption , dough development time and dough stability was seen . it was also noticed that stability and dough development time are related with each other , and increase in development time caused increased stability , thus leading to stronger dough . samples with maximum wheat bran concentration caused increase in water absorption with whole wheat flour ( whole wheat flour ) having maximum absorption ( 65.9% ) ( table 4 ) which was also noticed by and , lowest for refined wheat flour ( comb1 ) . reported that the differences in water absorption are mainly caused by the greater number of hydroxyl group which exist in the fibre structure and allow more water interaction through hydrogen bonding . alveograph is used to measure the viscoelastic properties ( strength and extensibility ) of the gluten protein that correlate 's well with the firmness and springiness of cooked pasta . gluten strength and tenacity / extensibility ratio pl is a good predictor of cooking quality , thus making it relevant for pasta - making ability . figure 2 results obtained from the alveograph of the various samples of flour . regarding the bread making and pasta characteristics of the flour under examination , the most dramatic effect of additives addition on semolina was observed when the biaxial properties of the wheat dough were assessed in the alveograph . the addition induced a significant modification of the alveograph parameters , where a steady increase of the tenacity ( p ) besides to a significant decrease in dough extensibility ( l ) . overall effect on tenacity and extensibility led to a significant increase of the curve configuration ratio ( pl ) . similar results were obtained by bonet et al . where he noticed the same , with increase in go ( glucose oxidase ) concentration . the same was also noticed in regard to go by . but the only change was seen in the deformation energy ( w ) where a steady decrease was noticed in this case . this could be attributed to the addition of a percent of refined wheat flour as the raw material . as the percent of refined wheat flour reduced , the pl ratio increased which was very similar to that of control . thus leading to better pasta quality . whereas bonet et al . noticed a steady increase as there was no presence of refined wheat flour and also the addition of go cause additional protein cross - links resulting in larger pl values . colour of pasta is a key quality because of the vital impact on the point of sale . in pasta products made with semolina , the higher the value , the more desirable the product . among l , a , and b parameters , the first two are considered more important as colour attributes . hunter colour parameters ( l , a , b ) of raw samples of durum pasta ( control ) the other raw materials used along with the sa and the comb pasta are shown in the table 5 . the higher values are noticed in table 5 , which shows increased levels of pasta quality and the presence of no adulteration in the pasta samples proving high levels of economical advantage as well as appearance . the lightness is seen lower in the all the samples , this may be attributed due to the alteration with different wheat milled products which has a lower lightness index compared to the control , thereby lower carotenoid pigments which contribute to the colour of durum pasta . yellowness in all samples was seen lower than the control , and this is due to the same factors affecting the lightness index . it is noted that the comb3 blend shows the highest lightness , but the comb2 blend gives the highest yellowness when compared to the control . it is also noted that the influence of the additives did not cause any major difference in both indexes . but it is seen that the influence of additives has reduced the lightness but has increased the yellowness . the cooking characteristics of all the raw materials and the trails along with the comb blends compared to the control are presented in figure 3 and the photographs of raw , and cooked samples compared to the control are shown in figure 4 . high - quality pasta has a good cooking resistance and firmness , does not release amount of organic matter into the cooking water , and does not show stickiness . the cooking loss of all the comb samples is much lesser when compared to the control . the lowest cooking loss is seen in ( figure 3 ) where it is 2.88% compared to the control , which is 4.05% . the quality of the cooked pasta can be better explained on the basis of the interactions between starch and gluten whose intensity is strongly dependant on the drying conditions , as reported by many authors . if the coagulated gluten structure lacks compactness and elasticity , the starch granules structure swell up easily during cooking and lose more soluble materials into the cooking water . starch was the main component ( 63.1% ) on dry basis of the cooking liquor when 100% semolina was used in spaghetti and vermicelli samples . high - temperature drying strengthens the gluten matrix , which protects starch granules from rupturing during cooking . furthermore , high temperature drying reduces water permeability and a crake in packaging & arrangement of starch granules , contributing to reduced cooking looses and increased cooked firmness . a reconstitution study indicates that the gluten quality is the major factor determining the cooking quality . this could be very well noticed in the comb samples where gluten was added , thus decreasing the cooking loss . this is also noticed in the comb samples , where the presence of hydroxypropyl - methylcellulose ( hpmc ) added as an additive is noted to increase water solubility and also at high temperatures forms a gel creating a temporary network of hydrocolloid chains , thus maintaining the structure . the main criteria generally accepted to access the overall quality of the cooked pasta are based on the textural evaluation . the work done by the probes to cut the pasta cooked firmness was greater for semolina than refined flour or whole wheat flour as seen in figure 5(a ) . whole wheat pasta was reported to have lower cooked firmness than traditional semolina . when cooked to optimum , firmness was seen the greatest in semolina ( semolina ) obtained from t. aestivum wheat but is deemed not suitable for the consumer as it fails at the other parameters . it is also noticed that the addition of additives has resulted in firmness , which is similar to that of control . this can be explained as gluten present is seen to improve the protein content , thus improving firmness of pasta . good - quality pasta should be al dente ; that is , it should have high degrees of firmness and elasticity . comb3 pasta has moisture values higher than that of control , and at the same time , its firmness showed increased values . this would suggest that the substitution of additives to 100% semolina contributes to structural strength . in this case of comb3 , this hypothesis could also be related to the low values obtained for cooking losses , indicating a well - formed structure from which small amounts of solids are released during cooking . longer cooking times resulted in an increased water absorption that led to moisture migration into the centre of the pasta . hence , we hypothesize that moisture migration into the centre of the pasta diminishes the resistance of the sample to cutting , since the plasticizing action of water increases the mobility of biopolymers chains . another factor that could have affected the texture was the more prolonged exposure to heat while drying that changed the structural conformation of the protein - starch network . this phenomenon may have caused loss of rigidity in the structure with a consequent decrease in firmness . a product , even if it is highly nutritious , but does not taste good , will not be accepted in the society , thus making sensory evaluation very important and a crucial criteria in the formulation of pasta . based on the different properties of importance , it is characterised and formulated as shown in table 6 . it was noticed that the overall score when compared to the control is slightly less compared to all the other samples . refined wheat flour considered being the alternate for semolina as a raw material is considered to be the second control . the main aim of the present study to find a suitable alternate for the control ( durum semolina ) thus needs an overall score to at least at par to be acceptable . it is noticed that additives improved the sensory scores especially the mouth feel character . the increase in mouth feels score ( table 6 ) , which is one of the main attributes for pasta sensory evaluation , may also be attributed to the increased water absorption ( 103.8% , 94% , and 104% ) , respectively , for the comb blends . comb3 mixture of semolina of t. aestivum and t. durum gives the highest overall score , which is very much similar to the control . being very essential for the bowel movement and the digestion in the body , dietary fiber levels are much noted in the present world . remarkable changes in the levels of fiber are noticed in the comb2 and 3 samples , where they are 4.25 and 4.1 , respectively ( figure 5(b ) ) . higher levels of dietary fiber are mostly recommended in the present world , owing to the increase in diet - conscious people and the increased digestive problems faced in today 's society . the link between dietary fiber and human health is well explained by where they explain that higher levels of dietary fiber increase cancer prevention , lower risk of chronic disease and help in diabetes management and prevention . it was also noticed by that incorporation of a combination of blends of wheat bran by - products increased the dietary fiber content . two views of sem showing the cut section and the network of the pasta are presented in figure 6(b ) . these micrographs are all presented in 100x and 3000x , respectively , for all of the samples . micrographs of the control pasta samples show the protein - starch matrix to be well formed , with strong and continuous protein strands entrapping large starch granules . the starch granules within the pasta appear to be slightly swollen and regular in shape and size , perhaps indicating a level of gelatinization during the extrusion process . it is also noticed that the control has smaller pores when compared to the comb blends . this supports the higher cooking loss ( 4.05% ) and lower cooking time ( 6.5 min ) as boiling water can reach the deeper core faster and stay in the cavities . the addition of refined wheat flour to the semolina is seen to disrupt the continuity of the protein matrix . the protein - fiber matrix within the pasta containing a portion of refined wheat flour appears to be less developed than the control resulting in an open appearance with discrete starch granules , which is uncovered and exposed to enzymatic attack . this may be explained by the dilution of the gluten protein , thus showing reduced firmness ( 1.757 n ) and increased water absorption ( 103.8% ) , making it a more elastic structure . the addition of semolina with refined wheat flour shows a protein starch matrix similar to that of the control . this sample is seen to have a gelatinized structure when compared to the other blends , thus forming a more compact structure , which enable reduced water absorption ( 92% ) during cooking . unswollen starch granules can still be found inside the durum wheat , thus reducing the water absorption during cooking due to the layer of gelatinized starch . use of whole semolina shows a higher network of protein - starch matrix , thus resulting in a higher degree of firmness ( 2.43 n ) and improved chewiness . the rate of starch digestion and absorption seems to be a determinant of the metabolic response to a meal . the gi is defined as the postprandial incremental glycemic area after a test meal , expressed as the percentage of the corresponding area an equi - carbohydrate portion of a reference food ( glucose or white bread ) . a recent joint fao / who expert consultation recommended increased consumption of low - glycaemic index ( gi ) foods . there is increasing evidence that a low -glycaemic - index diet can be beneficial in that , it improves metabolic control of hyperlipidaemia in diabetic patients as well as in healthy subjects . the rate and extent of starch digestion instigate a number of physiological functions that have different effects on health , including reduction of the glycemic and insulinaemic responses to a food , hypocholesterolemic effects , and protective effects against colorectal cancer . it is interesting to note that the addition of refined wheat flour as a raw material to the pasta sample increased the starch released in the pasta where pure refined wheat flour as pasta shows the highest level of starch release ( 44.90% ) . this is because as refined wheat flour consists of only the endosperm of the wheat grain , thus removing the nutritious bran and germ content ( wikipedia , refined wheat flour ) . goi et al . , have reported a total starch content of 74 2.24 for spaghetti , but the starch content obtained for the control pasta is approximately half of the value said above . this discrepancy may probably be due to the use of raw pasta for our analysis whereas goi et al . used the cooked samples of the pasta for their analysis . methodological differences in determination of total starch may also be a reason for the discrepancy . there are many factors that may influence the rate of starch digestion , including the nature of starch , the starch - protein interaction , the presence of fiber and antinutrients such as lectins , phytates and enzyme inhibitors and method and time of cooking . the present study revealed that pasta could be made using different wheat - milled products , which are economically viable and also have beneficial nutritional applications . different combinations of mixtures had been experimented to produce viable alternates where different concentrations of refined wheat flour , whole - wheat flour , and semolina of different varieties of wheat were used to prepare pasta . pasta made from refined wheat flour was seen to have reduced cooking loss , colour , firmness and sensory score . pasta made from a combination of t. durum ( 50% ) semolina and t. aestivum ( 50% ) has acceptable levels of all the parameters , but due to increase in financial requirements , it is not suitable . from all results obtained , it was seen that semolina t. durum ( 50% ) mixed with refined wheat flour t. aestivum ( 50% ) can be used as alternate for making pasta for use as a low glycemic index snack product with acceptable physical and sensory properties . the addition of additives to the pasta has made it almost equivalent to the standard of original 100% semolina pasta . acceptable cooking quality parameters were obtained in the pasta samples containing a mixture of both semolina and refined wheat flour , as measured by cooked weight , cooking loss , and so forth during cooking . however the incorporation of additives in the mixture of 50% semolina ( t. durum ) along with 50% refined wheat flour ( t. aestivum ) was finally concluded as the optimal alternate , because of its almostequivalent properties to the 100% t. durum semolina pasta , thus making it a economically nutritious alternate ' resulting as a staple food in developing countries .
this study is aimed to assess the suitability of t. aestivum wheat milled products and its combinations with t. durum semolina with additives such as ascorbic acid , vital gluten and hpmc ( hydroxypropyl methyl cellulose ) for pasta processing quality characteristics such as pasta dough rheology , microstructure , cooking quality , and sensory evaluation . rheological studies showed maximum dough stability in comb1 ( t. aestivum wheat flour and semolina ) . colour and cooking quality of comb2 ( t. durum semolina and t. aestivum wheat flour ) and comb3 ( t. aestivum wheat semolina and t. durum semolina ) were comparable with control . pasting results indicated that t. aestivum semolina gave the lowest onset gelatinization temperature ( 66.9c ) but the highest peak viscosity ( 1.053 bu ) . starch release was maximum in comb1 ( 53.45% ) when compared with control ( 44.9% ) as also proved by microstructure studies . firmness was seen to be slightly high in comb3 ( 2.430 n ) when compared with control ( 2.304 n ) , and sensory evaluations were also in the acceptable range for the same . the present study concludes that comb3 comprising 50% t. durum semolina and 50% t. aestivum refined wheat flour with additives would be optimal alternate for 100% t. durum semolina for production of financially viable pasta .
1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusion
the objective of the present study is to find suitability of t. aestivum milled products ( refined wheat flour , semolina , and whole wheat flour ) in the place of durum semolina for preparation of pasta , thereby reducing the cost of production , maintaining or improving the quality of the product , and then study in detail the rheological properties of the pasta dough , chemical composition , nutritional profile , and quality of developed pasta products . it was observed that the refined wheat flour ( refined wheat flour ) is much finer than the control ( semolina ) . proximate analysis of all the raw materials ( refined wheat flour , whole wheat flour , semolina ) used were shown in the table 3 . it is noticed that comb1 ( t. aestivum wheat flour & semolina with additives ) with the presence of additives has moisture of 10.87% is very much higher compared to the control ( 9.8% ) . it is noticed that all the comb samples have lower ash content when compared to the control , where comb3 ( semolina ( t. aestivum ) and semolina ( t. durum ) ) ( table 3 ) has 0.596% . the maximum increase is noticed in comb2 ( refined wheat flour and semolina ( t. durum ) ) which is 15.40% ( table 3 ) when compared to the control which has 12.8% ( table 3 ) of protein . regardless of samples , refined wheat flour had higher peak heights as shown in figures 1(b ) and 1(c ) , but semolina had higher peak width as shown in figures 1(a ) , 1(d ) , and 1(e ) . based on the mixograms obtained , refined wheat flour had higher dough stability when compared to semolina as evidenced by the rapid decline of the mixogram curves after 3.5 min . it is seen from ( figure 1(d ) ) , that the addition of refined wheat flour along with semolina has given the flour additional properties showing a higher peak height when compared to control ( figure 1(a ) ) , thus aiding in better pasta making ability . it is well noticed from the results that the samples with a higher concentration of refined wheat flour exhibit stronger dough characteristics ( increased water absorption , dough development time and dough stability ) , that is , refined wheat flour & comb1 in contrast to weak dough development . when cooked to optimum , firmness was seen the greatest in semolina ( semolina ) obtained from t. aestivum wheat but is deemed not suitable for the consumer as it fails at the other parameters . the main aim of the present study to find a suitable alternate for the control ( durum semolina ) thus needs an overall score to at least at par to be acceptable . the increase in mouth feels score ( table 6 ) , which is one of the main attributes for pasta sensory evaluation , may also be attributed to the increased water absorption ( 103.8% , 94% , and 104% ) , respectively , for the comb blends . comb3 mixture of semolina of t. aestivum and t. durum gives the highest overall score , which is very much similar to the control . this may be explained by the dilution of the gluten protein , thus showing reduced firmness ( 1.757 n ) and increased water absorption ( 103.8% ) , making it a more elastic structure . it is interesting to note that the addition of refined wheat flour as a raw material to the pasta sample increased the starch released in the pasta where pure refined wheat flour as pasta shows the highest level of starch release ( 44.90% ) . different combinations of mixtures had been experimented to produce viable alternates where different concentrations of refined wheat flour , whole - wheat flour , and semolina of different varieties of wheat were used to prepare pasta . pasta made from refined wheat flour was seen to have reduced cooking loss , colour , firmness and sensory score . pasta made from a combination of t. durum ( 50% ) semolina and t. aestivum ( 50% ) has acceptable levels of all the parameters , but due to increase in financial requirements , it is not suitable . from all results obtained , it was seen that semolina t. durum ( 50% ) mixed with refined wheat flour t. aestivum ( 50% ) can be used as alternate for making pasta for use as a low glycemic index snack product with acceptable physical and sensory properties . acceptable cooking quality parameters were obtained in the pasta samples containing a mixture of both semolina and refined wheat flour , as measured by cooked weight , cooking loss , and so forth during cooking . however the incorporation of additives in the mixture of 50% semolina ( t. durum ) along with 50% refined wheat flour ( t. aestivum ) was finally concluded as the optimal alternate , because of its almostequivalent properties to the 100% t. durum semolina pasta , thus making it a economically nutritious alternate ' resulting as a staple food in developing countries .
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as indicated by otto warburg many years ago and now accepted as a hallmark of cellular transformation , cancer cells entirely reprogram their metabolism to sustain hyperproliferation and growth also in particular environmental conditions . in particular , differently from normal cells , cancer cells rely mostly on glycolysis rather than oxidative phosphorylation ( oxphos ) for atp production [ 2 , 3 ] . tumor environment , oncogenes , and tumor suppressor mutations have an important role in this energetic shift to aerobic glycolysis [ 4 , 5 ] . another important feature of metabolic reprogramming of transformed cells is their reduced or strongly impaired mitochondrial function [ 3 , 6 ] . despite that , mitochondria cover an important role also in cancer cells , that is , through the maintenance of mitochondrial potential and oxidative equilibrium , necessary for cell viability and apoptosis control , and for the different anabolic processes that use precursors produced in this organelle such as lipid , amino acids , and nucleotides synthesis . there is a series of compounds targeting mitochondria , named mitocans , that are being tested as anticancer drugs . they usually lead to cancer cell death by inducing mitochondria destabilization with a consequent increase of reactive oxigen species ( ros ) and activation of apoptotic signals [ 7 , 8 ] . different classes of mitocans exist and can be classified into eight groups , more specifically hexokinase inhibitors , bcl-2 homology-3 ( bh3 ) mimetics , thiol redox inhibitors , drugs targeting the voltage - dependent anionic channel ( vdac ) or the adenine nucleotide translocator ( ant ) , agents interfering with the electron transport chain ( etc ) , lipophilic cations targeting the inner membrane , agents interfering with the mitochondrial dna , and drugs acting on not well - defined sites . among the compounds acting on the etc , vitamin e analogues that in particular target complex ii have been tested as anticancer agents . complex i inhibitors have shown anticancer properties as well , for example the acetogenins , such as rollinistatin and bullatacin , and also rotenone itself , which exhibits antitumor activity in animal models . on the other hand , cancer cells for their peculiar metabolism are particularly sensitive to treatments inhibiting glycolysis and to glucose deprivation [ 11 , 12 ] , since in both circumstances they lose hyperproliferative ability and ultimately die [ 1215 ] . therefore , combined treatment targeting both glycolysis and mitochondria , exploiting peculiar tumor features , may be lethal for cancer cells . in this regard it has been shown that cancer cells , like osteosarcoma cells , treated with etc inhibitors , are induced to switch over to glycolysis becoming hypersensitive to the glycolytic inhibitors . equally , it has been shown that inhibition of glucose metabolism , for example , by using 2-deoxyglucose ( 2-dg ) , can make tumor cells more dependent on oxphos and therefore more sensitive to treatment with etc inhibitors . however , glycolytic inhibitors , like 2-dg , could be potentially toxic for tissues like the brain , retinae , and testis that use glucose as the main energy source . in addition , they are also not very potent and must be used at high concentrations . in a previous study it has been shown that treatment of cancer cells with dichloroacetate ( dca ) , a tca cycle inducer , is able to redirect their metabolism from glycolysis to oxidative phosphorylation and hence to lead them towards apoptosis . therefore , it has been supposed that induction of a reversion of the warburg effect coupled to a treatment able to interfere with mitochondrial activity could specifically kill cancer cells . recently we have shown that exogenous activation of pka pathway can improve several mitochondrial parameters , leading to a warburg effect reversion , in k - ras cancer cells , where the protein kinase a ( pka ) pathway is generally deregulated . in fact , cancer cells treated with forskolin ( fsk ) , an activator of adenylate cyclase , show an increase of complex i activity , an increase of mitochondrial atp production , a decrease of ros generation , and an increase of mitochondria interconnections , that may lead to survival under glucose depletion . since nutrient deprivation widely exists in solid tumors because of the poor blood supply [ 21 , 22 ] , we decided to study the effects on cancer cells of glucose depletion , mimicking physiological tumor condition , instead of glycolysis inhibitors , combined with treatments with oxphos complex i inhibitors . as results we demonstrate that in low glucose availability different cancer cell lines , in a way dependent on their glycolytic metabolism , become sensitive to short treatment with low doses of complex i inhibitors as compared to optimal glucose condition . interestingly , in such a glucose - depleted condition , we also find evidence that stimulation of mitochondrial activity by fsk can further sensitize cancer cells to complex i inhibitors by enhancing cancer cell death . altogether our findings indicate that stimulation of respiratory chain activity , in low glucose availability , makes glycolytic cancer cells more sensitive to oxphos inhibitors . breast cancer cells mda - mb-231 , mouse fibroblasts nih3t3 normal and transformed , pancreatic cancer cells mia paca-2 , and lung cancer cells a549 were routinely cultured in dulbecco 's modified eagle 's medium ( dmem ) containing 4 mm l - glutamine , 100 u / ml penicillin , and 100 mg / ml streptomycin ( complete medium ) , supplemented with 510% fetal bovine serum ( human cells ) or 10% newborn calf serum ( mouse cells ) . for the experiments cells were plated in complete growth medium . after 16 hours cells were washed twice with phosphate buffer saline ( pbs ) and incubated in growth medium ( time 0 ) without glucose and sodium pyruvate , supplemented with 25 or 1 mm glucose . treatments and analyses were performed at 48 hours ( mda - mb-231 and a549 ) or 72 hours ( nih3t3 and mia paca-2 ) after time 0 . all reagents for media were purchased from life technologies ( carlsbad , ca , usa ) . ( st . louis , mo , usa ) . capsaicin and piericidin a were purchased from vinci - biochem ( florence , italy ) . cell viable count was performed by staining cells with trypan blue 0.4% ( life technologies ) . propidium iodide ( pi)/annexin v - fitc staining was performed using apoptosis assay kit from immunological sciences ( rome , italy ) and analyzed by facscan flow cytometer ( becton - dickinson , franklin lakes , nj , usa ) with cellquest software ( becton - dickinson ) . flow cytometric data were then carried out using the freely available winmdi software . for the evaluation of pi incorporation 5 10 cells were harvested and stained with 5 g / ml pi and 5 g / ml hoechst ( sigma - aldrich inc . ) in pbs for 15 min at r.t . after staining , cells were mounted on a microscope slide with 50% glycerol and analyzed under a nikon eclipse 90i fluorescence microscope ( nikon , tokyo , japan ) equipped with a b / w ccd camera ( hamamatsu - coolsnap , hamamatsu corporation , hamamatsu city , japan ) . the images were acquired using the imaging software metamorph 7 and then visualized and processed in image j ( freely available ) . for each sample after 12 days colonies were fixed with pbs - formaldehyde 5% , stained with crystal violet 1% , and then counted . intracellular atp levels were measured using celltiter glo luciferin - luciferase assay ( promega , madison , wi , usa ) as described in . mitochondrial potential was analyzed by staining cells with 20 nm jc-1 ( 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide , life technologies ) for 10 minutes . after staining , flow cytometric analysis was performed acquiring fl1 ( jc-1 monomers , low potential ) and fl2 ( jc-1 aggregates , high potential ) signals . for each sample d - glucose levels in culture medium were determined using a spectrophotometric assay kit ( r - biopharm , darmstadt , germany ) as specified by manufacturer 's datasheet . for the analysis of cleaved caspase 3 and actin b expression , cells were harvested and lysed in laemli buffer ( 50 mm tris - hcl ph6.8 , glycerol 6% , sds 2% , -mecaptoethanol 5% , bromophenol blue 0.05% ) . samples were then resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane , which was incubated overnight with antibodies for cleaved caspase 3 ( cell signaling technology inc . , danvers , ma , usa ; 1 : 1000 ) and actin b ( abcam , cambridge , uk ; 1 : 1000 ) . oxygen consumption was determined using seahorse xf24 extracellular flux analyzer ( seahorse bioscience , north billerica , ma , usa ) . cells were seeded in the 24-well xf24 cell culture plate in the culture medium containing 25 or 1 mm glucose , as described above . where indicated , cells were also treated with fsk . culture media were exchanged for base media ( unbuffered dmem supplemented with 10 mm sodium pyruvate and 20 mm glucose for cells grown in high glucose or only 10 mm sodium pyruvate for cells grown in low glucose ) 1 hour before the assay and for the duration of the experiment . selective inhibitors were injected during the measurements to achieve final concentrations of rotenone 3 nm and piericidin a 5 nm . the baseline ocr was defined as the average of the values measured from time points 1 to 5 ( 045 min ) during the experiments . due to some variations in the absolute magnitude of ocr measurements in different experiments after the analysis the cells were fixed , stained with crystal violet , and dosed at spectrophotometer after colorant solubilization with acetic acid 10% ; all ocr values obtained by the instrument were normalized on cell density . mda - mb-231 human breast cancer cells , like several other cancer cells , use mainly glycolysis instead of mitochondrial respiration to generate atp and other anabolic substrates necessary for their proliferation and survival . in fact , in low glucose availability , these cells show a reduced proliferation and an increase of cell death because of their inability to maximize the use of oxphos especially for energetic use . in this scenario , we tested the ability of rotenone , an inhibitor of oxphos , to increase their sensitivity to glucose depletion . rotenone is a natural compound that has been used to interfere with mitochondrial respiration , in particular with complex i activity , and hence to reduce intracellular atp levels especially in oxphos - dependent cell lines [ 25 , 26 ] . in order to evaluate their ability to proliferate and survive under oxphos inhibition , we treated proliferating mda - mb-231 cells , grown for 48 hours in low glucose ( 1 mm glucose , 4 mm glutamine ) or high glucose ( 25 mm glucose , 4 mm glutamine ) , with rotenone . the treatment was executed at 48 hours of culture because , despite a comparable proliferation rate in the two different glucose concentrations ( figure 1(a ) ) , in low glucose condition external medium analysis indicated that this carbon source was almost completely depleted at this time point ( figure 1(b ) ) . in addition , measurement of the basal cellular oxygen consumption rate ( ocr ) by seahorse xf analyzer indicated a 40% increase of cellular respiration rate in cells grown in low glucose ( figure 1(c ) ) , suggesting that mda - mb-213 cells , in absence of glucose as main substrate for glycolysis , partially shifted from glycolysis to mitochondrial respiration . short treatment with a low concentration of rotenone ( 3 nm for 4 hours ) , known to be ineffective on normal cell mitochondria activity [ 2729 ] , in glucose - depleted condition induced a reduction of cell viability , as confirmed by morphological analysis ( figure 1(d ) , circle and floating cells ) and by trypan blue viable cell count ( figure 1(e ) ) . in fact , after treatment trypan blue - positive cells increased from 10.2% to 22.3% . this effect on cell survival was also supported by clonogenic assays ( figures 1(f ) and 1(g ) ) , which showed that treated cells , replated in high glucose condition ( 25 mm ) , formed less colonies ( about 50% of reduction ) as compared to untreated control . such an assay shows that the short treatment is enough to reduce cancer cell ability to form a large colony and proliferate , suggesting that rotenone , inhibiting the alternative mitochondrial energetic route of these cancer cells upon glucose deprivation , heavily affects their viability . rotenone outcome on mitochondrial activity was evaluated by determination of ocr , mainly due to mitochondrial respiration , mitochondrial potential , and intracellular atp levels . in particular , 3 nm rotenone was injected by the instrument into the cells and its effect analyzed between 30 minutes and 1 hour after the injection . as shown in figure 1(h ) , ocr was reduced to ~30% of the baseline rates , indicating that 3 nm rotenone is able to decrease mitochondrial respiration . also mitochondrial potential was reduced by rotenone ( figure 1(i ) ) , confirming the direct effect of the treatment on mitochondrial function . in the same experimental setting , rotenone induced also a ~25% decrease of the intracellular atp levels ( figure 1(j ) ) . importantly , rotenone treatment in nonlimiting glucose condition had no effect on cell survival , as confirmed by trypan blue viable cell count ( figure 1(k ) ) and clonogenic assay ( figure 1(l ) ) . moreover , rotenone had no effect on intracellular atp levels ( figure 1(m ) ) , suggesting that in high glucose availability atp is generated essentially by glycolysis . it has been proposed that warburg effect reversion , gained by mitochondrial reactivation , may be a promising method for promoting naturally encoded programmed cell death and hence kill cancer cells . given that , we sought to investigate whether the combination of fsk , able to restore mitochondrial activity , and rotenone could synergistically enhance the killing of cancer cells in glucose depletion . first , we performed such an analysis on nih3t3 mouse fibroblasts ( immortalized cells , normal ) , an oxphos - dependent cell line , and nih3t3 mouse fibroblasts expressing an oncogenic k - ras gene ( transformed ) [ 15 , 24 ] . the latter cellular model of transformation is suitable since it presents a transcriptional profile and different metabolic features , such as the warburg effect , comparable to several human cancer cells harboring an oncogenic k - ras gene , like , for instance , mda - mb-231 cells [ 23 , 24 , 30 , 31 ] . the cells grown for 72 hours in both initial glucose concentrations were incubated with rotenone and fsk alone or in combination . as shown in figures 2(a ) and 2(b ) , in nonlimiting glucose condition rotenone had no effect on proliferation of both cell lines , confirming that such a low rotenone concentration does not inhibit mitochondrial respiration of normal cells ( figure 2(a ) ) and does not induce cell death in mouse transformed cells ( figure 2(b ) ) , as previously observed in mda - mb-231 cells . on the contrary , cells grown in low glucose for 72 hours , the time point at which both cell lines have completely consumed the glucose in the culture medium , showed a different response to the treatments with rotenone and/or fsk ( refer to figure 2(c ) for treatments schedule ) . normal cells were found to be insensitive to rotenone either alone or in combination with fsk ( figure 2(d ) ) . in contrast , transformed cells showed 17% of cell death in basal condition , 22% upon fsk treatment , 29% upon rotenone , and 42% when the two compounds were used in combination ( figure 2(e ) ) . these data indicate that normal mouse cells , relying especially on mitochondrial respiration , are less responsive to low doses of rotenone as well as to the combined treatment with fsk . on the contrary , transformed mouse cells , forced to use mitochondrial respiration by glucose deprivation or fsk treatment , become more sensitive to the complex i inhibitor . since a previous study has indicated that mda - mb-231 cells are responsive to fsk treatment as well as mouse k - ras - transformed fibroblasts , we treated mda - mb-231 cancer cells , grown in low glucose , with rotenone and fsk alone or in combination ( schedule is shown in figure 2(c ) ) . in order to evaluate cell death upon single or combined treatments , the cells were analyzed through trypan blue viable count ( figure 3(a ) ) or pi / annexin v staining followed by facs analysis ( figure 3(b ) and supplementary figure 1 in the supplementary material available online at http://dx.doi.org/10.1155/2013/243876 ) . as shown in figures 3(a ) and 3(b ) , both analyses indicated an increased cell death in the samples subjected to the combined treatment as compared to either untreated or rotenone - alone - treated samples . in particular , trypan blue staining indicated an increase of positive cells from 22% for rotenone alone to 33.6% in presence of fsk . similar values were observed by pi / annexin v staining ( 18% rotenone versus 30% rotenone + fsk ) . importantly , the combined treatment further reduced cancer cell ability to form colonies as compared to rotenone alone ( figure 3(c ) ) . the increase of cell death was associated with a reduction of about 50% of intracellular atp levels ( figure 3(d ) ) and of around 20% of mitochondrial potential ( figure 3(e ) ) as compared to untreated samples . to confirm a role of fsk in inducing a positive effect on mitochondrial activity , next we measured basal ocr , as previously described , in untreated or 2-to-4-hour fsk - treated cells . the interval of treatment was chosen since in our assays the cells were treated with fsk ( pretreatment plus combination with oxphos inhibitors ) for a maximum time of 5 hours . as shown in figure 3(f ) , fsk - treated samples showed an increase of around 30% of ocr as compared to untreated samples , suggesting that the formers are more respirative than the latter ones . moreover we did not observe differences in ocr values obtained in 2 and 4 hours - treated samples . altogether these findings indicate that , upon glucose depletion , the stimulation of respiratory chain activity makes cells more sensitive to oxphos inhibitors . to further confirm the role of mitochondrial inhibition in the cell death mechanism upon glucose depletion , and more specifically the role of complex i , we used two other inhibitors of this complex , namely piericidin a and capsaicin . importantly piericidin a , differently from rotenone that at higher concentration may affect cell cycle [ 33 , 34 ] , does not interfere with the cell cycle execution . as shown in figures 4(a ) and 4(b ) , upon 2 hours of treatment , both inhibitors , as previously observed with rotenone , did not induce cell death when added to mda - mb-231 grown in high glucose . on the contrary , their addition to glucose - depleted cells led to an increase of mda - mb-231 cell death that was much stronger in the samples treated with piericidin a ( 52% ) ( figure 4(c ) ) than with capsaicin ( 28% ) ( figure 4(d ) ) . notably , combined treatment with fsk further increased the percentage of cell death that reached a value of 80% in the sample piericidin a + fsk ( figure 4(c ) ) and 39% in the sample capsaicin + fsk ( figure 4(d ) ) . since cell viability was greatly affected by piericidin a , we evaluated also the potential of this molecule in combination with fsk in clonogenic assays ( figure 4(e ) ) . obtained data indicated a significant reduction of colonies number after treatment with piericidin a , reduction that was further increased upon combination with fsk as compared to untreated and fsk - treated sample ( figure 4(e ) ) . notably , ocr measurement indicated that piericidin a was able to decrease mitochondrial respiration ( figure 4(f ) ) as well as previously observed with rotenone ( figure 1(h ) ) , confirming its effect on cellular mitochondrial respiration . glucose deprivation has been shown to kill cells either by necrosis or through the mitochondrial pathway of apoptosis . similarly , prolonged treatment with mitochondrial oxphos inhibitors also lead to necrotic cell death . in order to assess whether single or combined treatments could induce necrosis or apoptosis , we performed experiments of pi incorporation followed by microscopy analysis and of western blot . as shown in figures 5(a ) and 5(b ) , complex i mitochondrial inhibitors ( rotenone and piericidin a ) caused an increase of pi incorporating cells as compared to untreated or fsk - treated samples . as expected from previous results , fsk treatment further enhanced the percentage of pi incorporating cells , supporting the notion that cells were experiencing a necrotic cell death process ( figures 5(a ) and 5(b ) ) . such a cell death mechanism was confirmed by morphology analysis indicating cell detachment ( data not shown ) and plasma membrane damage without nuclear condensation ( figure 5(a ) , hoechst staining ) . in addition , this cell death process was not associated with activation of caspase 3 ( figures 5(c ) and 5(d ) ) and poly(adp - ribose ) polymerase ( parp ) ( data not shown ) , both considered apoptotic markers , as it was seen with thapsigargin treatment for 6 hours ( figures 5(c ) and 5(d ) ) . to examine whether other cancer cell lines showed similar sensitivity to complex i inhibitors alone and combined with fsk , we examined the effect of the treatments on two different human cancer cell lines with a different dependence on glucose availability . previous data , in fact , have shown that mia paca-2 ( pancreatic cancer cell line ) and a549 ( non - small - cell lung cancer cell line ) are both sensitive to glucose deprivation , in particular mia paca-2 , undergoing cell death , and , in different extend , are both protected by fsk treatment because of its ability to restore complex i activity . moreover , both cell lines express an oncogenic k - ras that has been shown to promote cell metabolic rewiring and in particular glycolysis [ 24 , 37 ] . as shown in figures 6(a ) and 6(b ) piericidin a treatment , used at concentration of 10 nm for 8 hours , did not affect survival of both cell lines grown in 25 mm glucose , as measured by trypan blue vital staining . on the contrary , as previously observed for mda - mb-231 cells , its addition in glucose depletion led in both cell lines to an increase of cell death that was stronger in more glycolytic cells mia paca-2 ( 37.9% ) ( figure 6(c ) ) than in a549 ( 23.9% ) ( figure 6(d ) ) . notably , combined treatment with fsk further increased the percentage of cell death , reaching a value of 52.6% in mia paca-2 ( figure 6(c ) ) and 33.4% in a549 ( figure 4(d ) ) . interestingly , in mia paca-2 cells an increase of cell death was already observed in cells grown in low glucose as compared to high glucose ( 22.6% versus 10.8% ) , confirming their major dependence on glucose availability as compared to a549 cells . to further confirm the major sensitivity of cancer cells to inhibitors of mitochondrial function in a condition of low glucose mda - mb-231 cells are considered a cell line with a high glycolytic rate and a low level of respiration [ 15 , 24 ] . for this reason we supposed that under treatment with oligomycin these cells could undergo a limited effect in high glucose and a significant effect in low glucose , since the latter condition is characterized by acute stimulation of respiration . mda - mb-231 cells , cultured in 25 mm glucose , were treated for 1 hour with 5 m oligomycin and then analyzed . as shown in figures 7(a ) and 7(b ) both cell viability and total intracellular atp were not changed by the treatment , whilst a slight decrease of mitochondrial potential and a consistent decrease of colony formation ability were observed ( figures 7(c ) and 7(d ) ) . on the other hand , mda - mb-231 cells grown in low glucose and treated with oligomycin showed a strong increase of cell death as indicated by trypan blue viable count ( figure 7(e ) ) . moreover , a complete depletion of intracellular atp ( figure 7(f ) ) , associated with a partial reduction of mitochondrial potential ( figure 7(g ) ) , was observed . all these parameters were accompanied by a further decrease in the ability to form colonies as compared to cells grown in high glucose ( figure 7(h ) ) . taken together , these data indicate that in glucose shortage glycolytic cancer cells show a stronger and forced dependence on mitochondrial respiration that make them very sensitive to inhibitors of mitochondria function . in fact , since this organelle is central both as producer of cellular atp and as central regulator of apoptosis , it may be considered a good therapeutic hit . the primary metabolic function of mitochondria is oxphos , an energy - generating process that couples the oxidation of respiratory substrates with atp production . besides atp synthesis , mitochondria are involved in several other key metabolic processes such as oxidative decarboxylation of pyruvate , tricarboxylic acid cycle , and fatty acids oxidation . in addition , mitochondria take part in intracellular homeostasis of calcium and phosphate as well as in the balance of nad / nadh . besides their central role in metabolic activity , more recently mitochondria have been shown to have a central role also in the cascade of events that leads to programmed cell death . in fact mitochondria represent a central checkpoint of this process by integrating various signals coming from endogenous factors ( ions , metabolites , second messengers ) , from endogenous signaling proteins ( kinases and phosphatases ) , and from exogenous factors ( nutrients , oxygen ) . therefore the strong interconnection between metabolism and apoptosis and the central role of mitochondria in both processes have led to an explosion of interest in connecting such pathways to the pathophysiology of cancer . in this report , we have decided to utilize the main metabolic alterations of cancer cells , namely hyperglycolytic phenotype ( warburg effect ) and mitochondria dysfunction , as targets for combined treatments aimed to specifically kill cancer cells . in fact , as shown by a number of groups , cancer cell proliferation and tumor aggressiveness correlate with an enhanced glycolysis and a low mitochondrial respiratory chain activity [ 3 , 31 ] , and positive or negative modulation of oxphos activity , depending on the metabolic state of cancer cells , appears to reduce tumor growth [ 39 , 40 ] . herein by using a glycolytic human breast cancer cell line , namely mda - mb-231 , grown in limiting glucose availability , and some natural inhibitors of mitochondria activity , we show that complex i inhibition associated with an acute stimulation of respiration , due to glucose depletion , induces specifically necrotic cancer cell death ( figures 1 , 3 , and 5 ) . notably , this finding has been observed by using three different complex i inhibitors that strongly support our results ( figures 4 and 5 ) . moreover , we show that this cell death process , induced by the treatment with complex i inhibitors , is activated also in other three cancer cell lines , mouse k - ras transformed fibroblasts , human mia paca-2 pancreatic cancer cells , and human a549 lung adenocarcinoma cells . importantly , such cell death mechanism is strongly dependent on glycolytic rate of the cancer cells . in fact mia paca-2 cells , known to have a higher glycolysis as compared to a549 , appear to be more sensitive to the treatment with inhibitors ( figure 6 ) . our results are interesting also because our and previously published data indicate that , at the used concentrations , rotenone ( 3 nm ) , capsaicin ( 100 m ) , and piericidin a ( 5 nm ) have no effect on immortalized fibroblasts ( figure 2 ) , on normal pancreatic cells and on dopamine neurons , respectively . this reduced or absent effect on normal cells is an important characteristic for exploiting these compounds for cancer therapy . regardless of the mechanism of action of the three molecules , we show that the sensitivity to them increases upon glucose depletion that reflects the dependency of the cells on glycolysis . we suppose that less glycolytic cells , such as immortalized fibroblasts or normal cells , will be also less sensitive to these treatments . since we observed a necrotic cell death ( figure 5 ) , we attribute the synergistic effects more to atp depletion than a rapid decrease of mitochondrial potential . however we can not exclude an increase of ros levels , as shown by other authors , as a consequence of complex i inhibition [ 17 , 25 ] . in fact , it is possible that stimulation of mitochondrial activity upon glucose depletion in the deranged respiratory system of malignant cells results in an increased production of oxidants , which may overwhelm cellular antioxidant protections and lead to cell death . further experiments exploring this point will be addressed in the future . from our studies , in particular from results obtained with fsk , an important point emerges : stimulation of mitochondrial activity and restoration of an atp generating mechanism more similar to nonmalignant cells , might be an efficient tool in anticancer strategy . in particular , shifting cellular metabolism towards mitochondrial atp production might overcome the positive effects on glycolytic pathway of oncogenes like k - ras , akt and hif-1 . the idea is not completely new , since other authors have addressed this point by redirection of pyruvate towards oxidation in the mitochondria . in fact , inhibition of pyruvate dehydrogenase kinase ( pdk ) by dca , or lactate dehydrogenase ( ldh ) by rnai , has been shown to shift metabolism from glycolysis to glucose oxidation and to strongly reduce cancer cell viability and tumor growth [ 18 , 43 ] . our results with fsk suggest a similar mechanism in which reactivation of the mitochondrial function associated with glucose depletion and mitochondrial complex i inhibition strongly affects cancer cell survival . therefore , strategies involving the manipulation of both glycolytic and mitochondrial pathways might be useful to eradicate cancer cells . other information in such a direction was derived also by experiments with oligomycin , an inhibitor of mitochondrial atp synthase . we show that it is able to enhance cancer cell death in low glucose as compared to high glucose ( figure 7 ) . in fact , we observed that in normal glucose conditions it does not induce a strong reduction of cell viability , although it is able to reduce the capability to form colonies . we can suppose that such a long - term effect is due to the inhibition of the reverse action of atpase that in fact is reflected in the mitochondrial potential decrease not accompanied with loss of atp . on the other hand , the stronger effect of oligomycin on mda - mb-231 cells in glucose deprivation is experimental evidence of their forced dependence on oxphos in such condition , exploitable by mitochondrial targeting therapies . moreover , from another point of view , these data could suggest that inhibition of residual mitochondrial activity of cancer cells will further upregulate glycolysis and hence lead to their death by glucose depletion . this finding has been observed in lung carcinoma , in which oligomycin treatment upregulates glycolysis , increasing their dependence on this metabolic pathway . taken together our results provide a rationale for the use of mitochondrial inhibitors in cancer cells exploiting cancer cell fragility versus glucose depletion . in addition they point to an energetic switch from glycolysis to oxphos as an important therapeutic approach , since normal cells appear to be resistant to such combined treatments .
cancer cells generally rely mostly on glycolysis rather than oxidative phosphorylation ( oxphos ) for atp production . in fact , they are particularly sensitive to glycolysis inhibition and glucose depletion . on the other hand mitochondrial dysfunctions , involved in the onset of the warburg effect , are sometimes also associated with the resistance to apoptosis that characterizes cancer cells . therefore , combined treatments targeting both glycolysis and mitochondria function , exploiting peculiar tumor features , might be lethal for cancer cells . in this study , we show that glucose deprivation and mitochondrial complex i inhibitors synergize in inducing cancer cell death . in particular , our results reveal that low doses of complex i inhibitors , ineffective on immortalized cells and in high glucose growth , become specifically cytotoxic on cancer cells deprived of glucose . importantly , the cytotoxic effect of the inhibitors on cancer cells is strongly enhanced by forskolin , a pka pathway activator , that we have previously shown to stimulate oxphos . taken together , we demonstrate that induction in cancer cells of a switch from a glycolytic to a more respirative metabolism , obtained by glucose depletion or mitochondrial activity stimulation , strongly increases their sensitivity to low doses of mitochondrial complex i inhibitors . our findings might be a valuable approach to eradicate cancer cells .
1. Introduction 2. Material and Methods 3. Results 4. Discussion
in particular , differently from normal cells , cancer cells rely mostly on glycolysis rather than oxidative phosphorylation ( oxphos ) for atp production [ 2 , 3 ] . on the other hand , cancer cells for their peculiar metabolism are particularly sensitive to treatments inhibiting glycolysis and to glucose deprivation [ 11 , 12 ] , since in both circumstances they lose hyperproliferative ability and ultimately die [ 1215 ] . therefore , combined treatment targeting both glycolysis and mitochondria , exploiting peculiar tumor features , may be lethal for cancer cells . therefore , it has been supposed that induction of a reversion of the warburg effect coupled to a treatment able to interfere with mitochondrial activity could specifically kill cancer cells . in fact , cancer cells treated with forskolin ( fsk ) , an activator of adenylate cyclase , show an increase of complex i activity , an increase of mitochondrial atp production , a decrease of ros generation , and an increase of mitochondria interconnections , that may lead to survival under glucose depletion . since nutrient deprivation widely exists in solid tumors because of the poor blood supply [ 21 , 22 ] , we decided to study the effects on cancer cells of glucose depletion , mimicking physiological tumor condition , instead of glycolysis inhibitors , combined with treatments with oxphos complex i inhibitors . as results we demonstrate that in low glucose availability different cancer cell lines , in a way dependent on their glycolytic metabolism , become sensitive to short treatment with low doses of complex i inhibitors as compared to optimal glucose condition . interestingly , in such a glucose - depleted condition , we also find evidence that stimulation of mitochondrial activity by fsk can further sensitize cancer cells to complex i inhibitors by enhancing cancer cell death . on the contrary , transformed mouse cells , forced to use mitochondrial respiration by glucose deprivation or fsk treatment , become more sensitive to the complex i inhibitor . to further confirm the role of mitochondrial inhibition in the cell death mechanism upon glucose depletion , and more specifically the role of complex i , we used two other inhibitors of this complex , namely piericidin a and capsaicin . to examine whether other cancer cell lines showed similar sensitivity to complex i inhibitors alone and combined with fsk , we examined the effect of the treatments on two different human cancer cell lines with a different dependence on glucose availability . previous data , in fact , have shown that mia paca-2 ( pancreatic cancer cell line ) and a549 ( non - small - cell lung cancer cell line ) are both sensitive to glucose deprivation , in particular mia paca-2 , undergoing cell death , and , in different extend , are both protected by fsk treatment because of its ability to restore complex i activity . in this report , we have decided to utilize the main metabolic alterations of cancer cells , namely hyperglycolytic phenotype ( warburg effect ) and mitochondria dysfunction , as targets for combined treatments aimed to specifically kill cancer cells . in fact , as shown by a number of groups , cancer cell proliferation and tumor aggressiveness correlate with an enhanced glycolysis and a low mitochondrial respiratory chain activity [ 3 , 31 ] , and positive or negative modulation of oxphos activity , depending on the metabolic state of cancer cells , appears to reduce tumor growth [ 39 , 40 ] . herein by using a glycolytic human breast cancer cell line , namely mda - mb-231 , grown in limiting glucose availability , and some natural inhibitors of mitochondria activity , we show that complex i inhibition associated with an acute stimulation of respiration , due to glucose depletion , induces specifically necrotic cancer cell death ( figures 1 , 3 , and 5 ) . moreover , we show that this cell death process , induced by the treatment with complex i inhibitors , is activated also in other three cancer cell lines , mouse k - ras transformed fibroblasts , human mia paca-2 pancreatic cancer cells , and human a549 lung adenocarcinoma cells . regardless of the mechanism of action of the three molecules , we show that the sensitivity to them increases upon glucose depletion that reflects the dependency of the cells on glycolysis . in fact , it is possible that stimulation of mitochondrial activity upon glucose depletion in the deranged respiratory system of malignant cells results in an increased production of oxidants , which may overwhelm cellular antioxidant protections and lead to cell death . our results with fsk suggest a similar mechanism in which reactivation of the mitochondrial function associated with glucose depletion and mitochondrial complex i inhibition strongly affects cancer cell survival . therefore , strategies involving the manipulation of both glycolytic and mitochondrial pathways might be useful to eradicate cancer cells . we show that it is able to enhance cancer cell death in low glucose as compared to high glucose ( figure 7 ) . on the other hand , the stronger effect of oligomycin on mda - mb-231 cells in glucose deprivation is experimental evidence of their forced dependence on oxphos in such condition , exploitable by mitochondrial targeting therapies . taken together our results provide a rationale for the use of mitochondrial inhibitors in cancer cells exploiting cancer cell fragility versus glucose depletion .
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the discovery of somatotropin - release inhibitory factor , or somatostatin ( sst ) , in hypothalamic extract in the early 1970s , led to important advancements in the comprehension of the regulation of growth hormone ( gh ) secretion and offered the opportunity to develop drugs mimicking the actions of sst . the synthesis of the first somatostatin analog ( ssa ) , octreotide ( oct ) , the discovery of 5 sst receptor ( sstr ) subtypes , and the development of additional sstr ligands offered a great opportunity for the medical treatment of acromegaly and neuroendocrine neoplasms ( nens ) . with an annual incidence of 5 cases/100,000 in the usa,1 nens are rare tumors composed of multipotent neuroendocrine cells able to produce , store , and secrete biologically active substances which can cause if functioning distinct clinical syndromes . both functioning and nonfunctioning tumors can also produce symptoms due to mass effects , and even if the majority of these tumors exhibit long periods of relatively small growth , in some cases , they can show massive growth and can be associated with distant metastases . in > 50% of cases , tumors originate in the gastrointestinal system or the pancreas ( gastroenteropancreatic neuroendocrine neoplasms [ gep - nens ] ) . treatment of nens requires a multimodal approach , involving both tumor debulking and management of symptoms . tumor diameter is one of the most important parameters in the decision - making process for nonfunctioning forms . in fact , while surgery represents the treatment of choice for larger nonfunctioning tumors , small lesions can be treated conservatively . locally advanced and metastatic disease can also be treated with extended resections , considering tumor grading , size , ki-67 proliferation index , and the presence of extra - abdominal disease . other approaches include pharmacological treatment ( ssas , interferon , antiangiogenic agents , tyrosine kinase inhibitors , mammalian - target - of - rapamycin inhibitors , chemotherapy ) , radionuclide therapy , and chemo- or radioembolization . in particular , ssas , which were initially used to control hormonal syndromes associated with nens , have been successfully proposed as antiproliferative agents , able to control tumor growth.2,3 moreover , long - acting formulations of ssas have demonstrated their efficacy as antineoplastic agents in the treatment of gep - nens.4 long - acting formulations of ssas ( oct lar , slow - release lan , and lan autogel ) assure improved patient compliance with weekly up to monthly injections . in this review , we focus on the clinical utility of lan autogel in the treatment of gep - nens . human sst , isolated in 1973 and soon identified as a hypothalamic inhibitor of gh,5,6 has been subsequently found in several other tissues ( central nervous system , endocrine system , and gastrointestinal tract ) . sst is formed by proteolytic processing of larger precursor molecules : prepro - sst and pro - sst.7 prepro - sst is a 116-aminoacid precursor which is encoded by a single gene located on chromosome 3q28 , and it is processed to pro - sst ( 96 amino - acids ) . pro - sst undergoes tissue - specific enzymatic degradation to produce 2 bioactive proteins : the predominant , but functionally less active 14-aminoacid molecule called sst-14 and the larger and more potent 28-aminoacid form sst-28 ( figure 1).8 sst isoforms have endocrine , paracrine and autocrine inhibitory effects as well as on exocrine glands . the mechanisms by which sst and ssas inhibit the neuroendocrine cells are complex and poorly understood.9 once sst binds to its cell- and tissue - specific receptors , the downstream effects elicited by the ligand receptor formation are site - specific : in the central nervous system , sst acts as a neurotransmitter , while in the hypothalamic pituitary region , it acts as a neurohormone.10 sst is also known to inhibit several cellular functions . in particular , in the digestive tract , sst reduces gastrointestinal motility , inhibits gallbladder contraction , decreases portal blood flow , and suppresses the secretion of gastrointestinal hormones ( insulin , glucagon , gastrin , cholecystokinin , vasoactive intestinal peptide , and secretin)11 and that of other exocrine gastrointestinal cells ( gastric acid , intestinal fluid , and pancreatic enzymes).12 moreover , it inhibits proliferation of both normal and tumor cells.13 circulating sst has a short half - life ( ~2 minutes ) because both bioactive isoforms ( sst-14 and sst-28 ) contain multiple enzymatic cleavage sites causing a rapid degradation . this not only makes the analysis of their physiological activity difficult but also represents a serious limitation for their application in clinical practice . synthetic ssas have been developed by reducing the polypeptide chain , leading to an increased affinity for sstrs and to a longer half - life.9 structure activity studies of sst-14 showed that the aminoacid residues phe , trp , lys , and thr , which comprise a -turn , are necessary for biological activity . compound sms 201 - 995 ( oct ) exhibits a 3-fold potency in the inhibition of insulin secretion and a 19-fold potency in gh secretion inhibition compared to native sst.14 the introduction of d - phe at the n - terminal and l - thr at the c - terminal end , and the substitution of l - trp by d - trp in position 8 make the peptide resistant to degradation . of the many hundreds of ssas synthesized , 4 are currently used in clinical settings : oct , lan , vapreotide ( vap ) , and pasireotide ( pas ; som 230 ) ( figure 2 ) . oct and lan ( first - generation ssas ) have been quickly considered first - line medical therapeutic options for the treatment of acromegaly and for the control of hormonal symptoms in patients with nens.11 oct and lan show high - affinity binding to sstr 2 and 5 , with half - lives of 2 hours and < 1 hour , respectively . rebound hypersecretion of hormones does not occur.14 both drugs have a small volume of distribution and a low clearance that result in a longer duration of exposure and long - lasting biological activity compared with sst . oct and lan are administered by multiple subcutaneous injections or by continuous subcutaneous infusion or by the intravenous route ( either as a single injection or as a continuous infusion over many hours or days ) . the oct lar formulation has been obtained by combining oct with microspheres of carboxymethylcellulose which increase its therapeutic action to 2442 days.15 two lar / slow - release formulations have been developed : a slow - release lan obtained by combining lan with microspheres of lactide / glycolide copolymers , which allows for administration of every 728 days,16 and lan autogel which is a viscous aqueous formulation supplied in ready - to - use prefilled syringes administered every 2856 days.17 pas ( som 230 ) , a ssa developed using biodegradable polymers by a method similar to that of oct lar,18 is a multireceptor - targeted sst generated by the introduction of 4 synthetic and 2 essential amino acids of sst in a novel cyclohexapeptide structure . the biological effects of sst are mediated by its interaction with 5 sstrs ( sstr 1 , sstr 2 , sstr 3 , sstr 4 , and sstr 5 ) belonging to the family of g - protein - coupled membrane receptors . each receptor , encoded by genes localized on different chromosomes,19 consists of a single polypeptide chain with 7 transmembrane - spanning domains : the extracellular domain exhibits the ligand - binding sites , while the intracellular domain provides linkage to second messenger activation.20 all 5 sstrs have been identified in the central nervous system , gastrointestinal tract , endocrine glands , exocrine glands , and inflammatory and immune cells21 ( figure 3 ) . sst-14 and sst-28 have approximately equivalent affinity for all the receptor subtypes , except for sstr 5 which has a 10-fold higher affinity for sst-28 suggesting a potentially different role for this receptor.22 the interaction between sst and its receptor subtypes activates a number of intracellular cascades , including the inhibition of adenylate cyclase activity and the activity of calcium channels , as well as stimulation of phosphotyrosine phosphatase or mapk activity.19 while all these pathways suppress secretion processes , the activation of phosphotyrosine phosphatase or mapk by sst may play a role in the regulation of cell proliferation.23,24 in addition , sstrs may affect the activity of phospholipase c , cyclic guanosine monophosphate , and phospholipase a2 all involved in signal transduction.25 however , the current understanding of sst / sstr intracellular signaling is based on in vitro models , and its in vivo relevance remains to be elucidated . the antiproliferative mechanisms of sst are different and dependent on the sstr subtype and target cell type . they involve hyperphosphorylation of the retinoblastoma gene product and g1 cell cycle arrest , but can also be mediated by sstr 3-induced apoptosis.26 moreover , sst may exert an indirect antiproliferative effect by inhibiting the release of growth factors and trophic hormones ( gh , igf1 , insulin , gastrin , epidermal growth factor ) both from neoplastic cells and from the surrounding tumor matrix.9 the latter mechanism also involves the antiproliferative effects on tumor angiogenesis which plays a critical role in tumor progression . sst shows antiangiogenic properties by inhibiting the production and release of pro - angiogenic factors as well as expression of their receptors . in particular , in the pancreatic cancer cell line pc-3 , sstr 2 expression correlates with expression of vegf and matrix metalloproteinase-2.27 as well as the normal cell lines in sst - target tissues , most tumors originating from these tissues express a high density of sstr . this is the case of nens deriving from the neuroendocrine cells of the gastrointestinal and bronchopulmonary systems , as well as of pituitary tumors , medulloblastomas , medullary thyroid carcinomas , and adenocarcinomas of the breast , ovary , and colon.7,28 on the other hand , poorly differentiated or undifferentiated tumors express sstr at a lower density than their corresponding well - differentiated neoplasias.28 the synthetic ssas oct , lan , and vap bind preferentially to sstr 2 and sstr 5 , and with moderate affinity to sstr 3 and low affinity to sstr 1 and sstr 4;29 in contrast , the multireceptor ligand pas ( som 230 ) binds with high affinity to subtypes sstr 1 , sstr 2 , sstr 3 , and sstr 5.30 compared with oct , pas displays a 40- , 30- , and 5-fold higher binding affinity for sstr 5 , sstr 1 , and sstr 3 , respectively , and a 2.5-time lower binding affinity for sstr 2 . moreover , pas has a 106-fold higher affinity for sstr 5 in comparison with lan.29 both oct and lan have potent activity against gep - nens and inhibit cell proliferation by several mechanisms . a direct antitumor effect may result from the activation of sstrs on tumor cells leading to modulation of intracellular signaling pathways . immunohistochemistry and autoradiography reveal that sstr proteins are highly expressed in gastrinomas , insulinomas , and carcinoid tumors , and their metastases . the majority of the tumors express sstr 2 , followed by sstr 1 , sstr 5 , and sstr 3 , while sstr 4 is expressed in a minority of cases.31 the frequency and pattern of expression of each subtype can , however , vary in different tumor types and in each patient.32 indeed , sstrs are expressed in lower density in undifferentiated gep - nens than well - differentiated tumors . the largest expression of sstr 2 on pancreatic endocrine or carcinoid tumors is the reason for the successful clinical application of oct and lan in controlling symptoms related to hormonal hypersecretion.33 furthermore , ssas may also produce an indirect antitumor effect by inhibiting mitogenic growth factors ( such as igf ) and by inhibiting tumor angiogenesis through interaction with sstrs on endothelial cells and monocytes . in immortalized human dermal microvascular endothelial cells , expression of vegf and vegf receptor-2 and vegf release are inhibited by sstr 1 agonists.34 human sst , isolated in 1973 and soon identified as a hypothalamic inhibitor of gh,5,6 has been subsequently found in several other tissues ( central nervous system , endocrine system , and gastrointestinal tract ) . sst is formed by proteolytic processing of larger precursor molecules : prepro - sst and pro - sst.7 prepro - sst is a 116-aminoacid precursor which is encoded by a single gene located on chromosome 3q28 , and it is processed to pro - sst ( 96 amino - acids ) . pro - sst undergoes tissue - specific enzymatic degradation to produce 2 bioactive proteins : the predominant , but functionally less active 14-aminoacid molecule called sst-14 and the larger and more potent 28-aminoacid form sst-28 ( figure 1).8 sst isoforms have endocrine , paracrine and autocrine inhibitory effects as well as on exocrine glands . the mechanisms by which sst and ssas inhibit the neuroendocrine cells are complex and poorly understood.9 once sst binds to its cell- and tissue - specific receptors , the downstream effects elicited by the ligand receptor formation are site - specific : in the central nervous system , sst acts as a neurotransmitter , while in the hypothalamic pituitary region , it acts as a neurohormone.10 sst is also known to inhibit several cellular functions . in particular , in the digestive tract , sst reduces gastrointestinal motility , inhibits gallbladder contraction , decreases portal blood flow , and suppresses the secretion of gastrointestinal hormones ( insulin , glucagon , gastrin , cholecystokinin , vasoactive intestinal peptide , and secretin)11 and that of other exocrine gastrointestinal cells ( gastric acid , intestinal fluid , and pancreatic enzymes).12 moreover , it inhibits proliferation of both normal and tumor cells.13 circulating sst has a short half - life ( ~2 minutes ) because both bioactive isoforms ( sst-14 and sst-28 ) contain multiple enzymatic cleavage sites causing a rapid degradation . this not only makes the analysis of their physiological activity difficult but also represents a serious limitation for their application in clinical practice . synthetic ssas have been developed by reducing the polypeptide chain , leading to an increased affinity for sstrs and to a longer half - life.9 structure activity studies of sst-14 showed that the aminoacid residues phe , trp , lys , and thr , which comprise a -turn , are necessary for biological activity . compound sms 201 - 995 ( oct ) exhibits a 3-fold potency in the inhibition of insulin secretion and a 19-fold potency in gh secretion inhibition compared to native sst.14 the introduction of d - phe at the n - terminal and l - thr at the c - terminal end , and the substitution of l - trp by d - trp in position 8 make the peptide resistant to degradation . of the many hundreds of ssas synthesized , 4 are currently used in clinical settings : oct , lan , vapreotide ( vap ) , and pasireotide ( pas ; som 230 ) ( figure 2 ) . oct and lan ( first - generation ssas ) have been quickly considered first - line medical therapeutic options for the treatment of acromegaly and for the control of hormonal symptoms in patients with nens.11 oct and lan show high - affinity binding to sstr 2 and 5 , with half - lives of 2 hours and < 1 hour , respectively . rebound hypersecretion of hormones does not occur.14 both drugs have a small volume of distribution and a low clearance that result in a longer duration of exposure and long - lasting biological activity compared with sst . oct and lan are administered by multiple subcutaneous injections or by continuous subcutaneous infusion or by the intravenous route ( either as a single injection or as a continuous infusion over many hours or days ) . the oct lar formulation has been obtained by combining oct with microspheres of carboxymethylcellulose which increase its therapeutic action to 2442 days.15 two lar / slow - release formulations have been developed : a slow - release lan obtained by combining lan with microspheres of lactide / glycolide copolymers , which allows for administration of every 728 days,16 and lan autogel which is a viscous aqueous formulation supplied in ready - to - use prefilled syringes administered every 2856 days.17 pas ( som 230 ) , a ssa developed using biodegradable polymers by a method similar to that of oct lar,18 is a multireceptor - targeted sst generated by the introduction of 4 synthetic and 2 essential amino acids of sst in a novel cyclohexapeptide structure . the biological effects of sst are mediated by its interaction with 5 sstrs ( sstr 1 , sstr 2 , sstr 3 , sstr 4 , and sstr 5 ) belonging to the family of g - protein - coupled membrane receptors . each receptor , encoded by genes localized on different chromosomes,19 consists of a single polypeptide chain with 7 transmembrane - spanning domains : the extracellular domain exhibits the ligand - binding sites , while the intracellular domain provides linkage to second messenger activation.20 all 5 sstrs have been identified in the central nervous system , gastrointestinal tract , endocrine glands , exocrine glands , and inflammatory and immune cells21 ( figure 3 ) . sst-14 and sst-28 have approximately equivalent affinity for all the receptor subtypes , except for sstr 5 which has a 10-fold higher affinity for sst-28 suggesting a potentially different role for this receptor.22 the interaction between sst and its receptor subtypes activates a number of intracellular cascades , including the inhibition of adenylate cyclase activity and the activity of calcium channels , as well as stimulation of phosphotyrosine phosphatase or mapk activity.19 while all these pathways suppress secretion processes , the activation of phosphotyrosine phosphatase or mapk by sst may play a role in the regulation of cell proliferation.23,24 in addition , sstrs may affect the activity of phospholipase c , cyclic guanosine monophosphate , and phospholipase a2 all involved in signal transduction.25 however , the current understanding of sst / sstr intracellular signaling is based on in vitro models , and its in vivo relevance remains to be elucidated . the antiproliferative mechanisms of sst are different and dependent on the sstr subtype and target cell type . they involve hyperphosphorylation of the retinoblastoma gene product and g1 cell cycle arrest , but can also be mediated by sstr 3-induced apoptosis.26 moreover , sst may exert an indirect antiproliferative effect by inhibiting the release of growth factors and trophic hormones ( gh , igf1 , insulin , gastrin , epidermal growth factor ) both from neoplastic cells and from the surrounding tumor matrix.9 the latter mechanism also involves the antiproliferative effects on tumor angiogenesis which plays a critical role in tumor progression . sst shows antiangiogenic properties by inhibiting the production and release of pro - angiogenic factors as well as expression of their receptors . in particular , in the pancreatic cancer cell line pc-3 , sstr 2 expression correlates with expression of vegf and matrix metalloproteinase-2.27 as well as the normal cell lines in sst - target tissues , most tumors originating from these tissues express a high density of sstr . this is the case of nens deriving from the neuroendocrine cells of the gastrointestinal and bronchopulmonary systems , as well as of pituitary tumors , medulloblastomas , medullary thyroid carcinomas , and adenocarcinomas of the breast , ovary , and colon.7,28 on the other hand , poorly differentiated or undifferentiated tumors express sstr at a lower density than their corresponding well - differentiated neoplasias.28 the synthetic ssas oct , lan , and vap bind preferentially to sstr 2 and sstr 5 , and with moderate affinity to sstr 3 and low affinity to sstr 1 and sstr 4;29 in contrast , the multireceptor ligand pas ( som 230 ) binds with high affinity to subtypes sstr 1 , sstr 2 , sstr 3 , and sstr 5.30 compared with oct , pas displays a 40- , 30- , and 5-fold higher binding affinity for sstr 5 , sstr 1 , and sstr 3 , respectively , and a 2.5-time lower binding affinity for sstr 2 . moreover , pas has a 106-fold higher affinity for sstr 5 in comparison with lan.29 both oct and lan have potent activity against gep - nens and inhibit cell proliferation by several mechanisms . a direct antitumor effect may result from the activation of sstrs on tumor cells leading to modulation of intracellular signaling pathways . immunohistochemistry and autoradiography reveal that sstr proteins are highly expressed in gastrinomas , insulinomas , and carcinoid tumors , and their metastases . the majority of the tumors express sstr 2 , followed by sstr 1 , sstr 5 , and sstr 3 , while sstr 4 is expressed in a minority of cases.31 the frequency and pattern of expression of each subtype can , however , vary in different tumor types and in each patient.32 indeed , sstrs are expressed in lower density in undifferentiated gep - nens than well - differentiated tumors . the largest expression of sstr 2 on pancreatic endocrine or carcinoid tumors is the reason for the successful clinical application of oct and lan in controlling symptoms related to hormonal hypersecretion.33 furthermore , ssas may also produce an indirect antitumor effect by inhibiting mitogenic growth factors ( such as igf ) and by inhibiting tumor angiogenesis through interaction with sstrs on endothelial cells and monocytes . in immortalized human dermal microvascular endothelial cells , expression of vegf and vegf receptor-2 and vegf release are inhibited by sstr 1 agonists.34 literature regarding gep - nens has considerably increased during the past 2030 years , with changes in classifications , grading systems , and proposed treatments . over the years , classification systems have aimed at classifying nens on the basis of their differentiation and grade . the grading system currently used for the classification of all gep - nens is based on the ki-67 proliferation index or mitotic count.35 grading includes site - specific tumor node metastasis staging , referring to the extent of tumor spread.36 current and previous nen classifications are reported in table 1.37,38 clinical manifestations of gep - nens are very heterogeneous and cover a wide spectrum : from remaining asymptomatic for several years to causing obstructive symptoms ( such as abdominal pain , nausea , vomiting , and cholestasis ) , and from presence of metastases at the time of diagnosis to presenting signs and symptoms due to hormonal hypersecretion . in most cases , because of vagueness and nonspecificity of symptoms , the diagnosis is delayed ( 310 years on average ) , with an increased risk of developing metastases.39 in cases of functioning gep - nens , a specific syndrome develops due to hormonal hypersecretion . moreover , gep - nen cells , which have neuroendocrine differentiation , express both specific neuroendocrine markers such as chromogranin a ( cga)40 and synaptophysin , and less specific markers including cd56 and neuron - specific enolase ( nse).41 although these biomarkers are not associated with specific syndromes , they can be monitored in both functional and nonfunctional nens in relation to disease progression and response to treatment . in particular , the use of cga is recommended in clinical practice , while the utility of serum nse as a marker of tumor aggressiveness needs to be evaluated by further studies before it can be recommended for routine monitoring . the 5-hydroxyindoleacetic acid ( 5-hiaa ) is the main urinary metabolite of human serotonin , and its determination in 24-hour urine collection has a sensitivity of over 90% and a specificity of 90% for advanced carcinoid syndrome ( cs).42 various blood serotonin assays have been proposed , but their actual accuracy has not been established and serotonin determination is not recommended in clinical practice . serum gastrin determination is crucial in the diagnosis of gastrinoma and related syndrome ( zollinger ellison syndrome ) . simultaneous measurement of gastric ph is needed to rule out secondary hypergastrinemia due to other causes . for example , in achlorhydria , pernicious anemia , or atrophic gastritis , high gastrin levels are usually associated with high ph values , while elevated serum gastrin levels combined with gastric ph < 2 are virtually diagnostic of zollinger the occurrence of symptomatic hypoglycemia with non - suppressed endogenous insulin levels is suspicious for insulinoma . other specific markers include serum glucagon concentrations for the diagnosis of glucagonoma , associated with a characteristic clinical syndrome ( diabetes mellitus and cutaneous manifestations , such as migratory necrolytic erythema , nail dystrophies , and stomatitis ) , and vasointestinal peptide ( vip ) for vip - secreting tumors which cause the verner morrison syndrome , characterized by watery diarrhea , hypokalemia , achlorhydria , weight loss , metabolic acidosis , hypercalcemia , glucose intolerance , and flushing . different from the hypersecreting nens , which can present with specific endocrine syndromes , tumors which do not secrete biologically active substances may be present for years without ever displaying signs or symptoms , except for vague abdominal pain . the most important clinical manifestations of nens are related to mechanical complications ( pain , obstruction , and bleeding ) or to the bioactive factors secreted . the cs is characterized by signs and symptoms associated with hypersecretion of vasoactive substances by nens ( serotonin , histamine , tachykinins , and prostaglandins ) . the symptoms of cs include cutaneous flushing ( which occurs in 84% of patients ) , gastrointestinal hypermotility and diarrhea , heart disease , bronchial constriction , myopathy , and an abnormal increase in skin pigmentation.39 lan autogel is available at doses of 60 mg , 90 mg , or 120 mg.17 the recommended dose for the treatment of gep - nens is 120 mg administered every 4 weeks by deep subcutaneous injection allowing to reach steady - state concentrations after 45 injections . even if the use in the geriatric population is associated with differences in pharmacokinetics , no dose adjustment is required because of the wide therapeutic window of lan . on the other hand , there is no experience with lan in the pediatric population , in which its use should be avoided . since in preclinical studies lan has shown embryocidal effects , in the absence of adequate and well - controlled reproductive studies in humans , its use in pregnancy should be considered with particular care for the potential risk to the fetus . since lan may cause a reduction in heart rate , patients affected by underlying cardiac conditions should have their heart rate monitored prior to starting lan . in a group of 81 patients affected by gep - nens treated with lan autogel , the incidence of heart rate < 60 bpm was 23% ( vs 16% in placebo group ) , while the incidence of episodes of heart rate < 50 bpm as well as adverse event of bradycardia was 1% in each group . this finding can be explained by the activity of the bulbospinal neurons in the rostral ventrolateral medulla ( rvlm ) , which are known to be critical for the maintenance of sympathetic vasomotor tone and normal cardiovascular reflex function . in particular , rvlm presympathetic neurons that express sstr 2a are essential for maintaining and potentially generating sympathetic vasomotor tone.43 in rats , microinjection of either sst or lan into the rvlm causes a dose - dependent sympathoinhibition , hypotension , and bradycardia that is blocked by the sstr 2 antagonist.43 preclinical and clinical pharmacological studies show that lan , such as sst and other ssas , inhibits the secretion of insulin and glucagon . hence , patients treated with lan may experience hypoglycemia or hyperglycemia . for this reason , glucose levels should be monitored during treatment with lan , especially in diabetic patients who may require adjustments in their antihyperglycemic therapy.17 in the gastrointestinal system , lan significantly reduces the levels of pancreatic polypeptide , motilin , and gastric - inhibitory peptide , as well as postprandial gastrin secretion , without affecting secretin . moreover , lan may reduce the intestinal absorption of drugs and may reduce gallbladder motility leading to gall stone formation.17 lan clearance is reduced by 30% in patients with moderate - to - severe hepatic impairment , but the effects of lan in patients with hepatic failure have not been studied . at the dose of 120 mg , the clearance of lan is not modified in patients with mild - to - moderate renal impairment , while patients with severe renal impairment have not been studied . lan autogel has been approved for the long - term treatment of patients with acromegaly and for the treatment of gep - nens in order to delay disease progression in patients with g1 or a subset of g2 ( equivalent to ki-67 < 10% ) , unresectable , locally advanced or metastatic disease.17 the efficacy of lan autogel has been shown by studies demonstrating a benefit in progression - free survival ( pfs ) , even if there is no evidence of an overall survival benefit ( table 2 ) . moreover , lan autogel is widely recognized as effective in controlling tumor - related symptoms in the majority of patients affected by gep - nens ( table 2 ) . the rationale of using ssas as medical therapy in patients with nens is based on the expression of sstrs on the cell surfaces of the majority of these tumors . lan and oct , the most used ssas in this context , bind with high affinity to receptor subtypes 2 and 5 , inhibiting the signal - transmission pathways , causing a reduction in secretion of hormone and amine which can ameliorate tumor - related syndromes and stabilize tumor growth . moreover , compared with the earlier formulation of ssas , newer long - acting formulations reduce the number of injections required , increasing patients compliance . since the introduction of lan autogel in the clinical practice , several studies have compared the therapeutic equivalence between lan autogel formulation ( injected every 4 weeks at a dose of 60 mg , 90 mg , or 120 mg ) and lan microparticles ( injected every 714 days at the dose of 30 mg , or every 1428 days at the dose of 60 mg ) in gep - nens . in a phase iii randomized clinical trial , involving 46 patients who completed the study , lan autogel ( 120 mg/6 weeks ) demonstrated the same efficacy of lan microparticles ( 30 mg/3 weeks ) in terms of reduction of tumor markers and tumor size , offering the possibility to use a more delayed formulation of ssa.44 the efficacy , safety , and tolerability of lan autogel have been evaluated in metastatic , well - differentiated nens in an italian retrospective evaluation performed by bianchi et al.45 the study included 23 patients affected by metastatic nens , and in ~65% of cases , tumor was localized in the gastrointestinal system . functional tumors were 43.5% . in this clinical study , lan autogel 120 mg , given once a month by deep subcutaneous injection for at least 24 months , was well tolerated and induced long - lasting responses in terms of clinical symptoms . a partial response in terms of tumor reduction was observed only in 2 out of 5 lung tumors , but not in gep - nens , which remained either stable or showed progression ( table 2 ) . prospective evaluations investigating the antiproliferative effects of lan have showed an effective tumor stabilization and a pfs > 12 months in patients with progressive nens ineligible for surgery or chemotherapy ( table 2).46 the clarinet study represents a fundamental contribution to the evaluation of efficacy of lan autogel in gep - nens ( table 2 ) . this randomized , double - blind , placebo - controlled , 96-week study assessed the effects of lan autogel 120 mg / monthly in patients with advanced , well - differentiated , nonfunctioning , sstr - positive , g1 or g2 gep - nens and documented disease - progression status . tumors originated in the pancreas , midgut , hindgut , or unknown primary location . compared to placebo , lan autogel at a dose of 120 mg / monthly was associated with significant increase in pfs and a 53% reduction in the risk of tumor progression.47 quality of life did not differ significantly between treatment groups . moreover , in patients with baseline levels of cga above the upper limit of the normal range , lan autogel induced a significantly greater reduction compared to placebo . the overall incidence of adverse events was similar between lan autogel and placebo ( ~90% ) , but half the patients treated with lan autogel experienced drug - related adverse events ( diarrhea , hyperglycemia , or cholelithiasis ) . the decrease of cga levels during treatment with lan autogel from baseline was associated with a significant reduction of the hazard of disease progression , confirming the utility of this marker in the clinical follow - up of patients with nens.48 recent data from the open - label extension of the clarinet study confirm that lan autogel maintains favorable risk / benefit profile , providing new evidence of the lan antitumor benefits in indolent and progressive gep - nens ( table 2).49 the ki-67 proliferation index represents a good marker in predicting tumor response to ssas treatment ( table 2 ) . in patients with well - differentiated gep - nens , a ki-67 proliferation index of up to 5% , stable disease prior to treatment , and low - to - moderate hepatic tumor involvement ( 25% ) have been associated with tumor control during lan autogel treatment.50 the antiproliferative effects of long - acting ssas according to ki-67 index have been recently evaluated by an italian multicenter observational study using both oct lar 30 mg/28 days and lan 120 mg/28 days.51 objective response and tumor stability were not significantly different between g1 and g2 nens , and between locoregional disease and distant metastases . interestingly , the clinical benefit ( improvement of symptoms ) was significantly greater in patients with locoregional disease than in those with distant metastases as well as in patients with gep - nens than those with other primary tumors . although pfs was longer in g1 than g2 nens , the difference was not significant . however , in the subgroup of nens with ki-67 < 5% , the pfs was significantly longer compared to nens with ki-67 5% , consistent with previous data.46 furthermore , while pfs was not different between gep and thoracic nens , it was longer in gep and thoracic nens compared to those with unknown primary tumors.51 some functional nens release peptides and amines which produce a characteristic set of symptoms of the cs . this syndrome occurs in ~10% of patients with metastatic nens , and it is most prevalent in those with nens of the small intestine ( ~20% ) . it is caused by the release of serotonin , which is no longer metabolized in the liver , and other substances , such as tachykinins , prostaglandins , and bradykinins.52 the predominant signs and symptoms of cs are flushing ( 90% ) , diarrhea ( 70% ) , and abdominal pain ( 40% ) ; less frequent events are lacrimation , profuse sweating , telangiectasias , cardiac fibrosis , and cutaneous pellagra - like manifestations due to lack of niacin.39 these can be very distressing for patients and have a negative impact on their quality of life . ssas are currently considered the standard of care for symptom control in cs , demonstrating a significant reduction of flushing and diarrhea since the first day of treatment . moreover , a reduction of cga and 5-hiaa can be detected ( table 2).53 a recent 16-week , randomized , double - blind , phase iii trial involving 115 patients evaluated the response of cs symptoms to treatment with lan autogel 120 mg/4 weeks . in this study , patients had access to short - acting oct as rescue medication . results showed that the proportion of patients requiring oct rescue was significantly lower in the lan - treated group than in the placebo ( table 2).54 satisfaction with diarrhea control and flushing control and a good impact on the quality of life have also been reported in an open - label observational study ( table 2).55 moreover , the once - monthly dosing regimen of lan is associated with improved patient adherence and patient perception of lan injection.56,57 although in the majority of patients with metastatic carcinoids and pancreatic endocrine tumors treatment with ssas induces a rapid improvement of clinical symptomatology related to hormonal hypersecretion , most patients can develop desensitization within weeks to months.58,59 the potential mechanisms responsible for this phenomenon , as well as for the considerable variability in the duration of the responses to medical therapy , are not known at present . potential mechanisms of resistance to ssas therapy in patients with sst - positive tumors are the possibility of receptor downregulation as well as reduction in the number and/or affinity of sstrs . moreover , a decrease in responsiveness due to receptor uncoupling from second messenger activation can determine desensitization . other potential causes could be a non - homogeneous expression of sstrs in tumors , outgrowth of sst - negative cell clones , absence of sstr subtypes with high affinity for ssas , tachyphylaxis of the inhibitory effect of ssas on indirect tumor growth - promoting mechanisms , and mutations in sstr genes leading to the absence of functional receptor proteins.7 to date , ssas represent the main symptomatic therapeutic approach for the management of nens . in this context , there are no differences between lan microparticulate and lan autogel in their ability to control tumor growth and tumor hypersecretion . their long - acting formulation , which allows up to once - monthly administration regimens , increases the compliance of patients with nens . although several studies have been conducted since the approval of lan autogel , their conclusions are often difficult to compare due to differences in study design , eligibility criteria , and study end points . nonetheless , the majority of the studies indicate that the effects of lan autogel are mainly on improving symptoms and stabilization of the disease progress , and even if a benefit in pfs has been demonstrated , there is no evidence of an overall survival benefit . this may partly be explained by the fact that nens are frequently tumors showing slow progression and an indolent behavior . therefore , more data must be derived by the extension of studies that have already been published . in addition , the molecular bases of tumor response to ssas are still unclear , although the application of proteomics to this field has led to promising results.60 finally , the ongoing drug research has shown that lan above a critical assembly concentration of 20 mm spontaneously forms hollow nanotubes when solubilized in pure water.61,62 moreover , a recent study evaluated the utility of a self nano - emulsifying delivery system for model peptide lan which provided a protective effect toward thiol disulfide exchange reactions and can be useful to overcome sulfhydryl barrier of the gastrointestinal tract.63 therefore , lan is an interesting self - assembly model that can provide insights on how these mechanisms can be controlled , and applied to nanotechnology and drug delivery.64
somatostatin analogs ( ssas ) , which were initially used to control hormonal syndromes associated with neuroendocrine neoplasms ( nens ) , have been successfully proposed as antiproliferative agents , able to control tumor growth in patients affected by gastroenteropancreatic ( gep)-nens . the development of long - acting formulations of ssas which require only weekly or monthly injections can improve patient compliance . in particular , lanreotide ( lan ) autogel , which is a viscous aqueous formulation supplied in ready - to - use prefilled syringes , can be administered every 2856 days . since its introduction in the clinical practice , several studies evaluated the clinical utility of lan autogel in the medical treatment of gep - nens . although there is no evidence of an overall survival benefit , these studies confirm the efficacy of lan autogel in terms of benefit in progression - free survival , and in more than half of cases , a reduction of tumor markers can be observed during treatment with this drug . moreover , lan autogel is widely recognized to be effective in controlling tumor - related symptoms in the majority of patients affected by gep tumors , especially in patients affected by carcinoid syndrome , improving considerably patients quality of life .
Introduction Molecular basis of SSAs action SST and SSAs SSTRs and SSA actions Clinical features of GEP-NENs Lanreotide Autogel Use of LAN Autogel in GEP-NENs Conclusion
in particular , ssas , which were initially used to control hormonal syndromes associated with nens , have been successfully proposed as antiproliferative agents , able to control tumor growth.2,3 moreover , long - acting formulations of ssas have demonstrated their efficacy as antineoplastic agents in the treatment of gep - nens.4 long - acting formulations of ssas ( oct lar , slow - release lan , and lan autogel ) assure improved patient compliance with weekly up to monthly injections . the oct lar formulation has been obtained by combining oct with microspheres of carboxymethylcellulose which increase its therapeutic action to 2442 days.15 two lar / slow - release formulations have been developed : a slow - release lan obtained by combining lan with microspheres of lactide / glycolide copolymers , which allows for administration of every 728 days,16 and lan autogel which is a viscous aqueous formulation supplied in ready - to - use prefilled syringes administered every 2856 days.17 pas ( som 230 ) , a ssa developed using biodegradable polymers by a method similar to that of oct lar,18 is a multireceptor - targeted sst generated by the introduction of 4 synthetic and 2 essential amino acids of sst in a novel cyclohexapeptide structure . the oct lar formulation has been obtained by combining oct with microspheres of carboxymethylcellulose which increase its therapeutic action to 2442 days.15 two lar / slow - release formulations have been developed : a slow - release lan obtained by combining lan with microspheres of lactide / glycolide copolymers , which allows for administration of every 728 days,16 and lan autogel which is a viscous aqueous formulation supplied in ready - to - use prefilled syringes administered every 2856 days.17 pas ( som 230 ) , a ssa developed using biodegradable polymers by a method similar to that of oct lar,18 is a multireceptor - targeted sst generated by the introduction of 4 synthetic and 2 essential amino acids of sst in a novel cyclohexapeptide structure . lan autogel has been approved for the long - term treatment of patients with acromegaly and for the treatment of gep - nens in order to delay disease progression in patients with g1 or a subset of g2 ( equivalent to ki-67 < 10% ) , unresectable , locally advanced or metastatic disease.17 the efficacy of lan autogel has been shown by studies demonstrating a benefit in progression - free survival ( pfs ) , even if there is no evidence of an overall survival benefit ( table 2 ) . moreover , lan autogel is widely recognized as effective in controlling tumor - related symptoms in the majority of patients affected by gep - nens ( table 2 ) . in a phase iii randomized clinical trial , involving 46 patients who completed the study , lan autogel ( 120 mg/6 weeks ) demonstrated the same efficacy of lan microparticles ( 30 mg/3 weeks ) in terms of reduction of tumor markers and tumor size , offering the possibility to use a more delayed formulation of ssa.44 the efficacy , safety , and tolerability of lan autogel have been evaluated in metastatic , well - differentiated nens in an italian retrospective evaluation performed by bianchi et al.45 the study included 23 patients affected by metastatic nens , and in ~65% of cases , tumor was localized in the gastrointestinal system . the decrease of cga levels during treatment with lan autogel from baseline was associated with a significant reduction of the hazard of disease progression , confirming the utility of this marker in the clinical follow - up of patients with nens.48 recent data from the open - label extension of the clarinet study confirm that lan autogel maintains favorable risk / benefit profile , providing new evidence of the lan antitumor benefits in indolent and progressive gep - nens ( table 2).49 the ki-67 proliferation index represents a good marker in predicting tumor response to ssas treatment ( table 2 ) . in patients with well - differentiated gep - nens , a ki-67 proliferation index of up to 5% , stable disease prior to treatment , and low - to - moderate hepatic tumor involvement ( 25% ) have been associated with tumor control during lan autogel treatment.50 the antiproliferative effects of long - acting ssas according to ki-67 index have been recently evaluated by an italian multicenter observational study using both oct lar 30 mg/28 days and lan 120 mg/28 days.51 objective response and tumor stability were not significantly different between g1 and g2 nens , and between locoregional disease and distant metastases . nonetheless , the majority of the studies indicate that the effects of lan autogel are mainly on improving symptoms and stabilization of the disease progress , and even if a benefit in pfs has been demonstrated , there is no evidence of an overall survival benefit .
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immunoglobulin a ( iga ) nephropathy is characterized by mesangial iga deposits and expansion of glomerular mesangial matrix with proliferation of mesangial cells . its clinical manifestation is extremely variable , ranging from asymptomatic microscopic hematuria to rapidly progressive renal failure . it is considered as the most common primary glomerulonephritis worldwide and 36% of adult patients with iga nephropathy ( igan ) progress to end - stage renal failure within 20 years in china . however , the pathogenesis of igan is obscure and its current therapy remains unsatisfactory . activation of inflammatory and immune system is one of the important causal factors in the pathogenesis of chronic renal disease . it is becoming clear that toll - like receptors ( tlrs ) are directly involved in the pathogenesis of chronic kidney diseases related to inflammatory responses . tlrs are evolutionarily conserved receptors that bind to pathogens and initiate inflammatory responses which might trigger renal injuries . in humans , tlrs are expressed on a wide range of cell types including renal mesangial cells and tubular epithelial cells . among the eleven human tlrs , tlr4 has been demonstrated to be related to mesangial cell injury and renal fibrosis by induction of proinflammatory cytokines , profibrotic molecules , and transforming growth factor-1 in chronic kidney diseases , including igan , renal ischemia - reperfusion injury , acute kidney injury , and transplantation rejection . in vitro experiments have confirmed that tlr4 expression is increased in circulating mononuclear cells of patients with igan and tlr4 signaling is implicated in the activation of iga - stimulated mesangial cells . however , at present , there are few in vivo researches concerning tlr4 and igan . coppo et al . revealed that the upregulation of tlr4 in circulating mononuclear cells of patients with igan was associated with heavy microscopic hematuria and proteinuria . he et al . observed that the expression of tlr4 message ribonucleic acid ( mrna ) and protein in renal tissue was significantly increased in igan rats established by oral and intravenous immunization with bovine serum albumin ( bsa ) for 12 weeks . although these researches mentioned above suggested that the expression of tlr4 was increased in the serum or kidney of patients or rats with igan , they could not demonstrate what role tlr4 might play in the progression of igan . peroxisome proliferator - activated receptor- ( ppar- ) is a member of the nuclear hormone receptor family which expresses in many tissues including the kidney . in addition to regulating the metabolism of glucose and lipid , ppar- also exerts anti - inflammatory effects on kidney diseases including igan . our groups and others have noted that thiazolidinediones , a ppar- agonist , could attenuate inflammatory responses through the angiotensin ii signaling pathway by suppressing the activation of angiotensin receptor 1 , extracellular regulated protein kinases , and nuclear factor - kappa b ( nf-b ) in the in vitro experiments . as it has been proved , nf-b is the key factor of the downstream of tlr4 signaling pathway . it has been reported that pioglitazone exerted its anti - inflammatory effect through suppression of the expression and activity of tlr4 in many in vitro experiments of other disease models . so far , the interaction between ppar- and tlr4 and the plausible mechanism in igan have not been fully studied . the current study was conducted to assess whether tlr4 signaling pathway was involved in the pathogenesis and progression of igan in vivo . we hypothesized that the ppar- agonist exerted its role on alleviating the severity of igan by interfering with tlr4-dependent signaling pathway and impressed the increase of inflammatory mediators . with this aim , we evaluated the renal expression of inflammatory cytokine interleukin-1 beta ( il-1 ) , and tlr4 mrna and protein in rats before and after the establishment of igan and the changes of these factors after treated with tlr4 inhibitor , toll - like receptor 4 inhibitor ( tak242 ) . moreover , we tested the alterations of renal tlr4 mrna and protein in rats after gavaged by pioglitazone with a focus on the interactive roles between tlr4 and ppar- and how this interaction on the severity of experimental igan . bovine gamma globulin ( bgg ) was purchased from sigma chemical co. ( st . louis , mo , usa ) . antibodies were rabbit anti - rat tlr4 , rabbit anti - rat il-1 ( all from santa cruz biotechnology , santa cruz , ca , usa ) , and goat anti - rat iga ( lifespan biosciences , seattle , ca , usa ) . all procedures performed in studies involving animals were in accordance with the ethical standards of the ethics committee of huadong hospital for animal studies and the national institute of health guidelines for the care and use of laboratory animals . a total of 54 male , 3-week - old lewis rats weighing 45 5 g were purchased from weitonglihua animal technical co. , ltd . , in beijing , china , and housed in an specific pathogen - free room at the animal experimental center of fudan university with a room temperature of 25c and a 12-h light - dark cycle . the animals were provided standard rodent chow and water to drink ad libitum for 1 week before the initiation of the study . because of the different treating time of tak242 and pioglitazone , the animals were sacrificed at different time intervals . hence , 54 lewis rats were randomly divided into six groups with the method of simple randomization : controltak242 , igantak242 , tak242 , controlpio , iganpio , and pio groups . experimental igan was induced in 4-week - old lewis rats by continuous oral immunization with 0.1% bgg in 6 mmol / l hcl as drinking water for 9 weeks , followed by intravenous injection of 1 mg bgg ( dissolved in 0.3 ml 6 mmol / l hcl ) into the tail vein daily for 3 successive days . tak242 , dissolved in 20% lipovenoes at 3 mgkgd ( 7.5 ml / kg ) , was injected into rats with igan for 8 days after completing the model - creation ( tak242 group ) . experimental igan rats receiving intravenous injection of the same amount of lipovenoes for 8 days by tail veins were named igantak242 group . controltak242 group was normal rats who drank 6 mmol / l hcl for 9 weeks , and then injected with 1 mg 6 mmol / l hcl into the tail vein daily for three successive days followed by injection with 20% lipovenoes at 7.5 mlkgd for 8 days . pioglitazone , dissolved in saline at 1 mg / ml , was administered to rats after the model - creation by intragastric injection at 10 mgkgd for 4 weeks until the rats were killed ( pio group ) . rats of experimental igan receiving intragastric injection of saline at 10 mlkgd for 4 weeks were named iganpio group and controlpio group referred to normal rats who drank 6 mmol / l hcl for 9 weeks , and then were given intravenous injection of 1 mg 6 mmol / l hcl into the tail vein daily for three successive days followed by intragastric injection of saline at 10 mlkgd for 4 weeks . albumin - to - creatinine ratio ( acr ) was used to express the degree of albuminuria . the urine was centrifuged at 600 revolutions per minute for 5 min and the sediment was examined microscopically for erythrocytes . a diagnostic criterion for hematuria was more than 10 red cells on average per high - power field . serum creatinine ( scr ) and blood urea nitrogen ( bun ) were measured at the clinical laboratory at huadong hospital affiliated to fudan university . blood samples were drawn through the abdominal aorta after intraperitoneal anesthesia with 2% pentobarbital sodium to detect serum il-1 by enzyme - linked immunosorbent assay kits ( multisciences biotech co. , ltd . , the cortex from half of the kidney was fixed in 4% paraformaldehyde and paraffin - embedded for histological observation . the other half was snap frozen in liquid nitrogen and stored at 80c for total rna and protein extraction . three - micrometer thick paraffin - embedded kidney sections were deparaffinized with xylene and then rehydrated through a descending gradient of ethanol . the sections were stained with h and e. glomerular damage and mesangial hypercellularity were determined by examining 25 glomeruli in each sample . immunofluorescence examination of the paraffin - embedded renal sections was performed by staining with fluorescing isothiocyanate - labeled , specific antibodies against rat iga ( 1:16 ) at 4c overnight . the anti - rat iga antibody was visualized as bright green color using the dako envision plus system ( dako , carpinteria , ca , usa ) . the nephrologist who examined the specimens was unaware of the group assignments of the individual animals . total rna was extracted from renal tissues using the trizol reagent kit ( life technologies , usa ) according to the method described in the manufacturer 's protocol . thereafter , 1 ng of total rna was reverse transcribed using the primescript reverse transcription kit and random primers ( takara biotechnology co. , ltd . , real - time polymerase chain reaction amplification was carried out in an abi prism 7500 sequence detection system . primer sequences for rat tlr4 are forward 5-tgacagacctcaggcagattgt-3 , reverse 5-aatagtgcaatcgatagaaggaaca-3 ; il-1 forward 5-ccgtggcacattctggtca-3 , reverse 5-gctgtgcactggtccaaattc-3 ; and -actin forward 5-agattactgccctggctcctag-3 , reverse 5-catcgtactcctgcttgctga-3. -actin was used as an endogenous control . protein extracted from the renal tissues was analyzed by western blotting for tlr4 and il-1 in the following manners . after homogenization , the solution was centrifuged at 10,800 g at 4c for 5 min . protein was separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane . the membrane was incubated with bsa , and then with following primary antibodies including rabbit antibody to il-1 and tlr4 . detection was used by enhanced chemiluminescence , and the light signals were exposed to x - ray film . data were analyzed using spss software version 20 ( ibm , new york , usa ) . differences between groups were analyzed by one - way analysis of variances ( anova ) . bovine gamma globulin ( bgg ) was purchased from sigma chemical co. ( st . louis , mo , usa ) . antibodies were rabbit anti - rat tlr4 , rabbit anti - rat il-1 ( all from santa cruz biotechnology , santa cruz , ca , usa ) , and goat anti - rat iga ( lifespan biosciences , seattle , ca , usa ) . all procedures performed in studies involving animals were in accordance with the ethical standards of the ethics committee of huadong hospital for animal studies and the national institute of health guidelines for the care and use of laboratory animals . a total of 54 male , 3-week - old lewis rats weighing 45 5 g were purchased from weitonglihua animal technical co. , ltd . , in beijing , china , and housed in an specific pathogen - free room at the animal experimental center of fudan university with a room temperature of 25c and a 12-h light - dark cycle . the animals were provided standard rodent chow and water to drink ad libitum for 1 week before the initiation of the study . because of the different treating time of tak242 and pioglitazone , the animals were sacrificed at different time intervals . hence , 54 lewis rats were randomly divided into six groups with the method of simple randomization : controltak242 , igantak242 , tak242 , controlpio , iganpio , and pio groups . experimental igan was induced in 4-week - old lewis rats by continuous oral immunization with 0.1% bgg in 6 mmol / l hcl as drinking water for 9 weeks , followed by intravenous injection of 1 mg bgg ( dissolved in 0.3 ml 6 mmol / l hcl ) into the tail vein daily for 3 successive days . tak242 , dissolved in 20% lipovenoes at 3 mgkgd ( 7.5 ml / kg ) , was injected into rats with igan for 8 days after completing the model - creation ( tak242 group ) . experimental igan rats receiving intravenous injection of the same amount of lipovenoes for 8 days by tail veins were named igantak242 group . controltak242 group was normal rats who drank 6 mmol / l hcl for 9 weeks , and then injected with 1 mg 6 mmol / l hcl into the tail vein daily for three successive days followed by injection with 20% lipovenoes at 7.5 mlkgd for 8 days . pioglitazone , dissolved in saline at 1 mg / ml , was administered to rats after the model - creation by intragastric injection at 10 mgkgd for 4 weeks until the rats were killed ( pio group ) . rats of experimental igan receiving intragastric injection of saline at 10 mlkgd for 4 weeks were named iganpio group and controlpio group referred to normal rats who drank 6 mmol / l hcl for 9 weeks , and then were given intravenous injection of 1 mg 6 mmol / l hcl into the tail vein daily for three successive days followed by intragastric injection of saline at 10 mlkgd for 4 weeks . albumin - to - creatinine ratio ( acr ) was used to express the degree of albuminuria . the urine was centrifuged at 600 revolutions per minute for 5 min and the sediment was examined microscopically for erythrocytes . a diagnostic criterion for hematuria was more than 10 red cells on average per high - power field . serum creatinine ( scr ) and blood urea nitrogen ( bun ) were measured at the clinical laboratory at huadong hospital affiliated to fudan university . blood samples were drawn through the abdominal aorta after intraperitoneal anesthesia with 2% pentobarbital sodium to detect serum il-1 by enzyme - linked immunosorbent assay kits ( multisciences biotech co. , ltd . , the cortex from half of the kidney was fixed in 4% paraformaldehyde and paraffin - embedded for histological observation . the other half was snap frozen in liquid nitrogen and stored at 80c for total rna and protein extraction . three - micrometer thick paraffin - embedded kidney sections were deparaffinized with xylene and then rehydrated through a descending gradient of ethanol . the sections were stained with h and e. glomerular damage and mesangial hypercellularity were determined by examining 25 glomeruli in each sample . immunofluorescence examination of the paraffin - embedded renal sections was performed by staining with fluorescing isothiocyanate - labeled , specific antibodies against rat iga ( 1:16 ) at 4c overnight . the anti - rat iga antibody was visualized as bright green color using the dako envision plus system ( dako , carpinteria , ca , usa ) . the nephrologist who examined the specimens was unaware of the group assignments of the individual animals . total rna was extracted from renal tissues using the trizol reagent kit ( life technologies , usa ) according to the method described in the manufacturer 's protocol . thereafter , 1 ng of total rna was reverse transcribed using the primescript reverse transcription kit and random primers ( takara biotechnology co. , ltd . , real - time polymerase chain reaction amplification was carried out in an abi prism 7500 sequence detection system . primer sequences for rat tlr4 are forward 5-tgacagacctcaggcagattgt-3 , reverse 5-aatagtgcaatcgatagaaggaaca-3 ; il-1 forward 5-ccgtggcacattctggtca-3 , reverse 5-gctgtgcactggtccaaattc-3 ; and -actin forward 5-agattactgccctggctcctag-3 , reverse 5-catcgtactcctgcttgctga-3. -actin was used as an endogenous control . protein extracted from the renal tissues was analyzed by western blotting for tlr4 and il-1 in the following manners . after homogenization , the solution was centrifuged at 10,800 g at 4c for 5 min . protein was separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane . the membrane was incubated with bsa , and then with following primary antibodies including rabbit antibody to il-1 and tlr4 . detection was used by enhanced chemiluminescence , and the light signals were exposed to x - ray film . data were analyzed using spss software version 20 ( ibm , new york , usa ) . differences between groups were analyzed by one - way analysis of variances ( anova ) . as shown in figure 1a and 1b , urinary acr and serum il-1 level were significantly increased in igantak242 rats compared to controltak242 rats ( 4.45 1.33 mg / mmol vs. 2.89 0.96 mg / mmol , p < 0.05 ; 48.28 13.49 furthermore , treatment with tak242 significantly reversed the elevations of acr and serum il-1 ( 1.34 0.33 mg / mmol vs. 4.45 1.33 mg / mmol , p < 0.05 ; 32.43 7.42 pg / ml vs. 48.28 13.49 pg / ml , p < 0.05 ) . similarly , urinary acr was significantly increased in iganpio rats compared to controlpio rats ( 1.72 0.41 mg / mmol vs. 1.27 0.15 mg / mmol , p < 0.05 ) , which was reversed by pioglitazone ( 1.13 0.44 mg / mmol vs. 1.72 0.41 mg / mmol , p < 0.05 ) [ figure 1c ] . compared to iganpio group , there was a tendency of reduction of serum il-1 levels in pio group , but the difference did not reach the statistical difference ( 39.06 17.92 pg / ml , p > 0.05 ) [ figure 1d ] . however , urine from normal rats , igan rats , rats received treatment with tak242 or pioglitazone all revealed sporadic hematuria and the difference among groups did not reach the statistical significance . there was no significant difference in bun or scr between various groups ( data not shown ) . ( d ) serum concentration of il-1 of all groups detected by elisa kits . * compared to controltak242 , p < 0.05 ; compared to igantak242 , p < 0.05 ; compared to controlpio , p < 0.05 ; compared to iganpio , p < 0.05 . acr : urinary albumin - to - creatinine ratio ; igan : immunoglobulin a nephropathy ; pio : pioglitazone ; tak242 : toll - like receptor 4 inhibitor ; il-1 : interleukin-1 beta ; elisa : enzyme - linked immunosorbent assay . figure 2a indicates that there is hardly no clear green fluorescence seen in the glomeruli of controltak242 and controlpio groups . in contrast , bright mass - like green fluorescence was found in the glomeruli of igantak242 and iganpio rats . morphological changes observed by immunofluorescence and h and e staining ( n=9 in each group ) . ( a ) contrastive immunofluorescence of iga deposition in the glomeruli of kidney sections from normal rats and rats with iga nephropathy ( 450 ) . ( b ) representative h and e staining of glomeruli in kidney sections from rat of each group ( 400 ) . ( c ) glomerular cell number expressed as cells per glomerular cross - section of all groups stained by he . * compared to controltak242 , p < 0.05 ; compared to igantak242 , p < 0.05 ; compared to controlpio , p < 0.05 ; compared to iganpio , p < 0.05 . igan : immunoglobulin a nephropathy ; pio : pioglitazone ; tak242 : toll - like receptor 4 inhibitor . the kidney sections were stained with h and e. renal pathologic structure of controltak242 and controlpio groups was found without any glomerular or tubular injuries . the histological changes of rats in igantak242 and iganpio groups were both characterized by glomerular hypercellularity , moderate proliferation of the mesangial cells , and hyperplasia of mesangial matrix . the changes mentioned above were apparently ameliorated in rats of tak242 and pio groups [ figure 2b ] . the number of glomerular cells of cross section in experimental rats was higher than those in normal rats ( 51.39 3.58 vs.41.24 2.24 , p < 0.01 ) . compared to igantak242 group , the number of glomerular cells of cross section was much lower in tak242 group ( 35.47 3.38 vs.51.39 3.58 , p < 0.01 ) . in addition , there was a significant decrease of glomerular cells in pio group compared to iganpio group ( 45.53 6.37 vs.50.12 8.43 , p < 0.05 ) [ figure 2c ] . we studied the expression of il-1 and tlr4 mrna as well as protein of each sample from renal tissues . our findings illustrated that tlr4 , il-1 mrna , and protein in igantak242 group were dramatically higher than those in controltak242 group ( tlr4 mrna : 1.36 0.54 vs. 1.03 0.24 , p < 0.05 ; il-1 mrna : 1.38 0.62 vs. 0.88 0.37 , p < 0.01 ; tlr4 protein : 0.81 0.18 vs. 0.30 0.07 , p < 0.01 ; il-1 protein : 1.10 0.33 vs. 0.74 0.19 , p < 0.05 ) . in contrast , the levels of tlr4 , il-1 mrna , and protein in tak242 group were much lower than those in igantak242 group ( tlr4 mrna : 0.92 0.26 vs. 1.36 0.54 , p < 0.01 ; il-1 mrna : 0.95 0.45 vs. 1.38 0.62 , p < 0.01 ; tlr4 protein : 0.43 0.30 vs. 0.81 0.18 , p < 0.01 ; il-1 protein : 0.60 0.33 vs. 1.10 0.33 , p < 0.01 ) . likewise , there was a significant increase of tlr4 , il-1 mrna , and protein in iganpio group compared to controlpio group ( tlr4 mrna : 1.72 0.45 vs. 1.58 0.37 , p < 0.05 ; il-1 mrna : 1.53 0.19 vs. 1.32 0.37 , p < 0.05 ; tlr4 protein : 0.21 0.05 vs. 0.16 0.06 , p < 0.05 ; il-1 protein : 0.66 0.16 vs. 0.46 0.21 , p < 0.05 ) . in addition , the increase was reversed in pio group : the expression of tlr4 , il-1 mrna , and protein in pio group was dramatically decreased compared to iganpio group ( tlr4 mrna : 1.22 0.28 vs. 1.72 0.45 , p < 0.05 ; il-1 mrna : 1.27 0.41 vs. 1.53 0.19 , p < 0.01 ; tlr4 protein : 0.12 0.03 vs. 0.21 0.05 , p < 0.05 ; il-1 protein : 0.37 0.20 vs. 0.66 0.16 , p < 0.05 ) [ figure 3a and 3b ] . expression of il-1 and tlr4 in renal tissues of all groups of rats ( n=9 in each group ) . ( a ) the levels of il-1 and tlr4 in the kidney tissues determined by reverse transcription polymerase chain reaction . ( b ) the levels of il-1 and tlr4 in the kidney tissues determined by western blots . compared to controltak242 , p < 0.05 ; compared to igantak242 , p < 0.05 ; compared to controlpio , p < 0.05 ; compared to iganpio , p < 0.05 . igan : immunoglobulin a nephropathy ; pio : pioglitazone ; tak242 : toll - like receptor 4 inhibitor ; il-1 : interleukin-1 beta ; mrna : message ribonucleic acid ; tlr4 : toll - like receptor 4 ; gapdh : glyceraldehyde-3-phosphate dehydrogenase . as shown in figure 1a and 1b , urinary acr and serum il-1 level were significantly increased in igantak242 rats compared to controltak242 rats ( 4.45 1.33 mg / mmol vs. 2.89 0.96 mg / mmol , p < 0.05 ; 48.28 13.49 furthermore , treatment with tak242 significantly reversed the elevations of acr and serum il-1 ( 1.34 0.33 mg / mmol vs. 4.45 1.33 mg / mmol , p < 0.05 ; 32.43 7.42 pg / ml vs. 48.28 13.49 pg / ml , p < 0.05 ) . similarly , urinary acr was significantly increased in iganpio rats compared to controlpio rats ( 1.72 0.41 mg / mmol vs. 1.27 0.15 mg / mmol , p < 0.05 ) , which was reversed by pioglitazone ( 1.13 0.44 mg / mmol vs. 1.72 0.41 mg / mmol , p < 0.05 ) [ figure 1c ] . compared to iganpio group , there was a tendency of reduction of serum il-1 levels in pio group , but the difference did not reach the statistical difference ( 39.06 17.92 pg / ml , p > 0.05 ) [ figure 1d ] . however , urine from normal rats , igan rats , rats received treatment with tak242 or pioglitazone all revealed sporadic hematuria and the difference among groups did not reach the statistical significance . there was no significant difference in bun or scr between various groups ( data not shown ) . ( d ) serum concentration of il-1 of all groups detected by elisa kits . * compared to controltak242 , p < 0.05 ; compared to igantak242 , p < 0.05 ; compared to controlpio , p < 0.05 ; compared to iganpio , p < 0.05 . acr : urinary albumin - to - creatinine ratio ; igan : immunoglobulin a nephropathy ; pio : pioglitazone ; tak242 : toll - like receptor 4 inhibitor ; il-1 : interleukin-1 beta ; elisa : enzyme - linked immunosorbent assay . figure 2a indicates that there is hardly no clear green fluorescence seen in the glomeruli of controltak242 and controlpio groups . in contrast , bright mass - like green fluorescence was found in the glomeruli of igantak242 and iganpio rats . morphological changes observed by immunofluorescence and h and e staining ( n=9 in each group ) . ( a ) contrastive immunofluorescence of iga deposition in the glomeruli of kidney sections from normal rats and rats with iga nephropathy ( 450 ) . ( b ) representative h and e staining of glomeruli in kidney sections from rat of each group ( 400 ) . ( c ) glomerular cell number expressed as cells per glomerular cross - section of all groups stained by he . * compared to controltak242 , p < 0.05 ; compared to igantak242 , p < 0.05 ; compared to controlpio , p < 0.05 ; compared to iganpio , p < 0.05 . igan : immunoglobulin a nephropathy ; pio : pioglitazone ; tak242 : toll - like receptor 4 inhibitor . the kidney sections were stained with h and e. renal pathologic structure of controltak242 and controlpio groups was found without any glomerular or tubular injuries . the histological changes of rats in igantak242 and iganpio groups were both characterized by glomerular hypercellularity , moderate proliferation of the mesangial cells , and hyperplasia of mesangial matrix . the changes mentioned above were apparently ameliorated in rats of tak242 and pio groups [ figure 2b ] . the number of glomerular cells of cross section in experimental rats was higher than those in normal rats ( 51.39 3.58 vs.41.24 2.24 , p < 0.01 ) . compared to igantak242 group , the number of glomerular cells of cross section was much lower in tak242 group ( 35.47 3.38 vs.51.39 3.58 , p < 0.01 ) . in addition , there was a significant decrease of glomerular cells in pio group compared to iganpio group ( 45.53 6.37 vs.50.12 8.43 , p < 0.05 ) [ figure 2c ] . we studied the expression of il-1 and tlr4 mrna as well as protein of each sample from renal tissues . our findings illustrated that tlr4 , il-1 mrna , and protein in igantak242 group were dramatically higher than those in controltak242 group ( tlr4 mrna : 1.36 0.54 vs. 1.03 0.24 , p < 0.05 ; il-1 mrna : 1.38 0.62 vs. 0.88 0.37 , p < 0.01 ; tlr4 protein : 0.81 0.18 vs. 0.30 0.07 , p < 0.01 ; il-1 protein : 1.10 0.33 vs. 0.74 0.19 , p < 0.05 ) . in contrast , the levels of tlr4 , il-1 mrna , and protein in tak242 group were much lower than those in igantak242 group ( tlr4 mrna : 0.92 0.26 vs. 1.36 0.54 , p < 0.01 ; il-1 mrna : 0.95 0.45 vs. 1.38 0.62 , p < 0.01 ; tlr4 protein : 0.43 0.30 vs. 0.81 0.18 , p < 0.01 ; il-1 protein : 0.60 0.33 vs. 1.10 0.33 , p < 0.01 ) . likewise , there was a significant increase of tlr4 , il-1 mrna , and protein in iganpio group compared to controlpio group ( tlr4 mrna : 1.72 0.45 vs. 1.58 0.37 , p < 0.05 ; il-1 mrna : 1.53 0.19 vs. 1.32 0.37 , p < 0.05 ; tlr4 protein : 0.21 0.05 vs. 0.16 0.06 , p < 0.05 ; il-1 protein : 0.66 0.16 vs. 0.46 0.21 , p < 0.05 ) . in addition , the increase was reversed in pio group : the expression of tlr4 , il-1 mrna , and protein in pio group was dramatically decreased compared to iganpio group ( tlr4 mrna : 1.22 0.28 vs. 1.72 0.45 , p < 0.05 ; il-1 mrna : 1.27 0.41 vs. 1.53 0.19 , p < 0.01 ; tlr4 protein : 0.12 0.03 vs. 0.21 0.05 , p < 0.05 ; il-1 protein : 0.37 0.20 vs. 0.66 0.16 , p < 0.05 ) [ figure 3a and 3b ] . expression of il-1 and tlr4 in renal tissues of all groups of rats ( n=9 in each group ) . ( a ) the levels of il-1 and tlr4 in the kidney tissues determined by reverse transcription polymerase chain reaction . ( b ) the levels of il-1 and tlr4 in the kidney tissues determined by western blots . compared to controltak242 , p < 0.05 ; compared to igantak242 , p < 0.05 ; compared to controlpio , p < 0.05 ; compared to iganpio , p < 0.05 . igan : immunoglobulin a nephropathy ; pio : pioglitazone ; tak242 : toll - like receptor 4 inhibitor ; il-1 : interleukin-1 beta ; mrna : message ribonucleic acid ; tlr4 : toll - like receptor 4 ; gapdh : glyceraldehyde-3-phosphate dehydrogenase . the present study provided evidence that inflammatory responses and tlr4 signaling pathway are involved in the progression of igan . pioglitazone exerted its anti - inflammatory effects by interfering with tlr4 signaling pathway and reducing inflammatory cytokines to alleviate the progression of igan . research of igan has been handicapped due to the lack of a good animal model of igan for many years . since rifai et al . established igan model with balb / c mice for the first time in 1979 , many scholars have made various igan models based on the known pathogenesis . however , there were wide gaps in pathologic changes between the animal models and human igan . recently , the ddy strain of mouse was used as a spontaneous animal model for human igan . however , these mice showed mild proteinuria without hematuria at more than 40 weeks of age and the incidence of igan was highly variable . some investigators used bsa gavage and hypodermic injection of staphylococcal enterotoxin b plus lipopolysaccharides ( lps ) to establish igan models . however , lps itself is a high potent agonist of tlr4 which is apt to confound the interpretation of the subsequent inflammatory pathway . in 1983 , for the first time made igan models with balb / c mice by orally immunizing bgg . only part of the mice showed proteinuria and hematuria was not obvious . in 1992 , gesualdo et al . induced igan in different rat strains by oral bgg immunization and found that mesangial iga deposition was most severe in lewis rats . due to the mild histopathology even after prolonged observation of this model , lai et al . improved the model with lewis rats by immunization of bgg and followed by unilateral nephrectomy . this model had diffuse glomerular iga deposition , mesangial hypercellularity , high acr and obvious hematuria . although unilateral nephrectomy hastened the development of renal abnormalities , it may lead to acute kidney injury and other injuries not characteristic to igan which could confound the findings . taken together , our establishment of igan models with lewis rats by oral immunization of bgg for 9 weeks and then injection of bgg through tail vein for 3 successive days might be able to avoid those disadvantages mentioned above . although our models did not show obvious hematuria which was consistent with other studies , immunofluorescent assay showed granular and massive iga depositions and pathological observation revealed diffuse proliferation of mesangial cells and matrix . furthermore , our models showed obvious proteinuria . above all , igan rat model were successfully established and can be used to reflect the pathophysiological findings in human igan . il-1 is one of the most powerful moderators of inflammation and is closely involved in the development of mesangial cell proliferation and extracellular matrix production . in igan patients , il-1 has been shown to be produced locally in the glomeruli and interstitial . in our study , induction of igan is associated with increased renal expression of il-1 mrna and protein as well as serum il-1 which suggests that inflammatory responses participate in the pathogenesis of igan . there has been circumstantial evidence for the involvement of tlrs , notably tlr4 , in the pathogenesis of renal diseases . however , the reports about the effects of tlr4 on igan in vivo are incompletely elucidated . in our study , tak242 , a selective inhibitor of tlr4 intracellular signaling pathway , had been used in the current study to unravel whether the mechanisms of inflammatory response in igan were tlr4 dependent and , in such a case , to ascertain the specific role of tlr4 . we found that the expression of tlr4 mrna and protein significantly was decreased in rats treated with tak242 . this finding further supported the role of activated tlr4 signaling pathway in the pathophysiology of igan . in keeping with our findings , the study conducted by kwon et al . proved that in igan patients , the expression of renal tlr4 mrna was significantly elevated . the transcriptional level of tlr4 mrna in circulating mononuclear cells was significantly higher in patients with igan than those in healthy controls . what 's more , the increased expression of tlr4 in circulating mononuclear cells was significantly correlated with proteinuria or phases of clinical activity in patients with igan . binding with their ligands , tlrs initiate an intracellular signaling cascade , activate protein kinases , release cytokines , and enhance the expression of transcription factors which results in the generation of mediators including adhesion molecules , chemokines , cytokines , and inflammatory responses . the nuclear translocation of the nf-b favored the hyperplasia of b - cell and therefore increased iga synthesis . tlrs have been conformed to be involved in the switch from igm to iga production in b - cells in experimental animals . in addition , tlr4 was involved in the injuries of mesangial cells by induction of proinflammatory cytokines in igan and was constitutively expressed in podocytes to mediate glomerular injuries by modulating the expression of chemokines . all of the results mentioned above suggested that renal tlr4 signaling pathway may be a therapeutic target for the alleviation of igan . recently , increasing attention has been paid to the role of ppar- agonists in regulating inflammatory responses in kidney diseases . however , there were few reports on whether ppar- agonists exerted anti - inflammatory effects by interfering with tlr4 signaling pathway to improve igan in vivo . in the present study , activation of ppar- pathway with pioglitazone this confirmed that tlr4 pathway was involved in the renal protective effects of ppar- in vivo . our findings are consistent with that of dasu et al , who demonstrated that pioglitazone decreased the expression of tlr4 mrna and protein in a concentration - dependent manner and lowered the activity of nf-b , il-1 , tnf - alpha , and other inflammatory factors in human monocytes . in addition , in peritoneal macrophages of db / db mice , pioglitazone markedly suppressed the expression of myd88 ( myeloid differentiation factor 88 ) and trif ( tir - domain - containing adaptor inducing interferon- ) protein in the cytoplasm and reduced the phosphorylation of interleukin receptor - associated kinase and p38 , which subsequently inhibited the activation of tlr4 signaling pathway and nf-b . ji et al . revealed that ppar- agonist , rosiglitazone , could concentration dependently downregulate the expression of tlr4 mrna and protein in vascular smooth muscle cells of sprague dawley rats . the beneficial effects of rosiglitazone on inflammation were mediated through interference with tlr4 and its downstream signaling components such as trif , interferon regulatory factor 3 ( irf3 ) , and inducible protein-10 . first , we do not provide additional evidence that the alterations of tlr4 mrna and protein as well as inflammatory factors after using tlr4 agonist . second , our research is lack of data using specific ppar- antagonists for igan rats . it is unclear whether pioglitazone - induced anti - inflammatory effects are direct or indirect in in vivo experiment . one possible scenario is that pioglitazone as a ligand binds to the ppar- receptor and directly binds to the ppre of il-1 gene itself or genes which codes for its upstream mediators and inactivates them such as tlr4 . another possible scenario is whether pioglitazone itself or its metabolites are the active compound responsible for anti - inflammatory effects . in vitro researches with renal cells are recommended to dissect the precise mechanism of pioglitazone - induced anti - inflammatory effects in tlr4 signaling pathway in igan . in summary , our study indicated that tlr4 and inflammatory responses played a key role in the progression of igan . pioglitazone improved biochemical parameters , ameliorated renal histological damage , and alleviated iga deposits in glomeruli , at least in part , by negatively regulating the expression of tlr4 and blunted the activation of its downstream signal pathways involved in the expression of inflammatory factors . it suggested that either drug antagonizing tlr4 or ppar- agonists could have potently beneficial effects in the treatment of igan . this study was supported by a grant from the national natural science foundation of china ( no . this study was supported by a grant from the national natural science foundation of china ( no .
background : in vitro experiments have revealed that toll - like receptor 4 ( tlr4 ) pathway is involved in the progression of immunoglobulin a nephropathy ( igan ) by induction of proinflammatory cytokines . evidence showed that , in other disease models , peroxisome proliferator - activated receptor- ( ppar- ) agonists have been shown to exert anti - inflammatory effects through suppression of the expression and activity of tlr4 . however , the interaction between ppar- and tlr4 in igan has not been fully studied both in vitro and in vivo . in this study , we explored whether tlr4 pathway attributed to the progression of igan in experimental rats.methods:bovine gamma globulin was used to establish igan model . fifty - four lewis rats were randomly divided into six groups : controltak242 , igantak242 , toll - like receptor 4 inhibitor ( tak242 ) groups ( rats were administrated with tlr4 inhibitor , tak242 ) and controlpio , iganpio , pio groups ( rats were administrated with ppar- agonist , pioglitazone ) . urinary albumin - to - creatinine ratio ( acr ) , serum creatinine , and blood urea nitrogen were detected by automatic biochemical analyzer . renal histopathological changes were observed after hematoxylin - eosin staining , and the iga deposition in glomeruli was measured by immunofluorescence staining . real - time polymerase chain reaction and western blotting were used to detect tlr4 and interleukin-1 beta ( il-1 ) message ribonucleic acid ( mrna ) and protein expression in renal tissues . results were presented as mean standard deviation . differences between groups were analyzed by one - way analysis of variance.results:compared to normal rats , experimental rats showed higher acr ( 4.45 1.33 mg / mmol vs. 2.89 0.96 mg / mmol , p < 0.05 ) , obvious iga deposition with mesangial hypercellularity , hyperplasia of mesangial matrix accompanied by increased serum il-1 ( 48.28 13.49 pg / ml vs. 35.56 7.41pg / ml , p < 0.05 ) , and renal expression of il-1 and tlr4 . the biochemical parameters and renal pathological injury were relieved in both tak242 group and pio group . the expressions of renal tissue tlr4 , il-1 , and serum il-1 were decreased in rats treated with tak242 , and the expression of tlr4 mrna and protein was significantly reduced in pio group compared to iganpio group ( 1.22 0.28 vs. 1.72 0.45 , p < 0.01 , and 0.12 0.03 vs. 0.21 0.05 , p < 0.01).conclusions : our study proves that inflammation mediated by tlr4 signaling pathway is involved in the progression of igan in rat models . moreover , pioglitazone can inhibit the expression of tlr4 in igan .
I M Antibodies and reagents Experimental design Biochemical analyses of renal function Enzyme-linked immunosorbent assay Histological and immunofluorescence examination Quantitative real-time polymerase chain reaction and Western blotting Statistical analysis R Biochemical parameters Histology examination Renal expression of interleukin-1 beta and toll-like receptor 4 at the message ribonucleic acid and protein levels D Financial support and sponsorship Conflicts of interest
with this aim , we evaluated the renal expression of inflammatory cytokine interleukin-1 beta ( il-1 ) , and tlr4 mrna and protein in rats before and after the establishment of igan and the changes of these factors after treated with tlr4 inhibitor , toll - like receptor 4 inhibitor ( tak242 ) . hence , 54 lewis rats were randomly divided into six groups with the method of simple randomization : controltak242 , igantak242 , tak242 , controlpio , iganpio , and pio groups . as shown in figure 1a and 1b , urinary acr and serum il-1 level were significantly increased in igantak242 rats compared to controltak242 rats ( 4.45 1.33 mg / mmol vs. 2.89 0.96 mg / mmol , p < 0.05 ; 48.28 13.49 furthermore , treatment with tak242 significantly reversed the elevations of acr and serum il-1 ( 1.34 0.33 mg / mmol vs. 4.45 1.33 mg / mmol , p < 0.05 ; 32.43 7.42 pg / ml vs. 48.28 13.49 pg / ml , p < 0.05 ) . likewise , there was a significant increase of tlr4 , il-1 mrna , and protein in iganpio group compared to controlpio group ( tlr4 mrna : 1.72 0.45 vs. 1.58 0.37 , p < 0.05 ; il-1 mrna : 1.53 0.19 vs. 1.32 0.37 , p < 0.05 ; tlr4 protein : 0.21 0.05 vs. 0.16 0.06 , p < 0.05 ; il-1 protein : 0.66 0.16 vs. 0.46 0.21 , p < 0.05 ) . in addition , the increase was reversed in pio group : the expression of tlr4 , il-1 mrna , and protein in pio group was dramatically decreased compared to iganpio group ( tlr4 mrna : 1.22 0.28 vs. 1.72 0.45 , p < 0.05 ; il-1 mrna : 1.27 0.41 vs. 1.53 0.19 , p < 0.01 ; tlr4 protein : 0.12 0.03 vs. 0.21 0.05 , p < 0.05 ; il-1 protein : 0.37 0.20 vs. 0.66 0.16 , p < 0.05 ) [ figure 3a and 3b ] . as shown in figure 1a and 1b , urinary acr and serum il-1 level were significantly increased in igantak242 rats compared to controltak242 rats ( 4.45 1.33 mg / mmol vs. 2.89 0.96 mg / mmol , p < 0.05 ; 48.28 13.49 furthermore , treatment with tak242 significantly reversed the elevations of acr and serum il-1 ( 1.34 0.33 mg / mmol vs. 4.45 1.33 mg / mmol , p < 0.05 ; 32.43 7.42 pg / ml vs. 48.28 13.49 pg / ml , p < 0.05 ) . likewise , there was a significant increase of tlr4 , il-1 mrna , and protein in iganpio group compared to controlpio group ( tlr4 mrna : 1.72 0.45 vs. 1.58 0.37 , p < 0.05 ; il-1 mrna : 1.53 0.19 vs. 1.32 0.37 , p < 0.05 ; tlr4 protein : 0.21 0.05 vs. 0.16 0.06 , p < 0.05 ; il-1 protein : 0.66 0.16 vs. 0.46 0.21 , p < 0.05 ) . in addition , the increase was reversed in pio group : the expression of tlr4 , il-1 mrna , and protein in pio group was dramatically decreased compared to iganpio group ( tlr4 mrna : 1.22 0.28 vs. 1.72 0.45 , p < 0.05 ; il-1 mrna : 1.27 0.41 vs. 1.53 0.19 , p < 0.01 ; tlr4 protein : 0.12 0.03 vs. 0.21 0.05 , p < 0.05 ; il-1 protein : 0.37 0.20 vs. 0.66 0.16 , p < 0.05 ) [ figure 3a and 3b ] .
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cancer cachexia is a condition of progressive muscle wasting that develops as a secondary condition in response to tumour growth . while cachexia develops in a wide range of pathologies including anorexia nervosa , acquired immunodeficiency syndrome , amyotrophic lateral sclerosis , congestive heart failure and various malignant cancers , cancer cachexia has been reported to develop at a faster rate than any other cachectic condition . approximately half of all patients with cancer experience cachexia and almost a third of mortalities are estimated to result from cachexia . tumour growth induces a specific catabolic response in skeletal muscle that causes the accelerated loss of protein , characterised by the significant loss of body weight . animal tumour models have been used to investigate cancer cachexia , developing up to 30% loss in body weight after tumour cell implantation . nmri mice implanted with murine adenocarcinoma ( mac ) 16 present with solid tumour growth and cachexia , as have been shown in rats bearing the yoshida ah-130 hepatoma , characteristic of the human cancer cachectic condition . an abundance of evidence exists for the induction of the ubiquitin ( ub)-proteasome proteolytic pathway in the development of cancer cachexia ; however , evidence of protein oxidation in cancer cachectic patients suggests the involvement of reactive oxygen species ( ros ) . studies in both humans and animals with cancer cachexia have shown the presence of oxidised proteins . furthermore , cancer patients have been shown to present with elevated levels of serum ros and lower antioxidant levels , indicative of a pro - oxidative shift . similarly , increased levels of ros have been shown in cachectic tumour - bearing rats , with no compensatory response from antioxidant enzymes . although the catabolic mechanism leading to the severe muscle wasting associated with cancer development remains relatively unknown , there is evidence to suggest a role for ros . nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase ( nox ) is a multicomponent enzyme that when activated catalyzes the production of superoxide anion ( o2 ) . the nox2 enzyme consists of five subunits segregated into membrane bound , nox2 ( gp91 ) and p22 , and cytosolic , p67 , p47 and p40 , units . on stimulation , the protein components of the nox2 enzyme assemble , forming the active oxidase and the production of o2 . it has been apparent in recent years that non - phagocytes possess homologues to nox2 , and that these nox enzymes may activate in a similar manner to the well - established phagocyte nox2 enzyme . currently , there are five known nox homologues ( nox1 - 5 ) found to function in a variety of tissues , with the expression of both nox2 and nox4 enzyme subunits in skeletal muscle tissue , although its function in this particular cell system remains undefined . despite the unknown functionality of the nox enzymes in skeletal muscle tissue , o2 generated from nox the abundance of evidence for nox enzyme - generated o2 in disease , establishes its potential role in the catabolic skeletal muscle wasting in cancer cachexia . it is well - known that ros are generated in skeletal muscle as an essential by - product of cellular metabolism and oxidative enzymes . due to the high metabolic activity and oxidative capacity of skeletal muscle , the antioxidant system is a crucial component for the maintenance of cellular oxidative homeostasis . with the knowledge that ros are likely catabolic factors in the development of cancer cachexia , antioxidant enzymes are of equal importance for their crucial role in the maintenance of cellular oxidative homeostasis and the protection against harmful ros . o2 is dismutated by the o2 specific antioxidant enzyme , superoxide dismutase ( sod ) . three distinct forms of sod exist in the intracellular cytoplasmic compartment ( sod1 ) , mitochondria ( sod2 ) and extracellular matrix ( sod3 ) . the sod enzymes catalyse the conversion of o2 to h2o2 , which is further converted by catalase and glutathione peroxidise ( gpx ) scavenging . a depression in these antioxidant enzyme systems can consequently lead to an imbalance in cellular oxidation and oxidative tissue damage , which has been shown particularly in response to disease . endogenous antioxidants such as sod and gpx are low in cancer patients presenting with high levels of ros , indicating the importance of tight regulation from endogenous antioxidants . the ub - proteasome proteolytic pathway has been established in cancer - induced cachexia . in addition , tumour - derived factors such as tnf- and proteolysis - inducing factor ( pif ) are well recognised in the cancer cachectic condition . what is yet to be elucidated , however , are the downstream mediators of this complex system , in response to tumour growth and development of cancer cachexia . a study by russell et al . proposed a cachectic pathway of increased ub gene expression , downstream of nox - generated ros production , establishing a potential role for the nox enzymes in mediating skeletal muscle atrophy through the ub - proteasome proteolytic pathway . therefore , this study aims to investigate the o2 generating nox and antioxidant enzyme systems in a well - established animal model of cancer cachexia . in particular , this study aims to determine o2 levels and nox enzyme system expression ( nox2 , nox4 , p22 , p40 , p47 , p67 and rac1 ) in the skeletal muscle of cachectic ( mac16 ) tumour - bearing mice compared to non - cachectic ( mac13 ) tumour - bearing mice . furthermore , this study aims to determine levels of the primary antioxidants for o2 and h2o2 dismutation ( sod1 , sod2 , sod3 , catalase and gpx ) , in the skeletal muscle of tumour - bearing cachectic mice ( mac16 ) compared to tumour - bearing non - cachectic ( mac13 ) mice . all experimental procedures were carried out with approval from the victoria university animal ethics committee ( aeeth 07/05 ) . two weight - matched groups of female balb / c nu / nu mice were maintained under controlled environmental conditions , 12-h light / dark cycle , 21c 2c , 30% humidity , in conventional cages with ad libitum access to standard chow and water throughout the course of the study . as has previously been described , donor mice were used to establish and maintain a tumour line , which consistently produced tumour growth ( mac13 ) ( n = 3 ) and tumour growth with cachexia ( mac16 ) ( n = 4 ) . tumour fragments grown and maintained in donor mice were dissected and re - implanted subcutaneously into the flank of recipient mice with frequent monitoring and recording of body weight and tumour size . with cachectic weight loss evident at approximately 912 days post implantation , mice were anaesthetised , using pentobarbital sodium ( 70 mg / kg ) , for tissue collection before weight loss exceeded 25% of original body weight or before tumour growth exceeded 1,000 mm . skeletal muscle tissue was collected , weighed and immediately snap - frozen in liquid nitrogen and stored at 80c for later use . dhe ( 5 m ) was applied to quadriceps cross - sections ( 5 m ) and incubated in a light - protected oven at 37c for 30min . the sections were washed with pbs to remove excess dhe , and fluorescence was assessed by way of fluorescence microscopy ( axiocam hbo 50/ac , zeiss , germany ) . ethidium fluorescence density was detected from the whole section with mcid imaging software ( imaging research inc , australia ) and expressed as arbitrary units of fluorescence . rna was extracted from frozen quadriceps muscle using tri reagent ( molecular research centre ) , according to the manufacturer 's protocol . total rna concentration was determined spectrophotometrically at 260 nm . prior to reverse transcription ( rt ) , all rna samples were dnase - treated ( promega ) , and first - strand cdna was generated from 1-g rna using amv rt ( promega ) . pre - designed taqman gene expression assays ( applied biosystems ) were used containing specific primers and probes for the genes of interest . real - time pcr was performed using applied biosystems 7500 detection system and pcrs were performed using taqman gene expression master mix ( applied biosystems ) . to compensate for variations in input rna amounts and efficiency of reverse transcription , gapdh mrna was quantified and all results were normalised to these values . protein was extracted from frozen quadriceps muscle homogenised in ice - cold radio - immunoprecipitation assay buffer containing tris hcl ( 50 mmol / l ; ph 7.4 ) , nacl ( 150 mmol / l ) , np-40 ( 1% ) , sodium deoxycholate ( 0.5% ) and sds ( 0.1% ) and centrifuging at 13,000g for 15 min at 4c , to remove insoluble material . the protein concentration was determined by the bradford method ( bio - rad ) and equal amounts of protein were separated by sds - page and transferred to polyvinylidene difluoride membranes . the membranes were blocked for 2 h at room temperature in tris - buffered saline containing tris hcl ( 20 mm ; ph 7.6 ) nacl ( 137 mm ) and tween 20 ( 0.1% ) with 5% bsa and probed with primary antibodies for either nox2 ( gp91 ) , p40 , p67 or gapdh ( 1:200 ; santa cruz biotechnology ) overnight at 4c . antibody binding was detected using horseradish peroxidase - conjugated secondary antibody ( 1:50,000 ; santa cruz biotechnology ) . the protein bands were detected by supersignal west dura chemiluminescence reagents ( thermo scientific ) . the las 4000 imaging system ( fujifilm life science , usa ) was used to visualize protein bands , and densitometry was performed with multigauge software ( fujifilm life science , usa ) . to compensate for variation in protein loading , the relative density of immunoreactive bands were normalised to the density of the corresponding bands for gapdh . spectrophotometric assay kits were used to measure sod ( cayman-706002 ) , catalase ( cayman-707002 ) and gpx ( cayman- 703102 ) activity and hydrogen peroxide ( cayman-600050 ) levels , in muscle homogenates . frozen muscle pieces ( 100 mg ) were placed in ice - cold hepes buffer ( 20 mm ) containing egta ( 1 mm ) , mannitol ( 210 mm ) and sucrose ( 70 mm ) and adjusted to a ph of 7.2 ( 10 ml / g ) . muscle aliquots were homogenised in buffer , using a glass on glass homogeniser , and centrifuged at 1,500g for 5 min at 4c to remove insoluble connective tissue . for the detection of sod1 and sod2 , cytosolic and mitochondrial fractions the supernatant was centrifuged at 10,000g for 5 min at 4c , and the resulting supernatant , containing the cytosolic fraction , was collected for sod1 enzyme analysis . the remaining pellet containing the mitochondrial fraction was resuspended and homogenised in ice - cold hepes buffer ( 20 mm ) for sod2 enzyme analysis . the amount of enzyme activity and hydrogen peroxide levels were calculated and standardised for protein using the bradford method ( bio - rad ) . differences were determined by one - way anova with tukey hsd as posthoc to determine significant differences between groups , and results were considered statistically significant if p values were equal to or < 0.05 . all experimental procedures were carried out with approval from the victoria university animal ethics committee ( aeeth 07/05 ) . two weight - matched groups of female balb / c nu / nu mice were maintained under controlled environmental conditions , 12-h light / dark cycle , 21c 2c , 30% humidity , in conventional cages with ad libitum access to standard chow and water throughout the course of the study . as has previously been described , donor mice were used to establish and maintain a tumour line , which consistently produced tumour growth ( mac13 ) ( n = 3 ) and tumour growth with cachexia ( mac16 ) ( n = 4 ) . tumour fragments grown and maintained in donor mice were dissected and re - implanted subcutaneously into the flank of recipient mice with frequent monitoring and recording of body weight and tumour size . with cachectic weight loss evident at approximately 912 days post implantation , mice were anaesthetised , using pentobarbital sodium ( 70 mg / kg ) , for tissue collection before weight loss exceeded 25% of original body weight or before tumour growth exceeded 1,000 mm . skeletal muscle tissue was collected , weighed and immediately snap - frozen in liquid nitrogen and stored at 80c for later use . dhe ( 5 m ) was applied to quadriceps cross - sections ( 5 m ) and incubated in a light - protected oven at 37c for 30min . the sections were washed with pbs to remove excess dhe , and fluorescence was assessed by way of fluorescence microscopy ( axiocam hbo 50/ac , zeiss , germany ) . ethidium fluorescence density was detected from the whole section with mcid imaging software ( imaging research inc , australia ) and expressed as arbitrary units of fluorescence . rna was extracted from frozen quadriceps muscle using tri reagent ( molecular research centre ) , according to the manufacturer 's protocol . total rna concentration was determined spectrophotometrically at 260 nm . prior to reverse transcription ( rt ) , all rna samples were dnase - treated ( promega ) , and first - strand cdna was generated from 1-g rna using amv rt ( promega ) . pre - designed taqman gene expression assays ( applied biosystems ) were used containing specific primers and probes for the genes of interest . real - time pcr was performed using applied biosystems 7500 detection system and pcrs were performed using taqman gene expression master mix ( applied biosystems ) . to compensate for variations in input rna amounts and efficiency of reverse transcription , gapdh mrna was quantified and all results were normalised to these values . protein was extracted from frozen quadriceps muscle homogenised in ice - cold radio - immunoprecipitation assay buffer containing tris hcl ( 50 mmol / l ; ph 7.4 ) , nacl ( 150 mmol / l ) , np-40 ( 1% ) , sodium deoxycholate ( 0.5% ) and sds ( 0.1% ) and centrifuging at 13,000g for 15 min at 4c , to remove insoluble material . the protein concentration was determined by the bradford method ( bio - rad ) and equal amounts of protein were separated by sds - page and transferred to polyvinylidene difluoride membranes . the membranes were blocked for 2 h at room temperature in tris - buffered saline containing tris hcl ( 20 mm ; ph 7.6 ) nacl ( 137 mm ) and tween 20 ( 0.1% ) with 5% bsa and probed with primary antibodies for either nox2 ( gp91 ) , p40 , p67 or gapdh ( 1:200 ; santa cruz biotechnology ) overnight at 4c . antibody binding was detected using horseradish peroxidase - conjugated secondary antibody ( 1:50,000 ; santa cruz biotechnology ) . the protein bands were detected by supersignal west dura chemiluminescence reagents ( thermo scientific ) . the las 4000 imaging system ( fujifilm life science , usa ) was used to visualize protein bands , and densitometry was performed with multigauge software ( fujifilm life science , usa ) . to compensate for variation in protein loading , the relative density of immunoreactive bands were normalised to the density of the corresponding bands for gapdh . spectrophotometric assay kits were used to measure sod ( cayman-706002 ) , catalase ( cayman-707002 ) and gpx ( cayman- 703102 ) activity and hydrogen peroxide ( cayman-600050 ) levels , in muscle homogenates . frozen muscle pieces ( 100 mg ) were placed in ice - cold hepes buffer ( 20 mm ) containing egta ( 1 mm ) , mannitol ( 210 mm ) and sucrose ( 70 mm ) and adjusted to a ph of 7.2 ( 10 ml / g ) . muscle aliquots were homogenised in buffer , using a glass on glass homogeniser , and centrifuged at 1,500g for 5 min at 4c to remove insoluble connective tissue . for the detection of sod1 and sod2 , cytosolic and mitochondrial fractions the supernatant was centrifuged at 10,000g for 5 min at 4c , and the resulting supernatant , containing the cytosolic fraction , was collected for sod1 enzyme analysis . the remaining pellet containing the mitochondrial fraction was resuspended and homogenised in ice - cold hepes buffer ( 20 mm ) for sod2 enzyme analysis . the amount of enzyme activity and hydrogen peroxide levels were calculated and standardised for protein using the bradford method ( bio - rad ) . differences were determined by one - way anova with tukey hsd as posthoc to determine significant differences between groups , and results were considered statistically significant if p values were equal to or < 0.05 . female balb / c nu / nu mice were implanted with the mac13 cell line ( n = 12 ) that had previously developed a tumour in the same mouse model at approximately 912 days post implantation ( n = 3 ) and where the animal did not lose weight . another group of female balb / c nu / nu mice were implanted with the mac16 cell line ( n = 16 ) that had previously developed a tumour in the same mouse model at approximately 912 days post implantation ( n = 4 ) and lost approximately 1525% of their original body weight were used in this study . mouse body weight and skeletal muscle weights were recorded at tissue collection and evaluated as a measure of body mass and skeletal muscle cachexia for all mice . body weight and quadricep weights were significantly less in cachectic ( mac16 tumour - bearing mice ) when compared to mice with cancer alone ( bearing mac13 tumours ) ( p < 0.001 ; p = 0.003 , table 1 ) . to account for any differences in calorie intake between the groups , food intake was recorded throughout the course of the study and showed no differences between cachectic and cancer mice ( table 1).table 1mean body weight , quadriceps weight and food intake of mice with cancer and cancer cachexiaweightcancercachexiabody weight ( g)22.3 0.317.1 0.3*quadriceps ( mg)117.0 4.290.1 2.1*food intake ( g)3.6 0.33.6 0.5the values represent the mean sem*p < 0.003 , statistically significant differences mean body weight , quadriceps weight and food intake of mice with cancer and cancer cachexia the values represent the mean sem * p < 0.003 , statistically significant differences o2 levels were measured by histological dhe fluorescence in skeletal muscle sections from mice with cancer and cachexia . skeletal muscle o2 levels were significantly higher in cachectic mice ( 9.36 2.67 au ) when compared to mice with cancer ( 6.47 0.77 au ) alone ( p = 0.001 , fig the values represent the mean sem . statistically significant difference * p = 0.001 . a o2 levels detected by dihydroethidium ( dhe ) the values represent the mean sem . statistically significant difference * p = 0.001 . b histological dhe fluorescence in the skeletal muscle of mice with cancer and cancer cachexia o2 levels in the skeletal muscle of mice with cancer and cancer cachexia . statistically significant difference * p = 0.001 . a o2 levels detected by dihydroethidium ( dhe ) the values represent the mean sem . statistically significant difference * p = 0.001 . b histological dhe fluorescence in the skeletal muscle of mice with cancer and cancer cachexia the mrna level of the nox2 and nox4 enzyme systems previously shown to express in skeletal muscle to generate o2 were measured in mice with cancer and cancer cachexia . the mrna of the regulatory and catalytic nox2 enzyme subunits nox2 , p40 and p67 were lower in the skeletal muscle of cachectic mice when compared to mice with cancer alone ( p = 0.031 ; p < however , the mrna expression of the additional nox enzyme subunits , nox4 , p22 and p47 and rac1 were similar in skeletal muscle from mice bearing with cancer and cancer cachexia ( table 2 ) . the mrna level of sod antioxidant enzymes responsible for the dismutation of o2 were measured in mice with cancer and cancer cachexia . the mrna level of the sod1 and sod2 were significantly lower in the skeletal muscle from cachectic mice when compared to mice with cancer alone ( p = 0.007 ; p < 0.001 , table 2 ) . similarly , the mrna level of the h2o2 scavenging antioxidant enzyme gpx was lower in skeletal muscle from cachectic mice compared to mice with cancer alone ( p < 0.001 , table 2 ) . however , the antioxidant enzymes sod3 and catalase had similar mrna levels in both groups ( table 2).table 2gene expression in skeletal muscle of mice with cancer and cancer cachexiacancercachexiagene expression ( au ) nox211.8 2.35.0 1.1 * p22phox88.7 2.975.5 10.5 p40phox21.4 2.87.9 1.6 * p47phox5.7 1.27.3 1.4 p67phox367 92.715.6 3.3 * nox44.3 0.68.5 2.7 rac13.6 1.17.5 1.6 sod1350 36.5120 37.8 * sod2122 8.059.4 5.4 * sod32.6 0.31.8 0.3 gpx23.5 2.312.8 1.2 * catalase49.3 7.343.2 5.6the values represent the mean sem*p < 0.003 , statistically significant differences gene expression in skeletal muscle of mice with cancer and cancer cachexia the values represent the mean sem * p < 0.003 , statistically significant differences changes in the mrna levels of the key nox2 enzyme subunits nox2 , p40 and p67lead to further analysis of protein expression . the protein expression of the regulatory and catalytic nox2 enzyme subunits nox2 and p40 were lower in cachectic mice when compared to mice with cancer alone ( p = 0.021 ; p < 0.012 , fig . 2 ) . however , the protein expression of p67 was similar in skeletal muscle from mice in both groups ( fig . 2protein expression levels of nox2 enzyme subunits in the skeletal muscle of mice with cancer and cancer cachexia . statistically significant differences * p < 0.05 . a nox2 , bp40phox , cp67pho protein expression levels of nox2 enzyme subunits in the skeletal muscle of mice with cancer and cancer cachexia . statistically significant differences * p < 0.05 . a nox2 , bp40phox , cp67pho to validate changes in mrna and protein levels , sod1 , sod2 , catalase and gpx activity levels were measured in skeletal muscle of mice with cancer and cancer cachexia . sod1 and gpx activities were significantly lower in skeletal muscle from cachectic mice ( sod1 9.9 0.9 u / mg protein , gpx 0.74 0.22 mmol / min / mg protein ) when compared to mice with cancer alone ( sod1 11.2 0.8 u / mg protein , gpx 1.48 0.08 mmol / min / mg protein ) ( p < 0.003 , table 3 ) . however , levels of sod2 and catalase activity were similar in both groups of ( table 3 ) . h2o2 levels were similar in skeletal muscle from cachectic mice and mice with cancer alone ( table 3).table 3enzyme activity and h2o2 levels in skeletal muscle of mice with cancer and cancer cachexiacancercachexiasod1 ( u / mg protein)23.3 0.817.8 0.9*sod2 ( u / mg protein)11.2 0.89.9 0.8catalase ( mmol / min / mg protein)4.1 0.43.5 0.2gpx ( mmol / min / mg protein)1.48 0.080.74 0.22*h2o2 ( m)1.11 0.071.15 0.04the values represent the mean sem*p < 0.003 , statistically significant differences enzyme activity and h2o2 levels in skeletal muscle of mice with cancer and cancer cachexia the values represent the mean sem * p < 0.003 , statistically significant differences female balb / c nu / nu mice were implanted with the mac13 cell line ( n = 12 ) that had previously developed a tumour in the same mouse model at approximately 912 days post implantation ( n = 3 ) and where the animal did not lose weight . another group of female balb / c nu / nu mice were implanted with the mac16 cell line ( n = 16 ) that had previously developed a tumour in the same mouse model at approximately 912 days post implantation ( n = 4 ) and lost approximately 1525% of their original body weight were used in this study . mouse body weight and skeletal muscle weights were recorded at tissue collection and evaluated as a measure of body mass and skeletal muscle cachexia for all mice . body weight and quadricep weights were significantly less in cachectic ( mac16 tumour - bearing mice ) when compared to mice with cancer alone ( bearing mac13 tumours ) ( p < 0.001 ; p = 0.003 , table 1 ) . to account for any differences in calorie intake between the groups , food intake was recorded throughout the course of the study and showed no differences between cachectic and cancer mice ( table 1).table 1mean body weight , quadriceps weight and food intake of mice with cancer and cancer cachexiaweightcancercachexiabody weight ( g)22.3 0.317.1 0.3*quadriceps ( mg)117.0 4.290.1 2.1*food intake ( g)3.6 0.33.6 0.5the values represent the mean sem*p < 0.003 , statistically significant differences mean body weight , quadriceps weight and food intake of mice with cancer and cancer cachexia the values represent the mean sem * p < 0.003 , statistically significant differences o2 levels were measured by histological dhe fluorescence in skeletal muscle sections from mice with cancer and cachexia . skeletal muscle o2 levels were significantly higher in cachectic mice ( 9.36 2.67 au ) when compared to mice with cancer ( 6.47 0.77 au ) alone ( p = 0.001 , fig . the values represent the mean sem . statistically significant difference * p = 0.001 . a o2 levels detected by dihydroethidium ( dhe ) fluorescence expressed as arbitrary units of fluorescence . b histological dhe fluorescence in the skeletal muscle of mice with cancer and cancer cachexia o2 levels in the skeletal muscle of mice with cancer and cancer cachexia . the values represent the mean sem . statistically significant difference * p = 0.001 . a o2 levels detected by dihydroethidium ( dhe ) fluorescence expressed as arbitrary units of fluorescence . the values represent the mean sem . statistically significant difference * p = 0.001 . the mrna level of the nox2 and nox4 enzyme systems previously shown to express in skeletal muscle to generate o2 were measured in mice with cancer and cancer cachexia . the mrna of the regulatory and catalytic nox2 enzyme subunits nox2 , p40 and p67 were lower in the skeletal muscle of cachectic mice when compared to mice with cancer alone ( p = 0.031 ; p < however , the mrna expression of the additional nox enzyme subunits , nox4 , p22 and p47 and rac1 were similar in skeletal muscle from mice bearing with cancer and cancer cachexia ( table 2 ) . the mrna level of sod antioxidant enzymes responsible for the dismutation of o2 were measured in mice with cancer and cancer cachexia . the mrna level of the sod1 and sod2 were significantly lower in the skeletal muscle from cachectic mice when compared to mice with cancer alone ( p = 0.007 ; p < 0.001 , table 2 ) . similarly , the mrna level of the h2o2 scavenging antioxidant enzyme gpx was lower in skeletal muscle from cachectic mice compared to mice with cancer alone ( p < 0.001 , table 2 ) . however , the antioxidant enzymes sod3 and catalase had similar mrna levels in both groups ( table 2).table 2gene expression in skeletal muscle of mice with cancer and cancer cachexiacancercachexiagene expression ( au ) nox211.8 2.35.0 1.1 * p22phox88.7 2.975.5 10.5 p40phox21.4 2.87.9 1.6 * p47phox5.7 1.27.3 1.4 p67phox367 92.715.6 3.3 * nox44.3 0.68.5 2.7 rac13.6 1.17.5 1.6 sod1350 36.5120 37.8 * sod2122 8.059.4 5.4 * sod32.6 0.31.8 0.3 gpx23.5 2.312.8 1.2 * catalase49.3 7.343.2 5.6the values represent the mean sem*p < 0.003 , statistically significant differences gene expression in skeletal muscle of mice with cancer and cancer cachexia the values represent the mean sem * p < 0.003 , statistically significant differences changes in the mrna levels of the key nox2 enzyme subunits nox2 , p40 and p67lead to further analysis of protein expression . the protein expression of the regulatory and catalytic nox2 enzyme subunits nox2 and p40 were lower in cachectic mice when compared to mice with cancer alone ( p = 0.021 ; p < 0.012 , fig . 2 ) . however , the protein expression of p67 was similar in skeletal muscle from mice in both groups ( fig . 2).fig . 2protein expression levels of nox2 enzyme subunits in the skeletal muscle of mice with cancer and cancer cachexia . statistically significant differences * p < 0.05 . a nox2 , bp40phox , cp67pho protein expression levels of nox2 enzyme subunits in the skeletal muscle of mice with cancer and cancer cachexia . statistically significant differences * p < 0.05 . a nox2 , bp40phox , cp67pho to validate changes in mrna and protein levels , sod1 , sod2 , catalase and gpx activity levels were measured in skeletal muscle of mice with cancer and cancer cachexia . sod1 and gpx activities were significantly lower in skeletal muscle from cachectic mice ( sod1 9.9 0.9 u / mg protein , gpx 0.74 0.22 mmol / min / mg protein ) when compared to mice with cancer alone ( sod1 11.2 0.8 u / mg protein , gpx 1.48 0.08 mmol / min / mg protein ) ( p < 0.003 , table 3 ) . however , levels of sod2 and catalase activity were similar in both groups of ( table 3 ) . h2o2 levels were similar in skeletal muscle from cachectic mice and mice with cancer alone ( table 3).table 3enzyme activity and h2o2 levels in skeletal muscle of mice with cancer and cancer cachexiacancercachexiasod1 ( u / mg protein)23.3 0.817.8 0.9*sod2 ( u / mg protein)11.2 0.89.9 0.8catalase ( mmol / min / mg protein)4.1 0.43.5 0.2gpx ( mmol / min / mg protein)1.48 0.080.74 0.22*h2o2 ( m)1.11 0.071.15 0.04the values represent the mean sem*p < 0.003 , statistically significant differences enzyme activity and h2o2 levels in skeletal muscle of mice with cancer and cancer cachexia the values represent the mean sem * p < 0.003 , statistically significant differences evidence of this was found with increased levels of o2 detected by dhe fluorescence in the skeletal muscle of cachectic mice , when compared to non - cachectic mice . however , the nox enzyme systems do not appear to be the source of increased o2 in cancer - induced skeletal muscle cachexia . in fact , we observed a decrease in the expression of the nox2 enzyme subunits , nox2 , p40 and p67 in cachectic skeletal muscle . although this study would imply an additional source of o2 production in cancer - induced skeletal muscle cachexia , what may be of greater importance is the decrease in antioxidant enzyme function , primarily responsible for o2 dismutation . while this study may be indicative of a lack of compensation by sod1 and gpx , it may give evidence for their depression , in response to cancer induction and lack of dismutation of even normal cellular o2 production . the decrease in antioxidant gene expression and activity that function as part of the cellular defence system , for the elimination of o2 and h2o2 , may indicate antioxidant dysfunction in cachectic skeletal muscle , rather than an increase in ros production . cancer induces changes in skeletal muscle that lead to an imbalance in protein synthesis and degradation , resulting in muscle protein loss and function . interestingly , not all cancer patients develop this secondary condition , and it is this phenomena that has made the development of cancer cachexia relatively undefined [ 1 , 2 ] . in order to investigate this complex condition , we investigated a model that utilises two similar mac models , only one of which induces the secondary muscle wasting condition of cancer cachexia . the significant decline in body weight ( 1525% ) and skeletal muscle mass that was observed in mice bearing the mac16 tumour , demonstrated the development of cancer - induced skeletal muscle cachexia . while the mac16 cancer cachectic model is well - established in nmri mice , this study mimics the cachectic condition in mac16 tumour - bearing nude mice . this model of cancer and cancer cachexia establishes a direct comparison between a cancer control and cancer cachectic mouse model . contrasting studies have implicated the importance of calorie restriction in the development of cancer cachexia ; however , our study , like others , did not observe any change in food intake from cachectic mice . this study therefore suggests a more complex catabolic - mediated response to protein degeneration , in the development of cancer cachexia . the physical changes in skeletal muscle were mirrored by significant cellular oxidative changes in response to mac16 induction and cachectic development . in particular , the significant increase in o2 levels observed in cachectic skeletal muscle indicates that o2 is implicated in the cancer cachectic condition . previous studies have proposed a central mediating role for ros in the development of cancer cachexia [ 5 , 18 ] ; however , the source of ros in this process remains to be elucidated . the knowledge that the nox enzyme system functions primarily to produce o2 , recognises nox - generated o2 as more than a by - product of cellular metabolism , but rather a product of a regulated response to stimuli for a physiological purpose . this function of nox makes this enzyme system the potential source of o2 , vulnerable to changes associated with the cancer condition and development of cachexia . to our knowledge , this study is the first to investigate the nox enzymes in cancer cachectic skeletal muscle . interestingly , the nox2 enzyme has consistently been shown to increase in degenerative conditions ; however , this study does not support an increase in the nox2 enzyme in cancer cachexia . it is evident , however , from this study and others , that the absence of a functioning antioxidant enzyme system to dismutate o2 is present in cancer cachectic skeletal muscle . cellular oxidative stress has been implicated in skeletal muscle wasting conditions with marked attenuation following antioxidant induction . despite the increase in o2- in cachectic skeletal muscle that is indicative of a need for sod activity furthermore , our sod gene expression data would appear to suggest that the inability for sod to compensate for the increase in o2 is regulated at the gene level . alterations in this important protective system can lead to oxidative imbalance , and induce critical changes to cellular structure and function . antioxidant enzymes are valuable indicators of ros production as well as changes in cellular redox signalling and evidence of cell functionality . the consequences of sod antioxidant enzyme modifications have been demonstrated in studies investigating sod knockout models . muller et al . demonstrated an age - dependent loss of muscle mass in mice lacking sod1 , as well as a significant decrease in their average life span . furthermore , sun et al . investigated the effects of sod1 and sod2 overexpression in drosophila that demonstrated a decrease in cumulative oxidative damage and increased metabolic potential , with an increased life span by up to 37% and 75% , respectively . these models demonstrate a crucial role for sod1 shown through its significant contribution to oxidative damage and muscle degeneration . it is also not surprising that with a decline in o2 dismutation by sod , to convert o2 to h2o2 , the gene expression and activity of the h2o2 scavenging antioxidant enzyme gpx would also decrease . consequently , these important cellular systems would indeed contribute to significant changes in redox - sensitive signalling pathways in cancer cachexia . sod , not only has a crucial role in eliminating o2 accumulation , but also plays an important role in intracellular redox - sensitive signalling and regulation of oxidative systems . as ros have been described as important mediators of redox - sensitive intracellular signalling , so too are the antioxidants that regulate them . it is well - known that with the generation of intracellular o2 , sod1 functions to dismute o2 to h2o2 . h2o2 in particular , has been shown to be involved in numerous signalling cascades , and therefore , its cellular regulation , via sod , has the potential to influence a number of important cellular pathways . the changes in sod1 function would indeed have critical consequences to cellular function that is most certainly redox - related . a circulatory protein , pif was first described as a causative agent in cachexia when it was discovered in mice expressing the mac16 tumour . as this glycoprotein was initially discovered in the sera of mice bearing the cachectic mac16 tumour , but not in the sera of the mice bearing the non - cachectic mac13 tumours , it was regarded as an important factor in the development of cancer cachexia . the accelerated loss of skeletal muscle protein in cachexia has been attributed to ub - proteasome pathway activation [ 15 , 26 ] , and pif has been shown to induce this pathway of skeletal muscle atrophy . although the pif / ub pathway indeed plays an important role in the development of cancer cachexia , a number of additional mechanisms are most likely involved . preproteasomal mechanisms , mediators , receptor binding , signalling pathways and activation of specific transcription factors are all important considerations and ros have been implicated in these cellular processes . collectively , the results of this study suggest influential signalling involving o2 and antioxidant enzymes in cancer cachectic skeletal muscle . however , the signalling pathway(s ) leading to the decrease in the nox2 subunits and antioxidant enzyme expression , together with decreased antioxidant enzyme activity in cachectic skeletal muscle remains undefined . in response to ros , cells activate the expression of a number of genes via transcription factor regulation , leading to modifications in the gene expression of important proteins , including antioxidant enzymes and those involved in muscle protein synthesis and regeneration . furthermore , it is possible to speculate that the downregulation of the nox2 enzyme system is a compensatory response to o2 accumulation , induced primarily by sod and gpx antioxidant system dysfunction and is therefore a regulated response to o2 build - up in the cellular system . however , with the knowledge that ros can cause damage to cellular proteins , it is possible that the decrease in the nox2 enzyme subunits and antioxidant enzymes is a result of protein oxidation . with this in mind , it would be important to investigate these oxidative systems and the oxidative status of cachectic skeletal muscle during the progression of the disease . it is evident from cachectic studies that show the presence of oxidised proteins and attenuation following antioxidant administration that ros play an important role in the development of cachexia . although the exact mechanisms are poorly defined , experimental research in the nox enzyme systems have indicated a number of important roles for ros , in addition to direct oxidative tissue damage , for its involvement in redox - sensitive signalling pathways . while studies have implicated the involvement of ros in the pathogenesis of cachexia and have suggested a role for nox , this study suggests that the increase in o2 in cachectic skeletal muscle is a result of antioxidant dysfunction . however , what remains unclear is whether this result is a regulated response in the cellular system or an important contributor to the changes observed in skeletal muscle physiology . furthermore , with the well - established role for the ub - proteasome pathway in cancer cachexia , the increase in o2 levels in our study provides evidence of a signalling role for o2 in this pathway of skeletal muscle atrophy in cancer cachexia . while these results , along with the additional findings of this study indeed demonstrate complex changes in cachectic skeletal muscle , this multifactoral condition coupled with a multifunctional system , further demonstrates the complexity of skeletal muscle response(s ) to cancer induction . it is therefore important to understand further the role that these oxidative and antioxidative systems play in the skeletal muscle system and development of cancer cachexia .
backgroundcancer cachexia is the progressive loss of skeletal muscle protein that contributes significantly to cancer morbidity and mortality . evidence of antioxidant attenuation and the presence of oxidised proteins in patients with cancer cachexia indicate a role for oxidative stress . the level of oxidative stress in tissues is determined by an imbalance between reactive oxygen species production and antioxidant activity . this study aimed to investigate the superoxide generating nadph oxidase ( nox ) enzyme and antioxidant enzyme systems in murine adenocarcinoma tumour - bearing cachectic mice.methodssuperoxide levels , mrna levels of nox enzyme subunits and the antioxidant enzymes superoxide dismutase ( sod ) , glutathione peroxidise ( gpx ) and catalase was measured in the skeletal muscle of mice with cancer and cancer cachexia . protein expression levels of nox enzyme subunits and antioxidant enzyme activity was also measured in the same muscle samples.resultssuperoxide levels increased 1.4-fold in the muscle of mice with cancer cachexia , and this was associated with a decrease in mrna of nox enzyme subunits , nox2 , p40phox and p67phox along with the antioxidant enzymes sod1 , sod2 and gpx . cancer cachexia was also associated with a 1.3-fold decrease in sod1 and 2.0-fold decrease in gpx enzyme activity.conclusiondespite increased superoxide levels in cachectic skeletal muscle , nox enzyme subunits , nox2 , p40phox and p67phox , were downregulated along with the expression and activity of the antioxidant enzymes . therefore , the increased superoxide levels in cachectic skeletal muscle may be attributed to the reduction in the activity of endogenous antioxidant enzymes .
Introduction Methods Animal model of cancer-induced cachexia Detection of O Reverse transcription real-time PCR Protein electrophoresis and western blotting Antioxidant enzyme activity and hydrogen peroxide assays Statistical analysis Results Establishment of murine model of cancer and cancer cachexia O mRNA expression of pro-oxidant and antioxidant enzymes in skeletal muscle of cancer and cancer cachectic mice Protein levels of NOX2 enzyme subunits in skeletal muscle of cancer and cancer cachectic mice Antioxidant enzyme activity and hydrogen peroxide levels in skeletal muscle of cancer and cancer cachectic mice Discussion Conclusion
the mrna of the regulatory and catalytic nox2 enzyme subunits nox2 , p40 and p67 were lower in the skeletal muscle of cachectic mice when compared to mice with cancer alone ( p = 0.031 ; p < however , the mrna expression of the additional nox enzyme subunits , nox4 , p22 and p47 and rac1 were similar in skeletal muscle from mice bearing with cancer and cancer cachexia ( table 2 ) . however , the antioxidant enzymes sod3 and catalase had similar mrna levels in both groups ( table 2).table 2gene expression in skeletal muscle of mice with cancer and cancer cachexiacancercachexiagene expression ( au ) nox211.8 2.35.0 1.1 * p22phox88.7 2.975.5 10.5 p40phox21.4 2.87.9 1.6 * p47phox5.7 1.27.3 1.4 p67phox367 92.715.6 3.3 * nox44.3 0.68.5 2.7 rac13.6 1.17.5 1.6 sod1350 36.5120 37.8 * sod2122 8.059.4 5.4 * sod32.6 0.31.8 0.3 gpx23.5 2.312.8 1.2 * catalase49.3 7.343.2 5.6the values represent the mean sem*p < 0.003 , statistically significant differences gene expression in skeletal muscle of mice with cancer and cancer cachexia the values represent the mean sem * p < 0.003 , statistically significant differences changes in the mrna levels of the key nox2 enzyme subunits nox2 , p40 and p67lead to further analysis of protein expression . the mrna of the regulatory and catalytic nox2 enzyme subunits nox2 , p40 and p67 were lower in the skeletal muscle of cachectic mice when compared to mice with cancer alone ( p = 0.031 ; p < however , the mrna expression of the additional nox enzyme subunits , nox4 , p22 and p47 and rac1 were similar in skeletal muscle from mice bearing with cancer and cancer cachexia ( table 2 ) . however , the antioxidant enzymes sod3 and catalase had similar mrna levels in both groups ( table 2).table 2gene expression in skeletal muscle of mice with cancer and cancer cachexiacancercachexiagene expression ( au ) nox211.8 2.35.0 1.1 * p22phox88.7 2.975.5 10.5 p40phox21.4 2.87.9 1.6 * p47phox5.7 1.27.3 1.4 p67phox367 92.715.6 3.3 * nox44.3 0.68.5 2.7 rac13.6 1.17.5 1.6 sod1350 36.5120 37.8 * sod2122 8.059.4 5.4 * sod32.6 0.31.8 0.3 gpx23.5 2.312.8 1.2 * catalase49.3 7.343.2 5.6the values represent the mean sem*p < 0.003 , statistically significant differences gene expression in skeletal muscle of mice with cancer and cancer cachexia the values represent the mean sem * p < 0.003 , statistically significant differences changes in the mrna levels of the key nox2 enzyme subunits nox2 , p40 and p67lead to further analysis of protein expression . h2o2 levels were similar in skeletal muscle from cachectic mice and mice with cancer alone ( table 3).table 3enzyme activity and h2o2 levels in skeletal muscle of mice with cancer and cancer cachexiacancercachexiasod1 ( u / mg protein)23.3 0.817.8 0.9*sod2 ( u / mg protein)11.2 0.89.9 0.8catalase ( mmol / min / mg protein)4.1 0.43.5 0.2gpx ( mmol / min / mg protein)1.48 0.080.74 0.22*h2o2 ( m)1.11 0.071.15 0.04the values represent the mean sem*p < 0.003 , statistically significant differences enzyme activity and h2o2 levels in skeletal muscle of mice with cancer and cancer cachexia the values represent the mean sem * p < 0.003 , statistically significant differences evidence of this was found with increased levels of o2 detected by dhe fluorescence in the skeletal muscle of cachectic mice , when compared to non - cachectic mice .
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laparoscopic radical prostatectomy ( lrp ) and retropubic radical prostatectomy ( rrp ) are well - established procedures for the management of localized prostate cancer . recently , cancer treatment strategies have focused more on patient quality of life after surgery , thus requiring minimally invasive techniques . in 2002 , an initial experience with extraperitoneal laparoscopic radical prostatectomy ( elrp ) showed similar oncological outcomes to intraperitoneal prostatectomy without intraperitoneal complications . due to the low perioperative and postoperative mortality rate , postradical prostatectomy incontinence , which interferes with the patient 's quality of life , has become an issue . one - year continence rates are excellent after rrp and lrp in larger series . however , the early return of continence remains a challenge and has a major impact on the postoperative patient 's health - related quality of life . further understanding of the anatomy of adjacent structures of the prostate , such as the bladder neck , urethra , fascia , ligaments , and neurovascular bundle ( nbv ) , led to the development of nerve - sparing elrp ( nselrp ) and the most recent intrafascial nselrp , which shows a positive surgical outcome of early continence . in this study , we report our experience with 50 consecutive patients who underwent intrafascial nselrp by a single surgeon . among 62 patients , 50 patients with clinically localized prostate cancer at the time of preoperative magnetic resonance image ( mri ) with psa levels less than 10 ng / ml and gleason scores less than 7 ( 3 + 4 ) were included for intrafascial nselrp from november 2011 to april 2012 . if there were any suspicious lesions with extracapsular extension of tumors or high gleason scores ( 7 [ 4 + 3 ] to 10 ) , conventional radical prostatectomy and lymph node dissection were performed . a small group of patients underwent intrafascial nselrp despite their higher psa levels of more than 10 ng / ml because they had low gleason scores ( 6 ) . in short , the preparation of the space of retzius began with a 2-cm sized incision in the infraumbilical crease laterally to the midline , which was then carried down to the posterior rectus sheath where a balloon trocar was inserted and the preperitoneal space was developed . finally , the camera trocar was placed and further trocars were inserted with attention to the course of the epigastric vessels and peritoneum . next , the 12-mm assistant port , 12-mm operator port , and 5-mm operator port were placed sequentially . the fatty tissue surrounding the prostate and pelvic space was removed to expose the landmark structures such as the pubic arch , symphysis , endopelvic fascia , bladder , and prostate . the anterior surfaces of the bladder and prostate as well as the endopelvic fascia became visible to the operator . the removal of preprostatic fibro - fatty tissues facilitates more definite differentiation between the prostate and the bladder . the endopelvic fascia and pubo - prostatic ligament were not incised and the deep dorsal venous complex was not ligated at the beginning of the procedure . a bilateral incision of the periprostatic fascia was made medial to the puboprostatic ligament ( ppl ) and directed to the base of the prostate . the right plane of dissection was recognized when the surface of the prostate was completely smooth . as a result , the development of a plane between the prostate and overlaying fascia was possible and the operator could detach the prostate from its enveloping fascia . all lateral periprostatic fascia , endopelvic fascia , and puboprostatic fascia remained intact ( fig . careful coagulation - free dissection is necessary during bilateral seminal vesicle dissection because the pelvic plexus and nvb run in close proximity to the tip of the seminal vesicle . , somerville , nj , usa ) were used to ligate the seminal vesicular artery . when the vas deferens and seminal vesicle were both fully dissected , the denonvilliers fascia was visualized . the denonvilliers fascia was stripped down to find the correct plan for the intrafascial dissection of the prostate . after identification of the prostate capsule , the dissection was gradually directed toward the apex of the prostate in the midline to avoid injury of the nbvs . the prostate was fully detached from its surrounding fascias but was still attached by the pedicles and the apex . the assistant took the seminal vesicle and vas deferens and gently elevated them ventrally to allow clear sight of the prostatic pedicle . the prostatic pedicles were then clipped and cut in a step - by - step manner directly on the surface of the prostatic capsule without excessive traction or electrocautery . when dissecting the nbv , care must be taken to avoid damage to the nvb . after the correct plane was opened , the dissection was performed by using cold scissors in an essentially avascular plane . meticulous dissection is needed between the lateral side of the prostate and the lateral remnant of the periprostatic fascia toward the apex of the prostate to preserve the accessory nvb ( fig . the dissection was performed on the opposite side of the prostate pedicle and the nvb in the same fashion as that of the primary side . finally , the prostate was completely detached from the surrounding fascias , bladder , prostatic pedicles , and nvbs . sharp dissection of the prostate from the external sphincter and urethra at the site of the apex was performed . the assistant retracted the prostate to secure a clear view of the apex margin of the prostate . dissection and division of the external urethral sphincter and prostate were carefully made ( fig . dissection of the urethra was performed proximally very close to the prostate to preserve the urethral length as much as possible . vertical dissection was performed with cold scissors alone to achieve complete division of the prostate from the urethra . after identification of no contrast leakage at the anastomosis site , the foley catheter was removed . postoperative evaluation of continence was performed by evaluation of the number of pads used per day . postoperative follow - up was defined at 2 weeks , 6 weeks , and 3 months . complete continence was defined as usage of no pads and patient 's report of no urinary leakage . 18.0 ( ibm co. , armonk , ny , usa ) , with statistical significance considered at p<0.05 . spearman 's correlation test was used to evaluate the factors related to early continence recovery after nselrp . among 62 patients , 50 patients with clinically localized prostate cancer at the time of preoperative magnetic resonance image ( mri ) with psa levels less than 10 ng / ml and gleason scores less than 7 ( 3 + 4 ) were included for intrafascial nselrp from november 2011 to april 2012 . if there were any suspicious lesions with extracapsular extension of tumors or high gleason scores ( 7 [ 4 + 3 ] to 10 ) , conventional radical prostatectomy and lymph node dissection were performed . a small group of patients underwent intrafascial nselrp despite their higher psa levels of more than 10 ng / ml because they had low gleason scores ( 6 ) . in short , the patient was placed in the supine position with mild head - down tilt . the preparation of the space of retzius began with a 2-cm sized incision in the infraumbilical crease laterally to the midline , which was then carried down to the posterior rectus sheath where a balloon trocar was inserted and the preperitoneal space was developed . finally , the camera trocar was placed and further trocars were inserted with attention to the course of the epigastric vessels and peritoneum . next , the 12-mm assistant port , 12-mm operator port , and 5-mm operator port were placed sequentially . the fatty tissue surrounding the prostate and pelvic space was removed to expose the landmark structures such as the pubic arch , symphysis , endopelvic fascia , bladder , and prostate . the anterior surfaces of the bladder and prostate as well as the endopelvic fascia became visible to the operator . the removal of preprostatic fibro - fatty tissues facilitates more definite differentiation between the prostate and the bladder . the endopelvic fascia and pubo - prostatic ligament were not incised and the deep dorsal venous complex was not ligated at the beginning of the procedure . a bilateral incision of the periprostatic fascia was made medial to the puboprostatic ligament ( ppl ) and directed to the base of the prostate . the right plane of dissection was recognized when the surface of the prostate was completely smooth . as a result , the development of a plane between the prostate and overlaying fascia was possible and the operator could detach the prostate from its enveloping fascia . all lateral periprostatic fascia , endopelvic fascia , and puboprostatic fascia remained intact ( fig . careful coagulation - free dissection is necessary during bilateral seminal vesicle dissection because the pelvic plexus and nvb run in close proximity to the tip of the seminal vesicle . five - millimeter titanium clips ( ligaclip , ethicon inc . , somerville , nj , usa ) were used to ligate the seminal vesicular artery . when the vas deferens and seminal vesicle were both fully dissected , the denonvilliers fascia was visualized . the denonvilliers fascia was stripped down to find the correct plan for the intrafascial dissection of the prostate . after identification of the prostate capsule , the dissection was gradually directed toward the apex of the prostate in the midline to avoid injury of the nbvs . the prostate was fully detached from its surrounding fascias but was still attached by the pedicles and the apex . the assistant took the seminal vesicle and vas deferens and gently elevated them ventrally to allow clear sight of the prostatic pedicle . the prostatic pedicles were then clipped and cut in a step - by - step manner directly on the surface of the prostatic capsule without excessive traction or electrocautery . when dissecting the nbv , care must be taken to avoid damage to the nvb . after the correct plane was opened , the dissection was performed by using cold scissors in an essentially avascular plane . meticulous dissection is needed between the lateral side of the prostate and the lateral remnant of the periprostatic fascia toward the apex of the prostate to preserve the accessory nvb ( fig . the dissection was performed on the opposite side of the prostate pedicle and the nvb in the same fashion as that of the primary side . finally , the prostate was completely detached from the surrounding fascias , bladder , prostatic pedicles , and nvbs . sharp dissection of the prostate from the external sphincter and urethra at the site of the apex was performed . the assistant retracted the prostate to secure a clear view of the apex margin of the prostate . dissection and division of the external urethral sphincter and prostate were carefully made ( fig . dissection of the urethra was performed proximally very close to the prostate to preserve the urethral length as much as possible . vertical dissection was performed with cold scissors alone to achieve complete division of the prostate from the urethra . in short , the patient was placed in the supine position with mild head - down tilt . the preparation of the space of retzius began with a 2-cm sized incision in the infraumbilical crease laterally to the midline , which was then carried down to the posterior rectus sheath where a balloon trocar was inserted and the preperitoneal space was developed . finally , the camera trocar was placed and further trocars were inserted with attention to the course of the epigastric vessels and peritoneum . next , the 12-mm assistant port , 12-mm operator port , and 5-mm operator port were placed sequentially . the fatty tissue surrounding the prostate and pelvic space was removed to expose the landmark structures such as the pubic arch , symphysis , endopelvic fascia , bladder , and prostate . the anterior surfaces of the bladder and prostate as well as the endopelvic fascia became visible to the operator . the removal of preprostatic fibro - fatty tissues facilitates more definite differentiation between the prostate and the bladder . the endopelvic fascia and pubo - prostatic ligament were not incised and the deep dorsal venous complex was not ligated at the beginning of the procedure . a bilateral incision of the periprostatic fascia was made medial to the puboprostatic ligament ( ppl ) and directed to the base of the prostate . the right plane of dissection was recognized when the surface of the prostate was completely smooth . as a result , the development of a plane between the prostate and overlaying fascia was possible and the operator could detach the prostate from its enveloping fascia . all lateral periprostatic fascia , endopelvic fascia , and puboprostatic fascia remained intact ( fig . careful coagulation - free dissection is necessary during bilateral seminal vesicle dissection because the pelvic plexus and nvb run in close proximity to the tip of the seminal vesicle . five - millimeter titanium clips ( ligaclip , ethicon inc . , somerville , nj , usa ) were used to ligate the seminal vesicular artery . when the vas deferens and seminal vesicle were both fully dissected , the denonvilliers fascia was visualized . the denonvilliers fascia was stripped down to find the correct plan for the intrafascial dissection of the prostate . after identification of the prostate capsule , the dissection was gradually directed toward the apex of the prostate in the midline to avoid injury of the nbvs . the prostate was fully detached from its surrounding fascias but was still attached by the pedicles and the apex . the assistant took the seminal vesicle and vas deferens and gently elevated them ventrally to allow clear sight of the prostatic pedicle . the prostatic pedicles were then clipped and cut in a step - by - step manner directly on the surface of the prostatic capsule without excessive traction or electrocautery . when dissecting the nbv , care must be taken to avoid damage to the nvb . after the correct plane was opened , the dissection was performed by using cold scissors in an essentially avascular plane . meticulous dissection is needed between the lateral side of the prostate and the lateral remnant of the periprostatic fascia toward the apex of the prostate to preserve the accessory nvb ( fig . the dissection was performed on the opposite side of the prostate pedicle and the nvb in the same fashion as that of the primary side . finally , the prostate was completely detached from the surrounding fascias , bladder , prostatic pedicles , and nvbs . sharp dissection of the prostate from the external sphincter and urethra at the site of the apex was performed . the assistant retracted the prostate to secure a clear view of the apex margin of the prostate . dissection and division of the external urethral sphincter and prostate were carefully made ( fig . dissection of the urethra was performed proximally very close to the prostate to preserve the urethral length as much as possible . vertical dissection was performed with cold scissors alone to achieve complete division of the prostate from the urethra . all patients underwent cystography on the 4th postoperative day . after identification of no contrast leakage at the anastomosis site , postoperative evaluation of continence was performed by evaluation of the number of pads used per day . postoperative follow - up was defined at 2 weeks , 6 weeks , and 3 months . complete continence was defined as usage of no pads and patient 's report of no urinary leakage . statistical analysis was performed by using pasw ver . 18.0 ( ibm co. , armonk , ny , usa ) , with statistical significance considered at p<0.05 . spearman 's correlation test was used to evaluate the factors related to early continence recovery after nselrp . the mean operation time was 149.3 minutes ( range , 90 to 240 minutes ) . the mean hospitalization time was 6.3 days ( range , 4 to 19 days ) , and the mean catheterization time was 5.5 days ( range , 3 to 26 days ) . postoperative continence results are shown in table 2 . at the 2-week follow - up after surgery , 14 patients ( 28.0% ) achieved total continence and on average 2.3 pads were required for the other incontinent patients . a total of 35 patients ( 70.0% ) were continent at 6 weeks after surgery , 8 patients ( 16.0% ) had mild stress incontinence ( 1 to 2 pads ) , and 7 patients ( 14.0% ) required > 2 pads per day ( fig . 4 ) . a total of 31 patients were available for follow - up at 3 months after surgery , and 26 patients ( 83.9% ) were pad - free . compared with conventional nerve - sparing lrp , intrafascial nselrp showed early continence recovery ( table 2 ) . in patients with a relatively old age of above 65 years , preoperative low prostate volume and low gleason score were related with early continence at 6 weeks after surgery ( table 3 ) . signs of early recovery of potency ( morning erection or erection sensation ) were reported by 19 patients ( 38.0% ) at 6 weeks after surgery and the rate increased to 54.8% at 3 months after surgery . but from the 30 cases , the positive surgical margin rate decreased to 9.5% ( table 4 ) . two patients ( 4.0% ) with anastomosis site leakage were treated with prolonged catheterization for an additional 1 week . two patients ( 4.0% ) with acute urinary retention after catheter removal were treated with re - catheterization for 1 week . it has been shown that the incidence of postoperative incontinence depends on the urologist 's experience , patient 's age ( increased frequency after 70 years ) , and whether the operative technique includes minimal distal incision of the endopelvic fascia , preservation of the bladder neck , bilateral nerve - sparing surgery , or preservation of the ppl . in the american urological association guidelines , the reported risk of urinary incontinence ranges from 3 to 74% for radical prostatectomy . continence mechanisms involve many structures , including the ppl , denonvilliers ' fascia , levator muscle , endopelvic fascia , and internal and external sphincters . ventrally , the proximal prostate is covered by muscle fibers originating from the outer longitudinal bladder muscle and extending over the gland . the pubovesical / ppls ( pv / ppls ) are paired fibrous bands originating from visceral endopelvic fascia . they insert on the distal third of the posterior surface of the pubic bone adjacent and anterior to the urethral sphincter . the visceral component of the endopelvic fascia covers the pelvic organs including the prostate , bladder , and rectum , and it is fused with the anterior fibromuscular stroma of the prostate at the upper ventral aspect of the gland [ 12 - 14 ] . along the pelvic sidewall at the lateral aspect of the prostate and bladder , the parietal and the visceral components of the endopelvic fascia are fused . as a fascial condensation , this fusion is often recognizable as a whitish line and is named the fascial tendinous arch of the pelvis . access to the lateral prostate may be gained by incision of the endopelvic fascia either medial or lateral to this fusion . the pv / ppls stabilize the prostate , urethra , and bladder to the pubic bone and are considered an important part of the " suspensory system " of the continence mechanism [ 15 - 19 ] . some authors have suggested that preservation of these ligaments during radical prostatectomy may improve early recovery of urinary continence , but no definitive evidence has yet been established . preservation of the pv / ppls is facilitated by using the perineal and laparoscopic approach , whereas during open retropubic prostatectomy , the pv / ppls are more difficult to preserve . some authors have suggested that avoiding incision of the endopelvic fascia during radical prostatectomy , often combined with an intrafascial nerve - sparing procedure , might improve early recovery of urinary continence as well as improve postoperative erectile function , but definitive evidence has yet to be established . the incision of this fascia immediately lateral to the fascial tendinous arch incises the levator ani fascia ( laf ) and leaves the muscle fibers of the levator ani bare and the laf adherent to the prostate . an incision of the visceral endopelvic fascia medial to the fascial tendinous arch results in a dissection plane that leaves the levator ani muscle covered with its fascia without exposure of its fibers . the result is a prostate covered only by prostatic fascia ( pf ) , when present , and not by a layer of laf . this fascia is not a discrete single - layered structure stretching over the lateral surface of the prostate . laparoscopic surgery may offer an improved identification of these structures , resulting in less damage to the structures around the prostate . we could also identify these structures during the operation , which is related to recovery of postoperative urinary continence . in addition , others have showed that continence rates correlate with differences in the mean functional urethral length and the existing differences in the maximal urethral closure pressure postprostatectomy . reported an earlier return of continence with a ppl - sparing technique versus a nonsparing technique , but the final outcomes were equivalent . we believe that during mobilization of the prostate and especially during apical dissection , the intactness of the urethral supporting structures are of paramount importance because this avoids shear stress to the urethra as well as possible denervation . in our study , we observed an earlier return to continence in patients who underwent intrafascial nselrp compared with previous conventional nerve - sparing lrp . complete continence was achieved by 70.0% of patients who underwent nselrp at 6 weeks after surgery and by 83.9% at 3 months after surgery . in the control group , however , 55.2% of patients achieved complete continence at 6 weeks after surgery and 75.0% at 3 months after surgery . reconstruction methods such as periurethral suspension stitch , bladder neck reconstruction , and posterior reconstruction have also been shown to provide improved early continence recovery , but the results are still controversial . therefore , we focused on the preservation of normal structures rather than performing reconstruction after destruction of these structures . studies have reported that older age may be the only increasing risk factor for postprostatectomy incontinence , but in our study , increased prostate volume and gleason score were related with post - prostatectomy incontinence in the older aged group ( age over 65 years ) . we speculate that large prostate volume and old age may be related to preoperative bladder and external urethral sphincter dysfunction leading to interference with postprostatectomy recovery of continence . a preoperative urodynamic study may be helpful in proving the relationship between prostate volume and old age with postoperative incontinence . anatomical studies have illustrated the prostatic neuroanatomy in detail , detecting additional neural tissues to the nerve bundles on the anterior midpart and posterior surface of the prostate . costello et al . recently showed that most of the nvb descends posteriorly to the seminal vesicle . the nerves pass anteriorly and converge at the midprostatic level , and when they approach the apex , they diverge again . the anterior and posterior nerves of the nvb are separated by 3 cm at the level of the base of the prostate . at this anatomic site , the cavernosal nerves are not easily distinguished from the surrounding tissues and care should be taken during urethra - vesical anastomosis . walsh proposed that the nvb is enclosed within the two layers of the lateral pelvic fascia composed of the lateral layer of the levator fascia and the medial layer of the pf . kiyoshima et al . proved that the nvb was located on the posterolateral region of the prostate in 48% of their patients . in the remaining patients ( 52% ) , the nvb was widely distributed on the entire lateral aspect of the prostate without any specific localization . thus , the authors proposed performing wide dissection of the lateral aspect of the prostate during radical prostatectomy to preserve the nvb . therefore , meticulous dissection and disuse of electro - cauterization at the lateral and apex sides of the prostate are required to preserve the nvb by intrafascial nselrp . anastasiadis et al . reported potency rates of 30% and 41% at 12 months after lrp ( n=230 ) and open retropubic prostatectomy ( n=70 ) , respectively . after preservation of one or both nvbs , the potency rates increased from 37 to 44% with the retropubic approach and from 46 to 53% with lrp , respectively . patients younger than 60 years who underwent bilateral nvb preservation were reported to be potent in 72% and 81% of cases , respectively . reported rates of erections of 96.5% , 90.7% , and 84.3% and rates of intercourse of 69.0% , 52.8% , and 37.3% at 12 months after bilateral rrp in men < 55 years , 55 to 65 years , and > 65 years , respectively . in our study , the patients were generally old aged and were not sexually active ; thus , we could not obtain potency recovery data . however , 19 patients ( 38.0% ) reported subjective signs of potency recovery ( morning erection or erection sensation ) at 6 weeks after surgery , and at 3 months after surgery , the percentage was increased to 54.8% of patients . the oncologic data of 1,000 lrps at the montsouris institute revealed positive surgical margin rates of 6.9% for pt2a and 34% for pt3b tumors . in our study , the overall positive surgical margin rate was 34.0% . we consider this high positive margin rate as a " learning curve . " in fact , from 30 cases , positive surgical margin rates decreased compared with the first 30 cases ( 51.7% vs. 9.5% ) . the initial results are promising , providing favorable functional outcomes and oncologic results similar to other rp techniques .
purposewe present our initial experience and surgical outcomes for the most recent refinement of bilateral intrafascial nerve - sparing extraperitoneal laparoscopic radical prostatectomy ( nselrp).materials and methodsamong 62 patients who underwent laparoscopic radical prostatectomy , 50 patients underwent intrafascial nselrp by a single surgeon at pusan national university hospital from november 2011 to april 2012 . as part of the intrafascial technique , the dissection plane is directly on the prostatic capsule to preserve most of the periprostatic fascia containing small vessels and nerves , endopelvic fascia , neurovascular bundle , and puboprostatic ligament . postoperative continence recovery was established by daily consumption of pads . follow - up was done at 2 weeks , 6 weeks , and 3 months after surgery.resultsthe patients ' mean age was 66.56.2 years . the mean operation time and mean blood loss were 149.328.1 minutes and 155.4168.1 ml , respectively . the mean hospitalization time and mean catheterization time were 6.35.1 days and 5.54.7 days , respectively . two weeks after the operation , a total of 14 patients ( 28.0% ) were pad - free but the other incontinent patient group used on average 2.3 pads per day . after 6 weeks , 35 patients ( 70.0% ) achieved pad - free status and 7 patients ( 14.0% ) required more than 2 pads per day . at 3 months after surgery , a total of 31 patients were available for follow - up , and 26 patients ( 83.9% ) were pad-free.conclusionscompared with conventional laparoscopic prostatectomy , the intrafascial nselrp procedure enables the preservation of periprostatic structures that are essential to the recovery of surgical structures related to continence . as a result , early postoperative continence can be achieved .
INTRODUCTION MATERIALS AND METHODS 1. Patient selection criteria and evaluation 2. The surgical techniques Patient positioning and trocar placement Retroperitoneal preparation Initial approach and bladder neck dissection Preparation of the seminal vesicles Dissection of the prostatic pedicle and NBV Dissection of the urethra 3. Postoperative evaluation 4. Statistical analysis RESULTS DISCUSSION CONCLUSIONS
further understanding of the anatomy of adjacent structures of the prostate , such as the bladder neck , urethra , fascia , ligaments , and neurovascular bundle ( nbv ) , led to the development of nerve - sparing elrp ( nselrp ) and the most recent intrafascial nselrp , which shows a positive surgical outcome of early continence . in this study , we report our experience with 50 consecutive patients who underwent intrafascial nselrp by a single surgeon . among 62 patients , 50 patients with clinically localized prostate cancer at the time of preoperative magnetic resonance image ( mri ) with psa levels less than 10 ng / ml and gleason scores less than 7 ( 3 + 4 ) were included for intrafascial nselrp from november 2011 to april 2012 . all lateral periprostatic fascia , endopelvic fascia , and puboprostatic fascia remained intact ( fig . postoperative follow - up was defined at 2 weeks , 6 weeks , and 3 months . among 62 patients , 50 patients with clinically localized prostate cancer at the time of preoperative magnetic resonance image ( mri ) with psa levels less than 10 ng / ml and gleason scores less than 7 ( 3 + 4 ) were included for intrafascial nselrp from november 2011 to april 2012 . all lateral periprostatic fascia , endopelvic fascia , and puboprostatic fascia remained intact ( fig . the prostatic pedicles were then clipped and cut in a step - by - step manner directly on the surface of the prostatic capsule without excessive traction or electrocautery . a bilateral incision of the periprostatic fascia was made medial to the puboprostatic ligament ( ppl ) and directed to the base of the prostate . all lateral periprostatic fascia , endopelvic fascia , and puboprostatic fascia remained intact ( fig . the prostatic pedicles were then clipped and cut in a step - by - step manner directly on the surface of the prostatic capsule without excessive traction or electrocautery . postoperative follow - up was defined at 2 weeks , 6 weeks , and 3 months . the mean hospitalization time was 6.3 days ( range , 4 to 19 days ) , and the mean catheterization time was 5.5 days ( range , 3 to 26 days ) . at the 2-week follow - up after surgery , 14 patients ( 28.0% ) achieved total continence and on average 2.3 pads were required for the other incontinent patients . a total of 35 patients ( 70.0% ) were continent at 6 weeks after surgery , 8 patients ( 16.0% ) had mild stress incontinence ( 1 to 2 pads ) , and 7 patients ( 14.0% ) required > 2 pads per day ( fig . a total of 31 patients were available for follow - up at 3 months after surgery , and 26 patients ( 83.9% ) were pad - free . signs of early recovery of potency ( morning erection or erection sensation ) were reported by 19 patients ( 38.0% ) at 6 weeks after surgery and the rate increased to 54.8% at 3 months after surgery . it has been shown that the incidence of postoperative incontinence depends on the urologist 's experience , patient 's age ( increased frequency after 70 years ) , and whether the operative technique includes minimal distal incision of the endopelvic fascia , preservation of the bladder neck , bilateral nerve - sparing surgery , or preservation of the ppl . preservation of the pv / ppls is facilitated by using the perineal and laparoscopic approach , whereas during open retropubic prostatectomy , the pv / ppls are more difficult to preserve . some authors have suggested that avoiding incision of the endopelvic fascia during radical prostatectomy , often combined with an intrafascial nerve - sparing procedure , might improve early recovery of urinary continence as well as improve postoperative erectile function , but definitive evidence has yet to be established . in our study , we observed an earlier return to continence in patients who underwent intrafascial nselrp compared with previous conventional nerve - sparing lrp . complete continence was achieved by 70.0% of patients who underwent nselrp at 6 weeks after surgery and by 83.9% at 3 months after surgery . in the control group , however , 55.2% of patients achieved complete continence at 6 weeks after surgery and 75.0% at 3 months after surgery . however , 19 patients ( 38.0% ) reported subjective signs of potency recovery ( morning erection or erection sensation ) at 6 weeks after surgery , and at 3 months after surgery , the percentage was increased to 54.8% of patients .
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hepatic injury of various etiologies , such as chronic viral infections ( mainly hcv and hbv ) , excessive alcohol consumption , metabolic disorders , or autoimmune insults , leads to the development of liver fibrosis . fibrosis is a prolonged and exorbitant wound healing response causing the accumulation of redundant extracellular matrix ( ecm ) . ecm consists of a dense mesh of macromolecules , polysaccharides , and proteins , particularly -smooth muscle actin and different types of collagen , forming insoluble fibers and microfibrils . its main function is to support the structure and functioning of the tissue during healing processes . in the physiological state , balance between ecm deposition and degradation is controlled by numerous matrix metalloproteinases ( mmps ) , which are digesting / degrading the particular components of ecm . in the course of fibrosis , however , mmps are markedly inhibited by tissue inhibitors of mmps ( timps ) that are upregulated in response to the chronic liver insult . continuous scarring may eventually lead to the development of liver cirrhosis , end - stage liver disease , or hepatocellular carcinoma [ 1 , 2 ] ( figure 1 ) . these fibrogenic stimuli include reactive oxygen species ( ros ) , hypoxia , inflammatory and immune responses , hepatocytes apoptosis , and steatosis . response to these signals owing to persistent liver injury instigates the recruitment and transformation of the resident quiescent liver fibroblast ( hepatic stellate cells , hscs ) to the highly activated , proliferative , motile , and contractile myofibroblast phenotype ( figure 1 ) . . the activation process is initiated by the release of many growth factors such as platelet - derived growth factor ( pdgf ) and transforming growth factor ( tgf- ) , profibrogenic cytokines and chemokines by the injured hepatocytes , and inflammatory cells particularly macrophages and other nonparenchymal cells . deposition of the dense and complex net of scar tissue in the space of disse , where hscs reside , causes significant changes in the sinusoid architecture . fenestrations in the structure of liver sinusoidal endothelial cells ( lsecs ) are gone and hepatocytes lose their microvilli . although hscs remain the primary source of myofibroblasts , it has now become clear that other cell types can also contribute to myofibroblasts population including portal fibroblasts , bone - marrow derived cells , and possibly epithelial - mesenchymal transition ( emt ) and contribute to the liver scarring . first symptoms of the liver impairment in most of cases are indicating disease development into cirrhosis and this commonly occurs after 1520 years , when the prognoses of survival and recovery are dramatically reduced . a major difficulty in developing disease - specific therapy is the lack of accurate and established diagnostic techniques for long - term monitoring of disease progression and therapy responses and to optimize disease treatment strategies [ 3 , 4 ] . liver biopsy has been considered as the gold standard for the diagnosis and staging of liver fibrosis but is invasive and painful and has numerous limitations including risk of bleeding , sampling errors due to disease heterogeneity , and inter- and intraobserver variability [ 46 ] . moreover , liver biopsies only sample 1/50,000 of the liver , and undersized or fragmented samples may therefore underestimate hepatic fibrosis [ 4 , 5 , 7 ] . recently , guidelines by easl - aleh have been published summarizing and validating clinical use of noninvasive tests for evaluation of liver disease severity and prognosis . the tremendous advancement in the biomedical research over the last decade led to the development of novel , rapid blood tests for diagnosis of liver fibrosis . several commercial biochemical and serum tests classified into class i and class ii biomarkers are developed . class i biomarkers are associated with the mechanism of fibrogenesis , either as secreted matrix - related components or as a result of ecm synthesis or turnover , for example , hyaluronan . class ii biomarkers are indirect methods which are grouped into panels such as ( a ) european liver fibrosis test ( elf ) ( n - terminal propeptide of collagen type iii , hyaluronic acid , timp1 , and age ) , ( b ) fibrotest ( alpha-2-macroglobulin , haptoglobin , apolipoprotein a1 , gamma - glutamyl transpeptidase [ ggt ] , total bilirubin , and alanine transaminase ) , ( c ) fibrosis-4 index ( fib-4 ) combining standard biochemical tests ( platelets , alt , and ast ) and age , ( d ) hepascore ( age , sex , total bilirubin , gamma - glutamyl transferase , 2-macroglobulin , and hyaluronic acid ) , ( e ) aspartate and transaminase to platelet ratio ( apri ) , and ( f ) forns score ( platelet count , prothrombin index , ast , alpha-2-macroglobulin , ha , and blood urea ) which have been developed recently [ 913 ] . however , these tests rely on indirect markers and lack specificity as these markers can be influenced by unrelated diseases . nevertheless , recent studies indicate that the results from the serum panels might predict risk of decompensation and overall survival more accurately than biopsy [ 9 , 10 , 12 ] . number of emerging technologies have recently been developed for diagnosing and staging liver fibrosis over the past years such as ultrasonography ( us ) , computerized tomography ( ct ) , and magnetic resonance imaging ( mri ) . however , these imaging modalities are dependent primarily on structural and morphological alterations in the liver and these alterations are usually identified in advanced stage of fibrosis . currently , transient elastography ( te ) ( fibroscan , echosens , paris , france ) is the most widely used method for noninvasive and rapid measurement of liver stiffness . te uses a probe consisting of an ultrasonic transducer and a vibrator that emits low - frequency shear waves ( 50 hz ) propagating through the liver tissue . the speed of the shear waves is directly related to liver stiffness and can be expressed in kilopascal ( kpa ) . several studies have evaluated te for diagnosis of hepatic fibrosis and cirrhosis with relatively high specificity and sensitivity [ 1518 ] . point shear wave elastography ( pswe ) or acoustic radiation force impulse ( arfi ) involves mechanical excitation of tissue using short - duration acoustic pulses that produce shear waves , expressed in m / sec , which directly correlates with the extent of liver fibrosis [ 1925 ] . another promising technique , 2-dimensional shear wave elastography ( 2d - swe ) , is based on the combination of a radiation force induced in tissues by focused ultrasonic beams and a very high frame rate ultrasound imaging sequence capable of catching in real time the transient propagation of resulting shear waves . 2d - swe expressed either in m / sec or in kpa has an advantage of being implemented on a commercially available ultrasound machine . new magnetic resonance imaging ( mri ) based imaging techniques have recently gained substantial interest : magnetic resonance elastography ( mre ) , dynamic contrast - enhanced mr imaging ( dce - mri ) , perfusion weighted imaging ( pwi ) , and diffusion weighted imaging ( dwi ) [ 2729 ] . magnetic resonance elastography ( mre ) is similar to ultrasound based elastography techniques and can determine liver stiffness by analysis of mechanical waves propagating through the liver [ 3034 ] . diffusion weighted imaging ( dwi ) is a magnetic resonance technique that quantifies the diffusion of water molecules in tissues that can be quantified as apparent diffusion coefficient ( adc ) . collagen fibers in the liver would inhibit water diffusion thereby leading to a decrease in adc and therefore can be quantitatively used to assess liver fibrosis , but the technique has limitations since factors like steatosis can also affect adc . dynamic contrast - enhanced mr imaging ( dce - mri ) and mr perfusion weighted imaging ( mr - pwi ) rely on the intravenous administration of mr contrast agents that can more precisely reveal hepatic hemodynamic changes [ 3638 ] . however , these mri - based techniques are time - consuming and cost - ineffective . another novel and developing mr based imaging modality , molecular mr imaging , represents a unique implementation of mr modality to visualize , characterize , and measure biological processes at the cellular and molecular level with high spatial resolution . the specific contrast agents ( or probes ) can be endogenous and exogenous probes can be generated by encapsulating paramagnetic ( gadolinium ) or superparamagnetic ( iron - oxide ) metals in different nanoparticles . molecular mr imaging is based on the development of mr imaging probes composed of contrast generating materials , for example , gadolinium or iron - oxide , and molecular targets , for example , ecm binding probes such as collagen i ( ep-3533 ) , fibrin - fibronectin ( clt1-peptide ) , elastin ( emsa ) , and v3-integrin ( c(rgdyc)-uspio ) [ 39 , 40 ] . overall , no single method can provide the detailed information as histological examination but using noninvasive modalities can differentiate between mild and significant fibrosis and can potentially avoid unnecessary liver biopsy in a subgroup of patients . while these methods have provided some impressive results , there remains a paucity to validate their use in disease management or assessment of potential antifibrotic therapies . although molecular mri of liver fibrosis is currently developing , the conception of target specific molecular mri approach can open up new horizons and avenues for the diagnosis and effective management of this life - threatening disease . the term targeted therapy ( tt ) describes the set of treatment strategies aiming to inhibit or alter specific molecules or molecular pathways leading to certain disorders and diseases . some of the molecularly targeted agents exert the cytotoxic or cytostatic effects on the specific target cell types , while others inhibit the activity of the particular enzymes or proteins or boost the immune system activity against pathogenic mechanism . one of the main advantages of such approach is its specificity the principle of design is to affect only the pathologically transformed cells and processes , thus minimizing the adverse effects [ 41 , 42 ] . most important part of the targeted therapy development is to determine the appropriate molecular target proteins and enzymes , hormones , peptides , genes , and specific reactions involved in the pathological processes that , upon alteration , can lead to the disease resolution / reversion . three main types of the targeted therapy design can be distinguished : small molecule drugs : relatively small moieties which are able to target molecules and processes inside the cell [ 4346]monoclonal antibodies : large proteins produced by the immune cells that are able to highly specifically identify and bind with the targets on the cell surface or outside the cells [ 4749],targeted conjugates : delivery systems consisting of the therapeutic moiety , such as delivery vehicle or protein carrying therapeutic agent conjugated with the targeting ligands [ 5052]the antifibrotic therapeutic approaches are broadly classified among several categories : elimination of the primary cause of injury , for example , alcohol abstinence in alcoholic liver diseasesreduction of inflammation and immune response or inhibition of hepatocyte apoptosis / injury to avoid hsc activationresolution of fibrosis by inhibiting scar tissue formation , increasing matrix degradation , inhibiting hsc activation , or stimulating hsc apoptosisinhibition of signaling pathways ( extracellular and intracellular ) responsible for activation , contraction , and proliferation of hscs small molecule drugs : relatively small moieties which are able to target molecules and processes inside the cell [ 4346 ] monoclonal antibodies : large proteins produced by the immune cells that are able to highly specifically identify and bind with the targets on the cell surface or outside the cells [ 4749 ] , targeted conjugates : delivery systems consisting of the therapeutic moiety , such as delivery vehicle or protein carrying therapeutic agent conjugated with the targeting ligands [ 5052 ] elimination of the primary cause of injury , for example , alcohol abstinence in alcoholic liver diseases reduction of inflammation and immune response or inhibition of hepatocyte apoptosis / injury to avoid hsc activation resolution of fibrosis by inhibiting scar tissue formation , increasing matrix degradation , inhibiting hsc activation , or stimulating hsc apoptosis inhibition of signaling pathways ( extracellular and intracellular ) responsible for activation , contraction , and proliferation of hscs there is an intensified focus on the development of antifibrotic therapies for chronic liver diseases in the past years . advanced pathological and molecular understanding of the fibrosis pathogenesis has instigated identification of novel therapeutic and promising drugs in preclinical models . furthermore public health impact of liver diseases and novel diagnostic technologies for the assessment of fibrosis has resulted in increased clinical trials in this field . in this review , clinical studies concerning targeted therapies against liver fibrosis of diverse etiology are reviewed and summarized in table 1 . in general , the biggest emphasis is on the small molecule drugs ; so far these therapeutics were the most frequently investigated and the progress in this field is currently the most advanced . reviewed studies mostly are randomized trials on the parallel two or more groups of patients ( parallel assignment design ) ; less frequently there are also single group assignments . most of the studies are randomized and double - blinded to ensure the minimal risk of the results manipulation or bias . clinical trials are mostly performed on patients with nash ( nonalcoholic steatohepatitis ) , liver fibrosis , or cirrhosis with chronic hepatitis c infection and nafld ( nonalcoholic fatty liver diseases ) since these diseases are the most frequently occurring reasons for the development of liver fibrosis . clinical trials in chronic liver diseases present unique challenges , because clinical events that could be used as trial primary endpoints ( e.g. , histological assessment of fibrosis ) can vary depending on the etiology of the liver disease ; therefore the study outcomes largely rely upon noninvasive surrogates . current clinical trials are primarily based on pathological characterization of liver biopsy to assess fibrosis progression but now serum tests such as hepascore , elf , fibrotest and noninvasive imaging modalities like te or mr are characterized as surrogate endpoints . in the following list , liver histology necroinflammation : nafld activity score and knodell scorefibrosis : histopathological and immunohistochemical analysis necroinflammation : nafld activity score and knodell score fibrosis : histopathological and immunohistochemical analysis serum tests serum markers : alt , ast , alp , ggt , and albuminserum marker panels : elf test , apri , and fib-4lipidomic analysis serum markers : alt , ast , alp , ggt , and albumin serum marker panels : elf test , apri , and fib-4 liver function tests insulin sensitivityglucose toleranceindocyanine green clearance testsgalactose elimination tests indocyanine green clearance tests galactose elimination tests noninvasive tests liver stiffness measurement : transient elastography ( fibroscan ) ; shear wave elastography ; magnetic resonance elastography ; acoustic radiation force impulse ( afri)liver fat measurement : mri and spectroscopy ( mrs ) liver stiffness measurement : transient elastography ( fibroscan ) ; shear wave elastography ; magnetic resonance elastography ; acoustic radiation force impulse ( afri ) liver fat measurement : mri and spectroscopy ( mrs ) clinical scores meld scorechild - pugh scoreishak scoremetavir score . small molecule drugs are the group of the targeted therapeutic agents typically with molecular weight below 1000 da . they can be delivered intravenously or orally and , due to their small size , enter the target cells ( cross the cell membrane ) ; typically they are also able to penetrate the blood - brain barrier . the complex process of discovery and development of small molecule drugs mostly consists of two combined strategies : ( i ) knowledge - based design employing the knowledge about the structure of the target and its inhibitors / ligands and/or ( ii ) random high throughput screening of libraries of small molecules to search for the molecules with potential activity towards / against the target . following extensive screening , eventually , the prospective compounds are further investigated in vitro and in vivo for the therapeutic efficacy and , if applicable , enter further the clinical development phase [ 54 , 55 ] . some of the major clinically challenged targets of the small molecule drugs are mentioned below . activated hscs express a diverse group of nuclear receptors acting as transcription factors , for example , peroxisome proliferator - activated receptor ( ppar ) and farnesoid x receptor ( fxr ) , that play an important role in hsc regulation . ppar is highly expressed in the quiescent hscs and upon activation its expression diminishes . following treatment with ppar ligands / agonists , ppar expression is restored , and hsc activation and collagen expression are reduced in vitro . clinical trials using pioglitazone showed significant improvement in steatosis , inflammation , and insulin resistance in nash patients [ 59 , 60 ] ( table 1 ) , while clinical trials using ppar agonists farglitazar ( gi262570 ) [ 61 , 62 ] showed no effective treatment in patients with chronic hcv infection ( table 1 ) . fxr , another nuclear receptor , is highly expressed in the liver and small intestine . it is responsible for maintaining homeostasis of bile acids and cholesterol and regulates transcription of multiple genes involved in bile acids synthesis and transport . fxr is also expressed in hscs and activation of fxr in hscs is associated with significant decrease in collagen production . activation of fxr occurs via binding with bile acids such as deoxycholic or lithocholic acid , although many synthetic ligands are also known . however , most fxr ligands failed the preclinical and clinical assessment because of poor pharmacokinetics or toxicity issues . nevertheless , synthetic fxr agonists px-102 and px-104 , developed by phenex pharmaceuticals , showed promising safety and tolerability profile in healthy subjects ( px-102 , clinical trial nct01998659 ; nct01998672 ) and px104 is currently tested in a phase 2a study in patients with nafld ( nct01999101 ) . int-747 ( 6-ethyl chenodeoxycholic acid or 6-ecdca or obeticholic acid ) , semisynthetic fxr agonist , showed improvement of the histological and biochemical markers , ameliorated fibrosis , inflammation , and steatosis in nash patients . obeticholic acid is currently in clinical trials for long - term treatment of cholestatic liver diseases ( table 1 ) . ras is an important hormonal regulatory mechanism of the blood pressure and body fluid homeostasis . the key ras protein , angiotensin ii ( ang ii ) , is produced in the liver from its precursor angiotensin i by the proteolytic cleavage by angiotensin i converting enzyme ( ace ) . ang ii exerts its diverse biological effects by binding with one of its multiple receptors , particularly ang ii type 1 receptor ( at1-r ) , overexpressed in activated hscs . ang ii induces hsc activation , proliferation , and contraction , as well as increased tgf , timp1 expression , and collagen deposition . therefore , ang ii and its interaction with at1-r are considered to play an important role in liver fibrogenesis and its blocking by ace inhibitors ( acei ) or at1-r blockers ( arbs ) may be an effective therapeutic option for treatment of liver fibrosis and they are already in clinical trials , for example , losartan , irbesartan , and candesartan and moexipril ( table 1 ) . losartan , irbesartan , and candesartan share similarities in the chemical structure and they all are at1-r blockers , in contrast to moexipril , which is an ace inhibitor . clinical trials evaluating long - term losartan effects in chronic hepatitis c patients showed decreased inflammation , reduced expression of fibrogenic mediators , and decreased ecm ( collagen i ) accumulation [ 71 , 74 ] . furthermore , treatment markedly decreased ang ii induced oxidative stress in hepatic fibrosis [ 71 , 74 ] . prolonged exposure to the at1-r blocking treatment in patients with chronic hcv infection was proven to be safe and well tolerated . ras has been also shown to be associated with hypertension ; therefore , candesartan ( at1-r inhibitor ) , widely used for the therapy of hypertension and heart failure , has shown promising results in the clinical trials for alcoholic liver fibrosis in combination with ursodeoxycholic acid ( udca ) . it was demonstrated that candesartan significantly improved the treatment outcomes in comparison to udca and reduced the fibrosis scores and -sma positive fibrotic area in biopsies . relative expression of fibrogenic markers was downregulated and the arterial blood pressure was shown to be significantly reduced . however , long - term treatment with irbesartan ( arb and antihypertensive drug ) in severe fibrosis with chronic hepatitis c showed no substantial improvement in fibrosis scores , arterial pressure , and organ stiffness in the treated group , despite the fact that treatment was safe and well tolerated . in addition , ace inhibitor moexipril treatment did not show beneficial effects in primary biliary cirrhosis patients . furthermore , in halt - c cohort study , acei / arb therapy did not retard the progression of fibrosis . due to ambiguous results , further controlled studies are required to evaluate the long - term efficacy of arbs / acei . endocannabinoid system plays an important role in various liver diseases including viral hepatitis , nafld , and alcoholic liver disease . cannabinoid receptors cb1 and cb2 are upregulated in chronic liver diseases and several studies have convincingly demonstrated antagonism between cb1 and cb2 ; that is , cb1 promotes while cb2 suppresses liver damage [ 77 , 78 ] ; therefore cb1 antagonists and cb2 agonists were investigated as potential therapeutic approaches for liver diseases . clinically , daily cannabis ( cb1 and cb2 agonist ) promoted fibrosis progression in chronic hepatitis c . rimonabant cb1 antagonist was successfully tested in clinical trials in obese patients and showed reduction in body weight , improved metabolic function , and improved insulin resistance . however , depression and psychoactive side effects led to the termination of clinical rimonabant drug use . currently , efforts are directed towards development of novel cb1 antagonist with improved specificity that lacks neuropsychiatric adverse effects . other neurotransmitters , for example , opioids and serotonin ( 5ht ) , and their receptors are other potential therapeutic targets in liver fibrosis . opioid antagonist naltrexone and 5ht antagonist methiothepin have shown antifibrotic activity in animal models of liver disease [ 81 , 82 ] , but clinical trials are needed to demonstrate their long - term tolerability and efficacy . since inflammation promotes progression of liver fibrosis , use of anti - inflammatory drugs poses a potential and rationale therapeutic approach . corticosteroids ( e.g. , prednisone , prednisolone , methyl prednisone , and triamcinolone ) are used for the treatment of liver diseases , most commonly autoimmune hepatitis with improved outcome and survival . however , the adverse effects of long - term corticosteroid therapy are still the major causes of morbidity and mortality . another anti - inflammatory approach is to inhibit release of inflammatory cytokines or to neutralize it with receptor antagonists . upregulated tnf production is one of the initiating events in the liver injury leading to release of proinflammatory cytokines resulting in fibrosis . pentoxifylline ( ptx ) is a potent phosphodiesterase inhibitor , which suppresses tumor necrosis factor ( tnf ) production . ptx was also shown to be hepatoprotective since it reduces oxidative stress , which is important contributor in the hepatic pathologies and fibrogenesis . ptx has been registered for numerous clinical trials concerning its potential therapeutic efficacy in diverse fibrotic disorders [ 75 , 8588 ] . long - term treatment with ptx in nash patients demonstrated significant improvement of both histological features and significant improvement in the liver fibrosis in comparison to placebo - treated group . despite the fact that ptx activity is being associated with tnf inhibition , the study failed to demonstrate the tnf downregulation . finally , it was also concluded from the study that ptx treatment was safe and well tolerated by patients and there were no severe adverse side effects . following hepatocyte injury , hepatic macrophages secrete inflammatory chemokines or cytokines , for example , c - c chemokine ligand type 2 [ ccl2 or mcp1 ( monocyte chemoattractant protein-1 ) ] , driving the recruitment and migration of pro - ccr2 and ccr5 positive inflammatory monocytes to the liver . ccr2 and/or ccr5 antagonism has been suggested as a potential approach for the treatment of inflammatory diseases and fibrosis [ 91 , 92 ] . cenicriviroc ( cvc ) , a ccr2/ccr5 antagonist , is currently being evaluated for the treatment of nash and liver fibrosis ( centaur , nct02217475 , table 1 ) . cenicriviroc showed favorable safety profile in hiv - infected patients in a phase 2b study and in patients with hepatic impairment . another target molecule is galectin-3 ( gal-3 ) , pleiotropic -galactoside - binding lectin , that was shown to play an important role in the liver fibrosis . gal-3 possesses strong proinflammatory properties and is able to activate macrophages and stimulate their migration . gr - md-02 ( galactoarabino - rhamnogalacturonate ) is a potent inhibitor of galectin-3 that showed remarkable therapeutic effects in thioacetamide - induced liver fibrosis in rats and was submitted for 3 clinical studies concerning liver fibrosis . phase 1 study evaluating safety of gr - md-02 in patients with nonalcoholic steatohepatitis ( nash ) and advanced fibrosis is already completed [ 65 , 98 , 99 ] . results showed that the drug was safe and well tolerated in nash patients with liver fibrosis and demonstrated improvement in fibrosis and inflammation [ 100102 ] . two upcoming clinical trials will evaluate gr - md-02 efficacy for the treatment of liver fibrosis in patients with advanced fibrosis and cirrhosis originating in nash . oxidative stress or reactive oxygen species ( ros ) generation also plays an important role in initiation of fibrogenesis by activation of hscs ; therefore inhibition of oxidative stress or ros inhibits inflammation resulting in amelioration of liver fibrogenesis . hence , a number of antioxidants , for example , s - adenosyl - l - methionine ( same ) , silymarin , phosphatidylcholine , n - acetylcysteine ( nac ) , and vitamin e , are and have been tested in clinical trials ( refer to table 1 ) with beneficial effects . during liver fibrosis , a number of receptor tyrosine kinases , that is , pdgfr ( platelet - derived growth factor receptor ) , vegfr ( vascular endothelial growth factor receptor ) , fgfr ( fibroblast growth factor receptor ) , and egfr ( epidermal growth factor receptor ) , were significantly upregulated on activated hscs . many fibrotic and proliferative cytokines , for example , pdgf , tgf , fgf , and vegf , signal via these receptors tyrosine kinases resulting in the activation of intracellular signaling pathways resulting in differentiation and proliferation of quiescent hscs [ 2 , 103105 ] . antagonism of these pathways via tyrosine kinase inhibitors attenuates liver fibrosis in preclinical experiments on animal models . sorafenib , multitargeted tyrosine kinase inhibitor , was shown to attenuate liver cirrhosis , portal pressure , and angiogenesis . in a pilot clinical trial , recently , multicentered randomized clinical trial was carried out to study the effect of sorafenib on portal pressure in patients with cirrhosis ( nct01714609 , table 1 ) . erlotinib , egfr kinase inhibitor , attenuated liver fibrosis and hcc development in experimental animal models by suppression of egfr phosphorylation and inhibition of hsc activation . currently , clinical trial is ongoing to evaluate the effects of erlotinib in inhibition of fibrogenesis and hcc prevention ( nct02273362 , table 1 ) . apoptosis signal - regulating kinase 1 , ask1 , a serine / threonine kinase , promotes oxidative stress responsive pathway and leads to the activation of downstream p38 mitogen - activated protein kinases ( mapk ) and c - jun n - terminal kinase ( jnk ) , which stimulates inflammatory cytokines production , matrix remodeling genes expression , and abnormal cell proliferation . ask1 and p38 have been positively correlated with the fibrosis stage in patients with nafld . selonsertib ( or gs-4997 ) is a highly selective and potent ( ask1 ) inhibitor that inhibits ask1 by competitive binding to the catalytic domain of ask1 . in vivo in murine model , treatment with gs-4997 reduced fibrosis and steatosis , thus ameliorating the liver disease , thereby suggesting ask1 inhibition as the promising therapeutic approach . pharmacokinetics of gs-4997 have been already evaluated in phase 1 clinical study in adult with normal or impaired liver function ( nct02509624 ) and are currently registered in other clinical trials ( table 1 ) . mammalian target of rapamycin ( mtor ) is a serine / threonine protein kinase that is able to regulate cell growth and proliferation by controlling the protein translation . mtor performs its action by formation of mtor complexes 1 and 2 ( mtorc1 and 2 ) that further transmit the signal to the downstream effector proteins , that is , ribosome kinase p70s6 and 4e - bp1 , which are directly responsible for mrna translation . during fibrosis , mtor is highly dysregulated and was shown to be involved in the tgf responsiveness of the fibroblasts . mtor inhibitors were first shown to possess immunosuppressive properties and to date they are used as the immunosuppressive drugs preventing posttransplant organ rejection as well in autoimmune diseases ( e.g. , rheumatoid arthritis ) . the foremost mtor inhibitor is rapamycin ( or sirolimus ) ; however due to its stability and solubility issues , new derivatives have been developed with improved safety and pharmacokinetics . everolimus , one of the above - mentioned analogues , has been investigated in patients after liver transplantation [ 116 , 117 ] . monoclonal antibodies ( mabs ) are very relatively recent and becoming an essential element of the present pharmacotherapy [ 41 , 47 ] . utilization of mabs causes less adverse side effects and , alone or in combination with other drugs , can give remarkable results . there are many clinically approved mabs therapies for different types of diseases used either as monotherapies or as combined treatments ( e.g. , cetuximab , herceptin with docetaxel or paclitaxel ) . monoclonal antibodies are rather new approach in the liver fibrosis treatment ; therefore the development of the field is relatively in early stage . connective tissue growth factor ( ctgf ) is a heparin - binding ecm - associated protein , highly upregulated during liver injury . it stimulates ecm deposition ( particularly collagen i and fibronectin ) and is involved in ecm remodeling , important features of liver fibrosis . monoclonal antibody against ctgf ( fg-3019 ) was developed by fibrogen for treatment of the fibrotic disorders . fg-3019 was investigated in idiopathic pulmonary fibrosis ( ipf ) patients , and , after 2 years of the treatment , fg-3019 was proven safe and well tolerated in ipf patients . fg-30149 is recently being tested in phase 2 trials in subjects with liver fibrosis as a result of a chronic hepatitis b infection . vascular adhesion protein-1 ( vap1 ) is an endothelial glycoprotein that promotes leukocytes trafficking from the blood to the site of inflammation . upon injury and inflammation , vap1 translocates from intracellular storage to the cell surface . soluble form of vap1 ( svap1 ) is also able to initiate oxidative stress and secrete , via nfb , potent proinflammatory mediators . it was shown that serum levels of svap1 are markedly elevated in patients with chronic inflammatory liver diseases . blockade of vap1 inhibits inflammatory responses by attenuating leukocyte recruitment and oxidative stress [ 120 , 121 ] . btt-1023 , a human monoclonal antibody against vap1 , will be assessed in the clinical study in patients with primary sclerosing cholangitis . this autoimmune liver disease is characterized by the progressive destruction of the hepatic bile ducts , which in turn leads to liver fibrosis and cholestasis . preclinical studies showed efficient binding of the antibody with vap1 in the inflamed sites in vivo , as assessed by pet scans . elevated levels of lysyl oxidase - like-2 ( loxl2 ) expression were found in patient samples from liver fibrosis and primary biliary cirrhosis ; moreover it was found that the upregulation of loxl2 is limited to the fibrotic areas . loxl2 is an copper - dependent matrix metalloenzyme that enables collagen cross - linking thus creating a dense mesh of scar tissue . loxl2 is therefore an interesting target for the hepatic fibrosis treatment and numerous approaches to inhibit loxl2 have been developed . primarily , as it is copper - dependent enzyme , its activity can be impaired with the copper - binding ligands , such as d - penicillamine and -aminopropionitrile ( bapn ) [ 147 , 148 ] . nevertheless , the most promising approach is the use of monoclonal anti - loxl2 antibodies . they provide a specific allosteric inhibition of enzyme , by the binding with the scavenger receptor cysteine - rich ( srcr ) domains , which are the catalytic center of the molecule [ 146 , 147 ] . there were at least two types of antibodies reported : ab0023 , murine monoclonal antibody against loxl2 , used in in vivo studies [ 146 , 147 ] and simtuzumab ( sim or gs-6624 and formerly ab0024 ) , monoclonal antibody against human loxl2 . simtuzumab is currently registered in 11 clinical trials ( https://www.clinicaltrials.gov/ ) , from which 6 are related to fibrotic liver diseases . the preliminary results of the pilot study in patients with liver fibrosis reported that sim was well tolerated at the applied doses ( 10 mg / kg ) with no serious adverse effects and , due to its mechanism of action , provides a very promising antifibrotic therapy for patients with hepatic fibrosis . additionally , another clinical trial has been launched recently evaluating simtuzumab in combination with gs-4997 ( or selonsertib ) in patients with nonalcoholic steatohepatitis ( nash ) and fibrosis ( table 1 ) . targeted conjugates are the most diverse and the newest from of the described approaches ; however the concept is already more than a hundred years old , as is the idea of magic bullet by paul ehrlich . the targeted conjugates are combining the features attributed to small molecule drugs and monoclonal antibodies . it can consist of the delivery vehicle , for example , protein carrier ( hsa ) , liposome , polymeric nanoparticles , micelles , or nanoformulation , containing the active component , such as small molecule drug , small interfering rna ( sirna ) , micro rna ( mirna ) , cytokine , an active peptide , or a therapeutic protein . targeting ligand is attached to guide the delivery carrier to the specific site in the body , based on the specific ligand - receptor interactions . this allows the preferential accumulation of the conjugate in specific target cells , tissues , or organs . additional advantage of the targeted conjugates is that they provide the opportunity for theranostic approach ( therapy and diagnostics ) . application of the detectable moieties in the design , such as magnetic nanoparticles as the delivery vehicles or the fluorescent ligands on the surface of the conjugate , is to detect the accumulation of the particles in the target site . moreover , magnetic nanoparticles can serve as the contrast agents in magnetic resonance imaging and nanobubbles can provide the contrast enhancement in ultrasonography imaging . there are many strategies and nanoformulations explored for the treatment of liver fibrosis in preclinical animal models . the strategies are developed to target different receptors on different liver cell types , that is , hepatocytes , hepatic stellate cells , kupffer cells ( liver macrophages ) , and liver sinusoidal endothelial cells . the only representative of targeted conjugate in targeted therapies against liver fibrosis and a very promising drug which is currently under investigation in phase 1b/2 clinical trial is vitamin a - coupled lipid nanoparticle ( liposome ) containing sirna against collagen - specific chaperone heat shock protein 47 ( hsp47 ) [ 3 , 157 , 158 ] . hscs expresses retinol binding protein ( rbp ) receptor that regulates retinol ( vitamin a ) storage in hscs and is an interesting target for hsc - specific drug delivery . hsc - targeted liposomes ( nd - l02-s0201 ) carry sirna against hsp47 , which facilitates collagen secretion by ensuring triple - helix procollagen formation , and are implicated in translational regulation of procollagen synthesis [ 159 , 160 ] . downregulation of collagen production can result in the amelioration of fibrosis and reversion of cirrhosis . recently , lawitz et al . presented the preliminary results from the clinical trials performed on healthy subjects as well as on the patients with advanced liver fibrosis . nd - l02-s0201 was well tolerated in both groups of subjects without dose limiting toxicity neither in a single administration nor in multiple doses . furthermore , in the liver fibrosis patients , 6 out of 8 patients showed at least 1-stage improvement in the liver fibrosis suggesting beneficial effects of targeted approach . as presented in this review , the development of the targeted therapies against fibrotic diseases is in relatively advanced stage . numerous drugs are being assessed in the phase 2 clinical trials while some of them also reached phases 3 and 4 . multiple studies are currently ongoing , and the already completed trials revealed high potential of emerging drugs in ameliorating hepatic fibrosis of various etiology . however , besides the already investigated mechanisms and drugs , there are still some target proteins and pathways that remain to be elucidated . numerous promising molecular targets are currently under preclinical investigation and will be evaluated in the clinical trials . nevertheless , taken all together , there is remarkable improvement in the development of targeted therapies against fibrotic diseases and , in noninvasive technologies , many drugs are already being tested but many exciting targets still remain to be explored and further investigated . it is giving hope for the patients that clinically approved efficacious treatment will emerge soon .
hepatic fibrosis , characterized by excessive accumulation of extracellular matrix ( ecm ) proteins leading to liver dysfunction , is a growing cause of mortality worldwide . hepatocellular damage owing to liver injury leads to the release of profibrotic factors from infiltrating inflammatory cells that results in the activation of hepatic stellate cells ( hscs ) . upon activation , hscs undergo characteristic morphological and functional changes and are transformed into proliferative and contractile ecm - producing myofibroblasts . over recent years , a number of therapeutic strategies have been developed to inhibit hepatocyte apoptosis , inflammatory responses , and hscs proliferation and activation . preclinical studies have yielded numerous targets for the development of antifibrotic therapies , some of which have entered clinical trials and showed improved therapeutic efficacy and desirable safety profiles . furthermore , advancements have been made in the development of noninvasive markers and techniques for the accurate disease assessment and therapy responses . here , we focus on the clinical developments attained in the field of targeted antifibrotics for the treatment of liver fibrosis , for example , small molecule drugs , antibodies , and targeted drug conjugate . we further briefly highlight different noninvasive diagnostic technologies and will provide an overview about different therapeutic targets , clinical trials , endpoints , and translational efforts that have been made to halt or reverse the progression of liver fibrosis .
1. Liver Fibrosis: Mechanism and Pathogenesis 2. Assessment of Liver Fibrosis 3. Approaches for Targeted Therapy 4. Current Clinical Studies Overview 5. Developments in Targeted Therapy Related to Liver Fibrosis 6. Conclusions
fibrosis is a prolonged and exorbitant wound healing response causing the accumulation of redundant extracellular matrix ( ecm ) . response to these signals owing to persistent liver injury instigates the recruitment and transformation of the resident quiescent liver fibroblast ( hepatic stellate cells , hscs ) to the highly activated , proliferative , motile , and contractile myofibroblast phenotype ( figure 1 ) . molecular mr imaging is based on the development of mr imaging probes composed of contrast generating materials , for example , gadolinium or iron - oxide , and molecular targets , for example , ecm binding probes such as collagen i ( ep-3533 ) , fibrin - fibronectin ( clt1-peptide ) , elastin ( emsa ) , and v3-integrin ( c(rgdyc)-uspio ) [ 39 , 40 ] . three main types of the targeted therapy design can be distinguished : small molecule drugs : relatively small moieties which are able to target molecules and processes inside the cell [ 4346]monoclonal antibodies : large proteins produced by the immune cells that are able to highly specifically identify and bind with the targets on the cell surface or outside the cells [ 4749],targeted conjugates : delivery systems consisting of the therapeutic moiety , such as delivery vehicle or protein carrying therapeutic agent conjugated with the targeting ligands [ 5052]the antifibrotic therapeutic approaches are broadly classified among several categories : elimination of the primary cause of injury , for example , alcohol abstinence in alcoholic liver diseasesreduction of inflammation and immune response or inhibition of hepatocyte apoptosis / injury to avoid hsc activationresolution of fibrosis by inhibiting scar tissue formation , increasing matrix degradation , inhibiting hsc activation , or stimulating hsc apoptosisinhibition of signaling pathways ( extracellular and intracellular ) responsible for activation , contraction , and proliferation of hscs small molecule drugs : relatively small moieties which are able to target molecules and processes inside the cell [ 4346 ] monoclonal antibodies : large proteins produced by the immune cells that are able to highly specifically identify and bind with the targets on the cell surface or outside the cells [ 4749 ] , targeted conjugates : delivery systems consisting of the therapeutic moiety , such as delivery vehicle or protein carrying therapeutic agent conjugated with the targeting ligands [ 5052 ] elimination of the primary cause of injury , for example , alcohol abstinence in alcoholic liver diseases reduction of inflammation and immune response or inhibition of hepatocyte apoptosis / injury to avoid hsc activation resolution of fibrosis by inhibiting scar tissue formation , increasing matrix degradation , inhibiting hsc activation , or stimulating hsc apoptosis inhibition of signaling pathways ( extracellular and intracellular ) responsible for activation , contraction , and proliferation of hscs there is an intensified focus on the development of antifibrotic therapies for chronic liver diseases in the past years . clinical trials are mostly performed on patients with nash ( nonalcoholic steatohepatitis ) , liver fibrosis , or cirrhosis with chronic hepatitis c infection and nafld ( nonalcoholic fatty liver diseases ) since these diseases are the most frequently occurring reasons for the development of liver fibrosis . therefore , ang ii and its interaction with at1-r are considered to play an important role in liver fibrogenesis and its blocking by ace inhibitors ( acei ) or at1-r blockers ( arbs ) may be an effective therapeutic option for treatment of liver fibrosis and they are already in clinical trials , for example , losartan , irbesartan , and candesartan and moexipril ( table 1 ) . two upcoming clinical trials will evaluate gr - md-02 efficacy for the treatment of liver fibrosis in patients with advanced fibrosis and cirrhosis originating in nash . hence , a number of antioxidants , for example , s - adenosyl - l - methionine ( same ) , silymarin , phosphatidylcholine , n - acetylcysteine ( nac ) , and vitamin e , are and have been tested in clinical trials ( refer to table 1 ) with beneficial effects . many fibrotic and proliferative cytokines , for example , pdgf , tgf , fgf , and vegf , signal via these receptors tyrosine kinases resulting in the activation of intracellular signaling pathways resulting in differentiation and proliferation of quiescent hscs [ 2 , 103105 ] . apoptosis signal - regulating kinase 1 , ask1 , a serine / threonine kinase , promotes oxidative stress responsive pathway and leads to the activation of downstream p38 mitogen - activated protein kinases ( mapk ) and c - jun n - terminal kinase ( jnk ) , which stimulates inflammatory cytokines production , matrix remodeling genes expression , and abnormal cell proliferation .
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targeted drug delivery guided by molecular imaging approaches is a burgeoning area of clinical research , particularly for the treatment of cancer . this approach involves an optimized delivery of a therapeutic molecule and an imaging probe to the disease site , thereby using selective diagnosis and effective pharmacotherapy in unison for management of several diseases . successful utilization of this strategy requires integrated knowledge and versatile approaches in multidisciplinary fields such as cell and molecular biology , chemistry , material science , and physics and has opened up vast prospects in pharmacokinetics , therapeutic target discovery , drug delivery research , and quantification of multiple biomarkers in diseases . the major goal of this approach is to use molecular imaging to maximize effective therapy in diseased tissues and to minimize systemic drug exposure in order to reduce toxicities . in the past decade , innumerable studies have been reported on the synergistic use of molecular imaging with targeted drug delivery , and this strategy has now matured with promises to fulfill the vision of personalized medical treatment in the near future . in order to minimize the effects of toxicity and improve therapeutic effects , it is essential to deliver the therapeutic drugs to the right site , in the right time , and in the right concentration . ideally , the drug should act as a magic bullet that possesses perfect specificity to targeted lesions and has no side effect on the rest of the body . controllable and selective delivery of drugs improves bioavailability by preventing premature degradation and enhancing uptake , maintains drug concentration within the therapeutic window by adjusting the drug release rate , and reduces side effects by targeting to disease site and target cells . the ability to deliver therapeutic drugs locally , in a minimally invasive manner , has advanced drastically with the growth of molecular imaging techniques . molecular imaging approaches have been implemented in areas ranging from new therapeutic target discovery to effectively monitoring tumor pharmacokinetics and drug distribution to modulation of drug release at the target site . when molecular imaging probes are coadministered as part of the drug delivery system , it can help to achieve multiple goals , such as real - time and concurrent assessment of drug delivery efficiency / targeting , in vivo fate of drug and sites of localization / accumulation , modes of excretion , imaging , and monitoring the progress of drug treatment , in a single dosing . when an image - guided approach is not used , there is neither any means to track or image the in vivo fate nor the ability to measure the delivery efficiency of drugs . also , the bioavailability , therapeutic efficacy , and dose response of drug treatment has to be estimated based on separate sets of experiments which might render the process cumbersome and cost - ineffective . however , molecular imaging of the drug delivery process involves several challenges and is affected by several factors such as target expression , type of drug , in vivo accessibility of the receptor ( e.g. , vascular density , vascular permeability , and interstitial pressure ) , enhanced permeability and retention ( epr ) effect , receptor internalization , tracer protein dose , and timing of imaging . nevertheless , this approach has the potential for patient selection for targeted therapy and monitoring the therapeutic response after the drug is delivered . currently , several noninvasive image - guided modalities are being used in biomedical and clinical settings , which include magnetic resonance imaging ( mri ) , computed tomography ( ct ) , positron emission tomography ( pet ) , single photon emission computed tomography ( spect ) , optical imaging , and ultrasonography . among these , pet , spect , and optical imaging are regarded as quantitative or semiquantitative imaging modalities , whereas ct and mri are normally used for anatomical imaging . the relative advantages and limitations of these imaging modalities have been elaborately discussed in several review articles . in particular , pet offers picomolar sensitivity and is a fully translational noninvasive functional imaging technique with high sensitivity and accurate quantification and thus helps in measuring biological processes at the molecular and the metabolic levels in vivo . however , the limited spatial resolution of the pet images might sometimes make it difficult to accurately define the regions of interest ( rois ) . unnecessary radiation exposure to the nontargeted organs due to highly energetic -rays ( 511 kev ) emitted by the pet radioisotopes is also a cause of concern . nevertheless , one has to acknowledge that no single imaging modality can provide information on all aspects of structure and function . therefore , investigation of a subject using multiple imaging modalities is highly desirable and is rapidly gaining popularity . in this review , we aim to provide a timely and comprehensive overview of the pet image - guided drug delivery approaches reported to date , with focus on quantitative assessments of tumor - targeted therapeutic delivery , distribution , uptake , and response . the development of various carriers for site- and event - specific targeting and controlled drug release are summarized , and the great potential and intriguing opportunities for future development which might help in bringing this exciting research avenue closer to a clinical reality are discussed . the interest in using pet as a molecular imaging modality in clinical research has steadily grown during the last 23 decades , and has now gained considerable importance in routine hospital practices because of its ability to diagnose diseases in early stages and monitor therapeutic responses . in a typical scenario of pet imaging , a suitable compound is radiolabeled with positron - emitting radionuclides such as f , cu , ga , or zr and administered to a living subject . the positron that emerges from the radionuclide decay travels a short distance before being annihilated with an electron to release two 511 kev rays , which are approximately 180 apart . the 511 kev rays can be detected by a ring of detectors configured in the coincidence mode in the pet camera . the registered events are reconstructed into a three - dimensional image which provides information on the spatial distribution of the radioactivity as a function of time in the living subject . nowadays , pet is increasingly used in combination with ct as a hybrid imaging modality in clinical settings , to obtain higher resolution by fusing both functional and anatomical information at the same time . pet also plays an important role in the process of drug development and evaluation , whereby understanding drug action and establishing dosage regimens and treatment strategies have been most crucial . , o can replace the stable analogues in drugs and biomolecules , and hence it is possible to synthesize pet probes with the same chemical structure as the parent unlabeled molecules without altering their biological activity . low bioavailability , insufficient targeting , and poor localization in desired tissue / organ , adverse side effects , etc . targeted drug delivery systems have the potential to improve these undesirable features , and when used in conjunction with pet imaging , they are effective in increasing safety to efficacy ratio and decreasing dose , which in turn reduces adverse reactions and toxicity of drugs . pet can also provide information on the kinetics , dosimetry , and distribution of drugs in the diseased and normal tissues within the field of view as well as the clearance pattern in a biological system . pet image - guided drug delivery is expected to play an increasingly important role in realizing the full potential of the next generation of therapeutics . for this purpose , it is essential to choose radioisotopes of appropriate half - lives to match the pharmacokinetics of the drug carriers used . generally , for inorganic drug carriers ( such as silica nanoparticles , superparamagnetic iron oxide nanoparticle , gold nanoparticles , quantum dots etc . ) which are expected to have circulation half - lives of a few hours , short - lived or intermediate - lived radioisotopes such as ga ( t1/2 = 68 min ) , f ( t1/2 = 109.8 min ) , sc ( t1/2 = 3.9 h ) , ga ( t1/2 = 9.7 h ) , cu ( t1/2 = 12.7 h ) , etc . however , for organic drug carriers ( such as carbon nanotubes , polymeric nanoparticles , micelles , liposomes , etc . ) , which can circulate in vivo for more than 1 day , intermediate - lived or long - lived radioisotopes such as ga ( t1/2 = 9.7 h ) , cu ( t1/2 = 12.7 h ) , zr ( t1/2 = 78.4 h ) , or i ( t1/2 = 4.17 day ) would be the ideal choices for pet image - guided drug delivery . the choice of suitable radioisotopes is also governed by the conjugation strategies adopted for radiolabeling the drug carrier . the radiolabeled agent must demonstrate high in vitro and well as in vivo stability for successful use in pet image - guided drug delivery . the current revolution in targeted drug delivery is fueled by the innovations in material science , organic chemistry , functional genomics , and proteomics which have created carriers that are biodegradable ( which can be slowly dissolved in vivo by biological means ) , biocompatible ( which can remain in a biological system without causing any adverse effect ) , targeting , and stimulus - responsive ( which can control drug biodistribution in response to specific stimuli ) . in addition to increased selectivity against diseased cells , these delivery systems can also solve problems associated with drug instability in the biological environment as well as issues related to the modulation of drug . two different approaches are used for drug loading and delivery for pinpoint targeted treatment of cancer cells . in the first approach , chemotherapeutic drugs are loaded onto multifunctional drug carriers such as liposomes , micelles , nanoparticles , microparticles , microbubbles , dendrimers , copolymers , intestinal pathogen , etc . owing to the convenience in modifying the surface properties of these carrier systems , they can be conjugated with various targeting ligands such as monoclonal antibodies , antibody fragments , peptides , and other small molecules . the carriers are either directly conjugated to targeting ligands or derivatized for interactions with specific adapters that are conjugated to the targeting vectors . streptavidin / biotin interaction is one good example used for binding various carriers to targeting proteins and antibodies . in addition to delivery of chemotherapeutic drug molecules for therapy , these carriers also carry pet radionuclides or other contrast agents for diagnosis of the diseases . such drug delivery strategies are an important move toward achieving simultaneous diagnosis and therapy of diseases , which have recently been termed as drugs ( e.g. , therapeutic radionuclides ) are conjugated with the targeting ligands using suitable bifunctional linkers . unlike the first approach , here the drug and the imaging label ( pet radionuclide ) do not necessarily share the same delivery carrier . for diagnosis or monitoring therapeutic response , pet imaging is carried out separately in this case by conjugation of the targeting ligands with suitable pet radioisotopes . another striking difference between the two approaches is that , in the former , the delivery of the drug to the target tissue can be achieved by both passive and active targeting , while , in the latter , the drug is delivered primarily due to active targeting . in passive targeting , the drug carriers such as nanoparticles , liposomes , micelles , etc also , therapeutic concentrations can be much lower than optimal at the tumor site by simply relying on epr - mediated accumulation , and therefore passive targeting is generally not preferred for drug delivery . more efficient and selective uptake of drug into the target cells is achieved by active targeting wherein the drug carriers are conjugated with targeting ligands , as mentioned earlier . active targeting requires careful identification of tumor biomarkers , as well as selection of specific molecules that can bind to such markers in a selective and directed manner . targeted drug delivery vehicles can then be internalized by tumor cells via receptor - mediated endocytosis / phagocytosis , resulting in elevated concentration of drugs in tumor tissue . thus , the concept of theranostic agent is not just limited to chemotherapy but also has a relevant role to guide in radiation - based targeted therapies . various drug carrier systems have been radiolabeled with different positron emitter radionuclides for image - guided drug delivery , most of which are summarized in table 1 and discussed in the following text . albumin is an attractive macromolecular carrier that may be modified suitably for biomedical imaging applications . such carriers have also been studied for drug and gene delivery in vitro and in vivo , through cavitation . generally , albumin - based carriers are biodegradable , nontoxic , metabolized in vivo to produce harmless degradation products , nonimmunogenic , easy to purify , and soluble in water allowing ease of delivery by injection and thus ideal candidates for image - guided drug delivery procedures . a significant amount of drug can be incorporated in the albumin based carrier systems because of different binding sites present in the albumin molecule . owing to the defined albumin primary structure and high content of charged amino acids ( e.g. , lysine ) on the surface , albumin - based carriers offer the possibility of direct electrostatic adsorption of positively ( e.g. , ganciclovir ) or negatively charged ( e.g. , oligonucleotide ) molecules without the requirement of any other compound . in addition , these carriers can easily be prepared under mild conditions by coacervation , controlled desolvation , or emulsion formation . commercially , albumins are obtained with significant quantities from egg white ( ovalbumin ) , bovine serum ( bovine serum albumin , bsa ) , and human serum ( human serum albumin , hsa ) and also available from soybeans , milk , and grains . the chelator - free radiolabeling of macroaggregated human serum albumin with ga ( t1/2 = 68 min ) for pet imaging was first described by even et al . subsequently , this procedure was improved , and development of a kit for labeling macroaggregated human serum albumin with ga for pet imaging of liver anomalies was reported by okada et al . the kit was clinically tested and was found useful in the evaluation of the function of the reticuloendothelial system . in a similar study , maus et al . reported the radiolabeling of different commercially available human serum albumin kits with ga.in vivo pet imaging showed that ga - labeled human serum albumin was mainly retained in the lungs . no decrease in activity or migration of particles from the lungs was observed during the first 1 h ( 1 half - life of ga ) , which demonstrated the in vivo stability of the radiolabeled albumin over that period of time . also , no significant retention of ga - labeled human serum albumin particles in the liver was detected . the authors concluded that this approach could be used to estimate the liver - to - lung shunt and eliminate extrahepatic macroaggregate deposition in patients with primary and secondary liver malignancies , warranting y - based radioembolization therapy . in a recent development , liao et al . prepared albumin shelled microbubbles filled with perfluorocarbon ( c3f8 ) gas to enhance the contrast in ultrasound imaging . additionally , the microbubbles were radiolabeled with n - succinimidyl-4-[f]fluorobenzoate ( f - sfb ) and also conjugated with antibodies targeting vascular endothelial growth factor receptor 2 ( vegfr2 ) using avidin the radiolabeled microbubble shells could thus be used as dual - modality ( pet and ultrasound ) imaging agent . the f - labeled , albumin - shelled , vegfr2-targeted microbubbles had a lifetime of 30 min in the blood pool and demonstrated a highly specific adherence to tumor vessels in mice bearing human breast cancer . the size of the microbubbles was on the order of several micrometers and therefore should be retained in the tumor vasculature after intravenous injection . however , dynamic micropet imaging showed a relatively low tumor uptake of 1% id / g , even 1 h post injection . the low tumor uptake might be attributed to attachment of f - sfb on the surface of the microbubble , which might have influenced the targeting efficiency of the antibody . the targeted microbubbles accumulated rapidly in both the liver and lung and cleared slowly from the blood circulation . the trends found in micropet imaging were further corroborated by ex vivo biodistribution studies . the specificity of the binding of targeted microbubbles to endothelial vegfr2 was further validated by comparing the results of targeted and nontargeted contrast - enhanced ultrasound imaging . the authors concluded that the f - labeled albumin - shelled microbubbles can be used for targeted drug delivery to vegfr2 in breast cancer , guided by the dual - modality ( pet / ultrasound ) functional imaging approach . in all these studies , development of only imaging strategies using albumin - based platforms have been described without direct relation to drug delivery . however , there are several other reports on utility of drug - loaded albumin - based carriers and controlling drug release using ultrasound energy in such systems . therefore , it was expected that tracking disease progression would be analogous to tracking drug delivery using albumin - based carriers . liposomes are concentric , closed bilayer membranes of water - insoluble polar lipids that can that can be used to encapsulate biomolecules and drugs for targeted delivery while protecting their bioactivity . soluble drugs can be loaded in the aqueous core and the hydrophobic drugs partitioned in the lipid bilayer . they are widely used not only in delivery of a variety of anticancer drugs but also in delivery of antineoplastic agents , antimicrobial compounds , immunomodulators , anti - inflammatory agents , cardiovascular drugs , etc . the widespread interest in the use of liposomal systems for drug delivery stems from their biocompatibility , biodegradability , and nontoxicity and the ease of controlling their size during the preparation process . currently , there are several commercially available liposomal formulations for cancer therapy , including doxorubicin ( doxil ) , daunorubicin ( daunoxome ) , cytarabine ( depocyt ) , myocet , and vincristine ( onco - tcs ) . the recent advances in the use of radiolabeled liposomes for imaging as a tool in personalized medicine have been summarized in a recent review . generally , liposomal systems are coated with poly(ethylene glycol ) ( peg ) to increase the circulation time in blood and decrease uptake in the reticuloendothelial system ( res ) . radiolabeling of liposomes with pet radioisotopes generally requires the use of chelator molecules in the aqueous core or conjugation on the lipid bilayer . the radiolabeled liposomal systems employed in pet studies must be carefully designed as lower stability of radiolabeled agent might obscure image - based assessment of particle pharmacokinetics . seo et al . reported the development of a method for radiolabeling liposomes with cu for imaging and drug delivery monitoring using pet . bifunctional chelators , such as , 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane - n , n,n,n-tetraacetic acid ( bat ) , ( 6-(6-(3-(2-pyridyldithio)propionamido)hexanamido)benzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid ( teta - pdp ) , and 4-(2-(2-pyridyldithioethyl)ethanamido)-11-carboxymethyl-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane ( cb - te2a - pdea ) were radiolabeled with cu , and the radiolabeled conjugates were attached to maleimide lipids in the liposome . the radiolabeled liposomes were found to be stable in mouse serum even after 48 h of incubation . in vivo pet studies demonstrated that liposomal activity was high in the blood pool from 0 to 6 h and slowly cleared out through the res . the presence of the peg spacer between the chelator and the lipid did not significantly alter the labeling efficiency and the clearance rate of liposomes from the blood pool . the study was further extended by the same group of authors to characterize the in vivo clearance of cu - labeled distearoyl and dipalmitoyl lipids included within pegylated liposomes.in vivo pet imaging studies established that changes in lipidacyl chain length can result in desorption of lipid from the liposomal anchorage and interaction with blood components . therefore , this factor should be considered for liposomal pet studies as desorption can rapidly alter the apparent pharmacokinetics . in another study , peterson et al . developed a remote loading method using 2-hydroxyquinoline ionophore , to carry cu across the membrane of preformed liposomes and deliver it to an encapsulated copper - chelator . a highly efficient loading ( > 95% ) and retention stability ( > 99% ) was obtained adopting this approach . in vivo pet imaging studies demonstrated that a maximum tumor uptake of 5% id / g with high tumor to muscle ratio could be achieved . the cu - liposomes reached a maximum level in the liver and spleen after 4 h and subsequently remained at a constant level . also , the cu - liposomes remained in the blood pool for > 24 h. the method provided cu - labeled liposomes with excellent imaging properties due to the high concentration of cu inside the liposomes and restricted exchange of cu with the biological environment due to the protective barrier constituted by the liposomal membrane . the same group of authors investigated the suitability of cu - labeled liposomes for imaging somatostatin receptor expression in neuroendocrine tumor model . the peptide octreotate ( tate ) was covalently attached to the pegylated liposomes with an encapsulated positron emitter cu . this peptide is routinely used in clinic for imaging somatostatin receptor - positive tumors by scintigraphy.in vivo pet imaging and biodistribution studies revealed that the presence of tate on the liposomes resulted in a significantly faster initial blood clearance in comparison to control liposomes without tate . there was no significant difference in tumor uptake ( 5% id / g in both cases ) on using cu - labeled pegylated liposomes with or without tate , suggesting that the uptake was mainly due to passive targeting . however , cu - loaded pegylated liposomes with tate showed significantly higher tumor - to - muscle ( t / m ) ratio ( 12.7 1.0 ) than the control - liposomes without tate ( 8.9 0.9 ) . the tumor accumulation and t / m ratio achieved in this study suggest that lioposomal systems might be used as carriers of radionuclides for therapeutic use and also for delivery of chemotherapeutic drugs . tumor associated macrophages ( tams ) have been shown to play a major role in the growth and spread of several types of cancer . locke et al . reported pet imaging of tams in a mouse model of pulmonary adenocarcinoma , using mannose coated liposomes radiolabeled with cu.in vivo pet imaging and biodistribution studies revealed that radiolabeled mannosylated liposome accumulated in tams and exhibited little accumulation in remote lung areas at 6 h post injection . further , it was verified by confocal microscopy that the pet signal was due to liposome internalization by tams . urakami et al . developed a methodology for one - step labeling of liposomes with f. solid - phase transition method was utilized , and high labeling efficiency and visualization of liposomal trafficking in mice by real - time analysis were obtained by pet . the same group reported the development of an efficient method for preparation of f - labeled liposome - encapsulated hemoglobin . using the radiolabeled liposome , the oxygen transfer even in an ischemic brain could be monitored by dynamic pet . in another study , radiolabeling of pegylated liposomes with [ f]fluorodipalmitin ( [ f]fdp ) was reported by marik et al . radiolabeled diglyceride was synthesized by the incorporation of f into the lipid molecule by nucleophilic substitution of p - toluenesulfonyl moiety . while free [ f]fdp was rapidly taken by the liver , spleen , and lungs , liposome incorporated [ f]fdp was observed to circulate in blood vessels for nearly 90 min . adopting the previously reported procedures , f and cu - labeled liposomes were prepared by paoli et al . the liposomes were preconjugated with suitable fluorophores ( calcein or af-750 ) , for dual - modality pet / optical imaging . a model hydrophilic drug was encapsulated in the liposomal system and administered in mice bearing bilateral met-1 tumors . using in vivo pet imaging and ex vivo fluorescent imaging of tumors , the authors could demonstrate that the accumulation of the drug was increased by up to 177-fold by liposomal encapsulation . recently , oku et al . reported the radiolabeling of liposomes [ modified with peg or ala - pro - arg - pro - gly ( aprpg ) peptide ] with 1-[f]fluoro-3,6-dioxatetracosane , which enabled imaging of gliomas by pet with higher contrast than that obtained with [ f]fluorodeoxyglucose ( [ f]fdg ) . the liposomes did not accumulate in the normal surrounding brain tissue due to blood brain barrier protection , and using this approach , even a very small sized ( 1 mm ) brain tumor could be specifically imaged with the radiolabeled liposome ( figure 1 ) . mitchell et al . developed a series of liposomal systems with oligoethylene glycol spacers of differing lengths between the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ( dota ) chelator and the lipid headgroup . a suitable fluorophore , n-(fluorescein-5-thiocarbamoyl)-1,2-dihexa - decanoyl - sn - glycero-3-phosphoethanolamine triethylammonium salt , was attached to the liposome , which could also be chelated to gd ( for mri ) , in ( for spect ) , or cu ( for pet ) and used for multimodal imaging . the effective radiolabeling and noninvasive imaging strategies developed thus far might aid further research on pet image - guided drug delivery using liposomal carriers in the near future . pet imaging of brain tumor using peg - modified liposomes ( top panel ) and aprpg - modified liposomes ( middle panel ) , labeled with 1-[f]fluoro-3,6-dioxatetracosane . the other regions of the brain showed a low background . on the contrary , [ f]fdg imaged the whole brain , although the accumulation was higher in the tumor region ( bottom panel ) . miceller structures are important carriers for drug delivery because they can form relatively small and uniform size structures , be prepared from a variety of amphiphilic materials , increase solubility of hydrophobic molecules , and incorporate multiple functionalities into a single structure . when tagged with suitable contrast agents , these systems can also be used for molecular imaging as well as image - guided drug delivery . among the various miceller structures , the inner core is the hydrophobic part of the block copolymer , which encapsulates the water - insoluble drug . the outer shell or corona of the hydrophilic block of the copolymer is often composed of peg , and it protects the drug from the aqueous environment and also imparts particle stability and excellent dispersibility in an aqueous solution . owing to these characteristics , polymeric micelles have several advantages as drug carriers such as enhancing the aqueous solubility of hydrophobic drugs , prolonging the circulation time of the drug in the blood , improving the in vivo stability of the drug , providing both passive and active tumor targeting abilities , and reducing nonspecific uptake by the reticuloendothelial system . in vivo tumor targeting and drug delivery properties of polydiacetylene ( pda ) micelles ( diameter 10 nm ) were investigated by mackiewicz et al . in a breast cancer model . such small sized micelles can better diffuse through blood vessel walls and reach deeper tumor tissues due to the epr effect . the authors synthesized different micelles with coatings consisting of either nitrilotriacetic acids ( nta ) or peg chains of variable lengths and tested for their ability to passively target tumor . among them , 2 kda peg - coated micelle ( pda - peg2000 ) was identified as the most promising carrier in terms of longer blood residence time , higher tumor uptake , and better imaging contrast . fluorescence diffuse optical tomographic imaging indicated a tumor uptake of 3% of the injected dose of pda - peg2000 . the diffusion of pda - peg2000 micelles inside the tumor was further evidenced and quantified by pet imaging using f - fdg colocalization . drug delivery application of the cargo was also assessed using micelles loaded with paclitaxel , a hydrophobic anticancer drug , which showed good in vitro cytotoxicity and in vivo tumor growth inhibition . thus , the potential of pda - micelles for drug delivery could be successfully demonstrated in this study . in another study , cho et al . reported a novel drug delivery strategy using poly(ethylene glycol)-block - poly(-caprolactone ) ( peg - b - pcl ) micelles . three different drugs , namely , paclitaxel ( cytotoxic agent ) , cyclopamine ( hedgehog inhibitor ) , and gossypol ( bcl-2 inhibitor ) , were loaded on peg - b - pcl micelles and evaluated in xenograft models of ovarian cancer . multi - drug - loaded peg - b - pcl micelles were nanoscopic , fairly stable in aqueous solution , and capable of simultaneous as well as sustained release of each of the three drugs in vitro . in vivo studies based on bioluminescence imaging and 3-deoxy-3-f - fluorothymidine ( f - flt ) pet imaging revealed that multi - drug - loaded peg - b - pcl micelles had significantly less tumor burden than use of paclitaxel alone . also , f - flt - pet images clearly showed that multi - drug - loaded peg - b - pcl micelles significantly reduced tumor volumes over paclitaxel and vehicle controls and could thus prolong the overall survival . thus , the authors could establish that the strategy of concurrent delivery of drug combinations of cytotoxic agents and molecular targeted agents using a micellar - based drug delivery vehicle is effective for the treatment of ovarian cancer . recently , benezra et al . evaluated the potential of f - labeled dasatinib derivative ( ski249380 , a new - generation src and platelet - derived growth factor receptor ( pdgfr ) inhibitor ) loaded on micellar and liposomal carriers for drug delivery and uptake in xenograft models of high - grade glioma.in vivo pet imaging studies demonstrated a significantly higher tumor uptake for f - ski249380-loaded micellar formulations ( 4.9% id / g ) compared to control group ( 1.6% id / g ) . saturation studies using excess cold dasatinib showed marked reduction of tumor uptake values to levels in normal brain ( 1.5% id / g ) , consistent with in vivo binding specificity . the improved drug solubility , delivery , and kinetic behavior conferred by the use of these micellar f - ski249380 preparations might find utility in treatment of various types of gliomas . despite the excellent attributes of the polymeric micelles as carriers for drug delivery , such systems suffer from insufficient in vivo stability which is affected by the surrounding environment , especially the concentration of the amphiphilic block copolymers . upon dilution in the bloodstream , multimolecular polymeric micelles disassemble , leading to a burst release of drug and loss of tumor - targeting abilities . these limitations could be circumvented with the use of suitably engineered unimolecular micelles possessing excellent in vitro and in vivo stability . the synthesis of a multifunctional unimolecular micelle made of a hyperbranched amphiphilic block copolymer , boltorn h40-poly(l - glutamate - hydrazone - doxorubicin)-b - poly(ethylene glycol ) , for pet image - guided drug delivery was reported by xiao et al . the copolymer was conjugated with cyclo(arg - gly - asp - d - phe - cys ) peptides ( crgd ) for integrin v3 targeting and macrocyclic chelators ( 1,4,7-triazacyclononane - n , n,n-triacetic acid [ nota ] ) for cu - labeling and pet imaging . the anticancer drug doxorubicin ( dox ) was covalently conjugated onto the hydrophobic segments of the amphiphilic block copolymer arms via a ph - labile hydrazone linkage to enable ph - controlled drug release . in vivo pet imaging and biodistribution studies in u87 mg tumor - bearing mice showed higher tumor uptake for crgd - conjugated unimolecular micelles ( 7% id / g ) than nontargeted micelles ( 2.5% id / g ) ( figure 2 ) . additionally , crgd - conjugated unimolecular micelles exhibited a much higher cellular uptake in u87 mg human glioblastoma cells than nontargeted unimolecular micelles due to integrin v3 mediated endocytosis , thereby leading to a significantly higher cytotoxicity when the micellar systems were conjugated with dox . the same group of authors reported the synthesis of another unimolecular micelle formed by dendritic amphiphilic block copolymers poly(amidoamine)-poly(l - lactide)-b - poly(ethylene glycol ) conjugated with an anti - cd105 monoclonal antibody ( trc105 ) and nota for pet image - guided drug delivery . as observed in the previous study , cu - labeled targeted micelles exhibited a much higher level of tumor accumulation than cu - labeled nontargeted micelles , measured by serial noninvasive pet imaging and confirmed by biodistribution studies in murine breast tumor - bearing mice . thus , these multifunctional unimolecular micelles possessing passive and active tumor - targeting abilities , ph - controlled drug release , and pet imaging capabilities are potentially important drug delivery vehicles for image - guided therapy . ( a ) pet imaging of u87 tumor bearing mice at different time points post injection of cu - labeled unimolecular micelle loaded with dox ( h40-crgd - cu ) and cu - labeled unimolecular micelle conjugated with crgd and loaded with dox ( h40-dox - crgd - cu ) ( b ) ex vivo fluorescence imaging of u87 mg tumor , with the excitation and emission set for detecting dox fluorescence , harvested from mice injected with h40-dox - cu or h40-dox - crgd - cu . adapted with permission from ref ( 86 ) . enzyme / prodrug therapy is one of the most promising strategies where systemic toxicity can be minimized while maintaining the therapeutic efficacy . in this process , a drug - activating enzyme is targeted or expressed in cancer cells , following which a nontoxic prodrug is administered systemically . the enzyme converts the prodrug to an active anticancer drug , achieving high concentrations in the tumor and sparing the normal tissues . however , there are certain requirements for this strategy to work in clinical context . the enzyme should be non - human or expressed at very low concentrations in the normal tissue and should have high enzymatic activity . the prodrug should be a good substrate for the enzyme but should not be activated in nontumor tissues . while the prodrug should be nontoxic , the activated drug should be highly toxic and diffusible to be taken up by the adjacent cells for a bystander cell kill effect . currently , there are three major categories of enzyme / prodrug strategies : ( a ) delivery of genes that encode prodrug - activating enzymes into tumor tissue ( gene encoding prodrug activating enzyme therapy , gdept , and virus - directed enzyme prodrug therapy , vdept ) , ( b ) targeted delivery of active enzymes in tumor tissue where the therapeutic enzyme is conjugated with an antibody , small molecular ligand , or peptide that binds to antigens preferentially expressed on the surface of tumor cells or in the tumor vasculature or interstitium ( targeting group - directed enzyme / prodrug therapy , tdept ) , and ( c ) vasculature permeability - dependent enzyme / prodrug therapy ( vpdept ) in which the intratumoral delivery of the enzyme is realized through the higher permeability of tumor vasculature . pet image - guided enzyme / prodrug strategies have been extensively reviewed and hence will be discussed briefly in the following text . most of the studies in pet guided enzyme / prodrug based cancer therapy are based on the gdept approach . developed a strategy which combined gene therapy with aromatic l - amino acid decarboxylase ( aadc ) gene and a prodrug , dopamine . using this approach , the authors could synthesize and regulate the neurotransmitters involved in parkinson s disease . in vivo pet imaging using aadc tracer , 6-[f]fluoro - l - m - tyrosine ( fmt ) could measure the gene expression and thus establish the potential of enzyme / prodrug approach in delivery of therapeutic agents to the central nervous system . also , the extent of gene expression could be effectively used to predict the therapeutic response . this approach could be further validated in another study where pet imaging with i - labeled 2-fluoro-2-deoxy-1b - d - arabino - furanosyl-5-iodo - uracil ( i - fiau ) , a specific marker substrate for expression of the herpes simplex virus type-1 thymidine kinase ( hsv-1-tk ) gene , was used to identify the location , magnitude , and extent of vector - mediated gene expression in a phase i / ii clinical trial of gene therapy for recurrent glioblastoma . in this study , dynamic i - fiau - pet scans were done before gene transduction to assess the basal state of fiau - accumulation and washout of the tumor , and also after vector application to investigate whether specific fiau - accumulation did occur ( figure 3a ) . ganciclovir treatment ( 5 mg per kg twice a day over 14 days ) was done starting 4 days after vector infusion . treatment responses were recorded by repeated mri as well as pet with f - fdg and c - labeled methionine ( c - met ) . the same pet tracer ( i - fiau ) was used by hackman et al . to assess the potential of double prodrug activation gene therapy using the escherichia coli cytosine deaminase ( cd)-hsv-1-tk fusion gene ( cd / tk ) for treatment of different tumors . pet imaging was used for monitoring expression of the cd / tk fusion gene , and the different levels of cd / tk expression in tumor models could be imaged quantitatively . the results of these studies could be utilized to develop standardized gene therapy protocols adopting enzyme / prodrug strategy for human subjects . ( a ) pet imaging using f - fiau to identify the location , magnitude , and extent of vector - mediated gene expression in gene therapy for recurrent glioblastoma . the region of specific i - fiau retention within the tumor after hsv-1-tk - transduction ( white arrow ) showed the signs of necrosis ( cross hairs , right column and reduced methionine uptake [ met ] ) after ganciclovir treatment . ( b ) pet imaging of hsv-1-tk activity in tumors after sindbis / tk infection . tumor - bearing mice either received no vector treatment ( tumor + , sindbis / tk ) or received 3 sindbis / tk treatments via intraperitoneal injection far away from sites of tumor inoculation ( tumor + , sindbis / tk + ) . hsv-1-tk activity was determined after intravenous administration of f - feau as tracer . tumors on the right shoulder of scid mice are indicated by yellow arrows , and white arrows indicate activity in urinary bladder . adapated with permission from ref ( 102 ) . the synthesis of half - mustard prodrug , 4-[(2-chloroethyl)(2-ethyl ) amino]-phenoxycarbonyl - l - glutamic acid , by reductive alkylation of 4-[(2-chloroethyl)amino]-phenoxycarbonyl - l - glutamic acid was reported by malik et al . the prodrug was radiolabeled with c , and its potential for imaging antibody- and gene - directed enzyme prodrug therapy with pet could be established . the use of another prodrug , 1-(2-deoxy-2-fluoro--d - arabinofuranosyl)uracil ( fau ) , for treatment of tumors with high thymidylate synthase catalytic activity was reported by eiseman et al . pet imaging using f - fau was used to visualize tumors that have high thymidylate synthase catalytic activity . however , the authors did not observe high localization of f - fau in tumors compared with background , which might limit further utilization of this pet probe in clinical context . in another study , selective tumor targeting and quantitative in vivo monitoring using pet of a commonly applied gdept , based on hsv-1-tk and ganciclovir ( gcv ) , was reported by tseng et al . sindbis virus was used to deliver the hsv-1-tk suicide gene to tumor cells for subsequent gcv activation and tumor killing . pet imaging using f - labeled fluoro - ethyl - arabinosyluridine ( f - feau ) was used to monitor the hsv-1-tk activity in tumor cells after parenteral administration of sindbis virus ( figure 3b ) . high tumor uptake of f - feau ( 3% id / g ) proved that the sindbis vector efficiently targeted the hsv-1-tk enzyme gene into the infected tumor cells . also , pet imaging could be used to monitor hsv-1-tk activities after systemic sindbis vector treatments for determining the levels and tissue distribution of the vector and optimizing efficient prodrug activation for more accurate treatment planning and monitoring . this study was further extended by stelter et al . , where different molecular imaging strategies , such as bioluminescence , fluorescence molecular tomography , and pet , were used to evaluate sindbis virus mediated infection of tumor cells in vitro and in vivo . the authors concluded that the sindbis virus infection rates were not solely dependent on cellular laminin receptor expression and other factors such as cellular infection and viral replication might also be responsible . in another similar study , wang et al . evaluated the efficacy of 4 different radiotracers , i-5-iodo-29-fluoro-1-b - d - arabinofuranosyluracil ( i - fiau ) , 5-f - fluoro-29-deoxyuridine ( f - fudr ) , 2-f - fluoroethyl - l - tyrosine ( f - fet ) , and f - fdg for monitoring tumor responses using spect or pet during prodrug activation gene therapy with hsv-1-tk and gcv . based on tumor uptake of the radiotracers , f - fudr was identified as the most suitable radiotracer for assessment of responses in tumors undergoing hsv-1-tk and gcv prodrug activation gene therapy . the enzyme -glucuronidase ( -gus ) has recently been investigated as a target in prodrug therapy for cancer . in order to optimize -gus - based prodrug therapies , a pet tracer , f - labeled 1-o-(4-(2-fluoroethyl - carbamoyloxymethyl)-2-nitrophenyl)-o--d - glucopyronuronate ( f - feanga ) , was evaluated for imaging of -gus in tumor ( c6 gliomas ) and inflammation models.in vivo pet imaging and biodistribution studies showed high uptake of the radiotracer in tumor , high target to nontarget ratio , and rapid renal clearance . in inflammation model , the uptake of the radiotracer in inflamed muscle was significantly higher than in control muscle , thereby establishing the potential of this radiotracer to detect increased activity of -gus . the extent of -gus release in small c6 glioma tumors after a single treatment of doxorubicin ( dox ) , carmustine ( bcnu ) , and tumor necrosis factor ( tnf- ) with f - feanga pet was evaluated by the same group of authors . pet studies confirmed that -gus was released in vivo and the distribution volume of f - feanga in c6 gliomas was increased significantly . the results obtained in this study demonstrate the potential of a two - step chemotherapy prodrug approach , in which tumors are treated with a single dose of a cytostatic drug before prodrug treatment . recently , moon et al . reported the synthesis of f labeled 1-(3-furyl)-4-hydroxy-5-fluoro-1-pentanone ( f - f-4-im ) , which can be metabolized by the cyp4b1 enzyme and used for pet imaging of tumors and monitoring enzyme - activating anticancer prodrugs . biodistribution studies in normal rats showed that the uptake of f - f-4-im was high in the lung , where cyp4b1 gene is preferentially expressed . the results were further confirmed by in vitro cell assays , and the potential of f - f-4-im for imaging of cyp4b1-transfected tumor cells and monitoring cyp4b1 enzyme / prodrug interactions could be demonstrated . it is envisaged that further development of pet guided enzyme / prodrug protocols would significantly facilitate their clinical translation with high safety and reliability . in the past two decades , the applications of nanotechnology in cancer diagnostics and therapy have attracted widespread interest and a variety of functional nanoparticles have been developed and evaluated for drug delivery , diagnostic sensors , imaging agents , and labeling probes . nanoparticles used for this purpose vary with a size from 1 nm to few hundred nanometers and surface charge varying from negative to positive and even neutral . particularly as drug delivery vehicles and molecular imaging tools , targeted nanoparticle based systems hold significant promise by virtue of their controllable size , high surface area to volume ratio , and customized internal and external chemistries . the major advantages of using the engineered nanoparticle based systems for such applications include ( a ) the ease of particle functionalization for conjugation with suitable targeting vectors such as peptides or antibodies , ( b ) the ability to deliver a higher concentration of contrast agent for every targeted binding event to achieve higher detection sensitivity which might permit diagnosis of the disease in its very early stage , and ( c ) improved treatment effects when used as drug carriers by protecting entrapped drugs from degradation , enhancing tumor uptake through the enhanced permeability and retention effect as well as receptor - mediated endocytosis and thereby achieving increased exposure of the tumor to therapeutic drugs . a variety of drug delivery systems based on metallic nanoparticles , oxide nanoparticles , polymeric nanoparticles , carbon nanostructures , biodegradable nanoparticles , etc . have been developed for molecular imaging as well as drug delivery . for a given system , multiple factors determine the stability and fate of the delivery vehicle during storage and after administration , including size , rigidity , charge , solubility , and surface modifications of the nanoparticles . therefore , the choice of a nanoparticle based delivery system is guided by the biodistribution , types of drugs that can be delivered using that system , and the specificity and pharmacokinetics of delivery . the different nanoparticle based systems which can be radiolabeled with suitable positron emitting radioisotopes for pet image - guided drug delivery are discussed in the following text . among the various metallic nanoparticles reported to date , gold nanoparticles are most widely used for biomedical applications , including drug delivery and novel diagnostic and therapeutic approaches , due to their biocompatibility , small size , ease of characterization , and rich surface chemistry . the utilization of f - labeled gold nanoparticles for pet imaging was first reported by guerrero et al . , in which the gold nanoparticles of 12 nm were synthesized by citrate reduction of haucl4 . the nanoparticles were functionalized with two different peptides , ck and clpffd , and f - sfb was covalently bound to the nanoparticle conjugate . after intravenous administration of the radiolabeled nanoparticles in normal rats , in vivo pet imaging showed highest uptake of the radioactivity in the bladder . the lungs , liver , and spleen were the organs with the next highest levels of radioactivity , followed by the intestine , kidneys , and blood . the pancreas and brain , however , accumulated very low concentrations of radiolabeled nanoparticles . clearance of the nanoparticles from the biological system took place by both renal and biliary excretions . gold nanorods with suitable aspect ratios can absorb and strongly scatter light in the near - infrared region , which can be used for enhanced optical imaging and photothermal cancer therapy . the development of a multifunctional gold nanorod - based nanoplatform for targeted anticancer drug delivery and pet imaging of tumors was reported by xiao et al . the bare gold nanorods had a length and diameter of approximately 45 and 10 nm , respectively . an anticancer drug ( dox ) and tumor targeting agent ( crgd ) were conjugated to the pegylated gold nanorods . also , nota was attached onto the distal ends of the peg arms for complexation with cu . based on flow cytometry analysis , crgd - conjugated gold nanorods exhibited a higher cellular uptake and cytotoxicity than nontargeted ones in vitro . however , in vivo pet imaging and biodistribution studies showed that targeted and nontargeted gold nanorods had similar distribution pattern especially in the tumor ( figure 4a ) . despite this limitation , this initial attempt provided a suitable nanoplatform for possible integration of multifunctionality including molecular targeting , chemotherapy , and photothermal therapy , as well as multimodality imaging , which can potentially lead to improved therapeutic efficacy and cancer monitoring . to achieve a similar goal , xie et al . reported the preparation of cu - labeled gold nanoshells conjugated with crgd and studied the in vivo biodistribution and tumor specificity using pet . the nanoshell used in this study was composed of a silica core ( 120 nm in diameter ) and a gold shell ( 810 nm ) to absorb light at near - infrared wavelengths . in vivo pet imaging suggested that tumor targeting was improved by conjugation of gold nanoshells to crgd , which was advantageous over the previous study by xiao et al . both targeted and nontargeted gold nanoshells were cleared from the circulation by the liver and spleen . in the subablative thermal therapy study , enhanced biological effectiveness of targeted gold nanoshell the promising results obtained from this study might lead to advancement of gold nanoshells as theranostic platforms for effective cancer diagnosis and therapy . ( a ) targeting of integrin v3 expression in u87 mg tumor bearing mice by gold nanorods ( gnr ) conjugated with crgd . pet images at different time points post injection of cu - labeled gold nanorods conjugated with dox ( cu - nota - gnr - dox ) and cu - labeled gold nanorods conjugated with dox and crgd ( cu - nota - gnr - dox - crgd ) . ( b ) targeting of cd105 expression in 4t1 tumor - bearing mice by trc105-conjugated mesoporous silica nanoparticles . pet images at different time points post injection of cu - labeled mesoporous silica ( cu - nota - msio2 ) and cu - labeled mesoporous silica conjugated with trc105 ( cu - nota - msio2-trc105 ) . tumors were indicated by yellow arrowheads . adapted with permission from ref ( 142 ) . ( c ) targeting of lung endothelium in c57bl/6 mice by polymeric nanoparticles conjugated with anti - icam antibody . micro - pet images of mice at different time points post injection of cu - labeled nanoparticle conjugated with anti - icam antibody ( cu - dota - np - anti - icam ) and cu - labeled nanoparticle conjugated with anti - icam antibody after pretreating the mice with lipopolysaccharides ( cu - dota - np - anti - icam lps treated ) . ( d ) targeting of integrin v3-expression in u87 mg tumor bearing mice by crgd - functionalized single walled carbon nanotubes ( swnts ) . peg5400rgd observed in the u87 mg tumor ( first row ) and control experiment showing blocking of swnt the arrows point to the tumors . adapted with permission from ref ( 165 ) . another category of promising nanoplatforms which has drawn substantial interest recently is the oxide nanoparticles ( such as mesoporous silica and iron oxide nanoparticles ) due to their nontoxic nature , easily modifiable surface , and good biocompatibility . shell silica nanoparticles as targeted pet / optical multimodal imaging probes was reported by benezra et al . near - infrared fluorescent , cy5 dye - encapsulated , core shell silica - based nanoparticles were prepared and coated with peg as per the method reported by burns et al . the nanoparticle was conjugated with crgd and radiolabeled with i through a tyrosine linker . in vitro cell binding assays demonstrated the specificity of the nanoplatform toward intregrin v3 expression . in vivo pet / optical imaging and biodistribution studies showed a tumor uptake of 1.5% id / g at 4 h post injection , with high tumor to background ratio and rapid renal clearance . owing to their favorable characteristics , such as bulk renal clearance , favorable targeting kinetics , lack of acute toxicity , superior photophysical features , and multimodal ( pet / optical ) imaging capabilities , such nanoparticles have received the united states food and drug administration ( us fda)-investigational new drug approval for a first - in - human clinical trial . the synthesis of ultrasmall , monodisperse silica nanoconjugates for targeted dual - modal imaging of lymph nodes with metastatic tumors was reported by tang et al . the nanoparticles were functionalized with an aptamer derivative having high binding affinity for nucleolin , a protein that is overexpressed in the cytoplasm and on the plasma membrane of several cancer cells . also , a near - infrared ( nir ) dye and dota were conjugated on the surface of the functionalized nanoparticle . the aptamer functionalized silica nanoconjugate was radiolabeled with cu , and in vivo pet / optical imaging studies showed markedly enhanced uptake of the radiolabeled agent in lymph nodes with metastatic tumors in a murine breast tumor model . in a similar study , kim et al . shell silica nanoprobe for multimodal ( pet / optical / mri ) imaging of the sentinel lymph node . magnetic silica nanoparticles with cobalt ferrite core and silica shell were synthesized which encapsulated nir dye on the silica shell . the surface of the nanoparticle was modified with amino group and peg for conjugation with nota , which was used for chelating ga . the triple modality nanoprobe could be successfully utilized to visualize the sentinel lymph node in mice . thus , these multimodal silica nanostructures hold great potential for improving the accuracy of clinical tumor staging by serving as probes for efficient noninvasive targeted imaging of metastatic lymph nodes . different chemotherapeutic drugs can also be attached on the surface of the functionalized nanoconjugates for prevention of metastases . in an interesting study , di pascua et al . utilized commercially available mesoporous silica nanoparticles as a carrier material for the therapeutic radioisotope ho ( t1/2 = 26.8 h , emax = 1.84 mev ) . a lipophilic acetylacetonate complex of ho was incorporated in mesoporous silica nanoparticles ( 80100 nm in diameter ) , which were subsequently irradiated in a neutron flux to produce particles containing ho by ( n, ) reaction . these radioactive nanoparticles were utilized to deliver effective therapeutic doses for treating ovarian cancer metastases after intraperitoneal delivery in skov-3 ovarian tumor - bearing mice . in vivo spect imaging demonstrated that most of the ho - containing mesoporous silica nanoparticles administered to ovarian tumor - bearing mice were retained in the peritoneal cavity and selectively accumulated in the tumors ( 33% id / g after 24 h ) . radiotherapeutic efficacy was monitored using pet / ct using f - fdg which showed a decrease in tumor volume , which correlated with a marked increase in survival after treatment with 4 mbq of the radioactive nanoparticles . though the authors could not explain the reason for the high uptake of mesoporous silica nanoparticles in ovarian tumor , this strategy might find utility in incorporation with other therapeutic radionuclides in nanostructured materials for treatment of various types of cancer . in another approach , chen et al . reported the development of biocompatible functionalized mesoporous silica nanoparticles for actively targeted pet imaging and chemotherapeutic drug delivery . mesoporous silica nanoparticles were surface functionalized with thiol groups , pegylated , conjugated with nota chelator and trc105 antibody ( specific for cd105/endoglin ) , and radiolabeled with cu . in vivo pet imaging and biodistribution studies in 4t1 breast tumor bearing mice showed high tumor uptake ( 6% id / g ) at 5 h post injection ( figure 4b ) . the tumor uptake of radiolabeled nanoparticles not conjugated with trc105 was much lower than the tumor uptake observed with trc105 conjugated nanoparticles , indicating that active targeting was responsible for the enhanced tumor uptake . the authors also demonstrated the feasibility of enhanced tumor targeted drug delivery in vivo using trc105 conjugated mesoporous silica loaded with an anticancer drug , dox . the encouraging results obtained in this study hold promise for future image - guided drug delivery and targeted cancer therapy using this class of nanomaterials . recently , iron oxide nanoparticles have been actively investigated as nanoplatforms for multimodal molecular imaging . the conventional drug loading approach by covalent linkage on such nanoplatforms is inefficient and suboptimal for drug release . in order to circumvent this limitation , xie et al . synthesized iron oxide nanoparticles , modified their surface using dopamine , and encapsulated them into human serum albumin matrices , which are clinically utilized as drug carriers . the human serum albumin coated iron oxide nanoparticles were dually labeled with cu - dota and cy5.5 dye , and tested in a subcutaneous u87 mg xenograft mouse model . in vivo pet / optical / mr imaging showed a high tumor uptake ( 5% id / g at 4 h post injection ) with high tumor to background ratio . an inhomogeneous particle distribution pattern was observed with mri , but pet and optical imaging showed homogeneous intensities at the tumor area . the human serum albumin coated nanoparticles manifested a prolonged circulation half - life . adopting this strategy , small drug molecules can be coloaded with iron oxide nanoparticles into human serum albumin to yield theranostic agents . recently , chen et al . reported a chelator free approach for preparation of radioarsenic labeled iron oxide nanoparticles . the radiolabeled nanoparticle was used as pet / mri agent for dual - modality imaging in vivo and lymph node mapping . this strategy can be extended for radiolabeling iron oxide nanoparticles with as for radiotherapeutic applications . in another study , yang et al . reported the synthesis of crgd - functionalized , dox - conjugated , and cu - labeled iron oxide nanoparticles for targeted anticancer drug delivery and pet / mr imaging.in vivo pet imaging and biodistribution studies in u87 tumor bearing mice showed that crgd - conjugated iron oxide nanocarriers showed a much higher level of tumor accumulation ( 5% id / g ) than crgd - free ones ( < 2% id / g ) . also , crgd - conjugated nanocarriers induced a significant amount of cytotoxicity in the u87 mg tumor cells , suggesting that dox was released from the iron oxide nanocarrier and entered the cell nucleus . thus , the potential of iron oxide nanoparticles for combined tumor - targeting drug delivery as well as multimodal imaging could be amply demonstrated . in recent times , there has been widespread interest in the use of biocompatible and biodegradable polymer nanoparticles for drug delivery . reported the synthesis of nanoparticles using cyclodextrin - containing polycations and sirna sequence targeting luciferase mrna . a bifunctional chelator , dota , was conjugated to the 5 end of sirna and used for labeling with cu . a dual - modality ( pet / optical ) imaging approach was used to investigate the biodistribution and functional activity of sirna delivered by the nanoparticles . in vivo pet / ct imaging in mice bearing luciferase - expressing neuro2a tumors was used to analyze the biodistribution and tumor localization of the sirna nanoparticles . also , bioluminescent imaging was used before and after pet imaging to enable correlation of functional efficacy with biodistribution data . it was observed that both nontargeted and transferrin - targeted sirna nanoparticles exhibited similar biodistribution and tumor localization . however , transferrin - targeted sirna nanoparticles could reduce tumor luciferase activity by 50% relative to nontargeted sirna nanoparticles , 1 d after injection . compartmental modeling was used to demonstrate that the primary advantage of targeted nanoparticles was associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization . the authors inferred that optimization of internalization might be the key factor for effective targeted therapy using this class of nanoparticles . the utilization of pet to quantify the uptake of intercellular adhesion molecule 1 ( icam-1 ) targeted , cu - labeled polymeric nanoparticles by the pulmonary endothelium was reported by rossin et al.in vivo pet imaging and biodistribution studies showed a 3- to 4-fold higher uptake in the lungs of mice injected with icam - targeted nanoparticles compared to that of the control group ( figure 4c ) . the lung uptake could be further enhanced by pretreating the mice with lipopolysaccharides probably due to icam-1 upregulation . however , a considerable release of small cu - radiometabolites from the nanoparticles beginning as early as 1 h after injection was observed , suggesting poor in vivo stability of the radiolabeled conjugate . an improved strategy where the radiolabeled nanoparticle remained stable in vivo was reported by simone et al . the authors developed a polymeric nanoparticle using a poly(4-vinylphenol ) polymer backbone which could directly be radiolabeled with i. the polymeric nanoparticles were coated with monoclonal antibodies targeting endothelial determinants . the radiolabeled nanoparticles were used for imaging the pulmonary vasculature and also for tracking the nanoparticle pharmacokinetics . this approach might find utility in image - guided delivery of therapeutics to the pulmonary endothelium in patients with acute and chronic respiratory diseases . the synthesis and utilization of poly(n - vinylpyrrolidone)-b - poly(-caprolactone ) nanoparticles ( 100 nm diameter ) for drug delivery was reported by zhu et al . the nanoparticles were conjugated with a near - infrared fluorescent dye , nir-797 , for in vivo optical imaging . an anticancer drug , paclitaxel ( ptx ) , was loaded in the polymeric nanoparticles with high drug loading content ( > 25% ) and encapsulation efficiency ( > 85% ) . the antitumor effect of ptx - loaded nanoparticles was evaluated , both in vitro on three different cancer cell lines and in vivo on a hepatic h22 tumor bearing mouse model using optical imaging . the antitumor effects of the ptx - loaded nanoparticles were further visualized using f - fdg pet scans . by combining the tumor volumes and survival rate measurements , it could be confirmed that ptx - loaded nanoparticles exhibited superior in vivo antitumor effect than taxol ( commercially available formulation of paclitaxel ) . in a similar study , liu et al . reported the synthesis of poly(ethylene glycol)-poly(caprolactone ) nanoparticles ( 70 nm diameter ) and evaluated their efficacy for drug delivery . as in the previous case , the polymeric nanoparticles were conjugated with nir-797 dye for investigating the biodistribution of the drug - loaded nanoparticles using in vivo optical imaging . an anticancer drug , docetaxel ( doc ) , could be encapsulated into the polymeric nanoparticles with a high drug loading content ( 20% ) and encapsulation efficiency ( > 80% ) . in vitro cytotoxicity test showed that doc - loaded nanoparticles inhibited the murine hepatic carcinoma cell line h22 in a dose - dependent manner , which was similar to taxotere , the commercialized formulation of docetaxel . however , in vivo tumor evaluation using optical imaging and f - fdg pet scans demonstrated the superiority of doc - loaded polymeric nanoparticles over taxotere . therefore , it could be envisaged that these highly efficient and biodegradable nanoparticles might find clinical utility in pet image - guided anticancer drug delivery in the near future . in a pioneering study , zhou et al . reported the synthesis of poly(lactide - co - glycolide ) nanoparticles ( 70 nm diameter ) , which could be utilized as brain penetrating nanocarriers for the treatment of glioblastoma . in order to illustrate the translational potential of brain - penetrating nanoparticles , the authors conducted a screen of 2,000 compounds that were previously approved by us fda to inhibit patient - derived brain cancer stem cells and encapsulated the best agent ( dithiazanine iodide ) into the nanocarrier . the radiolabeled brain - penetrating nanocarriers were administered by convection - enhanced delivery in rats bearing brain cancer stem cell derived xenografts . in vivo pet imaging demonstrated accumulation of the nanoparticles in the brain . also , a significantly increased survival in rats bearing brain cancer xenografts was observed , which demonstrated the potential of such brain - penetrating nanoparticles for targeted image - guided drug delivery for treatment of brain tumors . with a different strategy , chen et al . employed anionic poly(l - glutamic acid ) as a carrier to covalently link with camptothecin ( an anticancer drug ) , enabling encapsulation into supramolecular nanoparticle vectors . approximately five camptothecin molecules were conjugated to each polymer chain by ester bond formation , which could be degraded via esterase - mediated hydrolysis to allow controlled release of camptothecin under physiological conditions . the authors synthesized nanoparticles of two different sizes ( 37 and 104 nm ) , both of which were radiolabeled with cu and administered in mice bearing lewis lung carcinoma xenografts . in vivo pet imaging and biodistribution studies revealed that the smaller sized ( 37 nm ) nanoparticles exhibited higher tumor accumulation due to the epr effect . the superior in vivo antitumor efficacy of the 37 nm supramolecular nanoparticles was further validated by tumor reduction / inhibition studies . despite the encouraging results , the tumor uptake of the supramolecular nanoparticles was not impressive , which might be a deterrent for their use as potential drug delivery vehicles . however , the tumor uptake might be improved on conjugation with suitable targeting ligands . homopolymers or copolymers have long been explored as potential carriers in targeted drug delivery . the synthesis and characterization of pegylated star - shaped copolymer nanoparticles ( 2570 nm size ) containing core shell morphology for in vivo pet imaging was reported by fukukawa et al . these copolymers possessed a hydrophilic inner shell bearing reactive functional groups , and a central hydrophobic core . dota was conjugated to the functional groups in the inner shell for cu labeling . in vivo pet imaging and biodistribution studies in normal rats showed that copolymers with increasing peg shell thickness showed increased blood circulation and low accumulation in excretory organs . this preliminary study suggested the potential of such systems as for in vivo tumor imaging and targeted drug delivery . the synthesis of n-(2-hydroxypropyl)methacrylamide ( hpma ) copolymers for pet imaging and image - guided chemotherapy of prostate cancer was reported by yuan et al . the hpma copolymer was conjugated with dota for cu labeling and also with crgd peptide for targeting v3 integrin in tumor neovasculature . the tumor localization of the radiolabeled copolymer was visualized by pet in a mouse model bearing human prostate cancer xenografts . a time - dependent increase in radioactivity uptake in tumor - bearing mice injected with the hpma - crgd - dota - cu copolymers was observed , but this phenomenon was not seen in mice injected with control hpma - dota - cu copolymers . however , the tumor uptake observed for the targeted copolymer ( 2.75% id / g ) at 3 h post injection was slightly higher than what was observed with the nontargeted copolymer ( 1.29% id / g ) , suggesting that , along with active targeting , passive epr effect also plays a partial role in tumor localization of the radiolabeled copolymer . the findings from these studies might set the stage for further optimization and evaluation of the copolymer constructs for image - guided drug delivery in various tumor models . owing to their unique physical and chemical properties , the use of functionalized carbon - based nanomaterials is gaining popularity in many areas of biomedical research , including molecular imaging and drug delivery . since their discovery , the carbon nanotubes have become the most widely used carbon - based nanomaterial for biomedical applications . pet imaging using pegylated single walled carbon nanotubes ( 15 nm diameter , 100300 nm length ) conjugated with rgd peptides was reported by liu et al . dota was attached to the termini of the peg chains and used to conjugate cu . in vivo pet imaging and biodistribution studies showed that peg5400 modified single walled carbon nanotubes conjugated with crgd exhibited a high tumor uptake of 1015% id / g , with high target to nontarget ratio ( figure 4d ) . high tumor uptake of the radiolabeled single walled carbon nanotubes was observed over long periods ( > 24 h ) . in another study , mcdevitt et al . studied the biodistribution pattern of y labeled carbon nanotubes ( 1 nm diameter , 50 nm length ) without peg modification in normal mice . the radiolabeled agent cleared from the blood within 3 h and distributed predominantly to the kidneys , liver , spleen , and bone . suitable peg modification of carbon nanotubes is of paramount importance in order to reduce reticuloendothelial system uptake and prolong blood circulation time of the single walled nanotubes for finding utility in drug delivery approaches . graphene is another structurally robust , yet highly flexible , nanoplatform with potential for use as a drug delivery vehicle . hong et al . reported the synthesis of covalently functionalized nanographene oxide sheets ( 1050 nm ) , which were pegylated and conjugated with anti - cd105 monoclonal antibody ( trc105 ) for imaging tumor angiogenesis . the pegylated graphene oxide was also conjugated with nota for cu labeling . in vitro studies using human umbilical vein endothelial cells ( huvecs , high cd105 expression ) demonstrated strong and specific cd105-binding by the trc105 conjugated nanographene . also , in vivo pet imaging and biodistribution studies in 4t1 tumor bearing mice showed high tumor uptake ( 6% id / g ) within 0.5 h post injection , which remained fairly stable over time . these studies were further validated by ex vivo histological analyses , and vasculature specific targeting with little extravasation of trc105 conjugated graphene oxide could be demonstrated . the same group further evaluated ga - labeled nanographene oxide conjugated with trc105 , and similar results were obtained . this work was further improved by using cu - labeled reduced graphene oxide conjugated with trc105 for in vivo tumor vasculature targeting . reduced graphene oxide has more desirable properties for photothermal therapy than the more hydrophilic graphene oxide used in the previous studies , due to its strong absorbance in the near - infrared range.in vivo pet imaging revealed rapid tumor uptake ( 5.5% id / g ) of cu - labeled nanoplatform with excellent tumor contrast . in all these studies , trc105 conjugated nanoparticles exhibited little extravasation in the 4t1 tumor , indicating the advantages of tumor vasculature targeting using such nanoplatforms . it can be envisaged that the promising results obtained in these studies can open up new avenues for image - guided drug delivery and cancer therapy using graphene oxide based nanoplatforms . the concept of theranostics has also played a vital role in radiation - based therapies , especially , using targeted radiopharmaceuticals . in this approach , a radiation dose is specifically administered to the cancerous lesions using peptides , proteins , or antibodies radiolabeled with suitable therapeutic radionuclides such as y , i , or lu . the same targeting ligands can also be conveniently radiolabeled with suitable positron emitters such as f , ga , cu , or zr , thereby providing exciting opportunities to guide such therapies using pet . this approach plays a dominant role in diagnosis of the disease in its early stage , validation of the targeting strategy , and development of novel therapeutic radiopharmaceuticals . thus , it facilitates better , faster , and cost - effective decision making , helping to eliminate failures in the targeted radiotherapy pathway and advance only with the promising candidates to receive such therapies . despite the availability of a wide variety of pet radiopharmaceuticals , f - fdg is still the most widely used radiotracer in cancer management , and the pivotal role of f - fdg - pet / ct in modern nuclear medicine needs hardly to be reiterated . excellent review articles have appeared in recent times which have summarized the clinical diagnosis and therapeutic response evaluation using f - fdg and other f - based radiotracers , and hence these are not discussed here further . another pet radioisotope which is gaining significant clinical attention in recent times is ga ( t1/2 = 68 min ) . the convenient availability of this radioisotope from ge / ga generators without the dependence on onsite cyclotrons makes it economical to use for a wide variety of pet scans . in particular , from the perspective of image - guided therapy , the radiolabeled peptides targeting somatostatin receptors , overexpressed in the majority of neuroendocrine malignancies , have a great potential for both imaging and therapy of tumors where other therapies fail . the development of ga - labeled somatostatin analogues such as dotanoc ( dota-1-nal - octreotide ) , dotatoc ( dota - d - phe - tyr - octreotide ) , or dotatate ( dota - d - phe - tyr - thr - octreotide ) for pet / ct imaging has significantly improved the diagnosis of neuroendocrine tumors . in a typical example , gains et al . investigated the efficacy of pet / ct using ga - dotatate to select children with primary refractory or relapsed high - risk neuroblastoma for treatment with lu - dotatate and also evaluated whether this is a viable therapeutic option for those children . imaging with ga - dotatate could indicate the expression of somatostatin receptors and was of paramount importance for guiding radiotherapy ( figure 5a ) . post - therapy of lu - dotatate , ga - dotatate scans was used to assess the response ( figure 5a ) , and a positive therapeutic outcome with a regression in some lesions and an apparent block of the metastatic activity could be observed in many cases . in a similar study , budiawan et al . investigated the application and role of ga - dotatate pet / ct imaging in non - radioiodine - avid refractory thyroid cancer patients who have undergone peptide receptor radionuclide therapy ( prrt ) with y / lu - dotatate . the authors concluded that prrt guided by pet imaging is an effective therapeutic option with minimal toxicity , good response rate , and excellent survival benefits . in a different study , wu et al . has reported the use of ga - labeled sir spheres as pet imaging surrogate for distribution assessment and radiation dose estimation of y - sir - spheres , which are currently used in the treatment of solid liver tumors . the authors observed that ga - sir - spheres had good in vivo stability and localization of the radiolabeled microparticles in the liver could be observed using pet imaging . similar results were obtained by avila - rodriguez et al . on using cu or y labeled sir spheres . ( i ) images showing ga - dotatate avid lesions in t4 vertebral body and 3 metastases in liver ( arrow ) . physiologic uptake is seen in pituitary , kidneys , bladder , stomach wall , liver , and spleen . ( ii ) images from repeated ga - dotatate pet / ct 1 y later after 3 administrations of lu - dotatate , showing metabolic partial response with reduction in suvmax of lesion in t4 and liver and no new lesions . adapted with permission from ref ( 190 ) . ( i ) a patient with liver and bone metastases , and ( ii and iii ) two patients with multiple bone metastases . a number of lesions have been specifically indicated by arrows . adapted with permission from ref ( 198 ) . another example , close to clinical translation , is represented by pet imaging of human epidermal growth factor receptor 2 ( her2 ) expressions . the overexpression of the her2 is observed in 15% of breast cancers and associated with poor prognosis in terms of overall survival . generally , y or i labeled trastuzumab is used for targeted radiotherapy of primary or metastatic breast cancer , in experimental , preclinical , or clinical settings . pet imaging of her2- positive lesions in patients with metastatic breast cancer was reported by dijkers et al . the antibody trastuzumab was radiolabeled with zr , a pet imaging surrogate for y. administration of zr - trastuzumab at appropriate doses allowed visualization and quantification of uptake in her2-positive lesions in patients with metastatic breast cancer by pet ( figure 5b ) . the authors concluded that pet imaging of her2 expression would aid in improving diagnosis , staging of the disease , guiding trastuzumab therapy , and monitoring the therapeutic response . a large number of other pet radiopharmaceuticals for molecular imaging and personalized cancer management have also been reported , details of which can be found in recent review articles . we would apologize to those whose work could not be presented here , mainly due to the vastness of the field and availability of enormous literature which could not be summarized in a single review . in the last several decades , major milestones have been reached in every area of cancer care and a variety of anticancer drugs have been developed which are now commercially available worldwide . though most of these anticancer agents have the potential to be effective at sufficiently high doses , they are often associated with severe systemic side effects that can not be tolerated by patients who are already weak due to effects of cancer . in a majority of the cases , the success of cancer therapy typically hinges upon circumventing the dose - limited toxicity while administering such drugs . additionally , many conventional therapeutic agents often fail due to their limited ability to reach the target tissue and their poor selectivity against cancerous lesions . ideally a drug should possess perfect specificity to cancerous cells and have no effect on the rest of the body . to achieve these objectives , a variety of drug delivery systems have been engineered for the targeted delivery and controlled release of therapeutic agents to specifically kill the cancerous cells . the full potential of the drug delivery systems extends beyond treatment , and several image - guided approaches have now been implemented in areas ranging from new therapeutic target discovery to effectively monitoring tumor pharmacokinetics and drug distribution to modulation of drug release at the target site . in particular , pet image - guided drug delivery provides a means for treating a variety of diseases with minimal systemic involvement while concurrently monitoring therapeutic efficacy . this minimally invasive approach provides a comprehensive answer to many challenges with conventional therapeutic approaches and is expected to lead to a paradigm shift in cancer patient care . the major advances in targeted drug delivery have been attributed to the recent progress in nanotechnology that has resulted in the development of nanosized drug delivery platforms having distinct advantages in cancer therapy . the versatile nanoplatforms provide opportunities for multifunctionalization so that a single platform can be used to detect and treat tumors , monitor treatment response , and thus guide therapeutic regimes . the nanoformulations can be functionalized to minimize clearance by the immune system and prolong circulation times , and also for attachment of suitable vectors ( peptides , proteins , antibodies , etc . ) targeting specific receptors , thereby enhancing tumor uptake through epr effect as well as receptor - mediated endocytosis . the nanomaterial based delivery systems also result in improved treatment effects by protecting entrapped drugs from degradation during their delivery . when two or more molecular imaging techniques are used in conjunction , they would provide synergistic information when compared with any single imaging modality . additionally , multiple therapeutic agents such as chemotherapy , antiangiogenic , or gene therapy agents can be simultaneously delivered by nanocarriers to tumor sites to enhance the effectiveness of therapy . despite these advantages , it must be admitted that the field of pet image - guided drug delivery is still in its infancy and more systematic studies to understand the mechanisms for targeting and drug delivery would be required in order to translate these novel discoveries into clinical impact . the potential challenges to clinical translation of the drug delivery systems are in vivo characterization of the drug carriers , preclinical validation of targeting and delivery , studies of biodistribution , pharmacokinetics , pharmacodynamics , and toxicity , and scale - up manufacturing of delivery systems . another major challenge lies in overcoming the biological barriers to deliver optimum amount of therapeutics into tumors and cells . moreover , the performance of the drug delivery carriers depends on several limiting factors which include the synthesis method adopted , their size and shape , internal structure of the drug carrier , drug loading methodology , surface functionalization and conjugation strategy adopted for attaching targeting ligands to the carrier platforms , etc . the issues related to toxicity of nanosized drug delivery platforms can be addressed by the use of biocompatible and biodegradable polymeric nanoparticles for drug delivery , which have received considerable attention in the recent times . also , stimulus - responsive polymeric nanomaterials can be synthesized which mimic the behavior of biological molecules , where external stimuli or changes in local environment can trigger a change in property to regulate drug release . such smart nanoparticle systems when coupled with suitable targeting ligands can probably best minimize off - target effects and maximize programmability , thereby offering the possibility of radically changing the practice of drug delivery . while numerous obstacles face all new technologies , materializing the opportunities presented by pet image - guided drug delivery requires addressing the significant interdisciplinary challenges and biological barriers . besides these , several other complex factors , such as considerable regulatory hurdles , limited potential market , lobbying by the manufacturers of established anticancer drugs , lack of reimbursement strategies by the insurance agencies for such novel strategies , etc . , might impede the bench to bedside translation of this promising approach . the concerted efforts of all stakeholders , which include scientists from academia as well as industries , progressive and technology savvy physicians , radiologists , surgeons , program advisory boards , regulatory authorities , and grant review panels , would be required , both to create enthusiasm for developing these new concepts and also to prevent adverse messaging based on myths , conjectures , hyperbole , and bias . this in turn would provide impetus to further research which might aid in clinical translation of pet image - guided drug delivery approaches and thus achieve the ultimate goal of personalized medicine in the near future .
positron emission tomography ( pet ) is an important modality in the field of molecular imaging , which is gradually impacting patient care by providing safe , fast , and reliable techniques that help to alter the course of patient care by revealing invasive , de facto procedures to be unnecessary or rendering them obsolete . also , pet provides a key connection between the molecular mechanisms involved in the pathophysiology of disease and the according targeted therapies . recently , pet imaging is also gaining ground in the field of drug delivery . current drug delivery research is focused on developing novel drug delivery systems with emphasis on precise targeting , accurate dose delivery , and minimal toxicity in order to achieve maximum therapeutic efficacy . at the intersection between pet imaging and controlled drug delivery , interest has grown in combining both these paradigms into clinically effective formulations . pet image - guided drug delivery has great potential to revolutionize patient care by in vivo assessment of drug biodistribution and accumulation at the target site and real - time monitoring of the therapeutic outcome . the expected end point of this approach is to provide fundamental support for the optimization of innovative diagnostic and therapeutic strategies that could contribute to emerging concepts in the field of personalized medicine . this review focuses on the recent developments in pet image - guided drug delivery and discusses intriguing opportunities for future development . the preclinical data reported to date are quite promising , and it is evident that such strategies in cancer management hold promise for clinically translatable advances that can positively impact the overall diagnostic and therapeutic processes and result in enhanced quality of life for cancer patients .
Introduction PET Imaging Carriers for PET Image-Guided Drug Delivery Summary and Future Perspectives
the major goal of this approach is to use molecular imaging to maximize effective therapy in diseased tissues and to minimize systemic drug exposure in order to reduce toxicities . in the past decade , innumerable studies have been reported on the synergistic use of molecular imaging with targeted drug delivery , and this strategy has now matured with promises to fulfill the vision of personalized medical treatment in the near future . when molecular imaging probes are coadministered as part of the drug delivery system , it can help to achieve multiple goals , such as real - time and concurrent assessment of drug delivery efficiency / targeting , in vivo fate of drug and sites of localization / accumulation , modes of excretion , imaging , and monitoring the progress of drug treatment , in a single dosing . currently , several noninvasive image - guided modalities are being used in biomedical and clinical settings , which include magnetic resonance imaging ( mri ) , computed tomography ( ct ) , positron emission tomography ( pet ) , single photon emission computed tomography ( spect ) , optical imaging , and ultrasonography . in this review , we aim to provide a timely and comprehensive overview of the pet image - guided drug delivery approaches reported to date , with focus on quantitative assessments of tumor - targeted therapeutic delivery , distribution , uptake , and response . the development of various carriers for site- and event - specific targeting and controlled drug release are summarized , and the great potential and intriguing opportunities for future development which might help in bringing this exciting research avenue closer to a clinical reality are discussed . targeted drug delivery systems have the potential to improve these undesirable features , and when used in conjunction with pet imaging , they are effective in increasing safety to efficacy ratio and decreasing dose , which in turn reduces adverse reactions and toxicity of drugs . , which can circulate in vivo for more than 1 day , intermediate - lived or long - lived radioisotopes such as ga ( t1/2 = 9.7 h ) , cu ( t1/2 = 12.7 h ) , zr ( t1/2 = 78.4 h ) , or i ( t1/2 = 4.17 day ) would be the ideal choices for pet image - guided drug delivery . the radiolabeled agent must demonstrate high in vitro and well as in vivo stability for successful use in pet image - guided drug delivery . in order to optimize -gus - based prodrug therapies , a pet tracer , f - labeled 1-o-(4-(2-fluoroethyl - carbamoyloxymethyl)-2-nitrophenyl)-o--d - glucopyronuronate ( f - feanga ) , was evaluated for imaging of -gus in tumor ( c6 gliomas ) and inflammation models.in vivo pet imaging and biodistribution studies showed high uptake of the radiotracer in tumor , high target to nontarget ratio , and rapid renal clearance . the different nanoparticle based systems which can be radiolabeled with suitable positron emitting radioisotopes for pet image - guided drug delivery are discussed in the following text . among the various metallic nanoparticles reported to date , gold nanoparticles are most widely used for biomedical applications , including drug delivery and novel diagnostic and therapeutic approaches , due to their biocompatibility , small size , ease of characterization , and rich surface chemistry . the encouraging results obtained in this study hold promise for future image - guided drug delivery and targeted cancer therapy using this class of nanomaterials . to achieve these objectives , a variety of drug delivery systems have been engineered for the targeted delivery and controlled release of therapeutic agents to specifically kill the cancerous cells . the full potential of the drug delivery systems extends beyond treatment , and several image - guided approaches have now been implemented in areas ranging from new therapeutic target discovery to effectively monitoring tumor pharmacokinetics and drug distribution to modulation of drug release at the target site . in particular , pet image - guided drug delivery provides a means for treating a variety of diseases with minimal systemic involvement while concurrently monitoring therapeutic efficacy . despite these advantages , it must be admitted that the field of pet image - guided drug delivery is still in its infancy and more systematic studies to understand the mechanisms for targeting and drug delivery would be required in order to translate these novel discoveries into clinical impact . this in turn would provide impetus to further research which might aid in clinical translation of pet image - guided drug delivery approaches and thus achieve the ultimate goal of personalized medicine in the near future .
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parental obesity increases the risk of obesity in offspring through genetic , biological , and environmental influences [ 13 ] . maternal obesity , weight gain , and increased body mass index ( bmi ) between pregnancies and gestational diabetes increase risks of offspring obesity , type 2 diabetes mellitus ( t2 dm ) , and cardiovascular disease ( cvd ) in the offspring [ 47 ] . consistent with the chronic low - grade inflammatory state of obesity , elevated concentrations of interleukin-6 ( il-6 ) and c - reactive protein ( crp ) have been observed in obese pregnant women . higher maternal levels of il-6 were predictive of the increased growth and adiposity in the offspring . maternal diet - induced obesity in sheep resulted in higher fetal triglyceride ( tg ) levels and upregulated inflammatory signaling . offspring of rats injected with il-6 throughout pregnancy had an altered inflammatory profile associated with greater body fat mass and reduced insulin sensitivity . epigenetic processes are important mediators of early - life environment , metabolism , and body composition in offspring . epigenetic changes in genes involved in the inflammatory response have been observed in response to maternal diet and adiposity . differences in methylation levels of the peroxisome proliferator - activated receptor alpha ( ppara ) and glucocorticoid receptor promoters have been observed in the liver of offspring of rats fed with protein - restricted diet during pregnancy [ 13 , 14 ] . methylation status of retinoid x receptor alpha ( rxra ) promoter in umbilical cord tissue dna was correlated with increased adiposity in children of mothers with lower carbohydrate intake . epigenetic mechanisms have been involved in inflammation and associated disorders including obesity [ 15 , 16 ] . bariatric surgery is known to improve glucose and lipid metabolism [ 1719 ] and prevent arterial hypertension and type 2 diabetes [ 1921 ] . bariatric surgery also decreases the low - grade inflammation associated with obesity . in previous studies , we demonstrated that children born after maternal bariatric surgery ( ams ) exhibited lower prevalence of severe obesity , greater insulin sensitivity , and improved lipid profile in comparison to siblings born before maternal surgery ( bms ) [ 23 , 24 ] . in addition , ams children demonstrated differences in a marker of chronic low - grade inflammation , namely the plasma c - reactive protein ( crp ) . this study was designed to analyze the relationship between maternal biliopancreatic diversion surgery and methylation levels of genes involved in immunologic and inflammatory pathways in bms and ams offspring . correlation analyses between gene methylation , expression , and plasma crp levels were conducted for the il-8 pathway . women from qubec city and surrounding areas ( administrative regions of capitale - nationale , mauricie , and chaudire - appalaches ) who had given birth before and after biliopancreatic diversion with duodenal switch for severe obesity were eligible for the current study . a subset of 20 unrelated mothers was recruited along with 50 siblings born before and after surgery ( 25 bms and 25 ams offspring ) . mothers and offspring attended the qubec heart and lung institute ( qubec city , qubec , canada ) or a regional hospital for assessment between july and october 2010 . there were 17 mothers with siblings born before and after surgery ( 23 bms and 24 ams ) , two with bms offspring only ( 2 bms ) , and one mother with only one ams offspring . this study was approved by the qubec heart and lung institute ethics committee . written informed consent was obtained from adults and mothers with assents from minors . percent body fat was determined for individuals at 6 years or more ( bms , n = 23 ; ams , n = 17 ) using bioelectric impedance analysis ( tanita , arlington heights , il , usa ) . weight , height , and resting systolic ( sbp ) and diastolic ( dbp ) blood pressure were obtained using standardized procedures and measured at the interview . bmi was calculated for mothers , and bmi percentile for children was obtained from the national health and nutrition examination survey 2000 charts . bmi z - score was calculated for children using charts from the centers for disease control and prevention . severe obesity in offspring was defined using age- and sex - corrected adiposity measurements ( bmi z - score > 3 ; bmi percentile > 98% ) . blood samples were collected from mothers and offspring after an overnight fast into bd vacutainer tubes containing edta and paxgene blood rna collection tubes ( qiagen , valencia , ca , usa ) . plasma tg , high - density lipoprotein cholesterol ( hdl - c ) , low - density lipoprotein cholesterol ( ldl - c ) , total cholesterol ( total - c ) , total - c / hdl - c ratio , glucose , and insulin concentrations were measured as previously described . the homeostatic model of insulin resistance ( homa - ir ) index was calculated as glucose insulin/22.5 . genomic dna was isolated from the blood buffy coat using the genelute blood genomic dna kit ( sigma , st . louis , mo , usa ) and quantified using both nanodrop spectrophotometer ( thermo scientific , wilmington , de , usa ) and picogreen dna methods . dna ( 1 g ) was bisulfite converted , and quantitative genome - wide methylation analysis was conducted using the infinium humanmethylation450 beadchip ( illumina , san diego , ca , usa ) . arrays were processed at the mcgill university and gnome qubec innovation centre ( montral , canada ) according to the manufacturer 's instructions ( illumina , san diego , ca , usa ) . the infinium humanmethylation450 beadchip array was designed for genome - wide methylation analysis with coverage targeted across gene regions with sites in the promoter region , 5utr , first exon , gene body , and 3utr . the beadchip interrogates more than 485 000 methylation sites at single - nucleotide resolution . visualization and analysis of methylation data were conducted using the genomestudio software version 2011.1 ( illumina inc . ) and the methylation module . methylation levels ( beta values ; ) were estimated as the ratio of signal intensity of the methylated alleles to the sum of methylated and unmethylated intensity signals of the alleles ( value = c/(t + c ) ) . internal control probe pairs were used for data correction ( background subtraction and normalization ) , and cpg sites with a detection p value > 0.05 were removed from analysis . differences in methylation levels between bms and ams groups ( mean values ) were tested using illumina custom model . false - discovery - rate - corrected ( fdr - corrected ) p values and diffscores were computed . diffscore is a differential methylation score calculated from p values and differences in ( delta ) between the two groups and was used here as the main statistical results for comparison of bms and ams siblings . differential methylation analysis between bms and ams siblings revealed 5698 genes ( 14 466 probes ) with significant differences in methylation levels ( fdr - corrected diffscore |13 distribution of differentially methylated probes obtained from comparison between bms and ams offspring is provided in supplementary table 1 available online at http://dx.doi.org/10.1155/2013/492170 . total rna was isolated and purified from whole blood for 46 offsprings ( 23 bms and 23 ams ) using paxgene blood rna kit ( qiagen ) following the manufacturer 's recommendations . the integrity of the purified rna was analyzed using both nanodrop ( thermo scientific , wilmington , de , usa ) and 2100 bioanalyzer ( agilent technologies , cedar creek , tx , usa ) . expression levels were measured using the humanht-12 v4 expression beadchip ( illumina inc . ) which contains more than 47 000 probes derived from ncbi refseq release 38 . microarray experiments were carried out using 250 ng of total rna and processed according to the manufacturer 's instructions at the mcgill university and gnome qubec innovation centre ( montral , canada ) . expression data were visualized and analyzed using the flexarray software ( version 1.6 ) , and the lumi algorithm was used for expression data analysis and normalization . probe detection analysis was conducted using the flexarray filter algorithm to generate a list of expressed probes . to be considered as significantly expressed , a probe had to show a detection p value 0.05 in at least 25% of the samples of a group . ( sam ) algorithm with unequal - variance ( welch 's ) t - statistic was used to assess differences between bms and ams siblings for significantly expressed probes . the sam algorithm uses permutations of the data set to generate an empirical null distribution and to assess the significance of observed effects , rather than using the hypothetical t - distribution . in addition , a cutoff of fold change 1.2 or 0.83 ( symmetrical fold change 1.2 or 1.2 ) was used . p values obtained from permutations and fold change cutoff values were then used to minimize the chances of false positives . differential expression analysis revealed a total of 862 probes differentially expressed with a symmetrical fold change |1.2| ( p 0.05 ; fold change < 0.83 or > 1.2 ) . gene expression microarray results were validated using real - time polymerase chain reaction ( rt - pcr ; applied biosystems gene expression assays ; applied biosystems , foster city , ca , usa ) . spearman correlations were computed for expression levels assessed by microarray and rt - pcr methods in a subset of genes ( bcl2 , nm_000633 ; ccnd2 , nm_001759 ; ncf2 , nm_000433 ; pik3r1 , nm_181523 ; prkch , nm_006255 ) for whom correlations between gene expression and methylation levels were observed . samples were analyzed in duplicate using predesigned probes ( hs00608023_m1 , hs00153380_m1 , hs01084940_m1 , hs00933163_m1 and hs00178933_m1 ) and calibrated to the actb housekeeping gene ( endogenous control ; actb : hs99999903_m1 ) . relative quantification estimations were performed on an applied biosystems 7500 real - time pcr system ( applied biosystems , foster city , ca , usa ) . analysis of potentially altered pathways was conducted using the knowledge base of the ingenuity pathway analysis ( ipa ) system . the lists of 14 466 differentially methylated and 862 differentially expressed probes produced from differential methylation and differential expression analysis , respectively , were submitted to ipa . ipa measured the likelihood that these genes participate in a particular pathway and calculated p values using a right - tailed fisher 's exact test for each pathway . clinical data were expressed as mean sd . the effect of bariatric surgery in mothers was assessed using a within - subject paired t - test . differences in anthropometric data between bms and ams siblings were tested using analysis of variance ( general linear model , type iii sum of squares ) with adjustments for the effects of sex and puberty . in the absence of tanner scores , we arbitrarily defined puberty as 12 years for girls and 14 for boys . differences in severe obesity between bms and ams siblings were evaluated using bmi percentile and bmi z - score and tested using fisher 's exact test . p values for crp were adjusted for the effects of sex , puberty and bmi percentile , an age- and sex - corrected adiposity measurement . pairwise pearson correlations between methylation , expression , and plasma crp were computed for the il-8 signaling pathway that was overrepresented in both differential methylation and expression analyses . partial pearson correlations were also computed for the il-8 signaling pathway after adjustments for age and sex . statistical analyses were conducted using the sas software version 9.2 ( sas institute inc . ) . a total of 20 mothers ( 41.0 5.3 years ; mean sd ) who had undergone biliopancreatic diversion were recruited . mean postoperative follow up was 12 years and 2 months at the time of the study . bariatric surgery induced dramatic weight loss in mothers . on average , the women weighted 121.5 19.2 kg ( bmi = 45.0 7.2 ) at the time of the surgery and 74.8 11.9 kg ( bmi = 27.6 4.8 ) at the recruitment interview . the mean weight loss of 46.7 16.1 kg was associated with significant improvements in plasma lipids ( p 0.01 for tg , hdl - c , ldl - c , total - c , and total - c / hdl - c ratio ) and reductions in insulin resistance ( homa - ir index ; p 0.001 ) and blood pressure ( sbp and dbp ; p 0.001 ) with a trend toward lower plasma glucose levels ( p = 0.06 ) . the 50 bms and ams offspring of 20 mothers were aged between 2 years and 8 months and 24 years and 11 months with similar sex distribution ( 40% males in both bms and ams ) . bms offspring were born 3 years and 5 months before and ams offspring were born 3 years and 7 months after maternal bariatric surgery . bms siblings were older ( mean age 14.9 y versus 9.6 y ) than ams siblings ( table 1 ) . following adjustments for the effects of sex and puberty , ams offspring showed a trend toward lower body fat percent ( p = 0.07 ) . there was a significantly lower prevalence of severe obesity in ams using bmi percentile ( p = 0.01 ) or bmi z - score ( p = 0.05 ) as indicators . ams offspring had improved fasting insulin levels ( p = 0.03 ) , lower homa - ir index ( p = 0.03 ) , and blood pressure . plasma crp levels were different in bms versus ams offspring ( 5.14 7.90 versus 3.58 11.09 ; p = 0.03 ) even after taking into account the sex effect ( p = 0.03 ) . these differences in plasma crp levels were no longer significant after further adjustments for the effects of puberty , and bmi ( bmi percentile ) . from the list of 5698 differentially methylated genes between bms and ams offspring , ipa revealed 160 pathways significantly overrepresented ( p < 0.05 ) . similar analysis conducted from the list of 862 differentially expressed genes between bms and ams offspring identified 68 overrepresented pathways . the list of the top 20 overrepresented inflammatory and immune pathways identified from differential methylation analysis along with corresponding results obtained from gene expression analysis is presented in table 2 . among them , 5 pathways were found to be overrepresented in both methylation and expression : il-8 signaling , icos - icosl signaling in t - helper cells , role of nfat in regulation of the immune response , b - cell receptor signaling , and glucocorticoid receptor signaling . among the 5 pathways listed above it was thus selected for further analyses to highlight the potential link between gene methylation , expression , and inflammation . among the 193 genes assigned to the il-8 signaling pathway by ipa , 70 genes represented by 214 methylation probes were found to be differentially methylated in bms versus ams ( supplementary table 2 ) . corresponding expression data were available for 102 of these probes ( 46 genes ) . among the 46 genes analyzed , correlations between dna methylation and expression were significant for 17 genes ( 23 methylation probes ; table 3 ) . further analyses were then focused on these 17 genes to correlate gene methylation and expression with crp levels . there were significant correlations between gene methylation and plasma crp levels for 16 genes . gene expression levels of 5 genes were found to correlate with plasma crp levels ( table 3 ) . all 5 genes with significant correlations between gene expression and plasma crp levels ( bcl2 , ccnd2 , ncf2 , pik3r1 , and prkch ) also demonstrated correlations between methylation and expression and between gene methylation and plasma crp levels ( table 3 ) . following adjustments for the effects of age and sex , all 17 genes initially showing correlation between gene methylation and expression levels demonstrated significant correlation after adjustments . in addition , the rhoh gene ( methylation probe cg26163153 ) reached significance level while initially showing a trend toward significant correlation ( p = 0.09 ) . from the 16 genes correlated with crp levels only one ( pik3r1 ) of the five genes with significant correlations between gene methylation , expression , and crp levels remained significant after adjustments , while trends toward significant correlation were found for bcl2 , ccnd2 , and ncf2 . validation of gene expression microarray data for these 5 genes demonstrated significant correlations between microarray and rt - pcr results for 4 of the genes assessed ( ccnd2 , r = 0.358 , p = 0.02 ; ncf2 , r = 0.574 , p < 0.0001 ; pik3r1 , r = 0.349 , p = 0.02 ; prkch , r = 0.462 , p = 0.001 ) , while bcl2 did not reach significance ( r = 0.223 , p = 0.14 ) . we demonstrated here for the first time that surgical treatment of severe maternal obesity results in sustained differences in the methylome and transcriptome of genes involved in inflammatory pathways in ams offspring compared to bms siblings . the proportion of 34.7% of differentially methylated genes ( 1976 of 5698 genes ) being identified by two probes or more strengthen , the validity of our results arguing for a regulation of methylation levels in offspring . differential analysis of gene methylation and expression levels revealed important differences between the sibling groups . pathway analysis from differentially methylated genes in bms versus ams revealed important regulation of inflammatory and immunity pathways : 29 inflammatory- or immunity - related pathways being identified among the top 50 overrepresented pathways ( data not shown ) . pathway analysis from differentially expressed genes between bms and ams revealed 5 overrepresented pathways among the top overrepresented pathways from differential methylation data ( table 2 ) . overrepresentation of genes from the il-8 signaling pathway was identified from both methylation and expression data , and significant correlations were found between gene methylation and expression levels . moreover , 5 genes from the il-8 pathway also demonstrated correlation of gene methylation and expression levels with plasma crp levels . despite the fact that the impact of dna methylation on gene expression seems to be site and location dependent , results obtained here for the il-8 pathway generally comply to the known phenomenon that intragenic methylation ( gene body , 5utr and 3utr ) correlates with increased gene expression , while promoter methylation is associated with decreased expression [ 30 , 31 ] . gene expression profiling in obese patients who had undergone weight loss or bariatric surgery demonstrated a regulation of inflammation - related transcripts and changes in inflammatory marker levels [ 32 , 33 ] . in the present study , we extend these data by reporting that metabolic improvements in mothers following biliopancreatic diversion surgery are reflected in gene methylation and expression levels of immunologic and inflammatory genes in offspring , indeed reinforcing the involvement of epigenetic differences in immunity and inflammatory genes in the determination of the offspring phenotypes . a limited number of epigenetic studies have evaluated the impact of maternal nutrition and obesity on methylation of immune and inflammatory genes in offspring . recently , human studies have revealed the implication of immune and inflammatory gene methylation in children with respect to adiposity . methylation levels of the rxra gene in umbilical cord tissue dna of healthy neonates were associated with maternal carbohydrate intake in early pregnancy and childhood adiposity at 9 years old . relton and collaborators reported associations between anthropometric indices ( bmi , fat mass , lean mass , and height ) in 9 years old children and methylation levels of selected immune and inflammatory genes in cord blood samples . with a different design involving siblings born under different maternal obesity condition , our study supports the role of epigenetic factors in immunity and inflammatory genes in the determination of offspring phenotypes . transcriptomic data revealed that improvements seen in mothers following bariatric surgery are reflected in gene expression levels of immunologic and inflammatory genes in offspring . animal studies have demonstrated a similar impact of maternal obesity and nutrition on immune and inflammatory genes in the offspring [ 3537 ] . these results are consistent with those obtained from the present gene expression analysis in bms offspring versus ams siblings and may provide potential mechanisms for higher levels of systemic inflammation found in bms offspring . we used dna extracted from blood as it is more convenient and acceptable than biopsies of target tissues , especially in children . although epigenetic signatures have been shown to differ between tissues , the overall impact is minor given the known similarities in methylation patterns . gene expression levels are also known to be tissue - or cell - type dependent but also correlated [ 4042 ] thus justifying the use of blood in clinical studies . differential gene expression analysis led to a limited number of differentially expressed genes ( 862 ) in comparison with the 5698 differentially methylated genes identified . this discrepancy may be attributable to differences in statistical algorithms , in corrections for multiple testing , or in the different cutoffs used ; a cutoff fold change 1.2 or 0.83 was applied for gene expression analysis . nonetheless , pathway analyses conducted from differentially methylated and expressed genes are concordant and demonstrate an overrepresentation of immune and inflammatory pathways . we did not stratify for sex in these gene methylation and expression analyses ; several studies did not find sex - specific autosomal methylation patterns [ 43 , 44 ] . owing to the before - after design of the study , we were unable to fully correct for the age difference between the sibling groups . this design and the young age of the ams offspring ( 80% being younger than 12 years old ) do not allow discussing the long - term effects in children . longitudinal studies are thus needed to further assess long - term effects of maternal bariatric surgery in the offspring . age - related differential methylation has been reported but was shown to be site- and location dependent [ 4547 ] and represented a very small proportion of cpg sites . furthermore , longitudinal studies in humans demonstrated relative stability of dna methylation over time . in regards to the general conclusion drawn from our results , the potential impact of maternal weight regain on offspring is limited in the current study considering the sustained weight loss achieved with the biliopancreatic diversion with duodenal switch [ 19 , 48 , 49 ] . analysis of maternal clinical files along with the lack of inflammation - related perioperative complications in biliopancreatic diversion with duodenal switches argues against the potential impact of specific inflammatory condition in mothers . the current study was not designed to analyze methylation and expression changes induced in mothers following bariatric surgery . however , based on a recent meta - analysis demonstrating a reduction in low - grade inflammation associated with obesity following bariatric surgery , it would be tempting to speculate that these changes may be accompanied by similar changes in methylation and expression levels . improvements in inflammatory profiles in mothers following bariatric surgery may be potentially responsible for the inflammatory effects seen in offspring through modification of the in utero environment and improved methylation profiles in ams offspring . using analysis of gene methylation and expression data , we reveal that improvements seen in mothers following bariatric surgery reflect on the expression and methylation levels of genes involved in immune and inflammatory pathways in the offspring . these results argue for a beneficial effect of maternal weight loss before pregnancy and provide potential mechanisms of action for physiological improvements through regulation of methylation and expression of genes involved in inflammatory and immune pathways . since inflammation is recognized as an independent risk factor for cardiovascular diseases ( cvd ) and is also associated with the development of the metabolic syndrome , insulin resistance , hypertension , and obesity [ 50 , 51 ] , it would be important to conduct longitudinal studies to examine the long - term effect of maternal bariatric surgery on offspring .
background . maternal obesity , excess weight gain and overnutrition during pregnancy increase risks of obesity , type 2 diabetes mellitus , and cardiovascular disease in the offspring . maternal biliopancreatic diversion is an effective treatment for severe obesity and is beneficial for offspring born after maternal surgery ( ams ) . these offspring exhibit lower severe obesity prevalence and improved cardiometabolic risk factors including inflammatory marker compared to siblings born before maternal surgery ( bms ) . objective . to assess relationships between maternal bariatric surgery and the methylation / expression of genes involved in the immune and inflammatory pathways . methods . a differential gene methylation analysis was conducted in a sibling cohort of 25 bms and 25 ams offspring from 20 mothers . following differential gene expression analysis ( 23 bms and 23 ams ) , pathway analysis was conducted . correlations between gene methylation / expression and circulating inflammatory markers were computed . results . five immune and inflammatory pathways with significant overrepresentation of both differential gene methylation and expression were identified . in the il-8 pathway , gene methylation correlated with both gene expression and plasma c - reactive protein levels . conclusion . these results suggest that improvements in cardiometabolic risk markers in ams compared to bms offspring may be mediated through differential methylation of genes involved in immune and inflammatory pathways .
1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusion
maternal obesity , weight gain , and increased body mass index ( bmi ) between pregnancies and gestational diabetes increase risks of offspring obesity , type 2 diabetes mellitus ( t2 dm ) , and cardiovascular disease ( cvd ) in the offspring [ 47 ] . in previous studies , we demonstrated that children born after maternal bariatric surgery ( ams ) exhibited lower prevalence of severe obesity , greater insulin sensitivity , and improved lipid profile in comparison to siblings born before maternal surgery ( bms ) [ 23 , 24 ] . this study was designed to analyze the relationship between maternal biliopancreatic diversion surgery and methylation levels of genes involved in immunologic and inflammatory pathways in bms and ams offspring . correlation analyses between gene methylation , expression , and plasma crp levels were conducted for the il-8 pathway . a subset of 20 unrelated mothers was recruited along with 50 siblings born before and after surgery ( 25 bms and 25 ams offspring ) . there were 17 mothers with siblings born before and after surgery ( 23 bms and 24 ams ) , two with bms offspring only ( 2 bms ) , and one mother with only one ams offspring . differential methylation analysis between bms and ams siblings revealed 5698 genes ( 14 466 probes ) with significant differences in methylation levels ( fdr - corrected diffscore |13 distribution of differentially methylated probes obtained from comparison between bms and ams offspring is provided in supplementary table 1 available online at http://dx.doi.org/10.1155/2013/492170 . total rna was isolated and purified from whole blood for 46 offsprings ( 23 bms and 23 ams ) using paxgene blood rna kit ( qiagen ) following the manufacturer 's recommendations . spearman correlations were computed for expression levels assessed by microarray and rt - pcr methods in a subset of genes ( bcl2 , nm_000633 ; ccnd2 , nm_001759 ; ncf2 , nm_000433 ; pik3r1 , nm_181523 ; prkch , nm_006255 ) for whom correlations between gene expression and methylation levels were observed . pairwise pearson correlations between methylation , expression , and plasma crp were computed for the il-8 signaling pathway that was overrepresented in both differential methylation and expression analyses . among the 46 genes analyzed , correlations between dna methylation and expression were significant for 17 genes ( 23 methylation probes ; table 3 ) . all 5 genes with significant correlations between gene expression and plasma crp levels ( bcl2 , ccnd2 , ncf2 , pik3r1 , and prkch ) also demonstrated correlations between methylation and expression and between gene methylation and plasma crp levels ( table 3 ) . from the 16 genes correlated with crp levels only one ( pik3r1 ) of the five genes with significant correlations between gene methylation , expression , and crp levels remained significant after adjustments , while trends toward significant correlation were found for bcl2 , ccnd2 , and ncf2 . we demonstrated here for the first time that surgical treatment of severe maternal obesity results in sustained differences in the methylome and transcriptome of genes involved in inflammatory pathways in ams offspring compared to bms siblings . overrepresentation of genes from the il-8 signaling pathway was identified from both methylation and expression data , and significant correlations were found between gene methylation and expression levels . moreover , 5 genes from the il-8 pathway also demonstrated correlation of gene methylation and expression levels with plasma crp levels . in the present study , we extend these data by reporting that metabolic improvements in mothers following biliopancreatic diversion surgery are reflected in gene methylation and expression levels of immunologic and inflammatory genes in offspring , indeed reinforcing the involvement of epigenetic differences in immunity and inflammatory genes in the determination of the offspring phenotypes . transcriptomic data revealed that improvements seen in mothers following bariatric surgery are reflected in gene expression levels of immunologic and inflammatory genes in offspring . animal studies have demonstrated a similar impact of maternal obesity and nutrition on immune and inflammatory genes in the offspring [ 3537 ] . nonetheless , pathway analyses conducted from differentially methylated and expressed genes are concordant and demonstrate an overrepresentation of immune and inflammatory pathways . improvements in inflammatory profiles in mothers following bariatric surgery may be potentially responsible for the inflammatory effects seen in offspring through modification of the in utero environment and improved methylation profiles in ams offspring . using analysis of gene methylation and expression data , we reveal that improvements seen in mothers following bariatric surgery reflect on the expression and methylation levels of genes involved in immune and inflammatory pathways in the offspring . these results argue for a beneficial effect of maternal weight loss before pregnancy and provide potential mechanisms of action for physiological improvements through regulation of methylation and expression of genes involved in inflammatory and immune pathways .
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for this study , we selected 20 patients at seoul national university hospital in south korea who had undergone hepatic resection due to hcc from july to september , 2010 . these patients have clinical evidence of chronic hepatitis b , such as serologic evidence of virus , more than 6 months of abnormal liver function , or radiologic evidence of hepatic fibrosis . there were 2 female and 18 male patients with ages ranging from 48 - 73 years ( with a median age of 58 ) . all patients had curative surgical resection ; 3 cases required liver transplantation and 17 needed partial resection . none of them underwent preoperative local treatment such as transarterial embolization , radiofrequency ablation and percutaneous ethanol injection therapy , nor preoperative systemic chemotherapy . there was one hepatitis c virus coinfection and one concomitant alcohol - related steatohepatitis with chronic hepatitis b. peritumoral tissues from each fresh liver specimen were sampled from the tumor border to a distance of 40 mm from the tumor . routine hematoxylin and eosin ( h&e ) and masson 's trichrome ( m - t ) stained slides were made as previously described.12,13 the adjacent pnlp was divided into eight 5 mm - sized columns , starting from the tumor border to a 40 mm distance from the tumor ( fig . the 5-mm column represents adjacent pnlp located between the tumor border and 5 mm distance from the tumor . the 10- , 15- , 20- , 25- , 30- , 35- , and 40-mm columns represent adjacent pnlp between 5 and 10 mm , 10 and 15 mm , 15 and 20 mm , 20 and 25 mm , 25 and 30 mm , 30 and 35 mm , and 35 and 40 mm from the tumor , respectively . the mean magnitude of each column was 15 mm ( width)5 mm ( length ) , and median number of portal tracts per column was 14 ( range , 3 to 17 ) . for comparison , background liver or remote pnlp was also taken farthermost from the tumor ( or at least > 40 mm from the tumor ) and treated similarly as the adjacent pnlp . parameters such as necroinflammatory activity of the hepatic parenchyma , fibrosis , bile ductular reaction , peliosis , perivenular inflammation , and steatosis were assessed by two pathologists ( k.b . lee and h.y . was applied as a grading system for necroinflammatory activity.14,15 spotty necrosis , periportal / periseptal interface hepatitis ( piecemeal necrosis ) , and portal inflammation were scored from 0 to 4 and confluent necrosis from 0 to 6 . the total modified hai score is the sum of all of these scores ( expected maximum score , 18 ) . in addition , the number of foci of spotty necrosis per 10 objective was counted at the most severely affected area . the first was scoring both h&e- and m - t - stained sections ( by light microscopy ) from 0 to 6 according to the ishak fibrosis staging system.15 in addition , collagen deposition shown by m - t staining was converted into a pixel count for an objective evaluation of fibrosis / cirrhosis . images of each column were taken using an olympus optical microscope ( bx50 , olympus , tokyo , japan ) with 4 objective and converted into jpeg format for analysis . 1f to include the majority of the samples so that we could exclude any selection bias . all images ( having mean image size of 15.98 mm ) were analyzed by image j software ( positive color : r , 0.8748878 ; g , 0.457825 65 ; b , 0.15829495 ; threshold : 0 to 290 ) . the ratio of collagen - positive pixel count to total pixel count was expressed in percentage terms . we defined peliosis - like lesions as blood - filled spaces , with or without sinusoidal lining , recognized with the 4 objective . the inflammatory activity around the central vein or the so - called hepatic venulitis was scored from 0 to 4 ( 0 , absent ; 1 , inflammatory cells accumulation in the lumen of hepatic venule ; 2 , focal inflammatory cells infiltration through the vascular wall ; 3 , continuous infiltration of infilammatory cells around the vascular wall ; and 4 , fibrinoid necrosis of the vascular wall ) . bile ductular proliferation was scored from 0 to 3 ( 0 , absent ; 1 , mild ; 2 , moderate ; 3 , severe ; or graded as <33 , 33 - 66% , and > 66% of the hepatic lobule by bile steatosis was graded 0 to 3 based on the percentage of hepatocytes in the biopsy ( 0 , none ; 1 , up to 33% ; 2 , 33 - 66% ; and 3 , > 66% ) , which is adopted from the nonalcoholic steatohepatitis clinical research network scoring system.16,17 the wilcoxon signed - rank test was used to compare pathologic characteristics of each column with those of adjacent columns , as well as remote parenchyma . tests having p - value less than 0.05 was considered as results with significant difference . for this study , we selected 20 patients at seoul national university hospital in south korea who had undergone hepatic resection due to hcc from july to september , 2010 . these patients have clinical evidence of chronic hepatitis b , such as serologic evidence of virus , more than 6 months of abnormal liver function , or radiologic evidence of hepatic fibrosis . there were 2 female and 18 male patients with ages ranging from 48 - 73 years ( with a median age of 58 ) . all patients had curative surgical resection ; 3 cases required liver transplantation and 17 needed partial resection . none of them underwent preoperative local treatment such as transarterial embolization , radiofrequency ablation and percutaneous ethanol injection therapy , nor preoperative systemic chemotherapy . there was one hepatitis c virus coinfection and one concomitant alcohol - related steatohepatitis with chronic hepatitis b. peritumoral tissues from each fresh liver specimen were sampled from the tumor border to a distance of 40 mm from the tumor . routine hematoxylin and eosin ( h&e ) and masson 's trichrome ( m - t ) stained slides were made as previously described.12,13 the adjacent pnlp was divided into eight 5 mm - sized columns , starting from the tumor border to a 40 mm distance from the tumor ( fig . the 5-mm column represents adjacent pnlp located between the tumor border and 5 mm distance from the tumor . the 10- , 15- , 20- , 25- , 30- , 35- , and 40-mm columns represent adjacent pnlp between 5 and 10 mm , 10 and 15 mm , 15 and 20 mm , 20 and 25 mm , 25 and 30 mm , 30 and 35 mm , and 35 and 40 mm from the tumor , respectively . the mean magnitude of each column was 15 mm ( width)5 mm ( length ) , and median number of portal tracts per column was 14 ( range , 3 to 17 ) . for comparison , background liver or remote pnlp was also taken farthermost from the tumor ( or at least > 40 mm from the tumor ) and treated similarly as the adjacent pnlp . parameters such as necroinflammatory activity of the hepatic parenchyma , fibrosis , bile ductular reaction , peliosis , perivenular inflammation , and steatosis were assessed by two pathologists ( k.b . lee and h.y . was applied as a grading system for necroinflammatory activity.14,15 spotty necrosis , periportal / periseptal interface hepatitis ( piecemeal necrosis ) , and portal inflammation were scored from 0 to 4 and confluent necrosis from 0 to 6 . the total modified hai score is the sum of all of these scores ( expected maximum score , 18 ) . in addition , the number of foci of spotty necrosis per 10 objective was counted at the most severely affected area . the first was scoring both h&e- and m - t - stained sections ( by light microscopy ) from 0 to 6 according to the ishak fibrosis staging system.15 in addition , collagen deposition shown by m - t staining was converted into a pixel count for an objective evaluation of fibrosis / cirrhosis . images of each column were taken using an olympus optical microscope ( bx50 , olympus , tokyo , japan ) with 4 objective and converted into jpeg format for analysis . 1f to include the majority of the samples so that we could exclude any selection bias . all images ( having mean image size of 15.98 mm ) were analyzed by image j software ( positive color : r , 0.8748878 ; g , 0.457825 65 ; b , 0.15829495 ; threshold : 0 to 290 ) . the ratio of collagen - positive pixel count to total pixel count was expressed in percentage terms . we defined peliosis - like lesions as blood - filled spaces , with or without sinusoidal lining , recognized with the 4 objective . the inflammatory activity around the central vein or the so - called hepatic venulitis was scored from 0 to 4 ( 0 , absent ; 1 , inflammatory cells accumulation in the lumen of hepatic venule ; 2 , focal inflammatory cells infiltration through the vascular wall ; 3 , continuous infiltration of infilammatory cells around the vascular wall ; and 4 , fibrinoid necrosis of the vascular wall ) . bile ductular proliferation was scored from 0 to 3 ( 0 , absent ; 1 , mild ; 2 , moderate ; 3 , severe ; or graded as <33 , 33 - 66% , and > 66% of the hepatic lobule by bile ductular proliferation , respectively ) . steatosis was graded 0 to 3 based on the percentage of hepatocytes in the biopsy ( 0 , none ; 1 , up to 33% ; 2 , 33 - 66% ; and 3 , > 66% ) , which is adopted from the nonalcoholic steatohepatitis clinical research network scoring system.16,17 the wilcoxon signed - rank test was used to compare pathologic characteristics of each column with those of adjacent columns , as well as remote parenchyma . tests having p - value dense inflammatory cell infiltration in the portal tracts adjacent to the tumor , which also lessened with distance , were observed as shown in fig . 1e ) columns . the severity of portal , periportal , and lobular activity scores in adjacent pnlp were illustrated for all cases in concentric graphs by color gradation and in bar graphs by mean of scores ( fig . the dark - colored centers of circular graphs indicate severe necroinflammatory activity near the tumor . the plotted mean value of the necroinflammatory activity as bar graph demonstrates a pattern of decreasing inflammatory cell infiltration in adjacent pnlp ( fig . however , at certain distances , the inflammation became stable and no longer differed from that of remote pnlp . moreover , the necroinflammatory activity in each column was compared using wilcoxon signed - rank test ( table 1 ) . portal inflammation was markedly severe in the 5-mm column ( mean score , 2.79 ) , and decreased with distance until the 25-mm column ( mean score , 1.84 ) ( p=0.011 between 5-mm and 10-mm column ; p=0.034 between 10-mm and 15-mm column ; p=0.046 between 20-mm and 25-mm column ) . therefore , in comparison to adjacent pnlp , the considerable cut - off point for portal inflammation could be 20 mm distance from the tumor . likewise , the foci of spotty necrosis were observed most frequently in the 5-mm column ( mean score , 2.65 ; mean count , 6.0 ) and decreased with distance until 25-mm column ( mean score , 1.45 ; mean count , 2.05 ) . both the score and the frequency of spotty necrosis did not change significantly beyond the 20 mm distance from the tumor . on the other hand , periportal inflammation were markedly severe in the 5-mm column ( mean score , 3.30 ) and significantly decreased in the 10-mm ( mean score , 2.55 ; p=0.001 ) and 15-mm column ( mean score , 2.20 ; p=0.008 ) . the modified hai , which is the sum of scores for portal , periportal inflammation and spotty necrosis , was markedly severe in the 5-mm column ( mean score , 8.75 ) and decreased until 15-mm column ( mean score , 6.10 ) , while it did not significantly change beyond 10 mm distance from the tumor . the lesions were irregularly dilated sinusoidal spaces filled with blood and fibrinoid material accompanied by damaged hepatocytes ( fig . 4a , b ) , especially starting from the tumor border towards the 10-mm column , while nearly no peliosis was found beyond the 10-mm column , denoted by a p - value of 0.018 between 10-mm and 15-mm column ( table 1 ) . apart from the usual occurrence of chronic viral hepatitis and neutrophic infiltration around the central veins , 10 cases of perivenular inflammation was observed in adjacent pnlp . perivenular inflammation was markedly severe from the tumor border until 10 mm distance from the tumor ( table 1 , fig . although all cases in this study resulted from cirrhosis or chronic hepatitis b backgrounds , the degree of fibrosis in pnlp varies with distance from the tumor ( fig . the ishak grade of fibrosis for all cases is illustrated in concentric circles ( fig . the mean value of the pixel fraction of collagen deposition for each column is presented as a bar graph ( fig . as shown in table 1 , the ishak grade of fibrosis was markedly higher in the 5-mm and 10-mm column ( p=0.007 between 5-mm and 10-mm column ; p=0.020 between 10-mm and 15-mm column ) . to quantify the amount of fibrosis , pixel fraction of collagen deposition this evaluation revealed that collagen deposition was much more severe in the 5-mm column than in more distant areas . thus , 10 mm could be the considerable cut - off point for the ishak grade of fibrosis , and 5 mm for pixel fraction of collagen deposition . bile ductular proliferation around the tumor was so severe that it was difficult to find remaining hepatocytes immediately adjacent to the tumor ( fig . 5c and d , its pattern was decreasing with distance but become stable beyond the 10 mm distance from the tumor border ( p=0.001 between 5-mm and 10-mm column ; p=0.008 between 10-mm and 15-mm column ) . however , there were no differences in steatosis between the columns ( data not shown ) . to confirm the gradation of histopathologic features in adjacent pnlp , we evaluated the same factors beyond 40 mm in remote pnlp , and then compared with each column of adjacent pnlp within 40 mm ( table 1 ) . parameters compared included portal inflammation , periportal inflammation , spotty necrosis score , spotty necrosis count , the modified hai score , peliosis , bile ductular proliferation and the ishak grade of fibrosis ( table 1 ) . the comparison demonstrated that most of the pathologic changes were markedly severe in 5-mm and 10-mm columns than those of those in remote parenchyma . these results were consistent with comparisons between adjacent columns for such parameters as periportal inflammation , the modifieid hai score , peliosis , bile ductular proliferation and the ishak grade of fibrosis , verifying that 10 mm distance from the tumor border could be the considerable cut - off point for those parameters . dense collagen deposition was observed within 5 mm pnlp , as opposed to remote pnlp . this was consistent with the result from the comparison between adjacent columns , suggesting this distance to be the considerable cut - off point for the collagen deposition . portal inflammation within 15 mm pnlp was markedly severe than that in remote pnlp , but the comparison between adjacent columns demonstrated changes fading from 20 mm . dense inflammatory cell infiltration in the portal tracts adjacent to the tumor , which also lessened with distance , were observed as shown in fig . 1e ) columns . the severity of portal , periportal , and lobular activity scores in adjacent pnlp were illustrated for all cases in concentric graphs by color gradation and in bar graphs by mean of scores ( fig . the dark - colored centers of circular graphs indicate severe necroinflammatory activity near the tumor . the plotted mean value of the necroinflammatory activity as bar graph demonstrates a pattern of decreasing inflammatory cell infiltration in adjacent pnlp ( fig . however , at certain distances , the inflammation became stable and no longer differed from that of remote pnlp . moreover , the necroinflammatory activity in each column was compared using wilcoxon signed - rank test ( table 1 ) . portal inflammation was markedly severe in the 5-mm column ( mean score , 2.79 ) , and decreased with distance until the 25-mm column ( mean score , 1.84 ) ( p=0.011 between 5-mm and 10-mm column ; p=0.034 between 10-mm and 15-mm column ; p=0.046 between 20-mm and 25-mm column ) . therefore , in comparison to adjacent pnlp , the considerable cut - off point for portal inflammation could be 20 mm distance from the tumor . likewise , the foci of spotty necrosis were observed most frequently in the 5-mm column ( mean score , 2.65 ; mean count , 6.0 ) and decreased with distance until 25-mm column ( mean score , 1.45 ; mean count , 2.05 ) . both the score and the frequency of spotty necrosis did not change significantly beyond the 20 mm distance from the tumor . on the other hand , periportal inflammation were markedly severe in the 5-mm column ( mean score , 3.30 ) and significantly decreased in the 10-mm ( mean score , 2.55 ; p=0.001 ) and 15-mm column ( mean score , 2.20 ; p=0.008 ) . the modified hai , which is the sum of scores for portal , periportal inflammation and spotty necrosis , was markedly severe in the 5-mm column ( mean score , 8.75 ) and decreased until 15-mm column ( mean score , 6.10 ) , while it did not significantly change beyond 10 mm distance from the tumor . the lesions were irregularly dilated sinusoidal spaces filled with blood and fibrinoid material accompanied by damaged hepatocytes ( fig . 4a , b ) , especially starting from the tumor border towards the 10-mm column , while nearly no peliosis was found beyond the 10-mm column , denoted by a p - value of 0.018 between 10-mm and 15-mm column ( table 1 ) . apart from the usual occurrence of chronic viral hepatitis and neutrophic infiltration around the central veins , 10 cases of perivenular inflammation was observed in adjacent pnlp . perivenular inflammation was markedly severe from the tumor border until 10 mm distance from the tumor ( table 1 , fig . although all cases in this study resulted from cirrhosis or chronic hepatitis b backgrounds , the degree of fibrosis in pnlp varies with distance from the tumor ( fig . the ishak grade of fibrosis for all cases is illustrated in concentric circles ( fig . the mean value of the pixel fraction of collagen deposition for each column is presented as a bar graph ( fig . as shown in table 1 , the ishak grade of fibrosis was markedly higher in the 5-mm and 10-mm column ( p=0.007 between 5-mm and 10-mm column ; p=0.020 between 10-mm and 15-mm column ) . to quantify the amount of fibrosis , pixel fraction of collagen deposition was evaluated . this evaluation revealed that collagen deposition was much more severe in the 5-mm column than in more distant areas . thus , 10 mm could be the considerable cut - off point for the ishak grade of fibrosis , and 5 mm for pixel fraction of collagen deposition . bile ductular proliferation around the tumor was so severe that it was difficult to find remaining hepatocytes immediately adjacent to the tumor ( fig . 5c and d , its pattern was decreasing with distance but become stable beyond the 10 mm distance from the tumor border ( p=0.001 between 5-mm and 10-mm column ; p=0.008 between 10-mm and 15-mm column ) . however , there were no differences in steatosis between the columns ( data not shown ) . to confirm the gradation of histopathologic features in adjacent pnlp , we evaluated the same factors beyond 40 mm in remote pnlp , and then compared with each column of adjacent pnlp within 40 mm ( table 1 ) . parameters compared included portal inflammation , periportal inflammation , spotty necrosis score , spotty necrosis count , the modified hai score , peliosis , bile ductular proliferation and the ishak grade of fibrosis ( table 1 ) . the comparison demonstrated that most of the pathologic changes were markedly severe in 5-mm and 10-mm columns than those of those in remote parenchyma . these results were consistent with comparisons between adjacent columns for such parameters as periportal inflammation , the modifieid hai score , peliosis , bile ductular proliferation and the ishak grade of fibrosis , verifying that 10 mm distance from the tumor border could be the considerable cut - off point for those parameters . dense collagen deposition was observed within 5 mm pnlp , as opposed to remote pnlp . this was consistent with the result from the comparison between adjacent columns , suggesting this distance to be the considerable cut - off point for the collagen deposition . portal inflammation within 15 mm pnlp was markedly severe than that in remote pnlp , but the comparison between adjacent columns demonstrated changes fading from 20 mm . in this study , we investigated that the gradual histologic changes from the tumor edges and tried to find reasonable cut - off points for adjacent and remote peritumoral parenchyma based on various morphologic characteristics that are believed to indicate microenvironmental changes . although the individual histologic parameters revealed several distances from the tumor edges , we found the area within 10 mm and beyond 20 mm distance from the tumor to be relatively constant . pnlp within 10 mm had significantly higher inflammatory cells infiltration around portal tract and hepatic parenchyma , bile ductular reaction , collagen deposition and peliosis than 10 mm distance from the tumor . moreover , pnlp beyond 20 mm had no distinct pathologic features compared with background liver parenchyma . hence , the parenchyma within 10 mm from the tumor would be considered the adjacent pnlp in which morphologic changes are believed to be directly affected by the tumor , and the parenchyma beyond 20 mm as the remote pnlp without tumor effect . the recurrence of hcc is one of the leading causes of death after curative liver resection , and the prognoses on cases of early and late recurrence are quite different.18 late recurrence , which is defined as that occurring more than 2 years after primary resection , has better prognosis , while early recurrence that develops within 2 year after resection carries a poor prognosis . early recurrence is strongly negatively correlated with patient 's survival.19 hence , many predictive or potential risk factors for early recurrence have been investigated , including vascular invasion , biliary tumor thrombi , intrahepatic metastasis , and positive surgical margin.18,20,21 these results imply that early recurrence is associated with local tumor factors rather than the background liver parenchyma . on the contrary , late recurrence of hccs represents the tumorigenic trait of the remaining liver parenchyma . currently , immunologic or inflammatory milieu of the adjacent peritumoral tissue , which is directly affected by the tumor , have been postulated to be important in tumor progression22 and early recurrence of hcc.7,23,24 jia et al.22 demonstrated that the colony - stimulating factor-1 receptor level of adjacent pnlp was associated with intrahepatic metastasis and early recurrence when the adjacent pnlp was defined as within 10 mm , there - by supporting our definition of adjacent pnlp as within 10 mm distance from the tumor . the remote pnlp , which is not affected by hcc , is the remaining hepatic parenchyma in patients after resection so it is related with multiple occurrence and late recurrence of hcc . it is reported in several studies that molecular signature of non - cancerous liver tissue can predict the risk for late recurrence of hccs and influence the clinical outcome of hcc patients . in this study , we defined the remote pnlp as beyond 20 mm distance from the tumor , which is consistent with the results of previous studies . okamoto et al.9 showed that specific gene - expression profiles of pnlp could predict the risk for multiple occurrence of hcc by using peritumoral liver tissue farthest from the tumor.9 hoshida et al.11 demonstrated that specific gene - expression profiles of pnlp predicted late recurrence of hcc using the peritumoral liver tissue that was available at the time of study . the morphological changes described here were based on the criteria for viral hepatitis , and might not be sufficient to detect all types of tumor effect on adjacent pnlp . for example , peliosis , hepatic venulitis and bile ductular proliferation could be caused by the elevation of sinusoidal and bile ductal pressure , and end up with hepatocyte damage.25 we revealed positive correlation between tumor size and the farthest distance from the tumor where the peliosis could be observed ( data not shown ; spearman correlation coefficient=0.556 ; p=0.009 ) . furthermore , this study was limited to a small sample size , so statistical analysis for parametric validation can not be conducted . however , this study is the first report on comparison between adjacent pnlp and remote pnlp based on several histologic parameters via semiquantitative and quantitative scoring systems . results of this study can be used as an objective histologic evidence that may help many hepatologists and reseachers select proper tissue specimen for research about primary hepatic tumors of patients with chronic liver disease . in conclusion , adjacent pnlp , especially that within 10 mm of the tumor border , showed severe inflammation , architectural changes , peliosis , and hepatic venulitis . however
backgroundthe molecular profile of peritumoral non - neoplastic liver parenchyma ( pnlp ) has recently been suggested as predictive factor of early and late recurrence of hepatocellular carcinoma ( hcc ) . however , there is no definite cut - off point for tumor - free pnlp in terms of either histological or molecular changes . therefore , our aim is to determine the numerical cut - off point for separating adjacent pnlp and remote pnlp in histopathologic perspective.methodsperitumoral tissues from 20 resected hcc patients were sampled from 0 to 40 mm distance from the tumor border ( divided into 5-mm columns ) . histopathologic parameters such as necroinflammatory activity , fibrosis , bile ductular reaction , hepatic venulitis , peliosis , and steatosis were compared between each column.resultsthe morphologic changes just adjacent to the tumor were notably severe and faded with distance . the parenchyma within 10 mm of the tumor showed significantly severe inflammation , fibrosis , peliosis and hepatic venulitis compared with those from farther areas . the histopathologic changes of the parenchyma became stable beyond 20 mm.conclusionsresults of this study revealed that the parenchyma within 10 mm distance from the tumor , or adjacent pnlp , has histopathologic changes that are directly affected by the tumor , and the parenchyma beyond 20 mm as the remote pnlp without tumor effect .
MATERIALS AND METHODS Patient characteristics Sampling of nonneoplastic liver tissue Grading and staging for histopathologic parameters Statistical analysis RESULTS Necroinflammatory activity in adjacent peritumoral hepatic parenchyma Peliosis and perivenular inflammation in adjacent peritumoral hepatic parenchyma Fibrosis, bile ductular reaction and steatosis in adjacent peritumoral hepatic parenchyma Comparison with adjacent and remote non-neoplastic hepatic parenchyma DISCUSSION
the 10- , 15- , 20- , 25- , 30- , 35- , and 40-mm columns represent adjacent pnlp between 5 and 10 mm , 10 and 15 mm , 15 and 20 mm , 20 and 25 mm , 25 and 30 mm , 30 and 35 mm , and 35 and 40 mm from the tumor , respectively . parameters such as necroinflammatory activity of the hepatic parenchyma , fibrosis , bile ductular reaction , peliosis , perivenular inflammation , and steatosis were assessed by two pathologists ( k.b . routine hematoxylin and eosin ( h&e ) and masson 's trichrome ( m - t ) stained slides were made as previously described.12,13 the adjacent pnlp was divided into eight 5 mm - sized columns , starting from the tumor border to a 40 mm distance from the tumor ( fig . the 10- , 15- , 20- , 25- , 30- , 35- , and 40-mm columns represent adjacent pnlp between 5 and 10 mm , 10 and 15 mm , 15 and 20 mm , 20 and 25 mm , 25 and 30 mm , 30 and 35 mm , and 35 and 40 mm from the tumor , respectively . parameters such as necroinflammatory activity of the hepatic parenchyma , fibrosis , bile ductular reaction , peliosis , perivenular inflammation , and steatosis were assessed by two pathologists ( k.b . therefore , in comparison to adjacent pnlp , the considerable cut - off point for portal inflammation could be 20 mm distance from the tumor . 5c and d , its pattern was decreasing with distance but become stable beyond the 10 mm distance from the tumor border ( p=0.001 between 5-mm and 10-mm column ; p=0.008 between 10-mm and 15-mm column ) . these results were consistent with comparisons between adjacent columns for such parameters as periportal inflammation , the modifieid hai score , peliosis , bile ductular proliferation and the ishak grade of fibrosis , verifying that 10 mm distance from the tumor border could be the considerable cut - off point for those parameters . therefore , in comparison to adjacent pnlp , the considerable cut - off point for portal inflammation could be 20 mm distance from the tumor . 5c and d , its pattern was decreasing with distance but become stable beyond the 10 mm distance from the tumor border ( p=0.001 between 5-mm and 10-mm column ; p=0.008 between 10-mm and 15-mm column ) . these results were consistent with comparisons between adjacent columns for such parameters as periportal inflammation , the modifieid hai score , peliosis , bile ductular proliferation and the ishak grade of fibrosis , verifying that 10 mm distance from the tumor border could be the considerable cut - off point for those parameters . pnlp within 10 mm had significantly higher inflammatory cells infiltration around portal tract and hepatic parenchyma , bile ductular reaction , collagen deposition and peliosis than 10 mm distance from the tumor . hence , the parenchyma within 10 mm from the tumor would be considered the adjacent pnlp in which morphologic changes are believed to be directly affected by the tumor , and the parenchyma beyond 20 mm as the remote pnlp without tumor effect . the recurrence of hcc is one of the leading causes of death after curative liver resection , and the prognoses on cases of early and late recurrence are quite different.18 late recurrence , which is defined as that occurring more than 2 years after primary resection , has better prognosis , while early recurrence that develops within 2 year after resection carries a poor prognosis . currently , immunologic or inflammatory milieu of the adjacent peritumoral tissue , which is directly affected by the tumor , have been postulated to be important in tumor progression22 and early recurrence of hcc.7,23,24 jia et al.22 demonstrated that the colony - stimulating factor-1 receptor level of adjacent pnlp was associated with intrahepatic metastasis and early recurrence when the adjacent pnlp was defined as within 10 mm , there - by supporting our definition of adjacent pnlp as within 10 mm distance from the tumor . in this study , we defined the remote pnlp as beyond 20 mm distance from the tumor , which is consistent with the results of previous studies . for example , peliosis , hepatic venulitis and bile ductular proliferation could be caused by the elevation of sinusoidal and bile ductal pressure , and end up with hepatocyte damage.25 we revealed positive correlation between tumor size and the farthest distance from the tumor where the peliosis could be observed ( data not shown ; spearman correlation coefficient=0.556 ; p=0.009 ) . in conclusion , adjacent pnlp , especially that within 10 mm of the tumor border , showed severe inflammation , architectural changes , peliosis , and hepatic venulitis .
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patients with retinal degeneration ( n = 5 ) and subjects with healthy vision ( n = 5 ) participated in this study ( table ) . all subjects had complete clinical ocular examinations , including best - corrected visual acuity . all testing was performed with dilated pupils . the tenets of the declaration of helsinki were followed , and informed consent and assent were obtained from all patients . the research was approved by the institutional review board at the university of pennsylvania . in the standard mp1 system , optical paths of the retinal imaging and stimulation systems partially overlap . for the retinal imaging system , infrared illumination enters the dilated pupil , and the proportion reflected by the retina is imaged by a camera running at 25-hz frame rate ( fig . a second light path allows the subject to focus onto an internal liquid crystal display ( lcd ) screen that shows targets locked to a specific retinal location based on tracking of retinal features ( fig . the scotopic version of the mp1 instrument ( mp1s ) adds a filter holder rail placed between the final lens and the lcd screen ( in addition to other software and hardware modifications ) in order to allow for two - color microperimetry under dim backgrounds . the modification used in the current work was designed to build further upon the mp1s and consisted of a custom - machined slide - in tray that inserted directly into the filter holder rail ( figs . the tray held an optical relay assembly and an external microprojector ( dlp lightcrafter with dlp3000 micromirror array ; texas instruments , dallas , tx , usa ) . the optical relay assembly consisted of two achromatic doublets ( l1 = 47 - 713 and l2 = 49 - 292 ; edmund optics , barrington , nj , usa ) and a front - surface mirror ( m1 , edmund optics ) . software control of 8-bit red , green , and blue ( r , g , b ) values provided > 2 log units of dynamic range of the dlp output controllable digitally . in addition , there were slots ( f2 and f4 ) for the introduction of absorptive neutral density ( nd ) filters ( e.g. , 65 - 822 , edmund optics ) to attenuate the light output by up to 9 log units . the scattered light from the internal lcd screen was blocked ( x1 ) . a long - pass filter ( f1 ) was mounted in front of the camera to block scattered light from the projector reaching the detector . for future studies intending to use higher levels of output , a short - pass ( < 625 nm ) filter ( f3 = 84 - 723 , edmund optics ) can be used to spectrally shape the light in order to avoid saturating the retinal imaging system . ( a ) schematic of the mp1 system and the light paths under the unmodified and modified conditions . the retina is illuminated with an infrared source and the reflected light from the retina is imaged with a camera ( light path shown with red arrows ) . in addition , a stimulus shown on the internal lcd screen is imaged on the retina ( light path shown with blue arrow ) . f1 is a long - pass filter in front of the camera in order to minimize the stray light originating from the external dlp ; this is relevant only when the modified mp1 is used under high - luminance conditions . the optical relay tray is introduced between the final lens and the lcd using the rail normally carrying the filters used for scotopic testing . the relay tray bends the light path 90 away from the lcd ( front - surface mirror m1 ) and focuses the dlp projector onto the retina along the stimulus path . two lenses , l1 and l2 , are used to form a virtual image at the same plane as the lcd . filters f2 , f3 , and f4 spectrally shape and attenuate the dlp light output . x1 is an opaque cover to prevent stray light from the internal lcd from reaching the eye . ( b ) photograph of the optical relay tray inserted into the mp1 scotopic filter slot . ( c ) judd chromaticity diagram of the dlp light coupled to the modified mp1 . red- , green- , blue- , yellow- , and white - filled circles correspond to r , g , b settings of ( 255 , 0 , 0 ) , ( 0 , 255 , 0 ) , ( 0 , 0 , 255 ) , ( 255 , 255 , 0 ) , and ( 255 , 255 , 255 ) , respectively . dashed lines delineate the final system gamut ( light yellow area ) within the standard observer . 1.7.8 ) tracks each frame of the video image compared to a reference image obtained at the start of each session ; the tracking result is an estimate , approximately every 40 ms , of the shift in degrees in two orthogonal directions . this estimate is used to shift the location of the stimulus presentation such that the same retinal location can be repeatedly stimulated . the standard software contains an option to send all the tracking data to a log file . for the current project , the manufacturer modified the standard mp1 software such that the stream consisting of the time stamp , and x and y values of shifts and tracking success , was sent through a serial communication link ( usb nmc-2.5 m ; ftdi ltd . , a laptop computer ( running ubuntu 15.04 linux ) was connected to the dlp via hdmi ( high - definition multimedia interface ) and usb ports and to the mp1 computer via a usb port acting as the serial communication link . the laptop was set up in a two - screen mode , and the second screen was defined as the projector connected through the hdmi port . custom software was written ( java 8 se ; oracle corp , redwood city , ca , usa ) to read retinal tracking information obtained from the mp1 computer through one usb port and control the dlp through commands sent via another usb port . three light - emitting diode ( led ) illuminators ( r = 624 nm , g = 526 nm , b = 454 nm ) of the dlp were run with the default current setting of 633 ma . in theory , the led illuminators can be run at up to 1.5 a each to increase illumination ; however , that approach requires active cooling and was not used in the current work . importantly , default settings of the dlp include a variety of color correction matrices such that specific changes to r , g , and b values do not necessarily correspond to equivalent changes in light output . in the current work , feasibility experiments were performed to help design and develop outcome measures in the future based on detection and discrimination thresholds for specific conditions and specific interventions . all thresholds used forced - choice methods where the presentation of a 0.5-second - long stimulus was paired with a sound and the subject was asked to respond following the sound . in a small subset of experiments , the subject indicated whether the stimulus was detected . in the great majority of experiments , the task was to discriminate between two possible alternatives representing the diagonal direction of a flashed grating . in this two - alternative forced - choice ( 2afc ) paradigm , the subject pressed one of two buttons of a modified numeric keyboard depending on the perceived direction of the stimulus , and the software recorded the response . appropriate for eventual outcome development for the clinic , there were 5 to 10 trials for each test condition depending on the difficulty of the task ; full psychophysical functions were not performed . after each experiment , all data were downloaded and analyzed offline together with a reference retinal image obtained at the start of each session . threshold was considered to correspond to the trial with the highest spatial frequency grating whose direction could be discriminated with no more than one incorrect answer . the mp1 uses a maxwellian illumination system where the retinal illuminance is the total power entering the dilated pupil divided by the retinal exposed area . to calculate the maximum retinal illuminance achievable with the modified mp1 system using the external dlp as the illumination source , the power entering the pupil was estimated by first measuring the irradiance in air at 50 cm with a calibrated radiometer ( il1700 ; international light , peabody , ma , usa ) and then multiplying this value by the area illuminated at that distance . the size of the dlp projector extent that is projectable onto the retina through the optical relay was nearly circular with a 32 diameter . thus the total power of 6.77-mw white light entering the pupil corresponded to the estimated maximum retinal illuminance of 0.1 mw.mm for an emmetropic eye . for this maximum estimate , transmission losses through the cornea and lens were not considered since they are rarely quantifiable in patients . the maximum achromatic light output was estimated to correspond to a photopic luminance of 6.3 log phot cd.m ( retinal illuminance of 6.8 log photopic trolands [ phot - td ] ) and a scotopic luminance of 6.7 log scot cd.m ( 7.1 log scotopic trolands [ scot - td ] ) . the gamut of stimulus chromaticities ( x , y judd chromaticity coordinates ) that can be produced with the modified mp1 are shown in figure 1c . for all of the feasibility experiments performed in the current work in human subjects , a 3 nd filter was always in the light path , substantially limiting the light output . feasibility experiments were performed in normal subjects and one bcm patient with stimuli consisting of unitary contrast grating patches superimposed on larger homogeneous background fields . the color and luminance of the background were varied to provide different levels of adaptation to photoreceptor systems . the color and peak luminance of the gratings were varied to provide different increments above the adapting backgrounds ; the troughs of the gratings were equal to the background . the gratings were oriented along one of two diagonals 45 from vertical and had a sinusoidal spatial profile perpendicular to the orientation . a radially varying sigmoid - shaped envelope with a flat top was applied to smoothly blend the border of the grating into the background . examples of achromatic ( white on white , wonw ) and chromatic ( blue on yellow , bony , and red on blue , ronb ) gratings demonstrate their appearance ( fig . ( a ) appearance of the achromatic grating on achromatic background ( white on white , wonw ) and chromatic gratings on chromatic backgrounds ( blue on yellow , bony ; and red on blue , ronb ) . inset right , schematic of a left eye showing the optic nerve and major blood vessels . fixation is to dark square , and gray circle with black outline shows the nasal localization of the gratings used . ( b , c ) spectra showing the background ( thick gray lines ) and grating increments ( thin black lines ) for achromatic and chromatic stimuli . the backgrounds are matched in terms of scotopic luminance ( b ) or photopic luminance ( c ) . all spectra are acquired under the same conditions and plotted on the same ( relative ) energy axis . ( d ) absorption spectra of the 1 nd filter used to attenuate the stimuli . the adapting background was on continuously , and gratings were flashed for 0.5 seconds following an auditory signal . in most experimental runs , the subject was asked to discriminate between the two directions of gratings by pressing one of two buttons in a 2afc testing paradigm . for each combination of grating and background , spatial frequency was varied to estimate the highest spatial frequency that could be correctly discriminated by the subject . the choice for testing along the spatial frequency ( as opposed to testing along the luminance / chromaticity ) axis was based on our interest in finding a single combination of grating and background for future outcome development . furthermore , in order to be able to perform the testing in a clinical setting quickly , a complete psychometric function was not used . instead , between 5 and 10 trials were presented ( depending on the difficulty of the discrimination task ) at each spatial frequency , progressing from lower to higher spatial frequencies . the highest spatial frequency with zero or one incorrect was considered to be the discrimination threshold . if the subject reliably discriminated the finest spatial frequency ( 4.7 cyc / deg ) available , the threshold was considered indeterminate . if the subject could not discriminate the coarsest spatial frequency ( 0.2 cyc / deg ) , the stimulus was presented without a grating ( zero spatial frequency ; a disc of peak intensity ) in order to determine the detectability of the increment under those conditions . gratings of 11 diameter were presented nasal to the optic nerve centered at an eccentricity of 22 ( fig . the fixation was to either four small squares brighter than background for scotopic conditions , or a single small square darker than the background for photopic conditions . the retinal illuminance in phot - td was estimated for each stimulus condition using measured values of illuminance in photopic lux at 100 mm . the retinal illuminance in scot - td was similarly estimated from measures of scotopic lux . the retinal illuminance in s - cone trolands ( s - td ) was estimated from phot - td values and irradiance spectra ( figs . whenever possible , the achromatic and chromatic gratings were tested in sets where the backgrounds were matched in terms of their effectiveness to stimulate a certain photoreceptor mechanism . for example , under scotopic conditions , wonw and bony sets were tested with rod - matched backgrounds ( fig . 2b ) , whereas wonw and red - on - blue ( ronb ) sets were tested with l / m - cone matched backgrounds ( fig . testing was performed over a large range of adaptation conditions from 3 log scot - td to 2.2 log phot - td with the use of nd filters . , the necessity of stacking of multiple nd filters would be expected to cause some shifts at the spectral extremes , but such effects should be minimal for photopic conditions where fewer nd filters are stacked . ( a ) eye movements of a 66-year - old rpgr - xlrp patient with end - stage retinal degeneration and light - perception visual acuity ( upper left ) , a 26-year - old bcm patient with 20/100 visual acuity ( lower left ) , a 13-year - old cnga3-achm patient with 20/125 acuity ( upper right ) , and an 18-year - old cngb3-achm patient with 20/150 acuity ( lower right ) . for each subject , eye movement data are shown as a chart recording for x and y directions ( main images ) . during the recording , the xlrp patient was trying to hold the direction of his gaze without any visual cues in a dark room ; the remaining patients , on the other hand , had a 1 diameter visible fixation target . the velocity of the eye movements was calculated and is shown as histograms ( insets ) . ( b ) schematic of the modified mp1 with its standard connection to the mp1 computer , which in turn is connected to an external projector computer that drives the projector coupled optically into the light path of the mp1 . also shown is the experimental system of a target , stimulus , and timer setup to measure the maximum time delay involved between tracking the target and displaying a stimulus at the correct spatial location . in this experimental system , a high - speed camera images the temporal sequence of the alternating target and the resulting stimulus movements . ( c ) first two and last two video frames from a representative sequence of 16 frames showing simultaneously the target ( t ) being tracked , the stimulus ( s ) that is driven by the modified mp1 , and the 1/100-second timer . patients with retinal degeneration ( n = 5 ) and subjects with healthy vision ( n = 5 ) participated in this study ( table ) . all subjects had complete clinical ocular examinations , including best - corrected visual acuity . all testing was performed with dilated pupils . the tenets of the declaration of helsinki were followed , and informed consent and assent were obtained from all patients . in the standard mp1 system , optical paths of the retinal imaging and stimulation systems partially overlap . for the retinal imaging system , infrared illumination enters the dilated pupil , and the proportion reflected by the retina is imaged by a camera running at 25-hz frame rate ( fig . a second light path allows the subject to focus onto an internal liquid crystal display ( lcd ) screen that shows targets locked to a specific retinal location based on tracking of retinal features ( fig . the scotopic version of the mp1 instrument ( mp1s ) adds a filter holder rail placed between the final lens and the lcd screen ( in addition to other software and hardware modifications ) in order to allow for two - color microperimetry under dim backgrounds . the modification used in the current work was designed to build further upon the mp1s and consisted of a custom - machined slide - in tray that inserted directly into the filter holder rail ( figs . the tray held an optical relay assembly and an external microprojector ( dlp lightcrafter with dlp3000 micromirror array ; texas instruments , dallas , tx , usa ) . the optical relay assembly consisted of two achromatic doublets ( l1 = 47 - 713 and l2 = 49 - 292 ; edmund optics , barrington , nj , usa ) and a front - surface mirror ( m1 , edmund optics ) . software control of 8-bit red , green , and blue ( r , g , b ) values provided > 2 log units of dynamic range of the dlp output controllable digitally . in addition , there were slots ( f2 and f4 ) for the introduction of absorptive neutral density ( nd ) filters ( e.g. , 65 - 822 , edmund optics ) to attenuate the light output by up to 9 log units . the scattered light from the internal lcd screen was blocked ( x1 ) . a long - pass filter ( f1 ) was mounted in front of the camera to block scattered light from the projector reaching the detector . for future studies intending to use higher levels of output , a short - pass ( < 625 nm ) filter ( f3 = 84 - 723 , edmund optics ) can be used to spectrally shape the light in order to avoid saturating the retinal imaging system . ( a ) schematic of the mp1 system and the light paths under the unmodified and modified conditions . the retina is illuminated with an infrared source and the reflected light from the retina is imaged with a camera ( light path shown with red arrows ) . in addition , a stimulus shown on the internal lcd screen is imaged on the retina ( light path shown with blue arrow ) . f1 is a long - pass filter in front of the camera in order to minimize the stray light originating from the external dlp ; this is relevant only when the modified mp1 is used under high - luminance conditions . the optical relay tray is introduced between the final lens and the lcd using the rail normally carrying the filters used for scotopic testing . the relay tray bends the light path 90 away from the lcd ( front - surface mirror m1 ) and focuses the dlp projector onto the retina along the stimulus path . two lenses , l1 and l2 , are used to form a virtual image at the same plane as the lcd . filters f2 , f3 , and f4 spectrally shape and attenuate the dlp light output . x1 is an opaque cover to prevent stray light from the internal lcd from reaching the eye . ( b ) photograph of the optical relay tray inserted into the mp1 scotopic filter slot . ( c ) judd chromaticity diagram of the dlp light coupled to the modified mp1 . red- , green- , blue- , yellow- , and white - filled circles correspond to r , g , b settings of ( 255 , 0 , 0 ) , ( 0 , 255 , 0 ) , ( 0 , 0 , 255 ) , ( 255 , 255 , 0 ) , and ( 255 , 255 , 255 ) , respectively . dashed lines delineate the final system gamut ( light yellow area ) within the standard observer . 1.7.8 ) tracks each frame of the video image compared to a reference image obtained at the start of each session ; the tracking result is an estimate , approximately every 40 ms , of the shift in degrees in two orthogonal directions . this estimate is used to shift the location of the stimulus presentation such that the same retinal location can be repeatedly stimulated . the standard software contains an option to send all the tracking data to a log file . for the current project , the manufacturer modified the standard mp1 software such that the stream consisting of the time stamp , and x and y values of shifts and tracking success , was sent through a serial communication link ( usb nmc-2.5 m ; ftdi ltd . , a laptop computer ( running ubuntu 15.04 linux ) was connected to the dlp via hdmi ( high - definition multimedia interface ) and usb ports and to the mp1 computer via a usb port acting as the serial communication link . the laptop was set up in a two - screen mode , and the second screen was defined as the projector connected through the hdmi port . custom software was written ( java 8 se ; oracle corp , redwood city , ca , usa ) to read retinal tracking information obtained from the mp1 computer through one usb port and control the dlp through commands sent via another usb port . three light - emitting diode ( led ) illuminators ( r = 624 nm , g = 526 nm , b = 454 nm ) of the dlp were run with the default current setting of 633 ma . in theory , the led illuminators can be run at up to 1.5 a each to increase illumination ; however , that approach requires active cooling and was not used in the current work . importantly , default settings of the dlp include a variety of color correction matrices such that specific changes to r , g , and b values do not necessarily correspond to equivalent changes in light output . in the current work , feasibility experiments were performed to help design and develop outcome measures in the future based on detection and discrimination thresholds for specific conditions and specific interventions . all thresholds used forced - choice methods where the presentation of a 0.5-second - long stimulus was paired with a sound and the subject was asked to respond following the sound . in a small subset of experiments , the subject indicated whether the stimulus was detected . in the great majority of experiments , the task was to discriminate between two possible alternatives representing the diagonal direction of a flashed grating . in this two - alternative forced - choice ( 2afc ) paradigm , the subject pressed one of two buttons of a modified numeric keyboard depending on the perceived direction of the stimulus , and the software recorded the response . appropriate for eventual outcome development for the clinic , there were 5 to 10 trials for each test condition depending on the difficulty of the task ; full psychophysical functions were not performed . after each experiment , all data were downloaded and analyzed offline together with a reference retinal image obtained at the start of each session . the threshold was considered to correspond to the trial with the highest spatial frequency grating whose direction could be discriminated with no more than one incorrect answer . the mp1 uses a maxwellian illumination system where the retinal illuminance is the total power entering the dilated pupil divided by the retinal exposed area . to calculate the maximum retinal illuminance achievable with the modified mp1 system using the external dlp as the illumination source , the power entering the pupil was estimated by first measuring the irradiance in air at 50 cm with a calibrated radiometer ( il1700 ; international light , peabody , ma , usa ) and then multiplying this value by the area illuminated at that distance . the size of the dlp projector extent that is projectable onto the retina through the optical relay was nearly circular with a 32 diameter . thus the total power of 6.77-mw white light entering the pupil corresponded to the estimated maximum retinal illuminance of 0.1 mw.mm for an emmetropic eye . for this maximum estimate , transmission losses through the cornea and lens were not considered since they are rarely quantifiable in patients . the maximum achromatic light output was estimated to correspond to a photopic luminance of 6.3 log phot cd.m ( retinal illuminance of 6.8 log photopic trolands [ phot - td ] ) and a scotopic luminance of 6.7 log scot cd.m ( 7.1 log scotopic trolands [ scot - td ] ) . the gamut of stimulus chromaticities ( x , y judd chromaticity coordinates ) that can be produced with the modified mp1 are shown in figure 1c . for all of the feasibility experiments performed in the current work in human subjects , a 3 nd filter was always in the light path , substantially limiting the light output . feasibility experiments were performed in normal subjects and one bcm patient with stimuli consisting of unitary contrast grating patches superimposed on larger homogeneous background fields . the color and luminance of the background were varied to provide different levels of adaptation to photoreceptor systems . the color and peak luminance of the gratings were varied to provide different increments above the adapting backgrounds ; the troughs of the gratings were equal to the background . the gratings were oriented along one of two diagonals 45 from vertical and had a sinusoidal spatial profile perpendicular to the orientation . a radially varying sigmoid - shaped envelope with a flat top was applied to smoothly blend the border of the grating into the background . examples of achromatic ( white on white , wonw ) and chromatic ( blue on yellow , bony , and red on blue , ronb ) gratings demonstrate their appearance ( fig . ( a ) appearance of the achromatic grating on achromatic background ( white on white , wonw ) and chromatic gratings on chromatic backgrounds ( blue on yellow , bony ; and red on blue , ronb ) . inset right , schematic of a left eye showing the optic nerve and major blood vessels . fixation is to dark square , and gray circle with black outline shows the nasal localization of the gratings used . ( b , c ) spectra showing the background ( thick gray lines ) and grating increments ( thin black lines ) for achromatic and chromatic stimuli . the backgrounds are matched in terms of scotopic luminance ( b ) or photopic luminance ( c ) . all spectra are acquired under the same conditions and plotted on the same ( relative ) energy axis . ( d ) absorption spectra of the 1 nd filter used to attenuate the stimuli . the adapting background was on continuously , and gratings were flashed for 0.5 seconds following an auditory signal . in most experimental runs , the subject was asked to discriminate between the two directions of gratings by pressing one of two buttons in a 2afc testing paradigm . for each combination of grating and background , spatial frequency was varied to estimate the highest spatial frequency that could be correctly discriminated by the subject . the choice for testing along the spatial frequency ( as opposed to testing along the luminance / chromaticity ) axis was based on our interest in finding a single combination of grating and background for future outcome development . furthermore , in order to be able to perform the testing in a clinical setting quickly , a complete psychometric function was not used . instead , between 5 and 10 trials were presented ( depending on the difficulty of the discrimination task ) at each spatial frequency , progressing from lower to higher spatial frequencies . the highest spatial frequency with zero or one incorrect was considered to be the discrimination threshold . if the subject reliably discriminated the finest spatial frequency ( 4.7 cyc / deg ) available , the threshold was considered indeterminate . if the subject could not discriminate the coarsest spatial frequency ( 0.2 cyc / deg ) , the stimulus was presented without a grating ( zero spatial frequency ; a disc of peak intensity ) in order to determine the detectability of the increment under those conditions . gratings of 11 diameter were presented nasal to the optic nerve centered at an eccentricity of 22 ( fig . the fixation was to either four small squares brighter than background for scotopic conditions , or a single small square darker than the background for photopic conditions . the retinal illuminance in phot - td was estimated for each stimulus condition using measured values of illuminance in photopic lux at 100 mm . the retinal illuminance in scot - td was similarly estimated from measures of scotopic lux . the retinal illuminance in s - cone trolands ( s - td ) was estimated from phot - td values and irradiance spectra ( figs . whenever possible , the achromatic and chromatic gratings were tested in sets where the backgrounds were matched in terms of their effectiveness to stimulate a certain photoreceptor mechanism . for example , under scotopic conditions , wonw and bony sets were tested with rod - matched backgrounds ( fig . 2b ) , whereas wonw and red - on - blue ( ronb ) sets were tested with l / m - cone matched backgrounds ( fig . testing was performed over a large range of adaptation conditions from 3 log scot - td to 2.2 log phot - td with the use of nd filters . , the necessity of stacking of multiple nd filters would be expected to cause some shifts at the spectral extremes , but such effects should be minimal for photopic conditions where fewer nd filters are stacked . expected eye movements and retinal tracking performance . ( a ) eye movements of a 66-year - old rpgr - xlrp patient with end - stage retinal degeneration and light - perception visual acuity ( upper left ) , a 26-year - old bcm patient with 20/100 visual acuity ( lower left ) , a 13-year - old cnga3-achm patient with 20/125 acuity ( upper right ) , and an 18-year - old cngb3-achm patient with 20/150 acuity ( lower right ) . for each subject , eye movement data are shown as a chart recording for x and y directions ( main images ) . during the recording , the xlrp patient was trying to hold the direction of his gaze without any visual cues in a dark room ; the remaining patients , on the other hand , had a 1 diameter visible fixation target . the velocity of the eye movements was calculated and is shown as histograms ( insets ) . ( b ) schematic of the modified mp1 with its standard connection to the mp1 computer , which in turn is connected to an external projector computer that drives the projector coupled optically into the light path of the mp1 . also shown is the experimental system of a target , stimulus , and timer setup to measure the maximum time delay involved between tracking the target and displaying a stimulus at the correct spatial location . in this experimental system , a high - speed camera images the temporal sequence of the alternating target and the resulting stimulus movements . ( c ) first two and last two video frames from a representative sequence of 16 frames showing simultaneously the target ( t ) being tracked , the stimulus ( s ) that is driven by the modified mp1 , and the 1/100-second timer . the main impetus for the development of the modified mp1 was to design a potential outcome measure for treatments designed for two cohorts of patients : those with severe vision loss due to end - stage degeneration retina - wide and those with cone photoreceptor diseases . a representative example of retinal movements expected from end - stage retina - wide photoreceptor degeneration and light perception 3a ) , a 66-year - old patient with x - linked retinitis pigmentosa due to rpgr mutation . the patient was instructed to gaze straight ahead , and eye movements were recorded directly from the retina under infrared illumination . there was small - amplitude nystagmus as well as a slow drift of the eyes in both x and y directions . examples of retinal movements in cone photoreceptor diseases were recorded in p1 , p2 , and p3 , representing patients with achm due to cnga3 or cngb3 mutations and bcm , respectively ( fig . all three patients would be potential candidates for gene augmentation therapy . in a dark room , the patients with cone diseases were instructed to fixate at a 1 diameter target that was adjusted to be visible . in the two achm patients , eye movements had both horizontal and vertical components , whereas in the bcm patient most of the movement was along the horizontal meridian . the estimated velocity of the eye movements varied in all four patients and could reach up to 100/s ; however , more than 84% of the samples had a velocity below 5/s ( fig . these examples illustrate the characteristics of the eye tracking needed for outcome measures in both types of patients . we first evaluated static performance of the retinal tracking by measuring the detection linearity of a tracked object across the area of the modified mp1 that could be illuminated . for this purpose , a trackable target was set up under external infrared ( ir ) illumination , and the modified mp1 was programmed to illuminate a small bright circular stimulus to overlay the target . in the worst - case scenario , static tracking error was found to be less than 1. important for stimulation of a fixed retinal locus in a moving eye is not only the static performance but also the dynamic performance of retinal tracking and stimulation system . the commercial unmodified mp1 system uses a video camera with 50-hz interlaced fields to track the retinal movements over time and produces a stream of lateral shift information in horizontal and vertical directions every 40 ms ( corresponding to the 25-hz frame rate ) , each with its own time stamp . we easily established that the external computer of the modified mp1 is able to read and process the stream of shifts without dropping any data by evaluating the consecutive time stamps of the samples . what is much more difficult to measure is the overall system delay associated with this stream of tracking data , that is , the delay between the time of the occurrence of a movement in the input and the resulting compensatory movement of the stimulus . a system was set up whereby spatial changes in input and output could be simultaneously visualized using a high frame rate ( 120 fps ) external video camera together with a high - resolution timer ( fig . the input to be tracked by the system was set up as two adjacent features : one darker than the background and one brighter than the background . the features could be electronically alternated , thus modeling an instantaneous saccade occurring faster than the frame rate of the mp1 imaging camera . the output was a small bright circular stimulus adjusted to fall immediately below the input target . the high - speed camera was placed such that the target , the light output , and a high - resolution timer could be simultaneously imaged . frame 1 is immediately before the dark target is switched to the right side , whereas frame 2 ( 8.3 ms later ) shows the dark target appearing . frames 3 through 15 show no change , demonstrating the stimulus remaining on the left side . at frame 16 the stimulus moves to the right , suggesting a delay of 14 frames corresponding to 117 ms . over several alternations , the mean time delay was 114 15 ms ( n = 11 ) , corresponding to approximately three samples of the tracking information obtained at 25 hz . next we estimated the proportion of this delay added by the modification as compared to the delay originating from the unmodified commercial mp1 system . for this measurement , the input was the same as described above , but the output was programmed by the commercial mp1 software to be a single goldman v - sized stimulus presented on the internal lcd screen ; the duration of the stimulus was set to the maximum allowed by the software ( 2000 ms ) to permit a long enough time to record its movement in response to several feature switch cycles representing several saccades . the high - speed camera was set up to view the input , the output , and a timer simultaneously . via frame - by - frame analysis , the time delay between the onset of the feature switch ( saccade ) and the corresponding onset of the stimulus movement was found to be 83 12 ms ( n = 5 ) , corresponding to approximately two samples of the tracking information obtained at 25 hz . thus , we conclude that our modification adds 31 ms , on average , to the tracking delay . the dominant contributors to this additional delay are likely to be the transfer of information between the two computers as well as the transfer between the external computer and the dlp . we used grating increments on dim backgrounds to evaluate spatial resolution of the peripheral rod system . the eyes were dark adapted , and the grating stimuli were expected to be visible to the normal rod system but below the threshold of the l / m- and s - cones in the periphery . the lower background level corresponded to 3 log scot - td , which was achieved by the insertion of a total of 8 log nd filters in the light path and digital selection of either a w ( r , g , b = 20 , 20 , 20 ) or a y ( r , g , b = 25 , 25 , 0 ) background . the spatial frequency of 0.6 cyc / deg first became perceptible with an increment of 0.7 log scot - td . greater increments above the background resulted in discrimination of finer spatial resolutions reaching 2.3 cyc / deg at 2.2 log scotopic increment ( fig . 4a , upper ) . ( a ) dark - adapted eyes on 3 log scot - td ( upper ) or on 2 log scot - td ( lower ) w or y backgrounds presented with wonw or bony gratings of a range of scotopic increments . ( b ) light - adapted eyes on 0.2 log phot - td ( upper ) or on 2.2 log phot - td ( lower ) w or b backgrounds presented with wonw or ronb gratings of a range of photopic increments . ( c ) light - adapted eyes on 0.1 log s - td ( upper ) or on 2.1 log s - td ( lower ) w backgrounds presented with wonw gratings of a range of s increments . ( d ) light - adapted eyes on 0.6 log s - td ( upper ) or on 2.6 log s - td ( lower ) y backgrounds presented with bony gratings of a range of s increments . symbols show the discrimination of the direction of the highest spatial resolution grating under each grating / background condition . results are from five normal subjects ( white symbols ) and one bcm patient ( black symbols ) . the x in the lower image ( b ) represents the condition tested in the bcm patient who did not detect this ronb grating . the higher background corresponded to 2 log scot - td and was achieved with a total of 7 log nd filters inserted into the light path ; the same set of achromatic and chromatic gratings / backgrounds were used as for the lower background . white - on - white or bony gratings of 0.8 cyc / deg first became perceptible with increments of 0.3 log scot - td . 4a , lower ) . with the largest scotopic increment available ( 2.1 log scot - td ) assuming peripheral cone thresholds to be near 2 log phot - td , all bony conditions and most wonw conditions would be expected to stimulate only the rod system . to evaluate the spatial resolution of the peripheral l / m - cone system , we took advantage of light adaptation and longer wavelength gratings on shorter - wavelength backgrounds with the chromatic stimuli . the lower of the two backgrounds corresponded to 0.2 log phot - td achieved by the insertion of a total of 5 log nd filters in the light path and either a w ( r , g , b = 53 , 53 , 53 ) or a b ( r , g , b = 0 , 0 , 255 ) background . wonw gratings of 2.1 cyc / deg first became perceptible with 0.1 log phot - td increments ( fig . 4b , upper ) . with greater increments near 0.7 log phot - td , both ronb and wonw gratings of 3.3 , all normal subjects could discriminate the 4.7 cyc / deg grating , which was the upper limit of the current system designed for low vision . the higher background corresponded to 2.2 log phot - td achieved with a total of 3 log nd filters inserted into the light path . with increments of 0.1 log phot - td , both the wonw and ronb gratings of up to 2.7 cyc / deg , most normal subjects could discriminate the 4.7 cyc / deg grating . considering that the maximum available r grating on the b background corresponded to an increment of 0.1 log s - td compared to 0.8 log phot - td , it is likely that ronb gratings were detected by the l / m - cone system in normal eyes . similarity of wonw results to those from ronb gratings suggest that the former are likely to be detected by the l / m - cone system also . to evaluate the spatial resolution of the peripheral s - cone system , we took advantage of light adaptation ( to saturate the rod system ) and shorter - wavelength gratings ( to preferentially stimulate the s - cones ) on middle- and longer - wavelength backgrounds ( to reduce the increments available to the l / m - cones ) . two y backgrounds ( r , g , b = 200 , 200 , 0 ) were used . the lower of the two backgrounds ( 0.6 log s - td ; 1.6 log phot - td ) was achieved by the insertion of a total of 5 log nd filters in the light path ( fig . 4d , upper ) and the higher background ( 2.6 log s - td ; 3.6 log phot - td ) with 3 nd filters ( fig . discrimination of grating directions was not possible for increments less than 0.4 log s - td at either background level . at 0.7 log s - td increment with the lower background , and at 0.4 log s - td increment with the higher background these bony conditions in the periphery under strong light adaptation likely represented the activity of s - cones considering that the maximum available b grating corresponded to an increment of 1.2 log s - td as opposed to 0.02 log phot - td . a 31-year - old bcm patient ( p4 ) ( table ) was evaluated with the modified mp1 to determine whether the combination of adapting conditions and chromatic gratings was able to discriminate between perceptions originating from different photoreceptor systems . the clinical , molecular , and retinal phenotype characteristics of the patient have been previously published . the dark - adapted bcm eye tested with a wonw achromatic grating at the 2 log scot - td background showed results qualitatively similar to those of normal subjects ( fig . ( 2.2 log phot - td ) , the bcm patient did not detect the highest available contrast ( 0.8 log phot ; 0.1 log s ) of chromatic ronb gratings and thus could not perform discrimination experiments ( fig . insensitivity to r stimuli was consistent with the severe abnormality of l / m - cone driven vision in bcm . achromatic wonw gratings were visible to the bcm patient under light - adapted conditions ( fig . 4c ) . for both the lower ( 0.1 log s - td ; 0.2 log phot - td ) and the higher ( 2.1 log s - td ; 2.2 log phot - td ) achromatic backgrounds , the bcm patient could discriminate the direction of gratings up to 1.6 cyc / deg with 0.7 log s increments ( fig . these spatial frequencies were substantially lower than the results obtained in normals with the same conditions ( fig . appear to confirm the conclusion that bony recordings are driven by s - cones in normal subjects . the main impetus for the development of the modified mp1 was to design a potential outcome measure for treatments designed for two cohorts of patients : those with severe vision loss due to end - stage degeneration retina - wide and those with cone photoreceptor diseases . a representative example of retinal movements expected from end - stage retina - wide photoreceptor degeneration and light perception 3a ) , a 66-year - old patient with x - linked retinitis pigmentosa due to rpgr mutation . the patient was instructed to gaze straight ahead , and eye movements were recorded directly from the retina under infrared illumination . there was small - amplitude nystagmus as well as a slow drift of the eyes in both x and y directions . examples of retinal movements in cone photoreceptor diseases were recorded in p1 , p2 , and p3 , representing patients with achm due to cnga3 or cngb3 mutations and bcm , respectively ( fig . all three patients would be potential candidates for gene augmentation therapy . in a dark room , the patients with cone diseases were instructed to fixate at a 1 diameter target that was adjusted to be visible . in the two achm patients , eye movements had both horizontal and vertical components , whereas in the bcm patient most of the movement was along the horizontal meridian . the estimated velocity of the eye movements varied in all four patients and could reach up to 100/s ; however , more than 84% of the samples had a velocity below 5/s ( fig . these examples illustrate the characteristics of the eye tracking needed for outcome measures in both types of patients . we first evaluated static performance of the retinal tracking by measuring the detection linearity of a tracked object across the area of the modified mp1 that could be illuminated . for this purpose , a trackable target was set up under external infrared ( ir ) illumination , and the modified mp1 was programmed to illuminate a small bright circular stimulus to overlay the target . in the worst - case scenario , static tracking error was found to be less than 1. important for stimulation of a fixed retinal locus in a moving eye is not only the static performance but also the dynamic performance of retinal tracking and stimulation system . the commercial unmodified mp1 system uses a video camera with 50-hz interlaced fields to track the retinal movements over time and produces a stream of lateral shift information in horizontal and vertical directions every 40 ms ( corresponding to the 25-hz frame rate ) , each with its own time stamp . we easily established that the external computer of the modified mp1 is able to read and process the stream of shifts without dropping any data by evaluating the consecutive time stamps of the samples . what is much more difficult to measure is the overall system delay associated with this stream of tracking data , that is , the delay between the time of the occurrence of a movement in the input and the resulting compensatory movement of the stimulus . a system was set up whereby spatial changes in input and output could be simultaneously visualized using a high frame rate ( 120 fps ) external video camera together with a high - resolution timer ( fig . the input to be tracked by the system was set up as two adjacent features : one darker than the background and one brighter than the background . the features could be electronically alternated , thus modeling an instantaneous saccade occurring faster than the frame rate of the mp1 imaging camera . the output was a small bright circular stimulus adjusted to fall immediately below the input target . the high - speed camera was placed such that the target , the light output , and a high - resolution timer could be simultaneously imaged . frame 1 is immediately before the dark target is switched to the right side , whereas frame 2 ( 8.3 ms later ) shows the dark target appearing . frames 3 through 15 show no change , demonstrating the stimulus remaining on the left side . at frame 16 the stimulus moves to the right , suggesting a delay of 14 frames corresponding to 117 ms . over several alternations , the mean time delay was 114 15 ms ( n = 11 ) , corresponding to approximately three samples of the tracking information obtained at 25 hz . next we estimated the proportion of this delay added by the modification as compared to the delay originating from the unmodified commercial mp1 system . for this measurement , the input was the same as described above , but the output was programmed by the commercial mp1 software to be a single goldman v - sized stimulus presented on the internal lcd screen ; the duration of the stimulus was set to the maximum allowed by the software ( 2000 ms ) to permit a long enough time to record its movement in response to several feature switch cycles representing several saccades . the high - speed camera was set up to view the input , the output , and a timer simultaneously . via frame - by - frame analysis , the time delay between the onset of the feature switch ( saccade ) and the corresponding onset of the stimulus movement was found to be 83 12 ms ( n = 5 ) , corresponding to approximately two samples of the tracking information obtained at 25 hz . thus , we conclude that our modification adds 31 ms , on average , to the tracking delay . the dominant contributors to this additional delay are likely to be the transfer of information between the two computers as well as the transfer between the external computer and the dlp . we used grating increments on dim backgrounds to evaluate spatial resolution of the peripheral rod system . the eyes were dark adapted , and the grating stimuli were expected to be visible to the normal rod system but below the threshold of the l / m- and s - cones in the periphery . the lower background level corresponded to 3 log scot - td , which was achieved by the insertion of a total of 8 log nd filters in the light path and digital selection of either a w ( r , g , b = 20 , 20 , 20 ) or a y ( r , g , b = 25 , 25 , 0 ) background . the spatial frequency of 0.6 cyc / deg first became perceptible with an increment of 0.7 log scot - td . greater increments above the background resulted in discrimination of finer spatial resolutions reaching 2.3 cyc / deg at 2.2 log scotopic increment ( fig . ( a ) dark - adapted eyes on 3 log scot - td ( upper ) or on 2 log scot - td ( lower ) w or y backgrounds presented with wonw or bony gratings of a range of scotopic increments . ( b ) light - adapted eyes on 0.2 log phot - td ( upper ) or on 2.2 log phot - td ( lower ) w or b backgrounds presented with wonw or ronb gratings of a range of photopic increments . ( c ) light - adapted eyes on 0.1 log s - td ( upper ) or on 2.1 log s - td ( lower ) w backgrounds presented with wonw gratings of a range of s increments . ( d ) light - adapted eyes on 0.6 log s - td ( upper ) or on 2.6 log s - td ( lower ) y backgrounds presented with bony gratings of a range of s increments . symbols show the discrimination of the direction of the highest spatial resolution grating under each grating / background condition . results are from five normal subjects ( white symbols ) and one bcm patient ( black symbols ) . the x in the lower image ( b ) represents the condition tested in the bcm patient who did not detect this ronb grating . the higher background corresponded to 2 log scot - td and was achieved with a total of 7 log nd filters inserted into the light path ; the same set of achromatic and chromatic gratings / backgrounds were used as for the lower background . white - on - white or bony gratings of 0.8 cyc / deg first became perceptible with increments of 0.3 log scot - td . 4a , lower ) . with the largest scotopic increment available ( 2.1 log scot - td ) , spatial frequencies of 3.2 cyc / deg could be discriminated . assuming peripheral cone thresholds to be near 2 log phot - td , all bony conditions and most wonw conditions would be expected to stimulate only the rod system . to evaluate the spatial resolution of the peripheral l / m - cone system , we took advantage of light adaptation and longer wavelength gratings on shorter - wavelength backgrounds with the chromatic stimuli . the lower of the two backgrounds corresponded to 0.2 log phot - td achieved by the insertion of a total of 5 log nd filters in the light path and either a w ( r , g , b = 53 , 53 , 53 ) or a b ( r , g , b = 0 , 0 , 255 ) background . wonw gratings of 2.1 cyc / deg first became perceptible with 0.1 log phot - td increments ( fig . 4b , upper ) . with greater increments near 0.7 log phot - td , both ronb and wonw gratings of 3.3 , all normal subjects could discriminate the 4.7 cyc / deg grating , which was the upper limit of the current system designed for low vision . the higher background corresponded to 2.2 log phot - td achieved with a total of 3 log nd filters inserted into the light path . with increments of 0.1 log phot - td , both the wonw and ronb gratings of up to 2.7 cyc / deg could be reliably discriminated ( fig . considering that the maximum available r grating on the b background corresponded to an increment of 0.1 log s - td compared to 0.8 log phot - td , it is likely that ronb gratings were detected by the l / m - cone system in normal eyes . similarity of wonw results to those from ronb gratings suggest that the former are likely to be detected by the l / m - cone system also . to evaluate the spatial resolution of the peripheral s - cone system , we took advantage of light adaptation ( to saturate the rod system ) and shorter - wavelength gratings ( to preferentially stimulate the s - cones ) on middle- and longer - wavelength backgrounds ( to reduce the increments available to the l / m - cones ) . two y backgrounds ( r , g , b = 200 , 200 , 0 ) were used . the lower of the two backgrounds ( 0.6 log s - td ; 1.6 log phot - td ) was achieved by the insertion of a total of 5 log nd filters in the light path ( fig . 4d , upper ) and the higher background ( 2.6 log s - td ; 3.6 log phot - td ) with 3 nd filters ( fig . discrimination of grating directions was not possible for increments less than 0.4 log s - td at either background level . at 0.7 log s - td increment with the lower background , and at 0.4 log s - td increment with the higher background these bony conditions in the periphery under strong light adaptation likely represented the activity of s - cones considering that the maximum available b grating corresponded to an increment of 1.2 log s - td as opposed to 0.02 log phot - td . a 31-year - old bcm patient ( p4 ) ( table ) was evaluated with the modified mp1 to determine whether the combination of adapting conditions and chromatic gratings was able to discriminate between perceptions originating from different photoreceptor systems . the clinical , molecular , and retinal phenotype characteristics of the patient have been previously published . the dark - adapted bcm eye tested with a wonw achromatic grating at the 2 log scot - td background showed results qualitatively similar to those of normal subjects ( fig . ( 2.2 log phot - td ) , the bcm patient did not detect the highest available contrast ( 0.8 log phot ; 0.1 log s ) of chromatic ronb gratings and thus could not perform discrimination experiments ( fig . insensitivity to r stimuli was consistent with the severe abnormality of l / m - cone driven vision in bcm . achromatic wonw gratings were visible to the bcm patient under light - adapted conditions ( fig . 4c ) . for both the lower ( 0.1 log s - td ; 0.2 log phot - td ) and the higher ( 2.1 log s - td ; 2.2 log phot - td ) achromatic backgrounds , the bcm patient could discriminate the direction of gratings up to 1.6 cyc / deg with 0.7 log s increments ( fig . these spatial frequencies were substantially lower than the results obtained in normals with the same conditions ( fig . appear to confirm the conclusion that bony recordings are driven by s - cones in normal subjects . perceptual testing is often performed under free - viewing conditions in which the subject either looks directly at the target with his or her best vision ( such as visual acuity testing ) or looks at a fixation target while the stimulus is presented elsewhere within the visual field ( such as computerized visual field testing ) . this approach often produces reliable , reproducible , and interpretable results as long as the subject has the ability to fixate steadily . on the other hand , there is much less certainty about the retinal location where perception is originating , and thus interpretation of the results , in patients who lack steady ( foveal ) fixation . for these patients , there is a long history of techniques used ( variably named fundus perimetry , gaze - controlled perimetry , or microperimetry ) coupling an objective estimate of the instantaneous gaze direction to the retinal location where the stimulus is being presented . earlier approaches have involved operator judgment for the localization of retinal landmarks to adjust the location of the stimulus presentation ( for examples , see refs . 3846 ) . more modern systems image the retina with infrared illumination , detect the movement of the retina compared to a reference image , and present a stimulus adjusted to the movement of the retina ( for examples , see refs . one of the modern systems is the nidek mp1 microperimeter , which has been used in a large number studies since its introduction more than 10 years ago ( examples include refs . modification of the hardware and the software has resulted in the scotopic version of mp1 primarily designed for dark - adapted two - color testing . chromatic microperimetry where spectrally opposing stimulus and background colors are used to preferentially probe different cone photoreceptor populations in cone diseases . here we report on a novel modification of the mp1 involving both hardware and software that allows the use of an external projector driven by an external computer as the source of light . further work will be directed at choosing a succinct set of conditions tapping perception originating from different photoreceptor systems and defining the repeatability of the measurements in order to develop the feasibility experiments reported here into an outcome measure . one of the key design features of the modified mp1 is the availability of high - luminance stimuli that may be necessary to activate optogenetic proteins . a clinical trial is currently under way ( nct02556736 , clinicaltrials.org ) to introduce channelrhodopsins into retinal ganglion cells of patients with severe loss of vision and determine whether there is any evidence of improved vision . assuming safety of the procedure , efficacy determination will require levels of retinal irradiance that will stimulate channelrhodopsin . the level of retinal irradiance that will be necessary in human eyes is currently unknown and is likely to be dependent on the expression level achieved . for any level of expression , it is thought that neural encoding of the stimuli will reduce the time - averaged retinal irradiance due to temporal sparsity of firing patterns applied at any given retinal location . however , success of efficiently activating optogenetic proteins in nonphotoreceptor cells with encoded patterns requires availability of high peak retinal irradiance that can be precisely controlled in space and time in response to a retina that is dynamically and unpredictably moving . we designed the optics to provide a maximum retinal irradiance of approximately 0.1 mw.mm , which is thought to fall within the bounds of retinal safety and potential effectiveness with first - generation optogenetic proteins . for comparison , this maximal irradiance is approximately 4.3 log units higher than that available from the unmodified mp1 , and approximately 2.8 log units higher than the standard ( 10,000 asb ) stimulus available in many computerized perimeters . the ability to repetitively stimulate the same region by tracking retinal features in a moving eye was an important prerequisite considered for choosing the mp1 as a starting platform . with the cooperation of the manufacturer , we were able to stream this retinal tracking information into an external computer and successfully use it to steer the stimuli presented by the external projector . however , there was an unavoidable delay between the retinal movement and the resulting stimulus movement . two - thirds of the delay originated from the standard mp1 , and one - third was added due to the modification . in eyes with stable fixation , the consequences of the delay are negligible . but in unstable eyes , the delay would be expected to result in errors between the intended and actual retinal locations to be stimulated , and the magnitude of the error would be proportional to the instantaneous velocity of eye movements . based on data from patients who could be enrolled in relevant clinical trials , we conclude that the average absolute value of such errors is < 0.25 , but the range can reach up to 4 with the current system . future improvements in speed of image processing and data transfer are likely to reduce the delay and the magnitude of this error but could not eliminate it . for patients with wandering eye movements , but not for patients with repetitive fast saccades , predictive methods of retinal tracking could also be considered in the future to partially compensate for the system delay . changes in the instantaneous velocity of retinal movements coupled with a fixed tracking delay could be hypothesized to reduce the effective modulation depth of gratings by smearing the stimulus . however , previous work in idiopathic infantile nystagmus syndrome comparing tachistoscopic flashes to constant illumination has suggested that smearing of gratings has no consequence for visual acuity determinations . applicability of those results to achm and bcm patients remains unknown . to minimize the effects of smearing on perception in patients , we used a grating flash duration that was long compared to both the integration time of the underlying rod and/or cone photoreceptors and the typical duration of a saccadic jerk in patients with abnormal eye movements . within the period of grating presentation , there were expected to be periods of eye movement with near - constant speed , which would correspond to a shift in retinal location stimulated ( due to tracking delay ) but no smearing when perception of highest spatial frequencies can occur . most commonly , standard high - contrast letters under free - viewing conditions are used in bright ambient lights to evaluate the resolving power of the l / m - cone system . in cone photoreceptor diseases , however , rod ( and sometimes s - cone ) photoreceptors can dominate low levels of vision even in daytime conditions . the overarching aim of gene therapies for cone photoreceptor diseases is to improve l / m - cone driven visual performance of patients . however , it is hypothetically possible , maybe even likely , that early - phase gene therapies will result in incremental improvements at best , and the detection of such improvement within the context of the native vision of these patients will require specialized outcome measures . the modified mp1 developed here had the luminance and chromaticity range to be applicable as such a specialized outcome measure . toward this aim , in preliminary experiments we measured grating visual acuities near the increment threshold in the nasal retina of normal subjects and a bcm patient . under conditions where grating discrimination is likely to be dominated by the peripheral rod system , we found spatial frequencies of 1 cyc / deg near luminance increment thresholds representing peak sensitivity . comparable were the limited results from the bcm patient and previous findings performed under similar but not identical conditions . red gratings on bright blue backgrounds were not detectable by the bcm patient even at the highest contrast levels , consistent with the expected pathophysiology in bcm . for these l / m - cone mediated conditions , spatial frequencies of 2 to 3 cyc / deg were measured near peak sensitivity with our flashed stimuli in normal subjects ; previously reported results in the periphery with drifting stimuli have been somewhat lower . and finally , blue gratings on bright yellow backgrounds tapped perception originating from s - cones and showed spatial frequencies of 1 to 1.5 cyc / deg near peak sensitivity in the nasal periphery . comparable results have been obtained from central s - cones near threshold or implied by suprathreshold results in perifovea or periphery . in conclusion , the modified mp1 described here provides an early step with promising potential toward presenting varied stimuli of a very wide range of luminance and chromaticity with precise spatiotemporal control locked to retinal features .
purposeto present stimuli with varied sizes , colors , and patterns over a large range of luminance.methodsthe filter bar used in scotopic mp1 was replaced with a custom slide - in tray that introduces light from an external projector driven by an additional computer . mp1 software was modified to provide retinal tracking information to the computer driving the projector . retinal tracking performance was evaluated by imaging the system input and the output simultaneously with a high - speed video system . spatial resolution was measured with achromatic and chromatic grating / background combinations over scotopic and photopic ranges.resultsthe range of retinal illuminance achievable by the modification was up to 6.8 log photopic trolands ( phot - td ) ; however , in the current work , only a lower range over 4 to + 3 log phot - td was tested in human subjects . optical magnification was optimized for low - vision testing with gratings from 4.5 to 0.2 cyc / deg . in normal subjects , spatial resolution driven by rods , short wavelength - sensitive ( s- ) cones , and long / middle wavelength - sensitive ( l / m- ) cones was obtained by the choice of adapting conditions and wavelengths of grating and background . data from a patient with blue cone monochromacy was used to confirm mediation.conclusionsthe modified mp1 can be developed into an outcome measure for treatments in patients with severe retinal degeneration , very low vision , and abnormal eye movements such as those for whom treatment with optogenetics is planned , as well as for patients with cone disorders such as blue cone monochromacy for whom treatment with gene therapy is planned to improve l / m - cone function above a normal complement of rod and s - cone function .
Methods Human Subjects Modification of the MP1S Microperimeter Hardware Modification of the MP1S Microperimeter Software External Computer and Software Psychophysics Used in Feasibility Experiments Maximum Retinal Illuminance Achromatic and Chromatic Gratings Results Eye Movements in Retinal Degenerations of Interest Retinal Tracking and Stimulation Performance of the Modified MP1 Spatial Resolving Properties of the Rod System Spatial Resolving Properties of the L/M-Cone System Spatial Resolving Properties of the S-Cone System Spatial Resolving Properties in Blue Cone Monochromacy Discussion
the modification used in the current work was designed to build further upon the mp1s and consisted of a custom - machined slide - in tray that inserted directly into the filter holder rail ( figs . in theory , the led illuminators can be run at up to 1.5 a each to increase illumination ; however , that approach requires active cooling and was not used in the current work . the maximum achromatic light output was estimated to correspond to a photopic luminance of 6.3 log phot cd.m ( retinal illuminance of 6.8 log photopic trolands [ phot - td ] ) and a scotopic luminance of 6.7 log scot cd.m ( 7.1 log scotopic trolands [ scot - td ] ) . testing was performed over a large range of adaptation conditions from 3 log scot - td to 2.2 log phot - td with the use of nd filters . the modification used in the current work was designed to build further upon the mp1s and consisted of a custom - machined slide - in tray that inserted directly into the filter holder rail ( figs . in theory , the led illuminators can be run at up to 1.5 a each to increase illumination ; however , that approach requires active cooling and was not used in the current work . the maximum achromatic light output was estimated to correspond to a photopic luminance of 6.3 log phot cd.m ( retinal illuminance of 6.8 log photopic trolands [ phot - td ] ) and a scotopic luminance of 6.7 log scot cd.m ( 7.1 log scotopic trolands [ scot - td ] ) . testing was performed over a large range of adaptation conditions from 3 log scot - td to 2.2 log phot - td with the use of nd filters . the eyes were dark adapted , and the grating stimuli were expected to be visible to the normal rod system but below the threshold of the l / m- and s - cones in the periphery . the lower of the two backgrounds corresponded to 0.2 log phot - td achieved by the insertion of a total of 5 log nd filters in the light path and either a w ( r , g , b = 53 , 53 , 53 ) or a b ( r , g , b = 0 , 0 , 255 ) background . with greater increments near 0.7 log phot - td , both ronb and wonw gratings of 3.3 , all normal subjects could discriminate the 4.7 cyc / deg grating , which was the upper limit of the current system designed for low vision . considering that the maximum available r grating on the b background corresponded to an increment of 0.1 log s - td compared to 0.8 log phot - td , it is likely that ronb gratings were detected by the l / m - cone system in normal eyes . for both the lower ( 0.1 log s - td ; 0.2 log phot - td ) and the higher ( 2.1 log s - td ; 2.2 log phot - td ) achromatic backgrounds , the bcm patient could discriminate the direction of gratings up to 1.6 cyc / deg with 0.7 log s increments ( fig . the eyes were dark adapted , and the grating stimuli were expected to be visible to the normal rod system but below the threshold of the l / m- and s - cones in the periphery . the lower of the two backgrounds corresponded to 0.2 log phot - td achieved by the insertion of a total of 5 log nd filters in the light path and either a w ( r , g , b = 53 , 53 , 53 ) or a b ( r , g , b = 0 , 0 , 255 ) background . with greater increments near 0.7 log phot - td , both ronb and wonw gratings of 3.3 , all normal subjects could discriminate the 4.7 cyc / deg grating , which was the upper limit of the current system designed for low vision . considering that the maximum available r grating on the b background corresponded to an increment of 0.1 log s - td compared to 0.8 log phot - td , it is likely that ronb gratings were detected by the l / m - cone system in normal eyes . for both the lower ( 0.1 log s - td ; 0.2 log phot - td ) and the higher ( 2.1 log s - td ; 2.2 log phot - td ) achromatic backgrounds , the bcm patient could discriminate the direction of gratings up to 1.6 cyc / deg with 0.7 log s increments ( fig . for these l / m - cone mediated conditions , spatial frequencies of 2 to 3 cyc / deg were measured near peak sensitivity with our flashed stimuli in normal subjects ; previously reported results in the periphery with drifting stimuli have been somewhat lower .
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we identified all patients with pe discharged from nongovernmental acute care hospitals in pennsylvania ( from 1 january 2000 to 30 november 2002 ) using the pennsylvania health care cost containment council ( phc4 ) database . this database contains information on demographic characteristics , insurance status , icd-9 clinical modification ( icd-9-cm ) diagnosis and procedure codes , hospital region and number of beds , and length of hospital stay for all patients . we included inpatients aged 18 years who were discharged with a primary diagnosis of pe based on the following icd-9-cm codes : 415.1 , 415.11 , 415.19 , and 673.2024 ( 7 ) . to ensure that we identified the most severely ill patients with pe as the primary reason for hospitalization , we also included inpatients with a secondary diagnosis code for pe and one of the following primary codes that may represent complications or treatments of this condition : respiratory failure ( 518.81 ) , cardiogenic shock ( 785.51 ) , cardiac arrest ( 427.5 ) , secondary pulmonary hypertension ( 416.8 ) , syncope ( 780.2 ) , thrombolysis ( 99.10 ) , and intubation or mechanical ventilation ( 96.04 , 96.05 , 96.7096.72 ) ( 7 ) . we excluded all other patients who had a secondary icd-9-cm code for pe or those who were transferred from another health care facility because the latter group of patients is more likely to have pe as a complication of hospitalization , and we did not know whether pe was diagnosed and treated before the patient was transferred . we excluded follow - up records for patients who were subsequently transferred to other hospitals , who had no identifiers required for linkage to the necessary clinical data , and for whom 30-day mortality information was not available . for this analysis , we also excluded patients without a documented serum glucose level at the time of presentation . patient demographic characteristics ( age , sex , and race ) and insurance status were abstracted from the phc4 database ( 7 ) . baseline clinical variables were obtained by linking eligible patients to the atlas database ( mediqual , marlborough , ma ) , which comprises clinical findings and laboratory parameters ( including serum glucose level ) at the time of presentation for all inpatients treated at nongovernmental acute care hospitals in pennsylvania ( 7 ) . the phc4 and atlas databases were matched by phc4 staff using unique patient identifiers ( patient date of birth , sex , and social security number ) ; we had no access to personal patient identifiers . we quantified severity of illness using the pulmonary embolism severity index ( pesi ) , a prognostic model for patients with pe that was developed and validated using these clinical data from the phc4 and atlas databases ( 7 ) . on the basis of the pesi , each patient is classified into one of five severity classes ( i v ) , with 30-day mortality ranging from 1.1 to 24.5% . to ascertain whether patients received thrombolytic therapy , we used icd-9-cm procedure codes ( 99.10 ) from the phc4 and atlas databases ( 7 ) . we abstracted the hospital region within pennsylvania , number of beds per hospital site , and annual number of pe admissions for each site from the phc4 database . we defined hospital teaching status based on data from the council of teaching hospitals of the association of american medical colleges . because 76% of teaching hospitals , but only 12% of nonteaching hospitals , had at least 350 hospital beds , we created a composite hospital - level variable for our statistical modeling based on teaching status and size ( i.e. , small nonteaching hospitals with fewer than 350 beds , large nonteaching hospitals with at least 350 beds , and teaching hospitals ) . we categorized admission serum glucose levels into five categories ( 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl ) according to previously published thresholds ( 11 ) . prior studies demonstrate that admission serum glucose levels > 110 mg / dl are significantly associated with mortality in patients with acute myocardial infarction and pneumonia ( 3,11 ) . we obtained mortality data by linking patients to the national death index with unique patient identifiers , including social security number , name , date of birth , and sex ( 1214 ) . the national death index has a sensitivity and specificity of > 97% for identifying mortality ( 14 ) . to ascertain our secondary outcome , hospital readmission for any reason to any acute care hospital in pennsylvania within 30 days of presentation , we used the phc4 database . to compare patient baseline characteristics across the five categories of serum glucose ( 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl ) , we performed tests for categorical variables and kruskal - wallis tests for continuous variables . p values for comparisons of baseline characteristics were adjusted using the holm method ( 15).we used survival analyses and the log - rank test to compare the cumulative 30-day mortality by serum glucose level . we used multivariable logistic regression to examine the independent association between categories of serum glucose and 30-day mortality , after adjusting for demographics ( age , sex , race , and insurance type ) , comorbid diseases ( history of cancer , chronic lung disease , heart failure , and diabetes mellitus ) , physical examination findings ( systolic arterial blood pressure < 100 mmhg , pulse 110/min , respiratory rate 30 breaths / min , body temperature < 36c , arterial oxygen saturation < 90% , and altered mental status ) , laboratory parameters ( hemoglobin level < 12 g / dl for women and < 13 g / dl for men , sodium 135 mmol / l , creatinine > 1.5 mg / dl , and troponin 0.1 ng / ml ) , administration of thrombolytics , and hospital characteristics ( region within pennsylvania , annual volume of pe , size , and teaching status ) . to account for patient clustering within hospital , we used random - intercept logistic regression with the two levels defined by patient and hospital site . to assess whether glucose - associated mortality risk differed in patients with and without known diabetes mellitus , we repeated multivariable analyses in patients with and without this condition . we used the same logistic regression model to examine the association between serum glucose level and readmission within 30 days in patients discharged alive . patients who were still hospitalized 30 days after admission and those without a documented readmission status were excluded from this analysis . all analyses were performed using stata 11.0 ( statacorp , college station , tx ) . we identified all patients with pe discharged from nongovernmental acute care hospitals in pennsylvania ( from 1 january 2000 to 30 november 2002 ) using the pennsylvania health care cost containment council ( phc4 ) database . this database contains information on demographic characteristics , insurance status , icd-9 clinical modification ( icd-9-cm ) diagnosis and procedure codes , hospital region and number of beds , and length of hospital stay for all patients . we included inpatients aged 18 years who were discharged with a primary diagnosis of pe based on the following icd-9-cm codes : 415.1 , 415.11 , 415.19 , and 673.2024 ( 7 ) . to ensure that we identified the most severely ill patients with pe as the primary reason for hospitalization , we also included inpatients with a secondary diagnosis code for pe and one of the following primary codes that may represent complications or treatments of this condition : respiratory failure ( 518.81 ) , cardiogenic shock ( 785.51 ) , cardiac arrest ( 427.5 ) , secondary pulmonary hypertension ( 416.8 ) , syncope ( 780.2 ) , thrombolysis ( 99.10 ) , and intubation or mechanical ventilation ( 96.04 , 96.05 , 96.7096.72 ) ( 7 ) . we excluded all other patients who had a secondary icd-9-cm code for pe or those who were transferred from another health care facility because the latter group of patients is more likely to have pe as a complication of hospitalization , and we did not know whether pe was diagnosed and treated before the patient was transferred . we excluded follow - up records for patients who were subsequently transferred to other hospitals , who had no identifiers required for linkage to the necessary clinical data , and for whom 30-day mortality information was not available . for this analysis , we also excluded patients without a documented serum glucose level at the time of presentation . patient demographic characteristics ( age , sex , and race ) and insurance status were abstracted from the phc4 database ( 7 ) . baseline clinical variables were obtained by linking eligible patients to the atlas database ( mediqual , marlborough , ma ) , which comprises clinical findings and laboratory parameters ( including serum glucose level ) at the time of presentation for all inpatients treated at nongovernmental acute care hospitals in pennsylvania ( 7 ) . the phc4 and atlas databases were matched by phc4 staff using unique patient identifiers ( patient date of birth , sex , and social security number ) ; we had no access to personal patient identifiers . we quantified severity of illness using the pulmonary embolism severity index ( pesi ) , a prognostic model for patients with pe that was developed and validated using these clinical data from the phc4 and atlas databases ( 7 ) . on the basis of the pesi , each patient is classified into one of five severity classes ( i v ) , with 30-day mortality ranging from 1.1 to 24.5% . to ascertain whether patients received thrombolytic therapy , we used icd-9-cm procedure codes ( 99.10 ) from the phc4 and atlas databases ( 7 ) . we abstracted the hospital region within pennsylvania , number of beds per hospital site , and annual number of pe admissions for each site from the phc4 database . we defined hospital teaching status based on data from the council of teaching hospitals of the association of american medical colleges . because 76% of teaching hospitals , but only 12% of nonteaching hospitals , had at least 350 hospital beds , we created a composite hospital - level variable for our statistical modeling based on teaching status and size ( i.e. , small nonteaching hospitals with fewer than 350 beds , large nonteaching hospitals with at least 350 beds , and teaching hospitals ) . we categorized admission serum glucose levels into five categories ( 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl ) according to previously published thresholds ( 11 ) . prior studies demonstrate that admission serum glucose levels > 110 mg / dl are significantly associated with mortality in patients with acute myocardial infarction and pneumonia ( 3,11 ) . we obtained mortality data by linking patients to the national death index with unique patient identifiers , including social security number , name , date of birth , and sex ( 1214 ) . the national death index has a sensitivity and specificity of > 97% for identifying mortality ( 14 ) . to ascertain our secondary outcome , hospital readmission for any reason to any acute care hospital in pennsylvania within 30 days of presentation to compare patient baseline characteristics across the five categories of serum glucose ( 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl ) , we performed tests for categorical variables and kruskal - wallis tests for continuous variables . p values for comparisons of baseline characteristics were adjusted using the holm method ( 15).we used survival analyses and the log - rank test to compare the cumulative 30-day mortality by serum glucose level . we used multivariable logistic regression to examine the independent association between categories of serum glucose and 30-day mortality , after adjusting for demographics ( age , sex , race , and insurance type ) , comorbid diseases ( history of cancer , chronic lung disease , heart failure , and diabetes mellitus ) , physical examination findings ( systolic arterial blood pressure < 100 mmhg , pulse 110/min , respiratory rate 30 breaths / min , body temperature < 36c , arterial oxygen saturation < 90% , and altered mental status ) , laboratory parameters ( hemoglobin level < 12 g / dl for women and < 13 g / dl for men , sodium 135 mmol / l , creatinine > 1.5 mg / dl , and troponin 0.1 ng / ml ) , administration of thrombolytics , and hospital characteristics ( region within pennsylvania , annual volume of pe , size , and teaching status ) . to account for patient clustering within hospital , we used random - intercept logistic regression with the two levels defined by patient and hospital site . to assess whether glucose - associated mortality risk differed in patients with and without known diabetes mellitus , we repeated multivariable analyses in patients with and without this condition . we used the same logistic regression model to examine the association between serum glucose level and readmission within 30 days in patients discharged alive . patients who were still hospitalized 30 days after admission and those without a documented readmission status were excluded from this analysis . all analyses were performed using stata 11.0 ( statacorp , college station , tx ) . of the 17,733 patient discharges that met our inclusion criteria , we excluded 323 with only a secondary code indicative of pe ( 1.8% ) , 767 patient transfers from another hospital ( 4.3% ) , 265 subsequent transfers to another hospital ( 1.5% ) , 777 discharges without a match to key clinical findings ( 4.4% ) , 70 without a linkage to the national death index ( 0.4% ) , and 1,910 ( 10.8% ) with an undocumented or erroneous admission serum glucose level . the study cohort comprised 13,621 patient discharges with a diagnosis of pe from 185 pennsylvania hospitals . on admission , 8,666 ( 63.6% ) patients had an elevated serum glucose level ( > 110 mg / dl ) . diabetes mellitus was known on admission in 1,889 ( 13.9% ) of all patients and in 1,685 ( 19.4% ) patients with an elevated serum glucose level . compared with the 13,621 enrolled patients , the 1,910 excluded because of an undocumented serum glucose level were significantly younger ( median age 63 vs. 67 years ; p < 0.001 ) and were less likely to have known diabetes mellitus ( 7.7 vs. 13.9% ; p < 0.001 ) , a history of heart failure ( 9.9 vs. 16.7% ; p < 0.001 ) , and a history of chronic lung disease ( 14.0 vs. 19.1% ; p < 0.001 ) . compared with eligible patients with a documented serum glucose level , these 1,910 excluded patients were also less likely to have a pulse 110/min ( 10.9 vs. 18.6% ; p < 0.001 ) , a systolic blood pressure < 100 mmhg ( 7.2 vs. 10.9% ; p < 0.001 ) , a respiratory rate 30/min ( 7.9 vs. 15.5% ; p < 0.001 ) , a body temperature < 36c ( 14.3 vs. 16.9% ; p = 0.005 ) , an altered mental status ( 5.9 vs. 7.5% ; p = 0.01 ) , and an arterial oxygen saturation < 90% ( 3.6 vs. 8.6% ; p < 0.001 ) at presentation . patients with higher admission serum glucose levels were older and more likely to have comorbid diseases ( cancer , chronic lung disease , heart failure , and diabetes mellitus ) and clinical and biological signs of disease severity ( tachycardia , hypotension , tachypnea , altered mental status , hypothermia , hypoxemia , anemia , hyponatremia , and elevated creatinine and troponin values ) ( table 1 ) . there was a higher proportion of patients in pesi risk classes iv and v among patients with higher serum glucose levels . baseline patient characteristics by level of admission glucose overall , 1,301 of 13,621 patients ( 9.6% ) died at 30 days . 170240 , and > 240 mg / dl had a cumulative 30-day mortality of 5.6 , 8.4 , 12.0 , 15.6 , and 18.3% , respectively ( p for trend < 0.001 ) ( fig . kaplan - meier estimates of 30-day mortality were 5.6 , 8.4 , 12.0 , 15.6 , and 18.3% for patients with serum glucose level 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl , respectively ( p for trend < 0.001 ) . admission glucose level ( mg / dl ) : * 110 , >110140 , >140170 , , elevated admission serum glucose levels remained significantly associated with 30-day mortality compared with patients with an admission serum glucose 110 mg / dl ( table 2 ) . the odds ratio ( or ) of dying increased from 1.19 ( 95% ci 1.001.42 ) for patients with a serum glucose level > 110140 mg / dl to 1.60 ( 1.262.03 ) for patients with a serum glucose level > 240 mg / dl . although higher admission serum glucose levels were significantly associated with an increase in the odds of dying in patients without diabetes , this association was not observed in patients with diabetes ( table 2 ) . other characteristics that were independently associated with higher adjusted odds of death included increasing age , the presence of comorbid diseases ( history of cancer , chronic lung disease , and heart failure ) , abnormal physical examination findings ( systolic arterial blood pressure < 100 mmhg , pulse 110/min , respiratory rate 30 breaths / min , body temperature < 36c , arterial oxygen saturation < 90% , and altered mental status ) , abnormal laboratory parameters ( hemoglobin level < 12 g / dl for women and < 13 g / dl for men , sodium 135 mmol / l , and creatinine > 1.5 mg / dl ) , the administration of thrombolytics , and hospital region within pennsylvania , size , and teaching status ( table 3 ) . independent association of admission glucose level with 30-day mortality in patients with and without diabetes mellitus independent associations of baseline characteristics with 30-day mortality ( n = 13,621 ) we estimated the 30-day readmission rate in 12,656 patients , after the exclusion of 836 patients who died in the hospital , 94 who were still hospitalized at 30 days after admission , and 35 with unknown readmission status . kaplan - meier estimates of 30-day readmission were 11.6 , 12.6 , 14.6 , 13.7 , and 14.7% for patients with a serum glucose level 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl , respectively ( p for trend = 0.001 ) . the adjusted odds of 30-day readmission was slightly higher in patients with admission serum glucose level > 140170 mg / dl ( or 1.21 [ 95% ci 1.021.44 ] ) but similar in patients with an admission serum glucose level > 110140 mg / dl ( 1.05 [ 0.911.20 ] ) , > 170240 mg / dl ( 1.06 [ 0.881.28 ] ) , and > 240 mg / dl ( 1.10 [ 0.871.38 ] ) compared with patients with a serum glucose level of 110 mg / dl . overall , the adjusted odds of readmission did not differ across serum glucose categories ( p = 0.31 ) . patients with higher admission serum glucose levels were older and more likely to have comorbid diseases ( cancer , chronic lung disease , heart failure , and diabetes mellitus ) and clinical and biological signs of disease severity ( tachycardia , hypotension , tachypnea , altered mental status , hypothermia , hypoxemia , anemia , hyponatremia , and elevated creatinine and troponin values ) ( table 1 ) . there was a higher proportion of patients in pesi risk classes iv and v among patients with higher serum glucose levels . 170240 , and > 240 mg / dl had a cumulative 30-day mortality of 5.6 , 8.4 , 12.0 , 15.6 , and 18.3% , respectively ( p for trend < 0.001 ) ( fig . kaplan - meier estimates of 30-day mortality were 5.6 , 8.4 , 12.0 , 15.6 , and 18.3% for patients with serum glucose level 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl , respectively ( p for trend < 0.001 ) . admission glucose level ( mg / dl ) : * 110 , >110140 , >140170 , , elevated admission serum glucose levels remained significantly associated with 30-day mortality compared with patients with an admission serum glucose 110 mg / dl ( table 2 ) . the odds ratio ( or ) of dying increased from 1.19 ( 95% ci 1.001.42 ) for patients with a serum glucose level > 110140 mg / dl to 1.60 ( 1.262.03 ) for patients with a serum glucose level > 240 mg / dl . although higher admission serum glucose levels were significantly associated with an increase in the odds of dying in patients without diabetes , this association was not observed in patients with diabetes ( table 2 ) . other characteristics that were independently associated with higher adjusted odds of death included increasing age , the presence of comorbid diseases ( history of cancer , chronic lung disease , and heart failure ) , abnormal physical examination findings ( systolic arterial blood pressure < 100 mmhg , pulse 110/min , respiratory rate 30 breaths / min , body temperature < 36c , arterial oxygen saturation < 90% , and altered mental status ) , abnormal laboratory parameters ( hemoglobin level < 12 g / dl for women and < 13 g / dl for men , sodium 135 mmol / l , and creatinine > 1.5 mg / dl ) , the administration of thrombolytics , and hospital region within pennsylvania , size , and teaching status ( table 3 ) . independent association of admission glucose level with 30-day mortality in patients with and without diabetes mellitus independent associations of baseline characteristics with 30-day mortality ( n = 13,621 ) we estimated the 30-day readmission rate in 12,656 patients , after the exclusion of 836 patients who died in the hospital , 94 who were still hospitalized at 30 days after admission , and 35 with unknown readmission status . kaplan - meier estimates of 30-day readmission were 11.6 , 12.6 , 14.6 , 13.7 , and 14.7% for patients with a serum glucose level 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl , respectively ( p for trend = 0.001 ) . the adjusted odds of 30-day readmission was slightly higher in patients with admission serum glucose level > 140170 mg / dl ( or 1.21 [ 95% ci 1.021.44 ] ) but similar in patients with an admission serum glucose level > 110140 mg / dl ( 1.05 [ 0.911.20 ] ) , > 170240 mg / dl ( 1.06 [ 0.881.28 ] ) , and > 240 mg / dl ( 1.10 [ 0.871.38 ] ) compared with patients with a serum glucose level of 110 mg / dl . overall , the adjusted odds of readmission did not differ across serum glucose categories ( p = 0.31 ) . our results demonstrate that a substantial proportion of patients with pe ( 63.6% ) have an elevated serum glucose level at the time of presentation . after adjusting for potential patient- and hospital - related confounders , and the administration of thrombolytic therapy , we found that patients with elevated serum glucose levels had a significantly higher short - term mortality . our findings are consistent with a retrospective analysis demonstrating an independent association between elevated mean glucose levels and inpatient mortality in intensive care unit patients with pe ( 16 ) . in contrast , we observed no significant association between serum glucose levels and the rate of hospital readmission . several mechanisms may explain the association between elevated serum glucose levels and mortality in patients with pe . first , elevated serum glucose levels have a procoagulant effect and decrease fibrinolysis ( 8,1719 ) . second , hyperglycemia is often accompanied by hyperinsulinemia , which may further inhibit fibrinolysis and increase the prothrombotic effect of hyperglycemia ( 8,18 ) . finally , it is possible that hyperglycemia is not a causal factor for adverse clinical outcomes but merely a marker of increased stress and severity of illness . in acute illnesses , such as pe , stress hormones ( i.e. , catecholamine , growth hormone , cortisol , and cytokines ) are released , increasing hepatic glucose production and insulin resistance ( 20 ) . although our study demonstrated a significant association between increasing glucose levels and mortality in patients without a diagnosis of diabetes mellitus , we did not find such an association in patients with known diabetes . a similar phenomenon has been observed in patients with stroke , with acute myocardial infarction , and in a mixed intensive care unit setting ( 4,11,21 ) . whether chronic hyperglycemia is protective of acute hyperglycemia - mediated damage or a lower intensity of stress is required to produce a similar degree of hyperglycemia in patients with diabetes mellitus remains unknown ( 5 ) . clinically , patients with pe who have hyperglycemia at presentation carry a higher risk of short - term mortality and may therefore potentially benefit from more intensive surveillance in the hospital and after discharge . whether laboratory markers of coagulation and fibrinolysis are correlated with the admission glucose level in patients with pe should be further examined . further research is also warranted to determine whether glucose - lowering treatment with insulin is associated with improved outcomes for patients with pe . currently , there is no evidence that insulin therapy to strictly control blood glucose in critically ill patients or patients with myocardial infarction or stroke improves mortality ( 22 ) . first , patients in the study sample were identified by use of icd-9-cm codes for pe rather than standardized radiographic criteria , and patient eligibility may therefore be subject to study selection biases due to hospital coding procedures . in a prior study , 96% of patients with specific codes for pe had objectively documented disease on the basis of chart review criteria ( 23 ) . second , our sample excluded 10.8% of younger , healthier , and less severely ill patients in whom serum glucose level was not measured at the time of admission . however , the exclusion of these lower risk patients , of whom probably a low proportion had hyperglycemia , is unlikely to change our study results . third , because measures of coagulation and fibrinolysis were not available in our database , we could not examine whether these factors are correlated with admission glucose levels . fourth , we were not able to adjust our results for other potential confounders that may influence glucose level and prognosis , such as concomitant inflammatory or infectious diseases , known metabolic syndrome , glucose intolerance , impaired fasting glucose , and hba1c levels . moreover , we had no information about insulin use or other glucose - lowering treatments during hospitalization and its effect on mortality . fifth , we had no information on serum glucose level after hospital admission and discharge ; thus , the prognostic implication of transient versus persistent elevated serum glucose level could not be analyzed . thus , we can not determine whether hyperglycemia has a direct effect on patient prognosis or is a mere marker of severity of illness and stress . in conclusion , in this large sample of patients hospitalized with acute pe , hyperglycemia at the time of presentation was associated with a significantly higher risk of 30-day mortality . elevated serum glucose levels may be a potential therapeutic target , and future studies should examine whether glucose - lowering treatments could improve outcomes in hyperglycemic patients with pe .
objectivealthough associated with adverse outcomes in other cardiopulmonary conditions , the prognostic value of elevated glucose in patients with acute pulmonary embolism ( pe ) is unknown . we sought to examine the association between glucose levels and mortality and hospital readmission rates for patients with pe.research design and methodswe evaluated 13,621 patient discharges with a primary diagnosis of pe from 185 acute care hospitals in pennsylvania ( from january 2000 to november 2002 ) . admission glucose levels were analyzed as a categorical variable ( 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl ) . the outcomes were 30-day all - cause mortality and hospital readmission . we used random - intercept logistic regression to assess the independent association between admission glucose levels and mortality and hospital readmission , adjusting for patient ( age , sex , race , insurance , comorbid conditions , severity of illness , laboratory parameters , and thrombolysis ) and hospital ( region , size , and teaching status ) factors.resultselevated glucose ( > 110 mg / dl ) was present in 8,666 ( 63.6% ) patients . patients with a glucose level 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl had a 30-day mortality of 5.6 , 8.4 , 12.0 , 15.6 , and 18.3% , respectively ( p < 0.001 ) . compared with patients with a glucose level 110 mg / dl , the adjusted odds of dying were greater for patients with a glucose level > 110140 ( odds ratio 1.19 [ 95% ci 1.001.42 ] ) , > 140170 ( 1.44 [ 1.171.77 ] ) , > 170240 ( 1.54 [ 1.261.90 ] ) , and > 240 mg / dl ( 1.60 [ 1.262.03 ] ) , with no difference in the odds of hospital readmission.conclusionsin patients with acute pe , elevated admission glucose is common and independently associated with short - term mortality .
RESEARCH DESIGN AND METHODS Patient identification and eligibility Patient and hospital characteristics Admission serum glucose level and diabetes mellitus Study outcomes Statistical analysis RESULTS Comparison of baseline patient characteristics by admission serum glucose level Association of admission serum glucose level and 30-day mortality Association of admission serum glucose level and 30-day readmission CONCLUSIONS
we used multivariable logistic regression to examine the independent association between categories of serum glucose and 30-day mortality , after adjusting for demographics ( age , sex , race , and insurance type ) , comorbid diseases ( history of cancer , chronic lung disease , heart failure , and diabetes mellitus ) , physical examination findings ( systolic arterial blood pressure < 100 mmhg , pulse 110/min , respiratory rate 30 breaths / min , body temperature < 36c , arterial oxygen saturation < 90% , and altered mental status ) , laboratory parameters ( hemoglobin level < 12 g / dl for women and < 13 g / dl for men , sodium 135 mmol / l , creatinine > 1.5 mg / dl , and troponin 0.1 ng / ml ) , administration of thrombolytics , and hospital characteristics ( region within pennsylvania , annual volume of pe , size , and teaching status ) . we used multivariable logistic regression to examine the independent association between categories of serum glucose and 30-day mortality , after adjusting for demographics ( age , sex , race , and insurance type ) , comorbid diseases ( history of cancer , chronic lung disease , heart failure , and diabetes mellitus ) , physical examination findings ( systolic arterial blood pressure < 100 mmhg , pulse 110/min , respiratory rate 30 breaths / min , body temperature < 36c , arterial oxygen saturation < 90% , and altered mental status ) , laboratory parameters ( hemoglobin level < 12 g / dl for women and < 13 g / dl for men , sodium 135 mmol / l , creatinine > 1.5 mg / dl , and troponin 0.1 ng / ml ) , administration of thrombolytics , and hospital characteristics ( region within pennsylvania , annual volume of pe , size , and teaching status ) . 170240 , and > 240 mg / dl had a cumulative 30-day mortality of 5.6 , 8.4 , 12.0 , 15.6 , and 18.3% , respectively ( p for trend < 0.001 ) ( fig . kaplan - meier estimates of 30-day mortality were 5.6 , 8.4 , 12.0 , 15.6 , and 18.3% for patients with serum glucose level 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl , respectively ( p for trend < 0.001 ) . kaplan - meier estimates of 30-day readmission were 11.6 , 12.6 , 14.6 , 13.7 , and 14.7% for patients with a serum glucose level 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl , respectively ( p for trend = 0.001 ) . the adjusted odds of 30-day readmission was slightly higher in patients with admission serum glucose level > 140170 mg / dl ( or 1.21 [ 95% ci 1.021.44 ] ) but similar in patients with an admission serum glucose level > 110140 mg / dl ( 1.05 [ 0.911.20 ] ) , > 170240 mg / dl ( 1.06 [ 0.881.28 ] ) , and > 240 mg / dl ( 1.10 [ 0.871.38 ] ) compared with patients with a serum glucose level of 110 mg / dl . 170240 , and > 240 mg / dl had a cumulative 30-day mortality of 5.6 , 8.4 , 12.0 , 15.6 , and 18.3% , respectively ( p for trend < 0.001 ) ( fig . kaplan - meier estimates of 30-day mortality were 5.6 , 8.4 , 12.0 , 15.6 , and 18.3% for patients with serum glucose level 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl , respectively ( p for trend < 0.001 ) . kaplan - meier estimates of 30-day readmission were 11.6 , 12.6 , 14.6 , 13.7 , and 14.7% for patients with a serum glucose level 110 , > 110140 , > 140170 , > 170240 , and > 240 mg / dl , respectively ( p for trend = 0.001 ) . the adjusted odds of 30-day readmission was slightly higher in patients with admission serum glucose level > 140170 mg / dl ( or 1.21 [ 95% ci 1.021.44 ] ) but similar in patients with an admission serum glucose level > 110140 mg / dl ( 1.05 [ 0.911.20 ] ) , > 170240 mg / dl ( 1.06 [ 0.881.28 ] ) , and > 240 mg / dl ( 1.10 [ 0.871.38 ] ) compared with patients with a serum glucose level of 110 mg / dl .
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vps4b , a member of the aaa ( atpases associated with diverse cellular activities ) protein family , plays an important role in the lysosomal degradation pathway , which functions in ligand - induced membrane receptor downregulation . in lysosomal degradation , endocytosed receptors are sorted into multivesicular bodies ( mvbs ) , which requires the sequential assembly of endosomal sorting complex required for transport i , ii , and iii ( escrt - i , -ii , and -iii ) on the endosomal membrane . vps4b functions to dissociate escrt - iii from endosomes for further rounds of endosomal sorting . expression of functionally inactive vps4b results in the accumulation of receptors on abnormally enlarged endosomes ( class e compartments ) and the abolishment of mvb biogenesis [ 36 ] . as an essential escrt - iii interacting protein , vps4b is also involved in protein trafficking among membrane compartments and in signal transduction [ 4 , 5 , 7 , 8 ] . epidermal growth factor- ( egf- ) mediated signaling is one of the most important signaling pathways for cell growth , proliferation , invasion , and metastasis in breast cancer . upon ligand binding , the egf receptor ( egfr ) is phosphorylated , promoting downstream signal transduction , while it is internalized and degraded within the mvb - lysosome . egfr signaling is initiated at the cell membrane ; however , late signaling propagation occurs on the endosomes . we and others have reported that dysfunction of vps4b not only leads to delayed egfr degradation , but also to prolonged and altered intracellular egfr signaling in abnormally enlarged endosomal compartments [ 46 , 12 ] . to further understand the role of vps4b dysfunction in breast cancer , we set out to determine the consequences of altered egfr signaling : changes in protein synthesis and degradation , as well as protein dynamics in vps4b downregulated breast cancer cells . altered protein synthesis and degradation of cancer - related proteins is involved in cellular transformation and cancer progression [ 1318 ] . understanding protein synthesis and degradation is essential to fully appreciate cellular dynamics and develop more effective strategies to treat cancer . traditional protein synthesis and degradation studies rely on radiolabel tracer labeling ( pulse ) followed by exposure to nonradioactive medium ( chase ) . however , these approaches are often designed to address the turnover of a specific protein . recently , high throughput synthesis and degradation mass spectrometry ( sdms ) has become a novel approach to study global protein turnover , in which protein degradation and synthesis can be measured simultaneously and unambiguously with minimal cell perturbation . in this approach , proteins are first metabolically labeled by stable isotope labeling with amino acids in cell culture ( silac ) followed by chasing in normal medium . as a result , the preexisting proteins are labeled with stable isotope amino acids , while the newly synthesized proteins only incorporate regular amino acids . these differential stable isotope - labeled proteins can be discriminated from each other and quantified by mass spectrometry based on the differences in masses of their peptides following enzymatic digestion . since pratt and coworkers introduced the use of mass spectrometry in protein synthesis and degradation studies ; this approach has been applied to numerous systems , such as escherichia coli , mycobacterium tuberculosis , mycoplasma pneumoniae , streptomyces coelicolor , saccharomyces cerevisiae , hela cells [ 25 , 26 ] , human adenocarcinoma cells , chicken skeletal muscle [ 28 , 29 ] , and mice [ 30 , 31 ] . one of the major assumptions of using a pulse - chase labeling experiment to study the dynamics of protein expression is that the level of protein expression is always under the steady state . in silac - based protein synthesis and degradation experiments , the degradation rate constant of targeted proteins can be readily derived from the decrease of relative isotope abundance ( ria ) or the percentage of preexisting labeled protein . on the contrary , it is more difficult to calculate the rate of protein synthesis in general since proteins are synthesized continuously in cultured proliferating cells , thus the rate of protein accumulation is not likely to be under steady state . in order to bypass such steady state assumption , the incorporation of isobaric tags for relative and absolute quantitation ( itraq ) or the introduction of an internal control , such as another set of labeled cells , permits the simultaneous measurement of protein synthesis , degradation , and expression under dynamic conditions , such as cell proliferation . dysfunction of vps4b leads to egfr accumulation and prolonged activation [ 46 , 12 ] , which could contribute to cell proliferation and growth . to estimate protein synthesis and degradation rates in the context of altered egfr signaling caused by downregulation of vps4b , we independently developed an approach called internal standard - assisted synthesis and degradation mass spectrometry ( isdms ) that normalizes protein abundance across different samples and time points and permits the comparison of protein synthesis , degradation , and expression measurements . a similar approach has also been developed recently by boisvert et al . to study global protein turnover in hela cells . through this isdms analysis , we compared the synthesis and degradation rates of more than 700 proteins between vps4b downregulated skbr3 cells and the parental skbr3 cells . we found that vps4b downregulation resulted in differential protein expression in energy metabolism pathways by altering the synthesis and degradation of related proteins , in which glycolysis proteins were downregulated , while mitochondrial fatty acid -oxidation proteins were upregulated . the adoption of fatty acid -oxidation as an alternative energy source could be an unrevealed survival mechanism for breast cancer cells with vps4b dysfunction . skbr3 cells were obtained from the american type culture collection ( rockville , md ) . vps4b knock - down skbr3 ( skbr3_shvps4b ) cells were generated by transducing skbr3 cells with a lentivirus harboring shrna against vps4b ( sa biosciences / qiagen , frederick , md ) as previously described . to study the role of vps4b downregulation on the dynamics of protein expression by isdms , skbr3_shvps4b , and the parental skbr3 cells were cultured in dmem silac medium ( pierce / thermo scientific ) supplemented with 28 mg / l c6-arginine ( arg6 , purity 9799% , cambridge isotope laboratories ) , 72 mg / l d4-lysine ( lys4 , purity 9698% , cambridge isotope laboratories ) , 10% dialyzed fetal bovine serum ( fbs , invitrogen ) , and 1% antibiotic antimycotic solution ( invitrogen ) . for skbr3_shvps4b cells , 2 g / ml after five passages , cells were starved in serum free silac medium for 18 hr . following three washes with dulbecco 's phosphate - buffered saline ( dpbs , invitrogen ) , cells were stimulated with 100 ng / ml egf and subsequently chased for 0 , 2 , 6 , 12 , and 24 hr in the presence of dmem light silac medium ( pierce / thermo scientific ) supplemented with light arginine ( arg0 ) , lysine ( lys0 ) ( sigma ) , 10% dialyzed fbs , and 1% antibiotic antimycotic solution . at the end of each chase time period , cell pellets were collected and frozen in liquid nitrogen and stored in 80c until analysis . to prepare the heavy- labeled internal standard , skbr3_shvps4b cells were metabolically labeled with c6n4-arginine ( arg10 ) and c6n2-lysine ( lys8 ) by culturing in dmem heavy silac medium supplemented with 28 mg / l c6n4-arginine ( arg10 , purity 9799% , cambridge isotope laboratories ) , 72 mg / l c6n4-lysine ( lys8 , purity 9799% , cambridge isotope laboratories ) , 10% dialyzed fbs , and 1% antibiotic antimycotic solution ( invitrogen ) . the incorporation of arg10 and lys8 in the internal standard skbr3_shvps4b cells was ~98% as determined by mass spectrometric analysis of tryptic peptides isolated from the arg10 and lys8 labeled skbr3_shvps4b cells . cell pellets were lysed and sonicated in 4% sds,100 mm tris - hcl ( ph 7.6 ) lysis buffer containing 5 u / ml benzonase nuclease ( novagen ) and complete edta - free protease inhibitor cocktail ( roche ) . after removal of cell debris by centrifugation at 10,000 g at room temperature for 10 min , protein concentration was measured in triplicate using the bca protein assay kit ( thermo scientific / pierce ) . 180 g of protein from each time point were mixed with 60 g of protein internal standard labeled with c6n4-arginine10 and c6n2-lysine8 . dithiothreitol ( dtt , sigma ) and tris ( 2-carboxyethyl ) phosphine ( tcep , thermo scientific / pierce ) were added to the mixture to final concentrations of 100 mm and 10 mm , respectively . after cooling to room temperature , the cell lysates were processed by the filter aided sample preparation ( fasp ) procedure using 30 k vivacon 500 filtration units ( sartorius biolab ) with modifications . briefly , cell lysates were first reduced by mixing with 200 l of ua buffer ( 8 m urea in 100 mm tris - hcl ph 8.5 ) containing 5 mm tcep , loaded into the filtration units , and centrifuged at 14,000 g for 15 min . at the end of the reduction reaction , the samples were washed three times in 200 l of ua buffer followed by centrifugation at 14,000 g for 15 min . alkylation was performed by adding 100 l of 50 mm iodoacetamide in ua buffer and incubating at room temperature for 30 min . excess iodoacetamide was eliminated by centrifugation , followed by three washes with 200 l of ub buffer ( 8 m urea in 100 mm tris - hcl , ph 8.0 ) . at the end of reductive alkylation reaction , samples were digested by sequential addition of proteases lys - c and trypsin in the filtration units . briefly , 2 g of endoproteinase lys - c ( roche ) in 40 l water were first added to the filtration units and kept at room temperature in a humidified chamber overnight for 16 hr . after lys - c digestion , samples were diluted with 200 l of 50 mm nh4hco3 , and 4 g of trypsin ( promega ) were added to the reaction mixture and incubated at 37c for another 68 hr . after digestion , the peptides were collected by centrifugation of the filtration units , followed by two washes with 50 l of 50 mm nh4hco3 . all peptides containing filtrates were pooled and acidified by the addition of trifluoroacetic acid ( tfa , sigma ) to a final concentration of 1% . acidified peptides were desalted by a sep - pak c18 cartridge ( waters ) and dried by speedvac ( thermo scientific ) . peptides were fractionated using stop and go extraction ( stage ) tips made in house with strong anion exchange ( sax ) disks as described in [ 3436 ] . the stage tips were assembled by stacking six layers of empore anion exchange membrane disks ( 3 m ) into a 200 l micropipet tip . britton and robinson buffer ( br buffer ) , composed of 20 mm acetic acid , 20 mm phosphoric acid , and 20 mm boric acid and titrated with naoh to the desired ph , was used in peptide separation . the tip was wet with methanol and washed with 1 m naoh , followed by equilibration with 100 l of br buffer ph 11 . peptides were loaded at ph 11 , and fractions were subsequently eluted with br buffer of ph 11 , 8 , 6 , 5 , 4 , and 3 , respectively . the flow through and all the fractions were acidified by adding tfa to a final concentration of 1% and desalted by a stage tip containing three layers of c18 membrane disks ( 3 m ) . peptides were dissolved in 0.5% acetic acid ( solvent a ) and separated on a 10 cm reverse - phase picofrit spray tip ( new objective , woburn , ma ) packed in house with sub-2 m c18 resin ( prospereon life science , il ) , using a nanoflow xtreme simple liquid chromatography system ( microtech / cvc ) coupled to a hybrid linear ion trap - orbitrap mass spectrometer ( ltq orbitrap , thermo scientific ) . peptides were loaded onto the column with solvent a at a flow rate of 0.6 l / min and eluted with a 180 min linear gradient at a flow rate of 0.2 l / min . a gradient of 260% solvent b ( 40% acetonitrile , 0.5% acetic acid ) was applied to the sax flow through , fractions eluted with ph 11 and ph 8 buffer , a 265% solvent b gradient was used for the ph 6 and ph 5 fractions , and a 570% solvent b gradient was used for the ph 4 and ph 3 fractions . after the linear gradient , the column was washed with 95% solvent b and reequilibrated with 95% solvent a. seven fractions from one sample were run sequentially followed by a 40 min wash with 80% acetonitrile with 0.5% acetic acid . mass spectra were acquired in the positive ion mode applying a data - dependent automatic switch between the survey scan and ms / ms acquisition . the survey ms1 scans were acquired in the orbitrap using a mass range of m / z 4001,600 at a resolution of 60,000 at 400 m / z . ms / ms scans were acquired in the linear ion trap on the 5 most intense ions in each survey scan with dynamic exclusion of previously selected ions ; repeat count 1 and exclusion duration 15 seconds . the fragmentation was performed by collision - induced dissociation ( normalized collision energy 35% ) with a target value of 30,000 . other mass spectrometric parameters that were spray voltage 1.35 kv ; no sheath and auxiliary gas flow ; ion transfer tube temperature 180c ; activation q = 0.25 ; activation time of 30 ms were applied in ms2 acquisitions . data were searched against a concatenated forward / reverse database , which was built based on a uniprot human database ( downloaded on oct . 18 , 2010 ) , using sequest ( bioworks 3.3.1 sp1 , thermo scientific ) . enzyme specificity was set to trypsin and allowed two missed cleavages . database search parameters were precursor peptide mass tolerance 50 ppm and fragment ion tolerance 1 amu . carbamidomethyl cysteine was set as a fixed modification , and oxidized methionine was set as a variable modification . additional variable modifications included arginine + 6.02013 , arginine + 10.00827 , lysine + 4.02510 , and lysine + 8.01420 . the peptides were initially filtered by the following criteria : xcorr 2.5 , 3.0 , and 3.5 for 2 + , 3 + , and 4 + peptides , respectively , and cn > 0.1 . the false discovery rate ( fdr ) was calculated by dividing the number of false positive peptides identified in the reverse database by the number of total identified peptides . in this study , the fdr was 0.94% . by adding the internal standard , three populations of a given peptide were present in the mixture : the peptide containing regular arginine ( arg0 ) or lysine ( lys0 ) ( light - labeled peptide ) , the peptide containing c6-arginine ( arg6 ) or d4-lysine ( lys4 ) ( medium - labeled peptide ) , and the peptide containing c6n4-arginine ( arg10 ) or c6n2-lysine ( lys8 ) ( heavy - labeled internal standard peptide ) . the relative abundance of light peptide ( al ) was defined as the ratio of the peak intensities of unlabeled peptide ( il ) to the intensities of internal standard peptide ( ih ) , ( 1 ) . similarly , the relative abundance of labeled peptide ( am ) was defined as the ratio of the peak intensities of labeled peptide ( i m ) to ih , ( 2 ) . an in - house silac - based mass spectrometry quantitation software , isoquant ( http://www.proteomeumb.org/mzw.html ) , was used to automatically integrate the isotopic peak intensities of each peptide and calculate al and am . the relative abundance of total peptide ( atot ) was calculated by summing the relative abundance of light unlabeled peptide ( al ) and labeled peptide ( am ) , ( 3 ) ( 1)al = ilih,(2)am= imih,(3)atot = al+am . since the silac labeled peptides were only subjected to degradation , the degradation rate constant could be derived from the changes in relative abundance of labeled peptides . since protein degradation rate follows first - order kinetics , the relative abundances of the preexisting labeled peptides over time ( am_t ) were fit to exponential decay curves to derive the degradation rate constant ( ) . at least four time points were required to find the best fitting curve . only am_t and t that were significantly correlated ( p < 0.05 ) with a coefficient of determination ( r ) greater than 0.8 were selected to derive the degradation rate constant from the exponential decay equation as follows : ( 4)am_t = aet , where a was the corrected - normalized initial peptide amount . peptide synthesis rate was defined as the rate of change of the relative abundance of the newly synthesized peptide over time . the relative abundance of the newly synthesized peptide can be calculated by the relative abundance of the unlabeled peptide directly , if the basal label efficiency reaches 100% . however , due to different cell types , cell culture conditions , and the purity of the stable isotope - labeled amino acids , the basal labeling efficiency was not 100% , which consequently resulted in the detection of unlabeled peptides at the beginning of the chase period . in this study , at the beginning of the chase period , ~10% and 30% of the peptides were unlabeled in skbr3_shvps4b and skbr3 cells , respectively . to calculate the relative abundance of the newly synthesized peptide , the relative abundance of the unlabeled peptide has to be corrected by subtracting the relative abundance of the preexisting unlabeled peptide at 0 hr , as well as the later time points . since both the preexisting unlabeled and labeled peptides were subjected to degradation , the preexisting unlabeled peptide at each time point ( apre_l_t ) was calculated by ( 5)apre_l_t = al_0et , where al_0 was the relative abundance of the unlabeled peptide at 0 hr . the relative abundance of the newly synthesized peptide at each time point ( al_t ) was calculated by ( 6)al_t=al_tapre_l_t , where al_t was the abundance of the total unlabeled peptide at each time point . the relative abundances of newly synthesized peptides over time al_t were fit to linear curves to derive the synthesis rates ( ) . p < 0.05 ) with a r greater than 0.8 were selected to derive the synthesis rate from the following linear equation : ( 7)al_t=t+b , where b was the corrected initial peptide amount . the protein synthesis rate , degradation rate constant , and relative protein abundance were calculated by the mean of their identified peptides ' synthesis rates , degradation rate constants , and total relative peptide abundance . our earlier report has shown that hypoxia leads to the abnormal accumulation of egfr and subsequent alteration of cell signaling in breast cancer . to further understand the role of vps4b dysfunction in global protein dynamics upon egf treatment , we decided to measure the rates of protein synthesis and degradation in vps4b downregulated skbr3 ( skbr3_shvps4b ) and the parental skbr3 cells using an internal standard assisted synthesis and degradation mass spectrometry ( isdms ) approach . as shown in figure 1 , both cultured skbr3_shvps4b and skbr3 cells were first metabolically labeled with stable isotopic amino acids arginine , and lysine ( arg6/lys4 , labeled in red ) , and then chased in medium containing regular arginine and lysine amino acids ( arg0/lys0 , labeled in blue ) in the presence of egf . as a result , one can monitor the rates of protein degradation simply by measuring the decrease of arg6/lys4-labeled proteins , while newly synthesized proteins can only be monitored by the incorporation of arg0 and lys0 amino acids . since the depletion of vps4b expression is likely going to have some effects on global protein expression , it is necessary to normalize the steady state protein profiling and differential gene expression between vps4b ablation and the parental cells . to overcome the issue of differential gene expression between the two cell lines , an internal standard - cell lysates metabolically labeled with arg10/lys8 ( labeled in green ) was added to each time point in order to normalize the relative abundance of arg6/lys4 or arg10/lys6-labeled peptides between skbr3_shvps4b and skbr3 cells . figures 2(a ) and 2(b ) are representative ms spectra of tryptic peptides , sllvnpegptlmr , derived from chain a of human fatty acid synthase ( fasn ) in skbr3 ( figure 2(a ) ) and skbr3_shvps4b cells ( figure 2(b ) ) identified in the chase time periods of 0 , 2 , 6 , 12 , and 24 hr . the decreasing abundance of the preexisting arg6/lys4-labeled peptides ( labeled in red ) between the 12 and 24 hr chase periods in both cell lines clearly indicates that the degradation of this peptide can be monitored by this type of approach . to determine the role of vps4b on protein turnover , a first - order exponential decay curve fitting was constructed ( see section 2.7 for more details ) . as indicated in figure 2(c ) , the peptide derived from fatty acid synthase ( sllvnpegptlmr ) clearly had a faster degradation rate in skbr3_shvps4b cells ( labeled in squares , degradation rate constant = 0.015 h , r = 0.91 ) than skbr3 cells ( labeled in diamonds , degradation rate constant = 0.045 h , r = 0.90 ) . on the other hand , the calculation of synthesis rate is much more difficult because the pool of unlabeled arg0/lys0 ( labeled in blue ) peptides consisted of both newly synthesized peptides during the chase period , and preexisting unlabeled peptides , which were present at the beginning of the experiment due to incomplete labeling of arg6/lys4 amino acids . since preexisting unlabeled ( arg0/lys0 ) peptides were being degraded during the course of the experiment , it is necessary to normalize the relative abundance of the remaining ( nondegraded ) preexisting unlabeled peptides at each time point based on their degradation rate constant(see section 2.8 for more details ) . figure 2(d ) is a representative time - course analysis of newly synthesized human fasn peptides ( sllvnpegptlmr ) . this result suggests that the fasn peptide ( sllvnpegptlmr ) had a slower synthesis rate in skbr3_shvps4b cells ( labeled in squares , synthesis rate = 0.055 h , r = 0.99 ) as compared to skbr3 cells ( labeled in diamonds , synthesis rate = 0.133 h , r = 0.98 ) . because isdms allows one to measure the rates of protein synthesis and degradation simultaneously , it is therefore possible to determine the dynamics of protein expression in vps4b - depleted cells in response to egf . figure 2(e ) is a representative time course analysis of fasn dynamic expression . in this study , the relative abundance of a given peptide ( atot ) of fasn ( sllvnpegptlmr ) from each time point was calculated and normalized by summing the relative abundance of unlabeled peptide ( al ) and labeled peptide ( am ) ( ( 3 ) , section 2 ) . it is clear that the relative abundance of fasn expression in skbr3_shvps4b cells ( figure 2(e ) , labeled in squares ) was drastically reduced after two hours of egf treatment . together with the result from the degradation study of fasn in figure 2(c ) , this observation suggests that the decrease of fasn expression is largely due to the increased turnover rate of fasn in skbr3_shvps4b cells ( figure 2(c ) , labeled in squares ) . interestingly , after 6 hr of egf treatment the rate of fan degradation was very similar between skbr3_shvps4b and skbr3 cells , indicating that the sudden increase of fasn turnover rate in skbr3_shvps4b cells could be potentially regulated by egf - related cell signaling pathways . vps4b is essential for the formation of mvb and has also been documented to play a pivotal role in regulating the degradation of various membrane receptors . therefore , we decided to examine the consequence of vps4b ablation on the dynamic proteome profile using our skbr3_shvps4b model system . briefly , both cultured skbr3_shvps4b and the parental control sbkr3 cells grown on silac medium , arg6/lys4 , medium were treated with 100 ng / ml of egf and chase labeled in silac light , arg0/lys0 , medium for 2 , 6 , 12 , and 24 hr as described in the section 2 . at the end of each chase period , skbr3_shvps4b and skbr3 cells were collected and analyzed by a fasp - based sax fractionation and lc - ms / ms analysis using a hybrid orbitrap mass spectrometer . ms / ms raw files were then subjected to database search and quantification analysis using our in - house software isoquant . however , in order to compare the degradation rate constants and synthesis rates of each peptide in skbr3_shvps4b and skbr3 cells at the proteome level , we required that the same peptides should be identified and quantified at each time point in both cell lines throughout the entire course of the experiment in all five time points . as indicated in figures 2(c)-2(d ) , the relative peptide abundance and the time period used to calculate both degradation rate constants and synthesis rates also need to be highly correlated ( p < 0.05 ; r > 0.8 ) with at least four time points in order to derive the rates . these criteria have significantly reduced the depth of proteome ; however , we were able to generate a highly statistically relevant dataset on the effects of vps4b in modulating proteome dynamics . overall , we have identified more than 15,000 total unique peptides assigned to more than 4,500 protein groups in both skbr3_shvps4b and skbr3 cells . over 70% of the proteins we obtained the degradation rate constants , synthesis rates , and total peptide profiles of 1623 peptides and 723 proteins . at the global level , the overall mean protein synthesis rate in skbr3_shvps4b cells was 0.058 0.010 h , which was similar to that of the skbr3 cells , 0.057 0.012 h(see supplemental figures 1(a ) and 1(b ) available online at http://dx.doi.org/10.1155/2013/291415 ) . this result suggests that vps4b does not affect global protein synthesis in general in response to egf treatment . vps4b depletion also does not have any systematic effect on global protein degradation , because the mean protein degradation rate constant was 0.033 0.012 h ( t1/2 = 20 hr ) in skbr3_shvps4b cells , which was only slightly higher than that of the skbr3 cells , 0.028 0.010 h ( t1/2 = 24 hr ) ( supplemental figures 1(c ) and 1(d ) ) . because the heavy silac arg10/lys8-labeled internal protein mixture was spiked into every time point , it is possible to analyze the effect of vps4b downregulation on global protein expression . to address the effect of vps4b downregulation on dynamic protein profiles , we defined increased and decreased protein synthesis , degradation , and relative protein abundance in skbr3_shvps4b cells as those values greater than 1.5-fold that of the skbr3 cells ( skbr3_shvps4b versus skbr3 ratio < 0.67 or > 1.5 ) . among 723 proteins , the majority did not show changes in their rates of protein synthesis ( 80.9% of proteins ) , degradation ( 77.2% of proteins ) , or relative protein abundance ( 90.2% of proteins ) ( supplemental table 1 ) . as expected , downregulation of vps4b has a rather limited effect on the alteration of overall protein synthesis , because only ~10% of proteins displayed changes in their rates of protein synthesis . on the other hand , ~20% of proteins had increased rates of degradation in skbr3_shvps4b cells , suggesting that vps4b depletion potentially affects and enhances the turnover of certain proteins . at the global level , our data suggest that vps4b downregulation does not change the dynamic protein profiles of most proteins in skbr3 cells . despite the vps4b - depleted cell line having a rather different growth phenotype and sensitivity to hypoxia as compared to the parental cells , we found that the overall distributions of relative protein abundance at 24 hr in skbr3 and skbr3_shvps4b cells are also very similar ( supplemental figures 1(e ) and 1(f ) ) . in this study , we found that among 33 proteins with increased relative abundance ( supplemental table 2 ) , 63.6% had increased synthesis only , 9.1% had decreased degradation only , and 9.1% had both ( table 1 ) . among 38 proteins with decreased relative abundance ( supplemental table 3 ) , 18.4% had decreased synthesis only , 15.8% had increased degradation only , and 65.8% had both ( table 2 ) . because the steady state accumulation of proteins is determined by their rates of synthesis and degradation , we therefore decided to further examine the relationship between the steady state protein expression and protein synthesis and degradation in response to the downregulation of vps4b expression . the relative protein abundance ratios between the two cell lines ( skbr3_shvps4b versus skbr3 ) were plotted against the ratios of protein synthesis rates ( supplemental figure 2(a ) ) and the ratios of protein degradation rate constants ( supplemental figure 2(b ) ) . as indicated in figure 3 , it is clear that the vps4b - mediated decrease of protein expression is largely due to increased protein degradation , while the increase of vps4b - mediated protein expression is directly related with increased protein synthesis . for instance , proteins with higher relative abundance in skbr3_shvps4b cells ( figure 3 , red diamonds ) were found to have both lower degradation rate constants and higher synthesis rates ( figure 3 , 2nd quadrant ) . conversely , those proteins with lower relative abundance have higher degradation rate constants and lower synthesis rates ( figure 3 , 4th quadrant , labeled in green diamonds ) . to further understand the biological consequences of vps4b ablation in egf - mediated cell signaling , a gene ontology and kyoto encyclopedia of genes and genome ( kegg ) pathway analysis was performed . as indicated in figure 4 , proteins involved in energy metabolism , in particular glycolysis and mitochondrial fatty acid -oxidation pathways , are significantly affected in skbr3_shvps4b cells upon egf treatment . in humans , glycolysis and fatty acid -oxidation are the two main sources of acetyl - coa for the tricarboxylic acid cycle ( tca ) to generate adenosine triphosphate ( atp ) . we found that the expression of many key glycolytic enzymes , such as glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) and l - lactate dehydrogenase b chain ( ldhb ) , fructose - bisphosphate aldolase a and c ( aldoa , aldoc ) , phosphoglycerate kinase 1 ( pgk1 ) , alpha - enolase ( eno1 ) , pyruvate kinase isozymes m1/m2 ( pkm2 ) , and l - lactate dehydrogenase a chain ( ldha ) , was drastically reduced in skbr3_shvps4b cells . as indicated in figure 4 , the decreased expression of these proteins is mainly caused by the simultaneously increased degradation and decreased synthesis rates of these proteins as the consequence of vps4b downregulation . interestingly , the expression of mitochondrial trifunctional protein alpha - subunit ( hadha / echa ) , mitochondrial trifunctional protein beta - subunit ( hadhb / echb ) , and mitochondrial hydroxyacyl - coenzyme a dehydrogenase ( hadh ) was increased more than 1.5-fold in skbr3_shvps4b cells . mitochondrial very long - chain specific acyl - coa dehydrogenase ( acadv ) and mitochondrial enoyl - coa hydratase ( echm ) were increased more than 1.25-fold . the increased expression of these mitochondrial proteins and proteins involved in fatty acid -oxidation are primarily caused by the increased protein synthesis rate , rather than caused by decreased protein degradation . on the other hand , fatty acid synthase ( fasn ) expression was decreased in skbr3_shvps4b cells with increased degradation and decreased synthesis rates . taken together , our results suggest that downregulation of vps4b expression can potentially alter the energy metabolism in breast cancer , suggesting that under either hypoxia or vps4b depletion , fatty acid tends to be oxidized to generate energy rather than being stored , consequently replacing glucose as a main energy source . dysfunction of vps4b results in altered endosomal trafficking of membrane receptors , such as egfr , as well as egfr - associated signaling molecules [ 38 , 12 ] . toward gaining a more thorough understanding of the consequences of altered egfr signaling caused by vps4b dysfunction , we developed an isdms method to identify changes in the protein synthesis , degradation rates , and dynamic protein expression , in breast cancer skbr3_shvps4b cells . in this study , we obtained dynamic profiles of more than 700 proteins in both skbr3_shvps4b and skbr3 cells in response to egf . most importantly , we have also identified many previously unidentified energy metabolism pathways that were altered as a result of vps4b downregulation in breast cancer . in particular , we found that downregulation of vps4b expression has a profoundly negative effect on the expression of several key proteins involved in glycolysis and fatty acid synthesis , suggesting that tca cycle and atp energy metabolism could be compromised . in order to overcome this defect in atp generation , we found that the expression of many proteins involved in fatty acid -oxidation is also elevated in the vps4b - depleted cells , indicating that the activation of fatty acid -oxidation could serve as a potential survival mechanism and ultimately lead to its resistance to chemotherapy and hypoxia . protein degradation rate constants vary among different cell types and tissues . in the same biological system , the degradation rate constants also vary widely among different proteins . in e. coli , protein degradation rate constants range from 0.017 h ( t1/2 = 40 hr ) to 0.058 h ( t1/2 = 11.9 hr ) , with a mean value of 0.03 h ( t1/2 = 23 hr ) . in human a549 adenocarcinoma cells , the degradation rate constants range from 2 10 ( t1/2 = 69,000 hr ) to 5.4 h ( t1/2 = 0.13 hr ) , with a mean of 0.081 h ( t1/2 = 8.5 hr ) . in our study , we found that among more than 700 proteins , protein degradation rate constants ranged from 0.012 ( t1/2 = 57 hr ) to 0.116 h ( t1/2 = 5.9 hr ) with a mean of 0.033 h ( t1/2 = 20 hr ) in skbr3_shvps4b cells and 0.01 to 0.087 h with a mean of 0.028 h ( t1/2 = 24 hr ) in skbr3 cells . due to the sensitive of our current isdms analysis , we were only able to examine the dynamic profiles of those proteins with t1/2 > 2 hr . generally , higher protein synthesis rate is related to higher protein expression , and higher protein degradation rate often leads to decreased protein expression . these trends are consistent with our results ( figure 3 and supplemental figure 2 ) . traditionally , increased protein expression has been considered as being mostly attributable to increased gene transcription and translation . however , several recent global dynamic protein profiling studies have indicated that increased protein expression can also be regulated by posttranscriptional and posttranslational control [ 2531 ] . oksvold et al . have suggested that posttranscriptional control and increased protein half life are two of the main mechanisms to maintain protein homeostasis and buffer cells from various transcriptional and gene expression noise in response to various environmental stimulations . in addition , several lines of evidence have also indicated that the abnormally elevated expression of many important receptors in cancer or drug resistant cancer cells is caused by decreased or delayed receptor degradation rather than being caused by altered gene expression [ 1318 ] . our results show that only ~10% of proteins with increased expression in vps4b have decreased degradation , while the increased expression of the majority of these proteins is attributable to changes in protein synthesis only . these results strongly suggest that increasing protein synthesis or increasing translational efficiency is the main method of increasing protein expression , whereas altered protein degradation is a more protein - specific approach to increasing protein expression in skbr3 cells . as indicated in figure 3 , we found that vps4b - dependent protein downregulation ( labeled in green diamonds ) is largely caused by the combination of decreased protein synthesis and increased protein degradation , suggesting that vps4b - mediated mvb dysfunction is playing a pivotal role in modulating protein homeostasis in breast cancer . the altered function of mvb - lysosomal degradation caused by vps4b depletion likely stimulates other cellular degradation pathways . vps4b plays an important role in protein degradation , especially in the lysosomal degradation of membrane receptors . it has been found that loss of vps4b function results in delayed egfr degradation and prolonged egfr retention on the limiting membrane of mvbs [ 46 ] . however , we were not able to obtain the dynamic expression profile for egfr and its related signaling molecules in this study due to the low abundance of endogenous egfr and the limited dynamic proteome profile coverage in skbr3 cells . surprisingly , we found that overall protein degradation rates at the proteomic level were similar in skbr3_shvps4b and skbr3 cells . this observation suggests that the vps4b downregulation - induced delayed lysosomal degradation of endocytosed cargo is likely to be cargo specific and explains why overall protein degradation is not affected . currently , we are in the process of identifying what protein cargos are specifically targeted and degraded by the vps4b - dependent mvb - lysosomal degradation system in breast cancer . identification of specific cargos that are delayed by vps4b - dependent target degradation is likely going to provide critical information on whether these proteins can be used as potential biomarkers for both the classification and progression of breast cancer . glycolysis and fatty acid -oxidation are two major metabolic pathways that cells utilize to generate energy . adaptation to different carbon and energy sources is an important cellular response to environmental or intracellular changes . our results indicate that dynamic protein expression of many enzymes involved in glycolysis and the tca cycle are coordinately regulated in response to vps4b depletion . recently , lin et al . have reported that there is a direct correlation between the downregulation of vps4b and the progression of breast cancer , and that decreased vps4b expression can be induced by hypoxia . although the molecular mechanisms underlying the hypoxia - induced vps4b - associated tumor angiogenesis and metastasis remain unknown , our results clearly indicate that there is a direct link between altered lipid metabolism and vps4b - mediated mvb dysfunction . it has been hypothesized that most cancer cells , including breast cancer cells , exhibit increased aerobic glycolysis and lead to the conversion of glucose to lactic acid , also known as the warburg effect , in part as a result of mitochondrial respiration injury and hypoxia . however , since the initial report of the warburg hypothesis , numerous reports also indicate that many cancer cells have higher glycolytic activity even under aerobic conditions . in addition , it has been suggested by gillies and colleagues that up - regulation of glycolysis can significantly provide many growth advantages for various cancers and the proposed conversion of glucose to lactate under aerobic conditions could be an important adaptation step for the cancer cells to survive under the intermittent hypoxic conditions during the early development of cancer ( see review by gatenby and gillies ) . our current study indicated that the glycolytic pathway is downregulated in vps4b - depleted skbr3 cells , suggesting a potential cross - talk between the abnormal glycolysis in cancer and mvb dysfunction . currently , we are exploring whether this attenuation of the glycolytic pathway by vps4b could also lead to the reduction of lactate - induced acidosis that is commonly associated with many cancers . interestingly , it has also been recently demonstrated that chronic acid - adapted mda - mb-231 cells are able to induce the accumulation of lc3 and the possible formation of autophagosomes . however , it is not currently clear whether the accumulation of lc3-positive autophagic vacuoles is due to the dysfunction of vps4b - mvb - mediated autolysosome formation or enhanced autophagic flux . although the main molecular and cellular events modulating this acid or environmental - induced autophagy formation in cancers are largely unknown , our systems approach described here provides a potential explanation of the role of vps4b in regulating the glycolytic pathway and the formation of autophagy under either hypoxia or acidosis . in addition to the downregulation of the glycolytic pathway and modulating the formation of autophagic vacuole formation , our findings also reveal that fatty acid -oxidation is significantly upregulated in vps4b - depleted cells . specifically , we found that the expression of mitochondrial trifunctional protein alpha subunit ( hadha / echa ) , mitochondrial trifunctional protein beta subunit ( hadhb / echb ) , and mitochondrial hydroxyacyl - coenzyme a dehydrogenase ( hadh ) is significantly elevated . the pathological consequences of abnormal fatty acid -oxidation activation are unclear . however , it is reasonable to postulate that the activation of fatty acid oxidation is part of the cellular compensatory response to vps4b - mediated downregulation of the glycolytic pathway . fatty acid -oxidation is known to be upregulated in many different cancers and other diseases [ 4449 ] . since mitochondria are known to be degraded by autophagy , we postulate that the vps4b - autophagy - mediated mitochondrial degradation could be impaired during the premetastatic stage of cancer or in vps4b - depleted cells and ultimately causes the activation of mitochondrial fatty acid -oxidation . finally , another important finding of our study is the decreased expression of fatty acid synthase ( fasn ) in vps4b - depleted cells . high levels of fasn are reported in many epithelial cancers , such as breast , colorectal , and prostate , and fasn overexpression is highly associated with a higher risk of both disease recurrence and death . most significantly , our results show that vps4b downregulation decreased the expression of fasn by simultaneously decreasing its rates of synthesis and increasing the rate of its degradation , suggesting that both de novo fatty acid synthesis and fatty acid -oxidation are tightly coupled in cancer . altogether , our study suggests the downregulation of vps4b causes the alteration of glucose metabolism and the glycolytic pathway , which ultimately leads to the activation of mitochondrial fatty acid -oxidation . here we have presented an approach , isdms , for the direct measurement of protein degradation and synthesis , as well as relative protein expression levels in skbr3 cells with downregulated vps4b expression . this approach can be feasibly applied to other cell culture systems to determine global protein dynamics under different genetic manipulations or environmental stimulations . as with other mass spectrometry - based approaches , isdms is limited by the depth and reproducibility of protein identification , especially when applied to multiple time points and multiple conditions . however , we are able to establish the dynamic protein profiling of many proteins involved in the central glucose and lipid metabolism that are preferentially regulated by the downregulation of vps4b expression . as downregulation of vps4b has been reported to be associated with certain high grade and recurring tumors , the adoption of fatty acid -oxidation as an alternative energy source could be a distinct feature of breast cancer cells with vps4b dysfunction . therapeutic interventions targeting fatty acid - oxidation energy metabolism could serve as alternative and complementary strategies to treat breast cancer .
the endosomal / lysosomal system , in particular the endosomal sorting complexes required for transport ( escrts ) , plays an essential role in regulating the trafficking and destination of endocytosed receptors and their associated signaling molecules . recently , we have shown that dysfunction and down - regulation of vacuolar protein sorting 4b ( vps4b ) , an escrt - iii associated protein , under hypoxic conditions can lead to the abnormal accumulation of epidermal growth factor receptor ( egfr ) and aberrant egfr signaling in breast cancer . however , the pathophysiological consequences of vps4b dysfunction remain largely elusive . in this study , we used an internal standard - assisted synthesis and degradation mass spectrometry ( isdms ) method , which permits the direct measurement of protein synthesis , degradation and protein dynamic expression , to address the effects of vps4b dysfunction in altering egf - mediated protein expression . our initial results indicate that vps4b down - regulation decreases the expression of many proteins involved in glycolytic pathways , while increased the expression of proteins with roles in mitochondrial fatty acid -oxidation were up - regulated in vps4b - depleted cells . this observation is also consistent with our previous finding that hypoxia can induce vps4b down - regulated , suggesting that the adoption of fatty acid -oxidation could potentially serve as an alternative energy source and survival mechanism for breast cancer cells in response to hypoxia - mediated vps4b dysfunction .
1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion
in lysosomal degradation , endocytosed receptors are sorted into multivesicular bodies ( mvbs ) , which requires the sequential assembly of endosomal sorting complex required for transport i , ii , and iii ( escrt - i , -ii , and -iii ) on the endosomal membrane . to further understand the role of vps4b dysfunction in breast cancer , we set out to determine the consequences of altered egfr signaling : changes in protein synthesis and degradation , as well as protein dynamics in vps4b downregulated breast cancer cells . recently , high throughput synthesis and degradation mass spectrometry ( sdms ) has become a novel approach to study global protein turnover , in which protein degradation and synthesis can be measured simultaneously and unambiguously with minimal cell perturbation . to estimate protein synthesis and degradation rates in the context of altered egfr signaling caused by downregulation of vps4b , we independently developed an approach called internal standard - assisted synthesis and degradation mass spectrometry ( isdms ) that normalizes protein abundance across different samples and time points and permits the comparison of protein synthesis , degradation , and expression measurements . we found that vps4b downregulation resulted in differential protein expression in energy metabolism pathways by altering the synthesis and degradation of related proteins , in which glycolysis proteins were downregulated , while mitochondrial fatty acid -oxidation proteins were upregulated . the adoption of fatty acid -oxidation as an alternative energy source could be an unrevealed survival mechanism for breast cancer cells with vps4b dysfunction . our earlier report has shown that hypoxia leads to the abnormal accumulation of egfr and subsequent alteration of cell signaling in breast cancer . to further understand the role of vps4b dysfunction in global protein dynamics upon egf treatment , we decided to measure the rates of protein synthesis and degradation in vps4b downregulated skbr3 ( skbr3_shvps4b ) and the parental skbr3 cells using an internal standard assisted synthesis and degradation mass spectrometry ( isdms ) approach . because isdms allows one to measure the rates of protein synthesis and degradation simultaneously , it is therefore possible to determine the dynamics of protein expression in vps4b - depleted cells in response to egf . because the steady state accumulation of proteins is determined by their rates of synthesis and degradation , we therefore decided to further examine the relationship between the steady state protein expression and protein synthesis and degradation in response to the downregulation of vps4b expression . as indicated in figure 3 , it is clear that the vps4b - mediated decrease of protein expression is largely due to increased protein degradation , while the increase of vps4b - mediated protein expression is directly related with increased protein synthesis . toward gaining a more thorough understanding of the consequences of altered egfr signaling caused by vps4b dysfunction , we developed an isdms method to identify changes in the protein synthesis , degradation rates , and dynamic protein expression , in breast cancer skbr3_shvps4b cells . in particular , we found that downregulation of vps4b expression has a profoundly negative effect on the expression of several key proteins involved in glycolysis and fatty acid synthesis , suggesting that tca cycle and atp energy metabolism could be compromised . in order to overcome this defect in atp generation , we found that the expression of many proteins involved in fatty acid -oxidation is also elevated in the vps4b - depleted cells , indicating that the activation of fatty acid -oxidation could serve as a potential survival mechanism and ultimately lead to its resistance to chemotherapy and hypoxia . as indicated in figure 3 , we found that vps4b - dependent protein downregulation ( labeled in green diamonds ) is largely caused by the combination of decreased protein synthesis and increased protein degradation , suggesting that vps4b - mediated mvb dysfunction is playing a pivotal role in modulating protein homeostasis in breast cancer . our results indicate that dynamic protein expression of many enzymes involved in glycolysis and the tca cycle are coordinately regulated in response to vps4b depletion . since mitochondria are known to be degraded by autophagy , we postulate that the vps4b - autophagy - mediated mitochondrial degradation could be impaired during the premetastatic stage of cancer or in vps4b - depleted cells and ultimately causes the activation of mitochondrial fatty acid -oxidation . most significantly , our results show that vps4b downregulation decreased the expression of fasn by simultaneously decreasing its rates of synthesis and increasing the rate of its degradation , suggesting that both de novo fatty acid synthesis and fatty acid -oxidation are tightly coupled in cancer . here we have presented an approach , isdms , for the direct measurement of protein degradation and synthesis , as well as relative protein expression levels in skbr3 cells with downregulated vps4b expression . as downregulation of vps4b has been reported to be associated with certain high grade and recurring tumors , the adoption of fatty acid -oxidation as an alternative energy source could be a distinct feature of breast cancer cells with vps4b dysfunction .
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the explicit encoding of the duration of events within the sub - second range is crucial for a number of everyday tasks from timing action such as deciding when to step onto an escalator or when to move off at traffic lights to picking up social signals encoded in the duration of mutual gaze . however , the mechanisms underlying the timing of events under 1 or 2 s are still obscure . a substantial number of studies have shown that the apparent duration of a visual stimulus in the order of milliseconds can be distorted not only by generic factors , such as stimulus novelty ( pariyadath and eagleman , 2007 , 2008 ) or attention ( tse et al . , 2004 ; cicchini and morrone , 2009 ) , but also by visually specific manipulations , such as adaptation to visual motion ( johnston et al . , 2006 , 2008 ; 2010 ) , contrast ( bruno and johnston , 2010 ) , and reduced illumination ( bruno et al . , 2011 ) . these observations have led some authors to question the ability of the classic pacemaker accumulator model ( creelman , 1962 ; treisman , 1963 ; treisman et al . , therefore , alternative models based on coding efficiency ( eagleman and pariyadath , 2009 ) or on a predict - and - compare strategy ( johnston , 2010 ) have recently been proposed . early studies revealed that moving stimuli tend to be seen as having a longer duration than stationary ones ( brown , 1931 ; roelofs and zeeman , 1951 ; goldstone and lhamon , 1974 ; lhamon and goldstone , 1974 ) . this observation , obtained with stimuli that were at most a few seconds long , was replicated for stimuli lasting several seconds ( brown , 1995 ) or even minutes ( leiser et al . , 1991 ) . some of these studies also showed a certain degree of dependency of duration estimation on stimulus speed : the faster the stimulus , the bigger the duration overestimation . in the sub - second range ( 2006 ) , using a variety of visual stimuli including squares , random dot patterns , expanding gratings , and flickering gaussian blobs , reported that apparent duration increased with stimulus temporal frequency , rather than speed , but only up to 48 hz . kaneko and murakami ( 2009 ) , using drifting gabors , observed that perceived duration is positively related to speed , rather than temporal frequency , and the effect does not seem to saturate at high speed levels . most of the above - mentioned studies tended to explain the effect of speed ( or temporal frequency ) on apparent duration by referring to a change model ( fraisse , 1963 ) . according to this view , the number of changes that occur within an interval provides a cue to the passage of time ( block and reed , 1978 ; poynter , 1983 , 1989 ; poynter and homa , 1983 ) . an interval containing fast motion or fast temporal change would be represented as having more temporal features than a slower stimulus and therefore would be perceived as having a longer duration . in contrast to this passive view of the effect of content on perceived duration , we can consider an active predict - and - compare content - dependant clock in which the content of the interval is intrinsic to the measurement of its duration ( johnston , 2010 ) . the aim of the current paper is to test the predictions of the change model , we measured perceived duration for static , drifting , or mixed ( where static and drifting intervals were interleaved ) stimuli that , according to the change model , should contain a different number of changes and therefore be judged as having different durations . we found that the mixed stimulus was consistently perceived as compressed relative to the drifting one but no different in duration to the static stimulus ( no change ) across duration ranges ( 600 , 1200 , and 2400 ms ) , without any substantial changes in the accuracy of the duration judgments . presenting a pair of stimuli sequentially can induce perceptual biases that depend on their position in the sequence . the observation that the magnitude of the first test is systematically underestimated dates back to fechner ( fechner , 1860 ; woodrow , 1951 ; woodworth and schlosberg , 1954 ) . consistent with these early results , compression has been reported in the duration domain for the first relative to the second of a pair of brief intervals ( jamieson and petrusic , 1975 ) . in the present study , we compared apparent duration and accuracy dependence on presentation order across a more extended range of durations . we observed that the difference between first and second tests increased with duration , while the difference in accuracy remained constant . stimuli were displayed , in a darkened room , on a 19 sony trinitron multiscan 500ps , with a refresh rate of 100 hz , driven by a vsg2/5 visual stimulus generator ( cambridge research systems ) . subjects fixated a spot in the center of the screen and judged the relative duration of two stimuli that were displayed sequentially in the near periphery ( see figure 1b ) . stimuli were vertically oriented sinusoidal gratings modulated in luminance ( spatial frequency = 1 cycle/ , diameter : 5 of visual angle , michelson contrast : 100% , centered 5 to the right and to the left of the center of the monitor ) . the duration of one of the stimuli ( the standard ) was fixed across trials , while the duration of the other stimulus ( the comparison ) varied across trials in order to generate a psychometric function . we used three standard durations in different sessions : 600 , 1200 , and 2400 ms . the duration of the comparison varied in seven steps , ranging from 0.2 standard duration to 2.67 standard duration . the comparison stimulus always drifted at 10/s across conditions ( its phase was randomized on a trial - by - trial basis ) . it was essential that the comparison stimulus was always the same across stimulus types ( static , drifting , mixed ) in order to have at least two direct comparisons ( static vs. drifting and mixed vs. drifting , in this case ) between stimuli with different numbers of temporal changes . we chose to use the drifting grating as comparison , however one of the other stimulus types would have served as well , since our analysis was focused on relative duration across the three conditions . the drifting vs. drifting condition provided us with a baseline condition , in which we compared stimuli with the same number of temporal changes . in different sessions the standard could be static , drifting at 10/s , or mixed ( see figure 1a ) . a static interval always followed a drifting one ( and vice versa ) and the total duration of static and drifting subintervals was always the same across trials ( 50% static , 50% drifting over the whole standard duration ) , but we assigned a duration between 100 and 200 ms to each subinterval , randomly , within , and across trials . a 500-ms blank interval separated the two tests and the relative spatial position of the stimuli was randomized . the presentation order of the two stimuli was also randomized and we saved the trials in which the standard was displayed first separately from those in which it was displayed second for later analysis . subjects had to report which of the two tests seemed to have the longer duration . the data were fitted with cumulative gaussian functions that were free to vary in position and width ( see figure 2c for example psychometric functions ) . the 50% point on the psychometric function ( point of subjective equality , pse ) provided an estimate of the perceived duration of the standard stimulus . the discrimination threshold was defined as the width of the error distribution . the value of is equivalent to the difference between the 50 and 84% point on the psychometric function . ( a ) space - time representation of the three standard stimulus types used in the experiment . the standard intervals contained luminance - modulated sinusoidal gratings that could be , in different sessions , static , drifting at 10 hz , or composed by an alternation of static and drifting subintervals whose duration varied from interval to interval ( see materials and methods for a more detailed description ) . ( b ) subjects had to keep fixation on the middle of the screen while two stimuli ( the standard , fixed duration , and the comparison , with variable duration across trials ) were sequentially displayed on either side of the fixation spot . presentation order and spatial positions of standard and comparison were randomized on a trial - by - trial basis . at the end of each trial , subjects had to report which stimulus appeared to stay on for the longer duration by pressing a button . ( a ) mean perceived duration ( calculated as the 50% point on the psychometric function , pse ) across 15 subjects for three standard durations and three stimulus configurations . ( b ) proportion change in perceived duration relative to the true standard duration [ calculated from the data plotted in ( a ) ] for the same conditions as in ( a ) . ( c ) example psychometric curves for a nave observer for the three stimulus configurations and for 600 ms standard duration . ( d ) average discrimination thresholds ( defined as the difference between the 50 and 84% point on the psychometric function / the standard duration ) for the same subjects and conditions as in ( a ) . stimuli were displayed , in a darkened room , on a 19 sony trinitron multiscan 500ps , with a refresh rate of 100 hz , driven by a vsg2/5 visual stimulus generator ( cambridge research systems ) . subjects fixated a spot in the center of the screen and judged the relative duration of two stimuli that were displayed sequentially in the near periphery ( see figure 1b ) . stimuli were vertically oriented sinusoidal gratings modulated in luminance ( spatial frequency = 1 cycle/ , diameter : 5 of visual angle , michelson contrast : 100% , centered 5 to the right and to the left of the center of the monitor ) . the duration of one of the stimuli ( the standard ) was fixed across trials , while the duration of the other stimulus ( the comparison ) varied across trials in order to generate a psychometric function . we used three standard durations in different sessions : 600 , 1200 , and 2400 ms . the duration of the comparison varied in seven steps , ranging from 0.2 standard duration to 2.67 standard duration . the comparison stimulus always drifted at 10/s across conditions ( its phase was randomized on a trial - by - trial basis ) . it was essential that the comparison stimulus was always the same across stimulus types ( static , drifting , mixed ) in order to have at least two direct comparisons ( static vs. drifting and mixed vs. drifting , in this case ) between stimuli with different numbers of temporal changes . we chose to use the drifting grating as comparison , however one of the other stimulus types would have served as well , since our analysis was focused on relative duration across the three conditions . the drifting vs. drifting condition provided us with a baseline condition , in which we compared stimuli with the same number of temporal changes . in different sessions the standard could be static , drifting at 10/s , or mixed ( see figure 1a ) . a static interval always followed a drifting one ( and vice versa ) and the total duration of static and drifting subintervals was always the same across trials ( 50% static , 50% drifting over the whole standard duration ) , but we assigned a duration between 100 and 200 ms to each subinterval , randomly , within , and across trials . a 500-ms blank interval separated the two tests and the relative spatial position of the stimuli was randomized . the presentation order of the two stimuli was also randomized and we saved the trials in which the standard was displayed first separately from those in which it was displayed second for later analysis . subjects had to report which of the two tests seemed to have the longer duration . the data were fitted with cumulative gaussian functions that were free to vary in position and width ( see figure 2c for example psychometric functions ) . the 50% point on the psychometric function ( point of subjective equality , pse ) provided an estimate of the perceived duration of the standard stimulus . the discrimination threshold was defined as the width of the error distribution . the value of is equivalent to the difference between the 50 and 84% point on the psychometric function . ( a ) space - time representation of the three standard stimulus types used in the experiment . the standard intervals contained luminance - modulated sinusoidal gratings that could be , in different sessions , static , drifting at 10 hz , or composed by an alternation of static and drifting subintervals whose duration varied from interval to interval ( see materials and methods for a more detailed description ) . ( b ) subjects had to keep fixation on the middle of the screen while two stimuli ( the standard , fixed duration , and the comparison , with variable duration across trials ) were sequentially displayed on either side of the fixation spot . presentation order and spatial positions of standard and comparison were randomized on a trial - by - trial basis . at the end of each trial , subjects had to report which stimulus appeared to stay on for the longer duration by pressing a button . ( a ) mean perceived duration ( calculated as the 50% point on the psychometric function , pse ) across 15 subjects for three standard durations and three stimulus configurations . ( b ) proportion change in perceived duration relative to the true standard duration [ calculated from the data plotted in ( a ) ] for the same conditions as in ( a ) . ( c ) example psychometric curves for a nave observer for the three stimulus configurations and for 600 ms standard duration . ( d ) average discrimination thresholds ( defined as the difference between the 50 and 84% point on the psychometric function / the standard duration ) for the same subjects and conditions as in ( a ) . we asked our subjects to compare the relative duration of two sequentially displayed temporal intervals ( containing sinusoidal gratings modulated in luminance , figure 1b ) . in different conditions ( figure 1a ) , we modulated the number of temporal changes contained in the standard test ( fixed duration across trials ) , which could be static , drifting , or mixed ( composed of an alternation of static and drifting intervals of randomly varied duration , thus containing an intermediate number of changes between the static and the drifting stimulus ) . the other test ( the comparison , variable duration across trials ) was always drifting . since we used three standard durations ( 600 , 1200 , and 2400 ms ) , we needed to transform the data in order to explore the effects of standard type and duration on apparent duration and discriminability . the values we obtained described the proportion of duration change relative to the standard duration : negative values indicated compression , positive values expansion , zero corresponds to the actual duration ( figure 2b ) . the discrimination thresholds were divided by the correspondent standard durations to give weber fractions . in figure 2a we plotted the actual pse and discrimination threshold values to facilitate an intuitive reading of the main results . statistical analyses conducted on the ratios revealed significant main effects of both standard stimulus type [ general linear model repeated measures , f(2 ) = 21.317 , p < 0.001 ] and standard duration [ f(2 ) = 4.726 , p = 0.017 ] on perceived duration ( pses , figure 1a ) . in general , we observed time compression for the static ( 10% ) and mixed conditions ( 15% ) relative to the actual duration , while the drifting standard appeared expanded ( +10% ) . when the different stimulus types were pooled together , we found the estimates for the 2400-ms condition to be veridical , but there was a slight compression for both the 600- ( 4% ) and 1200-ms data ( 6.5% ) . moreover , the effect of standard duration depends on stimulus type ( significant interaction , p = 0.039 ) . more specifically , we found that , for all the standard durations , the mixed stimulus appeared significantly compressed relative to the drifting stimulus ( paired t - tests , all p < 0.001 ) . no significant difference emerged from the comparison between the static and mixed conditions ( paired t - tests , all p > 0.1 , not significant ) . confirming previous reports ( brown , 1995 ; kanai et al . , 2006 ; kaneko and murakami , 2009 ) , the static standard looked shorter - lived than the drifting comparison for all standard durations ( all p < 0.001 ) apart from 2400 ms ( p = 0.102 , not significant ) . one might argue that the duration estimates for the mixed stimulus ( which contains static and drifting intervals in equal proportions ) could result from a linear combination of the estimates of its static and drifting components . however , the apparent duration of the mixed stimulus was found to be significantly less than the average of the static and drifting judgments for all standard durations ( paired t - tests , all p < 0.01 ) . a mild , but significant expansion was observed for the drifting conditions relative to the veridical standard duration ( one - sample t - tests conducted on the ratios against 0 , all p < 0.01 ) . this may be explained by the fact that we had to increase the range for long comparison durations , which , in an initial pilot experiment , were not reliably estimated for most of the subjects , especially in the 2400-ms standard duration . consequently , the mean of the comparison durations , which has been shown to influence subjects judgments ( nachmias , 2006 ; lapid et al . , 2008 ) , corresponded to a higher value than the true standard duration . analyses conducted on the weber thresholds ( discrimination threshold / standard duration ) , revealed no significant effect of the stimulus type on duration judgment accuracy . however , the average discrimination threshold for the 2400-ms standard duration ( 0.56 ) was higher than those for 600 and 1200 ms ( both 0.5 ) resulting in a significant main effect for standard duration [ general linear model repeated measures , f(2 ) = 4.278 , p = 0.024 ] and a significant interaction with stimulus type [ f(4 ) = 2.704 , p = 0.039 ] . no significant difference emerged from paired comparisons of static , drifting , and mixed conditions for the three standard durations separately . for all the conditions , we interleaved trials in which the standard was displayed first with trials in which it was displayed second . since it has been shown ( jamieson and petrusic , 1975 ; nachmias , 2006 ; lapid et al . , 2008 ) that the stimulus order has an influence on both apparent duration ( pse ) and discrimination threshold , we reanalyzed the data with presentation order as a variable with two levels ( standard first and standard second ) . figure 3a contains a graphic representation of this separation for the transformed pses [ pse/(standard duration ) 1 ] . consistent with previous reports ( jamieson and petrusic , 1975 ; nachmias , 2006 ; lapid et al . , 2008 ) , it is evident that duration estimates were significantly lower when the standard was displayed first relative to when it was displayed second [ general linear model , main effect for standard order , f(1 ) = 13.433 , p < 0.01 ] . the novel finding is that the difference between the two - stimulus orders significantly increases with standard duration [ interaction standard order standard duration , f(2 ) = 23.058 , p < 0.001 ] . also , the standard order has a different influence on apparent duration depending on stimulus type [ interaction standard order standard type , f(2 ) = 7.157 , p < 0.01 ] . in particular the difference between standard first and standard second seems less pronounced for the mixed condition relative to static or drifting . ( a ) proportion changes in perceived duration [ ( pse / standard duration ) 1 ] averaged across 15 subjects for three standard durations ( different panels ) and three stimulus types ( different symbols and colors ) . ( b ) mean discrimination threshold ( divided by standard duration ) for the same subjects and conditions as in ( a ) . in figure 3b the difference between presentation orders is clear : accuracy is statistically higher ( lower discrimination threshold ) for standard first condition [ general linear model , f(1 ) = 26.849 , p < 0.001 ] . however , in this case , this difference does not depend on standard type or duration ( all interactions not significant ) . we investigated the effect of the number of temporal changes and order on the apparent duration of visual intervals that were at most a few seconds long . we found that : the duration of a stimulus composed of a random alternation of static and drifting intervals ( mixed standard ) appeared compressed either relative to a drifting comparison stimulus or to the average of the static and drifting estimates . a static stimulus appeared shorter in duration than a drifting stimulus , confirming previous observations ( brown , 1995 ; kanai et al . however , lower discriminability was associated with 2400 ms relative to 600 and 1200 ms standard durations . the duration of the first of a pair of sequentially displayed stimuli appears compressed and the magnitude of the effect increases with standard stimulus duration . duration discrimination was significantly better for the first presented stimulus relative to the second regardless of standard duration . the dilation effect induced by speed or temporal frequency on the perceived duration of a visual stimulus has been traditionally seen as a supporting evidence for a change - based theory of time perception ( fraisse , 1963 ; poynter , 1989 ) . this model posits that our experience of time strongly relies on the amount of temporal information contained in the interval we want to estimate . more specifically , a prominent role is assigned to the extent to which an interval can be segmented into a sequence of temporal changes or features ( poynter , 1983 ; poynter and homa , 1983 ) . the changes in spatial position that occur within the trajectory of a visual stimulus in motion have been considered to provide a good basis on which to test of the predictions of this model . for instance , brown ( 1995 ) investigated duration estimates for stimuli that translated across a monitor along a non - linear trajectory at different speeds . he observed an apparent duration expansion for moving relative to stationary stimuli that increased with stimulus speed . according to the author , this result supports the change model , as faster stimuli change spatial location more frequently than slower ones within the same time interval . in our experiment , the number of changes associated with the mixed stimulus should arguably be intermediate between the stationary stimulus and the continuously drifting one . therefore , according to the change model , we should expect the duration estimates for the mixed stimulus to be somewhere halfway between those for the stationary and the drifting stimuli . however , this is not the case , as the mixed stimulus appears to be more short - lived than the mean of the other two conditions and is not perceived as significantly longer than the stationary stimulus . one might argue that our stimuli ( drifting gratings ) do not change in spatial location and therefore they might not be readily segmented into subunits . however , the findings that a similar increase in perceived duration to that observed for translating stimuli also occurs for flickering stimuli ( goldstone and lhamon , 1974 ; lhamon and goldstone , 1974 ; kanai et al . , 2006 ) and for drifting gabors ( kaneko and murakami , 2009 ) , which do not contain changes in spatial location , argue against this view . kaneko and murakami ( 2009 ) proposed a slightly different account for the speed - induced time expansion . according to their view , the rate at which our internal clock ( creelman , 1962 ; treisman , 1963 ; treisman et al . , 1990 ) produces ticks is increased with stimulus speed in order to increase the temporal resolution of the system . as far as our stimuli are concerned , the predictions of this model do not substantially differ from those of the classic change model , as the increase in the tick rate associated with the intervals containing drifting motion should be halved for the mixed stimulus ( which contains 50% drifting and 50% static intervals ) . interestingly , stimuli moving at a constant speed are not perceived as having the same duration as accelerating or decelerating stimuli with the same average speed ( matthews , 2011 ) , again arguing against the idea of duration as a reflection of an accumulation of temporal information . ( 2006 ) reported that the dependency of perceived duration of expanding gratings on temporal frequency tends to saturate quite quickly , in a range between 4 and 8 hz . the temporal frequency of our drifting stimuli ( 10 hz ) falls slightly outside this range , therefore it is arguable that different mechanisms might be at work for low relative to high temporal frequencies and we are maybe tapping into a range of frequencies that is not substantially influenced by the change rate within an interval . however , kaneko and murakami ( 2009 ) , using stimuli that were more similar to ours ( drifting gabors ) , reported that duration overestimation increased linearly with log speed even for values higher than 10/s . since the spatial frequency of our stimuli was always 1 cycle/ we can not discriminate between the effect of temporal frequency and speed on apparent duration . the inter - stimulus interval ( isi ) between standard and comparison stimuli was kept constant at 500 ms across trials and we did not use a mask to prevent visual aftereffects . however , the spatial position and the presentation order of the stimuli were fully randomized across trials . the standard and the comparison could be either displayed to the right or to the left of the fixation dot . therefore , if there is an influence of visual aftereffects on perceived duration in our setup , it can be argued that it should equally affect both the standard and the comparison stimuli . we decided to use only two ( right and left ) rather than many possible spatial positions to keep the task as simple as possible and avoid attention - related effects . judging duration is an attentionally demanding task that nave subjects in particular find quite challenging . first , observers have to allocate their attention to the considered stimulus interval in a given spatial position and then they have to sustain their attention until the very end of the interval , unlike in speed , or orientation judgments tasks , for example , in which attention is only required until a decision is made . therefore , the use of only two predictable spatial positions , allowed us to minimize the attentional resources required to relocate our spatial attention to unpredictable spatial positions . we instructed subjects to fixate the dot located at the center of screen for the whole duration of the experiment . however , one might argue that some of our conditions could have induced reflexive saccades , although reflexive saccades were not spontaneously reported by observers . a visual interval displayed at the time of a saccadic eye - movement appears compressed relative to an interval displayed well before the saccadic onset ( morrone et al . , 2005 ) . in our experiment , the condition which might attract most saccades is the continuously drifting one , which , for the aforementioned reasons , should therefore be perceived as compressed relative to , for example , the stationary condition . however , the opposite was true , as the drifting stimulus was actually perceived as longer than the static ( or the mixed ) one . also , the mixed stimulus does not appear more compressed than the static one , even though it might attract more reflexive saccades . therefore , the presence of reflexive saccades is likely to work against the pattern of results we observed . the stimuli we used in this study do not only differ in terms of the number of changes they contained , but also in terms of the predictability of subsequent frames of a motion sequence . a recent model proposed by johnston ( 2010 ) posits that the system might use a predict - and - compare strategy to determine the duration of a brief interval . in a predict - and - compare clock the clock produces a prediction of the visual world s appearance after a given time ( for example , 100 ms ) and then continuously compares it with the current appearance of the world that comes from the sensory input . once the current appearance of the world matches the prediction , the system knows that 100 ms have passed , it sends a tick to the accumulator and then it resets the prediction . clearly , the internal predictability of an interval in time plays an important role in the ability of the model to determine its duration . in the present study , we used some stimuli that could not benefit from a predict - and - compare strategy ( static ) , stimuli with a high degree of predictability throughout the interval ( drifting at 10/s ) or a low degree of predictability at transitions ( mixed ) . the poorly predictable stimulus was a balanced mixture of static and drifting subintervals ; each of them had a randomly chosen duration and a static interval always followed a drifting one . in other words , the model could not predict the appearance of the stimulus at the end of a segment solely on the basis of its temporal frequency or speed , therefore we expected it might provide an inaccurate estimate of duration . we observed that the poorly predictable stimulus ( mixed condition , figure 2a ) was perceived to be compressed relative to the highly predictable drifting stimulus . the difference in apparent duration between static and drifting conditions suggest the use of different mechanism in the two cases . critically , the duration of the mixed stimulus appeared reduced relative to the average of the drifting and static components , implying that the system does not simply integrate these intervals linearly to determine duration . the unpredictable change at the stimulus transition appears to introduce a small reduction in perceived duration that can not simply be attributed to combining the static and moving intervals . another recent model by ahrens and sahani ( 2011 ) uses the content of an interval to estimate time . this model assumes that the duration of an interval is derived from a bayesian combination of a sensory - based estimate of dynamic stimulus statistics with an internal estimate that is independent of stimulus properties . more specifically , changes in the stimulus detected by different stochastic sensory streams ( which correspond to retinal snapshots of the stimulus taken at different points in time ) are integrated to provide an estimate of duration that is based on the similarity between successive snapshots ( the bigger the change between them , the longer the time period that is estimated to separate them ) . therefore , the model predicts an underestimation of the duration of a static stimulus relative to a drifting one ( as is observed in our results ) , but it is not clear how it can account for the lack of a substantial difference between the static and the mixed stimulus . also , the model predicts a lower precision for the static ( the duration of which is determined solely on the basis of the internal estimate ) relative to the drifting stimuli ( which can also take advantage of the stimulus - driven estimate ) . this prediction was confirmed experimentally , however , we did not observe a difference in the discrimination threshold between the two - stimulus types in our study ( figure 2d ) . fechner was the first to notice that when a subject was asked to lift two weights sequentially , the magnitude of the first one was underestimated ( fechner , 1860 ; woodrow , 1951 ; woodworth and schlosberg , 1954 ) . this classic order effect was also replicated in the time domain by the demonstration that the first of a pair of brief visual stimuli was perceived as shorter in duration than the second one ( jamieson and petrusic , 1975 ) . interestingly , the ability to discriminate between two stimuli is also affected by the presentation order . more specifically , discrimination thresholds are lower for the first of a pair of sequentially displayed tests , regardless of whether the presentation order is blocked or randomized ( nachmias , 2006 ) . ( 2008 ) showed that this effect also holds for duration . using both auditory and visual stimuli , they found that the just noticeable difference ( jnd ) is smaller ( i.e. , higher discriminability ) when a stimulus is displayed first . the general explanation for these order effects on pses and jnds is that subjects tend to refer to an internal implicit standard for their judgments rather than to the presented standard , as suggested by the similarities observed in discriminability and perceived magnitude between single - stimulus tasks ( in which no standard was presented and , therefore , subjects had to refer to an internal standard ) and two - stimulus tasks when the standard was presented first . in all the conditions of the present experiments , we randomized the presentation order and collapsed together trials in which the standard was displayed first with trials where it was displayed second to minimize these biases . however , since we used quite a wide range of durations , we were also interested to investigate whether the time order effects vary with the presentation time . jamieson and petrusic ( 1975 ) used pairs of intervals in the range 2405515 ms , but they simply determined the proportion of correct responses rather than deriving a psychometric function . our finding that the standardized difference in pse ( pse / standard duration ) between standard first and standard second conditions significantly increased with standard duration ( figure 3a ) might suggest that that the remembered value of the first duration decays over time although , the observation that the time order effect declines as the isi increases ( jamieson and petrusic , 1975 ) seems to argue against this conclusion . it may be that both retaining a duration estimate and extracting a new duration estimate are attention demanding and that competition for limited resources generates a reduction in the capacity to sustain the perceived magnitude of the stored interval without any increase in the uncertainty about its magnitude . we also observed a smaller difference in pse between standard first and standard second for the mixed stimulus , suggesting that with a less predictable stimulus , subjects seemed to be less prone to ignore the presented standard and refer to an internalized reference to make a duration judgment . in conclusion , we showed that the number of temporal features that are contained within an interval are not necessarily the main factor determining the apparent duration of dynamic stimuli . therefore , speed - related changes in perceived time can not be explained in this way and we require a different means of incorporating content - dependence into a time estimation model , such as the ability to predict the appearance of a stimulus forward in time . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
the apparent duration of a visual stimulus has been shown to be influenced by its speed . for low speeds , apparent duration increases linearly with stimulus speed . this effect has been ascribed to the number of changes that occur within a visual interval . accordingly , a higher number of changes should produce an increase in apparent duration . in order to test this prediction , we asked subjects to compare the relative duration of a 10-hz drifting comparison stimulus with a standard stimulus that contained a different number of changes in different conditions . the standard could be static , drifting at 10 hz , or mixed ( a combination of variable duration static and drifting intervals ) . in this last condition the number of changes was intermediate between the static and the continuously drifting stimulus . for all standard durations , the mixed stimulus looked significantly compressed ( 20% reduction ) relative to the drifting stimulus . however , no difference emerged between the static ( that contained no changes ) and the mixed stimuli ( which contained an intermediate number of changes ) . we also observed that when the standard was displayed first , it appeared compressed relative to when it was displayed second with a magnitude that depended on standard duration . these results are at odds with a model of time perception that simply reflects the number of temporal features within an interval in determining the perceived passing of time .
Introduction Materials and Methods Apparatus Stimuli and procedure Results Discussion Conflict of Interest Statement
according to this view , the number of changes that occur within an interval provides a cue to the passage of time ( block and reed , 1978 ; poynter , 1983 , 1989 ; poynter and homa , 1983 ) . the aim of the current paper is to test the predictions of the change model , we measured perceived duration for static , drifting , or mixed ( where static and drifting intervals were interleaved ) stimuli that , according to the change model , should contain a different number of changes and therefore be judged as having different durations . we found that the mixed stimulus was consistently perceived as compressed relative to the drifting one but no different in duration to the static stimulus ( no change ) across duration ranges ( 600 , 1200 , and 2400 ms ) , without any substantial changes in the accuracy of the duration judgments . it was essential that the comparison stimulus was always the same across stimulus types ( static , drifting , mixed ) in order to have at least two direct comparisons ( static vs. drifting and mixed vs. drifting , in this case ) between stimuli with different numbers of temporal changes . in different sessions the standard could be static , drifting at 10/s , or mixed ( see figure 1a ) . the standard intervals contained luminance - modulated sinusoidal gratings that could be , in different sessions , static , drifting at 10 hz , or composed by an alternation of static and drifting subintervals whose duration varied from interval to interval ( see materials and methods for a more detailed description ) . it was essential that the comparison stimulus was always the same across stimulus types ( static , drifting , mixed ) in order to have at least two direct comparisons ( static vs. drifting and mixed vs. drifting , in this case ) between stimuli with different numbers of temporal changes . in different sessions the standard could be static , drifting at 10/s , or mixed ( see figure 1a ) . the standard intervals contained luminance - modulated sinusoidal gratings that could be , in different sessions , static , drifting at 10 hz , or composed by an alternation of static and drifting subintervals whose duration varied from interval to interval ( see materials and methods for a more detailed description ) . in different conditions ( figure 1a ) , we modulated the number of temporal changes contained in the standard test ( fixed duration across trials ) , which could be static , drifting , or mixed ( composed of an alternation of static and drifting intervals of randomly varied duration , thus containing an intermediate number of changes between the static and the drifting stimulus ) . in general , we observed time compression for the static ( 10% ) and mixed conditions ( 15% ) relative to the actual duration , while the drifting standard appeared expanded ( +10% ) . more specifically , we found that , for all the standard durations , the mixed stimulus appeared significantly compressed relative to the drifting stimulus ( paired t - tests , all p < 0.001 ) . however , the apparent duration of the mixed stimulus was found to be significantly less than the average of the static and drifting judgments for all standard durations ( paired t - tests , all p < 0.01 ) . for all the conditions , we interleaved trials in which the standard was displayed first with trials in which it was displayed second . , 2008 ) , it is evident that duration estimates were significantly lower when the standard was displayed first relative to when it was displayed second [ general linear model , main effect for standard order , f(1 ) = 13.433 , p < 0.01 ] . we found that : the duration of a stimulus composed of a random alternation of static and drifting intervals ( mixed standard ) appeared compressed either relative to a drifting comparison stimulus or to the average of the static and drifting estimates . the dilation effect induced by speed or temporal frequency on the perceived duration of a visual stimulus has been traditionally seen as a supporting evidence for a change - based theory of time perception ( fraisse , 1963 ; poynter , 1989 ) . in our experiment , the number of changes associated with the mixed stimulus should arguably be intermediate between the stationary stimulus and the continuously drifting one . the temporal frequency of our drifting stimuli ( 10 hz ) falls slightly outside this range , therefore it is arguable that different mechanisms might be at work for low relative to high temporal frequencies and we are maybe tapping into a range of frequencies that is not substantially influenced by the change rate within an interval . therefore , the model predicts an underestimation of the duration of a static stimulus relative to a drifting one ( as is observed in our results ) , but it is not clear how it can account for the lack of a substantial difference between the static and the mixed stimulus . also , the model predicts a lower precision for the static ( the duration of which is determined solely on the basis of the internal estimate ) relative to the drifting stimuli ( which can also take advantage of the stimulus - driven estimate ) . in conclusion , we showed that the number of temporal features that are contained within an interval are not necessarily the main factor determining the apparent duration of dynamic stimuli .
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