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we used mass spectrometry ( ms)based proteomics , as described by breci et al . and koller et al . proteins of individual ticks were reduced with 100 mmol / l dithiothreitol , alkylated with 50 mmol / l iodoacetamide , digested with trypsin at a final concentration of 0.01 g/l , and filtered through a c18 cartridge before being subjected to liquid chromatography ( lc ) with a 5%50% acetonitrile gradient in 0.1% formic acid , followed by tandem ms ( lc - ms / ms ; ltq thermoelectron , san jose , ca , usa ) . polyacrylamide gel electrophoresis , and gel slices were digested in situ with trypsin before using lc - ms / ms . upwards of 7,000 spectra were submitted to a protein identification algorithm for each pooled sample . the sequest search algorithm ( http://fields.scripps.edu/sequest ) with data - filtering criteria was used to identify sequence matches of output against databases of rabbit , sheep , deer , goat , mouse ( mus musculus ) , and tick proteins . we studied i. scapularis and the lone - star tick , amblyomma americanum , which is a vector of human monocytic ehrlichiosis in the united states . ticks were provided by the tick - rearing facility of the department of entomology and plant pathology of oklahoma state university ( stillwater , ok , usa ) . we examined pools of 15 a. americanum nymphs that had fed on sheep or rabbits as larvae and were 3 months postmolt . predominant vertebrate peptides in all pools were - and -globin chains of hemoglobin and immunoglobulins . sequences of these proteins corresponded to the source of the blood for the ticks . other mammalian proteins detected in pools from ticks fed on sheep or rabbits were histone h3 , histone h2 , mitochondrial malate dehydrogenase , glyceraldehyde-3-phosphate dehydrogenase , mitochondrial atp synthase , interferon regulatory factor , tubulin , tubulin , and transferrin . we then studied individual ticks that had fed as larvae on mice ( i. scapularis ) , rabbits ( a. americanum and i. scapularis ) , or sheep ( a. americanum ) and examined flat nymphs of a. americanum at 7 months postmolt and i. scapularis at 311 months postmolt . we also examined i. scapularis adults ( 2 males and 1 female ) that had fed as larvae and nymphs on mice and were 35 months postmolt . the figure shows representative lc - ms / ms spectra of an a. americanum nymph that had fed on a sheep as a larva . panel a shows the tandem mass spectrum for the singly charged peptide aavtgfwgk , corresponding to residues 816 of sheep hemoglobin -subunit ( p02075 ) . panel b shows the tandem mass spectrum for doubly charged vkvdevgaealgr , corresponding to residues 1729 of the same protein . these 2 peptides cover 15.2% of the protein sequence and differ from the orthologous sequence of rabbit ( p02099 ) at 8 of 22 positions . tandem mass spectra of 2 peptides from sheep hemoglobin -subunit identified in a nymph of an amblyomma americanum tick . the peaks are labeled in the conventional manner : b ions include the n - terminus of the peptide and y ions include the c - terminus , with subscripts indicating the number of amino acid residues in the fragment . there was no correlation between number of proteins detected and postmolt period . although some proteins , such as immunoglobulin and histone h3 , were detected in both species , other proteins distinguished between a. americanum and i. scapularis . cytochrome c - type heme lyase , which binds heme moieties and is transported from the cytoplasm to mitochondria of eukaryotes , was present in all samples of i. scapularis but not in a. americanum ( 2-sided p<0.001 , by likelihood ratio ) . digestion of a blood meal in ticks differs from what generally occurs in hematophagous insects . in ticks , digestion takes place gradually within cells of the intestinal tract after endocytosis , rather than by intraluminal enzymatic breakdown of blood cells and plasma components , as in insects ( 13 ) . the combination of slow assimilation and uptake of some host proteins into hemolymph may explain persistence of host blood proteins for months after feeding and molting . immunoglobulins in hemolymph have been demonstrated , but demonstration of several other proteins , including globin chains , histones , and mitochondrial enzymes , indicates that host protein persistence is not limited to 1 type of molecule . the success of our study depended on access to a large database of protein sequences for sheep , rabbits , and laboratory mice . for most host species for these ticks in nature , such as the white - footed mouse ( peromyscus leucopus ) thus , a priority before application of this approach is ms - based sequencing of highly prevalent proteins from the blood of p. leucopus and other host species . although lc - ms / ms of individual ticks is feasible and highly sensitive , its cost confines it to exploratory studies . high - throughput analysis of hundreds or thousands of specimens will likely require species - specific assays that use antibodies or aptamers for detection and identification of selected proteins . understanding contributions of different vertebrate hosts to pathogen maintenance is a prerequisite for effective monitoring , modeling , and disease prevention efforts that focus on natural reservoirs . unfortunately , this level of understanding has not been broadly achieved . a major impediment to success in this area for most tick - borne zoonoses has been the absence of reliable and reproducible methods for identification of the vertebrate source of the infection for the tick vector by characterizing residual blood components . similar data on uninfected ticks would establish the denominator for prevalence studies and indicate the relative competence of different host species .

mass spectrometry based proteomics of individual ticks demonstrated persistence of mammalian host blood components , including - and -globin chains , histones , and mitochondrial enzymes , in ixodes scapularis and amblyomma americanum ticks for months after molting . residual host proteins may identify sources of infection for ticks .

skin cancer is one of the most common malignancies among people , whose incidence exceeds the sum of other malignant tumors [ 13 ] . cutaneous melanoma ( cm ) is a member of skin cancers and is of high malignancy in clinical . it accounts for about 6.820% of skin cancers , with an increasing incidence of 38% every year . therapy for cm is mainly surgery , and sometimes chemo- or radio- therapy is also used [ 68 ] . because of its early metastasis and high mortality , it is important to diagnose and treat cm as early as possible . micrornas ( mirnas ) are a kind of noncoding rnas and consist of about 2123 nucleotides , which can regulate the gene expression at post - transcriptional level by specific binding to the messenger ribonucleic acids ( mrnas ) . they also play important roles on some other aspects , such as differentiation and apoptosis of cells , biological development , and disease processes [ 1214 ] . mir-137 , located on chromosome 1p22 , is a brain - enriched micro - rna with important roles during neurogenesis , including the proliferation and differentiation of neural stem cells , the regulation of dendritic morphogenesis , and synaptic plasticity . several existing reports confirmed that mir-137 participated in the development and processes of various cancers , such as gastric cancer , head and neck cancer , colorectal cancer , and brain tumors [ 1720 ] . bemis et al . first reported that mir-137 downregulated mitf , which is an important regulator in melanocyte development . recently , chen et al . demonstrated that mir-137 inhibited melanoma cell proliferation by down - regulation of mitf and cyclin - dependent kinase 6 . although considerable evidence has confirmed that mir-137 can serve as a cancer suppressor or candidate site for cancer therapies , proof of a link between mir-137 and its clinical value in cm is still lacking . therefore , additional studies are needed to elucidate the function of mir-137 in cm . in this study , we aimed to explore the mir-137 expression in cm tissues and paired normal tissues and evaluate its possibility as a prognostic biomarker for cm patients . a total of 97 patients , who were diagnosed with malignant cutaneous melanoma at guangdong general hospital , were enrolled in this study . among them , 55 were males and the others were females . patients selected in the study all signed consent forms . fresh cm tissues and paired normal tissues were reserved to use immediately after resection . the data , including age , sex , family history , tnm stage , ulcer , and occurrence site , were recorded in a database . the information about follow - up was updated every 3 months by telephone or questionnaire . total rna was isolated and purified from tissues by trizol reagent ( ambion , foster city ) . then the rna was used to synthesize the cdnas of mir-137 with m - mlv reverse transcriptase ( promega , madison , wi ) according to the manufacturer s directions . the relative expression of mir-137 was calculated with cycle threshold ( ct ) method and was normalized to u6 . the relationship of mir-137 expression and clinical characteristics was confirmed by chi - square testing . kaplan - meier survival analysis was performed to estimate the overall survival rate of cm patients , and the differences between the survival curves were tested by using the log - rank test . correlation between mir-137 expression and prognosis of cm patients was evaluated by cox regression analysis . a total of 97 patients , who were diagnosed with malignant cutaneous melanoma at guangdong general hospital , were enrolled in this study . among them , 55 were males and the others were females . patients selected in the study all signed consent forms . fresh cm tissues and paired normal tissues were reserved to use immediately after resection . the data , including age , sex , family history , tnm stage , ulcer , and occurrence site , were recorded in a database . the information about follow - up was updated every 3 months by telephone or questionnaire . total rna was isolated and purified from tissues by trizol reagent ( ambion , foster city ) . then the rna was used to synthesize the cdnas of mir-137 with m - mlv reverse transcriptase ( promega , madison , wi ) according to the manufacturer s directions . the relative expression of mir-137 was calculated with cycle threshold ( ct ) method and was normalized to u6 . all computations were carried out with spss 18.0 software . the relationship of mir-137 expression and clinical characteristics was confirmed by chi - square testing . kaplan - meier survival analysis was performed to estimate the overall survival rate of cm patients , and the differences between the survival curves were tested by using the log - rank test . correlation between mir-137 expression and prognosis of cm patients was evaluated by cox regression analysis . the relative expression of mir-137 in cm tissues was 1.590.43 ( mean sd ) , while that in normal tissues was 2.410.54 , indicating that mir-137 expression was lower in cm tissues ( figure 1 , p<0.05 ) . further detection was conducted to explore the potential relationship of the mir-137 expression and clinical characteristics . we manually divided the cm tissues into 2 groups : the low expression group had a mir-137 expression level of less than 1.35 , and others belonged to the high expression group . mir-137 expression was tightly associated with clinical characteristics , such as tnm stage , ulcer , and occurrence site ( p<0.05 ) . however , no significant relation was found between mir-137 expression and age , sex , and family history ( table 1 , p>0.05 ) . a follow - up was conducted to estimate the overall survival rate of cm patients . the follow - up lasted for about 60 months and 53 patients died . among the dead patients , 49 were in the low mir-137 expression group and the rest were in the high mir-137 expression group . kaplan - meier survival curve showed that the cm patients with low mir-137 expression presented significantly shorter survival time compared to those with high mir-137 expression ( figure 2 , p=0.000 ) . in addition , the correlation of mir-137 expression and prognosis of cm patients was evaluated by cox regression analysis and the results revealed that low expression of mir-137 was an independent prognostic marker of cm patients ( table 2 , hr=8.531 , 95% ci=2.95024.668 , p=0.000 ) . the relative expression of mir-137 in cm tissues was 1.590.43 ( mean sd ) , while that in normal tissues was 2.410.54 , indicating that mir-137 expression was lower in cm tissues ( figure 1 , p<0.05 ) . further detection was conducted to explore the potential relationship of the mir-137 expression and clinical characteristics . we manually divided the cm tissues into 2 groups : the low expression group had a mir-137 expression level of less than 1.35 , and others belonged to the high expression group . mir-137 expression was tightly associated with clinical characteristics , such as tnm stage , ulcer , and occurrence site ( p<0.05 ) . however , no significant relation was found between mir-137 expression and age , sex , and family history ( table 1 , p>0.05 ) . a follow - up was conducted to estimate the overall survival rate of cm patients . the follow - up lasted for about 60 months and 53 patients died . among the dead patients , 49 were in the low mir-137 expression group and the rest were in the high mir-137 expression group . kaplan - meier survival curve showed that the cm patients with low mir-137 expression presented significantly shorter survival time compared to those with high mir-137 expression ( figure 2 , p=0.000 ) . in addition , the correlation of mir-137 expression and prognosis of cm patients was evaluated by cox regression analysis and the results revealed that low expression of mir-137 was an independent prognostic marker of cm patients ( table 2 , hr=8.531 , 95% ci=2.95024.668 , p=0.000 ) . clinical manifestations include bleeding , itching , tenderness , and ulcer . because of its high malignancy , incidence , and mortality , previous studies have demonstrated that malignant melanoma patients have a very poor prognosis , with median survival of 610 months and a 5-year survival rate of < 5% . it is urgent to find new strategies and novel indicators to improve the diagnosis , treatment , and prognosis of cm patients . in recent years , mirnas have been recognized as important factors in various cancers , including cm . mirnas are single - stranded fragments with about 21 nucleotides and they participate in many kinds of cell regulation . each mirna may have several target genes or several mirnas that co - regulate the same gene . these agents make mirna tightly related to metabolism , cell cycle , cell differentiation , cell apoptosis , and development of individuals . the misregulation of mirnas has been verified to be related with cancer initiation , promotion , and progression . since mir-137 was first found as a regulator via targeting mitf , there has been increasing interest in the role of mir-137 in the development and progression of different tumors . many studies have reported that mir-137 presented significant down - regulation in various cancers . in the present study , we investigated the mir-137 expression in cm tissues and evaluated the relationship between mir-137 expression and the prognosis of cm patients . the results showed that the mir-137 expression level in cm tissues was lower than in paired normal tissues , which was in accord with existing reports . the result revealed that mir-137 expression was closely related with tnm stage , ulcer and occurrence site . zhang et al . verified that mir-137 acted as a tumor suppressor in non - small cell lung cancer . the survival curve showed that patients with low mir-137 expression had shorter survival time than those with high mir-137 expression . moreover , the cox regression analysis showed statistical significance between mir-137 expression and prognosis of cm patients , suggesting mir-137 was a target biomarker for cm . thus , we presumed that mir-137 might act as a prognostic factor for cm patients . although several targets of mir-137 have been identified in various cancers , the mechanism of mir-137 on cm remains unclear . shin et al . illustrated that mir-137 could regulate the p53 level in human keratinocytes . besides , some research outcomes indicated that c - met and yb1 were candidate targets of mir-137 in malignancy melanoma . interestingly , a recent study has shown that c - met expression was decreased by transfection of mir-137 in the melanoma cells . therefore , we conjectured that mir-137 might affect cm by regulating the expression of its candidate target genes like mitf , c - met and yb1 , or by regulating some signaling pathways . this hypothesis needs to be validated by more studies , which might provide directions for future research .

backgroundthe present study aimed to measure mir-137 expression in patients with cutaneous melanoma ( cm ) and to estimate the correlation of mir-137 expression and the prognosis of cm patients.material/methodsthe expression level of mir-137 was assayed by quantitative real - time pcr ( qrt - pcr ) and presented as mean sd . chi - square was used to evaluate the relationship between mir-137 expression and clinical characteristics . we used a kaplan - meier survival curve to determine the overall survival rate of cm patients . moreover , the correlation between mir-137 expression and the prognosis of cm patients was confirmed by cox regression analysis.resultsthe relative expression of mir-137 in cm tissue was 1.590.43 , while that in paired normal tissue was 2.410.54 , which was significantly higher . chi - square analysis showed statistical significance between mir-137 expression and clinical characteristics such as tnm stage , ulcer , and occurrence site ( p<0.05 ) . however , no association was found between mir-137 expression and age , sex , or family history ( p>0.05 ) . according to the survival curve outcome , patients with low mir-137 expression showed relatively higher mortality ( p=0.000 ) and multivariate analysis verified that low expression of mir-137 predicted poor prognosis of cm patients ( hr=8.531 , 95% ci=2.95024.668 , p=0.000).conclusionscompared with paired normal tissues , mir-137 expression was lower in cm tissues . patients with low mir-137 expression had higher mortality than those with high mir-137 expression , suggesting that low mir-137 expression indicated poor prognosis for cm patients .

sacroiliac screws ( siss ) has been used since vidal et al . introduced its use in 1973 . since that , sis has become a common technology in fixing pelvic posterior ring injuries . sis has made important progress in the treatment of posterior pelvic ring injury during the past 20 years . however , some clinical reports showed that conventional sis may not universally result in sufficiently stable fixation . currently , sis fixation represents the only minimally invasive technique to stabilize the posterior pelvic ring . for that reason , it is steadily gaining popularity , becoming one of the most commonly used techniques . some indications for this technique include sacroiliac joint dislocations , sacral fractures , certain iliac crescent fractures and combinations of those injuries . the sacrum , serving as the foundation of the spine , transmits the stress between spine and pelvis through sacroiliac joints . thus , the goal of surgical fixation is the reconstruction of the spine - pelvic - junction to allow early weight - bearing and to facilitate nursing care , particularly for multiple injured patients . to overcome the biomechanical limitations faced by the single iliac screw technique , the dual iliac screw technique was developed . have been demonstrated that the dual iliac screw technique provides good clinical results for patients with a partial or total sacrectomy with no iliac screw failure , confirming the biomechanical advantage of dual over single iliac screw in restoring the stability of the lumbo - iliac fixation construct in vertical and rotational planes . however , clinical practices caution that the dual iliac screw technique may increase bone stock loss , prominence of the instrumentation , and screw - rod connection difficulty as compared with the single iliac screw technique . the aim of this work is , through a nonexhaustive review of the literature expose current considerations about sis fixation technique . this technique obtains fixation by traversing bilateral sacroiliac joints and the sacral body , this technique can solve the problem of bilateral sacroiliac joint fractures and dislocations . in the past were commonly used two techniques , the oblique method of sis placement , in which the screw is obliquely aligned , directed inferiorly to superiorly and posteriorly to anteriorly , and the transverse alignment of the sis through the sacral ala on both the inlet and outlet views of the sacrum . with the first technique was an increased the risk of unrecognized anterior protrusion of screws beyond the sacral body , and with the second - technique results in a smaller secured area of the sacral isthmus bone . the current procedure technique is the sis under fluoroscopic observation with the standard matta projections anterior - posterior ( a.p . ) , inlet , outlet [ figures 1 and 2 ] . computed tomography scan three - dimensional of sacral luxation intraoperative radiography showing transverse alignment of sis fixation sacroiliac screw fixation is technically challenging and can be contraindicated depending on the shape of the sacrum . the surgeon has to decide if an extrapolated implant position will match a secure inner - bony position orienting on planar fluoroscopic images in different projections . in addition , image interpretation is aggravated by the high degree of shape variability of the upper sacrum . this circumstance unavoidably results in increased x - ray exposure for the patient and the surgeon due to more frequent application of image intensifier . aberrant screw or wire placement can lead to significant complications , including injury to the fifth - lumbar nerve root , sacral venous plexus , iliac vessels , or cauda equina . they permit iliac crest bone harvesting and have shown to have high - fusion rates , in long fusions and are valuable for the use in high - grade spondylolisthesis . as well , they offer advantages for correction of pelvic obliquity and for revision surgery and do not violate the sacroiliac joint . have been reported rates of implant malposition of up to 18 - 25% , which are potentially associated with iatrogenic neurovascular lesions . lengthened sis is more applicable to repair surgery after first failure of the sis fixation , but overall sis fixation is indicated in nondisplaced unstable sacroiliac joint injuries or sacral fractures . in the experimental setup , have been demonstrated that two ipsilateral screws provide more biomechanical stability than the one . however , using conventional fluoroscopy , many surgeons limit the placement of iliosacral screws to the pedicles of s1 , as those of s2 are narrow and difficult to visualize [ figure 2 ] . the screws are placed through the outer table of the ilium , through the s1 joint and into the lateral sacrum . this bone channel has been well - described and regularly used in pelvic trauma applications for screw placement . it has also been performed with an open and percutaneous technique . with the development of adjunctive devices for cotrel - dubosset instrumentation recently , pan et al . have introduced a sis fixation guide and evaluated its efficacy in fixation of sacroiliac joint fracture - dislocations , they found that the minimally invasive guide can eliminate discrepancies resulting from the surgeon 's own sensory input when inserting screws under the guidance of computed tomography ( ct ) , making percutaneous iliosacral screw fixation more accurate , safe and simple . as previously mentioned , is widely used the fluoroscopy control for the placement of the screws . this leads the technique being highly dependent on fluoroscopic technician , and on the operator 's ability not only to interpret the fluoroscopic images but also to control wire placement based on this interpretation . using the conventional fluoroscopy - based technique , the drilling can only be controlled in one projection at once ; thus , the position of the screws must be followed and adjusted under fluoroscopy in a.p . , inlet , outlet and lateral projections . all this result in increased radiation exposure for the patient and the surgeon . have been reported injuries to nerve roots and the gluteal vessels in up to 3 - 15% of cases using this method . navigation systems have been introduced for traumatological indications , especially in the field of spine and pelvic surgery with the aim to increase precision of screw positioning , various techniques for ct - guided / computer - navigated screw positioning were described . the potential advantages of ct - guided placement of percutaneous hollow screws are that it is a simple operation , the screws are accurately located , there is less hemorrhage , minimal injuries and strong fixation is achieved . however , in the emergency room ( er ) with patients in urgent need for early primary care , these time - consuming techniques are unsuitable . thus , fluoroscopy provides a straightforward and inexpensive approach to intraoperative visualization . some techniques , such as iso - c3d navigation demonstrates superiority to fluoroscopy navigation for sis fixation in an experimental set - up designed to assess the accuracy . this technique obtains fixation by traversing bilateral sacroiliac joints and the sacral body , this technique can solve the problem of bilateral sacroiliac joint fractures and dislocations . in the past were commonly used two techniques , the oblique method of sis placement , in which the screw is obliquely aligned , directed inferiorly to superiorly and posteriorly to anteriorly , and the transverse alignment of the sis through the sacral ala on both the inlet and outlet views of the sacrum . with the first technique was an increased the risk of unrecognized anterior protrusion of screws beyond the sacral body , and with the second - technique results in a smaller secured area of the sacral isthmus bone . the current procedure technique is the sis under fluoroscopic observation with the standard matta projections anterior - posterior ( a.p . ) , inlet , outlet [ figures 1 and 2 ] . computed tomography scan three - dimensional of sacral luxation intraoperative radiography showing transverse alignment of sis fixation sacroiliac screw fixation is technically challenging and can be contraindicated depending on the shape of the sacrum . the surgeon has to decide if an extrapolated implant position will match a secure inner - bony position orienting on planar fluoroscopic images in different projections . in addition , image interpretation is aggravated by the high degree of shape variability of the upper sacrum . this circumstance unavoidably results in increased x - ray exposure for the patient and the surgeon due to more frequent application of image intensifier . aberrant screw or wire placement can lead to significant complications , including injury to the fifth - lumbar nerve root , sacral venous plexus , iliac vessels , or cauda equina . they permit iliac crest bone harvesting and have shown to have high - fusion rates , in long fusions and are valuable for the use in high - grade spondylolisthesis . as well , they offer advantages for correction of pelvic obliquity and for revision surgery and do not violate the sacroiliac joint . have been reported rates of implant malposition of up to 18 - 25% , which are potentially associated with iatrogenic neurovascular lesions . lengthened sis is more applicable to repair surgery after first failure of the sis fixation , but overall sis fixation is indicated in nondisplaced unstable sacroiliac joint injuries or sacral fractures . in the experimental setup , have been demonstrated that two ipsilateral screws provide more biomechanical stability than the one . however , using conventional fluoroscopy , many surgeons limit the placement of iliosacral screws to the pedicles of s1 , as those of s2 are narrow and difficult to visualize [ figure 2 ] . the screws are placed through the outer table of the ilium , through the s1 joint and into the lateral sacrum . this bone channel has been well - described and regularly used in pelvic trauma applications for screw placement . it has also been performed with an open and percutaneous technique . with the development of adjunctive devices for cotrel - dubosset instrumentation recently , pan et al . have introduced a sis fixation guide and evaluated its efficacy in fixation of sacroiliac joint fracture - dislocations , they found that the minimally invasive guide can eliminate discrepancies resulting from the surgeon 's own sensory input when inserting screws under the guidance of computed tomography ( ct ) , making percutaneous iliosacral screw fixation more accurate , safe and simple . as previously mentioned , is widely used the fluoroscopy control for the placement of the screws . this leads the technique being highly dependent on fluoroscopic technician , and on the operator 's ability not only to interpret the fluoroscopic images but also to control wire placement based on this interpretation . using the conventional fluoroscopy - based technique , the drilling can only be controlled in one projection at once ; thus , the position of the screws must be followed and adjusted under fluoroscopy in a.p . , inlet , outlet and lateral projections . all this result in increased radiation exposure for the patient and the surgeon . have been reported injuries to nerve roots and the gluteal vessels in up to 3 - 15% of cases using this method . navigation systems have been introduced for traumatological indications , especially in the field of spine and pelvic surgery with the aim to increase precision of screw positioning , various techniques for ct - guided / computer - navigated screw positioning were described . the potential advantages of ct - guided placement of percutaneous hollow screws are that it is a simple operation , the screws are accurately located , there is less hemorrhage , minimal injuries and strong fixation is achieved . however , in the emergency room ( er ) with patients in urgent need for early primary care , these time - consuming techniques are unsuitable . thus , fluoroscopy provides a straightforward and inexpensive approach to intraoperative visualization . some techniques , such as iso - c3d navigation demonstrates superiority to fluoroscopy navigation for sis fixation in an experimental set - up designed to assess the accuracy . comparing the stability of lengthened sis and sis for the treatment of bilateral vertical sacral fractures , zhao et al . in a finite element model of tile c pelvic ring injury ( bilateral type denis ii fracture of the sacrum ) demonstrated that : the stability of one lengthened sis fixation in the s1 or s2 segment is superior to that of two bidirectional sis in the same sacral segmentthe stability of one lengthened sis fixation in s1 and s2 segments , respectively , is superior to that of two bidirectional sis fixation in s1 and s2 segments , respectivelythe stability of one lengthened sis fixation in s1 and s2 segments , respectively , is superior to that of one lengthened sis fixation in the s1 or s2 segmentthe stability of two bidirectional sis fixation in s1 and s2 segments , respectively , is markedly superior to that of two bidirectional sis fixation in the s1 or s2 segment and is also markedly superior to that of one sis fixation in the s1 segment and one sis fixation in the s2 segmentthe vertical stability of the lengthened sis or the sis fixation in s2 is superior to that of s1the rotational stability of the lengthened sis or sis fixation in s1 is superior to that of s2 . the stability of one lengthened sis fixation in the s1 or s2 segment is superior to that of two bidirectional sis in the same sacral segment the stability of one lengthened sis fixation in s1 and s2 segments , respectively , is superior to that of two bidirectional sis fixation in s1 and s2 segments , respectively the stability of one lengthened sis fixation in s1 and s2 segments , respectively , is superior to that of one lengthened sis fixation in the s1 or s2 segment the stability of two bidirectional sis fixation in s1 and s2 segments , respectively , is markedly superior to that of two bidirectional sis fixation in the s1 or s2 segment and is also markedly superior to that of one sis fixation in the s1 segment and one sis fixation in the s2 segment the vertical stability of the lengthened sis or the sis fixation in s2 is superior to that of s1 the rotational stability of the lengthened sis or sis fixation in s1 is superior to that of s2 . sacroiliac screw fixation is a commonly used procedure for iliosacral joint fracture - dislocations and sacral fractures . currently , the standard technique is the percutaneous iliosacral screw fixation under conventional c - arm fluoroscopy that often exposes the patient to prolonged radiation . unfortunately , even experienced surgeons can have a high rate of screw malposition and nerve and vessel injuries , being that the reason because sis fixation should be performed under better technology control , but in the er setting , this could be laborious and can finish in a loss of time for the patient treatment .

the sacral percutaneous fixation has many advantages but can be associated with a significant exposure to x - ray radiation . currently , sacroiliac screw fixation represents the only minimally invasive technique to stabilize the posterior pelvic ring . it is a technique that should be used by experienced surgeons . we present a practical review of important aspects of this technique .

primary amenorrhea ( pa ) refers to the absence of menarche by the age of 1618 years , although secondary sexual characters are developed . x chromosomal abnormalities ( deletions , iso x chromosomes ) , particularly monosomy x , are more common chromosomal abnormality seen in pa cases.[34 ] here we describe a rare karyotype with 45,x , rob(13;14)(q10;q10),inv(9)(p13;q13),i(xq)(qter --- q10::---qter ) in case of pa and short stature ( ss ) . a 14-year - old female was referred to cytogenetic laboratory for chromosomal analysis because of primary amenorrhea and short stature . her height was 122 cm ( < third centile ) and weight 23 kg ( < third centile ) . x - ray of right wrist and hand , right forearm revealed fused carpel bone centers , which is suggestive of bone age of 10 years . hormonal evaluation showed elevated levels of fsh ( 65.91 miu / ml ) and reduced levels of lh ( 22.31 miu / ml ) . the mother 's age at the time of birth was 20 years and father was 24 years . parental reproductive history showed no family history of abortions , bad obstetrics history and children with mental retardation . at least 50 well spread and good - banded metaphases were scored in each case . chromosomal analysis of the proband revealed 45,x , rob(13;14)(q10;q10),inv(9)(p13q33),i(xq ) ( qter --- q10::---qter ) [ figure 1a and b ] . father 's karyotype was 45,xy , rob(13;14)(q10;q10),inv(9)(p13q33 ) [ figure 1c and d ] . both the male siblings karyotype was same as father 's karyotype i.e. 45,xy , rob(13;14)(q10;q10),inv(9)(p13q33 ) . fluorescence in situ hybridization ( fish ) was performed using centromeric probe ( 14/22 , x ) and partial chromosome paint ( pcp ) ( xq , 9q ) [ figure 2a d ] . the fish results confirmed iso(xq ) with pcp xq probe and inv(9 ) with pcp . ( a ) proband metaphase showing rob(13;14),i(xq),inv(9 ) ( b ) karyotype showing i(x),(qter --- q10::---qter),rob(13;14 ) ( q10;q10),inv(9)(p13q33 ) . ( d ) karyotype showing rob(13;14)(q10;q10),inv(9 ) ( p13q33 ) ( a ) proband fish showing der(13 ) , i(xq ) with centromeric probes . chromosome aberrations such as 45,x , i(xp ) or i(xq ) , and xq deletions are frequently reported in patients with pa . the mosaicism of x chromosome including 45,x/46,xx or 45,x/46,x , iso(xq ) are also not uncommon in pa.[3467 ] fish has advantage in detection of x chromosomal abnormalities in interphase cells of pa cases , which are not detected with routine gtg - banding . in our case , combination of gtg - banding and fish investigation revealed 45,x , rob(13;14)(q10;q10),inv(9)(p13q33 ) , i(xq)(qter --- q10::---qter ) karyotype . this is the first case with multiple chromosomal abnormalities in a case of pa to our knowledge . genotypic and phenotypic correlation of the present case showed short stature and primary amenorrhea and this may be due to absence of short arm of x chromosome ( xp).[89 ] the direct phenotypic effect of t(13;14 ) and inv(9 ) not expressed as the translocation is balanced and inversion 9 is a common polymorphic variant . the inheritance pattern of the chromosome abnormality in the family showed interesting transmission [ figure 3 ] . in case of proband , i(xq ) was originated de novo and other two chromosome abnormalities were transmitted from her father . the father 's chromosomal analysis showed t(13;14 ) , inv(9 ) and found to be a carrier . the younger female sibling received only the inv(9 ) and other male siblings karyotype same as that of the father karyotype . the hormonal therapy may be helpful to achieve menstrual cycles as proband is a case of pa . chromosome aberrations [ t(13;14 ) , inv(9 ) ] were associated with infertility , repeated abortions , aneuploidy fetuses and bad obstetric history in general.[1013 ] the familial i(xq ) has not been reported in cases with pa or infertility . the cytogenetically abnormal pa cases are difficult to treat , since the present case has common chromosomal abnormalities ; some extent hormonal therapy such as , progesterone , estrogen can be given to improve reproductive life . however , proper genetic counselling and prenatal diagnosis for proband is essential for future pregnancies to rule out chromosomally abnormal fetuses . the cytogenetic investigations are essential in pa cases to detect sex chromosomal abnormalities , undetected mosaicism and multiple chromosome aberrations , which help in appropriate genetic counseling and management of the disease .

primary amenorrhea ( pa ) refers to the absence of menarche by the age of 1618 years although secondary sexual characters are developed . pa occurs in 13% of women in the reproductive age group . various factors such as anatomical , genetic and hormonal factors reported to influence pa . we report triple chromosomal abnormalities of rob(13;14)(q10;q10),inv(9)(p13q33 ) , i(xq)(qter --- q10::---qter ) in a case of pa and short stature . though proband has multiple chromosome aberrations , genotypic effect of only i(xq ) is evident as proband has pa and short stature . the rob(13;14 ) and inv(9 ) , which are paternally derived may have role in later reproductive age . therefore , chromosomal analysis is essential in such cases for the accurate diagnosis and management of the disease .

aging is the inescapable process by which individuals , from the age of about 30 years old onwards , gradually lose maximal functional capacity . some resilient individuals experience a slow decline lasting several decades , attaining ages past one hundred years old and more . these are exceptional centenarians who experience minimal physical impairment along with healthy minds and bodies [ 4 , 5 ] . however , many individuals experience more rapid functional declines in their 60 's or 70 's , sometimes afflicted with the frailty syndromedefined as a lack in general strength and unusual susceptibility to disease or to other infirmity and these individuals often suffer from multiple age - related morbidities such as cardiovascular disease , neurodegenerative diseases , diabetes , and cancer . the majority of individuals lie between these two extreme scenarios , with an average life expectancy of 81 years old in north america . in the past century , we have witnessed significant increases in life expectancy as more individuals live longer [ 9 , 10 ] . this increase in average life expectancy has undoubtedly resulted from advances in medical technologies and preventive medicine that prevent most ( > 80% ) early deaths due to acute illnesses ( e.g. , infections and injuries ) and prolong life of individuals afflicted with chronic life - threatening conditions ( e.g. , hiv / aids and cardiovascular disease ) . the incidence of age - related diseases has remained stable over the last decades , or even increased , which significantly contributes to health care costs . given that an unprecedentedly large proportion of the population is expected to reach 6080 years of age in the next two decades , changes in political and social health policy comprehensive frameworks including the panoply of factors capable of potently modulating the human aging process may be essential to address the impending social imperative of implementing health - enhancing strategies for the elderly , at minimal costs . a central question concerning longevity remains : why do some people live long whereas others die early ? another equally critical question concerns morbidity : why is aging associated with a greater incidence of almost every categorized disease including degenerative , metabolic , and malignant disorders ? since disease incidence , mortality , and longevity are all associated terms in the same aging equation , a more general question may be posed : what are the pathways that impact individuals ' rate of aging ? while it is well understood that both biological and psychosocial factors impact the aging process , it is still unclear how psychosocial factors influence cellular aging and translate into aging of the whole organism . below is a selective review focusing on physiological systems susceptible to constitute critical convergence points , acting as integrators of the interactive forces imposed by both biological and psychosocial factors . understanding how this physiological integration takes place will improve researchers ' means to develop multilevel interventions that optimize the decline in physical function associated with aging . physical health and function depends on the coordinated functioning of several organs and physiological systems that allow the organism in dynamic balance to adapt to perpetual environmental challenges . failure to adapt to challenges ( e.g. , healing wounds , increasing energy expenditure , and replenishing dying postmitotic cells ) may occur in aging . thus , senescence - induced loss of cell numbers and/or optimal functioning can result in suboptimal organ function . for this reason , markers of cellular aging , such as nuclear dna telomere length , these are the protective caps at the end of chromosomes , whose reduction in length is often used as a reliable and proximal indicator of cellular senescence . an important constituent of mammalian cells are mitochondria . these dynamic subcellular organelles contain their own circular dna , are the principal site of cellular adenosine triphosphate ( cellular energy currency ) synthesis , regulate cell death through apoptotic signalling , and are the major source of reactive oxygen species ( ros ) within the cell [ 16 , 17 ] . one of the most scrutinized hypothesis in aging research is the mitochondrial theory of aging which stipulates that , over time , mitochondrial dna accumulates oxidative damage from ros , which negatively impacts mitochondrial function , leading to cellular dysfunction , organ failure , and ultimately results in age - related disease . data supporting this theory has been obtained from transgenic animals with enhanced protection against mitochondrial oxidative damage [ 18 , 19 ] . these mice , which over - express a mitochondrial - targeted catalase , are resistant to age - related insulin resistance and have slightly increased lifespan . however , existing data render this theory imperfect [ 2023 ] and evidence supporting a direct role of ros in aging has largely been correlative . furthermore , examples exist in vertebrates ( e.g. , naked mole rat ) and invertebrates ( c. elegans [ 26 , 27 ] ) where the typical negative correlation between ros production and lifespan is uncoupled . although ros - induced damage has not consistently been causally linked to aging , a general shift in intra- and extracellular redox state towards more oxidized levels occurs in aging cells and in the blood of aged individuals , which could have important implications for redox - sensitive signalling pathways and their influence on the aging process [ 28 , 29 ] . in contrast , a general role of mitochondria in the aging process is supported by abounding experimental evidence [ 3037 ] . for example , animals with a deficient proof - reading version of the mitochondrial dna polymerase gamma ( polg mutator mice ) , a defect which leads to an abnormally rapid accumulation of mitochondrial dna mutations , exhibit several characteristics reminiscent of an accelerated aging phenotype ( e.g. , graying of fur , loss of muscle and brain mass , and kyphosis ) [ 38 , 39 ] . this indicates that mitochondrial dna damage is capable of causing aging - like symptoms such as organ dysfunction and early mortality . it must be noted that whether this model actually mimics natural human aging is uncertain [ 40 , 41 ] . nevertheless , although its exact cellular and physiological impact remain unclear , the integrity of mitochondrial dna is challenged during aging [ 42 , 43 ] and may contribute to cellular senescence and consequently , to the progressive functional changes in organs that characterize the aging process . further evidence supporting a role of mitochondria in the aging process comes from interventions that influence mitochondrial function . lifelong caloric restriction diminishes damage to mitochondrial dna and concomitantly decreases the age - related decline in muscle aerobic capacity [ 45 , 46 ] . of note , caloric restriction has also been reported to decrease the incidence of age - related illnesses ( e.g. , cancer ) in rodents , providing an interesting empirical link between mitochondrial integrity and age - related morbidity . moreover , evolutionarily inherited single nucleotide polymorphisms yielding genetic variants of mitochondrial dna , called haplogroups , may influence mitochondrial function and health outcomes in humans ( reviewed in ) . indeed , mitochondrial haplogroups have been associated with mitochondrial ros production and cellular oxidative capacity [ 4850 ] , resting metabolic rate and energy expenditure in humans , and disease incidence , progression , and longevity [ 14 , 5254 ] . collectively , this suggests that intrinsic mitochondrial factors ( e.g. , related to mitochondrial dna ) can indeed influence the aging process . like caloric restriction , physical activity and exercise are potent stimuli that increase mitochondrial content and function [ 5557 ] . physical activity reduces the age - related decline in function of different organ systems including brain and muscles . indeed , it is established that individuals who are more physically active exhibit lower incidences of age - related diseases and mortality [ 5860 ] as well as better control of existing chronic diseases . furthermore , endurance exercise prevents the premature aging - like characteristics of the polg mutator mice including mitochondrial abnormalities , skeletal muscle , and brain atrophy . physical inactivity leads to a reduction in mitochondrial content and function [ 56 , 62 ] and contributes towards insulin resistance ( i.e. , prediabetic state ) and enhanced metabolic risk . for example , older individuals ( 6370 years old ) who are sedentary , but not those who are active , have lower mitochondrial content than young individuals . finally , dietary lipid supply ( e.g. , virgin olive oil ) has been shown to impact membrane composition in brain mitochondria and to reduce oxidative damage to these organelles with aging in rats . thus , factors that impact mitochondrial function ( i.e. , levels of physical activity , caloric restriction , and diet ) can consequently impact age - related disease incidence , progression , and survival . the findings outlined in this section are consistent with the notion that biological mechanisms determine the aging process . additional arguments supporting this notion also exist . they notably include the loss of molecular fidelity with time as the major cause of aging and the recently discovered link between mitochondrial function , telomere length , and cellular senescence [ 68 , 69 ] . because each aspect outlined above appears to modulate the aging process in small yet sizeable ways , we must acknowledge that evidence suggests that the rate of aging is not solely determined by single biological factors , such as how many calories are ingested , which genetic polymorphism an individual has inherited , and how much physical activity is performed . rather , in real - life situations , the rate of aging for a given individual must ultimately be determined by the dynamic and reciprocal interplay of these and many other factors , as discussed below . despite the fact that aging research has generally been dissected using the biological scalpel , psychological and social variables are also important modulators of the aging process associated with mortality [ 7073 ] . for example , personality and lifestyle may influence longevity in humans . in a prospective study of patients with coronary heart disease , the authors found that pessimism and anxious personality traits were associated with adverse age - related health outcomes such as greater cancer incidence and all - cause mortality . degradation in negative affect ( i.e. , more negative emotions ) was also a strong prognostic indicator of long - term mortality in coronary heart disease patients , suggesting that negative emotions can adversely influence survival and resilience . on the other hand , centenarians with engaged lifestyle and certain personality traits ( e.g. , emotional stability , extraversion , and openness ) tend to have higher mental health status , a healthy sign of aging , when compared to those who do not possess these traits . a twenty - year prospective population study showed that individuals with more positive self - perceptions of aging tended to live about seven years longer than those with less positive perceptions of aging . srh , how an individual subjectively rates his / her health is one of the most powerful statistical predictor of morbidity and mortality [ 79 , 80 ] . of note , srh is often a more powerful statistical predictor of mortality than clinical and biological assessments of health . similarly , high socioeconomic status is associated with more positive multisystemic physiological profiles ( i.e. , allostatic load ) , which predict lower morbidity and mortality rates with aging [ 81 , 82 ] . there is also evidence that protective psychosocial factors such as control beliefs and quality of social support ( i.e. , emotional links with family and friends ) contribute to better maintenance of functional capacity with aging . although not directly supporting a causal link between psychosocial factors and longevity , these data strongly suggest that several psychosocial factors impact physiological pathways to aging and distal outcomes such as mortality and longevity . not living with a partner ( i.e. , being unmarried ) is associated with accelerated cellular aging , as evidenced by shorter telomere length in blood leukocytes of unmarried middle - aged men and women . similar reports by epel and colleagues demonstrate that psychological stress is associated with accelerated telomere shortening . likewise , depression has been linked with accelerated rates of aging and cellular senescence and mortality , demonstrating that psychosocial forces may accelerate cellular aging . collectively , these findings indicate that psychosocial forces can exert both negative and positive influences on the aging process , affecting both morbidity and mortality . as mentioned above , mitochondria influence cellular function [ 16 , 17 ] and impairments in mitochondrial function due to genetic variations / mutations or other stresses such as physical inactivity may accelerate the aging process . interestingly , several hormones including those involved in the body ' stress responses to psychosocial stressors modulate the synthesis of new mitochondria ( mitochondrial biogenesis ) and can modify important parameters of mitochondrial function . indeed , mitochondrial dna transcription and mitochondrial biogenesis are modulated by the glucocorticoid hormone cortisol downstream from the hypothalamic - pituitary - adrenal ( hpa ) axis , by catecholamines secreted by the sympathetic - innervated adrenal medulla ( epinephrine and norepinephrine ) , thyroid hormones , and by the steroid hormone estrogen , as well as by several cytokines ( e.g. , il-1 , il-1 , and tnf ) . in fact , the mitochondrial dna sequence contains putative response elements for several hormonal receptors ( e.g. , thyroid and glucocorticoid hormones , and insulin ) and some receptors for glucocorticoids , thyroid hormones , and estrogen have even been found in mitochondria of different cell types [ 90 , 91 ] . however , chronic exposure to elevated levels of these hormones , which can be induced by psychosocial stressors ( e.g. , social isolation , depression , and violent or abusive social environment ) , can lead to reductions in mitochondrial mass ( see figure 1 ) and concomitant increases in mitochondria - derived ros production [ 87 , 92 ] . these mitochondrial outputs synergistically damage cellular components and contribute to cellular senescence when chronically produced at high levels . beyond the direct effects that psychosocial stresses exert on mitochondrial function , psychosocial factors can also influence individual 's lifestyles , such as levels of physical activity and inactivity ( i.e. , sedentariness ) . for example , negative perceptions of one 's body image and negative influence from family and friends are associated with lower levels of physical activity [ 9597 ] . similarly , people who are depressed or suffer from mental illnesses also tend to be more physically inactive [ 98 , 99 ] . physical inactivity can in turn undermine physical and mental health , predisposing inactive young individuals to depression later in life . physical activity has positive effects on mitochondrial function and counteracts inflammatory processes and age - related chronic diseases . in fact , the beneficial effects of physical activity and exercise on the hormonal system ( e.g. , increases in interleukin 6 , growth hormone , brain - derived growth factor bdnf ) [ 103 , 104 ] , on psychological and cognitive aspects ( e.g. , decrease in stress levels and reactivity to stress , depression , improved well - being ) , as well as on metabolic regulation ( e.g. , increased mitochondrial mass and improved insulin sensitivity ) [ 65 , 106 ] , suggest that exercise and physical activity exert multisystemic protective effects which can prevent the deleterious consequences of chronic stress . indeed , improving physical fitness has been shown to decrease hormonal , physiological and psychological markers of chronic stress . psychosocial factors and physical activity can therefore interact to influence mitochondrial function and modulate the impact of chronic stress on the body . because mitochondria influence cellular aging and are responsive to stress hormone levels , they are especially well equipped to act as key integrators that synergistically influence biological and psychosocial factors ( figure 1 ) . as described above , work in the psychosocial sciences has unravelled important links between how individuals feel , their social contexts , and the effects of these factors on mortality and longevity . however , how these factors influence and interact with biological factors remains to be explored in more depth . the findings described above and many others bring new evidence coaxing researchers to focus on the interactions of biological and psychosocial forces that influence the aging process . conclusions derived from research not adopting an integrative approach risk being uni - dimensional and thus difficult to apply towards different real - life contexts , where individuals age under the collective influence of factors of different nature . interdisciplinarity and even transdisciplinarity have emerged as key necessities in the field of aging and others [ 108110 ] . both the national institutes of health ( nih ) in the usa and the canadian institutes of health research ( cihr ) funding agencies have opened institutes on / of aging promoting broad mandates that necessarily reach across traditional disciplinary boundaries . although some problems are best addressed with the approach of a single discipline , other issues require the integration of several disciplines to fully comprehend the complexity of the processes at play . likewise , the discipline of developmental psychopathology , driven by the impetus to grasp and impact the complexities of mental health at different stages of development , has evolved to become a deeply interdisciplinary field that seeks to elucidate the interplay among the biological , psychological , and social - contextual aspects of normal and abnormal development across the life course [ 112 , p.16 ] . the writings of cicchetti and toth are particularly enlightening regarding the conceptual , theoretical , and practical directions to take to achieve such a degree of integration within a discipline . they particularly stress the importance of cross - disciplinary dialogue , along with the need for an emphasis on the process of development naturally resulting from the interdependence over time of multiple biological and psychosocial factors . in this spirit , the aging process is influenced by several interactive forces inherent to the individual ( e.g. , genetic endowment , physical activity , diet , lifestyle , and personality ) and forces inherent to the psychosocial environment ( e.g. , sociocultural context , family , and life stress ) that continuously and progressively interact over long periods . aging must therefore be approached from an ontogenic perspective not solely focused on end outcomes ( i.e. , mortality , comparison of aged and young individuals ) or on the molecular factors predicting these outcomes . instead , aging research would benefit from being guided by a perspective focused on the changes and interactions among biological and psychosocial processes , which take place across stages of human development throughout the lifespan . table 1 lists five conceptual propositions elaborated by ryff and singer to advance hypothesis - testing research focused on healthy aging as an interdisciplinary process . in a similar way that mental health and disease emerge from cumulative life experiences in infancy , childhood , and adulthood [ 112114 ] , aging must also be a deeply experience - dependent process where the biology influences how a person responds to their experiences , but where the biology is also shaped by those experiences . the experience - dependent nature of aging has important implications for the research questions that are posed ( e.g. , how do specific events / factors at different life stages interact to modulate the rate of aging ? ) . this suggests that cumulative prenatal , earlylife , young adulthood , and later life circumstances impact pathways to aging . this could be mediated in part by alterations in the stress system across the life course : induced alterations in neurological substrates that signal stressful information [ 114 , 115 ] as well as neurobiological and allostatic processes involving inflammation and oxidative stress [ 72 , 116 ] . thus , as suggested by epel , psychological and metabolic stress may constitute a potent recipe for accelerated cellular aging . epigenetics , which involves the laying of relatively stable imprints on the genome that impact gene expression and cellular function over time [ 117 , 118 ] , is increasingly revealed as a candidate intersection point between biological and psychosocial processes in several age - related chronic diseases [ 119 , 120 ] . it is known that epigenetic marks are altered in aging [ 121 , 122 ] and in several age - related disease states such as cancer , neurodegenerative , and autoimmune diseases , as well as type 2 diabetes [ 124 , 125 ] . the altered epigenome could therefore mediate the experience - dependent modulation of the aging rate and age - related morbidities across the life span . further to that point , mitochondria themselves possess a plastic mtdna epigenome and have the potential to generate powerful signals capable of affecting the nuclear epigenome [ 127 , 128 ] , making these organelles well equipped to play a critical interfacing role between the environment and the genome . although this remains to be empirically supported , it is an hypothesis that integrates knowledge about the health consequences attributable to genetic variations , calorie intake , physical activity / inactivity , and neurobiological substrates of psychosocial stress into a unified framework for aging research . taken together , the reviewed literature indicates that an increasing number of biological factors ( e.g. , mtdna haplotypes , hormones , genetic polymorphisms affecting cellular signalling pathways , factors epigenetic imprints ) , behavioral and ( e.g. , diet / calorie intake , and exercise ) , and psychosocial ( e.g. , psychological stress , depression , personality , and marital status ) factors influence the aging process . the challenge lying ahead of researchers in this field lies in the exploration of the intersection points linking these multiple levels of analysis spanning several disciplines . for example , what physiological processes interact with the psychosocial effects of being married , of meditating regularly , or of experiencing psychological well - being and sense of purpose in life , which ultimately culminate in reduced telomere shortening ? what combination of elements lead to resilience and successful adaptation to aging ? and what are the combinations leading to age - related risk and ill - health ? interdisciplinary initiatives aimed at describing the interactive biopsychosocial processes that link these multiple levels will yield new knowledge of the pathways to aging , which in turn will inform effective prevention and intervention strategies . network perspectives inspired from systems biology [ 130 , 131 ] allowing modeling of complex nonlinear interactions among the studied variables may prove useful in this endeavour . similarly , building comprehensive theories of aging will require the combined efforts of researchers from different disciplines contributing diverse complementary expertise , perspectives , and approaches to study aging . the perfect individualization of treatment and prevention of age - related disorders may appear as an unattainable utopia at this point in time . this is particularly the case because up until now , our knowledge of the dynamic interplay between the different biological and psychosocial levels of analysis is still fragmentary , which impedes discoveries about the complex processes from which individual - specific pathways of aging emerge . likewise , the source of interindividual differences in aging trajectories undoubtedly lies in the interplay of several interdependent pathways of which there is no single universal means must be developed to distinguish between optimal ( i.e. , living to your full biological potential ) and suboptimal ( i.e. , dying or having disease sooner than your constitution should permit ) rates / trajectories of aging . from the onset , it can be established that optimal aging is characterized by a slow progressive decline in physiological functions , maintenance of well - being for the majority of the lifespan , and only a short period of very poor physical health leading to death . but what are the biomolecular ( i.e. , gene expression , mitochondrial function , and biomarkers ) sings of optimal adaptation to the passing decades ? what are the normative ontogenic trajectories , or healthy biological and physiological signatures of successful aging ? having answers to these questions will enable researchers to more accurately distinguish dysfunction from normal function in different aged organ systems . ryff , singer and colleagues have established biological correlates and a conceptual framework aimed at deciphering the biological and psychosocial underpinnings of resilience , positive health , and successful aging [ 71 , 132134 ] . building such a knowledge base of normal molecular , cellular , physiological , and psychosocial signatures of aging may also translate into more refined means to detect predisease or preclinical deviations from normal adaptation and to prevent age - related diseases . thus far , despite the fact that more resources are being invested to study specific aspects of the normal pathways leading to healthy aging [ 71 , 132134 ] , relatively little data is available to address pressing questions about healthy aging . a noteworthy exception is the macarthur studies of successful aging , which have collected a rich dataset spanning multiple biological and psychosocial levels over several years , thus providing an exceptional design for longitudinal evaluation of the biological - psychosocial interactions for a large cohort of elderly individuals . future smaller - scale ( i.e. , intervention trials and animal - based models ) integrative research initiatives should build from the strengths and experience of this and other such longitudinal endeavours . in conclusion , as a rejoinder to the question what are the pathways that impact individuals rate of aging ? , we ought to answer that there are surely several different pathways to healthy aging . these pathways must depend not on singular factors acting independently , but on interactive forces among multiple levels of function operating in synergy , including biological , behavioural , psychosocial and spiritual factors . identifying the developmental nature of pathways to aging is an interdisciplinary task inviting researchers in aging to join forces to discover and refine our comprehension of the intersections between our respective disciplines . biomedical scientists need to appreciate the complexity of biological - psychosocial interactions involved in health processes ; and psychosocial researchers need to appreciate the underlying biological factors susceptible to modulate individual responses to psychosocial challenges . this can be achieved , along with the ensuing collaborative interdisciplinary successes in research , by defining and empirically testing potential intersection points among biological and psychosocial disciplines . a deeper understanding of these intersections , and of the ensuing mind - body cross - talk , will enhance our appreciation of the multiple interacting facets that collectively determine optimal and suboptimal rates of aging for individuals . testing and defining inter- and transdisciplinary intersection points should also enhance our ability to translate health discoveries into applicable interventions to promote the health and quality of life of an increasingly old population .

compelling evidence suggests that both biological and psychosocial factors impact the process of aging . however , our understanding of the dynamic interplay among biological and psychosocial factors across the life course is still fragmentary . for example , it needs to be established how the interaction of individual factors ( e.g. , genetic and epigenetic endowment and personality ) , behavioral factors ( e.g. , physical activity , diet , and stress management ) , and psychosocial experiences ( e.g. , social support , well - being , socioeconomic status , and marriage ) in perinatal , childhood , and adulthood influence health across the aging continuum . this paper aims to outline potential intersection points serving as an interface between biological and psychosocial factors , with an emphasis on the mitochondrion . mitochondria are cellular organelles which play a critical role in cellular senescence . both chronic exposure to psychosocial stress and genetic - based mitochondrial dysfunction have strikingly similar biological consequences ; both predispose individuals to adverse age - related health disorders and early mortality . exploring the interactive nature of the factors resulting in pathways to normal healthy aging , as well as those leading to morbidity and early mortality , will continue to enhance our ability to translate research into effective practices that can be implemented throughout the life course to optimise the aging process .

malignant melanoma is one of the most aggressive cancers and its incidence has shown a rapid increase [ 1 - 3 ] . many treatments for melanoma , including chemotherapies , immunotherapies , and combined biochemotherapy regimens have been utilized but have failed to significantly improve outcome and the median overall survival ( os ) of metastatic melanoma is approximately 8 months . however , studies reported in recent years have resulted in many breakthroughs in the melanoma field . two extraordinary advances were recently achieved with positive results from two separate studies of new therapies , ipilimumab in immunotherapy and vemurafenib in molecular targeted therapy . ipilimumab ( yervoy , bristol - myers squibb ) is a fully human monoclonal antibody against the cytotoxic t - lymphocyte associated antigen 4 receptor . in phase iii clinical trials , treatment with ipilimumab resulted in a statistically significant improvement in os of patients with metastatic melanoma . a pooled analysis of 1,861 patients treated with ipilimumab showed long - term survival with a 3-year survival rate of 22% . in addition , many expanded access programs ( eap ) in various countries reported on the benefit of ipilimumab in a real world setting . compared to western countries , the incidence of melanoma in eastern counties is very low but has also increased . the most common subtype in western countries is cutaneous melanoma , while acral and mucosal melanoma are the most common subtypes in eastern countries [ 9 - 12 ] . ipilimumab has been approved in asian countries including korea ; however , data on the efficacy and safety of ipilimumab in asian patients with melanoma were limited . therefore , we reported the outcomes of ipilimumab in korean patients enrolled in the named - patient program ( npp ) cohort . in addition , we examined the association between response to ipilimumab and melanoma subtype , braf mutation , and blood parameters . patients who met the following eligibility criteria were enrolled in the ipilimumab npp ; a histologically confirmed stage iv malignant melanoma according to the american joint committee on cancer ( ajcc ) , and eastern cooperative oncology group ( ecog ) performance status ( ps ) 0 , 1 , or 2 . all patients received four cycles of 3 mg / kg ipilimumab every 3 weeks , unless severe adverse events ( aes ) or rapid progression of disease occurred . m - stage was defined according to site metastasis in combination with elevated lactate dehydrogenase ( ldh ) levels , as described previously . records of all patients who received at least one dose of ipilimumab were reviewed retrospectively . the study protocol was approved by the ethics committee from all six hospitals and this study was conducted according to the declaration of helsinki . the medical records of eligible patients were retrieved and included age , sex , ecog ps , previous therapies , melanoma subtype and disease stage , metastatic site , ldh , and braf and c - kit mutational status if available . treatment - related aes using the common terminology criteria for adverse events ver . 4.0 ; hematological and serum parameters including absolute neutrophil count ( anc ) , and lymphocyte count ; date of progression ; and reason for cessation of ipilimumab ( progressive disease , ae , and death ) were also collected . patients were reviewed routinely every 3 weeks during treatment and every 8 weeks during follow - up . radiological imaging was generally performed 6 - 9 weeks after ipilimumab induction and every 8 weeks thereafter . responses were scored according to response evaluation criteria in solid tumor ( recist ) ver . baseline , pre - treatment white blood cell count , anc , absolute lymphocyte count ( alc ) , neutrophil to lymphocyte ratio ( nlr ) , and serum ldh were analyzed . progression - free survival ( pfs ) was defined as the time from the start of ipilimumab to the onset of progression or death . os was defined as the time from the start of ipilimumab to death from any cause . univariate analysis for clinical and laboratory parameters was performed with respect to os and pfs . survival curves of categorical variables were calculated using the kaplan - meier method and compared using the log - rank test . multivariate models of patient and tumor characteristics in association with pfs and os were based on cox proportional hazards regression analyses . two - tailed patients who met the following eligibility criteria were enrolled in the ipilimumab npp ; a histologically confirmed stage iv malignant melanoma according to the american joint committee on cancer ( ajcc ) , and eastern cooperative oncology group ( ecog ) performance status ( ps ) 0 , 1 , or 2 . all patients received four cycles of 3 mg / kg ipilimumab every 3 weeks , unless severe adverse events ( aes ) or rapid progression of disease occurred . m - stage was defined according to site metastasis in combination with elevated lactate dehydrogenase ( ldh ) levels , as described previously . records of all patients who received at least one dose of ipilimumab were reviewed retrospectively . the study protocol was approved by the ethics committee from all six hospitals and this study was conducted according to the declaration of helsinki . the medical records of eligible patients were retrieved and included age , sex , ecog ps , previous therapies , melanoma subtype and disease stage , metastatic site , ldh , and braf and c - kit mutational status if available . treatment - related aes using the common terminology criteria for adverse events ver . 4.0 ; hematological and serum parameters including absolute neutrophil count ( anc ) , and lymphocyte count ; date of progression ; and reason for cessation of ipilimumab ( progressive disease , ae , and death ) were also collected . patients were reviewed routinely every 3 weeks during treatment and every 8 weeks during follow - up . radiological imaging was generally performed 6 - 9 weeks after ipilimumab induction and every 8 weeks thereafter . responses were scored according to response evaluation criteria in solid tumor ( recist ) ver . baseline , pre - treatment white blood cell count , anc , absolute lymphocyte count ( alc ) , neutrophil to lymphocyte ratio ( nlr ) , and serum ldh were analyzed . progression - free survival ( pfs ) was defined as the time from the start of ipilimumab to the onset of progression or death . os was defined as the time from the start of ipilimumab to death from any cause . univariate analysis for clinical and laboratory parameters was performed with respect to os and pfs . survival curves of categorical variables were calculated using the kaplan - meier method and compared using the log - rank test . multivariate models of patient and tumor characteristics in association with pfs and os were based on cox proportional hazards regression analyses . between september 2014 and july 2015 , 104 patients were treated with ipilimumab at six korean hospitals . the median age of patients was 58 years ( interquartile range , 50 to 66 ) and 51 patients ( 49% ) were male . the most common subtype was acral melanoma ( 33 patients , 31.7% ) , 27 patients ( 26% ) had mucosal melanoma , and 27 patients ( 26% ) had skin melanoma . all 104 patients had ajcc stage iv melanoma , and the majority of patients were stage m1c ( 76% ) . sixty - two patients ( 65.3% ) had elevated baseline ldh and 20 patients ( 19.2% ) had nlr 5 . braf and c - kit mutations were evaluated in 87 and 54 patients , respectively , and the incidence of braf and c - kit mutations was 18.4% and 13.0% , respectively . approximately 40% of patients received ipilimumab as first line treatment , and 28% patients had received at least three prior systemic therapies , median number of prior systemic treatments was two ( range , 1 to 5 ) . sixty - nine patients ( 66.3% ) received all four doses of ipilimumab , and nine patients received three doses , 11 patients received two doses , and 15 patients received one dose . of all patients , 68 patients ( 65.4% ) experienced treatment related aes of any grade ( table 2 ) , and the most common toxicities were pruritus ( 31.7% ) and skin rash ( 22.1% ) . the majority of treatment related aes were grade 1 or 2 , and were generally manageable and reversible without sequelae . ten patients ( 9.6% ) experienced grade 3 toxicities , and seven patients experienced treatment related aes , and three cases were considered to be disease related events . one patient suffered from grade 3 fatigue and grade 2 nausea after two cycles of ipilimumab and brain metastasis was detected on brain magnetic resonance imaging . one patient developed grade 3 elevation of aspartate aminotransferase ( ast)/alanine aminotransferase ( alt ) after three cycles of ipilimumab and showed progression of liver metastasis according to recist ver . one patient complained of grade 3 generalized pain , which improved to grade 1 after morphine treatment . no patient experienced grade 4 or 5 aes , and there were no immune response related serious aes . four patients ( 3.8% ) showed complete response ( cr ) and five patients ( 4.8% ) showed a partial response ( pr ) by recist . the overall response rate ( orr = cr+pr ) and disease control rate ( dcr = cr+pr+stable disease ) was 8.6% and 29.8% , respectively ( table 3 ) . the median follow - up duration was 7.1 months ( 95% confidential interval [ ci ] , 5.96 to 8.30 ) , and 85 patients ( 81.7% ) had progression of disease and 30 patients ( 28.8% ) had died by the time of this analysis . the median pfs was 2.73 months ( 95% ci , 2.67 to 2.85 ) in all patients ( fig . 1a ) , and significantly longer survival was observed for patients with disease control ( cr+pr+stable disease ) compared to those with progressed disease ( pd ) ( median pfs , 5.4 months vs. 2.2 months ; p there was no difference in orr , dcr , pfs , or os according to melanoma subtypes ( fig . pretreatment median anc was significantly higher in patients with pd compared to those who achieved disease control ( 4,017 cells/l vs. 3,183 cells/l , p=0.016 ) , while pretreatment median alc was significantly lower in patients with pd compared to those who achieved disease control ( 1,530 cells/l vs. 1,833 cells/l , p=0.049 ) . when anc and alc were incorporated in the nlr , significantly higher nlr was observed in patients who experienced pd than in those with disease control ( 2.63 vs. 1.90 , p=0.003 ) ( table 4 ) . in addition , we found that pfs and os were significantly longer in patients with low nlr compared to those with high nlr ( median pfs , 2.8 vs. 1.3 months ; p < 0.001 and median os , not reached vs. 2.6 months ; p < 0.001 ) ( fig . 1f and g ) . in univariate analysis between baseline characteristics and survival , ecog ps , ajcc m stage , liver metastasis , and nlr showed significant association with pfs , and ecog ps and nlr showed significant association with os . there was no difference in pfs and os according to the presence of brain metastasis , braf mutation , c - kitmutation , and ldh elevation before treatment , or the number of previous therapies . in multivariate analysis , ecog ps , liver metastasis , and nlr were independent prognostic factors for pfs , and ecog ps and liver metastasis were independent prognostic factors for os ( table 5 ) . for selection of patients likely to benefit from ipilimumab , the patients were divided into three groups based on independent prognostic factors for pfs and os , respectively . patients without adverse factors had longer pfs and os than patients with adverse factors ( fig . between september 2014 and july 2015 , 104 patients were treated with ipilimumab at six korean hospitals . the median age of patients was 58 years ( interquartile range , 50 to 66 ) and 51 patients ( 49% ) were male . the most common subtype was acral melanoma ( 33 patients , 31.7% ) , 27 patients ( 26% ) had mucosal melanoma , and 27 patients ( 26% ) had skin melanoma . all 104 patients had ajcc stage iv melanoma , and the majority of patients were stage m1c ( 76% ) . sixty - two patients ( 65.3% ) had elevated baseline ldh and 20 patients ( 19.2% ) had nlr 5 . braf and c - kit mutations were evaluated in 87 and 54 patients , respectively , and the incidence of braf and c - kit mutations was 18.4% and 13.0% , respectively . approximately 40% of patients received ipilimumab as first line treatment , and 28% patients had received at least three prior systemic therapies , median number of prior systemic treatments was two ( range , 1 to 5 ) . sixty - nine patients ( 66.3% ) received all four doses of ipilimumab , and nine patients received three doses , 11 patients received two doses , and 15 patients received one dose . of all patients , 68 patients ( 65.4% ) experienced treatment related aes of any grade ( table 2 ) , and the most common toxicities were pruritus ( 31.7% ) and skin rash ( 22.1% ) . the majority of treatment related aes were grade 1 or 2 , and were generally manageable and reversible without sequelae . ten patients ( 9.6% ) experienced grade 3 toxicities , and seven patients experienced treatment related aes , and three cases were considered to be disease related events . one patient suffered from grade 3 fatigue and grade 2 nausea after two cycles of ipilimumab and brain metastasis was detected on brain magnetic resonance imaging . one patient developed grade 3 elevation of aspartate aminotransferase ( ast)/alanine aminotransferase ( alt ) after three cycles of ipilimumab and showed progression of liver metastasis according to recist ver . one patient complained of grade 3 generalized pain , which improved to grade 1 after morphine treatment . no patient experienced grade 4 or 5 aes , and there were no immune response related serious aes . four patients ( 3.8% ) showed complete response ( cr ) and five patients ( 4.8% ) showed a partial response ( pr ) by recist . the overall response rate ( orr = cr+pr ) and disease control rate ( dcr = cr+pr+stable disease ) was 8.6% and 29.8% , respectively ( table 3 ) . the median follow - up duration was 7.1 months ( 95% confidential interval [ ci ] , 5.96 to 8.30 ) , and 85 patients ( 81.7% ) had progression of disease and 30 patients ( 28.8% ) had died by the time of this analysis . the median pfs was 2.73 months ( 95% ci , 2.67 to 2.85 ) in all patients ( fig . 1a ) , and significantly longer survival was observed for patients with disease control ( cr+pr+stable disease ) compared to those with progressed disease ( pd ) ( median pfs , 5.4 months vs. 2.2 months ; p < 0.001 ) ( fig . 1b and c ) . there was no difference in orr , dcr , pfs , or os according to melanoma subtypes ( fig . pretreatment median anc was significantly higher in patients with pd compared to those who achieved disease control ( 4,017 cells/l vs. 3,183 cells/l , p=0.016 ) , while pretreatment median alc was significantly lower in patients with pd compared to those who achieved disease control ( 1,530 cells/l vs. 1,833 cells/l , p=0.049 ) . when anc and alc were incorporated in the nlr , significantly higher nlr was observed in patients who experienced pd than in those with disease control ( 2.63 vs. 1.90 , p=0.003 ) ( table 4 ) . in addition , we found that pfs and os were significantly longer in patients with low nlr compared to those with high nlr ( median pfs , 2.8 vs. 1.3 months ; p < 0.001 and median os , not reached vs. 2.6 months ; p < 0.001 ) ( fig . 1f and g ) . in univariate analysis between baseline characteristics and survival , ecog ps , ajcc m stage , liver metastasis , and nlr showed significant association with pfs , and ecog ps and nlr showed significant association with os . there was no difference in pfs and os according to the presence of brain metastasis , braf mutation , c - kitmutation , and ldh elevation before treatment , or the number of previous therapies . in multivariate analysis , ecog ps , liver metastasis , and nlr were independent prognostic factors for pfs , and ecog ps and liver metastasis were independent prognostic factors for os ( table 5 ) . for selection of patients likely to benefit from ipilimumab , the patients were divided into three groups based on independent prognostic factors for pfs and os , respectively . patients without adverse factors had longer pfs and os than patients with adverse factors ( fig . ipilimumab improved survival in several clinical trials and many eaps in pretreated and treatment - nave patients with advanced melanoma . however , due to low disease incidence , clinical trials and eaps with ipilimumab have excluded asian patients with advanced melanoma , therefore efficacy and safety data on ipilimumab in the asian patient population has been limited . we report outcomes with ipilimumab in a real - world routine clinical setting in asian patients with different melanoma subtypes compared to western patients . although the median duration of follow - up was 7 months , ipilimumab showed consistent efficacy compared to western patients with advanced melanoma . it is the fifth most common cancer in the united states ; however , its incidence is relatively rare among africans , hispanics , and asians . the histologic subtype also differs , with the most common histology being superficial spreading melanoma in whites and acral lentiginous melanoma among blacks . according to recent data , the most common primary site in asians [ 9 - 12 ] was acral lentiginous melanoma . mucosal melanoma is the second most common primary site in patients in china and korea , although it is extremely rare in caucasians . in patients from western countries , there were no reported differences in efficacy of ipilimumab in patients with primary acral and mucosal melanoma compared with cutaneous melanoma . in the current study , the most common subtype was acral and mucosal melanoma ( 57.7% ) with no survival differences according to subtypes . although the follow up duration was not long , the median os was not yet reached and the median pfs was 2.73 months ( 95% ci , 2.67 to 2.85 ) . these results are comparable to those of western patients treated with ipilimumab , and the orr of 8.6% and dcr of 29.8% were similar to eap data in western countries . only two small studies of ipilimumab in asians have been reported . in a prospective phase ii trial in 20 previously untreated japanese patients , the second study was an eap study in 31 previously treated taiwanese patients , which reported a pfs of 4.1 months . therefore , ipilimumab is a feasible treatment option for asian patients with metastatic melanoma . for identification of predictive markers of efficacy for ipilimumab several blood parameters , including ldh , anc , alc , nlr , and c - reactive protein , have been suggested as associated markers with efficacy of ipilimumab [ 8,15,19,22 - 24 ] . in univariate analysis , ecog ps , ajcc m stage , liver metastasis , and nlr showed significant association with pfs , and ecog ps and nlr showed significant association with os . there was no difference in pfs and os according to brain metastasis or the number of previous therapies . in multivariate analysis , ecog ps , liver metastasis , and nlr were independent prognostic factors for pfs and ecog ps and liver metastasis were independent prognostic factors for os . in addition , patients without adverse factors had longer survival than those with adverse factors . therefore , ipilimumab could be administered in patients with good ps , lower nlr , and no liver metastasis . the safety of ipilimumab in the current study was similar to that reported in patients with cutaneous melanoma . there were no treatment - related deaths or withdrawal from the study due to drug - related aes . the most frequent toxicities were immune - related skin toxicities . despite development of colitis in seven patients ( 6.7% ) , there were no grade 3 or higher severe cases , and there was no immune related hepatitis . one patient with multiple liver metastases had elevated ast / alt of grade 3 after two cycles of ipilimumab , and computed tomography scan showed a further increase in size and numerous liver metastatic nodules and masses , replacing almost the entire liver . therefore , we considered that the hepatitis was related to disease progression , not ipilimumab . twenty - two patients ( 20.3% ) suffered from fatigue ; however , these patients had poor ps ( ecog ps 1 or 2 ) or heavy tumor burden at the baseline , thus this symptom could be disease - related . there are several limitations in this study , which are common in retrospective cohort studies , including potential patient selection bias , lack of strict methods of timing of assessments , and some missing data of toxicity profiles . among 104 patients , 69 patients ( 66.4% ) received all four doses of ipilimumab , and 15 patients completed only one treatment cycle . the most common reason for discontinuation of ipilimumab was rapid disease progression , because some patients with severe tumor burden who did not have other treatment options had to wait for approval of the npp before receiving treatment with ipilimumab . despite these limitations , because the npp population included patients who would have been excluded from a clinical trial due to their poor medical condition , our result may be more representative of advanced melanoma patients in the clinical setting . in the korean melanoma patients , ipilimumab showed a similar efficacy and tolerability compared with western patients , regardless of subtypes . all subtypes should benefit from ipilimumab with consideration of ps , liver metastasis , and nlr . the current npp cohort in korean patients with advanced melanoma showed that ipilimumab monotherapy had a manageable safety profile and antitumor activity . these findings are consistent with the safety and activity profile of ipilimumab in asian patients who had mainly acral and mucosal melanoma compared to patients with cutaneous melanoma from western studies .

purposeipilimumab improves survival in advanced melanoma patients . however , the efficacy and safety of ipilimumab has not been evaluated in asian melanoma patients with a high frequency of mucosal and acral melanoma subtypes.materials and methodsadvanced melanoma patients treated with 3 mg / kg ipilimumab in a korean multicenter named - patient program ( npp ) were evaluated between september 2014 and july 2015 . baseline characteristics and blood parameters including neutrophil to lymphocyte ratio ( nlr ) were assessed , and outcome and adverse events were evaluated according to subtypes.resultsa total of 104 advanced melanoma patients were treated . the primary sites were acral ( 31.7% ) , mucosal ( 26% ) , cutaneous ( 26% ) , uveal ( 9.6% ) , and unknown ( 6.7% ) . sixty - eight patients ( 65.4% ) experienced adverse events , and the most common toxicity was skin rash ( 22.1% ) , 10 patients ( 9.6% ) experienced adverse events of grade 3 or higher . the median progression - free survival ( pfs ) was 2.73 months ( 95% confidence interval , 2.67 to 2.85 ) , and there was no difference in pfs according to subtypes . poor performance status , liver metastasis , and nlr ( 5 ) were independent poor prognostic factors by multivariate analysis.conclusionin the korean npp cohort , ipilimumab showed similar efficacy and tolerability compared to western patients , regardless of subtypes . all subtypes should benefit from ipilimumab with consideration of performance status , liver metastasis , and nlr .

cerebrovascular accident ( stroke ) is one of the main causes of disability and mortality in the developed world1 , 2 . stroke victims experience a number of neurological deficits and disabilities , such as hemiparesis , communication disorder , cognitive impairment and visual - spatial perception disorder3 . moreover , approximately 50 to 60% of stroke victims experience some degree of motor impairment even after completing the standard rehabilitation protocol , and approximately 50% are at least partially dependent on others with regard to activities of daily living3 , 4 . according to wit et al.5 , the ability to walk following a stroke is often impaired due to muscle weakness , spasticity , compromised sensory - motor control and/or the loss of cognitive functions . most hemiparetic patients exhibit motor dysfunction , which affects their ability to walk , leading to an abnormal gait pattern . stroke victims exhibit coordination deficits that persist beyond the rehabilitation process , including altered trunk , pelvis , knee and ankle coordination in the standing position and during gait , leading to a reduction in walking endurance and velocity6 . a number of therapeutic methods are described in the literature for the motor recovery of limbs affected by spasticity , allowing improvements in strength , range of motion and function of patients with hemiparesis7,8,9,10 . equino - varus foot , which is common in these patients , shifts the weight support of the heel to the lateral - plantar surface of the foot , and may cause a loss of balance and a reduction in stride security . ankle - foot orthoses ( afo ) are prescribed to facilitate ankle control in cases of equinus and/or varus foot11 , and they reduce energy expenditure while walking12 . kinematic analysis has been employed for the evaluation of normal and pathological human gait and allows the analysis of spatiotemporal characteristics , such as step length , cadence ( number of steps per minute ) , stride duration and velocity . the spatiotemporal characteristics of gait describe the quantitative aspects of the movement pattern13 . according to leung and moseley14 , changes in step length , duration in the stance and swing phases , and double support time are related to reductions in gait symmetry and gait velocity , leading to the specific gait pattern exhibited by patients with hemiparesis : a relatively shorter step length , a longer stance phase and a shorter swing phase of the affected side . recovering the ability to walk is an important goal of the rehabilitation process for stroke patients . according to gok et al.15 , gait velocity , cadence and step length are diminished in hemiparetic gait and devices , such as an afo , can improve these aspects . an afo is generally prescribed to provide mediolateral stability of the ankle in the stance phase , facilitate gait in the swing phase and support the ankle . however , some researchers hold the view that an afo can prolong dependence on a mechanical device , leading to an increase in muscle disuse , especially the dorsiflexors of the ankle , with a consequent delay in functional recovery14 . the aim of the present study was to conduct a systematic review of the literature to determine the effect of an afo on spatiotemporal gait variables ( cadence and velocity ) of stroke patients . for the development of a systematic review , searches were performed for randomized controlled clinical trials that analyzed the effect of an afo on stroke patients ( acute / chronic ; ischemic / hemorrhagic ) . we searched for studies involving three - dimensional movement analysis of spatiotemporal gait variables ( cadence and velocity ) as well as measures of independent gait over short distances ( 6 and 10 meters ) . searches were carried out of the pubmed ( medline ) , lilacs , scielo and physiotherapy evidence database ( pedro ) databases using the following descriptors : 1 ) stroke ; 2 ) gait ; 3 ) stroke and gait ; 4 ) ankle foot orthosis ; 5 ) stroke and ankle foot orthosis ; and 6 ) spatiotemporal parameters . the papers identified in the initial search were evaluated based on the following inclusion criteria : 1 ) design : controlled , clinical trial ; 2 ) population : stroke patients ; 3 ) intervention : analysis of spatiotemporal variables of gait with an ankle - foot orthosis ; 4 ) control group with different intervention or no intervention ; and 5 ) outcome : improvements in gait velocity or cadence . the randomized controlled clinical trials selected were analyzed for methodological quality using the pedro scale , which has 11 items for the evaluation of internal validity and statistical information of randomized controlled trials . each adequately fulfilled item ( with the exception of item 1 , which addresses external validity ) receives 1 point and contributes to the score wich has a maximum of 10 points . if a paper analyzed was not found in one of the databases , the score was determined by two independent , blinded researchers . the full texts of the selected papers served as the reference and benchmark for the discussion and broadening of the concepts of the issue addressed . meta - analysis was not possible due to the lack of certain data necessary for statistical analysis as well as the use of different outcome measures . what follows is , therefore , a descriptive and comparative analysis of the findings . the initial search of the databases yielded 37 papers , 24 of which did not meet the inclusion criteria . thus , 13 papers were found addressing the use of an afo on hemiparetic gait , specifically improvements in the spatiotemporal variables of gait velocity or cadence ( fig . 1 . flowchart of studies included in review ) . the studies involved a total of 315 participants ( 216 men and 99 women ) , with a mean age of 56.8 years . the etiology of stroke was ischemic in 47 cases and hemorrhagic in 36 cases . a total of 114 patients had left - side hemiparesis and 126 had right - side hemiparesis . it is noteworthy that not all studies reported the type of stroke and prevalence of the affected side . flowchart of studies included in review the initial search of the databases yielded 53 titles addressing the use of an afo on hemiparetic gait . however , some papers did not meet the inclusion criteria and some did not achieve the necessary score on the pedro scale merit inclusion in the present review . other papers addressed alterations in other functional aspects , such as balance or muscle activity , and were therefore not selected . the final sample was made up of 13 papers that achieved a minimum of 3 points on the pedro scale and were therefore considered methodologically adequate ( table 1 table 1 . data on papers included in reviewnumber of papersauthor and year of publicationpedrotype of study1gok et al . 2006183/10clinical trial6bleyenheuft et al . , 2008194/10clinical trial7simons et al . , 2009203/10clinical trial8fatone et al . , 2010235/10clinical trial11chen et al . , 2010113/10clinical trial12erel et al . , 2011246/10clinical trial13de sse et al . , 2011256/10clinical trialand 2 table 2 . scores of papers included in reviewpedro12345678910111213eligibilitynnyyyyyyyyyyyrandomized allocationynyyyyyynyyyyconcealed allocationnnynnnnnnynyysimilar prognosisnyynyyyyynnyyblinded subjectsnnnnnnnnnnnnnblinded therapistsnnnnnnnnnynnnblinded ratersnynnnnnnnnnnnkey outcomesnnynnynnnnnyyintention to treatnnynnnnnnnnnncomparison between groupsyyyynnnyyyyyyprecision and variabilityyyyyyyyyyyyyy3/104/106/103/103/104/103/104/103/105/103/106/106/10y = yes ; n = no ) . in the present review , only outcomes regarding gait velocity and cadence ( number of steps per minute ) were considered . no analysis was performed regarding changes in stride or the step cycle or other aspects related to the benefits of the use of an afo , which have been widely discussed in the literature . the papers included in the present review report significant differences in gait velocity with the use of an afo , but divergent results regarding cadence ( table 3table 3 . , 200315n=12 stroke patientsanalysis of spatiotemporal gait variables , kinetic and kinematics using the 370 vicon system under 3 conditions : without afo , with plastic afo and with metal afo- no significant difference in cadence under any condition ; - significant difference in gait velocity with both afos ( 0.32 m / s without afo vs. 0.37 m / s with plastic afo p<0.05 ; 0.32 m / s without afo vs. 0.41 m / s with metal afo p<0.05)iwata et al . , 200316n=9 stroke patients ( afo group + tone inhibitory bar attached to afo ) and n=8 control group ( afo alone)cadence and gait velocity ( 10-m walk)- gait velocity increased by 13.8% ( p=0.0045 ) in comparison to control group ; - cadence increased by 6.1% ( p=0.0056 ) in comparison to control groupde wit et al . , 20045n=20 chronic stroke patients wearing afo for at least six months10-meter walk test , timed up - and - go ( tug ) test and stairs test performed with and without afostatistically significant mean difference of 4.8 cm / s in gait velocity ( 95% ci : 0.85/8.7 : p=0.02 ) , 3.6 s on tug ( 95% ci : 2.4/4.8 : p=0.000 ) and 8.6 s on stairs test ( 95% ci : 3.1/14.1 : p=0.004 ) favoring afowang et al . , 200517n=42 subjects with short - term hemiparesis ( 6 months ) and 61 subjects with long - term hemiparesis ( 12 months)balance and gait with and without afo ; static and dynamic balance activities evaluated using balance system ; functional balance evaluated using berg scale ; gait velocity and cadence measured during 10-m walk- significant improvement in gait velocity ( 0.58 m / s without afo vs. 0.69 m / s with afo p=0.028 ) and cadence ( 75.06 steps / min without afo vs. 87.26 steps / min with afo p=0.021 ) in group with short - term hemiparesis - no significant effects in group with long - term hemiparesisthijssen et al . , 200618n=27 chronic stroke patientsenergy expenditure , gait velocity and step length under 3 conditions : without afo , immediately after fitting of afo and after 3 weeks of afo usage- significant reduction in energy expenditure and increase in gait velocity immediately after fitting of afo ; - no significant difference in cadence ; - no significant changes in gait pattern after 3 weeks of familiarization.bleyenheuft et al . , 200819n=10 patients with chronic hemiparesis10-meter walk test , followed by instrumented gait test on treadmill under three conditions : without afo , with rigid afo and with dynamic chignon orthosismechanical work improved equally with both orthoses . chignon orthosis led to significant improvement in gait velocity in comparison to no orthosis ( 0.81 m / s vs. 0.64 m / s p<0.001 ) and segment kinematics of the ankle ; no significant differences in cadence ( p=0.79)simons et al . , 200920n=20 stroke patientsberg balance scale , timed up - and - go test , timed balance test , 10-m walk test and functional ambulation categories with and without afosignificant improvements in majority of functional tests with afo ; - significant difference in 10-m walk test ( 0.58 0.24 m / s with afo vs. 0.46 0.21 m / s without afo p=0.000)fatone et al . , 200921n=16 stroke patients ( mean age : 53.2 years ) wearing afo ; n=12 healthy individuals ( mean age : 57.1 years)gait analysis : kinetics , kinematics and gait variables- no significant difference in gait velocity between groups ( p=0.095)hiroaki et al . , 200922n=16 stroke patients with hemparesisspatiotemporal gait analysis with and without use of plastic afo walking on paper catwalk- significant increases in gait velocity , cadence , step length and stride length on affected and non - affected sides with use of afo in comparison to non - use ; - gait velocity : 18.1 m / s without afo vs. 22.9 m / s with afo ( p=0.0032 ) ; - cadence : 66.8 steps / min without afo vs. 73.3 steps / min with afo ( p=0.015)lewallen et al . , 201023n=13 chronic stroke patientsspatiotemporal gait analysis evaluated with rigid afo , articulated afo and without afo ( shoes alone)no significant differences among groupschen et al . , 201011n=14 stroke patients ; n=11 healthy individualsgait analysis : kinetics , kinematics and gait variables under three conditions ( without afo , with anterior afo and with posterior afo)- no significant differences in gait velocity , step length or duration of cycle in comparison to control grouperel et al . , 201124n=28 stroke patients with chronic hemiparesis allocated to an experimental group ( eg ) or a control group ( cg)both groups initially evaluated in shoes alone ; experimental group evaluated after three months of dynamic afo usage ; functional tests : functional reach , timed up - and - go test , timed up stairs , timed down stairs , gait velocity and physiological cost index- no significant differences between groups regarding variables analyzed ; - after 3 months , significant differences in timed up stairs ( 12 s in eg vs. 15 s in cg p=0.040 ) and gait velocity ( 0.99 m / s in eg vs. 0.72 m / s in cg p=0.001 ) ; - no significant differences regarding functional reach , timed up - and - go or timed down stairs ( p>0.05)de sse et al . , 201125n=28 stroke patients , 13 wearing chignon afo and 15 wearing standard afo ( control)10-meter walk test ( gait velocity ) and kinematic gait variables- significant increase in gait velocity in chignon group over control group immediately ( 27.2 36% vs. 0.8 17% p=0.006 ) , after 30 days ( 39.9 19% vs. 7.5 17% p=0.0004 ) and after 90 days ( 44.6 27% vs. 17.1 0.3% p=0.04 ) ) . according to verma et al.26 , impaired gait following a stroke is one of the most widely investigated aspects of neurological disorders , and the recovery of gait is one of the main goals of the rehabilitation process . a number of treatment methods have been developed to restore the walking ability of stroke patients . they include the use of an ankle - foot orthosis ( afo ) , which restricts the movement of the ankle , especially excessive plantar flexion , thereby enhancing stability in the stance phase and allowing greater freedom in the swing phase of the gait cycle14 , 27,28,29 . mean gait velocity of healthy individuals is around 1.3 m / s , but ranges from 0.23 to 0.73 m / s among individuals with hemiparesis26 . in the majority of studies analyzed , the use of a rigid or articulated afo offered important benefits with regard to gait velocity of stroke patients , regardless of the material with which the device was made . an afo seems to exert a positive influence on the joint alignment of the affected lower limb , with improvements in cadence and gait velocity . while these variables alone do not represent improved gait stability , the analysis of such aspects allows a better practical understanding of the effects of physical therapy and improvements in the motor skills of stroke patients30 . the findings of the present review revealed divergence in the results of different studies regarding changes in cadence with the use of an afo . while iwata et al.16 , wang et al.17 and hiroaki et al.22 report improvements in cadence , chen et al.11 , gok et al.15 , thijssen et al.18 and bleyenheuft et al.19 found no significant change in this variable . however , the same was true of gait velocity , as the majority of studies report significant improvements in gait velocity with the use of an afo5 , 15,16,17,18,19,20 , 22 , 24 , 25 . the exception was the study by lewallen et al.23 , but the authors compared gait velocity between use of rigid and articulated orthoses . gk et al.15 compared plastic and metal afos and found a significant increase in gait velocity in individuals with hemiparesis with both types of orthosis , but no significant changes in cadence . iwata et al.16 analyzed the use of an inhibitory bar attached to an afo worn by patients with hemiparesis and found an increase in gait velocity of approximately 13% as well as a significant increase in cadence ( 6% ) in comparison to a control group . bleyenheuft et al.19 compared gait velocity and cadence in the 10-meter walk test between a group wearing a rigid afo and another group wearing a chignon afo , and found significant differences in gait velocity , but no changes in cadence . in a similar study , de sse et al.25 compared the chignon afo to a standard polypropylene afo and found significant differences favoring the former immediately following fitting of the orthosis as well as after 30 and 90 days of usage . in contrast to the above - mentioned findings , chen et al.11 found no significant differences in spatiotemporal gait variables of stroke patients under three different conditions : 1 ) barefoot ; 2 ) wearing an anterior afo ; and 3 ) wearing a posterior afo . likewise , lewallen et al.23 found no significant differences in spatiotemporal gait variables of stroke patients wearing a rigid afo and those wearing an articulated afo . regarding the duration of use and adaptation to orthoses , a tendency toward an improvement in gait velocity and cadence was found with increased usage in the studies carried out by erel et al.24 and de sse et al25 . however , thijssen et al.18 only found an immediate increase in gait velocity with the use of an afo , there being no changes in either gait velocity or cadence after three weeks of familiarization with the orthosis . in the comparisons of with and without the use of an afo , or between an afo group and a control group ( without use of afo ) , significant improvements in gait velocity were found15 , 17 , 20 , 22 , 24 , and significant improvements in cadence were found in the studies by wang et al.17 and hiroaki et al22 . in a retrospective study by esquinazi et al.31 in a three - dimensional gait lab , moss rehabilitation engineering center , in the spatiotemporal aspects of gait of 42 patients were evaluated and a significant improvement in both the speed and cadence of gait of the patients using afo orthoses : ( 0.31 m / s ) speed with afo ( 0.41 m / s ) , p<0.001 ; and cadence without afo ( 63.3 steps / min ) cadence with afo ( 68.8 steps / min ) , p<0.001 . all studies11 , 21 comparing stroke patients wearing an afo with healthy individuals found no statistically significant differences between the groups in spatiotemporal gait variables , kinetics or kinematics . these findings demonstrate the benefits of an afo with respect to functional improvements in gait , with the values approaching those found in healthy individuals . based on the findings , all types of afo resulted in a significant improvement in gait velocity compared to a control group without the use of an afo . however , divergent results were reported for cadence with and without the use of an afo , as some studies report an improvement in this variable and others report no significant improvement . since cadence is the number of steps take in a certain time , these patients showed significant improvements in gait speed without changing the number of steps per minute . the main limitations encountered in this study were related to the nature of the data . most studies used ratings for immediate use , or afo types of with and without the same short - term , with neither a long - term analysis nor a convenience sample , without the sample calculation of sample size . this review notes only data related to speed and cadence of gait patients with stroke . although this study is relevant in some aspects , there are still questions of a primarily clinical character that need to be answered , such as the best type of afo to prescribe , the correct time to prescribe , and how long it should be used , among others .

[ purpose ] the aim of the present study was to analyze the effect of an ankle - foot orthosis on gait variables ( velocity and cadence ) of stroke patients . to do this , a systematic review was conducted of four databases . [ subjects and methods ] the papers identified were evaluated based on the following inclusion criteria : 1 ) design : controlled , clinical trial ; 2 ) population : stroke patients ; 3 ) intervention : analysis of spatiotemporal variables of gait with an ankle - foot orthosis ; 4 ) control group with different intervention or no intervention ; and 5 ) outcome : improvement in gait velocity or cadence . [ results ] thirteen controlled trials addressing the effect of an ankle - foot orthosis on gait variables of stroke patients were found . they exhibited methodological quality of 3 or more points on the pedro scale . [ conclusion ] while the findings suggest the benefits of an afo regarding gait velocity , the impact of this type of orthosis on cadence remains inconclusive . thus , there is a need for further well - designed randomized , controlled , clinical trials to establish better scientific evidence for the effects of afo usage on gait variables of stroke patients .

montmorency on mahelab root ) orchard in santaquin , utah ( gps : 395928.84n , 1114821.20w ) . within the orchard , two nest - box distributions ( treatments ; described below ) were established across six 1.2-ha plots ( treatments were paired across three geographically distinct blocks ; fig . because the typical foraging range of o. lignaria is about 60 m ( rust 1974 ) radius from the nest site , each treatment was separated by at least 75 m to minimize bee drift into neighboring experimental plots . bees were provided by watts bees ( bothell , wa ) in spring 2016 as loose - cell , overwintering adults in cocoons . they were stored at the usda - ars pollinating insects research unit in darkness at 4c until their release . at the onset of bloom ( 15 april 2016 ) , 825 female and 1,230 male just - emerging o. lignaria were released at the center of each plot from styrofoam and plastic emergence boxes ( 40 25 20 cm ) , situated 12 cm above ground . the emergence boxes each had a small , 2-cm exit hole at the bottom from which adult bees could crawl through and fly away . plastic , corrugated nest boxes were distributed in one of two different layouts ( or treatments ) within each experimental block concurrently with o. lignaria bee releases : ( 1 ) a mail tote / satellite ( mts ) treatment and ( 2 ) a uniform ( uf ) treatment ( fig . the mts treatment consisted of a large , centrally located mail tote ( described in cane 2006 ; fig . this layout included four small corner satellite nest boxes as well ( boxes described in artz et al . 2b ) , each containing 100 cardboard nest tubes ( 15.3 cm in length 7 mm inner diameter , each lined with paper straw inserts ) and situated 86 m from the central tote . the uf treatment consisted of 10 small nest boxes , distributed evenly across two central rows within each replicate and containing 100 nesting cavities each ( same as the previously described satellite boxes ; fig . all nest boxes were blue in color and installed at breast height with southeast - facing entrances . both treatments provided the same number of cavities ( n = 1000 ) to o. lignaria females in this experiment . a patented spray - on attractant ( pitts - singer et al . 2015 ) , which was identified from o. lignaria cocoons , was applied at a rate of 100 cocoon equivalents ( 105.77 g active ingredient = one cocoon equivalent ) per nest box by spritzing it directly onto the outer ends of the cardboard tubes . composed of a free fatty acid mixed into an ethyl acetate solvent , this attractant has been demonstrated to increase nesting when sprayed onto vacant cardboard tubes ( pitts - singer et al . dashed lines outline the uniform ( uf ) nest box distribution treatment ( n = 3 ) . solid lines outline the mts treatment ( n = 3 ) . dashed lines outline the uniform ( uf ) nest box distribution treatment ( n = 3 ) . however , o. lignaria were permitted to mate and provision their nests in the orchard through post - bloom until 17 may 2016 so that delicate , immature larvae would be able to develop to less vulnerable stages prior to their removal ( manipulation of early instar o. lignaria can dislodge them from their provision mass , resulting in mortality due to starvation ; bosch and kemp 2001 ) . no pesticide applications were made while the bees were nesting , and nest boxes were removed from the orchards prior to application of the first post - bloom nutritional spray ( 19 may 2016 ) . in addition to introduced o. lignaria , honey bees were present in the orchard at a standard rate of one hive per acre for the duration of bloom . upon their removal in mid - may , nest boxes were disassembled and the cardboard tubes were evaluated immediately for occupancy by visual inspection . fully or partially completed nests were determined to be occupied , and occupancy was compared between nest box distributions . starting 9 june 2016 , the nests with developing bees in cocoons from each treatment were x - radiographed to determine the number of successfully completed live cells ( or healthy cocoons ) , and offspring sex ratio ( by visual inspection of nest position and size ; fig . 1 ) . statistical comparisons between treatments were conducted using one - way anovas , blocked by replicate ( jmp , sas institute 2015 ) . despite the short bloom time of cherries ( about 2 wk in this orchard ) , o. lignaria nested successfully and in large numbers . regardless of treatment , reproduction of female o. lignaria surpassed the initial number of bees released into the orchard , resulting in a 1.6-fold increase in bees across treatments . in total , 19,336 live cells were created from the original 5,000 females released into the orchards . overall , an average 1.11 male to female sex ratio was obtained , which did not vary significantly by treatment ( f = 0.78 ; df = 5 ; p = 0.47 ) . while cavity occupancy did not reveal significantly higher nesting between treatments ( f = 3.71 ; df = 2 ; p = 0.194 ) , we did observe significantly more live cells ( f = 18.79 ; df = 1 ; p = 0.0494 ) produced in uf treatment over the mts treatment ( table 1 ) . this discrepancy results from a combination of a slightly higher number of cells per nest in the uf treatment ( 4.73 average cells per nest ) over the mts treatment ( 4.34 cells per nest ) and numerically higher occupancy in uf ( table 1 ) . block did not affect nesting occupancy ( f = 1.34 ; df = 2 ; p = 0.43 ) or live cell production ( f = 13.94 ; df = 2 ; p = 0.067 ) . in cherries , it appears best to distribute nest boxes uniformly throughout the orchard rather than rely on a central nesting location with satellite boxes on orchard block margins . table 1.mean ( se ) nest occupancy and live cell count of o. lignaria reproduction by treatmentnmean nest occupancy , % semean live cells , seaverage cells per nestuniform treatment380.6 8.23,812.0 562.54.73mts treatment360.7 2.12,633.3 501.64.34percent occupancy is a total percentage of all nest straws available at each site ( n = 1,000 per site ) . mean ( se ) nest occupancy and live cell count of o. lignaria reproduction by treatment percent occupancy is a total percentage of all nest straws available at each site ( n = 1,000 per site ) . previous attempts to incorporate o. lignaria as cherry orchard pollinators have reported extremely successful nesting of in - orchard bee populations and significantly increased cherry yield where they are used ( bosch et al . 2006 ) , although these earlier trials were limited to one small 1.4-ha orchard over several years . in addition , this smaller orchard received limited chemical inputs and was located in a semi - agricultural area . to our knowledge , our study is the first scientific report of successful o. lignaria propagation in conventional , commercially managed tart cherry orchards in utah , suggesting that it could be an excellent target crop for future o. lignaria application . as with many tart cherry varieties that are commercially produced , montmorency cherries are not entirely reliant on insect pollination , as they are partially self - compatible and capable of setting fruit without cross - pollination . however , this variety , along with many others that are grown commercially in the united states do benefit substantially from bee visitation ( lansari and iezzoni 1990 ) , which is why honey bee hives are frequently interspersed throughout these orchards . 1999 ) stocking rate of one hive per acre , o. lignaria may be a viable alternative or complimentary candidate for providing pollination services in this system . however , due to the sensitivity of o. lignaria to pesticide exposure ( e.g. , biddinger et al . 2013 , artz and pitts - singer 2015 ) and the practicalities concerning the timing of nest removal from orchards , the use of this pollinator requires care in cases where pesticides are routinely applied during or just after bloom . because the native range of o. lignaria overlaps with our study site , there is a possibility that some of the bees nesting in these orchards were from local , wild populations . however , due to the intensive management strategies employed in conventional commercial orchards ( including frequent agrochemical applications ) and short cherry bloom window , it is improbable that the local environment would be capable of sustaining large , stable populations of o. lignaria over time . on the day of nest removal from the orchard , no adult o. lignaria were observed . this is likely a consequence of limited available forage to the bees immediately following bloom . in the presence of alternative floral resources ( combined with careful pesticide use ) , the foraging window for o. lignaria can extend beyond crop bloom , which has been shown to have positive impacts on bee propagation ( sheffield et al . presently , the greatest challenge in using o. lignaria for commercial pollination is their limited supply . o. lignaria are primarily obtained by wild trapping , in which trappers place artificial nest cavities in regions where they are known to aggregate ( predominantly in high elevation areas of utah , idaho , and washington ) . at the end of nesting , wild - caught bees are sorted and processed for storage at 35c over the winter months as adults in cocoons . subsequently , they are sold and distributed to local farm stores or directly to orchardists . management practices to date are labor - intensive and expensive , which results in impractically high retail prices of o. lignaria for pollination ( typical 2016 costs were upwards of $ 1 per female usd ; j. watts , personal communication ) on a commercial scale . with a suggested stocking rate of 250 females per acre ( 618 females per ha ) ( bosch and kemp 2001 ) , commercial o. lignaria pollination will only be economically feasible if sustainable year - to - year reproduction within orchards is achieved . this study provides the kind of nuanced detail in o. lignaria management needed for improving bmps that will lead to sustainable o. lignaria reproduction within tart cherry orchards . overall , results obtained from this study are provocative , and further evaluation to examine pollination efficacy of o. lignaria in commercial cherry orchards is needed .

the blue orchard bee , osmia lignaria ( say ) , is a solitary bee that is an excellent pollinator of tree fruit orchards . due to the annual rising costs of honey bee hive rentals , many orchardists are eager to develop management tools and practices to support o. lignaria as an alternative pollinator . establishing o. lignaria pollination as a sustainable industry requires careful consideration of both bee and orchard management . here , we test the effect of artificial nest box distribution on in - orchard propagation of o. lignaria in utah commercial tart cherry orchards . two nest box distributions were compared across three paired , 1.2-ha plots . one distribution , traditionally employed by o. lignaria consultants , included a centrally located tote for mass - nesting with smaller , surrounding satellite nest boxes at orchard margins . the other distribution was composed of smaller , more equally distributed nest boxes throughout the 1.2-ha plots . significantly higher propagation of o. lignaria was observed in the latter nest box distribution , although all treatments resulted in bee return exceeding the number of bees initially released . these findings provide support for the use of o. lignaria in tart cherry orchards , and demonstrate how simple changes to bee set - up and management can influence propagation efforts .

sigma factors are a class of proteins constituting essential dissociable subunits that confer sequence - specific dna binding properties to rna polymerase ( rnap ) . within the rnap holoenzyme , sigma factors provide promoter recognition specificity to the polymerase and contribute to dna strand separation , then they dissociate from rnap core enzyme and transcription initiation follows . besides the vegetative sigma factor , which recognizes the standard promoter , many bacteria possess alternative sigma factors that confer altered promoter specificity upon rnap . they partially and/or temporarily replace the vegetative sigma factor and are thus crucial for many stress responses . sigma factors can be divided into two structurally unrelated families : the and the families . the family consists of primary sigma factors , as well as related alternative sigma factors [ 3 , 4 ] . these alternative sigma factors are further classified by the physiological processes that they control , for example , stress response and flagella biosynthesis [ 3 , 4 ] . the extracytoplasmic function factors ( ecf ) belong to the family of sigma factors , but are phylogenetically distinct . many ecf members are responsible for sensing and reacting to signals from the extracytoplasmic environment [ 35 ] . the family proteins generally contain four regions , region 1 to region 4 , but only region 2 and 4 are well conserved in all members [ 3 , 4 ] . rpos or , a member of the alternative sigma factors subfamily , is important for survival under various stress conditions including starvation , oxidative stress , osmotic shock , extreme temperature , low ph ( acidic stress ) , and exposure to uv ( dna damage ) [ 615 ] . v. alginolyticus is an opportunistic pathogen of marine fish , shellfish , as well as human . on the coast of southern china , v. alginolyticus is the dominant vibrio species found in the seawater and in farmed marine animals . v. alginolyticus zj-51 , isolated from diseased grouper , has shown colony phase variation between opaque / rugose ( op , opaque variant ) and translucent / smooth ( tr , translucent variant ) . the two variants exhibit different capability to form biofilms and to express genes involved in polysaccharide biosynthesis , flagellar synthesis , and the ai-2 quorum - sensing system . recently , genome sequences revealed that in addition to rpos , an rpos - like sigma factor was found in v. alginolyticus 12g01 . amino acid 10 to 277 contains an rpos - proteo region with three conserved sigma 70 regions . based on this , we wanted to identify this protein in v. alginolyticus zj-51 and study whether it has a role in biofilm formation and stress response in the two colony phase variants . zj - o is the opaque / rugose variant of the wild type zj-51 and zj - t is the translucent / smooth variant derived from zj - o . bacteria were routinely grown in trypticase soy broth ( tsb ) ( becton , dickinson and company ) , or 1.5% tsb agar plate ( tsa ) at 30c , with the following antibiotics : 100 g / ml ampicillin ( ap ) and 10 g / ml chloramphenicol ( cm ) . when necessary , luria - bertani ( lb ) and 1.5% lb agar plate ( la ) were used . biofilm growth medium ( bf medium ) contains minimal 1 m63 salts supplemented with 1% nacl , 1.5% casamino acids , 1% glucose , 1 mm mgso4 , and 10 g thiamine per liter . for selection of conjugants , thiosulfate citrate bile salts sucrose agar ( tcbs ) ( huankai microbial sci.&tech . , gd , china ) was used with cm at 10 g / ml . for sucrose selection , tsa and tsb containing 10% sucrose were used . to clone rpox from v. alginolyticus zj-51 , a pair of primers derived from the rpos - like sigma factor gene ( nz_aaps01000024 ) in v. alginolyticus 12g01 were designed as follows : rpox - i ( 5-ccccacctgctcacg-3 ) and rpox - ii ( 5-gagacacaagcgcaa-3 ) . pcr was performed at 95c for 5 min , 35 cycles at 94c for 30 sec , 30 sec at 55c , 1 min at 72c , followed by incubation at 72c for 10 min . a 1.199-bp dna fragment from the v. alginolyticus zj-51 genomic dna was amplified and sequenced . nucleotide and deduced protein sequences were analyzed with blast and clustal ( version 1.81 ) . to create null mutants of rpox in both op and tr backgrounds , an in - frame deletion was made by allelic exchanges as described previously . the first round of pcr was carried out to generate two fragments flanking the coding region of rpox . the first fragment was amplified using primers rpox - a ( atagatctccccacctgctcacg ) , which contains a sali site at the 5-end and rpox - b ( gggctaaaaatcttatgacagttcatcgt ) ; rpox - d ( atgtcgactcgagacacaagcgcaa ) , which contains a bglii site at the 5-end and rpox - c ( acggatgaactgtcataagatttttagccc ) were used to amplify the second fragment . both fragments contain a 10-bp overlapping sequence and were used as templates for the second pcr using the primers rpox - a and rpox - d . the resulting fragment was ligated into the suicide vector pdm4 that had been cut with sali and bglii to generate the plasmid pdmrpox . the recombinant suicide plasmid was then conjugated into v. alginolyticus zj - o and zj - t . conjugants were selected on the tcbs plates containing 10 g / ml cm , followed by sucrose selection . the in - frame deletions , where the wild - type coding sequence of rpox was completely removed by allelic exchange , were confirmed by sequencing . to construct complemented strains , the open reading frame ( orf ) of rpox and flanking dna cm- and ap - resistant transconjugants were selected , and the presence of the plasmid was confirmed by pcr analysis and sequencing . overnight cultures of the bacteria were inoculated in tsb with the initial od600 of 0.01 . strains of the op background were incubated statically in lb at 30c for 24 hours as described before . colony phase was observed and the variation rate was calculated as percentage of tr cells in the total number of cells . briefly , biofilms formed on sterilized glass coverslips that were placed in 50 ml centrifuge tubes containing 10 ml bf medium . after 6-hour statical incubation , the coverslips were removed and biofilm structures on the coverslip were observed with sem s-3400n . bacterial viability was quantitatively determined using the live / dead baclight bacterial viability kit for microscopy and quantitative assay ( molecular probes , or ) according to the manufacture instruction . fluorescence output was measured with a victor 3 multiple label microplate reader ( pe , usa ) with the excitation and emission wavelengths of 485 nm and 535 nm , respectively . before exposure to various stresses , overnight cultures were centrifuged and washed twice with 0.9% nacl . then the equal cell numbers , corresponding to an od600 of 0.5/ml , were diluted in 0.9% nacl containing either 18% ethanol ( ethanol challenge ) or 30 mm h2o2 ( oxidative challenge ) and 2.5 m nacl ( osmotic challenge ) . incubations were carried out at 30c . the heat challenge was performed in pre - warmed ( 45c ) 0.9% nacl . at selected time points , samples were withdrawn , centrifuged , and resuspended in the same volume of 0.9% nacl , then fluorescence was measured . a fragment of 1.199 bp in length was amplified from chromosomal dna of v. alginolyticus zj-51 using a pair of primers derived from the sequence of the v. alginolyticus 12g01 rpos - like gene . the sequence contained the orf of the rpox gene ( 840 bp ) and the flanking regions . blastn analysis showed a 73% identity of the dna fragment to the rpos - like gene of vibrio splendidus lgp329 ( fm954973 ) , in addition to a 98% identity to that of v. alginolyticus 12g01 ( nz_aaps01000024 ) . the sequence of the deduced protein was the same as the rpos - like sigma factor of v. alginolyticus 12g01 and showed 80% identity to the rpos - like sigma factor of vibrio sp . med222 and 79% identity to the rpos - like sigma factor of v. splendidus lgp32 . interestingly , rpoq of v. fischeri es114 and rpos of v. alginolyticus vib283 were also picked up in the search with 40% and 36% identity , respectively ( figure 1 ) . the predicted protein sequence of rpox contains three conserved domains : ( i ) sigma 70 region 1.2 ( amino acid 10 to 45 ) , ( ii ) sigma 70 region 2 ( amino acid 48 to 108 ) , and ( iii ) sigma region 4 ( amino acid 219 to 271 ) ( figure 2 ) . protein sequence alignment revealed that the reason for a 40% identity to the rpos of v. alginolyticus was due to the second conserved region , which belongs to the sigma 70 region 2 superfamily . in contrast to rpos and rpoq , rpox does not contain the sigma 70 region 3 , but it possesses sigma 70 region 1.2 , which is absent in the rpos - like sigma factors of vibrio sp . med222 and v. splendidus lgp32 ( figure 2 ) . bacterial growth was investigated for wild types , the rpox mutants , and the complemented strains . all the mutants showed the same growth rate as the wild types in both op and tr backgrounds ( figure 3 ) . deletion of rpox does not interfere with the cell growth . in the previous study , transition from tr to op has never been observed , thus variation from op to tr colony was examined only in the op background ( zj - o , rpox - o , and crpox - o ) . after incubation in lb without shaking for 24 hours , the switching rates from op to tr were 26.00% ( zj - o ) , 27.12% ( rpox - o ) , and 26.78% ( crpox - o ) , respectively ( figure 4 ) . biofilm formation was investigated for wild types , the rpox mutants , and the complemented strains . as shown in figure 5(a ) , all strains exhibited similar biofilm formation kinetics . the amount of attached cells peaked when the bacteria were in late exponential growth phase ( 6 hours ) . the peak was followed by a dramatic decrease to a basal level of attached cells . however , compared to their parental strains , rpox mutants in both backgrounds were reduced in their maximal attached biomass . the structure of the biofilms was observed using sem . as shown in figure 5(b ) , zj - o and rpox - o attached to the surface with single cells , but cells of zj - t and rpox - t were stacked together and formed structures resembling microcolonies . surprisingly , the rpox - t cell had a pilus - like structure on the cell surface , which is absent in the other three strains . the rpos mutants in vibrio species have been shown to be more susceptible to environmental stresses than the wild type [ 9 , 1215 ] . therefore we examined the effect of the rpox deletion in v. alginolyticus zj - o and zj - t on the survival of these cells under various stress conditions . as shown in figure 6(a ) , when challenged with heat ( 45c ) for 90 minutes , more than 60% of the cells had died in all strains . this suggests that rpox is not involved in the heat shock response in v. alginolyticus . when exposed to hydrogen peroxide ( 30 mm h2o2 ) , the rpox mutant in the op background was slightly better than the wild type to survive the first 60 minutes , but after 90 minutes , no differences could be observed . rpox in the tr background resulted in cells more sensitive to hydrogen peroxide , compared to wild type . interestingly , when exposed to 18% ethanol and/or to 2.5 m nacl ( hyperosmolarity ) , the rpox in the op background showed decreased survival rates than the wild type and complemented strain . however , deletion of rpox in the tr background resulted in higher survival ability to ethanol and nacl than the wild and complemented cells ( figures 6(c ) and 6(d ) ) . this suggests that the function of rpox is connected to the colony morphotype : in the op background , rpox is required for resistance towards ethanol and hyperosmolarity , whereas in the tr background , rpox may be detrimental , as its loss increases resistance . in this study , the gene rpox , an rpos - like sigma factor from v. alginolyticus zj-51 , was identified and sequenced . the nucleotide sequence showed no similarity with other genes , except for rpos of v. splendidus . in contrast the deduced protein exhibited a certain level of identity to rpos - like sigma factors in a few vibrio species . rpox contains three conserved domains : ( i ) sigma 70 region 1.2 , ( ii ) region 2 , and ( iii ) region 4 . all members of the family contain regions 2 and 4 [ 3 , 4 ] . region 2 is the most conserved region in the family since it contains both the 10 promoter recognition residues and the primary core rnap binding domain , which is essential for the specificity and initiation of transcription [ 4 , 23 ] . compared to rpos and rpoq , rpox does not have the sigma 70 region 3 . instead a sigma 70 region 1.2 is present , which is absent in the rpos - like sigma factors of vibrio sp . the region 1.2 is a subregion of region 1 and is found in the family except for the sigma b of myxococcus xanthus and the flia protein of salmonella typhimurium . this region is usually not involved in the recognition of promoter dna . but region 3 plays an important role in binding the core rna polymerase in the holoenzyme for some specific bacterial promoters that contain an extended 10 promoter element [ 4 , 23 ] . this region is weakly conserved or virtually absent from most of the sigma factors including the ecf subfamily . the difference in the conserved domains suggests that rpox may have functions other than those exerted by rpos . in a previous study we have shown that the tr variant produced copious biofilms with mature three - dimensional structures in contrast to the basic level of biofilm with mostly individual cells attached on the glass that is formed by the op variant . however , this gene could be involved in the attachment and not in the maturation of the biofilm , most likely through a change in cell surface properties . this theory is supported by accessory filamentous - like structures observed on the attached cells of rpox - t , though we do not yet know the role of these structures in the biofilm formation of v. alginolyticus . in comparison , the rpos gene was reported to be important for the maturation of a biofilm via regulation of the energy metabolism and motility . deletion of the rpos gene decreased the attachment of e. coli to the surface and the attached cells maintained as microcolonies and did not develop a mature biofilm . although the rpox gene is important for survival of v. alginolyticus in high concentrations of hydrogen peroxide , the effect is not as strong as in an rpos deletion [ 6 , 9 , 15 , 26 ] . this suggests that rpox might assist rpos to ensure survival in the presence of hydrogen peroxide . however , the rpox was not required for heat shock , although rpos is important for that . in the present study , rpox had no effect on the rate of colony phase variation , but interestingly , when the cells were treated with ethanol and or sodium chloride , the effect of the rpox gene on survival ability depended upon the strain background ( translucent versus opaque ) . in our study on valr , the master regulator of the quorum sensing signaling pathway , we observed that valr regulated flagella and exopolysaccharide biosynthesis genes in a colony phase dependent way ( submitted manuscript ) . sequence alignment revealed that rpox shared 43% identity with the rpos - like sigma factor ( rpoq ) of vibrio fischeri es114 . therefore , we suggest that the rpox gene is regulated by the quorum sensing system . in conclusion , the rpox sigma factor in v. alginolyticus has functions that are different from those of the rpos sigma factor . although both sigma factors are involved in biofilm formation and stress response , the regulatory mechanism of rpox and the relationship between rpox and rpos require further investigation . moreover , it is important to elucidate the effect of colony phase variation on the regulation of rpox , which may help this pathogen to survive changes in the environment .

vibrio alginolyticus zj-51 displays phase variation between opaque / rugose colonies ( op ) and translucent / smooth colonies ( tr ) . these colony variants show great differences in biofilm formation and motility . in this study , a gene encoding for an rpos - like sigma factor , rpox , has been cloned and characterized . the absence of rpox did not affect colony switching rate but did decrease biofilm formation in both the op and the tr variants . when challenged with hydrogen peroxide , the rpox in the op background showed a slightly higher survival rate compared with the wild type , whereas survival was decreased in the tr background . deletion of rpox in the tr background resulted in a higher ability to resist ethanol challenges and to survive hyperosmolarity challenges , and in the op background the opposite phenotype was observed . this indicates that the rpox gene is involved in biofilm formation and stress response but the effects are controlled by colony phase variation in v. alginolyticus .

parkinson 's disease ( pd ) is a movement disorder characterized by tremor , rigidity , slowness of movement , and postural imbalance . there is evidence of abnormalities in the vitamin d - endocrine system in pd patients , including low bone mineral density ( bmd ) , decreased vitamin d levels , and increased bone turnover makers ( bone alkaline phosphatase and urinary n - terminal telopeptide of type i collagen ) compared to controls . these factors , combined with balance problems , are the probable reasons for the high incidence of fractures , especially of the hip , reported in elderly women with pd . sunlight exposure can increase the bmd of pd by increasing serum 25-hydroxyvitamin d3 ( 25ohd ) levels . in another study , the bmd z - score of the trochanter was directly correlated to the degree of physical activity and total body bmd z - score correlated to the degree of rigidity . osteoporosis and osteopenia are common findings in pd patients , affecting up to 91% of women and 61% of men . decreased bmd , low concentrations of serum ionized calcium , and compensatory hyperparathyroidism increase the risk of hip fracture in pd patients . despite an abundance of correlational studies , it is unknown whether vitamin d deficiency is a cause or consequence of pd . in the present paper , significantly increased levels of mhc class ii expressions were detected in the cerebrospinal fluid ( csf ) monocytes of pd patients . human leukocyte antigen ( hla ) genes have also been implicated in pd , and large numbers of hla - dr - positive reactive microglia were detected in the substantia nigra ( sn ) and the nigrostriatal tract in pd patients [ 7 , 8 ] . hla - dr - positive microglia have also been found in these regions in a case of parkinson's - associated dementia in guam . conversely , calcitriol ( 1,25-dihydroxyvitamin d3 ) is known to stimulate phagocytosis but suppress mhc class ii antigen expression in human mononuclear phagocytes [ 9 , 10 ] . calcitriol also decreases interferon - gamma - induced hla - dr antigen expression on normal and transformed human keratinocytes [ 11 , 12 ] . these findings suggested that vitamin d may modulate mhc class ii antigen expression in pd . vdr and 1-hydroxylase , the enzyme responsible for the formation of active vitamin d in the human brain , are found in the large neurons of the sn , as well as in neurons and glial cells in the hypothalamus . vdr , a nuclear receptor , is restricted to the nucleus but 1-hydroxylase is distributed throughout the cytoplasm . the presence of these proteins in the cns suggests that vitamin d is active within the brain . genetic studies provide an opportunity to link molecular variations with epidemiological data , dna sequence variations , such as polymorphisms , exert subtle biological effects on the expression and functionality of proteins . vdr mrna was identified as a potential blood marker for pd . in a korean population , butler et al . reported that the vdr gene is a potential susceptibility gene for pd in the caucasian population . cyp superfamily of enzymes is responsible for the oxidation , peroxidation , and/or reduction of vitamins , steroids , and xenobiotics , as well as the metabolism of drugs . cyp2d6 , an important member of this superfamily , is expressed in neurons in the brain and gut and may be influenced by polymorphic expression . there is a higher prevalence of the poor - metabolizing cyp2d6 * 4 allele in pd patients compared with controls ( 20.7% versus 11.0% ) . in case - control and meta - analysis studies , the cyp2d polymorphism was found to be associated with pd [ 19 , 20 ] . other studies , however , did not find an association between the cyp2d6 polymorphism and pd in an asian population [ 21 , 22 ] . although the poor metabolizer genotype has a small but highly significant association with pd , it would be easily missed in studies with modest numbers of subjects . protein and enzymatic activity are completely absent in less than 1% of asian people and in up to 10% of caucasians with two null alleles . singh et al . reported the expression of cyp2d22 , an ortholog of human cyp2d6 , in mouse striatum and its modulation in mptp - induced pd phenotype and nicotine - mediated neuroprotection . cyp2d6 is a potential 25-hydroxylase , which converts vitamin d3 into 25ohd , and vitamin d 25-hydroxylase deficiency resulted in vitamin d deficiency . moreover , the cyp2d and pd loci are located on the same chromosome 22 [ 26 , 27 ] . deletion of chromosome 22q11 syndrome was reported to be associated with pd [ 28 , 29 ] . interestingly , patients with a deletion of chromosome 22q11 showed a reduced bmd , serum calcium , and pth levels ; 11% and 8% of these patients had serum 25ohd levels under 20 ng / ml and abnormal serum 1,25ohd levels , respectively . the primary function of the ras is to maintain fluid homeostasis and regulate blood pressure . several components of the ras and its receptors are found in the cns [ 3134 ] , suggesting that ras is important in the brain . csf levels of angiotensin - converting enzyme ( ace ) activity were reported to be decreased in pd patients and increased with dopaminergic treatment [ 35 , 36 ] . in addition , the ace inhibitor perindopril has been shown to exert beneficial effects on the dopaminergic system [ 37 , 38 ] . after four weeks of treatment with perindopril , patients with pd had faster improvement in motor response after l - dopa and a reduction in on phase the frequency of the homozygous dd genotype of the ace gene was significantly increased in patients with pd , and is also higher in pd patients with l - dopa - induced psychosis versus without psychosis [ 40 , 41 ] . however , other studies did not reveal any associations between ace polymorphisms , pd , and of l - dopa - induced adverse effects [ 42 , 43 ] . the use of ace inhibitors by the ace dd genotype has been shown to decrease the level of calcitriol . in addition , genetic disruption of the vdr resulted in overstimulation of the ras with increased production of renin and angiotensin ii , thereby leading to high blood pressure and cardiac hypertrophy . treatment with captopril reduced cardiac hypertrophy in vdr knockout mice , suggesting that calcitriol may function as an endocrine suppressor of renin biosynthesis . ho-1 is a stress protein that may confer cytoprotection by enhancing catabolism of pro - oxidant heme to the radical scavenging bile pigments , biliverdin , and bilirubin . the ho-1 gene can be upregulated by a host of noxious stimuli and is induced in cns tissues affected by neurological diseases . in the normal brain , basal ho-1 expression is low and restricted to small groups of scattered neurons and neuroglia . in the brains of pd patients , the ho-1 is highly overexpressed in astrocytes within the sn and in lewy bodies found in affected dopaminergic neurons . serum ho-1 levels are increased in pd patients but not in patients with alzheimer 's disease ( ad ) , suggesting a systemic antioxidant reaction to a chronic oxidative stress state that is unique to pd . similarly , calcitriol delayed of ho-1 immunoreactivity after the postlesional survival time of 12 hours concomitant with a reduction in glial fibrillary acidic protein immunoreactivity in remote cortical regions affected by a secondary spread of injury in glial cells of the focal cerebral ischemic , thereby supporting the protective role of calcitriol in postcellular injury . parp-1 is a nuclear protein that can promote either neuronal death or survival under certain stress conditions . overexpression of parp-1 has been reported in the dopaminergic neurons of the sn in pd . mptp is a neurotoxin that induces parkinsonian symptoms in human and animals , but mice lacking parp gene are spared from mptp neurotoxicity . therefore , parp inhibitors have proved to be valuable tools in pd model [ 55 , 56 ] . in addition , increased levels of vitamin d seem to downregulate parp-1 expression ; parp-1 levels decrease following calcitriol treatment in nb4 cells , which represent an acute promyelocytic leukemia cells . vitamin d exerts a concentration - dependent inhibitory effect on parp-1 in the human keratinocyte cells . vitamin d - induced downregulation of parp is further enhanced by nicotinamide in human myeloblastic leukemia cells . furthermore , parp is attenuated in the hippocampus of rats that received dexamethasone and vitamin d , suggesting that the anti - inflammatory effects of dexamethasone and vitamin d derives from their ability to downregulate microglial activation . these findings suggested that vitamin d may have a protective role in pd by downregulating parp . ntfs can exert protective or even restorative effects on pd animal models and dopaminergic cell cultures ; key examples include brain - derived neurotrophic factor ( bdnf ) , glial - derived neurotrophic factor ( gdnf ) , mesencephalic - astrocyte - derived neurotrophic factor ( manf ) , and cerebral dopamine neurotropic factor ( cdnf ) . receptors for these ntfs are expressed in the striatum and sn [ 67 , 68 ] . ntf expression is reduced in the sn of patients with pd [ 6971 ] . moreover , the c allele of an inotropic cdnf single nucleotide polymorphism ( rs7094179 ) has been suggested to infer susceptibility to pd in a korean population , and a allele of bdnf is associated with pd in a chinese han population . interestingly , calcitriol regulates the expression of the low - affinity neurotrophic receptor and increases striatal gdnf mrna and protein expression in adult rats . in vivo , calcitriol is also a potent inducer of gdnf in rat glioma cells . the brains of offspring from vitamin d - deficient dams are characterized by diminished expression of neurotrophic factors . furthermore , calcitriol protects against dopamine loss from 6-hydroxydopamine- ( 6-ohda- ) lesioned rats by increasing gdnf and partially restores tyrosine hydroxylase expression in sn and striatum [ 78 , 79 ] . sp1 transcription factor is a member of an extended family of dna - binding proteins that is acetylated in response to oxidative stress in neurons . the sp1 family of proteins plays an important role in controlling the expression of the dopamine transporter gene within dopaminergic neurons and also regulates expression of rat dopamine receptor gene . on the other hand , binding sites for the transcription factor sp1 in cultured human fibroblasts , the level of cyp24 ( 25-ohd 24-hydroxylase ) mrna plays a key role in the metabolism of 1,25ohd and increases up to 20,000-fold in response to calcitriol . two vitamin d - responsive elements ( vdres ) located upstream of the cyp24 gene are primarily responsible for the increased mrna levels , and sp1 acted synergistically with these vdres for the induction . the mvdr promoter is controlled by sp1 sites and functions as the transactivation component of the vdr / sp1 complex to trigger gene expression . moreover , the genes encoding sp1 and vdr were mapped to human chromosome 12q [ 75 , 76 ] . a link between dm and pd has been suggested in several reports , but the results have been inconsistent . insulin receptors and their mrnas are localized in the sn [ 83 , 84 ] . a high incidence of abnormal glucose tolerance has been reported in pd and seems to be exacerbated by l - dopa treatment . dm has been associated with the risk of developing pd [ 86 , 87 ] whereas others reported from protective to no associations with pd [ 8890 ] . human and experimental animal studies , however , demonstrated neurodegeneration associated with peripheral insulin resistance . in a 6-ohda model of pd , striatal insulin resistance was observed in the striatum , and patients with pd exhibited increased autoimmune reactivity to insulin . individuals newly diagnosed with pd display reduced insulin - mediated glucose uptake , which is hypothesized to be due to inhibit early insulin secretion and hyperglycemia after glucose loading . furthermore , chronic hyperglycemia decreased limbic extracellular dopamine concentrations and striatal dopaminergic transmission in streptozotocin - induced diabetic rats [ 96 , 97 ] . vitamin d levels may provide a link between the diseases ; serum 1,25ohd and 25ohd levels are low in diabetic patients [ 98 , 99 ] , and diabetic rats had an increased metabolic clearance rate of 1,25ohd . interestingly , a significant high prevalence of vitamin d insufficiency is reported in patients with pd [ 101 , 102 ] . a significant inverse association between serum vitamin d and pd was demonstrated and suggested that high vitamin d status might provide protection against pd . in diabetic - induced rats , vitamin d and insulin treatment markedly recovered the levels of altered gene ( cholinergic , dopaminergic , and insulin receptors ) expression and its binding parameters nearly to those of the control rats . maternal vitamin d deficiency was reported to alter the expression of genes involved in dopamine specification in the developing rat mesencephalon . calcitriol has been shown to protect dopamine neuronal toxicity induced by 6-ohda and the combination of l - buthionine sulfoximine and mptp , thereby restoring motor activity in the lesioned animals [ 106 , 107 ] . furthermore , vitamin d was reported to improve rigidity and akinesia and reduce levodopa dosage in a patient with pd . unlike most neurons in the brain , dopaminergic neurons function as autonomous pacemakers that rely on l - vscc to generate action potentials at a clock - like 24 hz in the absence of synaptic input . l - vscc activity during autonomous pacemaking increased the sensitivity of dopaminergic neurons to mitochondrial toxins in an animal model of pd . epidemiological data supports a link between l - vscc functioning and the risk of developing pd [ 111113 ] . pretreatment with the calcium channel antagonist nimodipine has been shown to block the development of mptp - induced neurotoxicity in animal models [ 114 , 115 ] . israpidine , another l - vscc antagonist , caused a dose - dependent reduction in l - dopa - induced rotational behavior and abnormal involuntary movements in the 6-ohda - lesioned rat model of pd . with respect to ad , amyloid- protein was reported to promote neurodegeneration by inducing l - vscc expression and suppressing vdr expression ; subsequent treatment with vitamin d protected neurons by preventing cytotoxicity and apoptosis , probably by downregulating l - vscc and upregulating vdr . calcitriol decreased l - vscc activity in aged rats and in neuronal vulnerability with particular impact on reducing age - related changes associated with ca dysregulation [ 118 , 119 ] . treatment with 24r , 25 dihydroxyvitamin d3 also reduced l - vscc activity in vascular smooth muscle in rats . ngf is a small secreted protein that is important for the growth , maintenance , and survival of certain target neurons . ngf has been implicated in maintaining and regulating the septohippocampal pathway , which is involved in learning and memory [ 121123 ] . ngf concentrations are decreased in the sn of the pd and in a rat model of pd [ 69 , 126 ] . ngf levels showed greater reductions in early states of the disease compared with advanced stages , implying that decreased ngf may be involved in the pathogenesis of pd . ngf is reported to protect dopamine neurotoxicity induced by mptp , rotenone , and 6-ohda via different pathways [ 127129 ] . the chronic infusion of ngf into the rat striatum resulted in cholinergic hyperinnervation and reduced spontaneous activity of striatal neurons . moreover , ngf increases survival of dopaminergic grafts , rescues nigral dopaminergic neurons , and restores motor dysfunction in a rat model of pd [ 131 , 132 ] . in addition , the brains of newborn rats from vitamin d - deficient dams showed reduced expression of ngf . in vitro , calcitriol regulated the expression of the vdr gene and stimulated the expression of the ngf gene in schwann cells . in mouse fibroblasts , calcitriol and vitamin d analogs are reported to enhance ngf induction by increasing ap-1 binding activity to the ngf promoter [ 134 , 135 ] . mmps are proteolytic enzymes responsible for extracellular matrix ( ecm ) remodeling and the regulation of leukocyte migration through the ecm , which is an important step for inflammatory processes . application of dopaminergic neurotoxins to two human neuroblastoma cell lines downregulates the transcription and translation of tissue inhibitor of metalloproteinase- ( timp- ) 2 effectively enhancing mmp activity . exendin-4 , which is an analogue of glucagon - like peptide 1 ( glp-1 ) , significantly attenuates the loss of sn neurons and the striatal dopaminergic fibers in the mptp - induced pd model , and inhibits the expression of mmp-3 . conversely , the vdr taqi polymorphism is associated with decreased production of timp-1 , a natural inhibitor of mmp-9 . calcitriol downregulates mmp-9 levels in keratinocytes and may attenuate deleterious effects due to excessive tnf--induced proteolytic activity associated with cutaneous inflammation . in addition , a vitamin d analogue was reported to reduce the expression of mmp-2 , mmp-9 , vegf , and parathyroid hormone - related protein in lewis lung carcinoma cells . these findings suggested that vitamin d plays a role in modulating mmp activation in pd . pge2 is a key product of cox-2 and is increased in the sn of patients with pd and mptp - induced pd in an animal model [ 145 , 146 ] . overexpression of cox-2 is reported in pd and an mptp - animal model [ 148 , 149 ] . similarly , regular use of cox-2 inhibiting nonsteroidal anti - inflammatory drugs ( nsaids ) , such as ibuprofen , has been associated with a decreased incidence of pd . calcitriol , which reportedly regulates the expression of several key genes involved in pg pathways , decreases pg synthesis . calcitriol and its analogues have also been shown to inhibit selectively the activity of cox-2 . these findings suggested that vitamin d has a role in anti - inflammatory processes in pd . analyses of postmortembrains from pd reveal evidence of increased lipid peroxidation in pd sn [ 155 , 156 ] . a selective superoxide dismutase ( sod ) is also increased in the sn of pd patients . calcitriol administration has been reported to exert a receptor - mediated effect on the secretion of hydrogen peroxide by human monocytes . in vitro , monocytes gradually lose their ability to produce superoxide when stimulated ; the addition of calcitriol , lipopolysaccharide , or lipoteichoic acid ( lta ) restored the ability of stimulated monocytes to produce superoxide and increases the oxidative capacity compared with unstimulated monocytes . calcitriol can also protect nonmalignant prostate cells from oxidative stress - induced cell death by preventing ros - induced cellular injuries . vitamin d metabolites and analogues were reported to induce lipoxygenase mrna expression , lipoxygenase activity , and ros production in a human bone cell line . vitamin d can also reduce lipid peroxidation and induce sod activity in the rat hepatic antioxidant system . nos is an enzyme involved in the synthesis of nitric oxide ( no ) , which has also been implicated in pd . in postmortem brains of pd , high levels of nos expression were found in the nigrostriatal region and basal ganglia . a significant increase in the nitrite content was reported in polymorphonuclear leukocytes of pd patients . inducible and neuronal no are increased in both 6-ohda and mptp animal models [ 165 , 166 ] . moreover , studies with nos inhibitors and nos knock - out animals have also confirmed the role of nos in neurodegeneration [ 167 , 168 ] . reduced and oxidized glutathione ( gsh ) were demonstrated in the sn of patients with pd . conversely , the activation of 1-hydroxylase in macrophages increases in 1,25ohd , which inhibits inducible nos ( inos ) expression and reduces no production by lipopolysaccharide- ( lps- ) stimulated macrophages . thus , calcitriol production by macrophages may provide protection against oxidative injuries caused by the no burst . calcitriol is known to inhibit lps - induced immune activation in human endothelial cells . in experimental allergic encephalomyelitis astrocytes play a pivotal role in cns detoxification pathways where glutathione ( gsh ) is involved in the elimination of oxygen and nitrogen reactive species , such as nitric oxide . calcitriol affects this pathway by enhancing intracellular gsh pools and significantly reduces nitrite production induced by lps . vitamin d may be beneficial in pd patients , as one patient showed improved rigidity and akinesia and was able to decrease their levodopa dosage after vitamin d therapy . genetic studies have provided opportunities to determine what proteins may link vitamin d to pd pathology . vitamin d can also act through a number of nongenomic mechanisms , including effects on protein expression , oxidative stress , inflammation , and cellular metabolism . among the many forms of vitamin d , calcitriol ( 1,25-dihydroxyvitamin d3 ) is an attractive therapeutic candidate , because it is a particularly active metabolite , and its receptor is expressed in the cns .

parkinson 's disease ( pd ) is the second most common form of neurodegeneration in the elderly population . clinically , it is characterized by tremor , rigidity , slowness of movement , and postural imbalance . a significant association between low serum vitamin d and pd has been demonstrated , suggesting that elevated vitamin d levels might provide protection against pd . genetic studies have helped identify a number of proteins linking vitamin d to pd pathology , including the major histocompatibility complex ( mhc ) class ii , the vitamin d receptor ( vdr ) , cytochrome p450 2d6 ( cyp2d6 ) , chromosome 22 , the renin - angiotensin system ( ras ) , heme oxygenase-1 ( ho-1 ) , poly(adp - ribose ) polymerase-1 gene ( parp-1 ) , neurotrophic factor ( ntf ) , and sp1 transcription factor . vitamin d has also been implicated in pd through its effects on l - type voltage - sensitive calcium channels ( l - vscc ) , nerve growth factor ( ngf ) , matrix metalloproteinases ( mmps ) , prostaglandins ( pgs ) and cyclooxygenase-2 ( cox-2 ) , reactive oxygen species ( ros ) , and nitric oxide synthase ( nos ) . a growing body of evidence suggests that vitamin d supplementation may be beneficial for pd patients . among the different forms of vitamin d , calcitriol ( 1,25-dihydroxyvitamin d3 ) is best indicated for pd , because it is a highly active vitamin d3 metabolite with an appropriate receptor in the central nervous system ( cns ) .

specificationsorganism / cell line / tissuehomo sapiens / mcf-7 cell line / mammary adenocarcinomasexfemalesequencer or array typemicroarray : affymetirx human gene 1.0 st and human gene 2.0 stchip - seq : illumina hi - seq 2000data formatmicroarray raw data : cel fileschip - seq raw data : fastq fileschip - seq processed data : wig files and bed filesexperimental factorspma treatment and sirna transfectionexperimental featuresmicroarray gene expression profiling to identify genes that are dependent on pkc - theta ( prkcq).chip - seq to map genomic sites that are bound by pkc - thetaconsentn / asample source locationn / a the mcf-7 cell line was cultured at 37 c in low glucose dmem ( gibco ) supplemented with fetal calf serum ( 10% ) , l - glutamine ( 2 mm ) , and penicillin and streptomycin ( 0.1% ) . non - stimulated ( ns ) mcf-7 cells were stimulated for 60 h with phorbol 12-myristate 13-acetate ( pma , 0.65 ng / ml , sigma - aldrich ) to generate the whole stimulated population ( wp ) before facs sorting using antibodies against cd44-apc ( 559942 , bd biosciences ) and cd24-pe ( 555428 , bd biosciences ) to give the cancer stem cell ( csc ) and non - cancer stem cell ( ncsc ) populations . hoechst 33258 ( 561908 , bd biosciences ) was used while sorting to exclude dead cells . the csc population was gated as cd44 high and cd24 low , while the ncsc population was the remaining stimulated cells . total rna was isolated from these cells using trizol ( invitrogen ) , labeled and hybridized to affymetrix hugene 1.0 st ( at the ramaciotti centre for genomics , nsw ) . mcf-7 cells were transfected with pkc- sirna ( sc-36252 ) or mock sirna ( fluorescein conjugate , sc-36869 ) ( 20 nm , santa cruz biotechnologies ) using lipofectamine 2000 ( invitrogen ) . this resulted in four samples ; mock non - stimulated ( mock ns ) , mock stimulated ( mock st ) , pkc sirna non - stimulated ( pkc ns ) , and pkc sirna stimulated ( pkc st ) . total rna was isolated from these cells using trizol , labeled and hybridized to affymetrix hugene 2.0 st . when robust multiarray average ( rma ) normalization ( affymetrix powertools ) was used on the hugene 1.0 st arrays , the ns sample values displayed a different distribution to the wp , csc and ncsc samples ( fig . loess normalization ( using the lpe package in r ) was used to correct the ns distribution ( fig . but this was expected as the other samples were all from stimulated cells . as the wp population consists of 89.5% of ncsc cells and 10.5% csc cells , its slightly higher correlation with the ncsc sample the two mock samples had lower correlation scores than the pkc- sirna samples , indicating that pkc theta plays an essential role in at least a proportion of the gene expression changes that occurred with stimulation ( fig . a comparison of the changes measured in the mock st samples compared to the in the pkc- sirna st sample , against the mock ns sample revealed that while pkc- was necessary for the increase and decrease of many genes it 's inhibition did not abrogate all changes ( fig . genes were judged to be changed in expression if their log2 difference was at least 0.5 . 1356 genes were identified as pkc- sensitive in that they were induced by stimulation in the mock sirna sample but were expressed less in the stimulated pkc- sirna sample than the stimulated mock sirna sample . this induction by pma and sensitivity to pkc- was confirmed for 10 ( of 10 tested ) transcripts by quantitative real - time pcr . to determine which of the pkc- sensitive genes were expressed more in csc we had to compare probesets on the hugene 1.0 st and 2.0 st arrays . the probesets were matched largely by gene symbol but were also considered to represent the same transcript if the chromosome , transcription start site and strand were identical . using this approach only 42% of the probesets on the hugene 2.0 st array had comparable probesets on the hugene 1.0 st array . first we compared the two stimulations , the mock ns and mock st comparison performed on the hugene 2.0 arrays and the ns and wp comparison performed on the hugene 1.0 st arrays ( fig . 672 were up - regulated with stimulation in both assays and this was 49.6% of those from the mock st to mock ns comparison . when the 2 sets of log2 differences were compared the pearson correlation coefficient was 0.583 . whether differences were due to biological variation , the different probes used or the mock transfection was unclear . of those represented on the hugene 1.0 st array , 134 ( 17.8% ) of pkc- sensitive gene cohort were enriched in csc compared to ncsc ( fig . thus together the two microrarray studies allowed us to identify genes associated with pkc- induced csc formation . non - stimulated ( ns ) and pma stimulated ( st ) mcf-7 cells were fixed with paraformaldehyde ( 1% ) for 10 min at room temperature and lysed with the upstate chip kit and sonication according to the manufacturer 's instructions . lysates were immunoprecipitated with anti - pkc- ( 5 g , sc-212 , santa cruz ) and protein a magnetic beads ( millipore ) . the resulting chip - dna ( 10 ng ) was used with the nebnext chip - seq library prep for illumina ( new england biolabs inc , neb#e6240l ) according to the manufacturer 's instructions . the kit consists of an end repair enzyme kit , klenow fragment for da - tailing , quick t4 dna ligase ( with nebnext adaptor primers ) and nebnext high - fidelity 2 pcr master mix , universal pcr primer ( 25 m ) , and index primer ( 25 m , new england biolabs inc , nebnext multiplex oligos for illumina , neb#e7335l ) for multiplexing . a total of 15 cycles were used for pcr amplification of the adaptor - ligated dna . fragments were size selected to be between 175 and 225 bp using ampure xp beads ( beckman coulter , inc . , part # : a63881 ) . the quality of the chip - dna library was assessed on a bioanalyzer ( agilent ) . a total of 2 nm was captured on the illumina flow cell for cluster generation and sequenced on the illumina hiseq2000 using a 50 bp run at the ramaciotti centre for genomics . total input ( ti ) samples were sequenced for both the ns and st samples . all quality controls were passed except for high sequence duplication levels in the pkc- st sample ( fig . 2a ) and a problem at base 37 was noted for one lane ( fig . the adapter sequences were trimmed using cutadapt ( http://code.google.com/p/cutadapt/ ) then mapped to the human genome ( hg19 ) using local alignment in bowtie 2 ( local -d 20 -r 3 -n 0 -l 20 -i s,1,0.50 ) . only reads that uniquely mapped to the genome ( 46% for the pkc- st samples ) were used for subsequent analysis ( obtained using awk at this point data from the 2 lanes were combined and duplicate reads were removed using picard ( http://picard.sourceforge.net , default parameters ) . as expected from the fastqc report the pkc- st sample had a high number of duplicate reads ( 81% ) , compared to the pkc- ns ( 40% ) and ti st ( 28.7% ) . the resulting reads totaled 14,108,692 and 4,645,493 from the ns and st mcf7 pkc- chip samples and 23,370,927 and 21,886,666 reads from the respective ti samples . pkc- enriched regions were called for the pkc- st sample against the ti st sample using a p - value cutoff of 0.05 for the zero inflated negative binomial algorithm ( zinba ) ( , extension = 200 , refine peaks = 1 , broad = f ) , a posterior probability cutoff of 0.999 for bayespeak ( , method = lowerbound ) and macs2 . for macs2 ( keep - dup all -g hs -s 50 bw 200 -q 0.05) but the resulting peaks were then filtered for q value < 0.01 . filtering on the q value after macs2 had been used resulted in more peaks than if q was set to 0.01 . for representation in ucsc , reads were extended by 200 bp and htseq was used to create wig files . of the 2945 regions called as enriched by both bayespeak and zinba , 795 ( 27% ) were also found by macs2 ( fig . however macs2 did not call a pkc- enriched region at the bhlhe40 promoter , which was found to give the strongest enrichment in chip - pcr . using the broad option for macs2 , and setting the p value cutoff to < 0.05 resulted in 20,218 peaks being called and a greater overlap with the other programs , especially bayespeak ( fig . however , the broad option still did not call the bhlhe40 promoter peak . to compare the ns and st samples , only the peaks called by both bayespeak and zinba from the st sample were used and the number of reads for each of these peaks in the two samples was counted in r. the number of reads per peak was normalized to the reads per million ( uniquely ) mapped reads . as noted in the resulting regression result was st = 1.241 ns + 0.9575 . the counts were not normalized further as chip - pcr for 6 ( of 8 tested ) of these regions confirmed a statistically significant increase in pkc- binding in st compared to ns , and biologically it was not expected that the samples would have similar pkc binding levels . as reported in overlay of the transcriptome and chip - seq data identified 62 direct pkc- targeted genes whose induction with pma was also dependent on pkc-. importantly , many of these genes are known key genes previously implicated in epithelial to mesenchymal transition and formation of csc . the mcf-7 cell line was cultured at 37 c in low glucose dmem ( gibco ) supplemented with fetal calf serum ( 10% ) , l - glutamine ( 2 mm ) , and penicillin and streptomycin ( 0.1% ) . non - stimulated ( ns ) mcf-7 cells were stimulated for 60 h with phorbol 12-myristate 13-acetate ( pma , 0.65 ng / ml , sigma - aldrich ) to generate the whole stimulated population ( wp ) before facs sorting using antibodies against cd44-apc ( 559942 , bd biosciences ) and cd24-pe ( 555428 , bd biosciences ) to give the cancer stem cell ( csc ) and non - cancer stem cell ( ncsc ) populations . hoechst 33258 ( 561908 , bd biosciences ) was used while sorting to exclude dead cells . the csc population was gated as cd44 high and cd24 low , while the ncsc population was the remaining stimulated cells . total rna was isolated from these cells using trizol ( invitrogen ) , labeled and hybridized to affymetrix hugene 1.0 st ( at the ramaciotti centre for genomics , nsw ) . mcf-7 cells were transfected with pkc- sirna ( sc-36252 ) or mock sirna ( fluorescein conjugate , sc-36869 ) ( 20 nm , santa cruz biotechnologies ) using lipofectamine 2000 ( invitrogen ) . this resulted in four samples ; mock non - stimulated ( mock ns ) , mock stimulated ( mock st ) , pkc sirna non - stimulated ( pkc ns ) , and pkc sirna stimulated ( pkc st ) . total rna was isolated from these cells using trizol , labeled and hybridized to affymetrix hugene 2.0 st . when robust multiarray average ( rma ) normalization ( affymetrix powertools ) was used on the hugene 1.0 st arrays , the ns sample values displayed a different distribution to the wp , csc and ncsc samples ( fig . loess normalization ( using the lpe package in r ) was used to correct the ns distribution ( fig . but this was expected as the other samples were all from stimulated cells . as the wp population consists of 89.5% of ncsc cells and 10.5% csc cells , its slightly higher correlation with the ncsc sample the two mock samples had lower correlation scores than the pkc- sirna samples , indicating that pkc theta plays an essential role in at least a proportion of the gene expression changes that occurred with stimulation ( fig . a comparison of the changes measured in the mock st samples compared to the in the pkc- sirna st sample , against the mock ns sample revealed that while pkc- was necessary for the increase and decrease of many genes it 's inhibition did not abrogate all changes ( fig . genes were judged to be changed in expression if their log2 difference was at least 0.5 . 1356 genes were identified as pkc- sensitive in that they were induced by stimulation in the mock sirna sample but were expressed less in the stimulated pkc- sirna sample than the stimulated mock sirna sample . this induction by pma and sensitivity to pkc- was confirmed for 10 ( of 10 tested ) transcripts by quantitative real - time pcr . to determine which of the pkc- sensitive genes were expressed more in csc we had to compare probesets on the hugene 1.0 st and 2.0 st arrays . the probesets were matched largely by gene symbol but were also considered to represent the same transcript if the chromosome , transcription start site and strand were identical . using this approach only 42% of the probesets on the hugene 2.0 st array had comparable probesets on the hugene 1.0 st array . first we compared the two stimulations , the mock ns and mock st comparison performed on the hugene 2.0 arrays and the ns and wp comparison performed on the hugene 1.0 st arrays ( fig . 672 were up - regulated with stimulation in both assays and this was 49.6% of those from the mock st to mock ns comparison . when the 2 sets of log2 differences were compared the pearson correlation coefficient was 0.583 . whether differences were due to biological variation , the different probes used or the mock transfection was unclear . of those represented on the hugene 1.0 st array , 134 ( 17.8% ) of pkc- sensitive gene cohort were enriched in csc compared to ncsc ( fig . thus together the two microrarray studies allowed us to identify genes associated with pkc- induced csc formation . non - stimulated ( ns ) and pma stimulated ( st ) mcf-7 cells were fixed with paraformaldehyde ( 1% ) for 10 min at room temperature and lysed with the upstate chip kit and sonication according to the manufacturer 's instructions . lysates were immunoprecipitated with anti - pkc- ( 5 g , sc-212 , santa cruz ) and protein a magnetic beads ( millipore ) . the resulting chip - dna ( 10 ng ) was used with the nebnext chip - seq library prep for illumina ( new england biolabs inc , neb#e6240l ) according to the manufacturer 's instructions . the kit consists of an end repair enzyme kit , klenow fragment for da - tailing , quick t4 dna ligase ( with nebnext adaptor primers ) and nebnext high - fidelity 2 pcr master mix , universal pcr primer ( 25 m ) , and index primer ( 25 m , new england biolabs inc , nebnext multiplex oligos for illumina , neb#e7335l ) for multiplexing . a total of 15 cycles were used for pcr amplification of the adaptor - ligated dna . fragments were size selected to be between 175 and 225 bp using ampure xp beads ( beckman coulter , inc . , part # : a63881 ) . the quality of the chip - dna library was assessed on a bioanalyzer ( agilent ) . a total of 2 nm was captured on the illumina flow cell for cluster generation and sequenced on the illumina hiseq2000 using a 50 bp run at the ramaciotti centre for genomics . total input ( ti ) samples were sequenced for both the ns and st samples . all quality controls were passed except for high sequence duplication levels in the pkc- st sample ( fig . 2a ) and a problem at base 37 was noted for one lane ( fig . the adapter sequences were trimmed using cutadapt ( http://code.google.com/p/cutadapt/ ) then mapped to the human genome ( hg19 ) using local alignment in bowtie 2 ( local -d 20 -r 3 -n 0 -l 20 -i s,1,0.50 ) . only reads that uniquely mapped to the genome ( 46% for the pkc- st samples ) were used for subsequent analysis ( obtained using awk at this point data from the 2 lanes were combined and duplicate reads were removed using picard ( http://picard.sourceforge.net , default parameters ) . as expected from the fastqc report the pkc- st sample had a high number of duplicate reads ( 81% ) , compared to the pkc- ns ( 40% ) and ti st ( 28.7% ) . the resulting reads totaled 14,108,692 and 4,645,493 from the ns and st mcf7 pkc- chip samples and 23,370,927 and 21,886,666 reads from the respective ti samples . pkc- enriched regions were called for the pkc- st sample against the ti st sample using a p - value cutoff of 0.05 for the zero inflated negative binomial algorithm ( zinba ) ( , extension = 200 , refine peaks = 1 , broad = f ) , a posterior probability cutoff of 0.999 for bayespeak ( , method = lowerbound ) and macs2 . ( keep - dup all -g hs -s 50 bw 200 -q 0.05) but the resulting peaks were then filtered for q value < 0.01 . filtering on the q value after macs2 had been used resulted in more peaks than if q was set to 0.01 . for representation in ucsc , reads were extended by 200 bp and htseq was used to create wig files . of the 2945 regions called as enriched by both bayespeak and zinba , 795 ( 27% ) . however macs2 did not call a pkc- enriched region at the bhlhe40 promoter , which was found to give the strongest enrichment in chip - pcr . using the broad option for macs2 , and setting the p value cutoff to < 0.05 resulted in 20,218 peaks being called and a greater overlap with the other programs , especially bayespeak ( fig . however , the broad option still did not call the bhlhe40 promoter peak . to compare the ns and st samples , only the peaks called by both bayespeak and zinba from the st sample were used and the number of reads for each of these peaks in the two samples was counted in r. the number of reads per peak was normalized to the reads per million ( uniquely ) mapped reads . as noted in the resulting regression result was st = 1.241 ns + 0.9575 . the counts were not normalized further as chip - pcr for 6 ( of 8 tested ) of these regions confirmed a statistically significant increase in pkc- binding in st compared to ns , and biologically it was not expected that the samples would have similar pkc binding levels . as reported in overlay of the transcriptome and chip - seq data identified 62 direct pkc- targeted genes whose induction with pma was also dependent on pkc-. importantly , many of these genes are known key genes previously implicated in epithelial to mesenchymal transition and formation of csc .

the protein kinase c ( pkc ) activator phorbol 12-myristate 13-acetate ( pma ) induces transition of the epithelial mcf-7 cell line to a mesenchymal phenotype . a subset of the resulting mesenchymal cells has surface markers characteristics of a cancer stem cell ( csc ) population . we profiled the transcriptome changes associated with the epithelial to mesenchymal transition and those that occurred in the csc subset . using a sirna knockdown strategy , we examined the extent to which these changes were dependent on the pkc family member , pkc-. the importance of the cytoplasmic signaling role of this kinase is well established and in this study , we have shown by pkc- chip - sequencing analysis that this kinase has a dual role with the ability to also associate with chromatin on a subset of pkc- dependent genes . in the associated manuscript ( zafar et al . , 2014 [ 5 ] ) we presented evidence for the first time showing that this nuclear role of pkc- is also important for gene induction and mesenchymal / csc phenotype . here we describe the analysis associated with the transcriptome and chip - seq data presented in zafar et al . ( 2014 ) [ 5 ] and uploaded to ncbi gene expression omnibus ( gse53335 ) .

despite extensive use of all ceramic systems today , metal - ceramic restorations are still considered the primary means of restoration due to their superior mechanical strength and their cost effectiveness . a considerable increase in the price of gold during the 1970s resulted in the development of alternative metallic systems . two decades ago physical properties of nickel - chromium ( ni - cr ) and cobalt - chromium ( co - cr ) base metal alloys were discussed and compared to those found in noble metal alloys . it was concluded that base metal alloys have higher melting temperatures , require more critical handling and care with the melting technique , and are more difficult to finish , when compared to noble metal alloys . even though base metal alloys have superior mechanical properties including higher modulus of elasticity , sag resistance , longevity , and higher melting temperature , there appears to be an investigative controversy about the bond strength of base metal alloy systems to porcelain . the metal substructure in a metal - ceramic restoration is ductile , bends under load , and has the ability to return to its original form . the fracture resistance of the metal , in combination with the esthetic nature of porcelain , has provided dentists with both durable and esthetic restorations . the bonding of porcelain to dental alloy occurs during porcelain firing , a process known as sintering . the metal - ceramic bond interface is critical in the functional and esthetic success of metal - ceramic restorations . four factors contribute to the strength of the metal - ceramic bond which includes chemical bond , mechanical interlocking , vander waals forces and compressive forces . chemical bond is dictated by the oxide layer formed on the metal substrate that forms metallic , ionic , and covalent bonds with oxides in the opaque ceramic layer . van der waals forces are based on molecular attraction between charges , and compressive forces are those based on the coefficient of thermal expansion . once bonded the underlying metal substructure provides support to porcelain thereby increasing the strength of the ceramic and placing its innermost layer adjacent to the metal substructure in compression . the most important of the four mechanisms is the formation of a chemical bond which results in a thermodynamic equilibrium between the metal and the porcelain by the formation of an intermediate oxide layer . the metal - ceramic bond is further enhanced by an actual physical interlocking of the ceramic with the metal . however , it can also lead to the formation of voids at the interface , which can adversely affect bonding mechanism . to a lesser extent van der waals 's forces contribute toward metal - ceramic bonding via inter - atomic forces . failure of the metal - ceramic bond has been shown to be dependent on many variables , including firing temperature of veneering porcelain and surface textures of the alloy systems . it is known that when a metal - ceramic system is loaded , failure occurs at the areas where bonding is the weakest , so that if the adhesive bond between the ceramic and metal is sufficient , the failure will be cohesive within the ceramic . factors such as impact and fatigue , occlusal forces , and incompatibility between physical properties of metal and porcelain may result in porcelain fracture , frequently of a cohesive nature . oxidation heat treatment of the metal substructure is used to remove the entrapped gas , eliminate surface contaminants , and form the metallic oxide layer . during porcelain firing cycle before flow of the ceramic begins , the fusing ceramic comes into immediate contact with an oxide layer rather than with a metal surface . the fusing ceramic dissolves the oxide originally formed and produces an interaction zone responsible for the formation of a bond . a sandblasted surface may have higher surface energy that allows increased wetting of the metal during ceramic application . however , literature on whether particle size has an effect on the bond strength of porcelain to metal is scanty . furthermore , it is known that the characteristic of the oxide layer is different before and after sandblasting . this article compares shear bond strength of feldspathic porcelain to ni - cr alloy when subjected to various surface treatments . the study was conducted at the department of prosthodontics and crown and bridgework , faculty of dental science , dharmsinh desai university , college road , nadiad , gujarat . the testing of specimens was done at the department of civil engineering , charotar university of science and technology , changa , gujarat . totally , 40 specimens of ni - cr alloy ( bellabond , bego , germany ) were fabricated with the dimensions 20 mm in length , 10 mm in width , and 5 mm in height . the measurements of specimens corresponded to the specifications of the universal testing machine ( tun 800 , miraj , india ) which was subsequently used to test the specimens . totally , 40 blocks of inlay casting wax ( surana , india ) were fabricated using a custom made metal mould . heated inlay wax ( 27c ) was filled in the mould and allowed to cool for the subsequent hour . the set inlay wax pattern was retrieved from the mould , and 2.50 mm wide prefabricated sprue former was attached ( sigma sprue wax , ambernath , india ) . the wax pattern was invested in a phosphate bonded investment material ( degudent , dentslpy , germany ) . mixing ratio of the powder and liquid recommended by the manufacturer wet asbestos ring liner ( gc , europe ) was used to allow for symmetric expansion . after performing the programmed burnout ( 850c ) as per the manufacturer 's instructions predominantly base metal alloy pellets in the form of ni - cr alloy ( bellabond , bego , germany ) were used for casting process in an induction casting machine ( fornex t , bego , germany ) . all forty specimens were prepared in a similar manner . of these , three castings were found to be incomplete which were discarded , and the whole process was repeated for them . a single operator carried out the above mentioned process to eliminate the bias and achieve standardization . the specimen were then steam cleaned ( ev 1 , silfradent , italy ) and immersed in an ultrasonic cleaner ( zhengzhou smile dental equipment co. , ltd . specimens were then subjected to sandblasting ( dual blaster , germany ) with different sized aluminum oxide ( al203 ) particles . the sandblasting was carried out for 10 s at a distance of 2 cm , under 2 bar pressure and an approximate angulation of 45. the specimen ( n = 40 ) were divided into four groups . each group ( n = 10 ) was sandblasted with a different grit size of aluminum oxide ( bego , germany ) . thus , the groups divided were as follows : group i - sandblasted with 50 aluminum oxidegroup ii - sandblasted with 110 aluminum oxidegroup iv - sandblasted with 250 aluminum oxide , followed by oxidation and again sandblasted with 250 aluminum oxide . group i - sandblasted with 50 aluminum oxide group ii - sandblasted with 110 aluminum oxide group iii - sandblasted with 250 aluminum oxide group iv - sandblasted with 250 aluminum oxide , followed by oxidation and again sandblasted with 250 aluminum oxide . group i , ii and iii were subjected to oxidation and the experimental surfaces were then coated with two thin layer of opaque feldspathic porcelain ( vita master , germany ) using a brush and fired separately , in a calibrated porcelain vacuum furnace ( ivoclar vivadent p300 ) to a temperature of 950c . body porcelain ( vita master ) was vibrated and condensed onto this fired opaque layer . the specimens were again fired under vacuum to 930c , to achieve a thickness of 1 mm of body porcelain . the thickness of the ceramic layer was measured with a digital vernier caliper ( insize , japan ) [ figure 1 ] . these measurements were again confirmed with the use of a stereomicroscope ( olympus , india ) . glazing was carried out ( vita , germany ) at 910c in the vacuum furnace ( ivoclar vivadent p300 ) . all steps and procedures the universal testing machine [ figure 2 ] is considered standard equipment for evaluating shear bond strength of two dissimilar materials which are conjoined together . the specimen was loaded on the assembly , and a crosshead speed of 0.5 mm / min was used to apply a compressive force at the junction of metal and feldspathic porcelain . the force application continued until adhesive fracture occurred , and the readings of the load applied to that particular specimen were recorded . the same process was repeated for all the 40 specimens , and all the readings of the load applications were expressed in megapascal . universal testing machine with specimen the shear bond strength values of the four groups were statistically analyzed by one - way analysis of variance ( anova ) and tukey 's honestly significant difference ( hsd ) post - hoc test . tables 1 and 2 indicate that the highest mean value of shear bond strength was recorded in group iii ( 337.81 16.97 ) , followed by group iv ( 237.08 4.33 ) , and group ii ( 233.16 3.85 ) . while the lowest mean value of shear bond strength was obtained for group i ( 226.92 1.67 ) . the difference between shear bond strength among all groups was found to be statistically highly significant using one - way anova test ( p < 0.001 ) [ table 3 ] . in order to statistically verify the significance of difference between the individual groups , statistical analysis of the data ( mean values ) of the different groups using tukey 's hsd post - hoc test , group iii and group i , group iii and group ii , and group iv and group iii indicate that the difference is significant . shear bond strength and mean the value of all groups mean , sd , se , minimum and maximum load values for each groups mean shear bond strength for all groups comparison of shear bond strength among all groups using one - way anova test ( p<0.0001 ) comparison of shear bond strength between the individual test groups by tukey 's hsd post - hoc test the purpose of conducting this study was to evaluate the shear bond strength of feldspathic porcelain to ni - cr alloy when subjected to various surface treatments which plays an important role in long - term success of metal - ceramic restorations . the study design gives in - depth understanding of the bonding mechanism which is essential for success of metal - ceramic restorations . it is suggested that the oxide composition and amount of oxide formed are influenced by different surface finishing procedures . an oxide layer forms on the surface of most dental porcelain fused to metal alloys when they were exposed to oxygen at high temperatures . certain metallic element oxides such as fe2o3 , in2o3 , sn2o3 have been shown to accumulate at the porcelain - metal interface . the concentration of these elements does not necessarily indicate a discrete oxide layer and may merely represent a solution of their ions at the metal porcelain interface . the fracture strength of repeatedly fired porcelain veneered to high noble and base metal alloy crowns were measured by barghi et al . they found that as the number of firings increased , the fracture strength of porcelain veneered to high noble alloys decreased but fracture strength for porcelain veneered to base metal alloy did not change significantly . sandblasting the finished surface is thought to remove furrows , overlaps and flakes of metal created during the grinding process . a sandblasted surface may have higher surface energy which increases wetting of the metal during ceramic application . evidence suggests that this roughened surface could also provide mechanical interlocking and increase the surface area for metal - ceramic bonding . in this study sandblasting with aluminum oxide 50 , 110 and 250 grit particle size the analysis of all the parameters used in assessing the bond strength between metal and porcelain has confirmed that the bond is strongest in the surface treated with sandblasting with 250 aluminum oxide particles . this is in contrast to the results obtained by pietnicki et al . in their study which demonstrated highest bond strength between metal and porcelain it would be noteworthy to mention that a study conducted by yadav et al . demonstrated that abrasion conducted with particle size 250 on titanium castings produced some degree of marginal loss . it should be noted that sandblasting with aluminum oxide particles followed by oxidation and again sandblasting significantly decreased the shear bond strength of the material . according to brantley et al . the most reliable evaluation of metal - ceramic bond strength should be based on minimal experimental variables and least residual stresses at metal - ceramic interfaces . cohesive failures occur within the layers of ceramic , even though , metal - ceramic bonding is adequate . in the present study , cohesive failures were considered as a success of metal - ceramic bonding mechanism even though it is clinically considered as failure of metal - ceramic restorations as only the bond strength was to be tested . although laboratory studies offer predictable guidance to a comprehensive selection of materials , clinical longitudinal studies must be encouraged to complement laboratory results and enhance clinical standards of fixed partial denture treatment . within the limitations of this research , it can be concluded that different surface treatments have an important role in determining bond strength of the metal - ceramic interface . the different aluminum oxide grit sizes affect the shear bond strength of metal - ceramic alloys.the shear bond strength is highest when metal alloy is sandblasted with 250 aluminum oxide particles.sandblasting of casting alloys , followed by oxidation and again sandblasting resulted in reduced bond strength as compared to conventional methods . it should be noted here that this comparison is between specimens sandblasted with 250 aluminum oxide particles only . both group iii and group iv specimens were sandblasted with 250 aluminum oxide particles . the results amply demonstrate that the bond strength reduced when sandblasting , followed by oxidation and again sandblasting was done . the different aluminum oxide grit sizes affect the shear bond strength of metal - ceramic alloys . the shear bond strength is highest when metal alloy is sandblasted with 250 aluminum oxide particles . sandblasting of casting alloys , followed by oxidation and again sandblasting resulted in reduced bond strength as compared to conventional methods . it should be noted here that this comparison is between specimens sandblasted with 250 aluminum oxide particles only . both group iii and group iv specimens were sandblasted with 250 aluminum oxide particles . the results amply demonstrate that the bond strength reduced when sandblasting , followed by oxidation and again sandblasting was done . therefore to achieve adequate bond strength between ni - cr alloy and feldspathic porcelain , the alloy surface should be preferably sandblasted with aluminum oxide particles of 250 .

purpose of the study : the purpose was to evaluate the effect of various surface treatments and sandblasting with different particle size on the bond strength of feldspathic porcelain with predominantly base metal alloys , using a universal testing machine.materials and methods : totally , 40 specimen of nickel - chromium alloy were prepared in an induction casting machine . the groups divided were as follows : group i - sandblasted with 50 al2o3 , group ii - sandblasted with 110 al2o3 , group iii - sandblasted with 250 al2o3 and group iv - sandblasted with 250 al2o3 , followed by oxidation and again sandblasted with 250 al2o3 . the dimensions of each specimen were adjusted so as to maintain the thickness of ceramic at 1 mm . the specimen were loaded on the assembly of the universal testing machine , and a cross head speed of 0.5 mm / min was used to apply a compressive force at the junction of metal and feldspathic porcelain . the force application continued until adhesive fracture occurred , and the readings of the load applied to that particular specimen were recorded.results:the means for shear bond strength for group i , ii , iii and iv were found to be ( 226.92 1.67 ) , ( 233.16 3.85 ) , ( 337.81 16.97 ) and ( 237.08 4.33 ) , respectively . means of shear bond strength among the groups were compared using one - way analysis of variance test . comparison between individual groups were made with tukey 's honestly significant difference post - hoc test.conclusion:different particle size and surface treatment have an important role on the bond strength of ceramic - metal interface . greater particle size demonstrated higher bond strength .

diabetes mellitus is the most frequent endocrine disease in developed countries estimated to have affected 366 million people worldwide and is expected to nearly double by 2030 owing to an increase in obesity , life span extension , and better detection of the disease . this global increase has a significant impact on the prevalence of diabetic complications among which diabetic retinopathy ( dr ) takes an important place [ 1 , 2 ] . dr is a leading cause of acquired blindness in working - age adults and has been estimated to represent 12% of blindness in developed countries [ 3 , 4 ] . the prevalence of retinopathy increases with the duration of diabetes and is related to hyperglycemia , hypertension , hyperlipidemia , pregnancy , nephropathy , and anemia [ 57 ] . diabetes causes damage to all the major cells of the retina , vascular cells ( endothelial cells and pericytes ) , and pigment epithelial cells . the vascular disruptions in dr are characterized by abnormal autoregulation of retinal blood flow caused by the loss of the pericytes that normally regulate vessel calibre , breakdown of the inner blood - retinal barrier , thickening of the capillary basement membrane , and damage and proliferation of endothelial cells . characteristic clinical manifestations are the result of four main processes : the appearance of microaneurysms , increased vascular permeability , capillary occlusion , and fibrous and neovascular proliferation . fluid leakage can range from microexudates to the most severe form , namely , macular edema , which can seriously reduce vision . another lesion characteristic of dr is capillary occlusion ( nonperfusion with retinal ischemia ) , which may lead to the proliferation of new vessels ( neovascularization ) , seeking out new routes to irrigate the ischemic area . these new vessels are often surrounded by fibrous tissue , and this fibrovascular complex may adhere to the posterior part of the vitreous body . traction on the vitreous which usually happens with age or with rapid eye movement during sleep can rupture the fragile structure of the new vessels and lead to vitreous haemorrhaging or even retinal detachment . new vessels and fibrous tissue can also close the anterior chamber angle which leads to neovascular glaucoma with severe elevations in intraocular pressure ( iop ) [ 8 , 9 ] . the primary goal of dr treatment is to improve or protect vision by reducing vascular leaking and macular edema formation , retinal ischemia , and growth of fragile new vessels and thereby preventing vitreous hemorrhages and tractional retinal detachment . however , it should be kept in mind that dr can progress towards advanced stages asymptomatically before actually affecting visual acuity [ 3 , 8 , 9 ] . the retina is a metabolically active tissue , and therefore hyperglycemia in diabetes with associated relative or absolute insulin deficiency is thought to adversely affect its normal physiology . various biochemical , hemorheological , and immunological mechanisms have been implicated to explain the vascular disruption in retinopathy [ 1013 ] . recently , numerous clinical and laboratory investigations have identified inflammation as an important factor in the development of dr [ 1417 ] . there is increasing evidence that inflammatory processes have a considerable role in the pathogenesis of dr with multiple studies showing an association of various systemic as well as local ( vitreous and aqueous fluid ) inflammatory factors and the progression of dr . inflammation is present in the development of both major causes of impaired vision in diabetes , namely , increased retinal vascular permeability ( diabetic macular edema ( dme ) ) and neovascularisation ( proliferative diabetic retinopathy ( pdr ) ) [ 1719 ] . one of these mediators is tumor necrosis factor - alpha ( tnf- ) , a proinflammatory cytokine which is known as an initiator of inflammatory reactions . high tnf- levels have been detected in vitreous , serum , and ocular fibrovascular membranes in patients with dr [ 2022 ] . similarly , interleukin-1 beta ( il-1 ) and its downstream signalling molecule caspase 1 are found in high concentrations in the vitreous and retina of diabetic patients [ 21 , 24 , 25 ] . interleukin-6 ( il-6 ) levels in the vitreous are significantly correlated with the severity and progress of dr [ 26 , 27 ] . in patients with pdr , increased vitreous concentrations of the il-1 , il-6 , soluble il-2 receptor ( sil-2r ) , and il-8 were found , whilst the serum of the same patients contained elevated levels of tnf- , il-6 , il-8 , and sil-2r [ 12 , 28 , 29 ] . the mean serum tnf- , il-8 , and sil-2r levels increased with the stage of diabetic retinopathy with the highest levels being detected in patients with the proliferative form . recently , some studies have pointed out the role of proinflammatory cytokine il-12 as a result of local retinal production in the development of dr [ 30 , 31 ] . chemokines such as ccl2 , ccl5 , cxcl8 , cxcl10 , and cxcl12 are also upregulated in the vitreous samples of patients with dr [ 3234 ] . in the serum of dr patients , levels of tnf- , il-1 , ccl5 , and cxcl12 are also found to be significantly increased [ 21 , 34 ] , suggesting that systematic inflammatory reactions are in some respect related to this disease . in addition to increases in the above - mentioned inflammatory mediators , increases in adhesion molecules such as intracellular adhesion molecule-1 ( icam-1 ) and vascular cell adhesion molecule-1 ( vcam-1 ) as well as activation of leukocytes have been found to be related to the progression of dr [ 34 , 35 ] . both molecules icam-1 and vcam-1 promote chemoattraction of leukocytes into the vascular walls and their migration into retinal tissues [ 35 , 36 ] . activated leukocytes via adhesion molecules attach to the vascular epithelium causing the release of inflammatory cytokines , growth cytokines , and vascular permeability factors and thus altering endothelial junctional proteins and allowing leukocytic diapedesis into the retina [ 14 , 17 ] . besides disrupting the inner retinal barrier and compromising the blood - retinal barrier ( brb ) , leukocytes promote angiogenesis . in all these activities , leukocytes further additionally contribute by producing vegf , an inflammatory mediator that increases vascular permeability and induces the expression of icam-1 and vcam-1 , respectively [ 35 , 36 ] . the first step in managing dr is to reduce the risk of its development and progression by controlling the underlying risk factors , namely , hyperglycemia , hypertension , and hyperlipidemia ( table 1 ) . the diabetes control and complications trial ( dcct ) and united kingdom prospective diabetes study ( ukpds ) have shown that intensive glycemic control is associated with a lower risk of retinopathy compared to conventional therapy in type 1 and type 2 diabetes [ 37 , 38 ] . the ukpds also determined that intensive blood pressure control reduces microvascular complications compared with less intensive control . lipid - lowering therapy particularly reducing the levels of serum cholesterol , low - density lipoprotein ( ldl ) , and triglyceride decrease the severity of retinal hard exudates as well as the risk of developing pdr [ 40 , 41 ] . in addition to controlling these modifiable risk factors , regular dilated eye examinations have been shown to reduce the incidence of blindness due to diabetic retinopathy through early detection and timely treatment . however , irrespective of all these efforts not all cases of dr can be prevented which thereby further highlights the need for effective treatment strategies . currently , laser photocoagulation is the primary method of treatment for patients with diabetic retinopathy who are at a high risk for vision loss but unfortunately is not always effective for improving vision . in many cases , when retinal damage and vision loss have already occurred , laser treatment can simply maintain vision and avoid further vision loss . the procedure is uncomfortable , and often repeated treatments are required . moreover , laser photocoagulation is an ablative destroying retinal tissue procedure , where scars always enlarge over time leading to decrease in night vision , colour vision , and peripheral vision as well as loss of 1 or 2 lines of visual acuity in some patients [ 43 , 44 ] . pars plana vitrectomy ( ppv ) is a microsurgical procedure designed to remove vitreous gel usually in order to achieve access to a diseased retina . it is the main method of treating severe complications of pdr such as severe persistent vitreous hemorrhages and tractional retinal detachment . despite standard intervention , vision loss due to dr still occurs at a frightening rate . considering the limitations and side effects of current treatments of dr , there has been a continuing attempt to understand the molecular mechanisms that contribute to the occurrence and changes seen in the diabetic retinas . thus , many researchers have directed their efforts towards better understanding of the microvascular changes in dr in order to develop more effective pharmacologic prevention and treatment as well as establishing new treatment strategies . currently , the three major classes of pharmacologic agents being studied are corticosteroids , vascular endothelial growth factor ( vegf ) antagonists , and agents that are involved in biochemical pathways ( polyol pathways activation , diacylglycerol , protein kinase c ( pkc ) pathway activation , stimulation of cellular oxidative stress , and changes in macromolecule structure and function via the formation of advanced glycation end - products ( age ) ) [ 46 , 47 ] . considering the involvement of the inflammatory processes of low - grade chronic inflammation in the pathogenesis of dr inhibiting the inflammatory pathway could be an appealing treatment option for dr in future practices [ 1619 , 48 ] . since inflammation is identified as a relevant mechanism for dr development , significant effort has been directed to the development of new concepts for the prevention and treatment of dr acting on the inflammatory processes ( table 2 ) . corticosteroids have been included in the treatment of dr and dme due to their anti - inflammatory and antiangiogenic effects . various inflammatory mediators that are upregulated in dr and play a significant role in its pathogenesis including tnf- , il-1 , and vegf are very well modulated with corticosteroids . they have been shown to reduce vascular permeability , reduce the breakdown of the blood retinal barrier , inhibit leukocyte adhesion to vascular walls , and inhibit vegf gene transcription and translation . they rapidly decrease macular edema ; however , their short term and transitory effectiveness limit their application . frequently , new injections are necessary at different time intervals when the antiedematous effects cease depending on the half - life of the steroid being used . systemic corticosteroids are excluded from dr therapy due to ocular complications such as cataract formation , iop , and glaucoma as well as systematic side effects including exacerbation of diabetes . currently , several different steroids are being used to treat dme , namely , triamcinolone , fluocinolone , and dexamethasone [ 4951 ] . intravitreal injection of triamcinolone acetonide ( ivta ) ( kenalog 40 ) , a slow - releasing steroid , is a promising therapy treatment for dme that suppresses inflammation , reduces vascular leakage , and inhibits fibrovascular proliferation . in eyes with dme , it causes a reduction in foveal thickness and shows improvement in visual acuity in several case series [ 53 , 54 ] . however , the treatment effect only lasts approximately 6 months ; therefore , repeated treatments may often be required with a risk limit on the safety due to multiple entries in the vitreous . the most significant complication of ivta is elevation of iop resulting in secondary open - angle glaucoma , which sometimes may be severe and intractable . elevation of iop up to 24 mm hg may occur in about 40% of patients , usually within 3 months . furthermore , cataract formation may become visually significant in about 50% of eyes within 1 year . the rates of injection - related endophthalmitis following ivta have been reported to range between 0.09% and 0.87% per injection . retinal detachment , lens trauma , and vitreous hemorrhage are rarely reported complications of ivta or other intravitreal injections . the use of peribulbar , rather than intravitreal , ta offers reduced risks of endophthalmitis and perhaps other complications ; however , this form of administration has some limitations and is less effective than ivta . the lack of efficacy for chronic use associated side effects and the need for reinjections have led to the development of novel sustained release intravitreal steroid delivery methods . these slow release formulations avoid the need for repeated injections , enabling the use of smaller amounts of corticosteroids with less secondary side effects [ 46 , 47 , 49 , 50 , 61 ] . a steroid drug delivery system in development for use in dme is the ta implant ( i - vation ) , which has already completed a phase i trial in the long - term treatment of dme , and a phase ii clinical trial is being planned . i - vation is composed of biodegradable polymers which slowly degrade over time , thereby bypassing the risk of secondary surgical complications upon removal as compared to nonbiodegradable devices . other corticosteroids which have been incorporated into these devices include dexamethasone and fluocinolone [ 61 , 62 ] . ozurdex is an extended - release biodegradable dexamethasone intravitreal implant that has been recently approved by the food and drug administration ( fda ) for the treatment of macular edema secondary to retinal vein occlusions ( rvo ) and noninfectious uveitis . a phase iii clinical trial of ozurdex use in the treatment of dme is currently underway . a previous study found that 700 g dexamethasone was well tolerated and produced statistically significant improvements in bcva and central retinal thickness at day 90 ; however , at day 180 no significant difference in visual acuity was found , and both treatments groups ( 350 g and 700 g ) had an increased incidence of elevated iop . the fluocinolone acetonide intravitreal implant ( retisert ) is fad - approved for the treatment of chronic , noninfectious posterior segment uveitis . a phase iii clinical trial conducted in patients with dme reported large cases of cataract and glaucoma . another potential new steroid delivery system is the sustained release fluocinolone acetonide nonbiodegradable intravitreal insert ( iluvien ) which is designed to release the drug gradually up to three years . since the device is miniature , it can be injected into the back of the eye with a 25-gauge needle creating a self - sealing hole with the procedure being similar to an intravitreal injection [ 62 , 64 ] . the two ongoing pivotal multicenter trials known collectively as fame study have shown improvement in best corrected visual acuity ( bcva ) at low - dose insert ( 0.19 mg total , approx . 0.23 g / day ) out of the two doses being studied [ 62 , 65 ] . vascular endothelial growth factor ( vegf ) , primarily isoform vegf164/165 , is a potent vasoactive cytokine and a key mediator of blood - retina barrier breakdown and angiogenesis in the ischemic retina . vegf is produced by the pigment epithelial cells , citespery , and endothelial cells of the retina in response to hypoxia from capillary loss and/or microaneurysm formation [ 4 , 8 , 66 , 67 ] . the vegf levels are significantly elevated in patients with dme when compared with nondiabetic eye conditions and its intravitreal concentration increases with the progression of dr from nonproliferative form to active pdr [ 66 , 67 ] . similarly , successful panretinal photocoagulation ( prp ) reduced intraocular vegf levels by 75% in patients treated for ocular neovascularization . these data suggest that specific inhibition of vegf activity may prevent retinal neovascularization and associated blood flow abnormalities . the role of vegf in retinal neovascularization has encouraged the development of drugs that directly inhibit the vegf molecule such as the anti - vegf aptamer , pegaptanib ( macugen ) , the monoclonal antibody fragment ranibizumab ( lucentis ) , and the full - length antibody bevacizumab ( avastin ) [ 46 , 47 ] . pegaptanib ( macugen ) is a selective aptamer directed against the vegf - a 165 isoform . it was the first united states fda - approved ophthalmologic anti - vegf agent for the treatment of choroidal neovascularization in age - related macular degeneration ( amd ) . in phases 1 , 2 , and 3 prospective clinical trial , pegaptanib appeared to improve anatomic and visual outcomes in patients with dme as well as regressing neovascularisation [ 69 , 70 ] . bevacizumab ( avastin ) , a full - length recombinant humanized antibody , is active against all isoforms of vegf - a . case reports and small , nonrandomized pilot studies have documented efficacy of using off - label intravitreal bevacizumab against exudative amd , macular edema from nonischemic central retinal vein occlusion , iris neovascularization as well as diffuse dme , and various complications of pdr [ 72 , 73 ] . the diabetic retinopathy clinical research network ( drcr.net ) in 2007 completed phase 2 of a prospective randomized multicenter clinical trial to determine the safety and possible benefits of this agent . ranibizumab ( lucentis ) , a recombinant humanized antibody fragment , is active against all isoforms of vegf - a . two pilot studies of ranibizumab demonstrated efficacy in the treatment of dme [ 76 , 77 ] . the results of phase 3 of two prospective randomized multicenter comparing clinical trials , one in patients with dme without pdr and another in patients with dme and pdr ( rise and ride ) , have been recently published . the outcome of phase 3 confirmed the results of phase 2 with the conclusion that ranibizumab rapidly and sustainably improved the vision , reduced the risk of further vision loss , and improved macular edema in patients with dme with low rates of ocular and nonocular impairments . notwithstanding all efforts , the results of multiple conducted researches have not yielded promising results for the use of anti - vegf treatments in dme such as in pdr or neovascular glaucoma . this clinical observation therefore indicates that the pathogenesis of dme is multifactorial , and many other factors beyond vegf may play a role in this process . nsaid are a group of drugs including aspirin and salicylate that exert anti - inflammatory activity by inhibiting cyclooxygenase ( cox ) enzyme - mediated eicosanoid formation . the early treatment dr study and the dipyridamole aspirin microangiopathy of diabetes study showed that although treatment of patients with advanced dr with a low dose of aspirin does not have any benefits , the development of retinal microaneurysms is significantly minimised in patients with early stage of dr when treated with a high dose of aspirin ( 900 mg / day ) . clinical trial of specific cox-2 inhibitors has been discouraged given that systemic cox-2 inhibitors increase incidence of heart attacks and strokes . however , in preclinical studies topical administration of cox-2 inhibitor was shown to reduce signs of dr similar to its systematic application without the side effects , and therefore it would be worthy to investigate its therapeutic benefits in future clinical trials . as a key mediator in inflammation , oxidative stress serves as an important target for anti - inflammatory therapy . in diabetic rats , supplements of antioxidants such as vitamins c and e attenuate the development of acellular capillaries and decrease the number of pericyte ghosts . in diabetic animals or models of ischemic retinopathy , blocking increased activity of nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase induced by diabetes prevents vascular leakage and pathological neovascularization , respectively . moreover , these studies also reveal that statin treatment effectively blocks upregulation of nadph oxidase activity in dr , suggesting that the benefit of statin treatment is at least partly due to its activity in blocking nadph oxidase . the ras could be a promising aim for dr treatment since this system is involved in both diabetes and hypertension - induced retinal inflammation and is a pathway that interrelates with multiple other pathways including oxidative stress and ages . specific blockade of the ras with at1r blocker ( losartan , candesartan ) and angiotensin - converting enzyme inhibitor ( enalapril ) has been shown to prevent oxidative stress , inflammation , and vascular damage in diabetic animal models [ 14 , 84 ] as well as reducing the risk and the progression of retinopathy [ 14 , 85 , 86 ] . however , further trials are necessary to resolve and to fully evaluate the beneficial effect of ras blockade before its clinical usefulness is fully understood . in addition to blockers of general inflammation , studies have been performed to determine whether targeting specific inflammatory molecules can be beneficial in dr treatment . as a key player in many inflammatory reactions , etanercept ( enbrel ) is a recombinant fusion protein having anti - tnf- property and is fda - approved for the treatment of psoriasis . a small series of patients with refractory dme were treated with intravitreal etanercept , but no statistically significant improvement was recorded . infliximab ( remicade ) is another tnf- antagonist that is fda - approved to treat crohn 's disease . an investigation of systemic treatment of dme with infliximab has led to a study of administration through intravitreal injection . diabetic retinopathy is a major cause of blindness in developed countries and remains one of the most serious complications of diabetes . thus , recently , many researchers have directed their efforts towards better understanding of the pathogenesis of dr in order to develop new and more effective preventative and treatment strategies with the emphasis being on pharmacologic therapy . however , at this time , primary prevention with intensive glycemic control , strict blood pressure regulation , and lipid - modifying therapy as well as local ocular treatment ( laser photocoagulation and pars plana vitrectomy ) still remain the proved treatment options addressing diabetic retinopathy . as prospective randomized clinical trials accumulate data , the role and guidelines of pharmacologic treatments will become clearer . the discovery that inflammation plays a critical role in the pathogenesis of dr opens up new pathways and methods for the development of novel improved treatments and strategies for this devastating disease . neither the precise mechanism by which the inflammatory cascade is initiated and involved in dr occurrence and development nor the individual roles of particular inflammatory molecules in the different stages of dr are currently fully understood . some inflammatory molecules have multiple functions and different actions on the various pathways of inflammatory processes which open up the challenge of finding an effective drug target . alternatively , drugs targeting several mechanisms simultaneously may be more efficient , and therefore more studies are necessary to understand the endogenous anti - inflammation processes in dr .

diabetic retinopathy ( dr ) , the most common microvascular complication of diabetes mellitus , is estimated to be the leading cause of new blindness in the working population of developed countries . primary interventions such as intensive glycemic control , strict blood pressure regulation , and lipid - modifying therapy as well as local ocular treatment ( laser photocoagulation and pars plana vitrectomy ) can significantly reduce the risk of retinopathy occurrence and progression . considering the limitations of current dr treatments development of new therapeutic strategies , it becomes necessary to focus on pharmacological treatment . currently , there is increasing evidence that inflammatory processes have a considerable role in the pathogenesis of dr with multiple studies showing an association of various systemic as well as local ( vitreous and aqueous fluid ) inflammatory factors and the progression of dr . since inflammation is identified as a relevant mechanism , significant effort has been directed to the development of new concepts for the prevention and treatment of dr acting on the inflammatory processes and the use of pharmacological agents with anti - inflammatory effect . inhibiting the inflammatory pathway could be an appealing treatment option for dr in future practices , and as further prospective randomized clinical trials accumulate data , the role and guidelines of anti - inflammatory pharmacologic treatments will become clearer .

the five , intracranial , recanalized aneurysms were retrospectively identified in five patients from our neurointerventional database who underwent coil embolization without stenting and had follow - up 3d dsa data . the aneurysms were regarded as having achieved complete occlusion according to the modified raymond classification . the mean size ( sd ) of the aneurysms before coil embolization was 7.4 mm ( 2.4 ) . the aspect ratios of the aneurysms were as follows : 1.87 in case 1 ; 1.43 in case 2 ; 2.74 in case 3 ; 1.14 in case 4 ; and 2.13 in case 5 . the recanalized aneurysms were located at the origin of the posterior communicating artery in cases 3 and 4 , at the basilar tip in case 1 , at the anterior communicating artery in case 2 and at the paraclinoid internal carotid artery ( ica ) in case 5 . all of the aneurysms showed recanalization during the follow - up period , and the mean follow - up period ( sd ) was 18.0 months ( 12.9 ) . mra is generally used for follow - up after coil embolization and if the mra shows recanalization of the treated aneurysm , 3d dsa is performed in order to confirm the recanalization . 3d dsa was obtained on axiom artis zee ( siemens ag , erlangen , germany ) , and the 3d data were available as dicom data after reconstruction on an x - workplace ( siemens ag , erlangen , germany ) . as we described in our previous study , we used a new cfd platform ( cfd 3.0 , prototype- not for diagnostic use ; siemens ag , erlangen , germany ) for cfd analysis . the dicom data were transferred to this platform . using this platform , all of the integral steps for producing a 3d model of the patient - specific vascular structure are available , including the initial dicom processing , segmentation , and surface - mesh generation . during the step of surfacemesh generation , we could create a paired model of one patient that consisted of a completely occluded aneurysm model and recanalized model based on patient - specific 3d dsa data using this research cfd platform . the virtual model of the completely occluded aneurysm was generated by artificially cutting off the recanalized aneurysm from the parent artery using this cfd platform . after this process , the virtual arterial wall under the identical conditions as the normal arterial wall was generated for the cross - section of the artificial cutting . in the next step , the inlet and outlets of the target arterial structure were designated in order to define the boundaries . the results were then saved in specific files in the stl format in order to progress to the next steps using this research cfd platform , although they could also be exported elsewhere . using this protocol , we could create two 3d models of vascular structures , i.e. a completely occluded aneurysm model and a recanalized aneurysm model , in the same patient , based on the dicom data of 3d dsa which were obtained so as to confirm the recanalization of the treated aneurysm . this new research cfd platform also includes the solver which is based on the " lattice boltzmann method " , a promising numerical scheme used for simulating complex fluid dynamics in the most varied applications , including models of the cerebral artery . the essential step of surface - mesh generation for the simulation domain was automatically performed and also integrated into the solver . calculations were performed using the following material constants : the density and dynamic viscosity of blood as 0.001 g / mm and 0.004 poiseuille ( pa s ) ; the number of cardiac cycles ( default , 2 ) ; and the output frequency ( default , 0.05s ) . the velocity profile of the inlet was def ined as pulsatile , and which corresponded to a pulse length of 0.87s and minimum and maximum flow velocities of 12.7 cm / s and 30.0 cm / sec , respectively . we used the average reynolds number which was within the range of normal blood flow in human arteries , as had been used in previous studies . in addition , the blood - vessel wall was assumed to be rigid with a no - slip boundary condition . the flow velocity magnitude and the wss at the systolic peak around the neck of the aneurysm were calculated , and the results were saved in specific files as vtk format for visualization . visualization of the results was performed using paraview 3.0 ( sandia corporation , kitware inc . ny , usa ) which could allow comparison of the two data sets at the same view and rotation angles by pixel - by - pixel bases . flow - velocity magnitudes around the neck of the aneurysms at the systolic peak were visualized with instantaneous streamlines as flow patterns in the 3d space using glyph rendering , and the wsss around the neck of the aneurysm were also visualized with a color map in the 3d space using surface rendering . these qualitative results of hemodynamic factors were obtained from cfd analysis of the completely occluded aneurysm model and recanalized aneurysm model in the same patient . finally , we evaluated the correlation between the hemodynamic factors and recanalization of the aneurysm following coil embolization . the five , intracranial , recanalized aneurysms were retrospectively identified in five patients from our neurointerventional database who underwent coil embolization without stenting and had follow - up 3d dsa data . the aneurysms were regarded as having achieved complete occlusion according to the modified raymond classification . the mean size ( sd ) of the aneurysms before coil embolization was 7.4 mm ( 2.4 ) . the aspect ratios of the aneurysms were as follows : 1.87 in case 1 ; 1.43 in case 2 ; 2.74 in case 3 ; 1.14 in case 4 ; and 2.13 in case 5 . the recanalized aneurysms were located at the origin of the posterior communicating artery in cases 3 and 4 , at the basilar tip in case 1 , at the anterior communicating artery in case 2 and at the paraclinoid internal carotid artery ( ica ) in case 5 . all of the aneurysms showed recanalization during the follow - up period , and the mean follow - up period ( sd ) was 18.0 months ( 12.9 ) . mra is generally used for follow - up after coil embolization and if the mra shows recanalization of the treated aneurysm , 3d dsa is performed in order to confirm the recanalization . 3d dsa was obtained on axiom artis zee ( siemens ag , erlangen , germany ) , and the 3d data were available as dicom data after reconstruction on an x - workplace ( siemens ag , erlangen , germany ) . as we described in our previous study , we used a new cfd platform ( cfd 3.0 , prototype- not for diagnostic use ; siemens ag , erlangen , germany ) for cfd analysis . the dicom data were transferred to this platform . using this platform , all of the integral steps for producing a 3d model of the patient - specific vascular structure are available , including the initial dicom processing , segmentation , and surface - mesh generation . during the step of surfacemesh generation , we could create a paired model of one patient that consisted of a completely occluded aneurysm model and recanalized model based on patient - specific 3d dsa data using this research cfd platform . the virtual model of the completely occluded aneurysm was generated by artificially cutting off the recanalized aneurysm from the parent artery using this cfd platform . after this process , the virtual arterial wall under the identical conditions as the normal arterial wall was generated for the cross - section of the artificial cutting . in the next step , the inlet and outlets of the target arterial structure were designated in order to define the boundaries . the results were then saved in specific files in the stl format in order to progress to the next steps using this research cfd platform , although they could also be exported elsewhere . using this protocol , we could create two 3d models of vascular structures , i.e. a completely occluded aneurysm model and a recanalized aneurysm model , in the same patient , based on the dicom data of 3d dsa which were obtained so as to confirm the recanalization of the treated aneurysm . this new research cfd platform also includes the solver which is based on the " lattice boltzmann method " , a promising numerical scheme used for simulating complex fluid dynamics in the most varied applications , including models of the cerebral artery . the essential step of surface - mesh generation for the simulation domain was automatically performed and also integrated into the solver . calculations were performed using the following material constants : the density and dynamic viscosity of blood as 0.001 g / mm and 0.004 poiseuille ( pa s ) ; the number of cardiac cycles ( default , 2 ) ; and the output frequency ( default , 0.05s ) . the velocity profile of the inlet was def ined as pulsatile , and which corresponded to a pulse length of 0.87s and minimum and maximum flow velocities of 12.7 cm / s and 30.0 cm / sec , respectively . we used the average reynolds number which was within the range of normal blood flow in human arteries , as had been used in previous studies . in addition , the blood - vessel wall was assumed to be rigid with a no - slip boundary condition . the flow velocity magnitude and the wss at the systolic peak around the neck of the aneurysm were calculated , and the results were saved in specific files as vtk format for visualization . visualization of the results was performed using paraview 3.0 ( sandia corporation , kitware inc . ny , usa ) which could allow comparison of the two data sets at the same view and rotation angles by pixel - by - pixel bases . flow - velocity magnitudes around the neck of the aneurysms at the systolic peak were visualized with instantaneous streamlines as flow patterns in the 3d space using glyph rendering , and the wsss around the neck of the aneurysm were also visualized with a color map in the 3d space using surface rendering . these qualitative results of hemodynamic factors were obtained from cfd analysis of the completely occluded aneurysm model and recanalized aneurysm model in the same patient . finally , we evaluated the correlation between the hemodynamic factors and recanalization of the aneurysm following coil embolization . the area with increased wss at the systolic peak of the cross - sectionally oriented neck of completely occluded aneurysms was observed in five cases ( fig . the flow - velocity magnitude at the systolic peak around the neck of the completely occluded aneurysm was not increased in all five cases . however , a small vortex formation with relatively low velocity near the neck of a completely occluded aneurysm was identified in four cases , except for case 5 ( fig . 3 ) . the increased wss and the small vortex formation developed at almost the same locations in four cases , except for case 5 . in addition , the recanalization points corresponded almost exactly to the location of the increased wss and/or the small vortex in all five cases . finally , the blood flow entered the sac of recanalized aneurysms through the impingement region and it formed a large and complex vortex in the recanalized aneurysmal sac ( fig . several studies have reported recanalization of a completely coiled aneurysm as modified raymond scale 1 , despite the best efforts of neurointerventionists . hemodynamic features around the perianeurysmal regions may have an important role in bringing about the recanalization of a coiled aneurysm , such as other risk factors including the aneurysm characteristics , patient characteristics , and technical problems . therefore , we performed cfd analysis of the local hemodynamic features both before and after recanalization using novel virtual models . our study shows that in completely coiled aneurysms that resulted in recanalization , there were increased wss and a small vortex formation with a relatively low velocity at the cross - sectionally oriented neck of coiled aneurysms . the previous studies regarding the cfd analysis of coil - embolized aneurysms were based on computer - generated aneurysm models , not on patient - specific aneurysm models , and which may result in a potential bias in the following computational simulation analyses . for example , some studies made the geometry of parent arteries and branches as straight without flexures and diameter variation and made the aneurysms with a smooth surface and almost globular shape . in order to overcome these limitations , recent cfd analysis studies of the hemodynamics around coil - packed aneurysms were based on patient - specific data derived from 3d dsa or 3d mra . since using the patient - specific data , the cfd analysis for hemodynamic factors has been made possible in various structures of parent arteries and aneurysms . however , a previous study that performed cfd analysis for identifying hemodynamic features on recanalization used the 3d dsa data of completely coiled aneurysms as the modified raymond scale . however , several studies have reported that detachable coils in the treated aneurysm may cause an artifact which sometimes makes it difficult to evaluate whether the tiny residual neck is seen on 3d dsa or 3d mra . therefore , we decided to create new virtual models of completely occluded aneurysms based on patient - specific 3d dsa data in order to remove the possible presence of a residual neck following coil embolization . several recent studies have focused on the hemodynamic patterns of embolized aneurysms with a residual neck using cfd analysis based on patient - specific imaging data . these studies reported that high wss and/or high blood flow velocity were identified near the residual neck of partially embolized aneurysms susceptible to future recanalization . however , there have only been a few studies which attempted to identify the hemodynamic patterns which lead to the recurrence of totally embolized aneurysms , using cfd analysis based on patient - specific imaging data . evaluated the hemodynamic features at the aneurysm neck using cfd analysis with patient - specific 3d dsa , while attempting to identify the hemodynamic risk factors leading to the recurrence of completely embolized aneurysms without residual lesions . they discovered that increased wss at the completely occluded aneurysm neck after embolization may be an important risk factor that contributes to recanalization of an aneurysm . in our study , there were points at which there was increased wss at the systolic peak near the neck of completely occluded aneurysms in all f i ve cases . in canine models , disruption of internal elastic lamina , loss of medial smooth - muscle cells , reduced proliferation of smooth - muscle cells , and loss of fibronectin were observed at the regions with higher wss than at the normal physiological levels . other studies also claimed that wss higher than the normal physiologic level may disrupt the normal function of endothelial cells . byun et al . also reported that high wss after coil embolization may disrupt thrombus formation in embolized aneurysms . finally , our study supported that increased wss near the neck of a completely embolized aneurysm can cause future recanalization probably via disruption of the neointima formation of the neck as well as impediment of thrombus formation into the aneurysmal sac . in our study , a small vortex formation with relatively low velocity near the neck of a completely occluded aneurysm was identified in four cases . this phenomenon indicates that there are certain amount of energy loss leading to a small vortex formation and subsequent stagnation of blood flow near the neck of a completely occluded aneurysm . several studies have reported that there was a significant relationship between the focal endothelial injury and the stagnation of blood flow . in addition , nakatani et al reported that the stagnation of blood flow may cause anoxic injury to endothelial cells and accumulation of platelets or leukocytes that damage intimal tissue . therefore , stagnation of blood flow may interfere with neointima formation of the neck of a completely embolized aneurysm . first , we obtained the control model for comparison by removing the coil mass and recurred aneurysm and which may differ from the initial vessel status after coil embolization . however , there is also an advantage to obtaining better cfd results by avoiding metal artifacts which usually makes cfd analysis difficult at the neck . second , our sample size was relatively small as only five patients were included in this study . however , this newly developed cfd platform could provide rapid processing and comparison of the results even though it was qualitative . fourth , several assumptions were made regarding the cfd analysis , including the newtonian flow condition , zero pressure boundary condition at all of the outlets , and the rigid with no - slip boundary condition of the arterial wall . however , discrepancies resulting from such an assumption can be compensated for paired cfd model comparison with the control . our study shows that increased wss and small vortex formation were identified in the cross - sectional area of the neck in completely occluded aneurysms using paired virtual cfd modeling methods . this observation may increase the evidence regarding the hemodynamic characteristics which can explain the recanalization mechanism in completely occluded aneurysms by coiling . cfd analysis using patient - specific 3d dsa may be helpful in order to identify coil - embolized aneurysms which have a risk of recanalization during the follow - up period .

purposehemodynamic factors are considered to play an important role in initiation and progression of the recurrence after endosaccular coiling of the intracranial aneurysms . we made paired virtual models of completely coiled aneurysms which were subsequently recanalized and compared to identify hemodynamic characteristics related to the recurred aneurysmal sac.materials and methodswe created paired virtual models of computational fluid dynamics ( cfd ) in five aneurysms which were initially regarded as having achieved complete occlusion and then recurred during follow - up . paired virtual models consisted of the cfd model of 3d rotational angiography obtained in the recurred aneurysm and the control model of the initial , parent artery after artificial removal of the coiled and recanalized aneurysm . using the cfd analysis of the virtual model , we analyzed the hemodynamic characteristics on the neck of each aneurysm before and after its recurrence.resultshigh wall shear stress ( wss ) was identified at the cross - sectionally identified aneurysm neck at which recurrence developed in all cases . a small vortex formation with relatively low velocity in front of the neck was also identified in four cases . the aneurysm recurrence locations corresponded to the location of high wss and/or small vortex formation.conclusionrecanalized aneurysms revealed increased wss and small vortex formation at the cross - sectional neck of the aneurysm . this observation may partially explain the hemodynamic causes of future recanalization after coil embolization .

in st - elevation myocardial infarction ( stemi ) the time of blood flow re - establishment in infarct area is crucial . in addition to fast diagnosis , immediate and accurate stratification of high - risk patients is also important in this situation . the 12-lead electrocardiogram ( ecg ) in emergency rooms is the most feasible and valuable test in patients with acute chest pain for diagnostic and therapeutic measures . stenosis of the proximal portion of rca and rv involvement may cause rv failure and poor prognosis ( 1 ) . considerable numbers of patients with highly suspected mi have normal on - admission 12-lead ecg , hence serial ecgs and also additional leads ( right and posterior leads ) may help physicians to deal with such cases ( 2 ) . many studies in this field suggested stepwise algorithms and criteria to predict the culprit artery , like fiol et alandtierala et al ( 3,4 ) . in this study we focused on fiol s algorithm and some other pre - specified criteria for prediction of occluded vessel in single vessel inferior stemi . in addition the relation of culprit lesion with st segment deviation in posterior and right precordial leads were also analyzed in terms of odds ratio and confidence intervals . in this retrospective study , of a total of 138 studied patients , 25 had 3-vessel disease and 37 had two occluded arteries . the remaining 76 patients were diagnosed with single vessel acute i - stemi , in which 56 ( 74% ) had right coronary artery ( rca ) occlusion [ 22 ( 29.3% ) proximal rca , 24 ( 32% ) middle rca , and 10 ( 13.3% ) distal occlusion of rca ] , 19(25% ) left circumflex artery ( lcx ) lesion and one middle left anterior descending ( lad ) artery occlusion . normal coronary was detected in one patient , which excluded based on the first decision of authors and the suggestion of similar studies . we enrolled 76 consecutive patients with acute single vessel myocardial infarction who admitted in tehran heart center ( thc ) from 2007 to 2012 and underwent clinically indicated percutaneous coronary interventions . all of them fulfilled following criteria : single - vessel occlusion , no sign of left bundle branch block and left ventricular hypertrophy in ecg , no history of coronary artery bypass graft and myocardial infarction . in emergency department ( ed ) of thc we routinely record 16-lead ecg ( conventional 12-lead ecg plus right ( v4r ) and posterior ( v7 to v9 ) leads ) . all ecg records and angiographic films were read and reported separately by two expert cardiologists without being aware of the patients condition . st changes were measured at 80 ms from j points with the tp segment fallen into the iso - electric line of the ecg . the st deviations < 0.5 mm in frontal leads and < 1 mm in precordial leads were regarded as isoelectric . significant stenosis was considered when > 70% narrowing of luminal was present . at the time of discrepancy , the maximum time between symptoms onset and coronary angiography in our patients was less than a week of hospitalization . categorical data were analyzed in terms of frequency and continuous data presented as means sd . dichotomous variables were compared by chi - square test and independent samples t - test used to compare means of continuous variables(age in sex groups ) . in order to explain the relation of variables , odds ratios and confidence intervals were also calculated . categorical data were analyzed in terms of frequency and continuous data presented as means sd . dichotomous variables were compared by chi - square test and independent samples t - test used to compare means of continuous variables(age in sex groups ) . in order to explain the relation of variables , odds ratios and confidence intervals were also calculated . the mean age of the patients was 56 10 ( maximum : 79 , minimum : 32 ) . female patients constituted 20% of the sample size with the mean age of 62 9 which was higher than male patients , 5510 . the age difference was significant in sex groups ( p=0.023).the youngest patient was a 32 year - old man and the oldest a 79 year - old man.angiographic data of all patients are displayed in table 1.univariate analysis of some traditional criteria , right and posterior criteria are shown in table 2 . we could not reach a significant multivariate regression model to predict the site of stenosis , due to lack of linearity of the effects for some criteria . * prior to the origin of the acute marginal artery , * * betweenacute marginal artery and the origin of the pda , * * * prior to first obtusmarginatum ( om1 ) we analyzed each step of fiol s algorithm in terms of specificity and sensitivity.fiols algorithm analysis is shown in fig 1 and table 3 . in step one , st depression in lead i , 38 patients were truly predicted as rca . the st was elevated in four patients in lead i which was associated with lcx occlusion in 3(75% ) . the other 26 patients , including one with lad occlusion , with the isoelectric st in lead i was assessed in the second step . before analyzing through the second step of fiol s algorithm , the site of the culprit artery in 41 of 75 patients was defined . in the second step nine patients had st elevation iiiii and in five of them lcx was the infarct - related artery ; three had rca as culprit lesion and one occlusion of mid lad . of these , 14 were truly predicted as rca but the remained two were in fact lcx . the patient with correctly diagnosed lcx occlusion ( based on this step ) had proximal lcx occlusion . the overall results of these 3 steps and each step of the algorithm are shown in table 3 and figure 1 respectively . we had 22 patients with prca lesion ( 17 with avl depression1 ) and 34 patients with non - proximal occlusion of rca ( 26 with avl depression1 ) . sensitivity and specificity of avl depression1 mm were 77% and 31% respectively for prca prediction ( p value=0.41 ) . this criterion was not able to define proximal vs. distal in rca and only significant to differentiate rca from lcx lesion ( p value=0.017 ) with sensitivity and specificity of 77% and 63% respectively . st elevation in 2 of 3 leads ii , iii , avf ( n=76 ) isoelectric or elevated v1 was another criterion , which was suggested by turhan ( 5 ) for prca prediction . although 86% of patients with prca occlusion had this criterion , in our analysis it was not statistically associated with the site of occlusion ( proximal , middle and distal ) within the rca ( p value=0.116 ) . in this study , sum of st - elevation in lead v5 and v62.5 mm had 63% and 91% sensitivity and specificity respectively for lcx prediction ( p value=0.044 ) . both sensitivity and specificity of this criterion were higher than those of tierala and fiol for lcx lesions . in clinical and therapeutic decision - making , the initial steps are to determine the culprit vessel and the location of the occlusion within the vessel ( 6 ) . many criteria and algorithms were proposedin recent studies but their specificity and sensitivity are limited . small sample size and confounding multi - vessel involvement multi vessel involvement ( two or three vessel disease ) is not discussed in this study however the authors believe that it would affect the results very much . in previous study by tierala et al ( 4 ) they included multi vessel involvement in addition to left main stenosis , which resulted in high degree accuracy of prediction ( from 12 lead ecg to angiographic results ) . for instance , in rca occlusion , the injury vector is toward the right side and results in st depression in leads i and avl . hira et al ( 7 ) suggested a new criterion based on mirror effect : st - t pattern in lead avl would be the mirror of lead iii , if the culprit artery is rca or distal lcx . hence lack of this mirror pattern is suggestive for proximal lcx . the magnitude of avr depression was another criterion based on reciprocal concept that literature ( 8) . as lead avr was placed in more opposite direction than avl , in cases of lcx occlusion , its magnitude of deviation had better chance distinguishing lcx as infarct related artery ( 3 - 8 ) . although it was not significant in our study , we recommend the use of this concept in future studies with a larger sample size . the reported sensitivity and specificity of fiol et al ( 3 ) study , for rca prediction , were 96% and 38% respectively ( reported by tierala ) . moreover fiol et al did not include inferior stemi due to apical mi and lad occlusion in their algorithm . one of our cases had stenosis in mid portion of lad that resulted in apical mi . tierala s algorithm ( 4 ) in previous study by hosseini et al ( 9 ) had 86% sensitivity and 50% specificiy which were both lower than fiol s algorithm study . wong et al ( 8) suggested the ratio of t - wave amplitude in lead iii to that of lead ii was more than 1 , as a criterion of rca vs. lcx . the 80% sensitive and 43% specific for rca detection in our analysis was not significant ( p=0.062 ) . although it is easier to measure t - wave amplitude than st elevation in leads iii and ii , but it was not a good substitute for data analysis . in addition , invert or negative - biphasic t - wave in their study was related to the prediction for rca , but this was not significant in our study ( p=0.4 ) . kanei et al ( 10 ) proposed avr depression 1 mm for prediction of lcx . nevertheless we had 14 patients with avr depression1 mm in which eight had rca occlusion ; and the remaining six , lcx was the culprit artery . sensitivity and specificity of this criterion for lcx prediction were 32% and 86% respectively ( p=0.094 ) . to define the proximal occlusion of rca , fiol et al ( 6 ) proposed avl depression1 mm as a criterion . the st segment elevation in v4r ( occluded before right ventricular branch ) was useful for identifying occlusion of rca . occlusion of proximal - rca in right dominant hearts , led to st elevation of right precordial leads due to rv involvement . however , in such dominant rca infarction of posterior wall could reciprocally depress st segment in right precordial leads . hence , the association between rv involvement and the site of rca stenosis could be a challenging phenomenon . although lcx obstruction and consequent mi could be missed by on - admission ecg but lcx can supplies the postero - lateral wall and posterior part of the inferoposterior septum . additionally the lcx can give rise to pda in a proportion of patients ( 8) thus , recording posterior leads will help identifying stemi due to lcx stenosis . the st segment elevation in posterior leads suggested to be related to lcx occlusion ( 11 ) . in this study we also found positive relationship between lcx stenosis and st deviation in posterior leads . actually , beat - to - beat variations in ecg can influence the reported magnitude by cardiologists . to deal with this issue we calculated the mean for the st elevation in three consecutive beats . in conclusion , adding other right and posterior leads to the conventional 12-lead ecg will improve decisions in ed , and also help taking faster therapeutic measures in high risk patients . benefit of adding posterior and right leads to 12-lead ecg is shown to be noteworthy in present study . prediction of the site of occlusion in lcx and also rv involvement will be more accurate when posterior and right precordial leads were taken into consideration .

background : in addition to diagnosing the acute myocardial infarction ( mi ) , stratifying high - risk patients and proper treatment strategies are important issues in managing patients complaining of chest pain and suspecting mi . many studies have been conducted to predict the occlusion site by interpreting the st segment deviations in electrocardiogram ( ecg).additional posterior and right precordial leads are suggested in literature to increase the sensitivity of prediction . the goal of this study was to determine the relation of st segment changes in ecg ( conventional 12 leads ecg besides right and posterior leads ) with the site of occlusion within the vessel . methods : retrospectively , from total 138 patients , 76 of them were analyzed as single vessel acute inferior st elevation myocardial infarction ( i - stemi)-st which 56 ( 74% ) had right coronary artery ( rca ) occlusion [ 22(29.3% ) proximal rca , 24(32% ) middle rca and 10(13.3% ) distal occlusion of rca ] , 19(25% ) had left circumflex artery ( lcx ) lesion and one had middle left anterior descending ( lad ) artery occlusion . on admission ecgs and coronary artery intervention films , were reported within maximum time of 6 days in hospital stay , and re - evaluated by two cardiologists . results : fiol s algorithm was 93% sensitive and 50% specific for predicting rca occlusion . the st elevation in lead iii was associated with rca stenosis ( odds ratio ( or ) : 12 , confidence interval ( ci ) : 2.2 - 68.9 ) , the association between st elevation in lead ii with lcx involvement was not significant . the v4r was a good marker for rv involvement on - admission , ( or=8 , ci : 1.6 - 37.5 ) . sum of st deviation in posterior leads ( v7 to v9 ) 2 mm had positive and significant relation to lcx stenosis ( or=4 , ci : 1.3 - 14 ) . conclusion : benefit of adding posterior and right leads to 12-lead ecg is shown to be noteworthy in present and prior studies , in identifying lcx stenosis and poor prognosis involvement .

quality of life is defined as one s perception style of his own situation in the system of his culture and values . in this concept , physical health , mental health , independency level , social relations , environmental factors and personal beliefs take part on the basis of subjectivity . quality of life brings a humanistic point of view to today s medicine which focuses on mechanic treatment of diseases and alleviation of symptoms ( 1 ) . since every researcher studies life quality according to their subject , there are many different definitions of quality of life and many different approaches of life quality emerged , as well . in previously conducted researches , data for quality of life indicators were tried to be collected generally in two separate sections as objective indicators and subjective indicators . being physically healthy , managing his own life and self - care are among the objective indicators of life quality . it is important for an individual to evaluate his own life and find it positive ( 4 , 5 ) . how socio - demographic factors effect life quality is indicated in a yugoslavian study . according to this study , it was determined that people living in towns had better life quality than people living in villages and educated elders had better life quality than uneducated youth ( 6 ) . foster et , al , were interested in subscales related to sleeping and pain . an important relation between sleeping disorder and relapse also , it was observed that pain was associated with using analgesic though direct relation could not be detected ( 7 ) . life quality includes many areas of life such as meeting of basic needs and social expectations of an individual and benefiting from the opportunities provided by the society in which he lives . first studies investigating life quality in mental disorders were in consultation liaison psychiatry and they were followed by studies on mood disorders , anxiety disorders , and schizophrenia and childhood - senility psychiatry ( 8 , 9 ) . rudolp et , al , observed that subjective life evaluation of the patients depended on their inner worlds rather than their outer worlds ( 10 ) . using the concept of life quality in medicine has been effective in changing the traditional , obsolete , narrow - minded views ( 8 , 11 ) . recently , the most common scale used to measure the quality of life has been short - form health survey titled as 36 ( sf36 ) . each of them contains 4 sub - groups ( i.e. total of 8) : physical functioning , limitations of function due to physical health problems , bodily pain , social functioning , general mental health , loss of function due to emotional problems , life and general health perception ( 6 ) . the purpose of this study was to investigate the prevalence of a group of mental health symptoms among health sciences students , and to determine the association between mental health symptoms prevalence and quality of life . data collection and samples from september to december 2015 , a descriptive survey was conducted university students attending at afyon health school . population of this study consists of 1250 students studying at afyon health school in the departments of nutrient and dietetics , physiotherapy and rehabilitation , nursing and health management . sample selection was not taken , all the students ( 429 ) that could be reached and that accepted to participate in the study were taken into scope of study . in order to conduct the study , written permission was taken from afyon kocatepe university medical faculty board of ethics and management of the school where the study was conducted . additionally , informed consent was taken from the students participating in the study voluntarily , and that if at any point they decided not to complete the questionnaire , they could withdraw from the study . in the study , brief symptom inventory and quality of life- sf-36 were employed to analyze psychological symptoms and general quality of life perception of young . data were collected by filling in the questionnaires in a determined class hour with the consent of instructor of the class and the students . socio - demographic characteristics , number and percentage of the students were given as average points of bsi and sf-36 scales . relation between their mental health and quality of life was evaluated by correlation and multi - linear regression analyses . statistical analyses were conducted by using spss for windows 15.0 and significance level was accepted as p<0.05 . descriptive form : it contains 18 questions in total about socio - demographic characteristics such as age , sex , marital status , department , grade , type of work , occupation and choosing the department willingly , satisfaction with the department , using cigarette and alcohol and success . brief symptom inventory ( bsi ) : it is a likert type symptom scanning scale which consisted of 53 items . the bsi was developed as a multidimensional test to measure current levels of psychological symptoms ( 12 ) . each item on a 5-point scale are evaluated between the points of 0 ( not at all ) to 4 ( extremely ) . subscales are , somatization , depression , anxiety , hostility and negative ego . global indices are global severity index ( gsi ) , positive symptom distress index ( psdi ) , and positive symptom total ( pst ) ( 12 ) . global indices are the parts that show actual functioning and level of indicator with different approaches ( 13 ) . dsi is obtained by dividing the total scores of all items , except for the blank ones , into 53 . if dsi is found above 1 , it shows psychopathological proneness in symptoms . in this study , tsi is the total score as a result of accepting all the items ( items that are positive ) as 1 except for the items marked as 0 . sri is obtained by dividing total scores of subscales into total of symptom ( 13 ) . turkish validation and reliability of were done for the samples formed by adults ( 1994 ) , adolescents and university students ( 2002 ) by ahin et , al , ( 14 ) . it is stated that bsi is a useful scale for measuring psychological problems in the studies of mental health screening ( 12 , 15 ) . quality of life scale - short form ( sf-36 ) : sf-36 which is a self - evaluation scale developed by ware ( 1987 ) was used in order to measure the life qualities of the young people related to health ( 16 ) . sf-36 is a general questionnaire which can be widely used for various populations and under various conditions and it was designed for individuals evaluating their health states from their own point of view ( 17 , 18 ) . turkish adaptation of sf-36 and its validity and reliability studies were conducted by pnar ( 19 ) , and its cronbach alpha value was found as 0.91 ( 19 ) . sf-36 scale contains 36 questions grouped as 8 sub - health states : physical function ( pf ) , role -physical ( rp ) , bodily pain ( bp ) , general health ( gh ) , energy / vitality ( v ) , social function ( sf ) , role- emotional ( re ) , mental health ( mh ) ( 17 ) . physical ( physical health component summary scale , pcs ) and mental ( mental health component summary scale , mcs ) health summary score is obtained by adding scores of sf-36 quality of life scale ( 17 ) . each health dimensions of sf-36 scale is scored between 0 - 100 and as the score increase , it means less limitation , better quality of life related to health or less pain ( 20 ) . the mean age of the students who participated in the study is 20.981.78 , mean of their body - mass index ( bmi ) is 21.612.85 and their cumulative grade point average ( cum - gpa ) is 2.630.38 . distribution of personal information of 429 college students forming the sample such as sex , age , msi , department , grade , satisfaction of department , income state are given in table 1 . numerical and percentage distribution of the profile of the students . of the students participating in the study ; 71.6 % is female , 100% is not married , 48% is at the age of 20 - 21 , 58.3% is staying at dormitory . it was discovered that 83.9% of the students chose their departments willingly , 56.9% of them had adequate monthly income , % 56.9 of them had adequate family monthly - income , and 47.3% of them had 3 - 4 siblings ( table 1 ) . it was found that of the students , 27.36% enjoyed listening to music , 25.44% enjoyed internet , 24.76% enjoyed going to cinema and theatre , 9.71 % enjoyed doing sport regularly and 7.8% enjoyed playing musical instruments . 42.03 % of the students often listens to rock , hip - hop music which is energetic , upbeat , renewed and consumed more quickly . additionally , 25.36% of the students listens to turkish classical - folk music , 20.65% of them listens to classical music and frequency of listening to arabesque music is at low level of 11.96% . it was discovered that of the students , 71.3% was not satisfied with their bodies , 85.75% had health problems , 82.3% did not use cigarettes , 87.2% did not use alcohol , 67.1% did not have sleeping problems and 91.6 % was not exposed to violence . while average score that students got from bsi was 57.0532.56 , it was found that , from the subscales , depression was 1.310.75 , hostility was 1.220.67 , anxiety was 1.000.65 , negative self - perception was 0.990.70 and somatization was 0.850.59 ( table 2 ) . according to the average of the scores , frequency of mental symptoms of the students is normal . among the global indices , while average of gsi ( 1.070.61 ) and sdi s ( 1.720.48 ) being above 1 shows that symptoms are at psychopathological level , it is seen that score of tsi ( 26.49 12.8 ) is at medium - level . bsi : brief symptom inventory , gsi : global severity index , sdi : symptom discomfort index , tsi : total symptom index , sf-36 : quality of life , pf : physical function , pr physical role , bp : bodily pain , gh : general health , vt : vitality , sf : social function , re : role emotional , mh : mental health , pcs : physical component summary , mcs : mental component summary . although average of their score which they got from sf-36 is 46.186.55 , when sub - scale scores were examined , it was found that pf was 50.098.63 , pr was 45.3411.17 , bp was 51.178.53 , gh was 47.327.92 , vt was 50.288.57 , sf was 45.0910.25 , re was 42.6413.38 , and mh was 43.679.50 . of the sf-36 indices , it was found that they got 48.927.93 from pcv and 43.449.52 from mcs . it was found that scores of sf-36 scale , sub - scales and indices were generally low ( table 3 ) . gsi : global severity index , sdi : symptom discomfort index , tsi : total symptom index , pcs : physical component summary , mcs : mental component summary , sf-36 : quality of life . the relation between the scores which students got from bsi , sub - scales of bsi and global indices and sf-36 and indices was given after evaluated by correlation analysis . according to results in table 5 , it was found that there was a medium - level negative relation between sub - scales of bsi and sf-36 , pcs and mcs . strong - level negative relation was found between msi which we use with the aim of general assessment of psychopathology and quality of life . as the mental problems of the students increase , their life qualities decrease . according to results in table 4 , there is significant relation between dsi global index which shows pathological level of mental state and quality of life ( p<0.005 ) . any significant relation between other global indices and quality of life could not be found ( p>0.05 ) . the relation between mental state and quality of life was found statistically significant ( f : 70.894 ; p<0.000 ) . among variables in addition , determination coefficient ( r ) was calculated as 0.441 and it can be said that % 44.1 of the changes in mental state depends on quality of life . mental state symptoms and quality of life related to health do not have the same meaning . mental health is defined as individual s perception of feeling well about physical , emotional , cognitive or psychosomatic states . on the other hand , quality of life ( or functional state ) the purpose of this study was to investigate the prevalence of a group of mental health symptoms among health sciences students , and to determine the association between mental health symptoms prevalence and quality of life . we thought that mental health symptoms and quality of life assessment among young people could provide unique insight into health status and help design appropriate interventions for happy life ( 21 ) . according to the scores that students got from bsi , it can be said that frequency of mental symptoms is at medium - level . global indices are the parts that show actual functioning and level of indicator with different approaches ( 13 ) . founding dsi above 1 shows the psychopathological proneness of mental symptoms . in this study , dsi score ( 1.07 ) was used in order to determine the psychopathological level of students mental states . according to study results , it can be said that mental symptoms of the students are at psychopathological level . in some studies conducted , psychopathological level of students mental states was found as 1.22 by yldrm et , al , ( 22 ) , 1.0 by tanrverdi and ekinci ( 23 ) and 1.05 by kartal et , al , ( 24 ) while mental symptom level of the students in this study has similarities with those studies , it is higher when compared to some study results . arslan et , al , ( 25 ) found the mental symptom level as 0.90 in their study , terakye ( 26 ) found it as 0.92 and demirel et . dsi s being above 1 makes us think that mental states of the students in the study group are at problematic level . average of mental symptom scores of the students was found as , from the highest to the lowest respectively , depression , hostility , anxiety , negative ego and somatization . the highest symptom in the average of mental symptom score is depression . in other studies conducted , it was seen that the order was different ( 27 , 28 , 29 , 30 ) . mental health is defined as the state of feeling well emotionally or cognitively or as the state of not being sick . of the mental health problems , frequency of seeing depression among young people is % 5 and % 20 . depression not only decreases the attention , concentration , memory , motivation , decision making skills but also it is a risk factor for attempting suicide ( 13 , 31 ) . hostility means sense of anger and hatred in the dimensions of thought , emotion and behaviors . it can be defined as an attitude that involves not liking others and causes them to evaluate others negatively ( 27 ) . these data emphasize the importance of the studies conducted on this subject in terms of discovering mental problems of young people early and preventing the formation of psychiatric disorders later in life ( 32 ) . in this study , sf-36 norm - based scores are given . among the sub - scales of sf-36 , the highest scores belong to bodily pain ( bp ) and physical function ( pf ) dimensions , the lowest scores belong to role - emotional ( re ) and mental health ( mh ) dimensions . average normalized sf-36 score , scores of re ( 41.6 13.6 ) and mcs ( 42.0 11.7 ) were parallel to our results and they were quite lower than 50 . in the study where domantay researched life qualities of medicine students , pf and bp had the highest scores while re had the lowest average score ( 34 ) . in the study which was conducted with turkish students by arslan et . al , for the students with no evidence of depression , the highest average score belonged to the sub - scales of pf , rp and bp while the lowest average scores belonged to the sub - scales of re , v and mh al , studied life qualities of 208 lebanon university students ; they stated that pf had the highest score ( 89.0 ; ss=17.2 ) and re had the lowest score ( 55.2 ; sd=42.4 ) ( 17 ) . in the study which was conducted by suleiman with 119 university students , the highest score belonged to pf ( 78.1 ; sd=23.0 ) and the lowest score belonged to vitality dimension ( 52.3 ; sd : 17.8 ) ( 18 ) . in the study of deberard et . al , it was seen that students got the highest scores for pf ( 92.1 ; sd=18.3 ) and lowest score for vitality ( 62.5 ; sd=19.8 ) ( 35 ) . in the same study , al researched quality of life of 561 medicine students and 332 students from other departments ( 36 ) . in the study of latas , general sf-36 scores and average scores of sub - scales were higher than ours . the reasons behind the differences between the results may be due to using different methodological approaches ( consecutive approach vs. distribution from all years ) and conducting the research with different samples ( only medicine students or nursing and physical education students ) ( 37 ) . health students from various departments and management students who are not directly linked to health participated in our study . in this study , it was discovered that there was a significant relation and a negative medium - level correlation between mental symptom level and quality of life . that is , it can be said that students whose quality of life decreases experience mental problems more . mental symptom scores determined by bsi scale try to define depression , hostility , anxiety , negative ego , somatization and these five mental states given . in this case , it can be stated that students with low quality of life are more likely to be prone to mental disorders such as depression , hostility , anxiety , negative ego and somatization . we found in this study that mental health symptoms were associated with poorer quality of life , and quality of life was explained by mental health symptoms . it is important to identify symptoms and offer early interventions to reduce mental health symptom burden and , thereby , improve quality of life of young people .

introduction : purpose of this descriptive study is to determine the mental health problems and quality of life levels of college students and examine the relationship between them.methods:the sample of the study consisted of 429 students continuing their education in health school at afyon kocatepe university . data were collected by using information form , brief symptom inventory ( bsi ) , sf 36 quality of life scale questionnaires . in the analysis of obtained data , descriptive statistics , t - test , anova and correlation analyses were used.findings:mental symptoms which the students got the highest scores from the subscales of bsi were respectively , depression ( 1.310.75 ) , hostility ( 1.220.67 ) and anxiety ( 1.000.65 ) . discomfort severity index which is the overall score of the scale was found 1.070.61 . relation between state of mind and quality of life was found statistically significant ( f : 70.894 ; p<0.000 ) . when quality of life summary scores examined , it was found out that physical health summary score ( 48.927.93 ) and mental health summary score ( 43.449.52 ) points were low.conclusions:consequently , according to averages of scores , it can be said that frequency of the mental symptoms of students are high but their quality of life is low . it was found that when students quality of life decreased , their mental symptoms increased .

alveolar bone resorption that occurs around two - piece implants following abutment attachment has been well documented [ 15 ] . in recent years , several investigations have been carried out in order to explain the changes observed in crestal bone height . location of the implant - abutment junction ( iaj ) in relation to crestal bone [ 68 ] , the bacterial colonization of the micro - gap at the iaj , the establishment of a biological width or mucosal barrier around dental implants [ 1 , 1012 ] and the stress - strain concentration due to occlusal loading [ 1317 ] are among factors that have been suggested as the most likely causes of these crestal bone - level changes . although this 1.52.0 mm of bone resorption is still clinically acceptable , the ability to reduce this crestal bone loss may have several advantages such as improved esthetics , higher bone to implant contact and better primary stability . . in order to minimize this crestal bone resorption , several techniques and procedures such as non - submerged technique , utilizing micro - roughness on implant neck surface and platform switching have recently been developed . the concept of platform switching was introduced by lazzara and porter , and refers to the use of a smaller - diameter abutment on a larger - diameter implant collar . through placement of the smaller prosthetic components on the implant platform , iaj is moved inward from the implant shoulder and further away from the crestal bone . hypothetically , platform switching may increase the distance between the inflammatory cell infiltrate and the adjacent alveolar crest that can limit crestal bone resorption around the restored two - piece implants . the results of several histomorphometric studies showed that platform switching can significantly help to maintain the peri - implant soft and hard tissue and may be especially beneficial in esthetically demanding locations that require strong soft tissue support [ 2529 ] . in the past two decades , finite element analysis ( fea ) has become an increasingly useful tool for prediction of the effects of stress on the implant and its surrounding bone , and has been used extensively in describing biomechanical performance of dental implant systems . however , excessive simplifications of geometry will result in considerable inaccuracy in fea results . to produce more accurate geometries , some methods starting from computed tomography ( ct ) or magnetic resonance data of actual human bones have been proposed . several studies on platform switching , using three - dimensional ( 3d ) finite element models , reported the biomechanical advantage of shifting stress concentration area away from the cervical bone implant interface [ 3134 ] . some fea studies , on the other hand , showed that platform switching may have a minimal effect on von - mises stress in the crestal region of the cortical bone . interestingly , a new fea investigation about platform switching demonstrated that the reduction in bone strain was mostly caused by increasing the diameter of the implant , instead of using the platform switching technique . reviewing the dental literature revealed that there are still some controversies about the biomechanical advantages of platform switching . therefore , the objectives of this three - dimensional fea study were to compare and analyze the biomechanical effects of platform switching on the crestal bone around the two different dental implant systems . computerized tomographic ( ct ) images of a human edentulous mandibular first molar area were acquired . the mandible was approximately 8.5 mm in width buccolingually and 24 mm in height inferosuperiorly . the cross - sectional image was extruded to create a 3d section of the mandible and then 3d finite element models were constructed . models were prepared with two implant systems : xive s plus ( dentsply friadent , gmbh , germany ) and 3i certain ( biomet 3i , florida , usa ) . these implant systems were assembled on the mandible creating six different models : model xive - a : 3.811 mm implant and 3.8-mm - diameter esthetic base abutment , model xive - b : 4.511 mm implant and 3.8-mm - diameter esthetic base abutment , model xive - c : 4.511 mm implant and 4.5-mm - diameter esthetic base abutment , model 3i - a : 4.011.5 mm implant and 4.1-mm - diameter certain abutment , model 3i - b : 5.011.5 mm implant and 4.1-mm - diameter certain abutment , model 3i - c : 5.011.5 mm implant and 5.0-mm - diameter certain abutment . platform - switching configuration was only assumed for groups xive - b and 3i - b ( fig 1 ) . the optical digitizing system atos ii ( gom , braunschweig , germany ) was used to digitize the implants and abutments with high accuracy . concord , ma , usa ) to construct the solid models that were analyzed by a three dimensional fe analysis package ( abaqus v6.7 - 1 ; simulia corp . , providence , usa ) . models were meshed with four - node tetrahedral solid elements , and were meshed between 90,765 and 102,795 nodes , and between 457,151 and 519,456 contact elements . the implants and bone used in the models were considered to be isotropic , homogeneous and linearly elastic . the elastic properties were adopted from the literature as shown in table 1 [ 37 , 38 ] . connectivity between the bone and implants were assumed to simulate 100% osseointegration and the abutments and implants were assumed to be completely bonded without any movement . as the boundary condition , the nodes at the mesial and distal surfaces of the mandibular bone were fixed in all directions . in order to evaluate the stress distribution in peri - implant bone tissue and on implants and abutments , a linear static analysis was performed on the prepared 3d solid models . loading was simulated by applying either axial or an oblique load ( in a buccolingual direction with 15 degrees of inclination to the alveolar longitudinal axis ) of 100 n on the top of the abutments in their central region [ 39 , 40 ] . finally , the finite element models were used to calculate von mises stresses in the crestal bone ( both compact and cancellous bone ) surrounding implants and in the implant - abutment interface area . moreover , stress distribution in the fe models were illustrated to compare the biomechanical differences between conventional and platform - switching models in two different implant systems . computerized tomographic ( ct ) images of a human edentulous mandibular first molar area were acquired . the mandible was approximately 8.5 mm in width buccolingually and 24 mm in height inferosuperiorly . the cross - sectional image was extruded to create a 3d section of the mandible and then 3d finite element models were constructed . models were prepared with two implant systems : xive s plus ( dentsply friadent , gmbh , germany ) and 3i certain ( biomet 3i , florida , usa ) . these implant systems were assembled on the mandible creating six different models : model xive - a : 3.811 mm implant and 3.8-mm - diameter esthetic base abutment , model xive - b : 4.511 mm implant and 3.8-mm - diameter esthetic base abutment , model xive - c : 4.511 mm implant and 4.5-mm - diameter esthetic base abutment , model 3i - a : 4.011.5 mm implant and 4.1-mm - diameter certain abutment , model 3i - b : 5.011.5 mm implant and 4.1-mm - diameter certain abutment , model 3i - c : 5.011.5 mm implant and 5.0-mm - diameter certain abutment . platform - switching configuration was only assumed for groups xive - b and 3i - b ( fig 1 ) . the optical digitizing system atos ii ( gom , braunschweig , germany ) was used to digitize the implants and abutments with high accuracy . concord , ma , usa ) to construct the solid models that were analyzed by a three dimensional fe analysis package ( abaqus v6.7 - 1 ; simulia corp . , providence , usa ) . models were meshed with four - node tetrahedral solid elements , and were meshed between 90,765 and 102,795 nodes , and between 457,151 and 519,456 contact elements . the implants and bone used in the models were considered to be isotropic , homogeneous and linearly elastic . the elastic properties were adopted from the literature as shown in table 1 [ 37 , 38 ] . connectivity between the bone and implants were assumed to simulate 100% osseointegration and the abutments and implants were assumed to be completely bonded without any movement . as the boundary condition , the nodes at the mesial and distal surfaces of the mandibular bone were fixed in all directions . in order to evaluate the stress distribution in peri - implant bone tissue and on implants and abutments , a linear static analysis was performed on the prepared 3d solid models . loading was simulated by applying either axial or an oblique load ( in a buccolingual direction with 15 degrees of inclination to the alveolar longitudinal axis ) of 100 n on the top of the abutments in their central region [ 39 , 40 ] . finally , the finite element models were used to calculate von mises stresses in the crestal bone ( both compact and cancellous bone ) surrounding implants and in the implant - abutment interface area . moreover , stress distribution in the fe models were illustrated to compare the biomechanical differences between conventional and platform - switching models in two different implant systems . the results from fea are demonstrated in stress maps with a color scale that makes it possible to compare the stress distribution in different structures ( implants and peri - implant bone tissues ) of all six models ( fig 2 ) . in order to compare stress distribution among the structures in different models more quantitatively , stress values are presented for equivalent stresses ( von mises ) ( table 2 ) . moreover , the maximum ( the first ) and minimum ( the third ) principal stresses at crestal bone are demonstrated in figs 4 and 5 . the positive values show tensile stress and the negatives demonstrate compressive stress.the pattern of stress distribution was almost similar under both loading situations and for both two implant systems . however , oblique loads produced more stress among the models than axial loads . in all models and under both loading situations , stress values were higher for the cortical bone than the cancellous bone ( table 2 ) . under 100 n oblique load , the maximum von mises stress in the cortical bone was from 15.06 mpa in model xive -b ( platform - switching model ) to 32.11 mpa in model 3i - a . moreover , the maximum von mises stress in the cancellous bone was from 2.49 mpa in model 3i - b ( platform switching model ) to 6.28 mpa in model xive - a . the results clearly showed that platform - switching reduced von mises stress values at the crestal bone in both implant systems ( fig 2 ) . in both 3i and xive implant systems , wide platform implants ( models 3i - c and xive - c ) presented lower stress values and more favorable patterns of stress distribution compared to regular platform implants ( models 3i - a and xive - a ) . for maximum principal stress , the tensile stress concentration was located on the buccal side of the cortical bone tissue that was on the opposite side of load application . for minimum principal stress , the location of compressive stress concentration was under the applied - load side of the models . in almost all models , tensile stress was less than compressive stress . in both implant systems and for both loading conditions , the platform switching models presented obvious decreases in tension and compression values in the third principal stress . however , for the first principal stress , wide platform models showed lower tension values , while platform - switching models showed lower compression values ( figs 4 and 5 ) . analyzing implant - abutment interface demonstrated that the stress concentration was around the periphery of the uppermost surface of the implant in the conventional models ( models a , c ) ( fig 3 ) , while this high stress area shifted toward the center of the implant in the platform - switching models . in both implant systems and for both loading conditions , moreover , wide platform implants ( models 3i - c and xive - c ) showed lower von mises stresses than implants with regular platforms ( models 3i - a and xive - a ) ( table 2 ) . the results from fea are demonstrated in stress maps with a color scale that makes it possible to compare the stress distribution in different structures ( implants and peri - implant bone tissues ) of all six models ( fig 2 ) . in order to compare stress distribution among the structures in different models more quantitatively , stress values are presented for equivalent stresses ( von mises ) ( table 2 ) . moreover , the maximum ( the first ) and minimum ( the third ) principal stresses at crestal bone are demonstrated in figs 4 and 5 . the positive values show tensile stress and the negatives demonstrate compressive stress.the pattern of stress distribution was almost similar under both loading situations and for both two implant systems . however , oblique loads produced more stress among the models than axial loads . in all models and under both loading situations , stress values were higher for the cortical bone than the cancellous bone ( table 2 ) . under 100 n oblique load , the maximum von mises stress in the cortical bone was from 15.06 mpa in model xive -b ( platform - switching model ) to 32.11 mpa in model 3i - a . moreover , the maximum von mises stress in the cancellous bone was from 2.49 mpa in model 3i - b ( platform switching model ) to 6.28 mpa in model xive - a . the results clearly showed that platform - switching reduced von mises stress values at the crestal bone in both implant systems ( fig 2 ) . in both 3i and xive implant systems , wide platform implants ( models 3i - c and xive - c ) presented lower stress values and more favorable patterns of stress distribution compared to regular platform implants ( models 3i - a and xive - a ) . for maximum principal stress , the tensile stress concentration was located on the buccal side of the cortical bone tissue that was on the opposite side of load application . for minimum principal stress , the location of compressive stress concentration was under the applied - load side of the models . in almost all models , tensile stress was less than compressive stress . in both implant systems and for both loading conditions , the platform switching models presented obvious decreases in tension and compression values in the third principal stress . however , for the first principal stress , wide platform models showed lower tension values , while platform - switching models showed lower compression values ( figs 4 and 5 ) . analyzing implant - abutment interface demonstrated that the stress concentration was around the periphery of the uppermost surface of the implant in the conventional models ( models a , c ) ( fig 3 ) , while this high stress area shifted toward the center of the implant in the platform - switching models . in both implant systems and for both loading conditions , moreover , wide platform implants ( models 3i - c and xive - c ) showed lower von mises stresses than implants with regular platforms ( models 3i - a and xive - a ) ( table 2 ) . bone resorption close to the first thread of the two - piece implants is frequently observed during initial loading [ 2 , 5 ] . to achieve and maintain stable osseointegration for implants in function , high stress concentration in the bone some studies showed that using abutments with a smaller diameter than the implant neck or platform - switching technique helps in reducing crestal bone resorption [ 4 , 2325 ] . the possible reasons for bone preservation with the platform switching technique include inward shifting of the location of the iaj or the stress concentration area between the abutment and implant [ 23 , 26 ] . the result of the present study revealed that the platform switching reduced von mises stress values at the crestal bone in both implant systems . it has been reported in the literature that crestal bone resorption is related to excessive load and damage of the supporting interfacial bone . stress concentration can lead to bone loss due to bone micro - damage and creation of crater - like bone defects around the implant [ 32 , 42 ] . tabata et al . reported that von mises , maximum and minimum principal stress were reduced in peri - implant bone tissue and implant when the platform - switching concept was used . in studies conducted by hsu et al . and schrotenboer et al . , their fe analyses showed that when the abutment diameter was reduced , less stress was transferred to the crestal bone . however , the results of the present study are in contrast to the results previously reported by pessoa et al . the possible reasons may be attributed to the fact that in their fe models , platform switching was defined as the circumferential horizontal mismatch of 0.5 mm between implant and abutment , while in the present study , this horizontal mismatch was 1 mm for 3i implants and 0.7 mm for xive implants . in another study , canay and akca revealed that the platform - switching concept is an effective factor on mechanical properties of implant - abutment complex rather than the load - induced stresses developed at the marginal bone around implants . however , those results were obtained in very simplified models that did not consider the internal geometry of implant - abutment junction in detail . in agreement with previous studies [ 32 , 44 , 45 ] , it was confirmed that wide platform implants show a lower stress value and more favorable stress distribution compared to regular platform implants . it seems that increasing the implant diameter enhances the contact between the implant and the bone that dramatically reduced stress concentration . interestingly , the platform - switching models of both implant systems still showed lower stress values than wide platform configurations . in other words , in a strain gauge analysis of immediately loaded implants concluded that bone strain was reduced more by increasing the diameter of the implant than by using platform switching . however , one should keep in mind that in the present study , similar to most of the previous fea studies , models of delay - loaded implants were used , in which there was an ideal osseointegration between the surface of the implant and the bone . in addition , some studies reported that the concentration of stress is greater around immediately loaded implants than around delay - loaded implants [ 26 , 27 ] . the present study also indicated that in both implant systems , the compressive and tensile stresses were lower in platform - switching models than conventional models at the compact bone in the vicinity of the implant neck . extensive compressive stress may increase the risk of bone resorption , since it can compromise the periosteal blood supply and may result in bone necrosis . thus , the platform - switched design of both systems can reduce the risk of bone resorption and loss of osseointegration . similarly , chang et al . showed higher compressive and tensile stresses in the conventional model than in the platform - switching model at the crestal bone around implants . in agreement with recently published studies [ 32 , 45 ] , the present investigation demonstrated that platform switching increased the stress concentration in the implant - abutment interface . it was suggested that the increased stress concentration at the implant - abutment interface in platform switching models can lead to mechanical problems such as screw loosening or fracture . however , according to a study performed by maeda et al . these increases in stress values may not result in any major complication , since the yield strength of titanium alloy ( 620 to 725 mpa ) and cobalt - chromium alloy ( 552 to 1,034 mpa ) is more than the stress values reported in the implant - abutment interface of both systems ( table 2 ) . to construct a finite element model , it is usually necessary to simplify the system by making some assumptions . the implants and bone used in the models were considered to be isotropic , homogeneous and linearly elastic and the occlusal forces were static . furthermore , in the present study , the bone - implant contact was 100% , but in vivo , bone - implant contact percentages usually range from 30% to 70% . thus , the final models represented an average clinical situation , and generalization of its results should be done with care . therefore , because the finite element models used in this study do not identically reproduce all clinical situations , the application of the results should be tempered with sound clinical judgment . within the limitation of this 3d finite element analysis study , the following conclusions were drawn : in both implant systems , platform - switching design reduced the stress concentration at the crestal bone and shifted it towards the area of implant - abutment interface.in the present study , this stress reduction was not related to increasing implant diameter . in both implant systems , platform - switching design reduced the stress concentration at the crestal bone and shifted it towards the area of implant - abutment interface . in the present study ,

objectives : the purpose of this study was to determine the influence of platform switching on stress distribution of two different implant systems using three - dimensional ( 3d ) finite element models.materials and methods : six 3d finite element models were created to replicate two different implant systems with peri - implant bone tissue , in which six different implant - abutment configurations were represented : model xive - a : 3.8-mm - diameter implant and 3.8-mm - diameter abutment ; model xive - b ( platform - switching model ) : 4.5-mm - diameter implant and 3.8-mm - diameter abutment ; model xive - c : 4.5-mm - diameter implant and 4.5-mm - diameter abutment ; model 3i - a : 4.0-mm - diameter implant and 4.1-mm - diameter abutment ; model 3i - b ( platform - switching model ) : 5.0-mm - diameter implant and 4.1-mm - diameter abutment ; model 3i - c : 5.0-mm - diameter implant and 5.0-mm - diameter abutment . vertical and oblique loads of 100 were applied to all models.results:while the pattern of stress distribution was similar for both loading situations , oblique loading resulted in higher intensity and greater distribution of stress than axial loading in both cortical bone and implant - abutment- interface . stress distribution at peri - implant bone was almost identical with similar magnitudes for all six models . in both implant systems , platform - switching models demonstrated lower maximum von mises stress in cortical bone than conventional models . however , in both implant systems and under both loading situations , platform - switching models showed higher stresses at the implant - abutment interface than conventional models.conclusion:in both implant systems , platform switching design reduced the stress concentration in the crestal bone and shifted it towards the area of implant - abutment interface .

mutations of the breast cancer susceptibility gene 1 ( brca1 ) are linked to familial breast and ovarian cancer [ miki et al . , 1994 ] . brca1 mutation carriers also experience a significantly increased risk for other hormone - responsive tumor types , including uterine , cervical , and prostate cancers [ thompson and easton , 2002 ] . however , this function does not explain the predilection of brca1 carriers to develop hormone - dependent cancers . we will discuss evidence that physiologic interactions between brca1 and steroid hormone receptors [ progesterone receptor ( pr ) and estrogen receptor ( er- ) ] contribute to the tissue - specific pattern of tumorigenesis in brca1 carriers . the pr , a transcriptional target of er- , plays a key role in mammary growth and development , especially during pregnancy . its role in breast cancer is not as well - established as for er- , but available data indicate that pr signaling can stimulate breast cancer development [ conneely et al . , 2003 ; lange et al . , 1999 ; schairer , 2002 ] . progesterone can exert a biphasic effect on the mammary epithelium , where growth stimulation is followed by inhibition , depending upon the context [ musgrove et al . , 1991 ] . it has been proposed that progesterone primes mammary epithelial cells to respond to other growth regulatory signals [ lange et al . , 1999 ] . studies in pr-/- mice have uncovered roles for pr in mammary ductal branching and lobulo - alveolar differentiation during pregnancy . a role in cancer is implied by the finding that pr-/- mice are resistant to carcinogen - induced mammary tumorigenesis [ conneely et al . , 2003 ] . in the human menstrual cycle , breast epithelial cell proliferation peaks during the luteal phase , when circulating progesterone levels are maximal , consistent with progesterone stimulation of proliferation in the adult breast [ lange et al . , epidemiologic studies have revealed a small but significant increase in breast cancer risk associated with menopausal hormone replacement therapy ( hrt ) using combined estrogen - progestin treatment , relative to estrogen alone [ schairer , 2002 ] . in contrast , combined hrt reduces the incidence of endometrial cancer , a tissue where progesterone has anti - proliferative effects . combined hrt is also associated with higher mammographic density , a marker of breast cancer risk [ mctiernan et al . , while most sporadic breast cancers ( 60 - 70% ) are hormone receptor positive , most brca1 mutant cancers are er- and pr negative [ lakhani et al . , 2002 ] . nevertheless , several lines of evidence suggest an important role for steroid hormones and their receptors in the genesis of brca1 mutant cancers . prophylactic bilateral oophorectomy confers a substantial reduction ( about 50% ) in breast cancer risk in brca1 mutation carriers [ narod , 2001 ; rebbeck et al . , 2002 ] ; and bilateral oophorectomy reduced the incidence of mammary cancer in mice with a mammary - targeted deletion of full - length brca1 [ bachelier et al . , in contrast to sporadic cancers , where early pregnancy has a risk - reducing effect , pregnancy increases the risk of breast cancer or accelerates cancer development in brca1 carriers [ narod , 2001 ] . early pregnancy is associated with high circulating levels of estrogen and progesterone , suggesting that steroid hormones may confer increased breast cancer risk in brca1 carriers . evidence supporting a hormonal etiology of brca1 mutant breast cancers is reviewed elsewhere [ rosen et al . , 2005 ] . a role for brca1 in mammary development is suggested by the finding that its expression in mice is increased in proliferating cellular compartments that are also undergoing differentiation , including the mammary epithelium during puberty and pregnancy [ lane et al . consistent with these findings , endogenous brca1 expression is increased during mammary epithelial cell differentiation in vitro ; and the differentiation process is enhanced by exogenous brca1 [ kubista et al . , 2002 ; rajan et al . , 1996 ] . furthermore , brca1 over - expression can inhibit estrogen and progesterone - stimulated mammary cancer cell proliferation in vitro [ ma et al . , 2006 ; xu et al . , 2005 ] , raising the possibility that brca1 may coordinate the balance of proliferation versus differentiation in mammary tissue . however , it should be noted that brca1-deleted animal models and women show normal mammary differentiation . previous studies indicate that brca1 physically interacts with er- and inhibits its transcriptional activity [ fan et al . , 2001 ; fan et al . , 2002 ; fan et al . , 1999 ; 2005 ] ; and inactivation of brca1 ( by mutation or knockdown ) confers activation of er- in the absence of ligand [ jones et al . , 2005 ; zheng et al . , we have extended the scope of these findings by showing that brca1 inhibits pr signaling in breast carcinoma cells [ ma et al . , thus , we found that in transient transfection assays using a progesterone responsive reporter ( mmtv - luc ) , exogenous wild - type brca1 [ but not a cancer - associated mutant ( t300 g ) ] inhibited the activity of the pr in progesterone - responsive human breast cancer cells ( mcf-7 and t47d ) . in cells lacking endogenous pr , brca1 inhibited the activity of both isoforms of pr ( pr - a and pr - b ) alone and in combination . knockdown of endogenous brca1 resulted in a four - fold increase in progesterone - stimulated pr activity . unlike er- this interaction differs from the brca1 : er- interaction in several respects : 1 ) er- contacts the n - terminus of brca1 , whereas pr can contact both n- and c - terminal sites on brca1 ; 2 ) brca1 binds to the af-2 ( activation function 2 ) domain of er- but to a different region on pr ; and 3 ) the nuclear receptor coactivator p300 rescues the brca1 repression of er- but not pr [ ma et al . , . over - expression of brca1 inhibited the expression of various progesterone - responsive genes just as it blocked expression of most estrogen - inducible genes [ xu et al . , 2005 ] ; and just as brca1 inhibited estradiol ( e2 ) stimulated cell proliferation in mcf-7 cells [ xu et al . , 2005 ] , it also inhibited progesterone - stimulated proliferation of t47d cells [ ma et al . , 2006 ] . we tested the effects of exogenous hormones in mice with a mammary - targeted deletion of the full - length brca1 isoform ( brca1co / commtv - cre ) . in adult mice with intact ovaries , exposure to a sustained release progesterone pellet for four weeks caused a significant increase in mammary gland volume and tertiary branching in brca1co / co mice but had little effect in wild - type non - transgenic mice [ ma et al . in ovariectomized mice , the combination of e2 plus progesterone caused an exaggerated proliferative response in brca1co / co mice , compared to wild - type mice ; and a four - week exposure to e2 alone caused a significant increase in mammary epithelial cell ( mec ) density in brca1co / co but not wild - type mice . these findings suggest that a brca1 deficiency abrogates the homeostatic mechanisms that limit the proliferative response to e2 or progesterone alone and enhances the response to the combination of e2 plus progesterone . in the setting of a heterozygous p53 mutation , brca1co / commtv - cre mice develop invasive mammary cancers [ xu et al . , 1999 ] . although these tumors are usually er- negative ( as are most mouse mammary cancers ) , a recent study suggests the development of these tumors is hormone - responsive . thus , implantation of a slow - release tamoxifen pellet significantly enhanced development of mammary cancers in brca1co / commtv - cre / p53+/- mice [ jones et al . , 2005 ] . tamoxifen also stimulated development of mammary hyperplasias , in which expression of er- was lost . consistent with these findings , knockdown of brca1 in cultured mcf-7 cells caused a significant increase in tamoxifen - stimulated er- activity , suggesting that brca1 may regulate the relative agonist versus antagonist activity of tamoxifen . based on these considerations , we propose a model for brca1 mutant breast cancer formation ( figure 1 ) . here , er / pr - positive mecs deficient for brca1 are hyper - sensitive to endogenous e2 and progesterone and secrete growth factors that stimulate proliferation of nearby er / pr - negative mecs . paracrine mechanisms of this type occur during normal mammary development [ conneely et al . , continual hormonal stimulation results in er / pr - negative hyperplasias . in the setting of genomic instability due to brca1 deficiency , these lesions eventually become autonomous and progress to invasive cancer . during the evolution of these tumors , the p53 gene becomes mutated , since brca1 mutant breast cancers exhibit a very high incidence ( approximately 80% ) of p53 mutations , suggesting this is an obligate event [ phillips et al . , 1999 ] . a value of this hypothesis is that it is testable in available animal models . alternatively , brca1 mutant hyperplasias and cancers may be derived from mecs that were originally er-/pr - positive and converted to er-/pr negativity as a consequence of hormone - stimulated cell proliferation , chromosomal instability due to loss of brca1 function [ tirkkonen et al . , a recent study supports the hypothesis of a paracrine interaction involving hormone receptor positive and negative cells in brca1 mutant cancer development . thus , pr expression was more common in normal mammary epithelial cells adjacent to an invasive breast cancer in brca1 mutant cancers than in sporadic cancers [ king et al . , 2004 ] . the wild - type brca1 allele was retained in the microdissected normal mammary epithelium cells . one possibility is that progesterone stimulates the pr+ surrounding non - cancer cells to secrete growth factors that stimulate proliferation of pr negative cells that become the cancer , but this remains to be proved . this model , if validated , provides a rationale for using an anti - progestin , alone or in combination with an anti - estrogen , for breast cancer prophylaxis in brca1 mutation carriers . the national surgical adjuvant breast and bowel project p1 breast cancer prevention trial , which showed a 50% overall risk reduction in women treated with tamoxifen , did not show a significant benefit for tamoxifen in brca1 carriers [ king et al . , it should be noted that the number of brca1 carriers in the nsabp - p1 trial was small ; and the efficacy of tamoxifen in preventing brca1 mutant breast cancers or as adjuvant treatment for brca1 mutant cancers is not settled [ rosen et al . , 2005 ] . a subset of sporadic breast cancers ( ? 30 - 40% ) show reduced or absent brca1 expression due to promoter methylation , loss of one brca1 allele , or other causes [ esteller et al . , 2000 ; loss of brca1 inhibition of pr activity could contribute to development of a subset of sporadic breast cancers , providing a rationale for the use of an anti - progestin to treat premenopausal breast cancers that are pr - positive and under - express brca1 . in the absence of functional brca1 , er-/pr positive mammary epithelial cells ( mecs ) are hypersensitive to e2 ( estradiol ) and progesterone and stimulate proliferation of hormone receptor negative cells ( eg . this results in er-/pr negative proliferative lesions ( hyperplasias or dysplasias ) , which ultimately progress to invasive cancer in the setting of genomic instability due to loss of brca1 function .

inherited mutations of the brca1 gene ( chromosome 17q21 ) , a tumor suppressor , lead to an increased risk of breast cancer , ovarian cancer , and several other hormone - responsive tumor types . over the last ten years , brca1 has been found to play major roles in dna damage signaling , repair , and cell cycle checkpoints . in addition , unfolding evidence suggests that brca1 functions as a co - regulator for steroid hormone receptors and modulates steroid hormone action . in this paper , we will briefly review this evidence and present a model to address the role of the progesterone and estrogen receptors in brca1 mutant mammary carcinogenesis . finally , we will consider some of the clinical implications of this model .

pancreatic cancer is the second most common gastrointestinal malignancy in the united states and the fifth most common malignancy in korea . by contrast , adenosquamous cell carcinoma of the pancreas is very rare and aggressive and accounts for 1 - 4% of all exocrine malignancies of the pancreas based on autopsy and surgical specimens . a rare case of pancreatic adenosquamous cell carcinoma with duodenal invasion is reported , and the literature is reviewed . a 46-year - old man presented with a 3-month history of dyspepsia and a 7-kg weight loss . direct tenderness was noted over the right upper quadrant area of the abdomen , but rebound tenderness and murphy 's sign were absent . the complete blood count showed a white cell count of 7,800/mm ( 70.9% neutrophils ) , hemoglobin 10.3 g / dl , hematocrit 29.2% , and platelet count of 283,000/mm . the blood chemistry showed a fasting glucose level of 107 mg / dl , urea nitrogen 19 mg / dl , creatinine 0.8 mg / dl , total protein 6.1 g / dl , albumin 4.0 g / dl , aspartate transaminase 13 iu / l , alanine aminotransferase 12 iu / l , alkaline phosphatase 238 iu / l , total bilirubin 0.6 mg / dl , total cholesterol 170 mg / dl , and triglyceride 64 mg / dl . the serum amylase was elevated slightly to 170 u / l , and the serum lipase to 338 u / l . the carcinoembryonic antigen ( cea ) was 3.3 ng / ml , -fetoprotein was 1.5 ng / ml , and the ca 19 - 9 was elevated to 566.7 ng / ml . gastroduodenoscopy showed a hard ulceroinfiltrative mass with a yellowish exudate that bled readily on touch at the second portion of the duodenum ( fig . initially , we thought that he had duodenal cancer . however , a biopsy of the duodenum showed adenosquamous cell carcinoma ( fig . abdominal computed tomography showed a 7.1 6.3-cm heterogeneously enhancing mass in the pancreatic head . multiple enlarged lymph nodes were seen around the left gastric vessel , greater omentum , and small bowel mesentery ( fig . fine - needle aspiration biopsy of the pancreatic mass revealed adenosquamous cell carcinoma , anaplastic type ( fig . we diagnosed primary adenosquamous cell carcinoma of the pancreas that had invaded the duodenum , resulting in an ulceroinfiltrative lesion . pancreatic cancer includes ductal adenosquamous carcinoma , endocrine neoplasms , cystic tumors , solid pseudopapillary tumors , acinar cell carcinoma , adenocarcinoma , squamous cell carcinoma , primary lymphoma of the pancreas , and metastatic lesions to the pancreas . pancreatic cancer originates mainly from the exocrine duct cells , with more than 80% showing exocrine adenomatous morphology . based on autopsy studies and surgical specimens , it accounts for 1 - 4% of all exocrine malignancies of the pancreas . a review of data taken from various cancer registries on 6,668 cases of exocrine pancreatic cancer treated between 1950 and 1985 revealed 36 cases ( 0.005% ) of squamous carcinoma and 68 cases ( 0.01% ) of adenosquamous carcinoma . there are no specific radiologic findings that distinguish this tumor from common ductal cell adenocarcinoma of the pancreas . . stated that the presence of central necrosis in a huge infiltrative pancreatic tumor was suggestive of adenosquamous carcinoma of the pancreas . the histogenesis of adenosquamous cell carcinoma of the pancreas remains uncertain , and several different hypotheses have been offered : 1 ) a preexisting adenocarcinoma undergoing squamous cell transformation ; 2 ) heterotopic squamous epithelium undergoing a malignant change ; and 3 ) a stem cell capable of differentiating into either a squamous or glandular cell undergoing a malignant change reported that the cytological features derived from a fine - needle aspiration biopsy are diagnostic of adenosquamous cell carcinoma of the pancreas . a few patients undergo surgery because most of them are reported to have stage iv disease at the time of presentation . our patient 's diagnosis was adenosquamous cell carcinoma of the pancreas based on a percutaneous needle aspiration biopsy . major surgery does not have a substantial benefit for patients with adenosquamous cell carcinoma of the pancreas , especially those with severe comorbidities . the prognosis of patients with adenosquamous carcinoma of the pancreas is less favorable than that of patients with common ductal cell carcinoma of the pancreas . reported that the survival averaged 5.7 months for 72 patients with adenosquamous carcinoma of the pancreas , and only five patients survived for more than 1 year . . reported that the overall survival was 12.5 months for patients treated with resection and adjuvant chemotherapy and 3.0 months for the patients who received no or palliative chemotherapy . in korea , seven cases ( including our case ) have been reported ( one case of squamous cell carcinoma , six cases of adenosquamous cell carcinoma ) [ 14 - 19 ] . these cases showed a male predominance ( m : f = 5:2 ) and a mean patient age of 61.7 11.1 years . the common symptoms were abdominal pain in seven ( 100% ) and weight loss in four ( 57.1% ) . lipase was elevated in three ( 42.9% ) , ca19 - 9 in two ( 28.6% ) , and cea in one ( 14.3% ) . on ct , the masses were located in the pancreas head in three ( 42.9% ) , body in one ( 14.3% ) , and tail in three ( 42.9% ) . central necrosis was observed in three ( 42.9% ) , and metastasis in six ( 85.7% ) . the median survival was 3.3 months , and the mean survival was 3.6 1.9 months . the prognosis in all cases was very poor , although one case was treated with adjuvant chemotherapy . multiple enlarged lymph nodes were seen around the left gastric vessel , greater omentum , and small bowel mesentery .

the most common pancreatic cancer is adenocarcinoma . primary adenosquamous cell carcinoma of the pancreas is very rare and aggressive . a 46-year - old man presented with a 3-month history of dyspepsia and a 7-kg weight loss . the physical examination showed tenderness of the right upper quadrant of the abdomen . there was no jaundice . amylase and lipase were elevated . ca 19 - 9 was elevated to 566.7 u / ml . gastroduodenoscopy showed a hard ulceroinfiltrative mass with a yellowish exudate that bled readily on touch in the second portion of the duodenum . abdominal computed tomography showed a 7.1 6.3-cm heterogeneously enhancing mass in the pancreatic head . the pancreatic mass had invaded the duodenum wall , gastric antrum , and gastroduodenal artery sheath . fine - needle aspiration biopsy of the pancreatic mass revealed adenosquamous cell carcinoma , anaplastic type . we concluded that an adenosquamous cell carcinoma of pancreas had invaded the duodenal mucosa causing ulceration .

this study was designed as a parallel , single - arm , and controlled study to evaluate the clinical and microbiological effects of st on the biodiversity of subgingival biofilm collected in smokers and non - smokers with chronic periodontitis . subjects included in the present study were examined , treated by st , and re - evaluated 21 days later . the study was approved by the institutional review board ( irb ) and patients received a detailed description of the proposed treatment and gave their informed and written consent . potential patients were selected from those referred to the graduate clinic of piracicaba dental school , university of campinas unicamp , brazil . all patients received a complete periodontal examination , including a full - mouth periodontal probing , radiographic examination and complete anamnesis . the study inclusion criteria were : diagnosis of chronic periodontitis , according to the criteria of the 1999 international classification ( 25);presence of at least 20 teeth;at least nine teeth presenting probing pocket depth ( ppd)5 mm with bleeding on probing ( bop ; being at least two with ppd7 mm);>35 years of age . diagnosis of chronic periodontitis , according to the criteria of the 1999 international classification ( 25 ) ; presence of at least 20 teeth ; at least nine teeth presenting probing pocket depth ( ppd)5 mm with bleeding on probing ( bop ; being at least two with ppd7 mm ) ; patients who : ( i ) were pregnant or lactating ; ( ii ) required antimicrobial pre - medication for the performance of periodontal examination and treatment ; ( iii ) were suffering from any other systemic diseases ( cardiovascular , pulmonary , liver , cerebral , or diabetes ) ; ( iv ) had received antimicrobial treatment in the previous 3 months ; ( v ) were taking long - term anti - inflammatory drugs ; and/or ( vi ) had received a course of periodontal treatment within the last 6 months ; were excluded from the study . a parallel , non - blinded and prospective study was designed to enroll the following two groups : non - smoker group ( n=10 ) : patients diagnosed with generalized and severe chronic periodontitis and that had never been smokers ; and smoker group ( n=10 ) : patients diagnosed with generalized and severe chronic periodontitis and that had a smoking habit for at least 10 years and smoked at least 20 cigarettes per day . sample size was determined by the bioestat program , using a standard deviation of 1.0 and a power value of 80% to detect a difference between groups of 1.0 mm . supragingival therapy ( st ) : after a full mouth examination and consent in participation , patients of both groups received a full mouth prophylaxis , supragingival calculus , and biofilm removal , using gracey curettes , an ultrasonic scaler , bicarbonate spray and floss , or interdental brushes , according to the interdental space . the patients were also individually instructed on how to perform oral self - care , including the bass technique , inter - dental flossing , and tongue brushing . all the subjects of the study received a standard fluoride dentifrice , toothbrushes , and floss as necessary ; and were asked to perform complete oral self - care hygiene at least twice a day . a week after this first instruction session , patients returned to a reinforcement of oral self - care instructions . twenty - one days after st , clinical re - evaluation was performed and subgingival samples were collected for microbiological analysis . the same individual ( dp ) was trained to perform st in all the patients , whereas another individual was trained to perform baseline and post - therapy clinical assessments ( tm ) . the following clinical parameters were assessed immediately before therapy : full - mouth plaque index ( fmpi ) , according to ainamo and bay ( 26 ) , and full - mouth bleeding score ( fmbs ) , according to mhlemann and son ( 27 ) ; these were calculated after assessing dichotomously the presence of dental biofilm at the site or bop from the bottom of the pocket when probing with a manual probe.1 the percentage of total sites that revealed the presence of plaque or bleeding was calculated . all teeth presenting at least one site with ppd5 mm were selected for clinical evaluation . these teeth did not present pulpal disease or furcation lesion ( in order to avoid bias ) . from those previously selected teeth , one site ( the deepest one ) was selected and followed - up during the experimental period , using an individually manufactured acrylic stent in which a groove was made to standardize the measurements . this selection resulted in a mean of 10.01.8 sites per patients evaluated during this study . for the sites selected , clinical parameters were : probing pocket depth ( ppd distance from the bottom of the pocket to the margin);relative clinical attachment level ( rcal distance from the bottom of the pocket to the stent margin);relative gingival margin position ( rgmp distance from the gingival margin to the stent margin ) . probing pocket depth ( ppd distance from the bottom of the pocket to the margin ) ; relative clinical attachment level ( rcal distance from the bottom of the pocket to the stent margin ) ; relative gingival margin position ( rgmp distance from the gingival margin to the stent margin ) . all parameters were evaluated using a periodontal probe at baseline and 21 days after st . for this calibration , three patients were selected and full mouth rcal and ppd were measured , twice , within 24 hours , with at least 1 hour between the examinations . the intra - class correlation was calculated for each parameter , resulting in 93.5% reproducibility for rcal and 94.3% for ppd . subgingival biofilm collection : after a full - mouth examination , all sites previously selected for clinical follow - up were included in the subgingival biofilm analysis , as described . following the careful removal of supragingival biofilm , the areas were washed with a water spray , isolated with cotton rolls and gently dried . a sterile paper point2 was inserted into the bottom of the periodontal pocket for 30 s. each paper point was placed separately in sterile plastic tubes containing 0.01 m tris edta solution , ph 8.0 ( te ) . dna collection and extraction for each patient , samples from the selected sites were pooled together to allow the 16s cloning sequencing . cloning and sequencing : first , the 16s rrna gene was amplified using a universal primer set ( 27f and 1492r ) as described by de lillo et al . ( 29 ) . cloning procedures were performed using a topo - ta cloning kit,3 following the manufacturer 's instructions . briefly , the amplicons resulting from universal amplification were cloned into escherichia coli and then cultured overnight in luria - bertani ( lb ) plates ( containing ampicilin and x - gal ) for posterior colony selection . after colony selection , each colony was separately cultivated overnight , in lb broth media.4 the lb broth samples were then centrifuged to form a bacterial pellet containing e. coli that received vectors / bacterial dna . after vector extraction , the products were purified5 and sequenced.6 after sequencing , a partial sequence of 600 bp was generated . these sequences were initially aligned and a similarity matrix was constructed from the alignments by the method of jukes and cantor ( 30 ) ( bioedit 7.0 program ( 31 ) ) . phylogenetic trees were constructed by the neighbor joining method ( dotur program ( 32 ) ) . sequences were compared using the homd database ( 33 ) applying a level of 98.5% sequence identity as cut - off . sequences presenting 98% or greater similarity within a genus were considered as the same species . clinical parameters were analyzed by student 's t test ( for baseline intergroup comparisons ) and repeated - measures anova / tukey ( for clinical changes occurring after st ) , using the proc glm procedure of the sas program.7 for microbiological data , a variance - stabilizing transformation , described by shchipkova et al . after data transformation , mann whitney and wilcoxon tests ( for inter and intragroup analysis , respectively ) were used for data analysis . for distribution and frequency analysis , the chi - square test was employed . a 5% level of significance this study was designed as a parallel , single - arm , and controlled study to evaluate the clinical and microbiological effects of st on the biodiversity of subgingival biofilm collected in smokers and non - smokers with chronic periodontitis . subjects included in the present study were examined , treated by st , and re - evaluated 21 days later . the study was approved by the institutional review board ( irb ) and patients received a detailed description of the proposed treatment and gave their informed and written consent . potential patients were selected from those referred to the graduate clinic of piracicaba dental school , university of campinas unicamp , brazil . all patients received a complete periodontal examination , including a full - mouth periodontal probing , radiographic examination and complete anamnesis . the study inclusion criteria were : diagnosis of chronic periodontitis , according to the criteria of the 1999 international classification ( 25);presence of at least 20 teeth;at least nine teeth presenting probing pocket depth ( ppd)5 mm with bleeding on probing ( bop ; being at least two with ppd7 mm);>35 years of age . diagnosis of chronic periodontitis , according to the criteria of the 1999 international classification ( 25 ) ; presence of at least 20 teeth ; at least nine teeth presenting probing pocket depth ( ppd)5 mm with bleeding on probing ( bop ; being at least two with ppd7 mm ) ; patients who : ( i ) were pregnant or lactating ; ( ii ) required antimicrobial pre - medication for the performance of periodontal examination and treatment ; ( iii ) were suffering from any other systemic diseases ( cardiovascular , pulmonary , liver , cerebral , or diabetes ) ; ( iv ) had received antimicrobial treatment in the previous 3 months ; ( v ) were taking long - term anti - inflammatory drugs ; and/or ( vi ) had received a course of periodontal treatment within the last 6 months ; were excluded from the study . a parallel , non - blinded and prospective study was designed to enroll the following two groups : non - smoker group ( n=10 ) : patients diagnosed with generalized and severe chronic periodontitis and that had never been smokers ; and smoker group ( n=10 ) : patients diagnosed with generalized and severe chronic periodontitis and that had a smoking habit for at least 10 years and smoked at least 20 cigarettes per day . sample size was determined by the bioestat program , using a standard deviation of 1.0 and a power value of 80% to detect a difference between groups of 1.0 mm . supragingival therapy ( st ) : after a full mouth examination and consent in participation , patients of both groups received a full mouth prophylaxis , supragingival calculus , and biofilm removal , using gracey curettes , an ultrasonic scaler , bicarbonate spray and floss , or interdental brushes , according to the interdental space . the patients were also individually instructed on how to perform oral self - care , including the bass technique , inter - dental flossing , and tongue brushing . all the subjects of the study received a standard fluoride dentifrice , toothbrushes , and floss as necessary ; and were asked to perform complete oral self - care hygiene at least twice a day . a week after this first instruction session , patients returned to a reinforcement of oral self - care instructions . twenty - one days after st , clinical re - evaluation was performed and subgingival samples were collected for microbiological analysis . the same individual ( dp ) was trained to perform st in all the patients , whereas another individual was trained to perform baseline and post - therapy clinical assessments ( tm ) . the following clinical parameters were assessed immediately before therapy : full - mouth plaque index ( fmpi ) , according to ainamo and bay ( 26 ) , and full - mouth bleeding score ( fmbs ) , according to mhlemann and son ( 27 ) ; these were calculated after assessing dichotomously the presence of dental biofilm at the site or bop from the bottom of the pocket when probing with a manual probe.1 the percentage of total sites that revealed the presence of plaque or bleeding was calculated . all teeth presenting at least one site with ppd5 mm were selected for clinical evaluation . these teeth did not present pulpal disease or furcation lesion ( in order to avoid bias ) . from those previously selected teeth , one site ( the deepest one ) was selected and followed - up during the experimental period , using an individually manufactured acrylic stent in which a groove was made to standardize the measurements . this selection resulted in a mean of 10.01.8 sites per patients evaluated during this study . for the sites selected , clinical parameters were : probing pocket depth ( ppd distance from the bottom of the pocket to the margin);relative clinical attachment level ( rcal distance from the bottom of the pocket to the stent margin);relative gingival margin position ( rgmp distance from the gingival margin to the stent margin ) . probing pocket depth ( ppd distance from the bottom of the pocket to the margin ) ; relative clinical attachment level ( rcal distance from the bottom of the pocket to the stent margin ) ; relative gingival margin position ( rgmp distance from the gingival margin to the stent margin ) . all parameters were evaluated using a periodontal probe at baseline and 21 days after st . the examinations were performed by a calibrated examiner ( tm ) . for this calibration , three patients were selected and full mouth rcal and ppd were measured , twice , within 24 hours , with at least 1 hour between the examinations . the intra - class correlation was calculated for each parameter , resulting in 93.5% reproducibility for rcal and 94.3% for ppd . subgingival biofilm collection : after a full - mouth examination , all sites previously selected for clinical follow - up were included in the subgingival biofilm analysis , as described . following the careful removal of supragingival biofilm , the areas were washed with a water spray , isolated with cotton rolls and gently dried . a sterile paper point2 was inserted into the bottom of the periodontal pocket for 30 s. each paper point was placed separately in sterile plastic tubes containing 0.01 m tris edta solution , ph 8.0 ( te ) . dna collection and extraction , samples from the selected sites were pooled together to allow the 16s cloning sequencing . cloning and sequencing : first , the 16s rrna gene was amplified using a universal primer set ( 27f and 1492r ) as described by de lillo et al . ( 29 ) . cloning procedures were performed using a topo - ta cloning kit,3 following the manufacturer 's instructions . briefly , the amplicons resulting from universal amplification were cloned into escherichia coli and then cultured overnight in luria - bertani ( lb ) plates ( containing ampicilin and x - gal ) for posterior colony selection . after colony selection , each colony was separately cultivated overnight , in lb broth media.4 the lb broth samples were then centrifuged to form a bacterial pellet containing e. coli that received vectors / bacterial dna . after vector extraction , the products were purified5 and sequenced.6 after sequencing , a partial sequence of 600 bp was generated . these sequences were initially aligned and a similarity matrix was constructed from the alignments by the method of jukes and cantor ( 30 ) ( bioedit 7.0 program ( 31 ) ) . phylogenetic trees were constructed by the neighbor joining method ( dotur program ( 32 ) ) . sequences were compared using the homd database ( 33 ) applying a level of 98.5% sequence identity as cut - off . sequences presenting 98% or greater similarity within a genus were considered as the same species . clinical parameters were analyzed by student 's t test ( for baseline intergroup comparisons ) and repeated - measures anova / tukey ( for clinical changes occurring after st ) , using the proc glm procedure of the sas program.7 for microbiological data , a variance - stabilizing transformation , described by shchipkova et al . after data transformation , mann whitney and wilcoxon tests ( for inter and intragroup analysis , respectively ) were used for data analysis . for distribution and frequency analysis , the chi - square test was employed . a 5% level of significance there were no statistical differences between groups regarding age or gender , as well as in relation to full mouth clinical parameters : plaque , bop , periodontal probing depth , and clinical attachment level ( p>0.05 ) . table 2 illustrates the effect of st on the clinical parameters assessed in the selected sites ( ppd5 mm ) . at baseline , intergroup analysis ( i.e. non - smokers vs. smokers ) showed no significant differences for any of the parameters assessed ( p>0.05 ) . the same results were seen after st , with no differences between the groups ( p>0.05 ) . in contrast , intragroup analysis ( i.e. baseline vs.after st , within the same experimental group ) demonstrated that st led to a significant reduction in pi , ppd , and rcal ( p<0.05 ) ; whereas no significant changes were found with respect to bop and gmp , in smokers and non - smokers ( p>0.05 ) . clinical and demographic characteristics of participants at baseline ( meanstandard deviation ) notes : fmpi , full mouth plaque index ; fmbop , full mouth bleeding on probing ; fmppd , full mouth periodontal probing depth ; fmcal , full mouth clinical attachment . clinical parameters in smokers and non - smokers , at baseline and after supragingival therapy ( st ) , at the selected sites notes : st , supragingival therapy ; pi , plaque index ; bop , bleeding on probing ; ppd , periodontal probing depth ; calr , clinical attachment level relative ; gmp , gingival margin position . statistical difference between baseline and st within groups ( repeated measures anova followed by tukey test , * p<0.05 , * * p<0.01 ) . in total , 1,800 clones were identified , representing 78 different species in non - smoker subjects at baseline and 73 after st , whereas in smokers 71 species were identified at baseline and 70 were observed after st . table 3 depicts the distribution of clones in the phylum and cultivation status ( cultivated and not - yet - cultivated ) . there was no difference in phylum distribution between smokers and non - smokers , neither at baseline nor after st . in regards to cultivation status , non - smoker subjects presented a higher prevalence of cultivated indexes compared with smokers at baseline , and at after st ( p<0.05 ) . in addition , 21 days after st , an increase in cultivated phylotypes was seen only in non - smokers ( p<0.05 ) . distribution by phylum ( total number of clones ) and culture condition ( % of cultivated and not - yet - cultivated phylotypes ) in smokers and non - smokers , at baseline and after supragingival therapy ( st ) notes : st , supragingival therapy . * indicates intergroup difference at baseline ; indicates intergroup difference after st ; indicates intragroup difference ( chi - square test , p<0.05 ) . figure 1 illustrates the distribution of 16s clonal analysis by genera in non - smokers and smokers , before and after st . as noted , at baseline , smokers demonstrated significantly greater levels of the genera fusobacterium and bacteroides , whereas non - smokers presented higher levels of eubacterium , synergistes , and streptococcus ( chi - square test , p<0.05 ) . distribution by genus ( percent total clones ) in smokers and non - smokers , at baseline and after supragingival therapy ( st ) . * indicates intergroup differences at baseline ; indicates intergroup differences after st ; indicates intragroup differences after st ( chi - square test , p<0.05 ) . moreover , st altered the genera distribution in both groups . in non - smokers , a significant reduction in some genera such as eubacterium , filifactor , tannerella , treponema , and fusobacterium was observed , whereas only filifactor and fusobacterium were reduced in smokers after st ( p<0.05 ) . the difference in genera distribution was still observed after st , where streptococcus was more present in non - smokers ( p<0.05 ) and bacteroides and porphyromonas were found more in smokers ( p<0.05 ) . the 20 most detected phylotypes were analyzed separately with regard to their frequency and proportions in the subgingival biofilm ( table 4 ) . significant differences were observed in levels of certain species between current and never - smokers . vincentii and bacteroidetes [ g-2 ] sp . oral taxon 274 clone au126 ( p<0.05 ) , whereas non - smokers showed higher levels of streptococcus constellatus and eubacterium [ g-6 ] nodatum oral taxon 694 . moreover , supragingival therapy led to a more expressive alteration in the subgingival composition in non - smokers . twenty - one days after st , non - smokers presented lower levels of filifactor alocis , t. forsythia , eubacterium [ xi][g-5 ] saphenum oral taxon 759 and eubacterium [ xi][g-3 ] brachy ( p<0.05 ) , whereas only t. forsythia presented a significant reduction after st . interestingly , a significant increase in synergistetes [ g-3 ] sp . oral clone bh017 and porphyromonas endodontalis oral clone aj002 was observed in smokers after st ( p<0.05 ) , highlighting the dissimilarities between smokers and non - smokers with regards to their microbial profile . top 20 most detected phylotype proportions ( x value mean ) in smokers and non - smokers , at baseline and after supragingival therapy notes : st , supragingival therapy * indicates statistical difference between non - smokers and smokers at baseline ; indicates statistical difference between non - smokers and smokers after st ; indicates statistical difference between baseline and after st within group ( mann whitney and wilcoxon tests , p<0.05 ) . there were no statistical differences between groups regarding age or gender , as well as in relation to full mouth clinical parameters : plaque , bop , periodontal probing depth , and clinical attachment level ( p>0.05 ) . table 2 illustrates the effect of st on the clinical parameters assessed in the selected sites ( ppd5 mm ) . at baseline , intergroup analysis ( i.e. non - smokers vs. smokers ) showed no significant differences for any of the parameters assessed ( p>0.05 ) . the same results were seen after st , with no differences between the groups ( p>0.05 ) . in contrast , intragroup analysis ( i.e. baseline vs.after st , within the same experimental group ) demonstrated that st led to a significant reduction in pi , ppd , and rcal ( p<0.05 ) ; whereas no significant changes were found with respect to bop and gmp , in smokers and non - smokers ( p>0.05 ) . clinical and demographic characteristics of participants at baseline ( meanstandard deviation ) notes : fmpi , full mouth plaque index ; fmbop , full mouth bleeding on probing ; fmppd , full mouth periodontal probing depth ; fmcal , full mouth clinical attachment . clinical parameters in smokers and non - smokers , at baseline and after supragingival therapy ( st ) , at the selected sites notes : st , supragingival therapy ; pi , plaque index ; bop , bleeding on probing ; ppd , periodontal probing depth ; calr , clinical attachment level relative ; gmp , gingival margin position . statistical difference between baseline and st within groups ( repeated measures anova followed by tukey test , * p<0.05 , * * p<0.01 ) . in total , 1,800 clones were identified , representing 78 different species in non - smoker subjects at baseline and 73 after st , whereas in smokers 71 species were identified at baseline and 70 were observed after st . table 3 depicts the distribution of clones in the phylum and cultivation status ( cultivated and not - yet - cultivated ) . there was no difference in phylum distribution between smokers and non - smokers , neither at baseline nor after st . in regards to cultivation status , non - smoker subjects presented a higher prevalence of cultivated indexes compared with smokers at baseline , and at after st ( p<0.05 ) . in addition , 21 days after st , an increase in cultivated phylotypes was seen only in non - smokers ( p<0.05 ) . distribution by phylum ( total number of clones ) and culture condition ( % of cultivated and not - yet - cultivated phylotypes ) in smokers and non - smokers , at baseline and after supragingival therapy ( st ) notes : st , supragingival therapy . * indicates intergroup difference at baseline ; indicates intergroup difference after st ; indicates intragroup difference ( chi - square test , p<0.05 ) . figure 1 illustrates the distribution of 16s clonal analysis by genera in non - smokers and smokers , before and after st . as noted , at baseline , smokers demonstrated significantly greater levels of the genera fusobacterium and bacteroides , whereas non - smokers presented higher levels of eubacterium , synergistes , and streptococcus ( chi - square test , p<0.05 ) . distribution by genus ( percent total clones ) in smokers and non - smokers , at baseline and after supragingival therapy ( st ) . * indicates intergroup differences at baseline ; indicates intergroup differences after st ; indicates intragroup differences after st ( chi - square test , p<0.05 ) . moreover , st altered the genera distribution in both groups . in non - smokers , a significant reduction in some genera such as eubacterium , filifactor , tannerella , treponema , and fusobacterium was observed , whereas only filifactor and fusobacterium were reduced in smokers after st ( p<0.05 ) . the difference in genera distribution was still observed after st , where streptococcus was more present in non - smokers ( p<0.05 ) and bacteroides and porphyromonas were found more in smokers ( p<0.05 ) . the 20 most detected phylotypes were analyzed separately with regard to their frequency and proportions in the subgingival biofilm ( table 4 ) . significant differences were observed in levels of certain species between current and never - smokers . vincentii and bacteroidetes [ g-2 ] sp . oral taxon 274 clone au126 ( p<0.05 ) , whereas non - smokers showed higher levels of streptococcus constellatus and eubacterium [ g-6 ] nodatum oral taxon 694 . moreover , supragingival therapy led to a more expressive alteration in the subgingival composition in non - smokers . twenty - one days after st , non - smokers presented lower levels of filifactor alocis , t. forsythia , eubacterium [ xi][g-5 ] saphenum oral taxon 759 and eubacterium [ xi][g-3 ] brachy ( p<0.05 ) , whereas only t. forsythia presented a significant reduction after st . interestingly , a significant increase in synergistetes [ g-3 ] sp . oral clone bh017 and porphyromonas endodontalis oral clone aj002 was observed in smokers after st ( p<0.05 ) , highlighting the dissimilarities between smokers and non - smokers with regards to their microbial profile . top 20 most detected phylotype proportions ( x value mean ) in smokers and non - smokers , at baseline and after supragingival therapy notes : st , supragingival therapy * indicates statistical difference between non - smokers and smokers at baseline ; indicates statistical difference between non - smokers and smokers after st ; indicates statistical difference between baseline and after st within group ( mann whitney and wilcoxon tests , p<0.05 ) . the habit of smoking has been reported to negatively affect periodontal tissues , cell defense , and host response . in addition however , very limited information is available with respect to the effect of st on the biodiversity of the subgingival biofilm in smokers with chronic periodontitis . with this in mind , it was hypothesized that st , consisting of standard methods used for supragingival biofilm removal and control , including supragingival biofilm / calculus removal and dental surface polishing , removal of biofilm retainers , oral hygiene instruction and reinforcement after 7 days , would affect subgingival biofilm profile in smokers with severe chronic periodontitis . subgingival biofilm biodiversity was assessed by the 16s gene cloning technique and clinical parameters were also used to illustrate clinical conditions at baseline and after st in smokers and non - smokers . data analyses demonstrated that : ( i ) there were significant differences in the subgingival biofilm composition , at baseline , in smokers versus non - smokers ; ( ii ) subgingival biodiversity was significantly affected by st in non - smokers , whereas only a slight effect was observed for smokers ; and ( iii ) clinical response was not affected by dissimilar microbiological outcomes to st in smokers versus non - smokers . with non - surgical subgingival therapy as the main treatment modality , most authors report greater reductions in probing depth in non - smokers , compared with smokers ( 19 , 3438 ) . it is , therefore , important to note that there is substantial evidence for the clinical improvement in smokers after treatment , indicating that smoking , as a risk factor , will compromise rather than prevent tissue healing . in non - smokers , st results in a reduction in gingival bleeding , probing depth , and a gain in attachment level ( 7 , 9 , 13 , 14 , 39 , 40 ) . in smokers , however , there was a lack of information on the impact of st on the periodontal tissues at untreated sites . gomes et al . , in 2007 ( 40 ) , were the only group to assess the changes in periodontal tissues after a supragingival periodontal therapy . in their study , st consisted of supragingival calculus removal with hand curettes , extractions , endodontic treatment , placement of temporary restorations , and prostheses . ( 40 ) demonstrated that a comparable clinical outcome was reached for smokers and non - smokers regardless of the severity of the disease . both groups featured a reduction in plaque and bleeding indices , as well as a reduction in probing depth , and gain in clinical attachment level after 30 days of st , which was maintained for up to 180 days ( 40 ) . in addition , as previously reported by others , in the present study , data analyses demonstrated that smokers presented a lower reduction in plaque index ( pi ) than non - smokers . moreover , as expected , deep pockets presented only a mild change after st : only a discrete reduction in bop index was observed in smokers and non - smokers . in summary , available clinical data suggest that smokers and non - smokers with severe chronic periodontitis may similarly benefit from st . in the present study , we aimed to determine whether or not st affected subgingival biofilm biodiversity in both groups . often , studies have focused on assessing , in smoking and non - smoking conditions , the effect of periodontal therapy on genera associated with periodontal disease , including p. gingivalis , treponema denticola , and t. forsythia ( 7 , 11 , 12 , 41 , 42 ) . however , an important study demonstrated that tobacco might also affect the levels of genera not always associated with periodontal disease ( 24 ) . in the present study , using an open - ended approach , a comparison between the subgingival biofilm composition of smokers and non - smokers at baseline ( before st ) , revealed a higher presence of certain genera , such as fusobacterium ( genus associated with periodontal disease ) and bacteroides ; and lower levels of streptococcus , synergistes , and eubacterium . these findings seem to confirm that tobacco exposure may lead to a subgingival biofilm composed not only in a higher proportion by pathogens associated with periodontal disease , but also some species , not commonly included as periodontal pathogens in target - ended or selective techniques . however , in addition to presenting a reduction in health - associated biofilm and an increase in disease - associated biofilm , in the present study , smokers showed a lower response to st , compared with non - smoker subjects as regards the microbiological composition of the subgingival biofilm . non - smokers had a significant reduction in five species commonly associated with periodontal disease following st , whereas in smokers only the levels of f. alocis were significantly reduced . in addition , after st in smokers , disease - associated species , including p. endodontalis , were found to be increased . new species associated with periodontal disease ( 43 ) , and the fact that p. endodontalis has additionally been shown to be reduced in subgingival biofilm when the smoking habit is given up ( 44 ) , suggesting this species as an important factor that might contribute to the pathogenesis of periodontal disease in smokers . the apparent reduction in some species in the subgingival biofilm after st may be explained by the intimate relationship between both supra- and subgingival environments ( 45 ) , and also as a result of the mild , but statistically significant , clinical benefits promoted by st after 21 days . the probing depth reduction may have led to significant changes in nutrients , oxygen , and microorganisms disposable in periodontal pockets , altering subgingival microflora . moreover , the reduced microbiological effect of st on smoker - subgingival microbiota , has been a common goal in previous studies . furthermore when subgingival scaling and root planing are performed , smokers appear to remain positive for periodontitis - associated species , including p. gingivalis , t. denticola , t. forsythia , p. intermedia , f. nucleatum , and parvimonas micra ( 41 , 46 , 47 ) . therefore , after st performance and the assessment of baseline and follow - up parameters , all the patients enrolled in this study were then treated by the conventional subgingival scaling and root planing approach . in conclusion , in smokers , st only slightly affected the subgingival biofilm biodiversity , as compared with non - smokers . however , clinically , the response in smokers and non - smokers was similar regardless of the differential impact of st on the subgingival biofilm composition in these groups of patients . there is no conflict of interest in the present study for any of the authors .

backgroundthe aim of this study was to assess subgingival microbiological changes in smokers versus non - smokers presenting severe chronic periodontitis after supragingival periodontal therapy ( st).methodsnon - smokers ( n=10 ) and smokers ( n=10 ) presenting at least nine teeth with probing pocket depth ( ppd ) ( 5 mm ) , bleeding on probing ( bop ) , and no history of periodontal treatment in the last 6 months were selected . clinical parameters assessed were plaque index ( pi ) , bop , ppd , relative gingival margin position ( rgmp ) and relative clinical attachment level ( rcal ) . subgingival biofilm was collected before and 21 days after st . dna was extracted and the 16s rrna gene was amplified with the universal primer pair , 27f and 1492r . amplified genes were cloned , sequenced , and identified by comparison with known 16s rrna sequences . statistical analysis was performed by student 's t and chi - square tests ( =5%).resultsclinically , st promoted a significant reduction in pi and ppd , and gain of rcal for both groups , with no significant intergroup difference . microbiologically , at baseline , data analysis demonstrated that smokers harbored a higher proportion of porphyromonas endodontalis , bacteroidetes sp . , fusobacterium sp . and tannerella forsythia and a lower number of cultivated phylotypes ( p<0.05 ) . furthermore , non - smokers featured significant reductions in key phylotypes associated with periodontitis , whereas smokers presented more modest changes.conclusionwithin the limits of the present study , st promoted comparable clinical improvements in smokers and non - smokers with severe chronic periodontitis . however , in smokers , st only slightly affected the subgingival biofilm biodiversity , as compared with non - smokers .

hashimoto 's thyroiditis ( ht ) is one of the most common human autoimmune diseases responsible for considerable morbidity [ 1 , 2 ] . it is characterized by diffuse lymphocytic infiltration of the thyroid gland , elevated levels of the serum antithyroid antibodies , clinical evidence of goitrous or atrophic gland , and frequent thyroid dysfunction in varying degrees . subacute thyroiditis ( sat ) is an acute inflammatory disorder of the thyroid gland most likely due to viral infection , characterized by painful toxic goiter with systemic inflammation . acute - phase reactants have been implicated for their involvement as proinflammatory molecules in various inflammatory diseases . serum c - reactive protein ( crp ) , the classical acute - phase protein of hepatic origin , is a nonspecific marker of inflammation in the body [ 6 , 7 ] . as changes in serum crp levels reflect the presence and intensity of inflammation , crp has been used as a clinical marker to assess the inflammatory status in many diseases . crp estimations have not been routinely used to monitor thyroid disease , although many thyroid conditions involve inflammation . saliva has been shown to contain systemically - derived biomarkers of infectious diseases [ 810 ] . saliva assay systems require necessary sensitivities for the detection of biomarkers present at low , but still pathophysiologically relevant , concentrations in saliva . in the present study , a newly developed enzyme - linked immunosorbent assay ( elisa ) method with a low limit of detection for crp was used for its estimation in saliva . previous studies have reported marked variations in serum crp levels with different thyroid disease entities . but data on crp levels in saliva of ht and sat patients are lacking in literature . the objective of the study was to estimate and compare salivary crp levels in hashimoto 's thyroiditis and subacute thyroiditis . the patients were recruited from k. s. hegde charitable hospital , deralakatte , mangalore , karnataka , india . the diagnosis was based on increased antithyroglobulin antibody ( tg - ab ) , antithyroid peroxidase antibody ( tpo - ab ) titers , thyroid function tests , and fine - needle aspiration ( fna ) cytology results . it consisted of 15 diagnosed patients who were negative for tpo - ab and tg - ab . exclusion criteria for the study were the existence of any comorbid cardiac , autoimmune , infectious , musculoskeletal , or malignant disease , oral disease , and a recent history of operation or trauma . serum total t3 ( tt3 ) , total t4 ( tt4 ) , and tsh levels were measured by electrochemiluminescence ( elecsys 2010 , germany ) . tg - ab and tpo - ab were measured by solid phase enzyme immunoassay ( hycore biomedicals ltd , germany ) . normal ranges in our laboratory are as follows : tt3 , 0.81.8 ng / ml ; tt4 , 4.511.5 g / dl ; tsh , 0.405.5 miu / liter ; tg - ab , less than 225 iu / ml ; tpo - ab , less than 35 u / ml ; esr , less than 20 mm / h ( female ) , 10 mm / h ( male ) . the procedures followed were in accordance with the ethical standards of the institutional human ethics committee and with the helsinki declaration . unstimulated whole saliva was collected by passive drooling as described previously at least 2 hours after any food intake . briefly , after 3 - 4 rinses of the mouth with water , saliva was allowed to accumulate in the floor of the mouth for approximately 2 minutes and repeatedly expectorated into an ice - chilled polypropylene vial to collect about 2 ml . the concentration of crp in saliva was estimated by elisa method using salimetrics c - reactive protein elisa kit ( pa , usa ) . data were analyzed with spss-17.00 using one - way analysis of variance ( anova ) method and turkey multiple comparison test . chi - square test was used for comparison of gender differences between the study groups . there was no significant difference in terms of age , gender , between the ht , sat , and control groups ( table 1 ) . the tsh titers of the ht patients were significantly higher when compared to sat and control subjects . mean tt3 and tt4 levels were below the normal lower limit in ht group indicating that the patients were in hypothyroid state . mean tt3 and tt4 levels were above the normal upper limit in sat group indicating that the patients were in hyperthyroid state . the esrs with a mean value of 61.82 mm / h were significantly higher in the sat group when compared to the control and ht groups ( table 1 ) . in sat group , there exist epithelioid cell granulomas with multinucleate giant cells , acute and chronic inflammatory cells , follicular epithelial cells with degenerative changes , and cellular debris in the background ( figure 1 ) . in ht group , there exist oxyphilic epithelial cells ( askanazy cells ) , moderate to large number of lymphocytes , few plasma cells admixed with and surrounding the epithelial cells , variable number of epithelioid histiocytes , scanty or no colloid , and hemorrhagic background ( figure 2 ) . mean crp level in saliva of ht ( hypothyroid ) group was not significantly different when compared to the euthyroid control group ( table 2 ) . however , mean crp level in saliva of sat ( hyperthyroid ) group was significantly higher when compared to the euthyroid control group ( table 2 ) . mean salivary crp level of sat ( hyperthyroid ) group was observed to be significantly higher than the ht ( hypothyroid ) group ( table 2 ) . the results of our study demonstrated higher salivary crp levels in sat patients who presented with clinical features of hyperthyroidism when compared to ht patients who presented with clinical features of hypothyroidism and euthyroid control subjects . this is not surprising because the hallmark of the laboratory evaluation of sat is a markedly elevated esr and elevated serum interleukin-6 ( il-6 ) levels , as well as other inflammatory markers . crp secretion is increased in response to a complex network of cytokines , especially il-6 and either il-1 or tumor necrosis factor ( tnf- ) . these results of crp in saliva are in concordance with previously reported findings by pearce et al . that serum crp levels were elevated in patients with painful subacute thyroiditis , which conceivably reflects the presence of an underlying systemic inflammatory process . there are few data in the literature regarding the relationship between serum and saliva levels of crp . crp in human saliva measured by application of a microchip assay system has been shown to positively correlate with serum crp estimated by elisa . elevated serum and salivary crp levels have been reported in patients with psoriasis [ 14 , 15 ] . positive correlation between porcine serum and salivary crp has been reported . in all those studies , however , the above - mentioned previous reports [ 8 , 1416 ] , findings of elevated serum crp levels in sat patients reported by pearce et al . , and the demonstration of increased saliva crp levels in sat patients of the present study indirectly suggest the possibility of the existence of a positive correlation between saliva and serum crp levels in the subjects . however , further studies need to be done to establish the association between serum and saliva levels of crp . the concentrations of most molecules present in saliva are usually one tenth to one thousandth of those in blood . in the present study , the estimated mean concentration of salivary crp in normal controls ( 0.837 g / l ; table 2 ) is observed to be approximately 1000-fold lower than the reported normal levels for serum crp ( 08 mg the estimated mean concentration of salivary crp in the present study in ht ( 0.817 g / l ; table 2 ) is observed to be approximately 3000-fold lower than the reported mean concentration of serum crp in ht by pearce et al . ( 2.80 mg / l ) . according to the knowledge of the authors , there are no other reports in literature on estimation of crp in saliva of ht or sat patients . mean crp level in saliva of ht ( hypothyroid ) group of the present study was not significantly different when compared to the euthyroid control group . these results are in concordance with studies by pearce et al . , sacide et al . , and kon and degroot which reported that serum crp levels are not significantly increased in patients with ht . . involved ht subjects at euthyroid status while patients of our ht group were in hypothyroid status . but the study by kon and degroot included patients with painful ht who presented with hypothyroidism . therefore , crp levels in ht appear to be independent from thyroid function status . regarding the association between thyroid hormone levels and serum crp , previous studies have reflected conflicting observations . had shown a clear association between hypothyroidism and raised serum crp [ 20 , 21 ] . reported higher serum crp and il-6 levels in patients with ht who presented with clinical features of subclinical hypothyroidism ( sch ) when compared to healthy controls . had shown that patients with ht , short - term hypothyroidism , and postpartum thyroiditis at different stages ( thyrotoxic , euthyroid , or hypothyroid ) had similar serum crp as compared to their euthyroid controls . . also showed no difference in serum crp levels between patients with sch and euthyroid individuals . christ - crain et al . reported that serum crp levels did not correlate with thyroid hormone levels in groups of overtly and subclinically hypothyroid subjects and that treatment of subclinical hypothyroidism did not alter serum crp values . with respect to the matter of whether systemic inflammation exists in ht , taddei et al . hypothetically thought the inflammatory process in ht to be related to the increased tsh levels and discussed the probability of chronic activation of the immune system due to ht . demonstrated that patients affected by ht without other apparent autoimmune disorders have a generalized activation of the immune system . the study by sacide et al . revealed low - grade systemic inflammation with extremely high values for another acute phase protein , serum amyloid a ( saa ) in ht patients ( euthyroid ) when compared to the controls . therefore , although salivary crp level is theoretically promising as a way to assess inflammation , it appears to have only a limited role in ht . the acute phase reaction is in most circumstances a good indicator of inflammatory activity and tissue damage . crp is a direct and quantitative measure for the acute phase reaction and , due to its fast kinetics , provides adequate information of the actual situation . the esr , on the contrary , is in fact an indirect measure of the acute phase reaction . it does react much slower to changes of inflammatory activity and is influenced by a number of other factors . the major advantages for using saliva - based assays have been described in some detail previously [ 810 ] . most importantly , collection of saliva may be done by procedures that are considered to be noninvasive , painless , and convenient . consequently , these methods may be performed several times a day . the use of saliva for crp estimation in sat patients could therefore allow the evaluation of their inflammatory status by using non - invasive and minimally stressful sampling methodology . in conclusion , crp levels estimated in saliva of sat patients were observed to be significantly increased compared to ht patients and euthyroid controls in our study . the rise in saliva crp levels in sat patients conceivably reflects the presence of an underlying systemic inflammatory process . saliva crp levels appear to serve as inflammatory markers in sat patients and , therefore , may aid clinical evaluation of the patients .

c - reactive protein ( crp ) , an acute - phase reactant , has been identified as a saliva - based biomarker of inflammation . the objective of the study was to estimate and compare salivary crp levels in hashimoto 's thyroiditis ( ht ) and subacute thyroiditis ( sat ) . the study included 30 ht patients who presented with clinical features of hypothyroidism , 15 sat patients who presented with clinical features of hyperthyroidism , and 20 healthy age- and sex - matched euthyroid controls . crp levels in saliva were estimated using an enzyme - linked immunosorbent assay method with enhanced sensitivity . in ht , the mean salivary crp levels did not differ significantly from controls . sat patients had significantly elevated salivary crp levels compared to ht patients and controls . the rise in salivary crp levels in sat patients conceivably reflects the presence of an inflammatory process . saliva crp levels appear to serve as inflammatory markers in sat patients and may aid their clinical evaluation .

in vertebrates , viral double - stranded rnas ( dsrnas ) in the cytoplasm bind and activate rig - i ( retinoic acid - inducible gene i)-like cytoplasmic viral sensor proteins ( rlrs ) ( for review , see takeuchi and akira , 2010 ) . the known features that these sensors use to discriminate virus and pathogen rnas from host cytoplasmic rnas include the presence of dsrna ends and 5 triphosphates . rlrs translocate on dsrna , and some rlrs may scan dsrna to help distinguish between host and virus molecules . cytoplasmic viral rnas usually lack modifications , because most viruses do not encode modifying enzymes . it has been proposed that nucleic acid modifications of cellular rnas help innate immune sensors to avoid aberrant activation by host nucleic acids ( gehrig et al . , 2012 ; karik et al . , 2005 ; vitali and scadden , 2010 ) . consistent with this idea , transfection of in - vitro - transcribed rna into cultured cells generates an innate immune response . however , if the rna is synthesized to contain naturally occurring modified bases , then the modified rna does not cause innate immune induction ( karik et al . . suppression of responses of innate immune rna sensors by modifications normally present in host rnas could act as thresholding mechanisms to help prevent aberrant responses . response thresholding mechanisms may be required because some cellular rnas do contain rna duplexes or have 5 triphosphates . rna duplexes in host rnas are particularly hazardous ; some alu hairpins are still present in 3 utrs of mature mrnas in the cytoplasm ( capshew et al . , 2012 ) . transcription also occurs over most of the human genome , which inevitably generates further dsrna that may reach the cytoplasm ( kapranov et al . , 2007 ) . the rig - i and mda5 ( melanoma differentiation associated gene 5 , ifih1 ; interferon induced with helicase c domain 1 ) sensors are activated by binding rna duplexes and signal through the mavs ( mitochondrial antiviral signaling ) adaptor protein to activate nfb , irf3/7 , and ap1 . this activates transcription of genes encoding type i interferon ( ifn ) and proinflammatory cytokines . secreted type i ifn binds to cell - surface type i ifn receptors to amplify and spread the antiviral response , inducing transcription of a large set of antiviral , ifn - stimulated genes ( isgs ) . aberrant or chronic activation of ifn - stimulated defense processes is very damaging to the host . experiments in cultured cells do not test the overall importance of rna modification for restraining innate immune responses in whole - model organisms or in human diseases . however , recent findings on human gene mutations causing autoimmune diseases are consistent with possible significant roles for rna modifications in autoimmune diseases . for instance , mutations in the adar1 gene encoding one rna - editing adenosine deaminase acting on rna cause aicardi - goutires syndrome ( ags ) ( rice et al . , 2012 ) . ags is a fatal childhood encephalopathy with aberrant ifn expression and symptoms resembling those caused by congenital virus infection . similar rare mutations in adar1 also cause dystonia due to bilateral striatal neurodegeneration associated with ifn overproduction ( livingston et al . over 100 mutations in adar1 have been identified in east - asian patients with dyschromatosis symmetrica hereditaria ( dsh ) , a mild genodermatosis with mostly unknown ifn status , in which the dominant phenotype appears to be often due to adar1 haploinsufficiency ( liu et al . , 2006 ) . adars catalyze the deamination of adenosine to inosine , which is the most common base modification known to occur in mammalian rna ( gerber and keller , 2001 ) . inosine is readily detected in transcriptome sequence data because inosine prefers to base pair with cytosine , leading to replacement of genome - encoded adenosine ( a ) by guanosine ( g ) at edited positions in cdna sequences . adars edit particular adenosine residues at specific positions in short rna duplexes in protein - coding transcripts , and they also edit numerous adenosines promiscuously in longer dsrnas ( for review , see heale and oconnell , 2009 ) . site - specific rna - editing events in transcript open reading frames that generate new isoforms of encoded proteins represent the best - understood mechanism of adar action . vertebrates have two enzymatically active adar proteins : adar1 ( adar ) and adar2 ( adarb1 ) . editing of the critical q / r site in the transcript encoding the key ampa receptor subunit is performed by adar2 . mutant adar2 mice die from seizures within 3 weeks of birth , and seizures and death are prevented by knocking in an editing - equivalent a to g mutation in the ampa receptor subunit gene ( higuchi et al . , 2000 ) . adar1 mutant mice die by embryonic day e12.5 with massive overproduction of ifn , loss of embryonic liver hematopoietic cells , liver disintegration , and widespread apoptosis ( hartner et al . cultured adar1 mutant embryonic fibroblasts are also highly sensitive to stress - induced apoptosis ( hartner et al . , research has focused on identifying a key adar1 target transcript analogous to the ampa receptor transcript . however , among the approximately 60 known editing events that recode open reading frames , many of the key events are catalyzed primarily by adar2 , and none appears likely to account for the adar1 mutant embryonic lethal phenotype . however , only 0.4% of human a - to - i editing occurs within protein - coding sequences ( peng et al . , 2012 ) ; the vast majority of known a - to - i - editing sites , now estimated at over 100 million in the human genome ( bazak et al . , 2013 ) , have been found in rna duplex - forming pairs of alu elements embedded in inverted orientations near each other in introns and 3 utr regions of transcripts . editing of cellular dsrna leads to formation of i - u wobble base pairs that cause bending and alter the properties of the dsrna helix ; multiple sequential i - u base pairs and high levels of promiscuous editing destabilize dsrna . editing of endogenous rna duplexes may lower the risk that they aberrantly induce innate immune responses . the constitutive adarp110 isoform is a shuttling protein that accumulates mainly in the nucleus and edits dsrna before nuclear export ( desterro et al . , 2003 ) . the n - terminally extended adarp150 isoform , expressed from a late ifn - inducible promoter , is predominantly cytoplasmic and has been shown to edit viral rnas ( samuel , 2011 ) . transfected dsrna oligonucleotides containing inosine - uracil ( i : u ) base pairs have been shown to bind to rlrs competitively with poly i : c and to suppress activation of innate immune responses ( vitali and scadden , 2010 ) . we reveal that loss of adar1 rna - editing activity and the resulting loss of inosine bases in rna are critical in producing aberrant rlr - mediated innate immune responses in adar1 mutant mice and cultured mouse cells . we characterize the immune response - blocking actions of human adar1 protein in adar1 mutant mouse cells and show that most ags - associated adar1 mutant proteins have impaired rna - editing activity . we wished to investigate whether aberrant immune responses are critical to the adar1 mutant embryonic lethal phenotype in mice . preventing type i ifn signaling is sufficient to fully rescue embryonic lethality in embryos mutant for dnaseii ( yoshida et al . , 2005 ) that also die with type i ifn overproduction . we constructed mouse strains that generate adar1 ; ifnar1 ( ifn- and - receptor 1 ) double - mutant embryos in crosses . no live adar1 ; ifnar1 progeny were obtained ( table s1 ) . staged embryo collections from crosses of adar1 ; ifnar1 parents suggest that the embryonic lifespan of the double homozygous adar1 ; ifnar1 embryos was extended due to ifnar1 ( table 1 ; figure 1 ) ; the mixed c57bl6/129 strain background in these crosses had little effect ( table s1 ) . responses to secreted type i ifn are not the main reason for adar1 mutant embryonic lethality . although embryonic death is delayed , histological analysis of the adar1 ; ifnar1 embryos revealed that the rescued embryos die with defects similar to those seen in the adar1 mutant ( figure s1a ) . rescue is incomplete and variable at e14.5e15.5 ( figures 1a1f ) , and some embryos still have major defects in liver structure with apoptotic nuclei both in hepatocytes and hematopoietic cells ( figures 1e1h ) . liver , heart , and lungs are underdeveloped in adar1 ; ifnar1 embryos compared to wild - type ; there are fewer densely staining hematopoietic cells in the adar1 ; ifnar1 liver , and there is an apparent lack of peripheral blood in or around any of the organs ( figure 1 ) . in the e15.5 adar1 ; ifnar1 liver , 58% of the erythrocytes are still nucleated , versus 5% in the wild - type ( figure s1b ) . a highly similar phenotype was observed in adar1 ; stat1 embryos , with hematopoietic defects and subsequent lethality occurring around e15.5 ( figure s1c ; table s1 ) . stat1 is a key mediator of systemic ifn responses ( stark and darnell , 2012 ) . embryonic lethality occurs earlier in adar1 mutant than in the dnaseii mutant and does not depend on the amplifying effect of high type 1 ifn secretion . because adar1 ; ifnar1 double - mutant embryos do survive for 34 additional days , this suggested that rescue of adar1 mutant embryonic lethality might be achieved with a mutation giving a more potent , cell autonomous block to aberrant immune antiviral activation , ifn production , and apoptosis . if cytoplasmic dsrna in the adar1 mutant triggers an aberrant antiviral response by activating rlrs , then the key adaptor protein in that signaling pathway is the mitochondrial antiviral signaling protein mavs . to investigate the effect of blocking signaling from rlrs , we generated strains that produce adar1 ; mavs double - mutant embryos in crosses . the adar1 ; mavs double - mutant embryos have a significantly extended survival up to live birth of pups ( table 1 ; figure 2a ) . histological sections through a newborn adar1 ; mavs pup show apparently normal liver , heart , and other internal organs ( figure 2a ) . the adar1 ; mavs double - mutant pups are able to feed , but they die within a day of birth . preliminary data suggest that the newborn pups still show heightened ifn and interleukin 1 ( il-1 ) expression in blood ( data not shown ) . this rescue shows that adar1 mutant embryonic lethality is largely due to aberrant rlr / mavs pathway signaling triggering apoptosis . to investigate innate immune responses in more detail , six primary mouse embryonic fibroblast ( mef ) cultures were established representing all possible adar1 genotypes in mavs mutant or wild - type genetic backgrounds . constitutive and polyriboinosinic : polyribocytidylic acid ( poly i : c)-induced levels of ifn- and interleukin-6 ( il-6 ) proteins were measured in early - passage mef culture supernatants by elisa . adar1 ; mavs mefs show a detectably elevated basal level of ifn- and respond to poly i : c by producing higher levels of both ifn- and il-6 than the wild - type ( adar1 ; mavs ) mefs . surprisingly , heterozygous adar1 mefs behave somewhat like homozygous adar1 mutant mefs and respond vigorously to poly i : c treatment with heightened expression of both ifn- and il-6 . this suggests that prevention of aberrant immune responses in adar1 mefs is very sensitive to levels of adar1 , consistent with evidence of adar1 haploinsufficiency in dsh patients ( liu et al . , 2006 ) . confirming that mavs mutation blocks the aberrant innate immune responses caused by the adar1 mutation , aberrant expression of type i ifn and proinflammatory cytokines observed in adar1;mavs mefs is prevented in the adar1 ; mavs double - mutant mefs ; these mefs also do not induce type i ifn or il-6 in response to poly i : c treatment ( figure 2b ) . to investigate whether there is an altered transcriptional profile in adar1 mutant embryos quantitative rt - pcr ( qrt - pcr ) analyses were performed to determine the expression levels of 12 interferon - stimulated gene ( isg ) , transcripts in total rna from e11.5 whole embryos . some isg transcripts are highly elevated in adar1 mutant whole embryos compared to wild - type embryos and levels return to normal in adar1 ; mavs and adar1 ; ifnar embryos ( figure 3a ) , though the adar1 ; ifnar1 double - mutant embryo does not normalize isg transcript levels as fully as adar1 ; mavs . the levels of these isg transcripts in the mavs and ifnar1 mutant embryos are also somewhat different from wild - type levels ( figure s2 ) , and therefore the relevant background has been subtracted from the double - mutant levels ( figure 3a ) . the qrt - pcr data confirm the elisa data on cultured fibroblasts showing that the abnormal ifn responses in adar1 mutant embryos are mediated by aberrant immune signaling through the mavs adaptor protein . to characterize alterations in gene expression across the whole transcriptome in adar1 mutant adar1 ; mavs double - mutant embryos , we sequenced ribosomal rna - depleted total rna from whole e11.5 embryos . overall , more transcripts decrease in whole adar1 embryos than increase ( figure 3b ) ; this may be related to losses of hematopoietic cells or to more widespread defects consistent with apoptosis observed in adar1 mutant embryos . we identified a set of 61 protein - coding transcripts that are upregulated at least 3-fold in adar1 mutant embryos and restored to near - wild - type levels in adar1 ; mavs embryos ( table s2 ) . gene ontology term analysis on the upregulated transcripts confirms enrichment of immune and antiviral response transcripts ( table s2 ) . to confirm the increased expression of immune gene transcripts in the adar1 mutant , changes in proinflammatory cytokine transcript expression in adar1 ; ifnar1 and adar1 ; mavs embryos were calculated relative those in ifnar1 and mavs mutants , which were again slightly different from wild - type levels ( figure s2 ) . consistent with the sequencing data , levels of certain isgs and proinflammatory cytokines were highly increased in the adar1 mutant compared with wild - type ( figures 3a and 3b ) . altered expression levels of immune gene transcripts in the adar1 mutant embryo are restored close to wild - type levels in both adar1 ; ifnar1 and adar1 ; mavs double - mutant embryos . the increase in the expression of the transcription factor stat1 is very striking in the adar1 embryo at e11.5 and the stat1 transcript is reduced in both adar1 ; ifnar1 and adar1 ; mavs double - mutant embryos . stat1 protein is critical to the cellular response to extracellular ifn ( for review , see stark and darnell , 2012 ) ; however , consistent with the similarly limited effect of the ifnar1 mutation , generating an adar1 ; stat1 double mutant also does not rescue adar1 mutant embryonic lethality fully ( figure s1c ) . the stronger rescue of embryonic lethality by mavs mutation may not be due mainly to stronger effects on transcription , but rather the mavs mutation may also block apoptosis or other nontranscriptional effects of aberrant rlr signaling . together both the qrt - pcr and elisa data confirm that the abnormal responses in adar1 mutant embryos are mediated by immune signaling through mavs . to examine transcription in more detail in embryonic hematopoietic cells , we sequenced ribosomal rna - depleted total rna from e15.5 embryonic livers of adar1 ; mavs and mavs sibling embryos ; liver is the main site of hematopoiesis at this stage . adar1 ; mavs e15.5 fetal livers appear normal and comparisons of rna sequencing ( rna - seq ) data from adar1 ; mavs and mavs livers could not identify any protein - coding transcripts expressed at significantly differing levels . the lack of aberrant transcription in adar1 ; mavs livers shows that all observed alterations in levels of protein - coding transcripts in the adar1 mutant are effects of aberrant rlr signaling and interferon production . it has been proposed that rna editing could increase turnover of edited dsrna due to cleavage by an inosine - specific ribonuclease , but the physiological effectiveness of this turnover process is not known ( scadden , 2005 ) . if increased levels of repetitive transcripts with dsrna - forming potential do arise in the adar1 mutant , these might cause the aberrant antiviral response . if repetitive transcripts increase as a direct result of the absence of adar1 , the aberrant repetitive transcript levels should be detected also in the adar1 ; mavs double mutant . to assess expression of transcripts likely to form dsrna , a detailed analysis of repetitive sequence expression in adar1 ; mavs and mavs e15.5 liver total rna - seq data was performed ( figure 4 ) . increased expression of individual members of erv and iap subfamilies was observed ( table s3 ) , and qrt - pcr analyses confirmed increased expression of mmervk10c transcripts ( figure 4e ) . overall , however , changes in repetitive transcripts parallel those seen for protein - coding transcript ; i.e. , levels of most repetitive elements did not change between adar1 ; mavs and mavs . this suggests that a general dramatic increase in levels of repetitive transcripts due to slowed turnover is not the initiating event in the aberrant immune response in adar1 mutants . changes in some individual repetitive transcripts could contribute to initiating aberrant immune responses in the adar1 mutant , but the reduction in inosine levels within cellular dsrna is perhaps more critical . to determine whether the aberrant antiviral response in adar1 mutant cells is due to reduced inosine modification in intracellular rnas , we established mef cell cultures from adar1 ; p53 double - mutant embryos . adar1 mefs could not be cultured long term due to cell death ( figure s3 ) , and adar1 ; mavs double - mutant mefs are not useful because the aberrant innate immune response is blocked . when compared with p53 mutant mefs , elevated expression of isgs is observed in adar1 ; p53 mefs when cultures are stressed by nutrient starvation ( figure 5a ) ; without stress , isg expression is very low in adar1 ; p53 mefs . we chose the p53 mutant background because this prevents cell death associated with a range or other mutations ( dittmer et al . , the reduction in aberrant isg expression in adar1 ; p53 versus adar1 mefs is consistent with the idea that preventing cell death is also an important feature of the mavs mutant rescue . to test for rescue of adar1 mutant phenotypes by adar protein variants , we next established stable adar1 ; p53 mef lines expressing wild - type or mutant human adar1 proteins from stably integrated piggybac constructs that we previously characterized ( heale et al . , 2009 ) . the adar1 ; p53 piggybac mefs were starved for 72 hr to stress the cells , total rna was isolated and qrt - pcr was performed to measure isg transcript levels . expression levels of isg transcripts were normal in p53 mutant cells and highly elevated in adar1 ; p53 cells ( figure 5a ) . when either the predominantly nuclear adar1 p110 isoform or the ifn - inducible mainly cytoplasmic adar1p150 isoform were expressed , isg transcripts were significantly reduced ( figure 5a ) . expressing the adar1p150 ( e912a ) mutant that inactivates these data indicate that robust suppression of isg transcription requires a catalytically active adar capable of deaminating adenosine to inosine into rna . expressing human adar2n , a nuclear localization mutant of adar2 that aberrantly accumulates in the cytoplasm ( wong et al . , 2003 ) , in adar1 ; p53 mefs also significantly reduced expression of the isgs ( figure 5a ) . mef cells already express endogenous , active , adar2 in the nucleus , and site - specific editing of protein - coding transcripts usually involves transient exon - intron rna duplexes formed before splicing . adar2 proteins have different specific site preferences from adar1 proteins ( keegan et al . , 2011 ) . the adar2n is more likely to rescue nonspecific , promiscuous editing events lost in the adar1 mutant than it is to edit some transcript containing a highly adar1-specific site . the sufficiency of either catalytically active adar for strong rescue is consistent with the idea that loss of promiscuous editing in intracellular dsrna is critical to the adar1 mutant phenotype . if cellular dsrna lacking inosine in adar1 mutant cells is sufficient to induce an aberrant innate immune response , then isolating rna from adar1 mutant cells and transfecting it into wild - type cells might replicate this effect . therefore , we extracted total rna from livers of adar1 ; mavs e15.5 embryos and transfected this into rig - i reporter cells but could not detect any difference between the responses induced by adar1 ; mavs and wild - type rna samples . because the majority of a total rna sample is rrna or trnas , this experiment may not mimic in vivo conditions . we decided to perform instead the converse experiment , which was to determine if inosine - containing rnas interfere with immune induction by transfected rna . to elucidate if transfecting dsrna oligonucleotides containing inosine - uracil base pairs suppress aberrant isg expression in adar1 mutant phenotype , we established a test system using adar1 ; p53 mefs . to partially induce innate immune responses in adar1 ; p53 mefs , we transfected these cells with levels of an in - vitro - transcribed fluc mrna that has been shown previously to induce a modest innate immune response in hela cells ( vitali and scadden , 2010 ) . transfecting similar concentrations of fluc rna into the hypersensitive adar1 ; p53 mef cells caused a dramatic increase in expression of isg transcripts ( figure 5b ) , as in the case of serum starvation ( figure 5a ) . this strong response of adar1 ; p53 mefs to transfected rna is likely to reflect the aberrant immune induction due to the adar1 mutation . transfecting dsrna oligos alone into adar1 ; p53 mefs has very little effect on immune induction at the concentrations used ( figure 5c ) . dsrna oligonucleotide ( c - dsrna ) or iu - dsrna that contained four inosine - uracil base pairs were then cotransfected with 500 ng fluc rna to look for specific inhibitory effects of iu - dsrna oligonucleotides on isg transcript induction ( figure 5c ) . iu - dsrnas further reduce isg transcript expression below reductions caused by control c - dsrnas ( figure 5d ) . these reductions in isg transcript levels were maintained for 24 hr with the iu - dsrna , whereas the c - dsrna was unable to reduce the isg response at this time point ( data not shown ) . the background inhibitory effect of the control c - dsrna oligonucleotide in these experiments could arise if the 20-mer dsrna oligonucleotides bind only one rlr molecule each , thus impeding oligomerization of rlr card domains ( peisley et al . , 2014 ) . it is clear that transfection of i - u dsrna specifically inhibits aberrant immune induction further . both this specific effect of i - u base pairs and the general suppressive effect of 20-mer dsrna oligonucleotides indicate that aberrant rna complexes formed by rlrs are central to the adar1 mutant phenotype . most of the mutations in adar1 occurring in patients with ags change residues on the surface of the catalytic domain at the c terminus of the protein ( figure 6 ) , close to where dsrna is predicted to bind . previously , we tested the effects of these ags mutations on enzymatic activity in adar1p110 ( rice et al . , 2012 ) . constructs expressing adar1p110 were transiently cotransfected into hek293 cells with a construct expressing a known editing substrate for adar1 and editing activity was measured . surprisingly , most of the ags mutant proteins , with the exception of the adar1 g1007r mutation , still exhibited robust editing activity , though with statistically significant reductions . we now tested the effects of the same ags mutations in adar1p150 in a similar manner ( figure 6a ) . each of the ags mutations causes a significant decrease in editing activity of adar1p150 that is greater than the effect of the same mutation in the adar1p110 isoform . this large difference between mutant effects in the two isoforms was not anticipated because the ags mutations are predominantly in the c - terminal domains shared by the two isoforms . we provide a report of mutations in adar1 that have different effects on the two isoforms , revealing a significant separation of roles for the two isoforms . these findings also imply that the effects of ags mutations in adar1 are mediated particularly through adar1p150 and primarily involve reductions in rna - editing activity . loss of the ifn - inducible cytoplasmic adar1 isoform may be important in ags and dystonia patients . clinical observations have suggested that symptoms may develop following an infection when type i ifn expression should have led to later expression of active adar1p150 ( livingston et al . , 2014 ) adar1 mutant proteins found in ags patients were also tested for effects on rna - editing activity in hek293 cells when expressed in combinations matching those observed in the ags patient cohort ( figure 6b ) . evidence for interactions between different ags mutations in the same adar1 protein and for interactions between different adar1 mutant proteins in heterozygotes was obtained . for instance , we found that the adar1p150 p193a mutant exhibits a significant decrease in editing activity ( figure 6a ) . the p193a mutation is expressed only in the n - terminally extended adar1p150 isoform and increases the effect of other ags mutations in the same protein ( figure 6b ) . the crystal structure of the adar1 n - terminal z - alpha domain complex with z - rna has been solved , and the p193a mutation is predicted to change a highly conserved amino acid involved in nucleic acid contact ( schade et al . , 1999 ) . this variant is enriched in the ags cohort but is also present in 41 of 6,553 control patients in the exome variant server database . an instance of strong interaction between different adar1 protein variants in ags heterozygote patients is seen with the adar1p110 g1007r mutant that binds dsrna but is catalytically inactive and which exerts a dominant - negative effect on editing by adar1p110 wild - type protein ( rice et al . , 2012 ) ( figure 6b ) . this study elucidates a key conserved role for adars in preventing aberrant ifn responses by cytoplasmic antiviral rlr innate immune rna sensors . mouse adar1 mutant embryos die by embryonic day e12.5 with aberrant ifn expression , failure of hematopoiesis , and degeneration of the embryonic liver ( hartner et al . we obtained partial rescues of adar1 mutant embryonic lethality by combination with ifnar1 or stat1 mutations and a much more substantial rescue to live birth by combination with a mavs mutation . the mavs rescue , in particular , indicates that the adar1 embryonic lethal mutant phenotype is largely due to an aberrant antiviral response . some mutations affecting rlrs have phenotypes similar to adar1 mutants , for example , a mutation in the mouse ifih1 gene encoding mda5 that causes constitutive ifn signaling ( funabiki et al . , 2014 ) , giving rise to a lupus - like autoimmune disorder . mutations in mavs but not ifnar1 also rescued the aberrant immune response in those ifih1 mice . in addition , with striking similarity to adar1 , mutations in human ifih1 also cause ags ( rice et al . , 2014 ) . our preliminary data indicate , however , that combining ifih1 with adar1 does not rescue the lethality of the adar1 mutant mouse embryo ( data not shown ) , suggesting that mda5 is not the sole mediator of the adar1 mutant phenotype . loss of the late ifn - inducible cytoplasmic adar1 p150 isoform , in particular , may be critical to the adar1 mutant phenotype . this isoform interacts in a complex way with viruses and may participate in the resolution phase of the ifn response by editing residual viral rnas ( ward et al . , 2011 ) . adars have been reported as being proviral because adars facilitate replication of viruses in cultured cells ( samuel , 2011 ) . however , proviral effects are unusual among proteins induced by ifn , and these experiments in cell cultures may be somewhat misleading . studies on adar1 mutant mice lacking only the adar1 p150 isoform instead suggest an overall negative effect of adar1 p150 on measles virus propagation , illustrating the importance of whole - animal studies ( ward et al . , hepatitis c virus encodes a protease that cleaves mavs , and it would be interesting to determine whether this virus or protease rescue aspects of adar1 mutant phenotypes . we show that signaling from antiviral innate immune sensors through mavs is inhibited by dsrna oligonucleotides containing inosine - uracil base pairs . transfection of i - u dsrna corresponding to an adar1-edited section of an artificial dsrna substrate into adar1 mutant mefs suppresses the aberrant antiviral response . the data suggest that the aberrant antiviral response in the adar1 mutant results from loss of inosine in cytoplasmic dsrna that normally inhibits antiviral sensor activation . other examples of how changes in modification states of rnas determine innate immune responses come from viruses encoding ribose 2-o - methylation enzymes that methylate viral mrna cap structures to match host mrna caps so that they avoid activating the mda5 sensor ( zst et al . , 2011 ) . vertebrate trnas also have specific modifications at certain positions in their structures that allow innate immune sensors to discriminate between them and bacterial trnas ( gehrig et al . , 2012 ) . modifications that occur naturally in host rnas also prevent activation of other innate immune sensors such as pkr and toll sensors ( nallagatla et al . , 2008 ) . because transfection of i - u dsrna blocks the aberrant immune activation in adar1 ; p53 mefs , it is most likely that the aberrant immune activation is due to an endogenous dsrna that activates rlrs . we can not exclude the possibility that a site - specific editing event contributes , but , if the adar1 mutant aberrant immune induction was due to loss of editing in a transcript encoding an innate immune protein or any other indirect effect , then inhibiting rlrs at the level of rna interaction would be unlikely to rescue . in the case of i - u dsrna inhibition of rlrs , the significant effect of sequential i - u wobble base pairs bending and destabilizing dsrna ( mclaughlin and keegan , 2014 ) makes it possible to envisage why the sensors are able to detect edited dsrna ( figure s4 ) . i - u dsrna binds the rlrs competitively with activating dsrna ( vitali and scadden , 2010 ) , probably making grossly similar contacts to the helicase domains and carboxy - terminal domains . rlrs are suitable to assess dsrna structure because they surround the dsrna using multiple protein domains to make many contacts to both strands ( kowalinski et al . , 2011 ; the hel2i domain , in particular , could have a role in scanning the dsrna minor groove to detect i - u base pairs and other imperfections ( figures s4c and s4d ) , possibly leading to alterations in dynamic rlr domain rearrangements required for signaling . natural rna duplexes formed by repetitive elements in transcripts will usually be imperfectly paired , and rna editing will further reduce helical regularity . formation of regular filaments by rlrs on dsrna facilitates formation of card domain complexes activating mavs signaling ( kowalinski et al . , 2011 ; peisley et al . , rlr signaling may be exquisitely sensitive to mismatches and rna - editing events present in rna duplexes that indicate the dsrna is not a direct product of virus replication . in adar1 ; p53 mutant mefs , the catalytically inactive adar1 protein is the least effective at reducing elevated isg transcripts though it is still rna binding competent , demonstrating that rna - editing activity is required . catalytically active adar1 proteins and even a cytoplasmically mislocalized adar2 mutant protein ( wong et al . , 2003 ) this emphasizes the role of rna - editing activity , argues against a dominant role for adar1-specific protein - protein or protein - rna interactions , and suggests that promiscuous editing by adars is involved rather than a highly adar1-specific editing event . no adar1 protein fully suppressed isg transcripts to levels observed in the p53 mutant , but the reintroduced adar1 proteins are not expressed under the control of the ifn - inducible adar1 promoter itself . because transfection of general dsrna oligonucleotides containing inosine - uracil base pairs significantly reduces elevated isg levels in adar1 ; p53 mefs , the importance of inosine in rna in preventing the innate immune response is supported in two distinct ways . several of the ags mutations alter residues on the surface of the deaminase domain close to where rna is predicted to bind ( rice et al . , 2012 ) , and it is possible that the d1113h mutation , which affects editing activity the least , alters adar1 function in other ways such as by perturbing protein and/or rna interactions required for suppression of innate immunity . in addition to the catalytically active adar1 and adar2 proteins , two other vertebrate adars lack catalytic deaminase activity ( for review , see heale and oconnell , 2009 ) . therefore , editing - independent adar roles are maintained and could involve protein interactions on dsrna or sequestration of dsrna . while this manuscript was under review , wang and colleagues showed a substantially stronger editing - independent role of adar1 in decreasing innate immune responses in hek293 ( yang et al . , 2014 ) . we also observe some editing - independent rescue , and we suspect that the difference is due to our use of adar1 ; p53 mefs completely lacking adar1 and their use of hek293 cells . we do not know why adar1 ; mavs newborn pups die within a day of birth , but preliminary data on cytokine expression in blood from these pups implicate some residual inflammatory effects . activated rlrs can bypass mavs and induce inflammasome responses via an alternative pathway ( lucioli et al . , cell death is a very prominent effect in the adar1 mutant , and possible mavs - bypass signaling to cell death pathways in some cells in adar1 ; mavs newborn pups also remains to be investigated . our findings suggest , however , that preventing the aberrant systemic ifn response may not always be sufficient to treat these diseases . when the important cellular modified nucleic acids that modulate innate immune responses have been defined , they may point the way to new therapeutic approaches to autoimmune and neurodegenerative diseases . adar1 mice in the c57bl6n background were obtained from m. higuchi and p. seeburg ( strain c192 ) ( hartner et al . , 2004 ) . these mice were crossed to the ifn receptor subunit mutant ( ifnar1 ) in the 129 strain background ( b&h animal suppliers ) . two sublines breed adar1 in the ifnar1 background , and crosses of these mice generate adar1 ; ifnar1 embryos . two further sublines generate heterozygous adar1 animals with the mixed b6/129 strain background , and crosses of these generate control adar1 embryos without the ifnar1 mutation . the cardif ( mavs , ips-1 , or visa ) mutant line generated by the tschopp group in lausanne was obtained from caetano reis e sousa in london . all experiments on mice were performed in accordance with edinburgh university rules and national guidelines on animal experimentation . cdnas encoding adar proteins were subcloned into the piggybac expression system ( system biosciences ) and transfected into adar1 ; p53 mefs using lyovec ( invivogen ) . cells were grown for 2 weeks , and those with successful integration were isolated by fluorescence - activated cell sorting ( gfp positive ) . the taqman array mouse immune panel ( cn : 4367786 ; life technologies ) was used for quantitative gene expression analysis of the immune response signatures in e11.5 whole embryos . the taqman array card was loaded with the first strand cdna sample and taqman universal pcr master mix ii ung ( life technologies ) according to the manufacturer s protocol . the amplification was performed on an upgraded version of the applied biosystems 7900ht fast real - time pcr system , and the analysis of the data was performed as described above . n.m.m . performed experiments and assisted with writing the manuscript ; s.m.g . performed experiments and assisted with writing the manuscript ; r.y . performed bioinformatic analyses of mouse sequences ; s.c . performed experiments ; j.b . performed genotyping of mouse strains ; d.r . performed genotyping of mouse strains ; c.n . performed bioinformatic analyses of mouse sequences ; c.v . performed experiments ; c.p.p . assisted with bioinformatic analyses of mouse sequences ; p.j.m . performed rna - protein structural analyses , m.f.j . designed experiments ; j.d . advised on innate immunity and mouse genetics ; i.r.a . performed bioinformatic analyses of repeats ; a.d.j.s . designed experiments and provided reagents ; m. . assisted with writing the manuscript ; l.p.k . performed experiments , designed experiments , and wrote the manuscript ; and m.a.o . designed experiments and wrote the manuscript .

summarythe adar rna - editing enzymes deaminate adenosine bases to inosines in cellular rnas . aberrant interferon expression occurs in patients in whom adar1 mutations cause aicardi - goutires syndrome ( ags ) or dystonia arising from striatal neurodegeneration . adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day e12.5 . we demonstrate that adar1 embryonic lethality is rescued to live birth in adar1 ; mavs double mutants in which the antiviral interferon induction response to cytoplasmic double - stranded rna ( dsrna ) is prevented . aberrant immune responses in adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing - active cytoplasmic adars . we propose that inosine in cellular rna inhibits antiviral inflammatory and interferon responses by altering rlr interactions . transfecting dsrna oligonucleotides containing inosine - uracil base pairs into adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response . adar1 mutations causing ags affect the activity of the interferon - inducible cytoplasmic isoform more severely than the nuclear isoform .

hidradenoma papilliferum ( hp ) is a benign neoplasm involving anogenital mammary - like glands ( mlg ) and occurs most commonly in the vulva or perianal region of adult white women . the lesions are usually solitary , well - demarcated papules or nodules covered with normal skin , and are generally less than 10 mm in diameter . when these tumors are not located in the anogenital area , they are termed ectopic hp . mlg have been documented in ectopic areas of the skin such as the ear , face , chest , and scalp . most of all are excised without suspicious . as a result , its imaging findings are not well known . then , the purpose of our report was to provide imaging findings of ectopic hp in axillary tail of the breast as rare location . a 46-year - old woman presented with a 5-year history of a slowly growing , palpable mass on the left breast . physical examination revealed a 4 cm , soft and movable , brown colored mass at the 2 o'clock location in the axillary tail region of the left breast . an ultrasound ( iu22 unit ; philips medical systems , bothell , usa ) of the left breast revealed grouped , 1 - 2 cm , well - circumscribed , round solid masses with internal cystic components in the dermis that abutted the subcutaneous layer of the left breast ( figure 1 ) and were located in the axillary tail . radiologically , these lesions were considered to be breast imaging reporting and data system ( bi - rads ) category 3 . grossly , part of the nodules was well encapsulated , lobular masses with purple - brown cut surface that abutted but did n't penetrate the skin ( figure 2 ) . hematoxylin and eosin ( h&e ) sections , 1 ( figure 3a ) and 200 ( figure 3b ) revealed dermally based nodules with complex architecture and uninvolved epidermis . the nodules consisted of a cyst with large arborizing papillae which had fibrovascular core . the non - neoplastic breast tissue underneath the tumor showed focal apocrine metaplasia . microscopically , the lesions are cystic papillary tumors and are composed of proliferation of papillary fronds made of loose fibrous connective tissue lined by pseudostratified bland - looking epithelial cells . the immunohistochemical staining for estrogen receptor ( er ) or progesterone receptor ( pr ) were all positive and her2 protein was negative . hp is a skin appendage tumor that is most commonly reported on the anogenital region of women , particularly the vulva . however , the observed similarities of hp with intraductal papillomas of the breast indicate that mlg is the true origin of hp . hp primarily affects caucasian females , typically during the third to fifth decades of life . ectopic hps occur on non - anogenital sites in older patients , with an equal incidence in males and females . mlg are located in the subepidermal stroma , larger than apocrine or eccrine glands , frequently branched and sometimes truly lobulated , lined by basophilic rather than eosinophilic cells and are er or pr positive . mlg have been documented in ectopic areas of the skin such as the ear , face , chest , and scalp . therefore , the ectopic hp could occur in these regions and 19 cases of ectopic hps have been reported in the english language literature ( table 1 ) . the classification of cutaneous sweat gland lesions is very complex , as these lesions have a wide histological spectrum , and the pathogenesis and exact origin of many of these lesions is still under investigation and is unclear . eccrine sweat glands are widely distributed almost everywhere in the skin , but apocrine lesions of the skin are rare and are found mainly in body folds including the axillary , groin , and anogenital regions . occasionally , the tumors can be large and elevated , forming a reddish - brown mass with an ulcerated and bleeding surface . the incidence of multiple lesions is reported as 5% and all multiple lesions have been described as unilateral . histologically , it is a well - circumscribed , solid or cystic , dermal nodular lesion , not connected to the epidermis . recurrence of anogenital and ectopic hp is unusual and is typically attributed to incomplete excision of the primary tumor . hence it is possible that estrogen stimulation may play a role in the pathogenesis of hp in women . thus , er and pr seem to be reliable markers for distinguishing female anogenital glands from conventional sweat glands . it is important to remember that er and pr positivity has been described in a subset of breast cancers , including papillary neoplasms . accordingly , er and pr immunohistochemical staining should not be used to distinguish breast tumors from hp . less than 0.5% of cutaneous eccrine and apocrine neoplasms are associated with positive her2 immunohistochemical staining , whereas 20% of invasive breast adenocarcinomas are her2 positive . complex cystic breast masses demonstrate both anechoic ( cystic ) and echogenic ( solid ) components on ultrasound . moreover , hp has features analogous to an intraductal papilloma of the breast which can lead to misdiagnosis . but , most papillomas are located in breast parenchyma and are related to ducts , although some peripheral type of papilloma looks like only solid mass . adnexal tumor appears as a fairly well - demarcated cystic - solid mass in the dermis and hps have similar findings . due to the rarity of this tumor , and its common removal without imaging studies because of small size , typical radiological findings have not yet been well established . in conclusion , skin adnexal tumors , specifically hp , should be kept in the differential diagnosis when evaluating a subcutaneous breast tumor . to our knowledge , this is the first case report of hp developed in the breast skin of middle - aged woman .

hidradenoma papilliferum ( hp ) is a benign neoplasm arising from mammary - like glands which typically involves the dermal layer of the female anogenital area . the prognosis for hp is good . recurrence is unusual and is typically attributed to incomplete excision of the primary tumor . malignant transformation is rare and hp of the breast has not yet been reported . ectopic hp is usually solitary , small , and asymptomatic . it appears as a well - circumscribed , complex cystic mass in the dermis on ultrasound . we present a case of hp arising from the axillary tail of the breast .

aziridines , the smallest saturated aza - heterocycles , are important and common synthetic intermediates in organic chemistry . the small bond angles and associated ring strain inherent in aziridines affords high reactivity toward ring - opening reactions with carbon and heteroatom nucleophiles , providing functionalized amines with stereocontrol . aziridines participate in a range of additional transformations that take advantage of the ring strain , including cycloaddition reactions and rearrangements , which have been employed particularly in the synthesis of other nitrogen heterocycles.c - halogen - substituted aziridines introduce additional structural complexity and a further range of reactivity . halo - aziridines bearing chlorine have been most widely investigated , especially gem - dihalogenated aziridines due to their ease of preparation by the addition of dichlorocarbene to imines , and other suitable methods . these have been used as important building blocks in the synthesis of heterocyclic compounds due to their high reactivity toward both ring - opening and ring expansion reactions . the preparation and isolation of monohalogenated aziridines is less common and more challenging due to rearrangement chemistry dominating their reactivity . pioneering work in the formation of monochloroaziridines was reported by deyrup and co - workers ; a monochloroaziridine was generated by the reaction of dichloromethyllithium with n - benzylideneaniline at low temperatures , affording the cis - chloroaziridine ( figure 1a ) . since this seminal investigation , -chloroaziridines have been accessed via reductive halogenation from gem - dihalogented aziridines , addition of acid chlorides across 2h - azirines , nitrene addition to chloroalkenes , and trapping of metalated aziridines with an electrophilic source of chlorine ( figure 1b ) . chlorinated aziridines have been shown to undergo nucleophilic displacement of chloride with a variety of nucleophiles including naome , nacn and lialh4 , leaving the aziridine intact . ziegler first disclosed a 4:1 cis / trans mixture of bromoaziridines , formed via a barton decarboxylation - bromination sequence ( figure 1c ) . these bromoaziridine products were used to regenerate the radical from the c br bond to promote intramolecular cyclization , toward the synthesis of mitomycin - like antitumor agents . more recently , yudin adopted a nitrogen - transfer approach , generating nitrenes from n - aminophthalimide with phi(oac)2 in the presence of bromoalkenes ( figure 1d ) . huang has formed bromoaziridines through a multiple electrophilic addition of tsnbr2 to ene - ynoates . bromoaziridines have also been reported as intermediates , formed by the reaction of a silyldibromomethyllithium with imines , from which bromide was displaced in situ ( rmgx , lialh4 ) leaving the aziridine intact . dibromoaziridines have been recently reported by li through addition of bromoform to imines followed by cyclization . . there has been significant recent interest in the functionalization of intact aziridine rings as a divergent route to aziridine derivatives . to date this has largely been achieved through the formation of aziridine anions followed by reaction with electrophiles . deprotonation of unstabilized monosubstituted aziridines can occur regio- and stereoselectively : occurring trans to the substituent at the least hindered position for alkyl - aziridines , or at the benzylic position for aryl - aziridines . functional group exchange has also been demonstrated to be an effective method for generating aziridinyl anions , which react with electrophiles at the predefined position . in addition , the palladium - catalyzed cross - coupling of intact aziridines has recently been achieved ; separately vedejs , and ourselves have reported the cross - coupling of aryl halides with aziridine metal species formed by bu3sn li exchange and tolso mg exchange respectively . in both examples , the cross - couplings proceeded via transmetalation to zinc and afforded retention of stereochemistry at the reacting center . we envisaged that iodo - substituted aziridines would offer potential for functionalization of the intact ring via a variety of methods in a regio- and stereoselective fashion . we proposed that an efficient preparation of iodoaziridines would open possibilities for new complementary reactivity , with nucleophilic or electrophilic reagents , or via cross - coupling . we recently communicated the first examples of the iodoaziridine functional group bearing an n - boc group through the reaction of diiodomethyllithium with n - boc - imine sulfinic acid adducts ( figure 1e ) . this was successful with aromatic imine substrates , proceeding via a gem - diiodide intermediate in a highly diastereoselective manner , to afford aziridines bearing the aryl and iodo groups in a cis - relationship . here we disclose the full study into the preparation of a new class of alkyl and aromatic substituted iodoaziridines bearing an n - ts group , isolated with excellent cis - diastereoselectivity , in high yields in one step from n - tosylimines and n - tosylimine we report in detail the development of the reaction to form n - ts iodoaziridines , and their differing reactivity and stability to the n - boc iodoaziridines . the present methodology extends the reaction scope , being successful for alkyl as well as aryl imine substrates , and we also report the diastereoselective reaction with a stereochemically pure n - sulfinyl imine . in addition , we report a protocol for determining the optimal stationary phase to use in chromatography for the purification of potentially unstable compounds , which resulted in increased yields for the iodoaziridines . the selective transformation of an iodoaziridine to novel -iodo - n - ts - imine and -iodo - aldehyde functional groups is also reported . we proposed iodoaziridines could be accessed by an addition - cyclization protocol involving the reaction of n - ts - imines with diiodomethyllithium , analogous to the aza - darzens reaction . the aza - darzens reaction involves the addition of a carbon nucleophile bearing a leaving group to an imine to form a -haloamine intermediate that undergoes cyclization to afford the aziridine ( scheme 1 ) . commonly the carbene equivalent reagent is stabilized by an electron - withdrawing group , often an ester ( e.g. , r = co2r in scheme 1 ) . in these cases , the diastereoselectivity in the aziridine product is determined in the initial addition , which is followed by a stereospecific cyclization . concelln has reported the enantioselective preparation of terminal aziridines using iodomethyllithium and enantioenriched imines . diiodomethylmetal reagents ( mchi2 ) differ from both above scenarios , being unstabilized and substituted , and importantly as symmetrical nucleophiles , the initial addition step is not diastereodetermining . consequently the cyclization step determines the diastereochemistry of the aziridine product through selecting one of two potential iodide leaving groups . since mchi2 reagents were first described by seyferth and lambert in 1973 , they have had relatively little use in synthesis , possibly due to the required low temperatures . charette recently described improved conditions for the formation of diiodomethane anions ( lichi2 and nachi2 ) at 78 c generating gem - diiodoalkanes by alkylation with primary alkyl iodides , and ( e)--aryl vinyl iodides from benzyl bromides by alkylation / elimination . initial investigations were undertaken on the addition of mchi2 to phenyl n - ts imine , chosen due to ready availability and stability . the tosyl group was expected to be an appropriate electron - withdrawing group to stabilize charge in the intermediate ( nn*(s o ) ) , and prevent elimination of iodide from the iodoaziridine products , while also providing a degree of steric bulk sufficient to engender diastereocontrol in the cyclization step . in this study , mchi2 reagents were preformed by deprotonation of ch2i2 for 20 min at 78 c , prior to addition of the imine , and the reaction quenched after 1 h at 78 c to avoid any potential product decomposition on warming . the initial choice of solvent conditions ( a mixture of thf : et2o ) was selected due to the stabilizing effect on the carbenoid reagent . early studies varied the base used for deprotonation and workup procedures to ensure stability of the products . the use of lihmds as base to afford lichi2 was shown to be productive , affording a mixture of products , of which three components were identified : amino gem - diiodide 3a , the desired iodoaziridine 4a , as the cis - isomer , as well as aminal 2a formed by the direct addition of lihmds to the imine ( table 1 , entry 1 ) . these products exhibited highly characteristic h nmr signals : amino gem - diiodide 3a characterized by signals at 5.34 , 5.27 , and 4.51 ppm ( nh ; chi2 ; chph ) ; cis - iodoaziridine 4a , characterized by doublets at 4.89 and 3.89 ppm ( chi ; chph ) ; aminal 2a displayed doublets at 5.85 and 5.19 ppm ( nh ; chph ) . both gem - diiodide 3a and aziridine 4a were observed at 78 c , indicating cyclization was occurring at low temperature . it was notable that elimination to afford the vinyl iodide was not observed , even though this was the major side product in the n - boc derivatives . the ratio of products was determined by h nmr of the crude reaction mixture in the presence of an internal standard . reaction conditions : imine 1a ( 0.50 mmol ) , ch2i2 , lihmds , thf / et2o . yield of 2a , 3a or 4a determined by h nmr spectroscopy with reference to an internal standard ( 1,3,5-trimethoxybenzene ) . we were concerned with minimizing the formation of the undesired aminal product 2a , and aimed to optimize for the combined yield of diiodide 3a and iodoaziridine 4a . the formation of aminal 2a suggested that deprotonation of ch2i2 was incomplete under these conditions , which was also implied by deuteration studies , leaving lihmds in solution . increasing the equivalents of lihmds led to increased formation of 2a . indeed , in the absence of diiodomethane , treatment of imine 1a with lihmds at 78 c afforded aminal 2a in almost quantitative yield . the formation of aminal 2a was irreversible under the reaction conditions , and as a result varying the reaction time at 78 c had no effect on the formation of 2a . increasing the deprotonation time to 40 min and 1 h caused a dramatic drop in the combined yield of 3a and 4a . increasing the excess of diiodomethane employed to 10 equiv did afford a significant increase in the formation of 3a and 4a ( entry 2 ) , though the formation of aminal 2a remained at similar levels . reaction concentration and ratio of thf : et2o were found to be important to the product distribution and these were thoroughly explored under these conditions , but with little overall increase in yield . interestingly , reducing the concentration of the reaction , led to a notable increase in the proportion of the diiodide that underwent cyclization in the time frame of the reaction ( compare entries 2 and 3 ) . to reduce the excess diiodomethane employed , we examined the effect of lewis basic additives ( compare entries 1 and 47 ) . the use of hmpa was detrimental , whereas tmeda afforded an increase in the formation of 2a , but promoted cyclization . the addition of 1 equiv dmpu afforded the highest combined yield of 3a and 4a ( entry 6 ) . we found that reducing the concentration under these conditions afforded an increase in cyclization product 4a ( entry 7 ) . despite extensive further investigation using dmpu as an additive instead we examined the effect of increasing the equivalents of diiodomethane and base ( up to 4 equiv lihmds ) at a higher concentration , which gave rise to an increased combined yield of 3a and 4a ( entries 810 ) , presumably due to an increase in the amount of lichi2 present in solution . finally , it became apparent that the addition of diiodomethyllithium to the imine was rapid and the iodoaziridine product was decomposing under the reaction conditions . we continued with 3 equiv lihmds and it was identified that warming the reaction mixture to 0 c was sufficient to promote rapid and complete cyclization , with minimum decomposition ( entry 11 ) . the precise mixture of thf : et2o was optimized , with a mixture of of 2.5:1 thf / et2o giving the maximum yield . finally , reducing the time under the reaction conditions to a minimum , that is , warming the reaction as soon as addition of the imine was complete , afforded the highest yield of 4a , with complete cyclization of any diiodide intermediate ( entry 12 ) . this provided our standard conditions for further study , affording an 81% h nmr yield , which corresponded to a 76% isolated yield of iodoaziridine 4a , exclusively as the cis - diastereoisomer . throughout the optimization , only the cis - diastereoisomer of the iodoaziridine was observed , assigned on the basis of the magnitude of the coupling constant between char and chi protons ( j = 6.1 hz ) . these assignments are consistent with the coupling constants observed for cis - n - boc iodoaziridines isolated by ourselves , and for cis - n - boc bromoaziridines by ziegler and co - workers . to rationalize the excellent cis - diastereoselectivity for the reaction , we invoke the steric properties of the bulky so2tol group to discriminate between three possible conformations ( figure 2 ) . the r and sulfonyl groups will align in an anti conformation to avoid the eclipsing interactions that make conformer c - trans unfavorable . placing the n - group and iodide in an antiperiplanar fashion appropriate for cyclization therefore provides two possible conformations a - cis and b - trans . in the transition state the pyramidalization of n will position the toluenesulfonyl group to one side of the ring , which clashes with other ring substituents . we propose that an unfavorable interaction between the nondisplaced iodide and the toluenesulfonyl group is dominant . in the preferred conformation , a - cis , the iodide is positioned away from the bulk of the tolyl - sulfonyl group leading to the cis - iodoaziridine . in conformation b - trans , the unfavorable interaction between the nondisplaced iodide and the large toluenesulfonyl group results in the conformation being disfavored . rationale of diastereoselectivity in cyclization to afford cis - iodoaziridines . to explore the scope of the iodoaziridination reaction a range of of n - ts imines 1 and n - ts imine adducts 5 were prepared by literature procedures , with some minor modifications ( scheme 2 and scheme 3 ) . adducts , due to the increased stability to hydrolysis and enamine formation compared to the imines . with a series of imines in hand we examined the scope of the iodoaziridination reaction under our optimized set of reaction conditions . initially we examined the addition of diiodomethyllithium to aromatic imines under the reaction conditions optimized for 4a , forming the iodoaziridines in high yields with exclusive cis - diastereoselectivity ( table 2 ) . method a : imine ( 0.50 mmol ) , nbuli ( 1.50 mmol ) , hmds ( 1.50 mmol ) , ch2i2 ( 1.70 mmol ) , thf : et2o ( 0.16 m at deprotonation ) , 78 to 0 c . method b : identical to method a but reaction warmed to rt for 20 min after addition of imine . where > 95:5 stated , only the cis - diastereoisomer could be observed by h nmr . electron - donating groups were tolerated under the reaction conditions , as shown by the 4-me and 4-tbu - phenyl examples ( table 2 , entries 2 and 3 ) . it is notable that while phenyl iodoaziridine was stable to chromatography on silica , these more electron - rich examples were not ; purification on deactivated basic alumina ( activity iv ) afforded the yields stated . the remainder of the scope was purified by chromatography using basic alumina ( activity iv , see below for further discussion ) . ortho - substituted aromatic substrates were warmed to rt as they required an increased temperature to induce full cyclization from the intermediate -amino gem - diiodides ( entries 4 and 5 , denoted method b ) . for 2-tolyl imine 1d , a 9:5 ratio of iodoaziridine ( 4d)/amino gem - diiodide ( 3d ) was observed under the standard conditions . warming to rt by removing the reaction flask from the dry ice bath ensured complete cyclization ( > 19:1 ) to yield the corresponding cis - iodoaziridine in high yield . this was similarly successful with the 1-napthyl substituent affording an excellent yield of iodoaziridine 4e ( entry 5 ) . 4-fluoro- and 4-chloro - substituted aromatics were also well tolerated under the reaction conditions ( entries 6 and 7 ) . ortho - chlorophenyl imine 1h was subjected to the ortho - substituted reaction conditions ( method b ) but no cyclization was observed , and attempts to promote cyclization by additional warming of the reaction mixture led to decomposition . the lack of cyclization was attributed to stabilizing coordinating interactions of the lone pairs of chlorine in the postulated lithiated intermediate ( scheme 4 ) , preventing the required orientation for cyclization being achieved . the corresponding amino gem - diiodide 3h could be isolated in high yield by quenching the reaction at 78 c . subjecting isolated 3h to cyclization conditions previously developed for -n - boc diiodides to their corresponding iodoaziridines ( cs2co3 , dmf , rt ) , only afforded degradation of the starting material . in our previous work on n - boc iodoaziridines , pleasingly with the n - ts group , alkyl iodoaziridines could be successfully accessed , constituting a significant increase in reaction scope . using the cyclohexyl imine 1i , the reaction performed similarly to the aryl examples , that is , the diiodide formed rapidly and complete cyclization occurred to the iodoaziridine on warming the reaction mixture to 0 c . the cyclohexyl substituted iodoaziridine 4i was also obtained from the imine hso2tol adduct through the use of identical reaction conditions except for an additional equivalent of base and diiodomethane employed ( method c ) to form the imine in situ . the two methods returned cis - iodoaziridine 4i in comparable yields ( 68% from imine 1i vs 57% from imine adduct 5i , table 3 entries 1 and 2 ) . due to ease of synthesis and handling of the adducts , several of the alkyl imines were used in this form . a range of branched alkyl imine hso2tol adducts were examined under the modified reaction conditions ( entries 3 to 5 ) , each displaying complete cis - diastereoselectivity upon cyclization with good yields . using the -chiral imine generated from 5l afforded excellent cis : trans selectivity in the cyclization step , but only minimal diastereoselectivity in the addition step ( facial selectivity = 1.9:1 ) . alkene containing imine 1 m was also successful , but again without significant facial selectivity . the tbu - substituted imine 1n was submitted under the ortho - reaction conditions ( method b ) , due to concerns with the steric bulk of the tbu group affecting the degree of cyclization . remarkably , under these conditions , iodoaziridine 4n was isolated in an excellent yield of 70% , and only the cis - iodoaziridine was observed , despite significant eclipsing interactions between the tbu and iodide groups in the product . method a : imine ( 0.50 mmol ) , lihmds ( 1.50 mmol ) , ch2i2 ( 1.70 mmol ) , thf : et2o ( 0.16 m at deprotonation ) , 78 to 0 c . method b : identical to method a but reaction warmed to rt for 20 min after addition of imine . method c : imine ho2stol adduct ( 0.50 mmol ) , lihmds ( 2.00 mmol ) , ch2i2 ( 2.20 mmol ) , thf : et2o ( 0.16 m at deprotonation ) , 78 to 0 c . where > 95:5 stated , adducts did not perform well under the reaction conditions with npr and nhex side chains returning only 6 and 4% yields of the corresponding cis - iodoaziridines respectively ( scheme 5 ) . here aminal formation was the major product from the reaction as the reduced steric demands of these primary alkyl substrates allows the irreversible addition of the bulky lihmds , which is prevented in the branched substrates . attempts to increase the equivalents of diiodomethane , or of both diiodomethane and base , were unsuccessful and further optimization is required for this substrate class . the isolation and purification of potentially unstable compounds is an essential skill of the synthetic chemist . due to the acidic nature of silica gel , decomposition of compounds during silica chromatography can be a common occurrence . while there are several alternative materials that can be employed as stationary phases for chromatography , there is not a method to rapidly and quantitatively compare the performance of these alternatives with regard to the recovery of unstable compounds . whereas the majority of the n - boc iodoaziridines were stable to silica , it was quickly apparent in this study that the n - ts derivatives behaved significantly differently and compounds 4b and 4c underwent major decomposition . while 4a afforded a good recovery with respect to the yield determined by h nmr , purification of iodoaziridine 4b afforded < 50% of the expected recovery . we therefore used compound 4b to study the effect of different stationary phases on the recovery after purification . to achieve this , we developed a simple protocol for assessing the stability of potentially unstable compounds to chromatography , which enabled us to access the iodoaziridines in high yield . a sample of 4b was prepared as described above , and an internal standard added to obtain a yield by h nmr prior to purification ( table 4 , entry 1 ) . to probe the stability of 4b on a range of stationary phases , we subjected crude 4b to conditions that model the experience of the compound during column chromatography , replicating both the solvent conditions and the length of time of a normal purification procedure . samples of 4b containing the standard were added to a slurry of the relevant stationary phase in etoac / hexane and stirred for 30 min . the slurry was then filtered and the filtrate analyzed by h nmr to assess the recovery of the iodoaziridine . on comparison of the yield determined by h nmr a selection of stationary phases were compared as indicated in table 4 , in addition to a control experiment where no stationary phase was added . yield determined by h nmr spectroscopy with reference to an internal standard ( 1,3,5-trimethoxybenzene ) . basic alumina ( activity i ) , oven - dried for 24 h prior to use . basic alumina ( activity iv ) prepared by addition of water ( 10% w / w ) to basic alumina ( activity i ) . the recovery of iodoaziridine 4b was dramatically affected by changing the stationary phase ( table 4 and figure 3 ) . exposure of 4b to bench silica ( entry 3 ) and base - doped silica ( 1% et3n , entry 4 ) caused major degradation of the iodoaziridine to iodo(phenyl)acetaldehyde 7 ( vide infra ) . neutral alumina and basic alumina ( activity i ) appeared to trap the aziridine , with poor yields being returned for iodoaziridine 4b ( entries 5 and 6 ) . however , the stability of the iodoaziridine was greatly enhanced using deactivated basic alumina ( activity iv vs activity i ) , with only a small drop in the recovery observed . pleasingly , using column chromatography on basic alumina ( activity iv ) afforded an isolated yield of 48% , which closely resembled that observed in the crude mixture . selected sections of h nmr indicating the stability of iodoaziridine 4b to stationary phases . by comparison , performing the analysis on phenyl analogue 4a displayed essentially quantitative recovery on all potential stationary phases ( table 5 ) , with the exception of neutral alumina and basic alumina ( activity i ) , which trapped the product ( entries 5 and 6 ) . the minor products on bench silica and base - doped silica were assigned to be the corresponding -iodo - aldehyde . this indicated that the method is appropriate , providing good recovery when the compound does not undergo degradation . as a consequence of these results , we used basic alumina ( activity iv ) for the remainder of the reaction scope above . we believe this protocol may be a useful approach to determine the optimal stationary phase for chromatography of other compounds unstable to silica . an additional advantage compared to directly performing chromotography on different stationary phases was that this protocol enables a more facile investigation into the identity of decomposition products , where these may be missed on collecting fractions . yield determined by h nmr spectroscopy with reference to an internal standard ( 1,3,5-trimethoxybenzene ) . basic alumina ( activity i ) , oven - dried for 24 h prior to use . basic alumina ( activity iv ) prepared by addition of water ( 10% w / w ) to basic alumina ( activity i ) . during the isolation of more electron - rich iodoaziridines 4b and 4c it was observed that they were subject to rearrangement to form -iodo imines ( scheme 6 ) . this rearrangement could be achieved in quantitative conversion by submitting neat cis - iodoaziridine 4b to mild heating under reduced pressure . we propose this occurs by unimolecular opening of the iodoaziridine and elimination of iodide to afford a benzylic cation , which is trapped by iodide ( scheme 6 ) . the rearrangement of more electron - rich aromatic n - ts iodoaziridines ( r = ( 4-tbu)c6h4 , 4c ) was also observed by h nmr , but the resulting iodo - imine could not be isolated due to rapid decomposition . following the observations made during the stability studies to silica above , we were keen to establish whether iodoaziridine 4b could be converted directly to the iodo - aldehyde , which was observed on stirring crude 4b with silica . indeed , treating a crude sample of iodoaziridine 4b with bench silica in a mixture of etoac / hexane and open to the air , afforded complete rearrangement / hydrolysis of the aziridine to iodoaldehyde 7 in 54% over the 2 steps following chromatography ( scheme 7 ) . we were keen to extend the current protocol of iodoaziridination to chiral n - protecting groups to provide facial selectivity in the initial addition to the imine . the use of ellman s auxiliary has received significant attention in the stereoselective synthesis of aziridines via the aza - darzens approach . attempts to use the comparable toluenesulfinyl group were unsuccessful , as this is known to undergo attack at sulfur with organometallic reagents . therefore , we investigated the tbu sulfinyl group , which has been shown to offer stabilizing interactions in the functionalization of aziridinyl anions . phenyl t - butyl sulfinyl imine 9 was prepared by direct condensation using ti(oet)4 ( scheme 8) . sulfinyl imine 8 was then subjected to the reaction conditions optimized for the n - ts imines ( method a , table 2 ) . pleasingly this successfully afforded the desired iodoaziridines 9a and 9b in a 59% yield with diastereoselectivity ( dr = 85:15 ) . characteristic h nmr signals for aziridine protons were observed for both products ; doublets at 4.54 and 3.71 ppm for the major diastereoisomer and 4.83 and 3.30 ppm for the minor diastereoisomer , all with j = 6.0 hz corresponding to the cis - isomer . given the well - established models for stereocontrol for the 1,2-addition of organolithiums to aldimines the major and minor diastereoisomers could be predicted ( scheme 8 , boxed ) . in this model , the organolithium approaches from the least hindered face of the imine via the lone pair of the sulfinyl group , affording diastereoisomer 9a as the major product . the dr obtained in the addition of diiodomethyllithium is comparable to that obtained for organolithium reagents attacking through an acyclic transition state , for example phli addition into n - tert - butylsulfinyl 4-chlorophenyl imine in thf at 78 c , dr = 73:27 . due to the differing nature of the n - sulfinyl protecting group , stability tests were run to determine the best stationary phase for flash column chromatography in the manner described above . here , the n - sulfinyl iodoaziridine was found show similar stability to 4a , with the best recovery obtained with basic alumina ( activity v , 15% w / w water added ) . applying the optimized conditions to tbu - sulfonyl imine 10 ( method a ) , prepared by oxidation of sulfinyl imine 8 , afforded the corresponding n - bus - iodoaziridine 11 in a moderate 45% yield ( scheme 9 ) . we have developed an effective method to install iodide functionality onto a range of alkyl and aromatic substituted n - tosylaziridines . adducts at low temperature , followed by warming , afforded cyclization to the corresponding cis - n - ts - iodoaziridines in a highly diastereoselective fashion and in good yields . the use of the n - ts protecting group has enabled the formation of alkyl iodoaziridines for the first time . these novel alkyl and aromatic substituted iodoaziridines provide fascinating structures and potential synthetic intermediates for functionalization of the intact aziridine ring . the formation of these iodoaziridines was achieved in conjuction with our new protocol for assessing the best stationary phase for purification of this new class of compound . rearrangement products of electron - rich aryl iodoaziridines were also discovered , and the formation of an enantioriched n - sulfinyl iodoaziridine was achieved for the first time . all nonaqueous reactions were run under an inert atmosphere ( argon ) with flame - dried glassware using standard techniques . anhydrous solvents were obtained by filtration through drying columns ( thf , et2o , ch2cl2 ) . flash column chromatography was performed using 230400 mesh silica or 50200 m brockmann basic alumina ( activity iv or activity v ) with the indicated solvent system according to standard techniques . analytical thin - layer chromatography ( tlc ) was performed on precoated , glass - backed silica gel plates . visualization of the developed chromatogram was performed by uv absorbance ( 254 nm ) , or aqueous potassium permanganate stain . infrared spectra ( max , ftir atr ) were recorded in reciprocal centimeters ( cm ) . chemical shifts for h nmr spectra are recorded in parts per million from tetramethylsilane with the solvent resonance as the internal standard ( chloroform , = 7.27 ppm ) . data is reported as follows : chemical shift [ multiplicity ( s = singlet , d = doublet , t = triplet , m = multiplet and br = broad ) , coupling constant in hz , integration ] . chemical shifts are reported in parts per million from tetramethylsilane with the solvent resonance as the internal standard ( cdcl3 : 77.0 ppm ) . chemical shifts are reported in parts per million referenced to the standard monofluorobenzene : 113.5 ppm . assignments of h / c spectra were made by the analysis of /j values , and cosy , hsqc , and hmbc experiments as appropriate . reagents : commercial reagents were used as supplied or purified by standard techniques where necessary . compound handling and storage : the n - ts iodoaziridines displayed sensitivity to light and during all handling , exposure of iodoaziridines to light was minimized . however , the n - ts iodoaziridines displayed a notable increase in stability to exposure to light in comparison to the n - boc derivatives . iodoaziridines were stored at 20 c neat for short periods or as a solution in ch2cl2 or chcl3 to prevent decomposition . for example , iodoaziridine 4i was stored in a cdcl3 solution for > 4 months without displaying noticeable decomposition . deactivated basic alumina : the activity of basic alumina was altered by the addition of water to commercial basic alumina ( activity i ) and evenly distributed ( activity iv : 10% w / w water ; activity v : 15% w / w).imines : imines 1a , c d , g , h i and imine l and 5o p were synthesized according to the method of chemla and co - workers . imine 1 m was synthesized by a modification of the method of chemla and co - workers . imines 1f and 1n by a modification of the method of proctor , and imines 1b , 1e by the method of stalick . the relevant aldehyde ( 10.0 mmol , 1.0 equiv ) was added to a solution of p - toluenesulfonamide ( 1.71 g , 10.0 mmol , 1.0 equiv ) and sodium p - toluenesulfinate ( 1.96 g , 11.0 mmol , 1.1 equiv ) in formic acid and water ( 1:1 , 30 ml ) . the mixture was stirred at rt for 24 h to 7 days at rt , then filtered under reduced pressure and washed successively with water ( 50 ml ) and hexane ( 50 ml ) . adduct was dissolved in ch2cl2 ( 100 ml ) and saturated aqueous sodium bicarbonate solution ( 100 ml ) was added . the organic layer was separated , dried ( na2so4 ) and the solvent was removed under reduced pressure to afford the imine , which was sufficiently pure or further purified where stated . prepared according to general procedure 1 described above , starting from benzaldehyde ( 1.02 ml , 10.0 mmol ) . purification by recrystallization ( etoac / hexane ) afforded imine 1a as colorless crystals ( 2.06 g , 79% ) : mp = 106108 c ( lit . mp = 107 c ) ; max ( film)/cm 3360 , 3263 , 3067 , 2925 , 2259 , 1599 , 1319 , 1155 , 1088 , 907 , 781 , 756 , 729 , 688 , 672 ; h nmr ( 400 mhz , cdcl3 ) 8.99 ( s , 1h , chn ) , 7.887.82 ( m , 4h , 2 so2tol - h and 2 ph h ) , 7.53 ( t , j = 7.4 hz , 1h , ph h ) , 7.40 ( t , j = 7.6 hz , 2h , 2 ph h ) , 7.28 ( d , j = 8.1 hz , 2h , 2 so2tol - h ) , 2.35 ( s , 3h , so2tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 169.9 ( chn ) , 144.4 ( so2tol - c quat . ) , 134.7 ( so2tolc - ch3 quat . and ph c ) , 131.9 ( ph c quat . ) , 130.9 ( 2 ph c ) , 129.5 ( 2 so2tol - c ) , 128.8 ( 2 ph c ) , 127.7 ( 2 so2tol - c ) , 21.3 ( so2tol - ch3 ) . observed data was consistent with that reported in the literature . 4-tolualdehyde ( 2.83 ml , 24.0 mmol , 1.2 equiv ) was added to a solution of p - toluenesulfonamide ( 3.42 g , 20.0 mmol , 1.0 equiv ) in toluene ( 50 ml ) . stark conditions for 48 h , after which the solvent was removed under reduced pressure . the crude imine was washed with hexane ( 50 ml ) , et2o ( 50 ml ) and then washed with 1 m naoh ( 50 ml ) to afford imine 1b as a brown solid ( 1.75 g , 32% ) : mp = 117118 c ( lit . mp = 118119 c ) ; max ( film)/cm 2922 , 1590 , 1558 , 1447 , 1413 , 1364 , 1315 , 1155 , 1086 , 1018 , 871 , 791 , 760 , 667 ; h nmr ( 400 mhz , cdcl3 ) 9.00 ( s , 1h , chn ) , 7.89 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.82 ( d , j = 8.1 hz , 2h , 2 tol - h ) , 7.36 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.30 ( d , j = 8.1 hz , 2h , 2 tol - h ) , 2.44 ( s , 6h , so2tol - ch3 and tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 169.9 ( chn ) , 146.3 ( tol - c quat . ) , 144.4 ( so2tol - c quat . ) , 135.2 ( so2tolc - ch3 quat . ) , 131.3 ( 2 tol - c ) , 129.8 ( 2 so2tol - c ) , 129.7 ( 2 tol - c and tolc - ch3 quat . ) , 127.9 ( 2 so2tol - c ) , 21.9 ( tol - ch3 ) , 21.5 ( so2tol - ch3 ) . observed data was consistent with that reported in the literature . prepared according to general procedure 1 described above , starting from 4-tert - butylbenzaldehyde ( 1.67 ml , 10.0 mmol ) . purification by recrystallization ( etoac / hexane ) afforded imine 1c as a white solid ( 1.72 g , 55% ) : mp = 114116 c ( lit . mp = 116117 c ) ; max ( film)/cm 2967 , 2366 , 1741 , 1598 , 1327 , 1159 , 1090 , 785 ; h nmr ( 400 mhz , cdcl3 ) 9.01 ( s , 1h , chn ) , 7.907.85 ( m , 4h , 2 so2tol - h and 2 tbuar h ) , 7.51 ( d , j = 8.3 hz , 2h , 2 tbuar h ) , 7.34 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 2.44 ( s , 3h , so2tol - ch3 ) , 1.34 ( s , 9h , c(ch3)3 ) ; c nmr ( 101 mhz , cdcl3 ) 170.0 ( chn ) , 159.3 ( tbu - car quat . ) , 144.4 ( so2tol - c quat . ) , 135.4 ( so2tolc - ch3 quat . ) , 131.3 ( 2 tbuar c ) , 129.8 ( tbuar c quat . ) , 129.7 ( 2 so2tol - c ) , 128.0 ( 2 so2tol - c ) , 126.2 ( 2 tbuar c ) , 35.4 ( c(ch3)3 quat . ) , 31.0 ( c(ch3)3 ) , 21.6 ( so2tol - ch3 ) . observed data was consistent with that reported in the literature . prepared according to general procedure 1 described above , starting from 2-tolualdehyde ( 1.15 ml , 10.0 mmol ) . purification by recrystallization ( etoac / hexane ) afforded imine 1d as a white solid ( 1.65 g , 60% ) : mp = 9395 c ( lit . mp = 9192 c ) ; max ( film)/cm 1588 , 1563 , 1321 , 1305 , 1290 , 1156 , 1089 , 818 , 755 , 673 ; h nmr ( 400 mhz , cdcl3 ) 9.36 ( s , 1h , chn ) , 8.02 ( d , j = 8.4 hz , 1h , tol - h ) , 7.90 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.517.45 ( m , 1h , tol - h ) , 7.36 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.327.25 ( m , 2h , 2 tol - h ) , 2.62 ( s , 3h , tol - ch3 ) , 2.45 ( s , 3h , so2tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 168.7 ( chn ) , 144.5 ( so2tol - c quat . ) , 142.3 ( tol - c quat . ) , 135.4 ( so2tolc - ch3 quat . ) , 134.6 ( tol - c ) , 131.6 ( tol - c ) , 130.7 ( tol - c ) , 130.4 ( tol - c quat . ) , 129.8 ( 2 so2tol - c ) , 128.0 ( 2 so2tol - c ) , 126.6 ( tol - c ) , 21.7 ( so2tol - ch3 ) , 19.7 ( tol - ch3 ) . observed data was consistent with that reported in the literature . a mixture of p - toluenesulfonamide ( 7.47 g , 43.6 mmol , 1.0 equiv ) and 1-naphthaldehyde ( 7.10 ml , 52.3 mmol , 1.2 equiv ) in toluene ( 100 ml ) was heated under dean stark conditions for 3 days . the resulting mixture was filtered and the solvent was removed under reduced pressure . purification by recrystallization ( etoac / hexane ) afforded imine 1e as yellow crystals ( 2.98 g , 22% ) : mp = 142144 c ( lit . mp = 139141 c ) ; max ( film)/cm 3063 , 2925 , 2257 , 1596 , 1564 , 1318 , 1309 , 1153 , 1087 , 804 , 774 , 729 ; h nmr ( 400 mhz , cdcl3 ) 9.63 ( s , 1h , chn ) , 9.01 ( d , j = 8.6 hz , 1h , ar h ) , 8.12 ( d , j = 8.2 hz , 1h , ar h ) , 7.997.91 ( m , 3h , ar h and 2 so2tol - h ) , 7.727.66 ( m , 1h , ar h ) , 7.647.56 ( m , 2h , 2 ar h ) , 7.37 ( d , j = 7.9 hz , 2h , 2 so2tol - h ) , 2.45 ( s , 3h , so2tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 169.8 ( chn ) , 144.5 ( so2tol - c quat . ) , 136.1 ( ar c ) , 135.4 ( so2tolc - ch3 quat . ) , 135.2 ( ar c ) , 133.8 ( ar c quat . ) , 131.8 ( ar c quat . ) , 129.8 ( 2 so2tol - c ) , 129.0 ( ar c ) , 128.9 ( ar c ) , 128.0 ( 2 so2tol - c ) , 127.6 ( ar c quat . ) , 127.0 ( ar c ) , 125.1 ( ar c ) , 124.3 ( ar c ) , 21.7 ( so2tol - ch3 ) . observed data was consistent with that reported in the literature . a mixture of p - toluenesulfonamide ( 1.71 g , 10.0 mmol , 1.0 equiv ) , toluene ( 20 ml ) , 4-fluorobenzaldehyde ( 1.07 ml , 10.0 mmol , 1.0 equiv ) and boron trifluoride thf complex ( 89 l , 0.80 mmol , 8 mol % ) was heated under reflux for 12 h. after cooling to rt the reaction mixture was quenched with 1 m naoh ( 20 ml ) and extracted with etoac ( 3 20 ml ) . the combined organic layers were washed with brine , dried ( na2so4 ) , and the solvent was removed under reduced pressure . the crude imine was then recrystallized ( etoac / hexane ) to afford imine 1f as a white solid ( 854 mg , 31% ) : mp = 110111 c ( lit . mp = 111 c ) ; max ( film)/cm 1598 , 1582 , 1509 , 1320 , 1236 , 1156 , 1089 , 814 , 769 , 670 ; h nmr ( 400 mhz , cdcl3 ) 9.01 ( s , 1h , chn ) , 7.997.94 ( m , 2h , 2 far h ) , 7.89 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.36 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.227.15 ( m , 2h , 2 far h ) , 2.45 ( s , 3h , so2tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 168.5 ( chn ) , 166.7 ( d , j = 258.4 hz , arc - f quat . ) , 144.6 ( so2tol - c quat . ) , 134.9 ( so2tolc - ch3 quat . ) , 133.7 ( d , j = 9.6 hz , 2 far c ) , 129.8 ( 2 so2tol - c ) , 128.7 ( d , j = 2.8 hz , far c quat . ) , 128.0 ( 2 so2tol - c ) , 116.5 ( d , j = 22.3 hz , 2 far c ) , 21.6 ( so2tol - ch3 ) . prepared according to the general procedure 1 described above , starting from 4-chlorobenzaldehyde ( 1.41 g , 10.0 mmol ) . purification by recrystallization ( etoac / hexane ) afforded imine 1 g as colorless crystals ( 700 mg , 24% ) : mp = 173174 c ( lit . mp = 175176 c ) ; max ( film)/cm 3067 , 2924 , 1592 , 1560 , 1487 , 1401 , 1316 , 1183 , 1160 , 1083 , 1011 , 869 , 820 , 786 , 706 , 692 , 656 ; h nmr ( 400 mhz , cdcl3 ) 9.00 ( s , 1h , chn ) , 7.927.80 ( m , 4h , 2 clar h and 2 so2tol - h ) , 7.47 ( d , j = 8.5 hz , 2h , 2 clar h ) , 7.36 ( d , j = 8.1 hz , 2h , 2 so2tol - h ) , 2.44 ( s , 3h , so2tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 168.6 ( chn ) , 144.8 ( so2tol - c quat . ) , 141.3 ( c - arcl quat . ) , 134.8 ( so2tolc - ch3 quat . ) , 132.3 ( 2 clar c ) , 130.7 ( clar c quat . ) , 129.8 ( 2 so2tol - c ) , 129.5 ( 2 clar c ) 128.1 ( 2 so2tol - c ) , 21.6 ( so2tol - ch3 ) . observed data was consistent with that reported in the literature . prepared according to general procedure 1 described above , starting from 2-chlorobenzaldehyde ( 1.13 ml , 10.0 mmol ) afforded imine 1h as a white solid ( 2.05 g , 70% ) : mp = 130131 c ( lit . mp = 128129 c ) ; max ( film)/cm 3090 , 1587 , 1560 , 1435 , 1318 , 1214 , 1154 , 1087 , 1051 , 863 , 804 , 786 , 759 , 707 , 666 ; h nmr ( 400 mhz , cdcl3 ) 9.49 ( s , 1h , chn ) , 8.14 ( dd , j = 7.9 , 1.6 hz , 1h , clar h ) , 7.90 ( d , j = 8.4 hz , 2h , 2 h ) , 7.45 ( dd , j = 8.1 , 1.2 hz , 1h , clar h ) , 7.387.31 ( m , 3h , clar h and 2 so2tol - h ) , 2.44 ( s , 3h , so2tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 166.7 ( chn ) , 144.8 ( so2tol - c quat . ) , 138.8 ( c - arcl quat . ) , 135.6 ( clar c ) , 134.5 ( so2tolc - ch3 quat . ) , 130.4 ( clar c ) , 130.1 ( clar c . ) , 129.8 ( 2 so2tol - c ) , 129.6 ( clar c quat . ) 128.2 ( 2 so2tol - c ) , 127.3 ( clar c ) , 21.6 ( so2tol - ch3 ) . observed data was consistent with that reported in the literature . cyclohexanecarboxaldehyde ( 5.45 ml , 45.0 mmol , 1.5 equiv ) was added to a stirred solution of p - toluenesulfonamide ( 5.14 g , 30.0 mmol , 1.0 equiv ) and sodium p - toluenesulfinate ( 6.41 g , 36.0 mmol , 1.2 equiv ) in formic acid and water ( 1:1 , 90 ml ) at 0 c . the reaction was then warmed to rt and stirred for 24 h. the reaction mixture was then filtered under reduced pressure and washed successively with water ( 100 ml ) and hexane ( 100 ml ) . the solid was then dissolved in ch2cl2 ( 300 ml ) and saturated aqueous sodium bicarbonate solution ( 300 ml ) was added . the resulting biphasic solution was vigorously stirred for 2 h at rt , after which the organic layer was separated , dried ( na2so4 ) and the solvent was removed under reduced pressure . the crude imine was then recrystallized ( etoac ) to afford imine 1i as white crystals ( 6.39 g , 80% ) : mp = 109110 c ( lit . mp = 106 c ) ; max ( film)/cm 3361 , 3265 , 2930 , 2856 , 1627 , 1600 , 1449 , 1317 , 1159 , 1093 , 901 , 814 , 676 ; h nmr ( 400 mhz , cdcl3 ) 8.48 ( d , j = 4.4 hz , 1h , chn ) , 7.81 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.34 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 2.462.42 ( m , 4h , cy - h and so2tol - ch3 ) , 1.921.63 ( m , 5h , 5 cy - h ) , 1.401.18 ( m , 5h , 5 cy - h ) ; c nmr ( 101 mhz , cdcl3 ) 181.0 ( chn ) , 144.5 ( so2tol - c quat . ) , 134.7 ( so2tolc - ch3 quat . ) , 129.7 ( 2 so2tol - c ) , 128.0 ( 2 so2tol - c ) , 43.6 ( ch ) , 28.3 ( 2 ch2 ) , 25.6 ( ch2 ) , 25.0 ( 2 ch2 ) , 21.6 ( so2tol - ch3 ) . 3-cyclohexene - carboxaldehyde ( 1.76 ml , 15.0 mmol , 1.5 equiv ) was added to a stirred solution of p - toluenesulfonamide ( 1.71 g , 10.0 mmol , 1.0 equiv ) and sodium p - toluenesulfinate ( 2.14 g , 12.0 mmol , 1.2 equiv ) in formic acid and water ( 1:1 , 30 ml ) at 0 c . the reaction was then warmed to rt and stirred for 24 h. the reaction mixture was filtered under reduced pressure and the filter cake washed successively with water ( 50 ml ) and hexane ( 50 ml ) . the solid was then dissolved in ch2cl2 ( 50 ml ) and washed rapidly with aqueous naoh solution ( 1 m , 50 ml ) . the organic layer was separated , dried ( na2so4 ) , and the solvent was removed under reduced pressure affording imine 1 m as a white solid ( 1.65 g , 63% ) : mp = 118119 c ; max ( film)/cm 3028 , 2922 , 1625 , 1597 , 1437 , 1320 , 1291 , 1156 , 1090 , 786 , 739 , 670 ; h nmr ( 400 mhz , cdcl3 ) 8.58 ( d , j = 4.3 hz , 1h , chn ) , 7.82 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.35 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 5.735.65 ( m , 2h , hc = ch ) , 2.762.68 ( m , 1h , ch ) , 2.45 ( s , 3h , so2tol - ch3 ) , 2.272.07 ( m , 4h , 2 ch2 ) , 2.001.93 ( m , 1h , ch ) , 1.661.55 ( m , 1h , ch ) ; c nmr ( 101 mhz , cdcl3 ) 180.6 ( chn ) , 144.6 ( so2tol - c quat . ) , 134.6 ( so2tolc - ch3 quat . ) , 129.7 ( 2 so2tol - c ) , 128.0 ( 2 so2tol - c ) , 127.0 ( hc = ch ) , 124.4 ( hc = ch ) , 40.0 ( ch ) , 26.7 ( ch2 ) , 24.5 ( ch2 ) , 23.8 ( ch2 ) , 21.6 ( so2tol - ch3 ) ; hrms ( esi / tof ) m / z calculated for c14h18no2s [ m + h ] : 264.1053 ; found : 264.1050 . a mixture of p - toluenesulfonamide ( 1.71 g , 10.0 mmol , 1.0 equiv ) , toluene ( 30 ml ) , pivaldehyde ( 1.14 ml , 10.5 mmol , 1.05 equiv ) and boron trifluoride thf complex ( 89 l , 0.80 mmol , 8 mol % ) was refluxed for 16 h. after cooling to rt the reaction mixture was quenched with 1 m naoh ( 20 ml ) and extracted with etoac ( 3 20 ml ) . the combined organic layers were washed with brine , dried ( na2so4 ) , and the solvent was removed under reduced pressure . the filtrate was concentrated under reduced pressure to afford the desired imine 1n as a white solid ( 916 mg , 38% ) : mp = 9094 c ( lit . c ) ; max ( film)/cm 3359 , 3261 , 1741 , 1530 , 1389 , 1305 , 1157 , 1098 , 905 , 816 , 696 ; h nmr ( 400 mhz , cdcl3 ) 8.33 ( s , 1h , chn ) , 7.66 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.19 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 2.26 ( s , 3h , so2tol - ch3 ) , 0.99 ( s , 9h , c(ch3)3 ) ; c nmr ( 101 mhz , cdcl3 ) 183.4 ( c = n ) , 144.1 ( so2tol - c quat . ) , 134.3 ( so2tolc - ch3 quat . ) , 129.3 ( 2 so2tol - c ) , 127.5 ( 2 so2tol - c ) , 37.3 ( c(ch3)3 quat . ) , 25.3 ( 3 c(ch3)3 ) , 21.1 ( so2tol - ch3 ) . observed data was consistent with that reported in the literature . a solution of imine 1a ( 130 mg , 0.50 mmol , 1.0 equiv ) in thf ( 2.0 ml ) was added dropwise over 5 min to a solution of lihmds ( 1.0 m solution in thf , 1.50 ml , 1.50 mmol , 3.0 equiv ) in thf ( 5.2 ml ) and et2o ( 2.7 ml ) at 78 c . the reaction mixture was then quenched by the addition of saturated aqueous sodium bicarbonate solution ( 40 ml ) . the aqueous mixture was extracted with ch2cl2 ( 3 30 ml ) . the combined organic layers were dried ( na2so4 ) and the solvent was removed under reduced pressure affording aminal 2a as a white solid ( 195 mg , 92% ) : mp = 110112 c ; rf 0.36 ( 15% etoac / hexane ) ; max ( film)/cm 3305 , 2959 , 1331 , 1252 , 1160 , 965 , 909 , 869 , 829 , 813 , 734 , 698 , 664 ; h nmr ( 400 mhz , cdcl3 ) 7.86 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.35 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.317.20 ( m , 5h , 5 ph h ) , 5.88 ( d , j = 7.7 hz , 1h , nh ) , 5.17 ( d , j = 7.7 hz , 1h , chn ) , 2.46 ( s , 3h , so2tol - ch3 ) , 0.15 ( s , 18h , si(ch3)2 ) ; c nmr ( 101 mhz , cdcl3 ) 143.3 ( so2tol - c quat . ) , 143.0 ( ph c quat . ) , 136.2 ( so2tolc - ch3 quat . ) , 129.6 ( 2 so2tol - c ) , 128.0 ( 2 so2tol - c ) , 127.4 ( ph c ) , 127.0 ( 2 ph c ) , 69.7 ( phcn ) , 21.5 ( so2tol - ch3 ) , 3.1 ( si(ch3)2 ) ; hrms ( ci ) m / z calculated for c20h36n3o2ssi2 [ m + nh4 ] 438.2061 ; found 438.2086 . diiodomethane ( 137 l , 1.70 mmol , 3.6 equiv ) in thf ( 1.0 ml ) was added dropwise to a solution of lihmds ( 1.0 m solution in thf , 1.50 ml , 1.50 mmol , 3.2 equiv ) in thf ( 4.2 ml ) and et2o ( 2.7 ml ) at 78 c in the dark . after 20 min at 78 c , a solution of imine 1h ( 137 mg , 0.47 mmol , 1.0 equiv ) in thf ( 2.0 ml ) was added dropwise to the reaction mixture . after a further 10 min at 78 c , the reaction was quenched by the addition of saturated aqueous sodium bicarbonate solution ( 40 ml ) . the aqueous solution was extracted with ch2cl2 ( 3 30 ml ) , the combined organic layers were dried ( na2so4 ) , and the solvent was removed under reduced pressure . purification by flash chromatography ( 50% et2o / hexane ) afforded amino gem - diiodide 3h as a white solid ( 191 mg , 72% ) : mp = 210211 c ; rf 0.20 ( 50% et2o / hexane ) ; max ( film)/cm 3237 ( nh ) , 1473 , 1437 , 1335 , 1281 , 1160 , 1081 , 1035 , 910 , 837 , 815 , 752 ; h nmr ( 500 mhz , cdcl3 ) 7.70 ( d , j = 8.1 hz , 2h , 2 so2tol - h ) , 7.37 ( dd , j = 7.8 , 1.5 hz , 1h , clar h ) , 7.30 ( dd , j = 8.0 , 1.4 hz , 1h , clar h ) , 7.26 ( td , j = 8.0 , 1.5 hz , 1h , clar h ) , 7.20 ( d , j = 8.1 hz , 2h , 2 so2tol - h ) , 7.15 ( td , j = 7.6 , 1.4 hz , 1h , clar h ) , 5.40 ( d , j = 8.1 hz , 1h , nh ) , 5.37 ( d , j = 3.2 hz , 1h , chi2 ) , 4.37 ( dd , j = 8.1 , 3.2 hz , 1h , chn ) , 2.37 ( s , 3h , so2tol - ch3 ) ; c nmr ( 125 mhz , cdcl3 ) 143.8 ( so2tol - c quat . ) , 136.3 ( so2tolc - ch3 quat ) , 135.5 ( clar c quat . ) , 132.3 ( clar c quat . ) , 129.8 ( clar c ) , 129.6 ( clar c ) , 129.5 ( 2 so2tol - c ) , 129.3 ( clar c ) , 127.5 ( 2 so2tol - c ) , 126.7 ( clar c ) , 62.6 ( chn ) , 21.5 ( so2tol - ch3 ) , 22.1 ( chi2 ) ; hrms ( esi / tof ) m / z calculated for c15h15cli2no2s [ m + h ] : 561.8596 ; found : 561.8592 . the relevant aldehyde ( 15.0 mmol , 1.5 equiv ) was added to a stirred solution of p - toluenesulfonamide ( 1.71 g , 10.0 mmol , 1.0 equiv ) and sodium p - toluenesulfinate ( 2.14 g , 12.0 mmol , 1.2 equiv ) in formic acid and water ( 1:1 , 30 ml ) at 0 c . after 3 days at rt , the reaction mixture was filtered under reduced pressure , washed successively with water ( 50 ml ) and hexane ( 50 ml ) affording the corresponding imine according to the general procedure 2 described above , starting from cyclohexanecarboxaldehyde ( 1.21 ml , 10.0 mmol ) afforded imine adduct 5i as a white solid ( 2.52 g , 60% ) : mp = 99102 c ( lit . mp = 101103 c ) ; max ( film)/cm 3268 , 2933 , 2860 , 1721 , 1600 , 1451 , 1331 , 1303 , 1290 , 1155 , 1081 , 906 , 812 , 731 , 705 , 661 ; h nmr ( 400 mhz , cdcl3 ) 7.65 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.45 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.23 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.17 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 5.17 ( d , j = 10.7 hz , 1h , nh ) , 4.47 ( dd , j = 10.7 , 2.9 hz , 1h , hc - nh ) , 2.45 ( s , 3h , so2tol - ch3 ) , 2.42 ( s , 3h , so2tol - ch3 ) , 2.402.34 ( m , 1h , cy - h ) , 2.071.99 ( m , 1h , cy - h ) , 1.801.59 ( m , 4h , 4 cy - h ) , 1.361.28 ( m , 2h , 2 cy - h ) , 1.100.95 ( m , 3h , 3 cy - h ) ; c nmr ( 101 mhz , cdcl3 ) 145.0 ( so2tol - c quat . ) , 143.5 ( so2tol - c quat . ) , 138.1 ( so2tolc - ch3 quat . ) , 134.1 ( so2tolc - ch3 quat . ) , 129.7 ( 2 so2tol - c ) , 129.5 ( 2 so2tol - c ) , 129.3 ( 2 so2tol - c ) , 126.6 ( 2 so2tol - c ) , 77.5 ( chn ) , 37.3 ( ch ) , 31.0 ( ch2 ) , 27.1 ( ch2 ) , 26.2 ( ch2 ) , 25.63 ( ch2 ) , 25.61 ( ch2 ) , 21.8 ( so2tol - ch3 ) , 21.6 ( so2tol - ch3 ) . observed data was consistent with that reported in the literature . prepared according to general procedure 2 described above , starting from isobutyraldehyde ( 0.91 ml , 10.0 mmol ) afforded imine adduct 5j as a white solid ( 2.00 g , 52% ) : mp = 8688 c ; max ( film)/cm 3283 , 2968 , 1598 , 1442 , 1332 , 1290 , 1160 , 1132 , 1082 , 890 , 813 , 779 , 702 , 670 ; h nmr ( 400 mhz , cdcl3 ) 7.70 ( d , j = 8.0 hz , 2h , 2 so2tol - h ) , 7.50 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.26 ( d , j = 8.0 hz , 2h , 2 so2tol - h ) , 7.18 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 5.38 ( d , j = 10.6 hz , 1h , nh ) , 4.52 ( dd , j = 10.6 , 2.7 hz , 1h , hc - nh ) , 2.71 ( dqq , j = 6.9 , 2.7 hz , 1h , ch ) , 2.45 ( s , 3h , so2tol - ch3 ) , 2.41 ( s , 3h , so2tol - ch3 ) , 1.05 ( d , j = 6.9 hz , 3h , ch3 ) , 0.87 ( d , 3h , j = 6.9 hz , ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.0 ( so2tol - c quat . ) , 143.4 ( so2tol - c quat . ) , 138.0 ( so2tolc - ch3 quat . ) , 133.9 ( so2tolc - ch3 quat . ) , 129.6 ( 2 so2tol - c ) , 129.4 ( 2 so2tol - c ) 129.2 ( 2 so2tol - c ) , 126.6 ( 2 so2tol - c ) , 77.5 ( hc - nh ) , 27.6 ( ch ) , 21.7 ( so2tol - ch3 ) , 21.5 ( so2tol - ch3 ) , 20.8 ( ch3 ) , 16.5 ( ch3 ) . prepared according to general procedure 2 described above , starting from 2-ethylbutyraldehyde ( 1.85 ml , 15.0 mmol ) afforded imine adduct 5k as a white solid ( 3.01 g , 73% ) : mp = 6364 c ( lit . mp = 5657 c ) ; max ( film)/cm 3265 , 2965 , 2876 , 1598 , 1450 , 1332 , 1154 , 1129 , 1084 , 1083 , 1067 , 885 , 810 , 703 , 676 ; h nmr ( 400 mhz , cdcl3 ) 7.70 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.52 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.28 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.20 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 5.23 ( d , j = 10.6 hz , 1h , nh ) , 4.68 ( dd , j = 10.6 , 2.0 hz , 1h , hc - nh ) , 2.46 ( s , 3h , so2tol - ch3 ) , 2.42 ( s , 3h , so2tol - ch3 ) , 2.102.01 ( m , 1h , ch ) , 1.891.79 ( m , 1h , ch2 ) , 1.571.47 ( m , 1h , ch2 ) , 1.151.03 ( m , 1h , ch2 ) , 0.970.90 ( m , 4h , ch2 and ch3 ) , 0.87 ( t , j = 7.3 hz , 3h , ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.0 ( so2tol - c quat . ) , 143.5 ( so2tol - c quat . ) , 138.0 ( so2tolc - ch3 quat . ) , 134.1 ( so2tolc - ch3 quat . ) , 129.7 ( 2 so2tol - c ) , 129.4 ( 2 so2tol - c ) 129.2 ( 2 so2tol - c ) , 126.5 ( 2 so2tol - c ) , 74.4 ( hc - nh ) , 41.4 ( ch ) , 22.7 ( ch2 ) , 22.0 ( ch2 ) , 21.7 ( so2tol - ch3 ) , 21.5 ( so2tol - ch3 ) , 11.9 ( ch3 ) , 11.7 ( ch3 ) . observed data was consistent with that reported in the literature . prepared according to general procedure 2 described above , starting from 2-methylbutyraldehyde ( 1.61 ml , 15.0 mmol ) afforded imine adduct 5l as a white solid ( 2.38 g , 60% ) : mp = 8687 c ; max ( film)/cm 3227 , 2929 , 1599 , 1454 , 1334 , 1292 , 1131 , 1083 , 813 , 763 , 669 ; h nmr ( 400 mhz , cdcl3 ) 7.69 ( d , j = 8.1 hz , 2h , 2 so2tol - h ) , 7.50 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.27 ( d , j = 8.1 hz , 2h , 2 so2tol - h ) , 7.19 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 5.19 ( d , j = 10.6 hz , 1h , nh ) , 4.63 ( dd , j = 10.6 , 2.0 hz , 1h , hc - nh ) , 2.462.42 ( m , 1h , ch(ch3)ch2ch3 ) , 2.46 ( s , 3h , so2tol - ch3 ) , 2.42 ( s , 3h , so2tol - ch3 ) , 1.211.09 ( m , 2h , ch(ch3)ch2ch3 ) , 1.05 ( d , j = 6.8 hz , 3h , ch(ch3)ch2ch3 ) 0.87 ( t , j = 7.3 hz , 3h , ch(ch3)ch2ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.0 ( so2tol - c quat . ) , 143.5 ( so2tol - c quat . ) , 138.0 ( so2tolc - ch3 quat . ) , 134.0 ( so2tolc - ch3 quat . ) , 129.7 ( 2 so2tol - c ) , 129.4 ( 2 so2tol - c ) 129.2 ( 2 so2tol - c ) , 126.5 ( 2 so2tol - c ) , 75.8 ( hc - nh ) , 33.9 ( ch ) , 27.4 ( ch2 ) , 21.8 ( so2tol - ch3 ) , 21.5 ( so2tol - ch3 ) , 14.1 ( chch3 ) , 11.5 ( ch2ch3 ) . hrms ( esi / tof ) m / z calculated for c19h24no4s2 [ m h ] : 394.1141 ; found : 394.1137 . prepared according to general procedure 2 described above , starting from butyraldehyde ( 1.34 ml , 15.0 mmol ) afforded imine adduct 5o as a white solid ( 3.42 g , 90% ) : mp = 118119 ( lit . mp = 119120 c ) ; max ( film)/cm 3214 , 2932 , 1597 , 1460 , 1441 , 1334 , 1291 , 1210 , 1166 , 1127 , 1078 , 922 , 815 , 667 ; h nmr ( 400 mhz , cdcl3 ) 7.68 ( d , j = 8.1 hz , 2h , 2 so2tol - h ) , 7.54 ( d , j = 8.1 hz , 2h , 2 so2tol - h ) , 7.28 ( d , j = 8.1 hz , 2h , 2 so2tol - h ) , 7.21 ( d , j = 8.1 hz , 2h , 2 so2tol - h ) , 4.96 ( d , j = 10.2 hz , 1h , nh ) , 4.59 ( dt , j = 10.2 , 3.8 hz , 1h , hc - nh ) , 2.45 ( s , 3h , so2tol - ch3 ) , 2.43 ( s , 3h , so2tol - ch3 ) , 2.172.08 ( m , 1h , ch2 ) , 1.701.62 ( m , 1h , ch2 ) , 1.451.38 ( m , 1h , ch2 ) , 1.291.19 ( m , 1h , ch2 ) , 0.87 ( t , j = 7.3 hz , 3h , ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.2 ( so2tol - c quat . ) , 143.6 ( so2tol - c quat . ) , 137.8 ( so2tolc - ch3 quat . ) , 132.7 ( so2tolc - ch3 quat . ) , 129.7 ( 2 so2tol - c ) , 129.6 ( 2 so2tol - c ) 129.5 ( 2 so2tol - c ) , 126.7 ( 2 so2tol - c ) , 73.6 ( hc - nh ) , 27.6 ( ch2 ) , 21.7 ( so2tol - ch3 ) , 21.5 ( so2tol - ch3 ) , 18.4 ( ch2 ) , 13.5 ( ch3 ) . observed data was consistent with that reported in the literature . prepared according to general procedure 2 described above , starting from 1-heptanal ( 2.09 ml , 15.0 mmol ) afforded imine adduct 5p as a white solid ( 2.67 g , 64% ) : mp = 7677 c ; max ( film)/cm 3261 , 2925 , 1597 , 1451 , 1334 , 1301 , 1161 , 1129 , 1081 , 1038 , 1010 , 901 , 813 , 676 ; h nmr ( 400 mhz , cdcl3 ) 7.71 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.55 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.30 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.22 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 5.11 ( d , j = 10.3 hz , 1h , nh ) , 4.53 ( dt , j = 10.3 , 3.6 hz , 1h , hc - nh ) , 2.45 ( s , 3h , so2tol - ch3 ) , 2.42 ( s , 3h , so2tol - ch3 ) , 2.192.01 ( m , 1h , ch2 ) , 1.691.60 ( m , 2h , 2 ch2 ) , 1.221.07 ( m , 7h , 7 ch2 ) , 0.84 ( t , j = 7.3 hz , 3h , ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.3 ( so2tol - c quat . ) , 143.6 ( so2tol - c quat . ) , 137.9 ( so2tolc - ch3 quat . ) , 132.6 ( so2tolc - ch3 quat . ) , 129.7 ( 4 so2tol - c ) , 129.5 ( 2 so2tol - c ) , 126.7 ( 2 so2tol - c ) , 73.8 ( hc - nh ) , 31.3 ( ch2 ) , 28.6 ( ch2 ) , 28.0 ( ch2 ) , 24.5 ( ch2 ) , 22.3 ( ch2 ) , 21.8 ( so2tol - ch3 ) , 21.5 ( so2tol - ch3 ) , 14.0 ( ch3 ) . the above compound is previously reported without characterization data . nbuli ( 1.50 mmol , 3.0 equiv ) was added dropwise to a solution of hexamethyldisilazane ( 315 l , 1.50 mmol , 3.0 equiv ) in thf ( 5.7 ml ) and et2o ( 2.7 ml ) at 78 c . after 30 min , diiodomethane ( 135 l , 1.70 mmol , 3.4 equiv ) in thf ( 1.0 ml ) was added dropwise to the reaction mixture at 78 c in the dark . after 20 min at 78 c , a solution of the appropriate imine ( 0.50 mmol , 1.0 equiv ) in thf ( 2.0 ml ) was added dropwise to the reaction mixture over 5 min . the reaction was then immediately warmed to 0 c in an ice bath and left at this temperature for 15 min . the reaction was then quenched by the addition of saturated aqueous sodium bicarbonate solution ( 40 ml ) . the aqueous solution was extracted with ch2cl2 ( 3 30 ml ) , then the combined organic layers were dried ( na2so4 ) and the solvent was removed under reduced pressure . purification by flash chromatography on deactivated basic alumina ( activity iv or activity v ) afforded the cis - iodoaziridine . identical to method a , except the reaction was warmed to rt for 20 min after addition of imine at 78 c . nbuli ( 2.00 mmol , 4.0 equiv ) was added dropwise to a solution of hexamethyldisilazane ( 420 l , 2.00 mmol , 4.0 equiv ) in thf ( 7.5 ml ) and et2o ( 3.5 ml ) at 78 c . after 30 min , diiodomethane ( 177 l , 2.20 mmol , 4.4 equiv ) in thf ( 1.5 ml ) was added dropwise to the reaction mixture at 78 c in the dark . ho2stol adduct ( 0.50 mmol , 1.0 equiv ) in thf ( 2.0 ml ) was added dropwise to the reaction mixture over 5 min . the reaction was then immediately warmed to 0 c in an ice bath and left at this temperature for 15 min . the reaction was then quenched by the addition of saturated aqueous sodium bicarbonate solution ( 40 ml ) . the aqueous solution was extracted with ch2cl2 ( 3 30 ml ) and the combined organic layers were dried ( na2so4 ) , and the solvent was removed under reduced pressure . purification by flash chromatography on deactivated basic alumina ( activity iv or activity v ) afforded the cis - iodoaziridine . prepared according to method a described above , starting from imine 1a ( 130 mg , 0.50 mmol ) . purification by flash chromatography ( 10% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded cis - iodoaziridine 4a as a yellow oil ( 152 mg , 76% ) : rf 0.24 ( 15% et2o / hexane ) ; max ( film)/cm 3035 , 2928 , 1600 , 1499 , 1453 , 1330 , 1157 , 1088 , 902 , 813 , 763 , 727 , 696 , 683 , 666 ; h nmr ( 400 mhz , cdcl3 ) 7.92 ( d , j = 8.5 hz , 2h , 2 so2tol - h ) , 7.427.33 ( m , 5h , 3 ph h and so2tol - h ) , 7.317.25 ( m , 2h , 2 ph h ) , 4.89 ( d , j = 6.1 hz , 1h , chi ) , 3.89 ( d , j = 6.1 hz , 1h , chph ) , 2.47 ( s , 3h , ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.3 ( so2c - tol quat . ) , 134.1 ( so2tolc - ch3 quat . ) , 132.9 ( phc quat . ) , 129.9 ( 2 so2tol - c ) , 128.7 ( ph c ) , 128.1 ( 2 so2tol - c ) , 127.8 ( 2 ph c ) , 44.9 ( phcn ) , 21.7 ( so2tol - ch3 ) , 16.4 ( chi ) ; hrms ( esi / tof ) m / z calculated for c15h15ino2s [ m + h ] 399.9863 ; found 399.9856 . prepared according to method a described above , starting from imine 1b ( 137 mg , 0.50 mmol ) . purification by flash chromatography ( 5% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded cis - iodoaziridine 4b as a yellow oil ( 100 mg , 48% ) : rf 0.16 ( 10% etoac / hexane ) ; max ( film)/cm 3022 , 2923 , 1616 , 1598 , 1517 , 1329 , 1292 , 1242 , 1158 , 1089 , 1037 , 1019 , 905 , 842 , 810 , 766 ; h nmr ( 400 mhz , cdcl3 ) 7.90 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.38 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.187.14 ( m , 4h , 4 tol - h ) , 4.87 ( d , j = 6.1 hz , 1h , chi ) , 3.84 ( d , j = 6.1 hz , 1h , char ) , 2.46 ( s , 3h , so2tol - ch3 ) , 2.35 ( s , 3h , tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.2 ( so2tol - c quat . ) , 138.6 ( tolc quat . ) , 134.2 ( so2tolc - ch3 quat . ) , 129.9 ( 2 so2tol - c ) , 129.8 ( tolc quat . ) , 128.9 ( 2 tol - c ) , 127.8 ( 2 so2tol - c ) , 127.4 ( 2 tol - c ) , 44.9 ( chn ) , 21.7 ( so2tol - ch3 ) , 21.2 ( tol - ch3 ) , 16.8 ( chi ) ; hrms ( esi / tof ) m / z calculated for c16h17ino2s [ m + h ] : 414.0019 ; found : 414.0037 . prepared according to method a described above , starting from imine 1c ( 158 mg , 0.50 mmol ) . purification by flash chromatography ( hexane to 5% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded cis - iodoaziridine 4c as a yellow oil ( 131 mg , 58% ) : rf 0.13 ( 15% etoac / hexane ) ; max ( film)/cm 2966 , 2908 , 2871 , 1602 , 1333 , 1242 , 1159 , 1089 , 1020 , 905 , 839 , 810 , 753 , 727 , 671 ; h nmr ( 500 mhz , cdcl3 ) 7.927.88 ( m , 2h , 2 so2tol - h ) , 7.407.35 ( m , 4h , 2 so2tol - h and 2 tbuar h ) , 4.88 ( d , j = 6.1 hz , 1h , chi ) , 3.85 ( d , j = 6.1 hz , 1h , char ) , 2.47 ( s , 3h , so2tol - ch3 ) , 1.32 ( s , 9h , c(ch3)3 ) ; c nmr ( 125 mhz , cdcl3 ) 151.8 ( tbu - car quat . ) , 145.2 ( so2tol - c quat . ) , 134.3 ( so2tolc - ch3 quat . ) , 129.9 ( 2 so2tol - c ) , 129.8 ( tbuar c quat . ) , 127.9 ( 2 so2tol - c ) , 127.2 ( 2 tbuar c ) , 125.1 ( 2 tbuar c ) , 44.9 ( chn ) , 34.6 ( c(ch3)3 quat . ) , 31.2 ( c(ch3)3 ) , 21.7 ( so2tol - ch3 ) , 16.6 ( chi ) ; hrms ( esi / tof ) m / z calculated for c19h23ino2s [ m + h ] 456.0489 ; found 456.0490 . prepared according to method b described above , starting from imine 1d ( 137 mg , 0.50 mmol ) . purification by flash chromatography ( hexane to 5% etoac / hexane ) on deactivated deactivated basic alumina ( activity iv ) afforded cis - iodoaziridine 4d as a yellow oil ( 141 mg , 68% ) : rf 0.13 ( 5% etoac / hexane ) ; max ( film)/cm 3030 , 2923 , 2860 , 1597 , 1492 , 1460 , 1331 , 1240 , 1158 , 1089 , 906 , 754 , 734 , 713 , 684 , 667 ; h nmr ( 400 mhz , cdcl3 ) 7.94 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.40 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.317.24 ( dt , j = 7.4 , 1.6 hz , 1h , tol - h ) , 7.227.09 ( m , 3h , 3 tol - h ) , 4.92 ( d , j = 6.0 hz , 1h , chi ) , 3.89 ( d , j = 6.0 hz , 1h , char ) , 2.47 ( s , 3h , so2tol - ch3 ) , 2.35 ( s , 3h , tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.3 ( so2c - tol quat . ) , 136.1 ( tol - c quat . ) , 134.2 ( so2tolc - ch3 quat . ) , 131.8 ( tol - c quat . ) , 130.0 ( 2 so2tol - c ) , 129.8 ( tol - c ) , 128.6 ( tol - c ) , 127.9 ( 2 so2tol - c ) , 127.4 ( tol - c ) , 125.7 ( tol - c ) , 44.5 ( tolcn ) , 21.7 ( so2tol - ch3 ) , 19.0 ( tol - ch3 ) , 15.3 ( chi ) ; hrms ( esi / tof ) m / z calculated for c16h17ino2s [ m + h ] 414.0019 ; found 414.0024 . prepared according to method b described above , starting from imine 1e ( 155 mg , 0.50 mmol ) . purification by flash chromatography ( 5% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded cis - iodoaziridine 4e as a yellow oil ( 176 mg , 78% ) : rf 0.40 ( 15% etoac / hexane ) ; max ( film)/cm 3287 , 3054 , 2926 , 2259 , 1922 , 1722 , 1600 , 1511 , 1330 , 1157 , 1088 , 905 , 801 , 779 , 758 , 725 , 682 , 667 ; h nmr ( 400 mhz , cdcl3 ) 8.017.95 ( m , 3h , ar h and 2 so2tol - h ) , 7.957.90 ( d , j = 8.1 hz , 1h , ar h ) , 7.87 ( d , j = 8.1 hz , 1h , ar h ) , 7.637.53 ( m , 2h , 2 ar h ) , 7.457.35 ( m , 4h , 2 ar h and 2 so2tol - h ) , 5.07 ( d , j = 6.0 hz , 1h , chi ) , 4.38 ( d , j = 6.0 hz , 1h , char ) , 2.48 ( s , 3h , so2tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.4 ( so2tol - c quat . ) , 134.2 ( so2tolc ch3 quat . ) , 133.1 ( ar c quat . ) , 130.7 ( ar c quat . ) , 130.0 ( 2 so2tol - c ) , 129.4 ( ar c quat . ) , 129.0 ( ar c ) , 128.8 ( ar c ) , 128.0 ( 2 so2tol - c ) , 126.7 ( ar c ) , 126.1 ( ar c ) , 125.8 ( ar c ) , 125.1 ( ar c ) , 122.6 ( ar c ) , 44.4 ( arcn ) , 21.7 ( so2tol - ch3 ) , 15.2 ( chi ) ; hrms ( esi / tof ) m / z calculated for c19h17ino2s [ m + h ] 450.0019 ; found 450.0024 . prepared according to method a described above , starting from imine 1f ( 139 mg , 0.50 mmol ) . purification by flash chromatography ( 5% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded cis - iodoaziridine 4f as a yellow oil ( 97 mg , 47% ) : rf 0.24 ( 15% etoac / hexane ) ; max ( film)/cm 3029 , 2933 , 2259 , 1905 , 1604 , 1512 , 1334 , 1240 , 1158 , 1089 , 904 , 835 , 814 , 735 , 706 , 680 , 666 ; h nmr ( 400 mhz , cdcl3 ) 7.89 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.40 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.287.22 ( m , 2h , 2 far h ) , 7.087.01 ( m , 2h , 2 far h ) , 4.85 ( d , j = 6.1 hz , 1h , chi ) , 3.85 ( d , j = 6.1 hz , 1h , char ) , 2.48 ( 3 h , s , so2tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 163.0 ( d , j = 248 hz , f - car quat . ) , 145.5 ( so2tol - c quat . ) , 134.1 ( so2tolc - ch3 quat . ) , 130.1 ( 2 so2tol - c ) , 129.4 ( d , j = 8.5 hz , 2 far c ) , 128.8 ( d , j = 3.1 hz , far c quat . ) , 128.0 ( 2 so2tol - c ) , 115.4 ( d , j = 22 hz , 2 far c ) , 44.3 ( arcn ) , 21.8 ( so2tol - ch3 ) , 16.5 ( chi ) ; f nmr ( 377 mhz , cdcl3 ) 112.3 ( ar f ) ; hrms ( esi / tof ) m / z calculated for c15h14fino2s [ m + h ] 417.9768 ; found 417.9783 . prepared according to method a described above , starting from imine 1 g ( 147 mg , 0.50 mmol ) . purification by flash chromatography ( 10% etoac / hexane ) afforded cis - iodoaziridine 4 g as a yellow oil ( 123 mg , 57% ) : rf 0.13 ( 10% etoac / hexane ) ; max ( film)/cm 3022 , 2923 , 1596 , 1493 , 1332 , 1305 , 1241 , 1158 , 1088 , 1036 , 1014 , 903 , 844 , 805 , 735 , 688 , 668 ; h nmr ( 400 mhz , cdcl3 ) 7.88 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.38 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.32 ( d , j = 8.5 hz , 2h , 2 clar h ) , 7.20 ( d , j = 8.5 hz , 2h , 2 clar h ) , 4.85 ( d , j = 6.1 hz , 1h , chi ) , 3.83 ( d , j = 6.1 hz , 1h , char ) , 2.46 ( s , 3h , so2tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.5 ( so2tol - c quat . ) , 134.7 ( clar c quat . ) , 133.9 ( so2tolc - ch3 quat . ) , 131.4 ( clar c quat . ) , 130.0 ( 2 so2tol - c ) , 128.8 ( 2 clar c ) , 127.8 ( 2 so2tol - c ) , 44.2 ( chn ) , 21.7 ( so2tol - ch3 ) , 16.1 ( chi ) ; hrms ( esi / tof ) m / z calculated for c15h15clino2s [ m + h ] : 433.9473 ; found : 433.9467 . prepared according to method a described above , starting from imine 1i ( 133 mg , 0.50 mmol ) . purification by flash chromatography ( 10% et2o / hexane ) afforded cis - iodoaziridine 4i as a colorless oil ( 137 mg , 68% ) : rf 0.18 ( 10% et2o / hexane ) ; max ( film)/cm 2926 , 2851 , 1598 , 1450 , 1330 , 1242 , 1158 , 1090 , 968 , 900 , 883 , 814 , 732 , 669 ; h nmr ( 400 mhz , cdcl3 ) 7.82 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.37 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 4.53 ( d , j = 6.0 hz , 1h , chi ) , 2.47 ( s , 3h , so2tol - ch3 ) , 2.26 ( dd , j = 9.4 , 6.0 hz , 1h , chcy ) , 1.841.71 ( m , 2h , 2 cy - h ) , 1.701.62 ( m , 2h , 2 cy - h ) , 1.601.53 ( m , 1h , cy - h ) , 1.330.99 ( m , 6h , 6 cy - h ) ; c nmr ( 101 mhz , cdcl3 ) 145.0 ( so2tol - c quat . ) , 134.4 ( 2 so2tolc ch3 quat . ) , 129.8 ( 2 so2tol - c ) , 128.0 ( 2 so2tol - c ) , 47.8 ( cy - chn ) , 40.1 ( cy - ch ) , 30.3 ( cy - ch2 ) , 28.3 ( cy - ch2 ) , 25.9 ( cy - ch2 ) , 25.2 ( cy - ch2 ) , 25.1 ( cy - ch2 ) , 21.7 ( so2tol - ch3 ) , 13.5 ( chi ) ; hrms ( esi / tof ) m / z calculated for c15h21ino2s [ m + h ] : 406.0332 ; found : 406.0328 . prepared purification by flash chromatography ( 5% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded cis - iodoaziridine 4j as a yellow oil ( 114 mg , 63% ) : rf 0.21 ( 10% etoac / hexane ) ; max ( film)/cm 2963 , 2931 , 2874 , 1597 , 1466 , 1403 , 1329 , 1244 , 1156 , 1089 , 1026 , 954 , 885 , 831 , 813 , 734 , 684 , 667 ; h nmr ( 400 mhz , cdcl3 ) 7.82 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.36 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 4.54 ( d , j = 5.9 hz , 1h , chi ) , 2.46 ( s , 3h , so2tol - ch3 ) , 2.20 ( dd , j = 9.7 , 5.9 hz , 1h , chn ) , 1.92 ( dqq , j = 9.7 , 6.7 , 6.7 hz , 1h , ch(ch3)2 ) , 0.99 ( d , j = 6.7 hz , 3h , ch(ch3)2 ) , 0.92 ( d , j = 6.7 hz , 3h , ch(ch3)2 ) ; c nmr ( 101 mhz , cdcl3 ) 145.1 ( so2tol - c quat . ) , 134.3 ( so2tolc - ch3 quat . ) , 129.8 ( 2 so2tol - c ) , 128.0 ( 2 so2tol - c ) , 49.3 ( chn ) , 31.5 ( ch(ch3)2 ) , 21.7 ( so2tol - ch3 ) , 20.0 ( ch3 ) , 17.9 ( ch3 ) , 13.8 ( chi ) ; hrms ( esi / tof ) m / z calculated for c12h17ino2s [ m + h ] : 366.0019 ; found : 366.0034 . prepared according to method c described above , starting from imine purification by flash chromatography ( 5% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded cis - iodoaziridine 4k as a yellow oil ( 123 mg , 63% ) : rf 0.31 ( 10% etoac / hexane ) ; max ( film)/cm 2963 , 2930 , 2877 , 1597 , 1458 , 1330 , 1244 , 1157 , 1089 , 976 , 887 , 835 , 813 , 731 , 667 ; h nmr ( 400 mhz , cdcl3 ) 7.82 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.36 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 4.55 ( d , j = 5.9 hz , 1h , chi ) , 2.46 ( s , 3h , so2tol - ch3 ) , 2.39 ( dd , j = 9.6 , 5.9 hz , 1h , chn ) , 1.531.20 ( m , 5h , 2 ch2 and ch ) , 0.97 ( t , j = 7.4 hz , 3h , ch3 ) , 0.84 ( t , j = 7.4 hz , 3h , ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.0 ( so2tol - c quat . ) , 134.3 ( so2tolc - ch3 quat . ) , 129.8 ( 2 so2tol - c ) , 127.9 ( 2 so2tol - c ) , 47.1 ( chn ) , 42.1 ( ch(ch2ch3)ch2ch3 ) , 23.7 ( ch(ch2ch3)ch2ch3 ) , 22.3 ( ch(ch2ch3)ch2ch3 ) , 21.7 ( so2tol - ch3 ) , 15.0 ( chi ) , 10.6 ( ch(ch2ch3)ch2ch3 ) , 10.1 ( ch(ch2ch3)ch2ch3 ) ; hrms ( esi / tof ) m / z calculated for c14h21ino2s [ m + h ] : 394.0332 ; found : 394.0332 . prepared according to method c described above , starting from imine purification by flash chromatography ( 5% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded a mixture of cis - iodoaziridines 4l ( 1.9:1 major : minor ) as a yellow oil ( 115 mg , 61% ) : rf 0.24 ( 10% etoac / hexane ) ; max ( film)/cm 2963 , 2926 , 1597 , 1459 , 1402 , 1330 , 1243 , 1157 , 1089 , 1019 , 940 , 871 . 813 , 733 , 686 ; majorh nmr ( 400 mhz , cdcl3 ) 7.82 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.36 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 4.58 ( d , j = 6.0 hz , 1h , chi ) , 2.46 ( s , 3h , so2tol - ch3 ) , 2.26 ( d , j = 9.7 , 6.0 hz , 1h , chn ) , 1.561.20 ( m , 3h , ch and ch2 ) , 0.96 ( t , j = 7.4 hz , 3h , ch2ch3 ) , 0.90 ( d , j = 6.7 hz , 3h , chch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.1 ( so2tol - c quat . ) , 134.3 ( so2tolc - ch3 quat . ) , 129.8 ( 2 so2tol - c ) , 128.0 ( 2 so2tol - c ) , 48.7 ( chn ) , 37.4 ( ch ) , 25.8 ( ch2 ) , 21.7 ( so2tol - ch3 ) , 17.0 ( ch2ch3 ) , 14.6 ( chi ) , 11.1 ( chch3 ) ; minorh nmr ( 400 mhz , cdcl3 ) 7.82 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.36 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 4.50 ( d , j = 6.0 hz , 1h , chi ) , 2.46 ( s , 3h , so2tol - ch3 ) , 2.29 ( dd , j = 10.3 , 6.0 hz , 1h , chn ) , 1.561.20 ( m , 3h , ch and ch2 ) , 0.97 ( d , j = 6.7 hz , 3h , ch2ch3 ) , 0.86 ( t , j = 7.4 hz , 3h , chch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.1 ( so2tol - c quat . ) , 134.3 ( so2tolc - ch3 quat . ) , 129.8 ( 2 so2tol - c ) , 127.9 ( 2 so2tol - c ) , 48.1 ( chn ) , 37.0 ( ch ) , 27.6 ( ch2 ) , 21.7 ( so2tol - ch3 ) , 14.5 ( chi ) , 13.9 ( ch2ch3 ) , 10.7 ( chch3 ) ; hrms ( esi / tof ) m / z calculated for c13h19ino2s [ m + h ] : 380.0176 ; found : 380.0186 . prepared according to method a described above , starting from imine 1 m ( 132 mg , 0.50 mmol ) . purification by flash chromatography ( 5% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded a mixture of cis - iodoaziridines ( 1.5:1 major : minor ) 4 m as a yellow oil ( 105 mg , 52% ) : rf 0.33 ( 10% etoac / hexane ) ; max ( film)/cm 3025 , 2920 , 1597 , 1436 , 1330 , 1242 , 1158 , 1090 , 1019 , 957 , 934 , 893 , 814 , 732 ; majorh nmr ( 400 mhz , cdcl3 ) 7.847.81 ( m , 2h 2 so2tol - h ) , 7.397.36 ( m , 2h , 2 so2tol - h ) , 5.735.56 ( m , 2h , hc = ch ) , 4.57 ( d , j = 6.0 hz , 1h , chi ) , 2.47 ( s , 3h , so2tol - ch3 ) , 2.38 ( dd , j = 9.7 , 6.0 hz , 1h , chn ) , 2.261.39 ( m , 7h , 3 ch2 and ch ) ; c nmr ( 101 mhz , cdcl3 ) 145.1 ( so2tol - c quat . ) , 134.2 ( so2tolc - ch3 quat . ) , 129.8 ( 2 so2tol - c ) , 128.0 ( 2 so2tol - c ) , 127.0 ( hc = ch ) , 125.2 ( hc = ch ) , 47.0 ( chn ) , 36.2 ( ch ) , 28.6 ( ch2 ) , 24.0 ( ch2 ) , 23.6 ( ch2 ) , 21.7 ( so2tol - ch3 ) , 13.2 ( chi ) ; minorh nmr ( 400 mhz , cdcl3 ) 7.847.81 ( m , 2h , 2 so2tol - h ) , 7.397.36 ( m , 2h , 2 so2tol - h ) , 5.735.56 ( m , 2h , hc = ch ) , 4.54 ( d , j = 6.0 hz , 1h , chi ) , 2.47 ( s , 3h , so2tol - ch3 ) , 2.40 ( dd , j = 9.7 , 6.0 hz , 1h , chn ) , 2.261.39 ( m , 7h , 3 ch2 and ch ) ; c nmr ( 101 mhz , cdcl3 ) 145.1 ( so2tol - c quat . ) , 134.2 ( so2tolc - ch3 quat . ) , 129.8 ( 2 so2tol - c ) , 128.0 ( 2 so2tol - c ) , 127.4 ( hc = ch ) , 124.2 ( hc = ch ) , 46.9 ( chn ) , 36.1 ( ch ) , 26.6 ( ch2 ) , 25.7 ( ch2 ) , 23.6 ( ch2 ) , 21.7 ( so2tol - ch3 ) , 13.7 ( chi ) ; hrms ( esi / tof ) m / z calculated for c15h19ino2s [ m + h ] : 404.0176 ; found : 404.0183 . prepared according to method b described above , starting from imine 1n ( 120 mg , 0.50 mmol ) . purification by flash chromatography ( 5% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded cis - iodoaziridine 4n as a yellow oil ( 132 mg , 70% ) : rf 0.17 ( 15% etoac / hexane ) ; max ( film)/cm 2964 , 2874 , 1600 , 1330 , 1258 , 1158 , 1089 , 953 , 931 , 852 , 734 , 677 , 667 ; h nmr ( 400 mhz , cdcl3 ) 7.82 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 7.36 ( d , j = 8.2 hz , 2h , 2 so2tol - h ) , 4.36 ( d , j = 6.4 hz , 1h , chi ) , 2.45 ( s , 3h , so2tol - ch3 ) , 2.43 ( d , j = 6.4 , 1h , chn ) 0.98 ( s , 9h , c(ch3)3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.0 ( so2tol - c quat . ) , 134.1 ( so2tolc - ch3 quat . ) , 129.7 ( 2 so2tol - c ) , 128.0 ( 2 so2tol - c ) , 50.2 ( hcc(ch3)3 ) , 31.4 ( c(ch3)3 quat . ) , 27.0 ( c(ch3)3 ) , 21.7 ( so2tol - ch3 ) , 6.6 ( chi ) ; hrms ( esi / tof ) m / z calculated for c13h19ino2s [ m + h ] 380.0176 ; found 380.0203 . prepared according to method c described above , starting from imine purification by flash chromatography ( 5% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded cis - iodoaziridine 4o as a yellow oil ( 11 mg , 6% ) : rf 0.23 ( 10% etoac / hexane ) ; max ( film)/cm 2960 , 2931 , 2873 , 1598 , 1331 , 1245 , 1160 , 1090 , 902 , 717 ; h nmr ( 400 mhz , cdcl3 ) 7.83 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.37 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 4.55 ( d , j = 5.9 hz , 1h , chi ) , 2.58 ( m , 1h , chn ) , 2.47 ( s , 3h , so2tol - ch3 ) , 1.651.34 ( m , 4h , ch2ch2 ) , 0.95 ( t , j = 7.3 hz , 3h , ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.1 ( so2tol - c quat . ) , 134.5 ( so2tolc - ch3 quat . ) , 129.8 ( 2 so2tol - c ) , 127.9 ( 2 so2tol - c ) , 43.7 ( chn ) , 33.5 ( ch2 ) , 21.7 ( so2tol - ch3 ) , 19.7 ( ch2 ) , 14.5 ( ch3 ) , 13.7 ( chi ) ; hrms ( esi / tof ) m / z calculated for c12h17ino2s [ m + h ] : 366.0019 ; found : 366.0031 . purification by flash chromatography ( 5% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded cis - iodoaziridine 4p as a yellow oil ( 9 mg , 4% ) : rf 0.24 ( 10% etoac / hexane ) ; max ( film)/cm 2955 , 2927 , 2858 , 1597 , 1458 , 1402 , 1334 , 1246 , 1161 , 1091 , 717 . h nmr ( 400 mhz , cdcl3 ) 7.83 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 7.37 ( d , j = 8.3 hz , 2h , 2 so2tol - h ) , 4.56 ( d , j = 6.0 hz , 1h , chi ) , 2.54 ( dt , j = 7.3 , 6.0 hz , 1h , chn ) , 2.47 ( s , 3h , so2tol - ch3 ) , 1.651.46 ( m , 2h , ch2 ) , 1.391.17 ( m , 8h , 4 ch2 ) , 0.88 ( t , j = 7.1 hz , 3h , ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 145.1 ( so2tol - c quat . ) , 134.5 ( so2tolc - ch3 quat . ) , 129.8 ( 2 so2tol - c ) , 127.9 ( 2 so2tol - c ) , 43.9 ( chn ) , 31.6 ( ch2 ) , 28.7 ( ch2 ) , 26.2 ( ch2 ) , 22.4 ( ch2 ) , 21.7 ( so2tol - ch3 ) , 14.5 ( ch3 ) , 14.0 ( chi ) ; hrms ( esi / tof ) m / z calculated for c15h23ino2s [ m + h ] : 408.0489 ; found : 408.0509 . neat cis - iodoaziridine 4b ( 95 mg , 0.23 mmol ) was stirred under reduced pressure ( 2 mbar ) for 5 h at 20 c and 1 h at 35 c , where cis - iodoaziridine 4b rearranged to -iodo - imine 6 ( 93 mg , 99% ) : rf 0.15 ( 10% etoac / hexane ) ; max ( film)/cm 3031 , 2921 , 1611 , 1512 , 1449 , 1320 , 1157 , 1088 , 910 , 812 , 786 , 731 , 675 ; h nmr ( 400 mhz , cdcl3 ) 8.81 ( d , j = 8.3 hz , 1h , chn ) , 7.82 ( d , j = 8.2 hz , 2h , so2tol - h ) , 7.397.35 ( m , 4h , 2 so2tol - h and 2 tol - h ) , 7.16 ( d , j = 7.9 hz , 2h , 2 tol - h ) , 5.90 ( d , j = 8.3 hz , 1h , chi ) , 2.45 ( s , 3h , so2tol - ch3 ) , 2.33 ( s , 3h , tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 170.4 ( c = n ) , 145.1 ( so2tol - c quat . ) , 139.7 ( tol - c quat . ) , 133.9 ( so2tolc - ch3 quat . ) , 132.1 ( tol - c quat . ) , 130.1 ( 2 tol - c ) , 129.9 ( 2 so2tol - c ) , 128.3 ( 2 tol - c ) , 128.2 ( 2 so2tol - c ) , 28.5 ( chi ) , 21.7 ( so2tol - ch3 ) , 21.3 ( tol - ch3 ) ; hrms ( esi / tof ) m / z calculated for c16h15ino2s [ m h ] : 411.9874 ; found : 411.9869 . crude iodoaziridine 4b was prepared by method a starting from imine 1b ( 0.50 mmol ) . the crude cis - iodoaziridine was dissolved in ch2cl2 ( 5 ml ) and then added to a stirred suspension of silica ( 150 g ) in a mixture of hexane / etoac ( 300 ml ) . the resulting suspension was stirred in the dark for 3 h , filtered , washed with ch2cl2 ( 100 ml ) and the solvent was removed under reduced pressure . purification by flash chromatography ( 5% etoac / hexane ) afforded iodo - aldehyde 7 as a yellow oil ( 70 mg , 54% over 2 steps ) : rf 0.16 ( 5% etoac / hexane ) ; max ( film)/cm 3024 , 2956 , 1715 , 1608 , 1511 , 1449 , 1383 , 1275 , 1181 , 1045 , 1004 , 813 , 772 , 715 , 672 ; h nmr ( 400 mhz , cdcl3 ) 9.46 ( d , j = 3.8 hz , 1h , cho ) , 7.38 ( d , j = 8.0 hz , 2h , 2 tol - h ) , 7.18 ( d , j = 8.0 hz , 2h , 2 tol - h ) , 5.58 ( d , j = 3.8 hz , 1h , chi ) , 2.34 ( s , 3h , tol - ch3 ) ; c nmr ( 101 mhz , cdcl3 ) 189.6 ( c = o ) , 139.4 ( tol - c quat . ) , 131.0 ( tol - c quat . ) , 129.9 ( 2 tol - c ) , 129.2 ( 2 tol - c ) , 35.4 ( chi ) , 21.3 ( tol - ch3 ) ; hrms ( ci ) m / z calculated for c9h13ino [ m + nh4 ] : 278.0036 ; found : 278.0049 . the crude cis - iodoaziridine was dissolved in ch2cl2 ( 16 ml ) and c.a . 30 mg of 1,3,5-trimethoxybenzene , as an internal standard , was added to the crude mixture . the mixture was then split into 2 ml portions and each portion was added to a slurry of a different stationary phase ( 30 g stationary phase/75 ml eluant ) and stirred for 30 min to replicate a purification procedure . after 30 min , the slurry was filtered , eluting with ch2cl2 ( 50 ml ) . the solvent was then removed under reduced pressure to afford the recovered sample , which was analyzed by h nmr against the internal standard to determine the recovery of the iodoaziridine . a typical screening process involved the following stationary phases : ( a ) control ( sample stirring in solvent system , 5% etoac / hex ) ; ( b ) silica gel ; ( c ) silica gel + 1% net3 ; ( d ) neutral alumina ; ( e ) basic alumina ( activity i ) ; ( f ) basic alumina ( activity iv ) ; ( g ) florasil . ti(oet)4 ( 2.05 g , 9.0 mmol , 3.0 equiv ) , benzaldehyde ( 306 l , 3.0 mmol , 1.0 equiv ) and ( r)-(+)-2-methyl-2-propanesulfinamide ( 364 mg , 3.0 mmol , 1.0 equiv ) were sequentially added to thf ( 6 ml ) . the reaction was stirred at rt for 24 h , after which brine ( 6 ml ) was added , while stirring vigorously . the resulting suspension was filtered through celite and was washed with etoac ( 100 ml ) . the filtrate was transferred to a separating funnel , water was added ( 10 ml ) and the aqueous layer was extracted with etoac ( 3 30 ml ) . the organic layers were washed with brine , dried ( na2so4 ) and the solvent was removed under reduced pressure to afford sulfinylimine 8 as a colorless oil ( 625 mg , 99% ) , which was used without further purification : rf 0.29 ( 25% etoac / hexane ) ; [ ]d 103.0 ( c 2.00 , chcl3 ) ; max ( film)/cm 3063 , 2961 , 2925 , 2868 , 1606 , 1573 , 1450 , 1363 , 1216 , 1171 , 1084 , 1026 , 855 , 759 , 729 , 691 ; h nmr ( 400 mhz , cdcl3 ) 8.60 ( s , 1h , chn ) , 7.897.84 ( m , 2h , 2 ph h ) , 7.567.45 ( m , 3h , 3 ph h ) , 1.28 ( s , 9h , c(ch3)3 ) ; c nmr ( 101 mhz , cdcl3 ) 162.7 ( c = n ) , 134.0 ( ph c quat . ) , 132.4 ( ph c ) , 129.4 ( 2 ph c ) , 57.8 ( c(ch3)3 quat . ) , 22.6 ( c(ch3)3 ) . observed data was consistent with that reported in the literature . prepared according to the procedure of ruano , mcpba ( 227 mg , 1.31 mmol , 1.1 equiv ) was added at rt in one portion to a solution of sulfinylimine 8 ( 254 mg , 1.19 mmol , 1.0 equiv ) in ch2cl2 ( 6 ml ) . after 5 min , the reaction was diluted with ch2cl2 ( 12 ml ) and then washed with saturated aqueous sodium bicarbonate solution ( 3 10 ml ) . the organic layer was dried ( na2so4 ) and the solvent was removed under reduced pressure , affording sulfonylimine 10 as a colorless oil ( 268 mg , 99% ) , which was used without further purification : rf 0.43 ( 30% etoac / hexane ) ; max ( film)/cm 2984 , 1608 , 1575 , 1479 , 1452 , 1397 , 1366 , 1299 , 1222 , 1176 , 1124 , 1074 , 1023 , 862 , 810 , 786 , 760 , 681 ; h nmr ( 400 mhz , cdcl3 ) 9.02 ( s , 1h , chn ) , 7.95 ( d , j = 7.8 hz , 2h , 2 ph h ) , 7.63 ( t , j = 7.6 hz , 1h , ph h ) , 7.51 ( t , j = 7.7 hz , 2h , 2 ph h ) , 1.48 ( s , 9h , c(ch3 ) ) ; c nmr ( 101 mhz , cdcl3 ) 172.8 ( c = n ) , 134.9 ( ph c ) , 132.3 ( ph c quat . ) , 131.0 ( 2 ph c ) , 58.2 ( c(ch3)3 quat . ) , 23.9 ( c(ch3)3 ) . observed data was consistent with that reported in the literature . prepared according to method a described above , starting from imine 8 ( 105 mg , 0.50 mmol ) . purification by flash chromatography ( 5% etoac / hexane ) on deactivated basic alumina ( activity v ) afforded a mixture of cis - iodoaziridines 9a ( major ; 85:15 ) and 9b ( minor ) as a yellow oil ( 103 mg , 59% ) : rf = 0.36 ( 25% etoac / hexane ) ; [ ]d 21.3 ( c 0.66 , chcl3 ) ; max ( film)/cm 2960 , 2867 , 1605 , 1495 , 1475 , 1454 , 1363 , 1312 , 1238 , 1170 , 1080 , 1026 , 906 , 847 , 817 , 791 , 758 , 699 , 676 ; majorh nmr ( 400 mhz , cdcl3 ) 7.437.33 ( m , 5h , 5 ph h ) , 4.54 ( d , j = 6.0 hz , 1h , chi ) , 3.71 ( d , j = 6.0 hz , 1h , chph ) , 1.20 ( s , 9h , c(ch3)3 ) ; c nmr ( 101 mhz , cdcl3 ) 133.8 ( ph c quat . ) , 128.5 ( ph c ) , 128.2 ( 2 ph c ) , 57.5 ( chph ) , 35.4 ( c(ch3)3 ) , 22.6 ( c(ch3)3 ) , 17.8 ( chi ) ; minorh nmr ( 400 mhz , cdcl3 ) 7.437.33 ( m , 5h , 5 ph h ) , 4.83 ( d , j = 5.9 hz , 1h , chi ) , 3.30 ( d , j = 5.9 hz , 1h , chph ) , 1.41 ( s , 9h , c(ch3)3 ) ; c nmr ( 101 mhz , cdcl3 ) 134.7 ( ph c quat . ) , 128.4 ( ph c ) , 128.0 ( 2 ph c ) , 58.6 ( chph ) , 38.6 ( c(ch3)3 ) , 23.3 ( c(ch3)3 ) , 15.4 ( chi ) ; hrms ( esi / tof ) m / z calculated for c12h17inos [ m + h ] 350.0070 ; found 350.0078 . prepared according to method a described above , starting from imine 10 ( 113 mg , 0.50 mmol ) . purification by flash chromatography ( 5% etoac / hexane ) on deactivated basic alumina ( activity iv ) afforded cis - iodoaziridine 11 as a yellow oil ( 82 mg , 45% ) : rf = 0.24 ( 15% et2o / hexane ) ; max ( film)/cm 2987 , 1457 , 1314 , 1249 , 1175 , 1126 , 905 , 862 , 765 , 730 , 699 , 670 ; h nmr ( 400 mhz , cdcl3 ) 7.467.35 ( m , 5h , 5 ph h ) , 4.90 ( d , j = 6.0 hz , 1h , chi ) , 3.86 ( d , j = 6.0 hz , 1h , chph ) , 1.58 ( s , 9h , c(ch3)3 ) ; c nmr ( 101 mhz , cdcl3 ) 133.1 ( ph c quat . ) , 128.9 ( ph c ) , 128.3 ( 2 ph c ) , 60.1 ( c(ch3)3 quat . ) , 43.3 ( chn ) , 24.0 ( c(ch3)3 ) , 19.0 ( chi ) ; hrms ( esi / tof ) m / z calculated for c12h17ino2s [ m + h ] 366.0019 ; found 366.0021 .

the preparation of c - iodo - n - ts - aziridines with excellent cis - diastereoselectivity has been achieved in high yields by the addition of diiodomethyllithium to n - tosylimines and n - tosylimine hso2tol adducts . this addition - cyclization protocol successfully provided a wide range of cis - iodoaziridines , including the first examples of alkyl - substituted iodoaziridines , with the reaction tolerating both aryl imines and alkyl imines . an ortho - chlorophenyl imine afforded a -amino gem - diiodide under the optimized reaction conditions due to a postulated coordinated intermediate preventing cyclization . an effective protocol to assess the stability of the sensitive iodoaziridine functional group to chromatography was also developed . as a result of the judicious choice of stationary phase , the iodoaziridines could be purified by column chromatography ; the use of deactivated basic alumina ( activity iv ) afforded high yield and purity . rearrangements of electron - rich aryl - iodoaziridines have been promoted , selectively affording either novel -iodo - n - ts - imines or -iodo - aldehydes in high yield .

filoviral hemorrhagic fevers are rare but highly lethal diseases associated with outbreaks in developing countries . the causative agents , ebola virus and marburg virus , are considered to be high level biothreat agents by the united states centers for disease control and prevention and the national institutes of health . there are no effective vaccines to be used as prophylactics and no effective antiviral interventions to manage the diseases . because the clinical symptoms of marburg and ebola hemorrhagic fevers are difficult to distinguish , a drug that is effective against either would be a first , and one that is effective against both the viruses would be an enormous breakthrough . filoviruses are non - segmented negative - strand rna viruses that produce filamentous enveloped virions . there are currently four known clinically relevant species of ebola virus and a single species of marburg virus . initial virus replication occurs in mononuclear cells and viremia is usually apparent within 2 days after infection . death can occur in up to 90% of the infections after 710 days of symptoms , usually due to hemorrhagic fevers . although the cell receptors for either ebola virus or marburg virus have not been fully characterized , the broad cell tropism of the viruses suggests a wide distribution of their receptors . in any regard , the trimeric envelope glycoprotein ( gp ) spikes of the filoviruses are believed to mediate their entry into host cells via endocytic pathways . within endo / lysosomal compartments , host endosomal cysteine proteases ( cathepsins ) cleave the filoviral gp1 protein to generate an entry intermediate comprising an n - terminal gp1 fragment and gp2 . recent work indicates that a cleaved form of ebola virus gp subsequently interacts with niemann - pick c1 , an endo / lysosomal cholesterol transporter , to trigger membrane fusion . as illustrated in table 1 , the pipeline of candidate anti - filovirus therapeutics is limited . however , promising work on inhibition of the virus entry into their host cells with small molecules , recombinant c type lectins or immuno - adhesion technology , disruption of viral rna capping and directly targeting viral rna with antisense oligos or sirnas have recently been reported . as table 1 also indicates , the oligonucleotide - based therapeutics appear to be the farthest along in development , reaching phase i clinical trials , but challenges remain and the extent to which these approaches will progress is uncertain . some of these challenges are inherent to the oligonucleotide approach and the lack of efficient cell - specific delivery technologies . although a morpholino modification or a lipid nanoparticle formulation has been used for these approaches in their anti - filovirus applications , specific targeted delivery of oligonucleotide into disease - relevant tissues and cells remains a major issue to address . only a few small molecule antivirals have been shown to have in vivo efficacy against filoviruses . fgi-103 , 104 , 106 and nsc62914 were discovered using cell - based high - throughput screening , and their antiviral mechanisms are largely unknown . the other two families of small molecules target host enzymes , the s - adenosyl - l - homocysteine hydrolase and endoplasmic reticulum ( er ) -glucosidases i and ii . the er -glucosidases are especially interesting because the broad - spectrum antiviral activities of their inhibitors have been extensively demonstrated with a variety of enveloped viruses from many families ( table 1 ) . in addition , more than 1000 us fda - approved drugs were tested for new antiviral indications . this effort resulted in the discovery that chloroquine could disrupt entry and replication of two or more viruses in vitro and protect mice against ebola virus challenge in vivo . as for many other enveloped viruses , the morphogenesis of filoviruses appears to depend upon er -glucosidases mediated processing of envelope glycoproteins . therefore , the amplification and propagation of filoviruses are sensitive to imino sugar derivatives that inhibit the er -glucosidases i and ii . due to their unusual antiviral mechanism , imino sugars should be complementary to the other antiviral approaches and have the advantage of being broadly active . medical management of virus infection can , in theory and has in practice , involve targeting either virus or host functions . targeting virus specified functions such as viral enzymes ( dna or rna polymerases , proteases , helicases , neuraminidases ) has been enormously effective and offered opportunities for great selectivity . on the other hand , drugs targeting host functions , upon which the viruses rely , have been approached with more skepticism because of concerns regarding selectivity and toxicity . table 1 shows a few examples that have been attempted for filoviruses , and discovery of the virus receptor may lead to a few more . the success of the interferon therapy makes the point that it is possible to identify host functions that can serve as targets of broad spectrum antivirals . however , there are , to date , very few examples of small molecule antivirals that target host functions and retain broad activity . it is a short list , including inhibitors of host cellular cyclophilins , inosine 5-monophosphate dehydrogenase , 3-hydroxy-3-methylglutaryl - coenzyme a reductase , s - adenosyl - l - homocysteine hydrolase and er -glucosidases . the er -glucosidase inhibitors are thus in a rare group , having been shown to have selective antiviral activity for multiple enveloped viruses in tissue cultures and , in several cases , in animal models ( table 2 ) . why viral functions are more sensitive to glucosidase inhibition than are host functions is becoming appreciated and touched on briefly below . briefly , as illustrated in figure 1 , the n - linked glycans of mammalian glycoproteins are processed ' with the sequential removal of their terminal glucose residues by the er - resident glucosidases i and ii , shortly after becoming glycosylated at specific asparagine residues . people can tolerate long - term ( months and years ) treatment with glucosidase inhibitors under conditions where there is substantial repression of the er enzymes . however , there are troubling adverse effects ( i.e. , gastric distress ) which must be taken into consideration . glucosidase inhibitors have been approved for the management of type ii diabetes , gaucher 's disease . glucosidase inhibitors had also advanced to phase ii human trials for management of hepatitis c virus and human immunodeficiency virus infection . thus , although mammalian cells and animals tolerate significant , and even total , repression of er glucosidases , many viruses can not . for example , viruses such as the hepadnaviruses , flaviviruses , filoviruses and influenza virus are significantly repressed by imino sugars under conditions where there is no detectable affect upon the host cells , or in the cases of animal experiments , only limited affect upon the host animals ( table 2 ) . the sensitivity of these viruses to glucosidase inhibitors is presumably because they possess envelope proteins that must oligomerize , and they have an apparent requirement for glucosidase processing of their glycoproteins presumably to enable time - sensitive proper calnexin mediated folding for their oligomerization . taken together , the trend is clear and has not been broken for any of the viruses that have been tested so far : enveloped viruses that bud from intracellular membranes and/or use calnexin dependent pathways are selectively sensitive to glucosidase inhibitors . this has been expanded to viruses from four families causing hemorrhagic fevers ( filoviridae , bunyaviridae , arenaviridae and flaviviridae ) ( table 2 ) . a wide variety of structurally diverse compounds , from both nature and synthesis , have been identified as glucosidase inhibitors . for example , inhibition of intestinal glucosidases digesting carbohydrates is therapeutic for the management of type ii diabetes . the imino sugar n - butyl - deoxynorjirimycin ( nbdnj ) is currently approved for the treatment of gaucher 's disease , with patients taking near gram amounts a day for many years . however , the molecular target of the imino sugar in gaucher 's disease is a glucocerebroside transferase , which is highly sensitive to nbdnj , and not er -glucosidases . nevertheless , these successes demonstrate the principle of tolerability of this category of drugs , as well as their practical therapeutic benefits . the benefits of imino sugar glucosidase inhibitors in the treatment of viral infections have been less clear . the currently available and human - tested glucosidase inhibitors are all limited by side effects and poor pharmacokinetic properties that make them less appealing for controlling chronic diseases . for example , although glucosidase inhibitor cellgosivir met the viral reduction milestone for the treatment of chronic hepatitis c in human studies , the drug was not advanced presumably because of gastritis and , frankly , the emergence of more potent direct acting antivirals . thus , a consistent problem has been maintaining serum concentrations of compounds that are antiviral without causing the intestinal upsets , due to the inhibition of intestinal glucosidases . there are now several reports showing that orally administrated imino sugar derivatives inhibited dengue virus and japanese encephalitis virus in mice , as well as woodchuck hepatitis virus in woodchucks . we note , however , that reduction of viremia by even 12 logs has been anticipated to significantly improve clinical outcomes by reducing the severity of disease and increasing survival rates . along these lines , we recently reported imino sugar compounds that significantly reduced mortality in mouse model of lethal derivatives inhibited dengue virus infection . taken together , these results make it reasonable to consider finding broadly active glucosidase inhibitors reaching therapeutic levels in people for multiple viral indications . however , as indicated , one major limitation for developing imino sugar antivirals has been the lack of potency and/or poor pharmacokinetic properties , which lead to difficulty in maintaining therapeutic drug concentrations in vivo . we have improved the antiviral potency of imino sugars from the platform ( nbdnj ) by more than 500-fold ( figure 2 ) . for example , based upon the encouraging in vivo efficacy results achieved with dengue mouse models , we have synthesized imino sugar glucosidase inhibitors , represented by tert butyl urea ( u ) dnj ( 19029 ) , with submicromolar antiviral activity against four families of hemorrhagic fever viruses in cultured cells . in addition , significant in vivo efficacy in ebola and marburg virus infected animals has also been achieved ( 70%80% protection ) . imino sugars , such as those with dnj head groups , are glucose mimetics competitive inhibitors of the n - glycan processing enzymes glucosidase i and ii ( figure 2 ) . as stated and shown in figure 1 , all n - linked glycans , following transfer to acceptor asparagine amino acids on nascent glycoproteins , are trimmed ' in the er by a series of sequentially active glycoprocessing enzymes . specifically , all nascent n - linked glycoproteins contain three terminal glucose residues at the distal termini of their n - glycans following transfer of the oligosaccharide to the protein by way of the enzyme oligosaccharyltransferase . immediately following this transfer , the terminal glucoses are removed , sequentially , by the action of er resident enzymes glucosidases i and then ii . the er chaperons , calnexin and calreticulin , recognize monoglucosylated n - glycans and then fold ' the nascent glycoprotein after which it is further processed by mannosidases and transferred to the golgi where it is further modified into its characteristic complex carbohydrate structures . underglucosylated polypeptides that have not been folded and have not transferred to the golgi may get second chances following reglucosylation and the possibility of calnexin and calreticulin interactions by the enzyme udp - glucose glycoprotein : glucosyltransferase . however , ultimately , misfolded or unfolded polypeptides are sent to the proteasomes where they are degraded . thus , glucosidase inhibitors prevent the interaction of nascent n - glycoprotiens with calnexin and calreticulin and cause their misfolding and degradation . it has been known for more than 20 years that many viruses are sensitive to glucosidase inhibition , and the sensitivity of sindbis virus , influenza virus and fowl plague virus envelope proteins was reported in the 1980s . viruses that depend upon calnexin and calreticulin would thus be expected to be the most sensitive to glucosidase inhibition . and we developed a biogenesis ' theory that viruses that bud from the endoplasmic reticulum would be sensitive to glucosidase inhibitors . this was not to say that viruses that did not bud from the er would not be sensitive to glucosidase inhibitors , and indeed the infectivity of retroviruses which bud from the plasma membrane is apparently greatly affected by glucosidase inhibitors . we would now offer a refinement of the biogenesis ' theory , in light of what is now known , to claim that viruses that depend upon the calnexin / calreticulin type pathway for morphogenesis will be sensitive to glucosidase inhibition . these predictions , for the most part , seem to be supported by experimental results . most strikingly is the example of hepatitis b virus and bovine viral diarrhea virus which are both sensitive to glucosidase inhibition , and are two completely different viruses . hepatitis b virus has a dna genome and is a human pararetrovirus that primarily infects hepatocytes . bovine viral diarrhea virus is a flavivirus with an rna genome and grows primarily in non - liver cells . both , we now know , also depend upon calnexin for the maturation of specific viral glycoproteins . as table 2 shows , both are also greatly inhibited by glucosidase inhibitors at concentrations that do not apparently affect cell viability . although imino sugars have been developed as host glucosidase inhibitors to disrupt viral glycoprotein folding and consequentially , virion morphogenesis , the compounds have additional biological activities due to either on - target suppression of er -glucosidases or off - target interference of other cellular components . for example , on - target inhibition of er glucosidase results in viral glycoprotein degradation has been shown to enhance major histocompatibility complex i presentation of epitopes derived from viral envelope glycoproteins . moreover , changes in the glycan structures associated with viral glycoproteins may also alter their interaction with c - type lectins and consequentially , inhibit pro - inflammatory cytokine production . in addition , some imino sugars may even have off target activities , repressing the virus by mechanisms that do not involve inhibition of er glucosidases . off - target activities are not unusual in compound development , and in the case of extra biological effects upon antigen presentation and cytokine function , could even be beneficial . however , it is essential that such activities be recognized and be part of the decision to either progress or disqualify a compound , and if compounds with off - target activity are advanced , these activities must be monitored . the imino sugars have , in the past , been proposed primarily for use in chronic infections such as human immunodeficiency virus and chronic viral hepatitis . however , they may be best suited to treat acute viral infections , such as filoviral hemorrhagic fevers which involve weeks of therapy , as opposed to chronic infections which may require years of use . moreover , the new generation of compounds being brought forward should be designed to avoid or reduce intestinal distress . finally , used for life - threatening infections such as the hemorrhagic fevers or severe influenza , either alone or in combinations with other management regimes , the glucosidase inhibitors could provide a powerful option in the treatment tool kit .

ebola virus and marburg virus are members of the family of filoviridae and are etiological agents of a deadly hemorrhagic fever disease . the clinical symptoms of ebola and marburg hemorrhagic fevers are difficult to distinguish and there are currently no specific antiviral therapies against either of the viruses . therefore , a drug that is safe and effective against both would be an enormous breakthrough . we and others have shown that the folding of the glycoproteins of many enveloped viruses , including the filoviruses , is far more dependent upon the calnexin pathway of protein folding than are most host glycoproteins . drugs that inhibit this pathway would be expected to be selectively antiviral . indeed , as we summarize in this review , imino sugars that are competitive inhibitors of the host endoplasmic reticular -glucosidases i and ii , which are enzymes that process n - glycan on nascent glycoproteins and thereby inhibit calnexin binding to the nascent glycoproteins , have been shown to have antiviral activity against a number of enveloped viruses including filoviruses . in this review , we describe the state of development of imino sugars for use against the filoviruses , and provide an explanation for the basis of their antiviral activity as well as limitations .

epigenetics participate not just in the normal functioning of the cell and its development , but also in diseases like neurological diseases and cancer . the importance of epigenetics in cancer has been recognized , and the field has emerged rapidly in recent years . epigenetic modifications precede genetic changes , and usually occur at an early stage in development of a neoplasm , but may also be involved in its invasion and spread . recent technological advances in genetics and epigenetics offer a better understanding of the underlying epigenetic alterations during initiation and in the progression process of the human tumors . techniques like next - generation sequencing allow studies of the dna methylation status of human cells at nucleotide resolution ; rna - seq , high - density microarrays for mirna profiling ; and chip - chip and chip - seq for detecting the transcriptome and identifying the binding sites of dna - associated proteins.1 the main classic and next generation hallmarks of cancer have been recognized and analyzed in recent years,24 although changes in the differentiation process have not yet been analyzed . , it will be necessary to know the heritable and somatic changes in gene expression not related to alterations in dna sequences , such as key processes of dna methylation , chromatin modifications , nucleosome positions , and alterations in noncoding rna profiles . the encyclopedia of dna elements ( encode ) project has enhanced our understanding of the principles of the genome , epigenome , and chromatin organization by identifying hundreds of thousands of potential regulatory regions and transcription factor - binding sites in normal cells . the encode project has provided some of the strongest and most detailed data regarding organization of the human genome . after understanding the principles of chromatin and epigenome organization in human primary cell types , the next step will be identification of changes ( particularly through ongoing epigenome mapping ) that have an important role in specific diseases.5 reprogramming of somatic cells can be used as a simplistic model to understand the cell differentiation process.6 takahashi and yamanaka et al devised an accessible model of cell reprogramming from the differentiated to the induced - pluripotent state , in which somatic to pluripotent states are reverted by re - establishing modification of epigenetic markers.7 similarly , in the last decade , many studies have highlighted the prominent role of altered environments ( epigenetic signals ) in regulation of gene expression in carcinogenesis . it seems evident that epimutations generally outnumber genetic abnormalities , and often occur much earlier in the development of cancer.1,8 these changes in the epigenetic landscape should be seen as responsive to aberrant signals coming from the environment and/or to abnormal interactions between the mesenchyme / stroma and the parenchyma / cells ( tissue organization field theory).9 there are substantial parallels between the molecular mechanisms that underpin the reprogramming events leading to generation of induced - pluripotent stem cells and those that lead to oncogenic transformation and phenotype switching , including epithelial mesenchymal transition ( emt ) and mesenchymal epithelial transition , and regeneration of tumor - initiating or cancer stem cell - like cells.10,11 oncogenesis is ontogenesis gone awry , and like the small subset of the cancer cell population responsible for tumor initiation and growth , cancer stem cells ( cscs ) may also have a similar kind of epigenetic reprogramming phenotype , leading to loss of expression of specific genes that maintain the differentiated state and regain or preserve stem cell - specific characteristics . reprogramming epigenetic mechanisms in cscs or in low - grade differentiation tumor cells by inducing them to differentiate from their pluripotent state to a mature differentiated form offers the potential for development of new therapeutic interventions.8,12 cancer represents a group of over 300 specific diseases that share a number of genetic , epigenetic , and pathological features . cancerous and precancerous cells present different epigenetic abnormalities that modify the transcription of cancer - associated genes through aberrant expression of oncogenes and underexpression of tumor suppressor genes . the main epigenetic alterations in cancer cells are : genome - wide hypomethylation ( in the gene body and in repetitive genomic regions ) , cpg island hypermethylation of gene promoter regions , activation and mobilization of retrotransposons , abnormal non - coding rna patterns ( micrornas have a prominent role ) , loss of genomic imprinting , modifications of histone and polycomb repressive complexes , chromatin remodeling , nucleosomal positioning and chromosomal looping changes , and accessibility of gene transcription regions . this is a hypermethylation pattern of cpg islands at gene promoter regions that is associated with the silencing of numerous cancer - related genes affecting a variety of vital cell processes . cimp is strongly associated with clinical outcome , suggesting it may be a predictive biomarker of the disease course . in recent years , technological advances have enabled genome - wide dna methylation analysis and unsupervised clustering of methylome data , which have clearly delineated cimp - positive groups , including breast , lung , colorectal , endometrial , bladder , oral , and renal cancers.13,14 modern evidence indicates that epigenetic changes in cancer are present in the normal - appearing tissue surrounding many tumors . different epigenetic aberrations and mechanisms are associated with premalignant , invasive , and metastatic spreading phenotypes . some of these epigenetic biomarker profiles may have diagnostic , prognostic , and predictive clinical utility.1,15 recent studies have shown that the dynamics of repetitive elements or transposable elements may have a positive function by contributing to tissue - specific transcriptional programs as enhancer - like elements . they may also modulate the regulation of higher order chromatin structure.16 these mobile dnas , also known as jumping genes , are widespread in nature and comprise an estimated 45% of the human genome ; the long interspersed element-1 ( line-1 , retrotransposon ) in particular , is capable of retrotransposing , not just of their own rna but also of nearby rnas by encoding reverese transcriptase . retrotransposition is a common contributor to human genetic variation.17 line-1 encodes reverse transcriptase , required for its own mobility , and its expression is inhibited in differentiated tissues , but active in tumors . experimental evidence indicates that inhibition of line-1-derived reverse transcriptase restores differentiation in cancer cells , inhibits tumor progression , and yields globally reprogrammed transcription profiles.18 epigenetic changes in line-1 occur early during carcinogenesis . in cancer cells , the methylation levels of a majority of line-1 loci is decreased ( 44% ) , with some loci remaining unchanged and a few being increased ( 56%61% ) when compared with normal cells . hypomethylation of line-1 loci is common in most types of cancer ; this increases their mobilization and can drive the mobilization of adjacent coding regions and regulatory elements to new locations . line-1 dissemination has been observed to wax and wane during evolution of tumors in humans . line-1 hypomethylation levels is a candidate tumor marker for cancer.19,20 aberrant epigenomics contributes to development of cancer by its involvement in initiation , promotion , invasion , metastasis , and resistance to chemotherapy . the proteins of these cancer - related genes participate , eg , in the transcriptional inactivation caused by promoter hypermethylation of genes involved in the major cellular pathways ( including dna repair , cell cycle control , cell differentiation , apoptosis , metastasis , detoxification , hormone response , vitamin response , and the p53 network ) or in dna hypomethylation of oncogenes and repetitive regions of the genome ( causing genomic instability ) . these genes also affect histone modification patterns related to gene expression , long noncoding rnas , and repositioning chromatin involved in complex cellular regulations . the key processes responsible for epigenetic regulation in eukaryotic cells are dna methylation and modifications of chromatin ( covalent alteration in core histones ) . early successes have been achieved with dna - demethylating drugs and agents supporting histone acetylation . dna methylation is an epigenetic event that regulates compacting of chromatin and repression of gene expression . in cancer cells , a variety of genes are abnormally silenced by dna methylation , including tumor suppressor genes and genes controlling the immune response and drug sensitivity . dna methylation is catalyzed by dna methyltransferases ( dnmts ) and involves the transfer of a methyl group from the methyl donor s - adenosylmethionine to the c-5 of cytosine in dna . dnmt1 is the most abundant dnmt and maintains methylation during dna replication , while dnmt3a and dmnt3b are responsible for establishing methylation during embryonic development . overexpression of the major dnmts may result in accumulated hypermethylation of dna for tumor - related genes . disrupted homeostasis of dna methylation is a hallmark of cancer , and acts as an alternative and complementary mechanism to gene mutation or deletion , leading to inactivation of specific tumor suppressor genes . the former class includes azacitidine and decitabine , of which low doses have shown clinical benefits.21 histone modifications include a multitude of covalent variations affecting the histone amino - terminal tails protruding from nucleosomes . the post - translational modifications comprise acetylation , methylation , phosphorylation , sumoylation , and adp ribosylation , which alter both the electrostatic charge of the histone and its interactions with multiprotein complexes , thus modulating the activity of the associated dna . among the post - translational modifications on histone tails , histone acetylation and methylation of specific lysine residues on histones h3 and h4 are the most extensively studied . histone acetylation is an important determinant of gene expression , acetylation is generally associated with elevated transcription , and histone deacetylation is often associated with gene repression . histone deacetylases ( hdacs ) are critical regulators of gene expression , removing the acetyl group from histones and non - histone proteins . four classical hdac classes , ie , i , ii , iii , and iv , containing eleven hdacs , have been classified according to their homology to yeast proteins , subcellular location , and enzyme activity . the biological outcome of hdac inhibition is dependent on the hdac specificity of the compound and intrinsic operation of cell - signaling pathways . hdac inhibitors ( eg , vorinostat and romidepsin ) exert their anticancer activity by promoting acetylation of histones , and are the best characterized anticancer drugs . these first - generation epigenetic inhibitors have a broad target specificity for the mechanisms of action of the eleven hdacs.22 over the last few years , many mutations have been identified in genes that modify the epigenome . epigenetic changes in tumors are generally of a clonal nature , and occur in the early generation of cancer cells . it is established that 5-methylcytosine residues are hot spots for mutations , because 5-methylcytosine is highly mutable by deamination . environmental and micro - environmental changes such as oxidative stress can contribute to tumor development not only through genetic mechanisms but also through epigenetic mechanisms.1 recently , new sequencing technologies have identified driver mutations in epigenetic regulators , providing a mechanistic link between the cancer epigenome and genetic alterations . many genetic alterations and post - translational modifications in cancer - targeted epigenetic regulators cause cancer - associated phenotypes via epigenetic dysfunction . most of the structural data of the epigenetic enzyme classes is well known and is used in the application of targetable molecules . mutations in epigenetic modifiers comprise a large proportion of all genetic events in many cancers . oncogenic activating mutations occur in a number of epigenetic regulators ( eg , idh1/2 , ezh2 , dnmt3a ) or inactivation mutations of tumor suppressors / chromatin modifiers ( eg , kdm6a , crebbp / ep300 , smarcb1 ) . modifier proteins with frequent driver mutations in cancer can be identified by specific function and target site : dna methylation and hydroxymethylation modifiers ( dnmt3a , tet2 , idh1/2 ) ; three covalent histone modifier types , ie , reader ( recognizes and recruits ) , writer ( adds modifications , eg , ezh2 , setd2 , mlls , nsd1/2 ; crebbp , ep300 ) , and eraser ( removes modifications , eg , kdm6a , other kdms ; hdacs ) ; and chromatin remodeling modifiers , such as the swi / snf complex ( arids , smarcs , pbrm1 ) . low recurrent mutations in genes encoding the histones have also been identified ( hist1h1b , hist1hic ) ; most striking is the location of these mutations at amino acid residues near critical sites for post - translational modifications on the histone tail ( eg , h3k27 , h3k36).22 several studies have shown that the mutations in epigenetic modifiers are both incredibly diverse and ubiquitous in different types of cancer . several neoplasms rely on few or many mutations in selected genes that drive tumorigenesis through an altered epigenome , at least in part . mutations in regulators of dna methylation have a strong cancer - specific prevalence , whereas genetic alteration in histone modifiers is widespread across many cancers . understanding how genetic alterations give rise to the cancer epigenome will offer new possibilities for developing better prognostic and therapeutic strategies . epigenomic alterations associated with these driver gene mutations could lead to the discovery of novel pathways involved in tumorigenesis , which may themselves possess unique vulnerabilities for therapeutic intervention.23 to better understand the interplay between etiological factors , cellular - molecular characteristics , and disease evolution , the field of molecular pathological epidemiology ( mpe ) has emerged as an interdisciplinary integration of molecular pathology and epidemiology . mpe is founded on the unique disease principle that each disease process results from a unique profile of exposomes , epigenomes , transcriptomes , proteomes , metabolomes , microbiomes , and interactomes in relation to the macro - environment and the tissue micro - environment . the mpe paradigm ( in cancer ) represents an integrative interdisciplinary science of molecular pathology ( genetic patterns of germlines and genomic patterns of somatic cells ) and epidemiology ( data on risk factors ) . the epigenetic patterns of tumor ( somatic ) cells have recently been added to this paradigm.24 the epigenetic epidemiology of cancer includes the study of variation in epigenetic traits and the risk and progression of disease using large - scale , population - based data . epigenotyping can classify cancers affecting different organs into distinct groups , with different clinical , pathological , and molecular characteristics . further , identification of diagnostic epimarkers that enable early detection of disease and its prognosis can be used to apply targetable treatments . risk factors associated with epigenetic diagnostic and prognostic biomarkers have been studied in lung , breast , colorectal , and bladder cancer.25 nowadays , the holistic mpe approach in cancer patients analyses the genome , methylome , epigenome , metabolome , transcriptome , proteome , microbiome , immunity , and the interactome . the holistic mpe approach enables us to link potential etiological factors to specific molecular pathologies , and gain novel pathogenetic insights on causality . the main epigenome analyses encompass methylome patterns , such as the promoter cimp , global methylation , and line-1 methylation epigenotypes as well as the histone post - translational profile , including acetylation / methylation of specific lysines of h3 and h4 . biomarkers detected using molecular biology tools allow molecular characterization of cancer signatures and provide information relevant for personalized treatment . molecular biomarkers based on the molecular profile of the tumor can be used as prognostic biomarkers or molecularly targeted therapy , and for evaluation of the toxic effects of anticancer drugs.26,27 molecular diagnostics have become an essential component in clinical cancer decision - making . however , tumor evolution and behavior can not be accurately predicted , despite numerous research studies reporting promising tumor biomarkers . to advance in molecular diagnostics , it is essential to have a better understanding of intratumor and intertumor heterogeneity , and the extracellular matrix non - neoplastic host cell interactions influenced by genomic variation , hormones , dietary , lifestyle , and environmental exposure . we can better classify tumors by molecular methods , and move closer toward precision medicine for cancer.28 recently , president obama announced a research initiative that aims to accelerate progress toward a new era of precision medicine and bring us closer to being able to cure diseases like cancer and diabetes . this initiative will encourage and support scientists efforts to develop creative new approaches for detecting , measuring , and analyzing a vast amount of biomedical information . these research activities will propel our understanding of diseases and their origins and mechanisms , and open up opportunities for prevention and treatment.29 conceptually , epigenetic changes are reversible , so they are rational targets to approach . to date , five epigenetic agents have been approved by the us food and drug administration , and comprise the first generation of epigenetic inhibitors ( table 2 ) , ie , two dnmt inhibitors and three hdac inhibitors . the dnmt inhibitors , 5-azacytidine ( vidaza ) and 5-aza-2deoxycytidine ( decitabine , dacogen ) , are analogs of cytidine and have demonstrated clinical utility in myelodysplastic syndrome and leukemia , and the hdac inhibitors , suberoylanilide hydroxamic acid ( vorinostat , zolinza ) , the natural product of the chromobacterium violaceum ( romidepsin , istodax ) , and pxd-101 ( belinostat , beleodaq ) have shown clinical utility in the treatment of rare cutaneous t - cell lymphoma.30 dnmt inhibitors act as irreversible covalent inhibitors of all catalytically active dnmt isoforms following incorporation into dna.31 hdac inhibitors are a diverse group of compounds that vary in structure , biological activity , and specificity , and novel therapeutics are now targeting zinc - containing hdac enzymes in the treatment of various types of cancer.32,33 many other dnmt inhibitors and hdac inhibitors are being used in clinical trials . because cross talk can occur between dna methylation and histone deacetylation , a combination of these two epigenetic modifications has been used too . the first generation of epigenetic inhibitors , being drug - like molecules , has shown significant but limited utility in hematological malignancies due to toxicity and off target effects ( table 2 ) . developing therapeutic strategies targeting epigenetic aberrations ( in much the same way as the successful development of the targeted protein kinase inhibitors currently used in the treatment of cancer ) keeps expanding and is guiding research efforts for many investigators . efforts to derive more selective and/or less pleiotropic inhibitor scaffolds are continuing , and may provide efficacy in a broader set of tumors . similarly , the development of a second generation of epigenetic inhibitors ( table 2 ) , which have a sufficient preclinical therapeutic index to allow clinical evaluation , is a growing area . these second - generation compounds hold more promise , because they have greater intrinsic selectivity for their molecular targets and will be developed in indications where the target is known to be a driver or a key mediator of the malignancy . second - generation epigenetic inhibitors are exciting new drugs that target other reader , writer , and eraser histones which represent epigenetic aberrations in tumor cells ( alterations in epigenetic proteins of tumors).34 most second - generation epigenetic inhibitors are : histone methyltransferase inhibitors such as g9a , responsible for the dimethylation of lysine 9 on histone h3 ( h3k9me2 ) ; ezh2 inhibitors that catalyze methylation of h3k27 and carry out its function as part of the polycomb repressive complex 2 ; dot1l inhibitors that catalyze the transfer of one , two , or three methyl groups to h3k79 ; lsd1 inhibitors that catalyze the demethylation of monomethylated and dimethylated h3k4 and h3k9 ; and jumonji inhibitors , which represent the largest group of lysine demethylases . other second - generation epigenetic inhibitors ( in the study phase ) are the epigenetic protein inhibitors , as the acetyl lysine readers ( bet subfamily members ) , and the methyl lysine readers.31 recently , a rapidly expanding list of reported somatic gene alterations ( recurrent point mutations and chromosomal translocations ) have been identified in both hematological and solid tumors . according to the model of cellular differentiation deregulation in carcinogenesis process , normal adult somatic stem cells and particularly cscs can be modified using molecular epigenetic interventionism targeting oncogenic signaling cascades . cscs represent into the tumor , a small subset of the cancerous population responsible for tumor initiation and growth . they possess the characteristic properties of quiescence , indefinite self - renewal , and intrinsic resistance to chemotherapy and radiotherapy . somatic stem cells and cscs share common signaling pathways for the retention of their stem cell properties . the most important self - renewal signaling pathways include wnt / b - catenin , notch , and hedgehog , each of which has multiple downstream target proteins and genes.12 the generation of cscs in the process of carcinogenesis involves epigenetic reprogramming mechanisms , which lead to loss of expression of genes ( specific to the differentiated states ) and regaining of stem cell - specific characteristics . in the reprogramming of cscs many epigenetic alterations in cscs modify structural patterns of dna methylation / chromatin remodeling and post - transcriptional regulation of non - coding rnas . most of the epigenetic alterations in cscs are caused by epigenetic enzyme alterations , which are frequently used as targets for anticancer therapy . epigenetic enzymes are often dysregulated in human tumors through mutation , altered expression , or inappropriate recruitment to certain loci . identification of these enzymes and their partner proteins has driven the rapid development of small - molecule inhibitors that target the cancer epigenome ( table 1 ) . regulation of histone post - translational modifications and dna methylation is controlled and catalyzed by many different classes of enzymes , the existence and functions of which have been elucidated with a rapid progression in the last decade . in the years to come , identification of somatic gene alterations highlighting epigenetic deregulation may be possible for many cancers , and in the same way as the successful development of the targeted protein kinase inhibitors currently in use , the possibility of developing personalized epigenetic medicine will be achieved . epigenetic modifications are reversible nuclear chemical reactions that occur due to the ability of enzymes to exercise opposing catalytic effects ( eg , using specific inhibitors ) . for now , the structural genomics consortium ( sgc ; http://www.thesgc.org ) , a non - profit organization , formed in 2004 to determine the three - dimensional structures of proteins of medical relevance , and in particular its epigenetics program , is working to generate pharmacological inhibitors of human proteins that regulate epigenetic signaling . the sgc develops chemical probes and antibodies to study the role that different protein domains play in histone modifications and dna methylation . sgc accelerates the development of new or not very well understood areas of human genome research , creating collaboration networks between nine global pharmaceutical companies , scientists , and universities.31 cancer pathologies have been associated to many different epigenetic changes . each type of cancer may have an epigenetic alteration profile , and patients with the same type of neoplasm may have a particular epigenetic profile . epigenetic aberrations arise early in the carcinogenesis process , preceding classical gene mutations identified in the specific pathophysiology of each of the 300 types of human cancer . epigenetic - related aberrations associated with changes in genomic , transcriptomic or signaling pathways ( particularly of the oncogenes and tumor - suppressor genes ) , should be used for epigenetic analysis and as epigenetic targets for anticancer therapy . for example , the estrogen receptor and human epidermal growth factor 2 are epidrug targets in the treatment of breast cancer ; chromosomal instability , mir-34b , mir-148-a , and cimp are targets in colorectal cancer ; and the epidermal growth factor receptor in non - small - cell lung carcinoma.35 nevertheless , different tumor types express csc - like markers , such as cd133 , cd44 , cd34 , and cd24 , and pluripotency genes such as oct3/4 , nanog , sox2 , and myc . also , other expressed markers of invasiveness such as vimentin , n - cadherin , snail , twist , and zeb1 as well as markers of drug resistance such as aldehyde dehydrogenase and abc transporters.12,36 different combinations of these markers are characteristic for identification of the different types of tumor tissues and are used to design epigenetic targets for anticancer therapy . on the other hand , the selection of specific anticancer epidrugs needs to transit through long analytic studies to to obtain clinical therapeutic utility . this developing research on epidrugs requires : target selection ( altered epigenetic proteins ) ; epigenetic chemical probes ; analyses of drug candidates or small - molecule inhibitors ; in vitro assays ( development of cells ) ; in vivo biological / pharmacological / toxicological assays ( novel animal models with specific epigenetic aberrations ) ; and studies progressing through phase i , ii , iii , and iv clinical trials.34,37 as with other cancer therapies , the deployment of epigenetic therapies will likely require development of a rational combination of chemotherapy agents , target kinase inhibitors , inmmunotherapies , or different classes of epigenetic drugs . epigenetic therapy should also consider the biological evolution of the specific cancer and the cytotoxic agents previously used in patients . this strategy has been used to reverse resistance to cisplatin in patients with ovarian cancer cells , and in other tumor types.31 this observation is based on the limited clinical utility of epidrugs in treating defined hematopoietic neoplasms , our better understanding of epigenetic modifications in normal and cancer cells , the development of promising second - generation epigenetic inhibitor , and data from preclinical and clinical studies . many regulatory proteins involved in abnormal dna methylation and chromatin coiling in cancer epigenome , including dnmts , hdacs , methyl - cpg - binding domains , polycomb - group , and other proteins , are targetable and represent active opportunities in cancer therapy.38 pathophysiological ( genomic and epigenomic ) and phenotypic changes are more numerous and complex in solid tumors than in hematopoietic tumors , so carcinomas and sarcomas have more targetable molecules that can be used as therapeutic targets . differing patterns of dna methylation abnormalities are emerging from deep sequencing analyses of different tumor types and subtypes ; and similar conditions may be identified in chromatin coiling . . a rational design of chemical epidrugs , in addition to rigorous preclinical and clinical analyses , will pave the way for clinical success . drugs targeting the epigenome are promising new cancer treatment modalities , but not all patients receive the same benefit from these drugs . in contrast with conventional chemotherapy , responses may not be apparent until several months after initiation of treatment . changes in specific epigenetic biomarkers in tumor cells ( eg , line-1 methylation pattern elements , histone acetylation patterns ) may be used as predictor clinical biomarkers in the assessment of anticancer responses . assessment of these epigenetic markers , together with specific subclinical cancer biomarkers , such as circulating tumor cells , or minimal residual disease ( gene expression signature of a specific solid tumor ) in treated cancer patients improve our ability to predict the clinical response.39,40 ideally , identification of good predictive biomarkers would allow selection of a personalized therapy , and thereby maximize the benefit of treatment.41 at present , despite our comprehensive knowledge of the biology and function of epigenetic therapies , the search for specific biomarkers for response and survival is still key . human gene expression patterns are controlled and coordinated by the activity of a diverse array of epigenetic regulators , including histone methyltransferases , acetyltransferases , and chromatin remodelers . deregulation of these epigenetic pathways can lead to genome - wide changes in gene expression . in recent years , research has suggested that cross talk between genomic and epigenomic factors may drive the etiology of both hematological malignancies and solid tumors . epigenetic alterations are associated with different stages of tumor formation and progression in many types of malignancy . epigenetic alterations are frequently observed in cancers associated with chronic inflammation and/or persistent infection with infection with viral or other pathogenic micro - organisms , and with cigarette smoking . to obtain a comprehensive picture of the standard epigenome profiles of normal tissues , the international human epigenome consortium ( http://www.ihec-epigenomes.org ) has set the ambitious goal of decoding at least 1,000 epigenomes in the years to come.42 the international human epigenome consortium coordinates the production of reference maps of human epigenomes for key cellular states that are relevant to health and disease . new epigenetic regulators , whose contribution to cancer initiation and progression has been identified , and many second generation epigenetic inhibitors are currently in preclinical and clinical studies.34,43 the experience with epigenetic therapy in hematopoietic and solid tumors to date has been restricted to dna - demethylating drugs ( decitabine , 5-azacitidine ) and hdac inhibitors ( romidepsin , vorinostat , belinostat ) . these first - generation epidrugs have had very modest anti - tumor efficacy as monotherapy in phase i and ii clinical trials in patients with solid tumors , and the combination of epidrugs with other therapies would enhance or restore sensitivity to such therapies . these drugs have shown very modest antitumor efficacy , with some responses seen in isolated cases ( 3% of patients responded to epidrugs alone , 20% of patients responded to epidrugs in combination with other chemotherapies ) . the disadvantage of epidrugs for the management of cancer is genome - wide effects , which may cause unwanted upregulation of , eg , prometastatic genes . development of gene - targeted epigenetic modifications in dysregulation of tumor - associated genes of specific tumors can provide a novel approach to prevent such unwanted events.38,45,46 at the present time , 72 clinical trials using first - generation and second - generation epidrugs , alone or in combination , as epigenetic cancer therapy are registered at clinicaltrials.gov ( http://www.clinicaltrials.gov ) . a review by nie et al of decitabine - based epigenetic therapy in patients with 12 different types of solid tumors showed that cervical carcinoma , pleural tumors , ovarian carcinoma , and colorectal cancer registered improved response rates ( 8%35%).47 in another interesting study , li et al showed that the strong immunomodulatory intracellular role of 5-azacitidine in cancer cell lines and in biopsies from carcinoma patients , this condition can restore functional expression of the components involved in the biosynthesis and assembly of the peptide / beta-2-microglobulin / human leukocyte antigen class i heavy chain complex.48,49 these changes in expression may be screened for in the future to identify and handle epigenetic priming for immune therapy . epigenetic therapy is emerging as a potentially effective therapy for solid tumors . in solid tumors , simultaneous multilayer / integrative omics analyses ( including genome , epigenome , and transcriptome abnormalities ) will be useful to elucidate the molecular background of pathogenesis and to explore possible therapeutic targets . this organization model has been implemented by the program for promotion of fundamental studies in health sciences at the national institute of biomedical innovation.42 as mentioned earlier , in the coming years , the international human epigenome consortium together with information from gene expression human maps will provide reference maps of human epigenomes and transcriptomes for key cellular types and states . also , specific gene sets participating in oncogenic signaling pathways , oncogenic phenotype cell characteristics , and/or cancer reprogramming cells should be analyzed to obtain a wide and specific landscape of cancer cells , particularly with regard to their epigenomic profile . each type of solid tumor has particular oncogenic phenotype characteristics based on a specific genomic / epigenomic program for initiation of its transformation . each patient with this type of malignancy may show a specific phenotypic or personalized subtype , which includes particular genomic / epigenomic / transcriptomic aberrations ( epigenome mechanisms show heterogeneity among tissues and cell lineages ) . this specific profile should be identified to determine the specific pathophysiology of the neoplastic process , which could be potentially applicable in disease prevention , diagnosis , and therapy . pathophysiological ( genomic / epigenomic / transcriptomic ) aberrations in solid tumor types may be suspected considering the biological behavior of the clinical history and the histopathological subtype image.38 for example , in most solid cancers , metastatic disease is the main cause of mortality and morbidity . metastasis is a complex process requiring tumor cells to invade the surrounding tissue , gain access to the vasculature , survive transport , exit the vasculature , and then grow in a foreign tissue environment . this is a cellular program orchestrated by a set of pleiotropic transcriptional factors such as twist , snail , slug , and zeb1/2 that together form an intricate transcriptional circuit which acts as a transcriptional repressor of e - cadherin and zona occludens-1 , leading to dissolution of adherens and tight junctions . induction of emt is accompanied by dynamic reprogramming of the epigenome , involving changes in dna methylation and several post - translational histone modifications . different epigenetic regulators involved in emt could be used in individualized epigenetic tumor therapy , eg , specific small - molecule inhibitors.50 as clinical response indicators , patients treated with epidrugs may show results several months after starting the treatment . in these patients , it is relevant to identify molecular biomarkers associated with positive or negative clinical response in a post - treatment stage . quantitative changes in signaling molecules in the oncogenic pathway can be used as predictive biomarkers , particularly tumor - associated gene expression and molecular epigenetic biomarkers , such as dna methylation patterns in tumor - associated genes , or histone post - translational changes like cimp in specific genes ; these have also been used as criteria for prognostication in patients.51 an example of assessment of tumor - associated gene expression was shown by li et al in response to use of the epidrug 5-azacitidine , surveying the differential determination of 15 immunomodulatory genes expression.48 a promising new second - generation of epigenetic inhibitors is being developed , and the support and collaboration of the structural genomics consortium or consortium - like organizations in the design , generation , and analysis of pharmacological inhibitors of epigenetic - targeted molecules , which will enhance the progress of testing preclinical and clinical assays . the application of genome engineering techniques in human gene - therapy will be critical to obtain improved therapeutic results ( eg , bacterial rna - guided crispr - cas system).52 induced - pluripotent stem cell technology can be employed in various genetic / degenerative diseases and types of cancer for disease modeling and gene therapy . use of induced - pluripotent stem cells is a better alternative to conventional tests of toxicology and drug research , since they provide an environment that is more similar to human physiological conditions.53 more clinical trials on epigenetic therapy for cancer are necessary for design and contribution to further progress of this therapeutic strategy . the proven clinical utility of dnmt inhibitors and pan - hdac inhibitors , as well as the rapid preclinical advancement of second - generation epigenetic modulators , offer an optimistic outlook for future epigenetic drug discovery and development . more clinical experience with these agents will serve to guide strategies for therapeutic application in targeted patient populations.34 csc - targeted therapeutic approaches might improve the chances of patient survival by reducing the frequency of tumor relapse . with the president s budget for 2016 , the national cancer institute will have increased funding to support clinical trials in order to link their genomic findings to clinical data , and to connect them with lifestyle risks and environmental conditions . the translational and therapeutic programs at the national cancer institute , such as the molecular analysis for therapy choice ( nci - match ) program , and others with similar purposes , will become stronger in their use of molecular methods to identify drugs that will deliver optimum results for treating tumors . the use of epigenetic inhibitors in the treatment of cancer has multiple therapeutic challenges , particularly with regard to identification of genome / epigenome signaling pathways in each tumor type and subtype and in personalized patient profiles . biomarkers and clinical criteria should be used , such as identification of class- and subclass - specific targets , pharmacological dose and dosage schedule , and use of clinical protocols . epigenetic therapy for each cancer patient should consider the tumor - profile specific type or subtype . the selection of the appropriate epidrug ( with minimal toxic effects ) will lead to an improved therapeutic response .

cancer is a complex disease with both genetic and epigenetic origins . the growing field of epigenetics has contributed to our understanding of oncogenesis and tumor progression , and has allowed the development of novel therapeutic drugs . first - generation epigenetic inhibitor drugs have obtained modest clinical results in two types of hematological malignancy . second - generation epigenetic inhibitors are in development , and have intrinsically greater selectivity for their molecular targets . solid tumors are more genetic and epigenetically complex than hematological malignancies , but the transcriptome and epigenome biomarkers have been identified for many of these malignancies . this solid tumor molecular aberration profile may be modified using specific or quasi - specific epidrugs together with conventional and innovative anticancer treatments . in this critical review , we briefly analyze the strategies to select the targeted epigenetic changes , enumerate the second - generation epigenetic inhibitors , and describe the main signs indicating the potential of epigenetic therapies in the management of solid tumors . we also highlight the work of consortia or academic organizations that support the undertaking of human epigenetic therapeutic projects as well as some examples of transcriptome / epigenome profile determination in clinical assessment of cancer patients treated with epidrugs . there is a good chance that epigenetic therapies will be able to be used in patients with solid tumors in the future . this may happen soon through collaboration of diverse scientific groups , making the selection of targeted epigenetic aberration(s ) more rapid , the design and probe of drug candidates , accelerating in vitro and in vivo assays , and undertaking new cancer epigenetic - therapy clinical trails .

up to 80% of all pregnant women experience some form of nausea and vomiting during their pregnancy.13 the international statistical classification of disease and related health problems , tenth revision , defines hyperemesis gravidarum ( hg ) as persistent and excessive vomiting starting before the end of the 22nd week of gestation and further subdivides the condition into mild and severe , with severe being associated with metabolic disturbances such as carbohydrate depletion , dehydration , or electrolyte imbalance.4 hg is a diagnosis of exclusion , characterized by prolonged and severe nausea and vomiting , dehydration , large ketonuria , and more than 5% body weight loss.5,6 affecting approximately 0.3%2.0% of pregnancies , hg is the commonest indication for admission to hospital in the first half of pregnancy and is second only to preterm labor as a cause of hospitalization during pregnancy.79 according to the hyperemesis education and research foundation , conservative estimates indicate that hg can cost a minimum of $ 200 million annually in in - house hospitalizations in the united states.10 taking into account other factors such as emergency department treatments , potential complications of severe hg , and the fact that up to 35% of women with paid employment will lose time from work through nausea , the actual cost of hg to the economy is significantly higher.3 in a related economic analysis , piwko et al projected that the united states spends nearly $ 2 billion in costs attributed to pregnancy - related nausea and vomiting ; 60% of this expenditure is a result of direct costs ( eg , drugs , hospital admission ) , and 40% is a result of indirect costs ( eg , time lost from work).11 to date , studies investigating the association between hg and adverse pregnancy outcomes and maternal morbidities have provided conflicting results.9,12 in all aspects of research involving hg , the interpretation of results and associations must be with caution , as the majority of the studies have been limited by retrospective study design,10,1315 small numbers,16 bias , lack of control for potential confounders , and variable definitions of hg.15,17,18 thus , to examine current clinical perspectives of hg , we performed a review of medline ( 1994january 2014 ) , embase ( 1994january 2014 ) , and the cochrane library . articles related to hyperemesis gravidarum and/or nausea and vomiting of pregnancy were considered for inclusion in our review . although the review focused on articles published in the last 10 years , a second search with unrestricted time limits was performed to identify key papers related to hg that were also considered in the review . hg is most likely a multifactorial condition and has been associated with many risk factors.19 women with hg are more likely to be younger , primiparous , persons of color , and less likely to drink alcohol.20,21 body mass index , smoking , and socioeconomic status do not appear to differ significantly between women with hg and those without.21 female infant sex has also been associated with hg.8,2225 paternal genes are not thought to play a role in the occurrence of hg . in contrast , maternal intergenerational effects have been observed , with increased odds of hg among women whose mothers also experienced hg during a previous pregnancy ( unadjusted odds ratio [ or ] , 3.2 ; 95% confidence interval [ ci ] , 1.66.4).26 moreover , although recurrence rates are higher in women with hg , they are not 100% , indicating a multifactorial process rather than purely maternal genetics.27,28 in a small pilot study , dorazio et al examined personality characteristics between 15 women with hg and 15 matched women without hg and did not detect any differences in personality , psychological , or somatic variables.29 mullin et al examined risk factors in 395 women with prolonged hg . women with prolonged hg were slightly younger and weighed more and had a history of allergies and a restrictive diet.30 of those women with hg with a significant weight loss ( > 15% of prepregnancy weight ) , hg tended to be more severe , with some symptoms , such as food aversion , continuing through the postpartum period.14 ethnicity may play a role , with one study in germany demonstrating that immigrants were 4.5 times more likely to be treated for hg than native germans . these women also scored higher on a somatization scale ( symptom checklist-90-revised ) , indicating a higher degree of psychological distress.31 asian ethnicity has also been reported as a risk factor.32 one observational study demonstrated that women with hg were more likely to have higher levels of pregnancy - associated plasma protein a ( papp - a ) and free human chorionic gonadotropin ( hcg ) in the first trimester compared with controls.34 maternal serum concentrations of hcg peak during the first trimester , when hg symptoms are often at their worst.35 similarly , symptoms of hg are often more severe in multiple pregnancies and molar pregnancies , which are conditions associated with excessively high hcg levels . however , conflicting reports exist,3436 and therefore a causal association between hg and hcg has not been established.36 infection with helicobacter pylori may play a role in the development of hg in some women . a meta - analysis examining h. pylori infection in women with hg reported a significant association ( or , 3.32 ; 95% ci , 2.254.90).37 the meta - analysis was limited by significant heterogeneity among studies . therefore , similar to hcg , a causal association between hg and h. pylori has not been established . other factors implicated in the etiology of hg include estrogen,38 stress , depression , and anxiety.21 hg has been reported to be associated with an increased risk for adverse pregnancy outcomes such as low birth weight , preterm birth , and small - for - gestational age infants.10,13,25 a recent systematic review identified no association with apgar scores , congenital anomalies , or perinatal death.25 several additional studies were not included in the aforementioned review either because of inclusion criteria or because of publication after the review search period . mccarthy et al performed a prospective cohort study of 3,423 nulliparous women.21 hg was defined as repeated vomiting in early pregnancy not resulting from other causes ( eg , gastroenteritis ) and requiring any of the following : inpatient admission , day stay with intravenous fluids , nasogastric feeding ( at home or in hospital ) , or vomiting associated with loss of more than 5% of her booking weight . secondary outcomes included spontaneous preterm birth , preeclampsia , birthweight , small - for - gestational age infants , and infant sex ratio . women with severe hg had an increased risk of having a spontaneous preterm birth compared with women without hg ( adjusted or , 2.6 ; 95% ci , 1.25.7).21 no significant associations were observed among other secondary outcomes . vikanes et al conducted a retrospective cohort study and identified 814 women with hg during a 10-year period in norway.39 relative to women without hg , no increased risk for adverse pregnancy outcomes or low birthweight was observed among women with hg . vandraas et al conducted a population - based cohort study of 2,270,363 births between 1967 and 2009 , using the norwegian birth registry.40 they reported a decreased odds of very preterm birth ( or , 0.66 ; 95% ci , 0.50.9 ) and large - for - gestational - age infants ( or , 0.9 : 95% ci , 0.80.9 ) among women diagnosed with hg . hastoy et al reviewed obstetric outcomes in a small cohort of 197 women hospitalized for hg in a tertiary maternity hospital in france.41 similar to vikanes et al,39 no significant associations were observed between hg and adverse perinatal outcomes . however , in contrast , hastoy et al did observe an increased risk for low birth weight ( adjusted relative risk [ rr ] , 1.7 ; 95% ci , 1.12.4).41 fejzo et al performed a study involving 819 women from an hg web site registry : 16% of babies were born prematurely , and 8% of the women reported infants born weighing less than 2,500 g.14 among women with extreme weight loss , 9.3% reported having a child with a behavioral disorder . as with other research in hg , the lack of a robust control group makes these results difficult to interpret . still , similar results have been reported in women with extreme starvation , suggesting similar underlying pathological processes.42 there is a paucity of data examining the long - term effects of hg throughout childhood and into adulthood.25 in a retrospective case - control study of 259 adults , psychological and behavioral disorders were more frequently reported among adults exposed to hg in utero ( or , 3.6 ; 95% ci , 1.96.9).43 notably , this risk estimate was based on a composite outcome of 17 different disorders because of small numbers for the majority of diagnoses under review ( often < 5 cases observed per individual disorder ) . nonetheless , individual analyses of anxiety , depression , and bipolarism revealed no increased odds of anxiety ; though in contrast , increased odds of depression and bipolarism were observed . although other research has reported an increased risk for psychological disorders in adulthood , as well as reduced insulin sensitivity in prepubertal children,44 prospective longitudinal investigations are warranted to better understand the underlying dynamics of these associations.45 hg can be extremely debilitating for women and , if inadequately managed , can cause significant morbidities , including malnutrition and electrolyte imbalances , thrombosis , wernicke s encephalopathy , depressive illness , and poor pregnancy outcomes such as prematurity and small - for - gestational - age fetuses.13,4649 mullin et al showed that those with hg were more likely to suffer from hematemesis , dizziness , fainting , and antiemetic treatment.30 bolin et al observed that women with hg have an increased risk for placental disorders , such as placental abruption , and that this risk was particularly marked among women presenting with hg in the second trimester.50 furthermore , after pregnancy , these women were more likely to develop posttraumatic stress disorder , motion sickness , and muscle weakness and to have infants with colic , irritability , and growth restriction.30 jrgensen et al33 demonstrated that the risk for any autoimmune disorder was significantly increased in women with hg ( rr , 1.41 ; 95% ci , 1.301.51 ) . in its extreme forms , hg may cause malnutrition and end organ damage manifesting as oliguria and abnormal liver function tests . reassuringly , permanent hepatic damage and associated death are rare in women with hg.51 in their large , prospective study on women with hg , mccarthy et al demonstrated that women with hg , particularly severe hg , were at increased risk for cognitive , behavioral , and emotional dysfunction in pregnancy.21 other studies have linked hg with an increased risk for depression , anxiety , and mental health difficulties,52,53 and as a result , some advocate psychiatric evaluation.54 one study reported women with hg meet criteria for anxiety and depression in 47% and 48% of cases , respectively.12 despite such associations , care must be taken not to stigmatize the condition of hg . it is important to emphasize that early assessment of nausea and vomiting in pregnancy is essential to prevent delay in diagnosis and management of hg . apart from hg , consideration should be given to other underlying complications associated with persistent vomiting , such as gastrointestinal conditions ( eg , hepatitis , pancreatitis , or biliary tract disease ) , pyelonephritis , and metabolic disorders ( eg , diabetic ketoacidosis , porphyria , or addison s disease).55 if such conditions are ruled out , adherence to obstetrical guidelines for the management of nausea and vomiting in pregnancy is encouraged,5558 although disconcertingly , this may not always be followed in practice.59 notably , diagnostic biomarkers for hg have produced inconsistent results . a recent systematic review and meta - analysis found that although ketonuria is often assessed as part of a clinical examination , the robustness of ketonuria as a diagnostic marker for hg remains unclear.60 future investigations examining ketonuria levels in the diagnosis and severity of hg are warranted . lymphocytes were typically higher in women presenting with hg , although the association between hg and hcg and thyroid hormones , leptin , estradiol , progesterone , and white blood count were less reliable.60 as previously discussed , h. pylori serology may be of diagnostic benefit.60 treatment strategies for hg include inpatient and outpatient care involving intravenous fluids , antiemetics , and dietary advice . a lack of support may prevent women from accessing timely and appropriate care.61 a recently published systematic review involving 37 trials and 5,049 women investigated interventions for the treatment for hg . interventions examined included acupressure , acustimulation , acupuncture , ginger , chamomile , lemon oil , mint oil , vitamin b6 , and several antiemetic drugs . again , the review was significantly limited by heterogeneity in study participants , interventions , comparison groups , and outcomes measured or reported . pharmacological agents including vitamin b6 and antiemetic drugs may help relieve mild or moderate nausea and vomiting.62 administration of promethazine and metoclopramide may yield comparable therapeutic effects.63 although research is limited , preemptive treatment with diclectin ( duchesnay , blainville , qubec , canada ) in women with a history of severe nausea and vomiting in pregnancy may decrease the onset of hg.64 overall , however , evidence is lacking as to which pharmacological agent is more effective and less dangerous to both mother and fetus.62,6568 the management of hg is therefore based on correcting electrolyte imbalance and dehydration , prophylaxis against recognized complications , and providing symptomatic relief . tan et al randomized women with hg to either treatment with 5% dextrose saline or normal saline for rehydration . outcomes were resolution of ketonuria and the woman s well - being.69 short - term benefits ( < 24 hours ) were observed in those treated with 5% dextrose , but these had dissipated by 24 hours . there is an understandable reluctance to prescribe antiemetics for symptomatic relief , but extensive data exist to show a lack of teratogenesis with dopamine antagonists , phenothiazines , and histamine h1 receptor blockers.7072 although most women respond well to rehydration , if necessary , enteral tube feeding may be initiated to serve as either as a supplemental or primary source of nutrition.73 consideration may also be given to total parenteral nutrition , although increased risk for infectious complications is a potential concern.73,74 day care has proven to be a beneficial and safe mode of care for women in other clinical settings.75 studies have demonstrated that day care management of women with nausea and vomiting during pregnancy appears acceptable and feasible,76 but no systematic reviews or randomized controlled trials have been performed that examine the effects of introducing day care on rates of hospital admission , duration of inpatient stay , and patient satisfaction . a randomized controlled trial comparing day patient and inpatient management has finished recruiting approximately 100 women and will soon publish its findings.77 further studies are needed that focus on safe alternative treatments , preventative measures in high - risk women , new biomarkers underlying the etiology of hg , and interventions that may reduce adverse pregnancy outcomes . further research is also required to determine whether the provision of emotional support for women with hg is beneficial . although studies are limited in this area , in general , there is a demand for support for women suffering from nausea and vomiting in pregnancy.51 as shown in a recent study evaluating a nausea and vomiting in pregnancy hotline in the united states , women primarily seek support in the management of the nausea and vomiting as well as understanding drug risks for the fetus.78 given that much of the information available on the internet uses complicated language , there is a clear need to improve web resources for hg ; this may be a complementary strategy to providing support for women.79 any new interventions , however , must be shown to be safe from both a maternal and fetal point of view , to be acceptable to mothers , and to be cost - effective . despite the prevalence and considerable morbidity associated with hg , good - quality research investigating the underlying etiology and interventions to treat and prevent hg remains scarce . exploring new pharmacological interventions in pregnant women for the prevention and treatment of hg remains elusive , and this may be a result of avoiding inducing unnecessary risk for the developing fetus . controversies such as that involving the administration of thalidomide to women with morning sickness , which subsequently resulted in significant congenital malformations , has likely discouraged researchers from investigating other interventions for hg.80 as a result , the current mainstay of treatment remains regular hydration and antiemetics . nonetheless , because of the prevalence and morbidity associated with this condition , safe , well - conducted , good - quality research is needed to investigate and clarify the etiology , prevention , and treatment of this condition .

hyperemesis gravidarum is a complex condition with a multifactorial etiology characterized by severe intractable nausea and vomiting . despite a high prevalence , studies exploring underlying etiology and treatments are limited . we performed a literature review , focusing on articles published over the last 10 years , to examine current perspectives and recent developments in hyperemesis gravidarum .

overweight ( body mass index ( bmi ) 25.029.9 kg / m ) and obesity ( bmi 30 kg / m ) are global public health problems [ 1 , 2 ] . all demographic sectors of the united states ( us ) population are affected , but african american ( aa ) women are disproportionately burdened [ 1 , 3 ] . as reported in 2012 ( national health and nutrition examination survey ( nhanes ) , 2009 - 2010 ) , approximately 82% of aa adult women in the us were classified as overweight or obese . this disparity is of particular concern given that overweight and obesity are associated with a number of serious chronic diseases [ 4 , 5 ] . the most common approach to obesity treatment includes lifestyle interventions that target both diet and physical activity ( pa ) and some form of behavioral self - management [ 69 ] . traditionally , aa women enrolled in behavioral lifestyle interventions lose less weight when compared to other subgroups [ 3 , 1016 ] although even modest weight reduction improves the cardiovascular risk profile [ 17 , 18 ] and decreases diabetes incidence . if weight loss is not sustained , the health benefits of weight reduction are attenuated [ 20 , 21 ] . this fact highlights the importance of understanding factors that support long - term weight control across populations . typically , individuals regain about 3035% of their initial weight loss within the first year following treatment , and more than half return to their baseline weight within five years [ 7 , 25 ] . data from nhanes ( 19992006 ) found that only 1 of 6 of the overweight / obese participants surveyed reported that they had ever maintained a weight loss of at least 10% for one year . contributors to weight regain include physiologic adaptations such as reduced resting energy expenditure and leptin concentrations [ 28 , 29 ] , increased ghrelin ( a gut peptide associated with hunger ) [ 30 , 31 ] , and exposure to an obesogenic environment [ 32 , 33 ] . weight regain may also be related to the distinct differences between weight loss and weight loss maintenance behaviors [ 7 , 22 ] . for example , during weight loss , foods that lead to weight gain are avoided , whereas with weight maintenance , food needs to be better managed , overall . despite these challenges , some individuals are successful at long - term weight maintenance . behaviors associated with successful weight loss maintenance identified through the use of the national weight control registry , which consists of more than 6000 adults who have lost at least 13.6 kg and maintained this loss for a minimum of one year , report that successful weight loss maintainers consume a low - fat diet , eat breakfast , weigh themselves regularly , and report high levels of both dietary restraint and pa [ 34 , 35 ] . although these data are encouraging , the registry is comprised predominately of caucasian women , making it a less representative sample . minorities , including aa women , are largely underrepresented in the behavioral lifestyle intervention literature , however , two systematic reviews addressing obesity treatment in minority populations were recently published [ 36 , 37 ] . reviews concluded that cultural adaptations , church - based studies , a low carbohydrate diet plan , individual sessions , family - centered programs [ 41 , 42 ] , and problem - solving skills [ 43 , 44 ] promoted both weight loss and maintenance in minority adults . a third review , focusing specifically on aa women , concluded that attention to cultural preferences , behavioral management strategies , and session attendance were important factors to successful weight loss . however , to the best of our knowledge , no studies have examined the existing behavioral lifestyle intervention literature to identify potential strategies that are effective in promoting long - term weight control specific to aa women . therefore , our objective was to systematically review , synthesize , and summarize the behavioral lifestyle intervention literature to evaluate the effectiveness of these interventions on weight loss maintenance in aa women . these results can then better inform the design of future weight management interventions for this population . note . african american describes a diverse group of people descended from many different cultures of africa and the caribbean including those whose families have lived in the us for centuries and those who more recently emigrated . the systematic review focused on the behavioral lifestyle intervention literature published between 1990 and 2011 . the year 1990 was chosen as a starting point because healthy people 2000 , which was the first comprehensive preventive health agenda for the us population , established specific goals for reducing the prevalence of overweight and obesity . the authors referred to the guidelines recommended by the preferred reporting items for systematic reviews and meta - analyses ( prisma ) checklist for collection , synthesis , and reporting of the data for the systematic review . references were identified through a search of medline via pubmed , cinahl plus , and academic search premier databases . the authors defined weight loss maintenance as period of at least six months , with or without inclusion of a formal maintenance program , following participation in an intensive behavioral lifestyle intervention in which weight was an outcome . search terms included a combination of the following : weight loss maintenance , long - term weight loss , weight regain , weight loss , dietary intervention , obesity , aa , and black . we also used the ancestry approach by reviewing the reference sections of pertinent papers as well as past review articles focused on weight loss maintenance . randomized and nonrandomized studies were included in the review if they met the following criteria : ( 1 ) english language papers published in peer - reviewed journals , ( 2 ) behavioral lifestyle interventions with a maintenance phase of at least six months ( both formal maintenance programs and non - contact periods ) in which weight was reported as an outcome , ( 3 ) studies conducted in the us ( due to potential country - specific differences in weight management practices ) , ( 4 ) adult participants at least 18 years of age , ( 5 ) inclusion of aa women , and ( 6 ) weight outcomes reported separately by ethnicity / race and sex . studies were excluded if they : ( 1 ) were published in a language other than english , ( 2 ) excluded aa women , ( 3 ) had a maintenance period less than six months ( both formal maintenance programs and non - contact periods ) , ( 4 ) included pregnant or postpartum women , ( 5 ) primarily focused on a surgical or pharmacological weight loss intervention , ( 6 ) provided prepared meals , ( 7 ) omitted weight outcomes for the maintenance phase , ( 8) were not an intervention study , or ( 9 ) included a pediatric sample . studies that used liquid meal replacements as the primary intervention were also excluded , although studies in which meal replacements were used as one component of an intervention were included . finally , due to the paucity of studies reporting on this topic , authors of eligible studies that did not report results by race / ethnicity and/or sex were contacted by email to inquire if such information could be provided . weight - related data by race / ethnicity and sex were obtained through this method for four studies [ 3 , 16 , 44 , 50 ] but could not be gleaned from the primary authors for four other interventions and were thus excluded [ 5154 ] . figure 1 presents the study attrition diagram and the number of publications included at each step during the search process . the initial search , utilizing the three databases , yielded 675 publications . after eliminating duplicates , the total was reduced to 476 papers . the authors l. m. tussing - humphreys , a. kong , and m. l. fitzgibbon completed an initial screening using article abstracts or full articles , where necessary , to determine eligibility . to avoid bias , the studies in which m. l. fitzgibbon was primary or coauthor , l. m. tussing - humphreys and a. kong reviewed the abstracts and articles for study inclusion . after reviewing the abstracts and/or full text from the primary search , 465 articles were excluded , leaving 11 papers . the secondary search , using the ancestry approach , resulted in the identification of 28 additional articles . the abstracts for these articles were reviewed as described previously and resulted in the inclusion of 5 additional articles . in total however , one article reported weight loss results for two separate multicenter hypertension trials ( hypertension prevention trial and the trials of hypertension prevention ) and thus was coded as two separate studies , resulting in a total of 17 trials . for each of the 17 studies , the primary author ( l. m. tussing - humphreys ) extracted the following data , using a standardized form , which are presented in tables 1 and 2 : ( 1 ) author and year of publication ; ( 2 ) study design , setting , and duration of the trial ; ( 3 ) participant characteristics including sample size , age , income , education , and health status ; ( 4 ) overarching intervention characteristics including use of a formal theoretical framework inclusion of cultural adaptations , duration of the maintenance phase , a priori criteria for entry into the maintenance period , and components targeted at weight loss maintenance ; ( 5 ) frequency , format , and dose of maintenance intervention sessions or contacts ; ( 6 ) mean baseline weight in kg ; ( 7 ) change in weight ( kg ) immediately following the active intervention phase ; ( 8) weight change in kg from baseline ; ( 9 ) % weight regain at several reported follow - up intervals ( 12 months , 18 months , and end of trial when available ) ( several time points were selected in an attempt to compare weight changes during the maintenance phase across studies ) ; and ( 10 ) adherence to maintenance sessions or components and study retention ( defined as % of participants available at designated postintervention follow - up time - point ) . where possible , missing variables were calculated or estimated from data reported or from figures presented in the paper or obtained directly from the study authors . percent weight regain was crudely calculated for all studies using available weight change data . for trials in which multiple articles were published ( e.g. , diabetes prevention program ( dpp ) , weight loss maintenance trial ( wlm ) , the hypertension prevention trial ( hpt ) , the trial of hypertension prevention ( tohp ) , the trial of hypertension prevention ii ( tohp ii ) , the trial of nonpharmacologic interventions in the elderly ( tone ) , and the treatment of obesity in underserved settings ( tours ) ) , we incorporated all relevant data regardless if the source was other than the study reporting weight loss by race / ethnicity and sex . data extracted from the 17 studies were reviewed by two of the coauthors for accuracy ( m. l. fitzgibbon and a. kong ) . to address study quality , we adapted the ranking system developed by whitt - glover and kumanyika which was designed to evaluate both randomized and nonrandomized studies . nonrandomized studies were included to allow for insight regarding potentially effective strategies utilized in studies with a less rigorous design and due to the paucity of literature published on the topic . the ranking system was 1 for uncontrolled studies , 2 for nonrandomized controlled studies , 3 for randomized controlled pilot studies , and 4 for full - scale randomized controlled trials ( rcts ) . full - scale rcts were deemed the highest - quality study design because ( 1 ) random assignment to treatment tends to minimize selection bias , ( 2 ) treatment and control groups are similar in characteristics and sample size , and ( 3 ) equality of treatment arms produces valid statistical tests . the ranking system was 1 for studies in which weight control was not a primary focus of the intervention and 2 for studies in which weight control was the primary focus . our working assumption was that studies in which the intervention content was focused on weight control would produce better weight change and maintenance outcomes than studies in which weight control was not the primary focus . the ranking system was 1 for interventions with no formal maintenance intervention and 2 for studies that included a formalized maintenance phase . the assumption was that interventions that included formal maintenance treatment would produce better long - term weight control for participants compared to interventions with minimal or no contact during the maintenance period . extended care following a period of intensive behavioral treatment has shown to be effective in producing long - term weight control . the ranking system was 1 for studies in which no cultural adaptations were reported , 2 for studies in which the only adaptation was limiting recruitment to aas , and 3 for studies reporting attempts at adapting intervention - related content and other adaptations including staff trainings and oversight committees . the working assumption was that cultural adaptations could affect acceptability , effectiveness , and retention . the 17 studies are ranked alphabetically according to date published and study quality which ranged between 5 and a maximum of 11 points ( tables 1 and 2 ) . weight was the primary outcome for the majority of studies ( 15 of 17 ) [ 3 , 11 , 1316 , 44 , 50 , 56 , 6062 , 65 , 69 ] . however , one trial focused on increasing pa and improving dietary quality and another focused on increasing daily steps . both trials reported weight outcomes , as a secondary endpoint , and were thus included in the review . the interventions were implemented in various settings including academic medical centers [ 3 , 11 , 13 , 44 , 50 , 56 , 65 ] , five of which were multi - institution collaboratives [ 11 , 13 , 44 , 56 , 65 ] , universities [ 14 , 60 , 62 ] , medical clinics [ 16 , 61 , 69 ] , and community - based locales [ 15 , 67 ] . one study did not report the intervention setting . the multi - institution rcts [ 11 , 13 , 44 , 56 , 65 ] and the pilot rct by tsai et al . recruited participants of mixed race / ethnicity and sex . notably , the weight loss treatment arms of the hpt , tohp , and tone trials included relatively small numbers of aa women ranging from just 28 to 46 women . six studies [ 6062 , 67 , 69 , 70 ] targeted recruitment specifically at aa women with sample sizes ranging from 21 to 366 women . two studies recruited both aa and caucasian women [ 14 , 15 ] , and two recruited aa men and women only [ 3 , 50 ] . the majority of the studies ( 16 of 17 ) enrolled aa women with mean ages between 40 to 60 years old . participants in the tone trial and study by banks - wallace were hypertensive , participants in the wlm trial were hypertensive and/or dyslipidemic , the dpp trial participants presented with impaired glucose tolerance , west et al . and mcnabb et al . recruited type 2 diabetics , and djuric et al . recruited breast cancer survivors . [ 3 , 11 , 44 , 56 , 60 , 67 , 74 , 75 ] . twelve studies [ 3 , 11 , 15 , 44 , 50 , 56 , 6062 , 67 , 69 ] reported incorporating some form of cultural adaptation salient to aas including recruitment of only aa participants [ 6062 , 67 ] , culturally specific diet and pa modifications [ 3 , 11 , 50 , 56 , 60 , 61 , 67 ] , cultural sensitivity training for research staff [ 44 , 56 ] , employing aa case managers and interventionists [ 3 , 11 , 50 , 60 , 67 ] , special attention to religion and spirituality [ 60 , 62 ] , aa community - focused field - trips to grocery stores , parks , and so forth , selection of study site in an aa community , and the formation of a minority implementation committee . across the 17 studies , weight changes for aa women following the intensive intervention phase ranged from + 0.5 to 8.5 kg . in the studies enrolling both aa and caucasian women [ 11 , 13 , 14 , 16 , 44 , 56 ] , initial weight loss for aa women ranged from 1.9 to 7.1 kg versus 3.4 to 10.7 kg for caucasian women . the weight loss plus sodium reduction arm of the tone trial was the only treatment arm across the 17 studies in which initial weight losses were similar between aa ( 3.9 3.6 kg ) and caucasian ( 3.9 3.9 kg ) women . only , two studies [ 3 , 56 ] reported inclusion criteria for entry into the maintenance phase . for the wlm trial , participants were required to have lost a minimum of 4 kg during the six - month active weight loss phase to be randomized to a maintenance treatment group . for the help study participants were required to attend the postphase 1 data collection to be randomized to the maintenance phase . common features of the maintenance interventions included some combination of didactic nutrition and pa sessions [ 3 , 11 , 1315 , 44 , 50 , 56 , 60 , 62 , 65 ] , promotion of adherence to the prescribed eating pattern or dietary modifications ( e.g. , calorie control , fat control , increased consumption of fruits , vegetables , and fiber ) [ 3 , 11 , 1315 , 44 , 50 , 56 , 60 , 62 , 65 ] , achieving a set amount of pa ( minutes or steps per day or week ) [ 3 , 11 , 1315 , 44 , 50 , 56 , 60 , 62 , 65 ] , and ongoing emphasis on behavioral modification strategies learned during the active intervention phase including self - monitoring of weight , dietary intake , and pa , goal - setting , problem solving , relapse prevention , and stimulus control [ 3 , 11 , 1315 , 44 , 50 , 56 , 60 , 62 , 65 ] . importantly , the extent to which these topics were reviewed was difficult to discern from the manuscripts , as the needs of the participants often dictate what content is featured during the maintenance sessions . additionally , supervised pa sessions were offered in three of the trials [ 11 , 50 , 60 ] , and a number of more unique maintenance components were also tested including use of an individualized tool box [ 11 , 76 ] , internet - based delivery , motivational interviewing [ 14 , 61 ] , spirituality counseling , and family and friend support . participants in seven studies [ 3 , 16 , 56 , 61 , 67 , 69 , 70 ] received no or minimal contact during the maintenance period . six studies delivered the maintenance intervention through a combination of group and individual in - person or phone - based sessions [ 13 , 14 , 44 , 50 , 60 , 65 ] with frequency ranging from twice weekly to bimonthly . three studies conducted individual in - person or phone - based maintenance sessions [ 11 , 15 , 62 ] . contact was made monthly in the personal contact arm of the wlm trial , at least monthly or as often as requested by participants in the dpp , and tapered from weekly , to biweekly , to monthly in the djuric et al . the group maintenance arm of the help study met solely in group sessions biweekly , during months 79 , and lessened to monthly thereafter . additionally , several studies mailed newsletters to participants at various times throughout the maintenance phase [ 13 , 15 , 50 , 60 , 62 ] . for the studies reporting dose of the maintenance sessions , participants , enrolled in four of the multi - institution rcts , reported modest to excellent adherence to maintenance sessions [ 11 , 13 , 56 , 65 ] . unfortunately , adherence was not reported separately for aa women , and three of the multi - institution rcts failed to report adherence to maintenance sessions altogether [ 13 , 44 ] . in four studies , which included a formal maintenance intervention in which only aa adults were enrolled , adherence to maintenance sessions was paltry [ 3 , 50 , 60 , 62 ] . in one of the single - site rcts , with a mixed race / ethnicity sample , women attended less than 60% of the offered sessions , and in another , total counseling contact time , for the extended care maintenance treatment groups , exceeded the a priori estimate of 8.7 hours ; results were not reported separately for aa women . the majority of studies , with a formal maintenance intervention , did not report adherence to specific maintenance activities such as self - monitoring of weight , dietary intake , or pa [ 3 , 11 , 13 , 44 , 50 , 60 , 62 , 65 ] . one study reported the mean number of food and activity diaries submitted throughout the intervention with submissions dropping from a mean of 15 ( sd 8) diaries during the intensive phase of the program to only 5 ( sd 9 ) diaries at 18-month follow - up . reported approximately 16 hours of journaling time for the extended care maintenance groups compared to just 10 hours in the self - directed group . the wlm trial reported that self - weighing was more frequent for aa women compared to caucasian women . however , like with session attendance , most studies with mixed race / ethnicity and/or sex sample [ 3 , 11 , 1315 , 50 , 56 ] failed to report adherence to maintenance components altogether or separately for aa women making it difficult to discern any disparities . the percentage of participants available for final assessment varied . five of the eight studies [ 11 , 13 , 44 , 65 ] with a mixed race / ethnicity and/or sex sample reported retention rates separately for aa women . retention rates for women randomized to an active intervention arm ranged from 48 to 97% for aa versus from 66 to 100% for caucasian . notably , retention rates were similar for the hpt , tohp , and tohpii studies while retention was lower for aa women in the tone and dpp trials . in the six trials enrolling only aa women [ 60 , 61 , 67 , 69 , 70 ] retention across treatment groups ranged from 63 to 92% . for the studies enrolling aa men and women [ 3 , 50 ] , retention for aa women ranged from 55% to 66% across treatments . table 2 reports the ratings for each quality category , a summary quality score , maintenance intervention characteristics , and % weight regain at 12 , 18 , and > 18 months ( calculated using available data ) . by summarizing the findings in this manner , we could more easily compare across interventions and determine if a particular set of maintenance intervention characteristics were more effective at promoting weight control for aa women . however , it is important to highlight the difficulty in comparing across studies given the heterogeneity in sample sizes , differences in duration of the maintenance phase , attrition rates , time interval in which weight outcomes were reported , and analysis approach ( intention to treat versus completers ) ; therefore , findings should be interpreted with some caution . with this acknowledgement , the 18 month weight outcomes were reported by a majority of the studies ( 14 of 17 ) [ 3 , 11 , 1315 , 44 , 50 , 6062 , 65 , 67 , 70 ] and will be used to make comparisons . at 18-month follow - up , % weight regain for aa women in studies with the highest quality ranking ( 11 points ) , enrolling only aa women or aa adults [ 3 , 50 ] , ranged from 0 to 49% . in studies with a lower quality ranking ( 10 or less ) , % weight regain at 18-months ranged from 15 to 138% . generally , the studies not focused on weight as an outcome [ 67 , 70 ] or lacking a formalized maintenance program [ 61 , 67 , 70 ] had the poorest outcomes . the highest ranking studies ( 11 points ) , enrolling both aa and caucasian women [ 11 , 44 ] , reported 18-month % weight regain ranging from 0 to 17% for aa women and from 12 to 17% for caucasian women . notably , in the tone study , % weight regain was lower for aa women in both weight treatment arms throughout the maintenance period and % weight regain was only slightly higher for aa compared to caucasian women in the dpp trial . in the lower ranking studies ( 10 points or less ) [ 1315 , 65 ] , 18-month % weight regain for aa women ranged from 19 to 89% and from 14 to 64% for caucasian women . the only instance , for which aa women had similar or lower 18-month % weight regain , was for those randomized to the self - directed maintenance arm of the tours study . however , the sample size of women randomized to this treatment was relatively small , and results should be interpreted with caution . cultural adaptations appeared to be an important component in multisite trials with a mixed race / ethnicity and gender sample as evidenced by less % weight regain for aa women in the tone , dpp , and wlm trials . inclusion of a formal maintenance program was associated with lower % weight regain for aa and caucasian women [ 3 , 11 , 1315 , 44 , 50 , 56 , 60 , 62 , 65 ] compared to programs without a formal maintenance intervention [ 16 , 61 , 67 , 70 ] . lastly , weight maintenance for the wlm trial was reported at 36-month follow - up only . both aa and caucasian women responded favorably to individualized sessions whereas aa women responded less favorably to the internet - based maintenance format . this paper reports on a systematic review of the behavioral lifestyle intervention literature published between 1990 and 2011 that reported weight outcomes , included a maintenance phase of at least six months , and enrolled or specifically targeted aa women . only 17 studies met the inclusion criteria , underscoring the limited research in this area . the studies reviewed differed in design , duration , and intensity of the maintenance interventions , sample size , and attrition rates , which led to the inevitable challenge of cross - study comparisons . generally , aa women lost less weight during the intensive weight loss phase and maintained a lower % of their weight loss compared to caucasian women in the behavioral lifestyle interventions reviewed [ 11 , 1315 , 44 , 65 ] . however , for studies reporting 18-month weight maintenance outcomes , in all but two [ 67 , 70 ] , aa women maintained some percentage of the weight loss achieved during the intensive weight loss phase . this is important given that small , sustained weight losses are associated with clinically meaningful health benefits [ 17 , 18 ] . the tone trial was the only study in which aa women had similar weight loss and maintenance as caucasian women . importantly , the sample of aa women in the tone trial was relatively small ( n = 46 ) , retention poorer than that for caucasian women , and women were older , overweight / obese , and hypertensive . the most remarkable finding was that the majority of studies failed to describe the specific strategies used in the delivery of the maintenance intervention , adherence to those strategies , and did not incorporate a maintenance phase process evaluation making it difficult to identify intervention characteristics associated with better weight control . also , many of the studies did not report a distinction between what similar or different behaviors were performed during the active and maintenance phase of the intervention . this may be due to the fact that often , the active intervention phase does not lead to sufficient weight losses to warrant an active maintenance phase . other than the wlm trial , a set amount of weight loss was not used as a criterion for participating in the maintenance phase of the other trials [ 3 , 11 , 1316 , 44 , 50 , 6062 , 65 , 67 , 69 , 70 ] . many individuals remain obese , even after one year of treatment , and continue to desire to lose . therefore , the maintenance phase , which is often arbitrarily set by the study investigators , may not truly reflect participants engaging in weight maintenance - type behaviors . furthermore , behaviors associated with successful weight management such as monitoring of food intake , limited intake of fast food , and sugar sweetened beverages , limited tv viewing , regular self - weighing , eating breakfast , and meal planning were not closely tracked or routinely reported or , when reported , distinctions were not made based on race / ethnicity and or sex . in a recent article , by barnes and kimbro , limiting fat intake , consuming less fast food , and monthly weighing were associated with better long - term weight control in aas who successfully reduced their weight by 10% and maintained the loss for at least one year . this further emphasizes that consistent documentation of these types of behaviors in the literature , and by race / ethnicity and sex when appropriate , can help to identify behaviors that lead to successful long - term weight control [ 83 , 84 ] . despite this significant caveat , we attempted to identify design components that influence the effectiveness of behavioral lifestyle interventions designed to promote weight maintenance specific to aa women . findings suggest that inclusion of cultural adaptations may result in more favorable weight maintenance outcomes for aa women and is consistent with the existing literature [ 3 , 45 ] . for example , in the multisite tone , wlm , and dpp trials , enrolling a mixed race / ethnicity and gender sample , inclusion of cultural adaptations resulted in superior weight outcomes compared to hpt , tohp , and tohp ii trials . however , it is hard to discern what specific cultural adaptations or combination of adaptations are most useful . what researchers consider to be cultural adaptations is often derived from qualitative studies [ 8587 ] , based on community input , based on researcher perception of sociocultural perspectives of aas , or , informal participant and community leader conversations . for example , aa women have cited inclusion of spirituality as a culturally salient adaptation to promote weight control . however , when tested empirically , in the trial by djuric et al . it may be that several rather than a single adaptation is necessary for a particular population or setting . however , assessment and comparison of a package of cultural adaptations presents an empirical challenge [ 91 , 92 ] . nonetheless , a clearer definition of what constitutes a cultural adaptation and a better understanding of the mechanistic relationship between cultural adaptations and the weight control process are needed . not surprising , inclusion of a formal maintenance program was largely associated with lower % weight regain for both aa and caucasian women [ 3 , 1315 , 44 , 50 , 56 , 60 , 62 , 65 ] compared to studies lacking a formalized program [ 16 , 61 , 67 , 70 ] . this finding is consistent with two other reviews investigating weight management in minority and nonminority populations [ 37 , 93 ] . however , in two of these trials [ 3 , 15 ] , aa women randomized to the self - directed or no contact maintenance arm had lower % weight regain at 18-month follow - up [ 3 , 15 ] . it is unclear why less contact resulted in better weight maintenance although the study authors speculated that the design , setting , or staffing or a failure to culturally adapt the maintenance intervention may have resulted in the outcomes observed . as for the more unique design features , aa and caucasian women randomized to motivational interviewing had lower % weight regain compared to women allocated to the attention control counseling , whereas internet delivery was less effective than personalized treatment , particularly for aa compared to caucasian women , in the wlm trial . a previous study found that randomization to internet maintenance resulted in greater weight regain as compared to in - person treatment . furthermore , at 12-month follow - up , 70% of internet participants reported that they would have preferred in - person contact , suggesting that a priori knowledge concerning an individual 's acceptability of treatment delivery mode may increase an intervention 's effectiveness . african american and caucasian women were more successful with weight maintenance when study participants were recruited for this purpose . it may be that aa women recruited for interventions where weight loss was secondary ( e.g. , walking intervention , sodium reduction ) [ 67 , 70 ] were less interested or motivated to lose weight . in a review of pretreatment predictors , self - motivation , general efficacy , and autonomy were all consistent pretreatment predictors of long - term weight success ( 1 year or more ) . therefore , designing an intervention that places the priority on weight loss throughout the trial ( i.e. , from recruitment to implementation and maintenance phases ) might improve weight outcomes . we included rcts , pilot rcts , and nonrandomized controlled and single group design trials . the small sample sizes of the nonrandomized trials and higher attrition rates in several of the studies may have introduced selection bias [ 3 , 11 , 13 , 14 , 50 , 61 , 67 , 69 , 70 ] . data obtained from study authors were for completers only which may have led to reporting bias [ 3 , 16 , 44 , 50 ] . similarly , many of the studies reported data from completers or persons with available follow - up data which could also lead to reporting bias . with a limited number of studies reporting racial / ethnic and sex differences , this paper did not fully capture differences in terms of the efficacy of behavioral lifestyle interventions on weight loss maintenance , among aa women . additional limitations include the exclusion of studies not published in english and of studies predating 1990 . we also intentionally did not explore the differential effects of food provision , surgery , or pharmaceutical intervention 's on weight loss maintenance in aa women . overall , our synthesis of the literature shows that aa women struggle unduly with both weight loss and maintenance . it may be that the inherent biology and social and environmental constraints of aa women , unfavorably impacts their adoption of these behaviors [ 45 , 96 ] . in terms of biology , studies suggest that aa women have several metabolic and physiologic factors that may account for their difficulty with weight management . these factors include less energy expenditure when sleeping , exercising , and in the resting state [ 97 , 98 ] ; alterations in fat oxidation consistent with increased fat storage ; higher steady - state ghrelin levels which leads to increased hunger ; lower pyy production after meal which could lessen satiety ; and decreased energy cost of activity following diet - induced weight loss . the biological aspects of weight regain are increasingly being studied and understood [ 102 , 103 ] . however , future studies should examine these biological factors within the context of weight loss / maintenance trials and test for racial / ethnic differences . in terms of aa women 's socioenvironment , several factors may hinder their adoption of behaviors shown to positively impact weight control . these factors include socioeconomic status [ 104 , 105 ] , availability and access to high quality foods [ 106 , 107 ] , availability and access to pa resources [ 108 , 109 ] , heightened exposure to unhealthy foods , neighborhood safety , stress , discrimination , and dysfunctional social networks . behavioral economic research suggests that these intertwining biological and contextual factors place eating and pa behaviors beyond an individual 's rational control . therefore , future research should evaluate how biologic and socioeconomic factors mediate diet and pa behavior change within a weight management trial . additionally , researchers might attempt to understand these pathways prior to developing interventions and utilize findings to inform future intervention design . the emergence of system - oriented and multilevel research will provide greater insight into the relational complexity of individual- and population - level factors affecting weight management . quantification of these factors ' influence on weight control and identification of the optimum level for intervention within subgroups of the population pose a complex set of research questions for investigators . cross - disciplinary , translational research addressing the intersection between individual behaviors , biology , social , and environmental contextual factors will allow researchers to more effectively design and evaluate interventions that simultaneously address multiple mechanisms of weight management . the ultimate goal of this research is to make the adoption of healthy eating and regular pa within everyday life the easier option . continued research that affords a more complete understanding of the complex connectedness of the behavioral , sociocultural , environmental , and biologic factors that lead to successful weight control in this population is warranted .

we performed a systematic review of the behavioral lifestyle intervention trials conducted in the united states published between 1990 and 2011 that included a maintenance phase of at least six months , to identify intervention features that promote weight loss maintenance in african american women . seventeen studies met the inclusion criteria . generally , african american women lost less weight during the intensive weight loss phase and maintained a lower % of their weight loss compared to caucasian women . the majority of studies failed to describe the specific strategies used in the delivery of the maintenance intervention , adherence to those strategies , and did not incorporate a maintenance phase process evaluation making it difficult to identify intervention characteristics associated with better weight loss maintenance . however , the inclusion of cultural adaptations , particularly in studies with a mixed ethnicity / race sample , resulted in less % weight regain for african american women . studies with a formal maintenance intervention and weight management as the primary intervention focus reported more positive weight maintenance outcomes for african american women . nonetheless , our results present both the difficulty in weight loss and maintenance experienced by african american women in behavioral lifestyle interventions .

since its introduction in 1996 by ulmsten et al , the tension - free vaginal tape ( tvt ) sling procedure has become a preferred treatment for female stress urinary incontinence ( sui ) worldwide as a result of its minimally invasive nature and high success rates . however , little is known about the long - term effects of the tvt sling on voiding and storage or on long - term patient satisfaction . although the tvt sling is placed with no or minimal tension under the midurethra , there appears to be a significant effect on voiding , and reported rates of urinary retention range from 4% to 20% . in addition , whereas preoperative urgency incontinence and overactive bladder symptoms have been reported to be resolved in 63.1 - 90.1% and 47.8 - 57.3% of patients after tvt , respectively , the rates of postoperative de novo urgency and urgency incontinence are reported to be 5 - 25% [ 7 - 10 ] after the procedure . postoperatively , a maximal urinary flow rate ( mfr ) < 10 ml / s and retention were found to compromise patient satisfaction , and de novo urgency was found to have a severe impact on quality of life in 14.5% of patients . the present report describes a 5-year follow - up study of female sui patients who underwent the tvt procedure . of 155 female patients who underwent the tvt procedure for sui in three institutions between march 1999 and june 2000 , the study retrospectively examined the clinical data of 134 who were followed up for more than 5 years . we previously reported long - term outcomes of the tvt procedure . in this study , pre - operative evaluation data available on all patients included detailed medical history , physical examination , neurological evaluation , uroflowmetry ( mms flowstar ) , post - void residual urine volume ( pvr ) as measured by using a bladder scan , a 3-day voiding diary , and a complete multichannel urodynamic study ( aquarius 120 filling cystometry ) . postoperative voiding symptoms were evaluated objectively as mfr and pvr , and storage symptoms were evaluated as urgency , frequency , and urgency incontinence by use of a 3-day voiding diary . the data from the uroflowmetry , pvr measurements , and 3-day voiding diary were re - analyzed . at 5 years postoperatively , the patients were asked to classify their global satisfaction level as very satisfied , satisfied , so - so , or dissatisfied . chicago , usa ) , and a p - value<0.05 was considered to indicate a significant difference . for statistical analysis , the correlation between patient satisfaction and 5-year postoperative voiding patterns was analyzed by using the chi - square test by percentage frequencies . the 134 study patients had a mean age of 52.39.3 ( range , 35 to 78 ) years , and the mean follow - up period was 67.04.6 ( range , 60 to 76 ) months . prior to surgery , 30 ( 22.4% ) women had symptoms of urgency and 25 ( 18.7% ) had symptoms of urgency incontinence . the preoperative sui symptom grades were 1 in 66 ( 47.8% ) patients , 2 in 63 ( 47.0% ) patients , and 3 in 5 ( 3.7% ) patients ( table 1 ) . among the patients , 7 ( 5.2% ) patients had undergone previous anti - incontinence surgery ( anterior vaginal wall sling for 4 , a raz operation for 2 , and bladder neck suspension for 1 ) , and 19 ( 14.2% ) the objective 5-year cure rate was 76.9% ( 103/134 ) , and the satisfaction rate was 86.6% ( 116/134 ) . the mean mfr decreased from 25.910.3 ml / s preoperatively to 20.48.6 ml / s at 1 month after the tvt procedure ( p<0.001 ) . however , at 1 year postoperatively , the mfr increased to 22.38.5 ml / s , and at 5 years postoperatively it recovered to 24.88.5 ml / s , which was similar to the preoperative flow rate ( p>0.05 ) . pvr increased significantly from 13.610.3 ml preoperatively to 24.432.9 ml at 1 month , 31.970.4 ml at 1 year , and 30.347.1 ml at 5 years postoperatively , with the three postoperative figures being not significantly different ( p>0.05 ) ( table 2 ) . of the 14 patients who showed a > 50% decrease in the mfr at 1 month postoperatively , 2 ( 14.3% ) still had a > 50% decrease in the mfr after 5 years . of the 29 patients who showed a 25 - 50% decrease in the mfr at 1 month postoperatively , 1 ( 3.4% ) had a > 50% decrease in the mfr at 5 years postoperatively . of the 38 patients who showed a < 25% reduction in the mfr at 1 month postoperatively , 2 ( 5.3% ) remained the same at 5 years postoperatively . a total of 63.2% ( 24/38 ) of patients with a < 25% decrease in the mfr at 1 month postoperatively showed < 25% decreases in the mfr at 5 years ( fig . 1 ) . the mean micturition frequency per day value at 5 years postoperatively was lower than the mean preoperative value ( 7.11.8 vs. 6.51.7 , p=0.004 ) ( table 2 ) . of the 43 patients with a preoperative micturition frequency per day of 8 , 13 ( 30.2% ) had a > 30% decrease , 4 ( 9.3% ) had an increase , and 26 ( 60.5% ) had no change after 5 years . of the patients with a preoperative micturition frequency per day of < 8 , 23 ( 34.3% ) had a decrease and 18 ( 26.9% ) had an increase after 5 years . surgery had no effect on the maximal voided volume ( 358.986.6 ml preoperatively , 324.969.7 ml at 1 month , 305.757.4 ml at 1 year , and 343.791.3 ml at 5 years postoperatively ; p=0.460 ) ( table 2 ) . of the 30 patients ( 22.4% ) with preoperative urgency , 14 ( 46.7% ) showed improvement at 5 years postoperatively . of the patients who did not show preoperative urgency , 16 ( 12.2% ) developed de novo urgency postoperatively . thus , the total number of patients with urgency remained similar over the 5 years . of the 25 ( 18.7% ) patients with preoperative urgency incontinence , 12 ( 48% ) showed improvement . of the patients who did not have preoperative urgency incontinence , 12 ( 11.0% ) developed de novo urgency incontinence postoperatively . the total number of patients with urgency incontinence at 5 years postoperatively was 24 ( 17.9% ) , which was similar to the preoperative figure ( fig . the proportion of patients reporting global satisfaction with the operation was 86.6% ( 116/134 ) at 5 years postoperatively . the satisfaction rate was found to correlate with the mfr , with a greater reduction in the mfr associated with greater dissatisfaction ( p=0.030 ) ( table 3 ) . in contrast , changes in the pvr over 5 years did not correlate with satisfaction . for the 43 patients with a preoperative micturition frequency per day of 8 , the satisfaction rates for those showing increased and decreased micturition frequency at 5 years postoperatively were 86.4% ( 19/22 ) and 93.6% ( 44/47 ) , respectively ( p>0.05 ) . the satisfaction rates for patients who developed de novo urgency and de novo urge incontinence were 68.8% ( 11/16 ) and 66.7% ( 8/12 ) , respectively , whereas the satisfaction rates for those with preoperative urgency and urgency incontinence that improved postoperatively were 78.6% ( 11/14 ) and 81.8% ( 9/11 ) , respectively . of the postoperative voiding parameters we examined , the most important factor affecting patient satisfaction was de novo urgency ( p=0.008 ) . the mean mfr decreased from 25.910.3 ml / s preoperatively to 20.48.6 ml / s at 1 month after the tvt procedure ( p<0.001 ) . however , at 1 year postoperatively , the mfr increased to 22.38.5 ml / s , and at 5 years postoperatively it recovered to 24.88.5 ml / s , which was similar to the preoperative flow rate ( p>0.05 ) . pvr increased significantly from 13.610.3 ml preoperatively to 24.432.9 ml at 1 month , 31.970.4 ml at 1 year , and 30.347.1 ml at 5 years postoperatively , with the three postoperative figures being not significantly different ( p>0.05 ) ( table 2 ) . of the 14 patients who showed a > 50% decrease in the mfr at 1 month postoperatively , 2 ( 14.3% ) still had a > 50% decrease in the mfr after 5 years . of the 29 patients who showed a 25 - 50% decrease in the mfr at 1 month postoperatively , 1 ( 3.4% ) had a > 50% decrease in the mfr at 5 years postoperatively . of the 38 patients who showed a < 25% reduction in the mfr at 1 month postoperatively , 2 ( 5.3% ) remained the same at 5 years postoperatively . a total of 63.2% ( 24/38 ) of patients with a < 25% decrease in the mfr at 1 month postoperatively showed < 25% decreases in the mfr at 5 years ( fig . the mean micturition frequency per day value at 5 years postoperatively was lower than the mean preoperative value ( 7.11.8 vs. 6.51.7 , p=0.004 ) ( table 2 ) . of the 43 patients with a preoperative micturition frequency per day of 8 , 13 ( 30.2% ) had a > 30% decrease , 4 ( 9.3% ) had an increase , and 26 ( 60.5% ) had no change after 5 years . of the patients with a preoperative micturition frequency per day of < 8 , 23 ( 34.3% ) had a decrease and 18 ( 26.9% ) had an increase after 5 years . surgery had no effect on the maximal voided volume ( 358.986.6 ml preoperatively , 324.969.7 ml at 1 month , 305.757.4 ml at 1 year , and 343.791.3 ml at 5 years postoperatively ; p=0.460 ) ( table 2 ) . of the 30 patients ( 22.4% ) with preoperative urgency , 14 ( 46.7% ) showed improvement at 5 years postoperatively . of the patients who did not show preoperative urgency , 16 ( 12.2% ) developed de novo urgency postoperatively . thus , the total number of patients with urgency remained similar over the 5 years . of the 25 ( 18.7% ) patients with preoperative urgency incontinence , 12 ( 48% ) showed improvement . of the patients who did not have preoperative urgency incontinence , 12 ( 11.0% ) developed de novo urgency incontinence postoperatively . the total number of patients with urgency incontinence at 5 years postoperatively was 24 ( 17.9% ) , which was similar to the preoperative figure ( fig . the proportion of patients reporting global satisfaction with the operation was 86.6% ( 116/134 ) at 5 years postoperatively . the satisfaction rate was found to correlate with the mfr , with a greater reduction in the mfr associated with greater dissatisfaction ( p=0.030 ) ( table 3 ) . in contrast , changes in the pvr over 5 years did not correlate with satisfaction . for the 43 patients with a preoperative micturition frequency per day of 8 , the satisfaction rates for those showing increased and decreased micturition frequency at 5 years postoperatively were 86.4% ( 19/22 ) and 93.6% ( 44/47 ) , respectively ( p>0.05 ) . the satisfaction rates for patients who developed de novo urgency and de novo urge incontinence were 68.8% ( 11/16 ) and 66.7% ( 8/12 ) , respectively , whereas the satisfaction rates for those with preoperative urgency and urgency incontinence that improved postoperatively were 78.6% ( 11/14 ) and 81.8% ( 9/11 ) , respectively . of the postoperative voiding parameters we examined , the most important factor affecting patient satisfaction was de novo urgency ( p=0.008 ) . although the tvt procedure is generally associated with universally excellent outcomes , concerns about postoperative voiding have recently been raised . dietz et al examined long - term mfr , pvr , and voiding symptoms after the tvt procedure . they reported improvement in voiding as reflected in reduced residual urine volumes , normalized voided volumes , and an increased mfr . the present study found that overall voiding function improved over time postoperatively , although it remained poor or worsened in some patients . of the patients showing a > 50% decrease in the mfr at 1 month postoperatively , 14.3% sustained this decrease to 5 years . thus , patients with a greatly reduced flow rate in the immediate postoperative period are more likely to retain this reduction in the long term than are patients with smaller reductions in the immediate postoperative flow rate . tvt procedures are known to have an obstructive effect on the urethra , which may affect voiding . our study showed that pvr increased during the follow - up period , which is consistent with previous reports . whereas the overall mfr was lower than preoperative levels at 1 month after the tvt procedure one hypothesis for this improvement in voiding patterns is that chronic obstruction results in progressive detrusor hypertrophy , allowing better emptying . others reported an increase in the prevalence of symptoms of urgency and urgency incontinence after the tvt procedure . in contrast , the present data indicate no change in the number of patients showing these symptoms over 5 years . we found that some patients with preoperative urgency or urgency incontinence showed postoperative improvement , whereas some patients developed de novo urgency or urgency incontinence . the prevalences of de novo urgency and urgency incontinence in our study ( 12.2% and 11.0% , respectively ) were within the ranges reported in previous studies [ 7 - 10 ] . we previously reported that a decline in the mfr to < 10 ml / s can compromise patient satisfaction with the tvt procedure . in the present study , we evaluated factors additional to the mfr to understand issues affecting global patient satisfaction in more detail . for patients with altered voiding patterns after surgery , satisfaction was impaired by a decreased mfr , de novo urgency , and de novo urgency incontinence . patient satisfaction was not found to correlate with increased pvr , micturition frequency , improved urgency , or improved urgency incontinence . the major factor affecting patient satisfaction was the development of de novo urgency ( p=0.007 ) . 0verall , 86.6% ( 116/134 ) of patients were satisfied with the operation , whereas only 80.0% ( 20/25 ) of those who developed de novo urgency were satisfied . a previous study reported on the assessment of 87 patients using a patient satisfaction questionnaire ( psq ) at 24 months postoperatively . that study found that 95.5% of patients were somewhat or completely satisfied with their progress , and that the majority of patients were satisfied despite the presence of irritating voiding symptoms . in contrast , the present study over 5 years showed that the development of de novo urgency reduced patient satisfaction . the present findings will likely assist clinicians in preoperative discussions with patients regarding potential changes in voiding patterns after surgery . a limitation with the present study was the lack of follow - up urodynamic data on patients who showed changed voiding patterns after the tvt procedure . a study by lin et al that included follow - up multi - channel urodynamic studies found that the mean preoperative values for average flow rate , mfr , pvr , and detrusor pressure at peak flow during voiding cystometry were not different from the values obtained at 3 , 6 , and 12 months after tvt surgery . any obstructive effect of the tvt procedure diminished over time in most patients , although a decrease in the mfr was sustained in some patients . concerning overactive bladder symptoms , therefore , the total number of patients with overactive bladder was maintained similarly during the 5 years .

purposewe assessed the long - term effects of the tension - free vaginal tape ( tvt ) procedure for stress urinary incontinence ( sui ) on voiding , storage , and patient satisfaction.materials and methodsthis retrospective study examined the records of 134 patients who had undergone the tvt procedure for sui and were followed up for more than 5 years . voiding function was evaluated by measuring maximum urinary flow rate ( mfr ) , post - void residual urine volume ( pvr ) , and storage function by using a voiding diary . patients were asked to describe their satisfaction with the operation.resultsmfr was lower at 1 month compared with the preoperative level , but had recovered to preoperative levels by 5 years postoperatively . however , some patients with > 50% , 25 - 50% , and < 25% decreases in the mfr at 1 month postoperatively showed a decrease in the mfr of > 50% at 5 years . pvr increased over the 5 postoperative years . of the patients with urgency and urgency incontinence , 43.8% and 48.1% showed improvement , respectively , whereas new patients developed postoperatively . thus , the total number of patients with urgency or urgency incontinence remained similar over the 5 years . in those with a changed voiding pattern , patient satisfaction was negatively affected by de novo urgency and urgency incontinence and decreased mfr.conclusionsany obstructive effect of the tvt procedure diminished over time in most patients , although a decrease in the mfr was sustained in some patients . with regard to overactive bladder symptoms , some patients were cured and some patients complained of de novo symptoms . the most major factor affecting patient satisfaction was de novo urgency .

a 55-year - old woman suddenly developed motor weakness of the right side and dysarthria 1 day prior to admission . on admission she had mild dysarthria , especially of the lingual components , hemiparesis of the right side ( motor grade iii ) , and right facial palsy . diffusion- and t2-weighted mr images obtained the same day revealed high signal intensities in the left pontine regions , consistent with an ischemic lesion ( fig . 1-a ) . the source images for both contrast - enhanced mr imaging and three - dimensional ( 3-d ) mr angiography displayed narrow segments of the basilar artery and double - lumen signs ( fig . these mr findings were consistent with a long intimal dissection or fenestration of the basilar artery . using virtual endoscopy ( rapidia ; infinitt , seoul , republic of korea ) , a diagnosis of an extreme fenestration because of the complete proximal and distal fusion shown by virtual endoscopy , we speculated that this finding was a basilar arterial fenestration of the true duplication type . two weeks after the onset of symptoms , hemiparesis of the right side improved ( motor grade v- ) and she was discharged . the reported incidence of fenestration of the basilar artery has varied from 0.3% in angiographic examinations to 5.26% in autopsy series.6,7 basilar artery fenestration is the most commonly observed form of fenestration of the cerebral arteries.7 the fenestration is usually short and occurs primarily in the proximal segment of the artery,8 with its clinical interest attributable to the possible association with aneurysms localized at the junctions of the fenestrated segments.9 there is known to be a defect in the media of the arterial wall that has been thought to be the predisposing factor for the prevalence of saccular aneurysms together with turbulent blood flow at the joining points.10 in the present case , the lesion was a true duplicationt - type fenestration , with each lumen sharing the medial wall . this finding was consistently observed from the joining portion of the two vertebral arteries to the top of the basilar artery , and each of the posterior cerebral arteries was branched from the adjoined portion of the basilar artery . there has been speculation about associations between either vertebrobasilar artery fenestrations and brainstem ischemia,10 or infarctions,5,11,12 although their relationships are controversial . among these reports , some authors showed basilar artery fenestration and associated brainstem infarctions.5,11 one study showed an arterial slit - type fenestration that was confirmed by virtual arterial endoscopy,11 and another study showed a true duplication - type that was confirmed by autopsy.5 the latter case was a true duplication - type , nonseparated form of fenestration that shared the medial wall that was confined to the caudal half portion of the basilar artery . the autopsy findings showed a partially organized thrombus that occluded both halves of the duplicated portion , suggesting possible hemodynamic disturbances and turbulent blood flow at the site of fenestration . another case of extreme fenestration of the basilar artery was reported in a retrospective mr angiographic study.4 in that study , 10 basilar artery fenestrations were observed from 600 brain reviews ( 1.7% ) and one case showed a total duplication . the shape of the fenestration was different in the proximal and distal parts of the basilar artery ; in the proximal two - thirds of the artery , a true duplication - type fenestration that shared the medial wall was noted , but in the distal one - third of the artery , the artery was fully separated , with each basilar artery terminating in a posterior cerebral artery . however , it was not mentioned whether this finding was combined with a brainstem infarction . because of the ' double - lumen sign ' in the present case , a basilar artery fenestration might be misinterpreted because it is known as a pathognomonic sign of arterial dissection.13 even though neuroimaging has improved in both resolution and 3-d capabilities , it remains difficult to diagnose fenestrations of intracranial arteries . this is consistent with the large discrepancy in the reported incidence of fenestration of the basilar artery between angiographic examination and autopsies.6,7 virtual endoscopy is a new diagnostic method that uses computer processing of 3-d image datasets , such as ct or mr imaging scans , to provide simulated visualization of specific organs.14 the ability to obtain an internal view of a vessel facilitates qualitative analyses and could improve the pretherapeutic visualization of vascular variants , stenosis , and obstruction of cerebral vessels.15 virtual endoscopy in patients with fenestration , as in the present case , has both improved the anatomical identification of vessels and prevented misdiagnoses of fenestration as either dissection or thrombus . in summary , we have reported a case of a patient with a duplication - type , nonseparated fenestration of the basilar artery and associated brainstem infarction . even though a causative linkage between arterial fenestration and cerebral ischemia was not established , a reliable diagnosis of the vascular condition in the affected lesion was important clinically . from this point of view , virtual arterial endoscopy may enhance diagnostic capabilities , especially in certain cases .

we present a case of a 55-year - old woman with pontine infarction and fenestration of the basilar artery that was demonstrated using virtual endoscopy . the patient had motor weakness of the right side with sudden onset . diffusion- and t2-weighted mr images revealed high signal intensities in the left pontine regions , and an mr angiographic examination showed the double - lumen sign of the basilar artery . an extensive duplication - type , nonseparated fenestration of the basilar artery was diagnosed by virtual endoscopy and craniocaudal communications with discrete openings were observed . we report a case of a patient with basilar artery fenestration and associated brainstem infarction .

anisakiasis is a human disease caused by the larval nematodes of anisakis species and pseudoterranova decipiens . dolphins or other marine mammals are hosts to adult anisakis worms , and their presence in coastal waters leads to increased larval contamination causing heavy infections in fish such as mackerel . human anisakiasis is peculiar because this parasite is not adapted to live in humans and infection is transitory . anisakis larvae will die within 14 days in a human body ; however , persistent inflammation and granuloma can remain . since anisakis larvae die in the course of time , enteric anisakiasis is generally alleviated by conservative treatment ; however , there have been reports of a worm body penetrating the intestinal wall into the abdominal cavity . a large study conducted in japan revealed that only 567 ( 4.5% ) of 12,586 anisakiasis cases involved the intestine . however , most patients with intestinal anisakiasis have needed hospitalization because it can cause intestinal obstruction , ileus , peritonitis or intestinal perforation [ 7 , 8 , 9 , 10 ] . the most effective treatment for intestinal anisakiasis has not yet been established . here , we present a case of intestinal anisakiasis that was diagnosed by abdominal ultrasound and was treated successfully with oral administration of prednisolone and olopatadine hydrochloride without hospitalization . a 43-year - old woman with a history of allergic rhinitis complained of intermittent severe abdominal pain and came to the clinic within 5 h after onset of this symptom . she and her brother had eaten the same food of pickled mackerel ( raw fish ) together 2 days before the onset , and her brother suffered from gastric anisakiasis ( fig . 1 ) , diagnosed the day before her onset . her blood pressure was 112/70 mmhg , pulse was 62 bpm , and body temperature was 36.4c . on physical examination , spontaneous pain and tenderness was noted on palpation over the periumbilical region ; however , muscle defense and rebound tenderness were not present . laboratory data showed slight increases in ige - rist level ( 236 iu / ml ; normal range < 170 iu / ml ) and the serum titer of anti - anisakis igg / a antibody ( 1.75 ; normal range < 1.50 ) . abdominal ultrasonography revealed marked local edema in a part of the jejunal wall ( 12 cm long , 9 mm thick ) , dilatation of the oral sided lumen with fluid accumulation , and ascites ( fig . 2 ) . in the edematous jejunal wall , mucosa and submucosal layers were thickened with a normal structure of layers . an anisakis body was not detected by ultrasonography . according to the clinical course and examinations , we advised her to seek hospital admission , but she rejected hospitalization and chose conservative ambulatory treatment as an outpatient . she was treated with oral administration of prednisolone 5 mg and olopatadine hydrochloride 10 mg daily . after starting the medication , the patient 's symptoms improved immediately . three days after the onset , ultrasound showed that the jejunal wall thickening was diminished , the luminal dilatation disappeared and ascites was reduced . prednisolone was administered for a total of 10 days and olopatadine hydrochloride for 14 days because anisakis can live for maximally 2 weeks , and its body could remain in the intestinal wall for several days even if it had died . two weeks later , the patient did not have any abdominal symptoms ; however , ultrasound showed a remaining unilateral wall thickening in the jejunum , approximately 3 cm long ( fig . therefore , olopatadine sulfate was administered for 28 additional days ( total , 42 days ) . six months after the treatment , the patient had no abdominal symptom , and the abdominal ultrasonography was normal . nausea , ascites and peritonitis are often observed , and intestinal obstruction and ileus have been seen in patients with intestinal anisakiasis . the annual incidence of intestinal anisakiasis in japan is estimated to be about 3.0 per 1 million people per year . patients with intestinal anisakiasis should be treated initially with conservative therapy . in several reports , patients have undergone fasting , fluid infusion , nasogastric tube or long tube insertion and received administration of antibiotics as conservative therapy [ 7 , 8 , 9 ] . however , in a previous report , the prevalence of ileus , perforation , bleeding and intussusception were 50 , 8 , 2 and 0.5% , respectively . the average lengths of stay were 9.6 and 13.2 days in non - surgical and surgical cases , respectively . human anisakiasis comprises mostly allergic and digestive symptoms , although extradigestive manifestations have also been observed occasionally . in the previous report , 5 patients with acute , established intestinal obstruction by anisakis simplex were treated with intravenous corticosteroids ( 6-methylprednisolone 1 mg / kg/24 h , 5 days ) , which resulted in a dramatic clinical and radiologic improvement in all 5 patients . in a case report , it was demonstrated that an antiallergic drug and oral administration of 5 mg prednisolone were useful as a conservative treatment in patients with gastric anisakiasis . the small intestine has a very thin wall and narrow lumen compared with the stomach , and thus , intestinal anisakiasis can cause severe complications . reported two patients with intestinal anisakiasis who needed surgical operation on the 23rd and 35th in the hospital . therefore , we decided that this patient needed strong and long - term ( over 5 weeks ) antiallergic therapy . subsequently , oral administration of prednisolone and olopatadine hydrochloride provided a dramatic improvement of her abdominal symptoms . in the outpatient clinic , a lower dose of prednisolone was easier to administer firstly and we administered antihistamine agent additionally . although there are some reports that show the effect of corticosteroid for anisakiasis , there has been no report of antihistamine for anisakiasis . a prospective study or reports of a larger number of patients is required to confirm the effect of antihistamine for anisakiasis . for this type of infection it has been reported that ultrasonography [ 14 , 15 ] and ct were useful for establishing a diagnosis . in this case , this case suggests that prompt administration of corticosteroid and an antiallergic agent for intestinal anisakiasis can improve abdominal symptoms immediately and help to avoid complications , such as ileus , peritonitis and perforation . however , the appropriate medication can not be predetermined because it might depend on patients individual condition . furthermore , ambulatory treatment should be limited to young or middle - aged patients without severe concomitant diseases , because complications could cause a critical situation in elderly patients . in conclusion , we reported that a case of intestinal anisakiasis was diagnosed by abdominal ultrasound and was successfully treated with administration of prednisolone and olopatadine hydrochloride without hospitalization . treatment with corticosteroid and an antiallergic agent could be an option for patients with intestinal anisakiasis .

the clinical characteristic of gastrointestinal anisakiasis is severe abdominal pain after eating raw fish . intestinal anisakiasis is more uncommon than gastric anisakiasis . most patients with intestinal anisakiasis need hospitalization because anisakiasis can cause intestinal obstruction , ileus , peritonitis or intestinal perforation . we report a case of intestinal anisakiasis . a 43-year - old woman presented with symptoms of intermittent abdominal pain 2 days after eating raw fish . her brother had eaten the same food and had been suffering from gastric anisakiasis . abdominal ultrasonography in this patient showed localized jejunal wall thickening with dilated lumen of proximal jejunum and ascites . according to the clinical course and examinations , she was diagnosed with intestinal anisakiasis . administration of prednisolone 5 mg / day and olopatadine hydrochloride 10 mg / day improved her symptoms quickly without hospitalization . prednisolone was administered for 10 days , and olopatadine hydrochloride was administered for a total of 6 weeks according to ultrasonographic findings . six months after the treatment , the abdominal ultrasonography demonstrated normal findings . this case demonstrates that ultrasonography was quite useful for the diagnosis and surveillance of intestinal anisakiasis . furthermore , treatment with corticosteroid and an antiallergic agent could be an option for patients with intestinal anisakiasis .

cardiovascular ( cv ) disease is a major comorbidity in patients with rheumatoid arthritis ( ra ) . ra patients experience two to three times the risk of cv disease compared with the non - ra population.1,2to accurately examine the important association of ra and cv disease , however , validated cv outcomes such as myocardial infarction ( mi ) , stroke ( ischemic or hemorrhagic ) , coronary artery bypass graft ( cabg ) surgery , and percutaneous coronary intervention ( pci)must be established . international classification of diseases , clinical modification , ninth revision ( icd-9-cm , hereafter referred to as icd-9 ) ; icd-9-procedure , and current procedural terminology ( cpt ) codes represent a convenient method of developing such cv outcomes . use of validated icd-9 , icd-9-procedure and cpt codes in electronic medical records ( emr ) can be convenient and requires fewer hours when compared to manual chart abstraction . however , there are some important gaps in the literature regarding validation of cv coding . first , previous studies report widely divergent positive predictive values ( ppvs ) for each individual icd-9 or cpt code . the ppvs for myocardial infarction icd-9 codes , for example , vary from 2.5 percent to 100 percent.3 second , prior literature tends to only offer estimates for the accuracy of administrative data for prevalent cv disease.4,5 when cv events are defined without explicitly defining a window of allowable time for the event around the date the code was assigned , it limits the validity of the administrative data as an outcome measure.68without an associated date , such coding algorithms are also unsuitable for time - to - event analysis . third , prior literature frequently does not give all of the details that might be desirable for different types of analysis . for example , many of the previously published manuscripts do not report the sensitivity or specificity of the codes used to identify incident disease 6,912 , and ppv has not been reported in the few studies regarding pci and cabg.10,1214fourth , all previously published works in this field 4,13,15 rely exclusively upon data from hospital discharges , which may introduce a significant bias by neglecting to account for cv events documented in outpatient records . to address these deficiencies , we evaluated the validity of icd-9 , icd-9 procedure , and cpt codes for cv events by examining a cohort of patients with well - established ra who were enrolled in the veterans affairs rheumatoid arthritis ( vara ) registry . using electronic medical record ( emrs ) of the vara patients , we determined the validity of cv - event codes within a six - month window using inpatient , outpatient , and inpatient + outpatient records . in contrast to prior investigation , our study focused on acute events ( those occurring within months of the billing code , as opposed to cv events occurring at any time ) , estimated sensitivity and specificity for the coding algorithms , evaluation of the value in using outpatient in addition to inpatient records , and clarification of the acceptable time window necessary to meet our case definition . the vara registry is a multicentered observational cohort at 11 va medical centers that has been fully described elsewhere.16,17 in brief , all patients with ra at participating sites are invited to enroll and provide informed consent for the collection of demographic and longitudinal clinical information as well as biologic samples ( sera , plasma , dna ) . for this study , we relied on a convenience sample of participants from enrollment sites for which we had direct access to medical records : dallas , texas ; denver , colo . ; and omaha , neb . we performed a validation study and utilized administrative data from inpatient and outpatient national va databases , based on clinical encounters from time of enrollment into vara ( initiated in 2003 ) until june 1 , 2010 or until the patient s most recent clinical encounter , whichever came first . we examined cv events subsequent to vara enrollment , as the additional attention of study coordinators , coinvestigators , and site principle investigators is likely to increase the odds of detecting cv events above the surveillance performed by primary care providers during usual care . these data include the following : age at time of registry enrollment ; gender ; age at time of death ; education ( years ) ; race / ethnicity ; tobacco use at time of enrollment ; rheumatoid factor ( rf ) antibody positivity ; anticyclic citrullinated protein ( anti - ccp);presence of rheumatoid nodules ; and ra disease duration from the time of diagnosis until enrollment in vara . in addition , 28-joint disease activity score ( das28 ) and health assessment questionnaire ( haq ) values were collected over the duration of enrollment in vara . das28 is a clinical measure of active disease and haq is a measure of disability used in clinical practice and for ra research . our study cohort included two populations : those with cv - event coding , which included icd-9 , icd-9-procedure , and cpt coding for mi ( 410.x , 411.x , 412.x , 413.x , 414.x , 429.2 , and v45.81 ) , stroke ( 433.11 , 433.91 , 434.91 , 435.x , 436.x , 437.9x , 438.x ) , cabg ( icd-9-procedure 36.1x ) , or pci ( icd-9-procedure 36.06 , 36.07 , 0.66 or cpt 92973 , 92980 , 92995 ) ; and those without cv - event coding ( see figure 1 ) . these codes were selected based on prior work for mi,4 stroke,4 cabg,13 and pci.13 individuals with and without coding for mi , stroke , cabg , and pci were randomly chosen based on a randomly assigned numeric identification code from the list of vara participants for medical chart review . cv - related codes were drawn from the inpatient and outpatient files of the va s corporate franchise data center , a national centralized computer - processing center . samples of 110 and 60 individuals for cv - related and no - cv - related codes were chosen a priori . prior work suggests that codes appearing in nonprimary positions ( i.e. , codes that were not the primary reason for hospitalization or outpatient visits ) often represent false positives.18 for this reason , we limited the coding position to the first position for in - hospital records , hospital discharge records , and outpatient records for icd-9 diagnostic codes ( mi , stroke ) . for icd-9-procedure and cpt codes ( cabg , pci ) , which were not anticipated to demonstrate high false positive rates , we accepted procedural codes in any position . the entire medical records of those individuals randomly chosen for medical chart review ( both in the cv - event coding and in the no - cv - event coding populations ) were abstracted using a structured chart abstraction instrument for mi , stroke , pci , and cabg . those with cv - event coding were specifically evaluated for cv events within a six - month window of the assignment of the code ( three months on either side of the date on which the code was entered into the emr ) . since many analyses ( such as time - to - event ) require that the timing of events be known , our case definition included this specified allowable time frame to characterize valid events . because these events may not be the patient s initial cv event ( i.e. , events occurring prior to introduction of the emr may not be captured ) , it is not entirely accurate to describe them as incident events.19 thus , we defined the first event since the introduction of the emr data as acute events in contrast to prevalent events . the case definition of a cv event required documentation by a clinician of a mi , stroke , pci , or cabg in progress notes , discharge summaries , or procedure notes . for mi and strokes , we accepted events described as likely , but did not accept those characterized as possible . for those in the no - cv - event population , the patient s entire medical record was reviewed for the presence of a cv event , from the time of enrollment into vara until june 1 , 2010 . the ppv was then determined for each individual diagnostic or procedural code . to assess a more sensitive approach , a composite coding algorithm was then created consisting of the sum of the individual codes with a ppv 50 percent for each clinical condition . the negative predictive value ( npv ) , sensitivity , and specificity of each of the individual codes and composite codes were also calculated . in order to account for the bias that may have resulted from our sampling method ( choosing from those who had cv coding and those who did not ) , we utilized the method described by weiner et al.20 to calculate sensitivity and specificity . students t - test and chi - square were used to determine differences in baseline variables , as appropriate . however the funding sources had no role in the study design ; in the collection , analysis and interpretation of data ; or in the writing of the report . the views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the u.s . this work was approved by the internal review board of the university of colorado and the veterans affairs medical center in denver , colorado . additionally , this project was approved by the scientific and ethics approval committee for the vara registry . the vara registry is a multicentered observational cohort at 11 va medical centers that has been fully described elsewhere.16,17 in brief , all patients with ra at participating sites are invited to enroll and provide informed consent for the collection of demographic and longitudinal clinical information as well as biologic samples ( sera , plasma , dna ) . for this study , we relied on a convenience sample of participants from enrollment sites for which we had direct access to medical records : dallas , texas ; denver , colo . ; and omaha , neb . we performed a validation study and utilized administrative data from inpatient and outpatient national va databases , based on clinical encounters from time of enrollment into vara ( initiated in 2003 ) until june 1 , 2010 or until the patient s most recent clinical encounter , whichever came first . we examined cv events subsequent to vara enrollment , as the additional attention of study coordinators , coinvestigators , and site principle investigators is likely to increase the odds of detecting cv events above the surveillance performed by primary care providers during usual care . participants in vara have baseline demographic information collected upon enrollment into the registry . these data include the following : age at time of registry enrollment ; gender ; age at time of death ; education ( years ) ; race / ethnicity ; tobacco use at time of enrollment ; rheumatoid factor ( rf ) antibody positivity ; anticyclic citrullinated protein ( anti - ccp);presence of rheumatoid nodules ; and ra disease duration from the time of diagnosis until enrollment in vara . in addition , 28-joint disease activity score ( das28 ) and health assessment questionnaire ( haq ) values were collected over the duration of enrollment in vara . das28 is a clinical measure of active disease and haq is a measure of disability used in clinical practice and for ra research . our study cohort included two populations : those with cv - event coding , which included icd-9 , icd-9-procedure , and cpt coding for mi ( 410.x , 411.x , 412.x , 413.x , 414.x , 429.2 , and v45.81 ) , stroke ( 433.11 , 433.91 , 434.91 , 435.x , 436.x , 437.9x , 438.x ) , cabg ( icd-9-procedure 36.1x ) , or pci ( icd-9-procedure 36.06 , 36.07 , 0.66 or cpt 92973 , 92980 , 92995 ) ; and those without cv - event coding ( see figure 1 ) . these codes were selected based on prior work for mi,4 stroke,4 cabg,13 and pci.13 individuals with and without coding for mi , stroke , cabg , and pci were randomly chosen based on a randomly assigned numeric identification code from the list of vara participants for medical chart review . cv - related codes were drawn from the inpatient and outpatient files of the va s corporate franchise data center , a national centralized computer - processing center . samples of 110 and 60 individuals for cv - related and no - cv - related codes were chosen a priori . prior work suggests that codes appearing in nonprimary positions ( i.e. , codes that were not the primary reason for hospitalization or outpatient visits ) often represent false positives.18 for this reason , we limited the coding position to the first position for in - hospital records , hospital discharge records , and outpatient records for icd-9 diagnostic codes ( mi , stroke ) . for icd-9-procedure and cpt codes ( cabg , pci ) , which were not anticipated to demonstrate high false positive rates , we accepted procedural codes in any position . the entire medical records of those individuals randomly chosen for medical chart review ( both in the cv - event coding and in the no - cv - event coding populations ) were abstracted using a structured chart abstraction instrument for mi , stroke , pci , and cabg . those with cv - event coding were specifically evaluated for cv events within a six - month window of the assignment of the code ( three months on either side of the date on which the code was entered into the emr ) . since many analyses ( such as time - to - event ) require that the timing of events be known , our case definition included this specified allowable time frame to characterize valid events . because these events may not be the patient s initial cv event ( i.e. , events occurring prior to introduction of the emr may not be captured ) , it is not entirely accurate to describe them as incident events.19 thus , we defined the first event since the introduction of the emr data as acute events in contrast to prevalent events . the case definition of a cv event required documentation by a clinician of a mi , stroke , pci , or cabg in progress notes , discharge summaries , or procedure notes . for mi and strokes , we accepted events described as likely , but did not accept those characterized as possible . for those in the no - cv - event population , the patient s entire medical record was reviewed for the presence of a cv event , from the time of enrollment into vara until june 1 , 2010 . the ppv was then determined for each individual diagnostic or procedural code . to assess a more sensitive approach , a composite coding algorithm was then created consisting of the sum of the individual codes with a ppv 50 percent for each clinical condition . the negative predictive value ( npv ) , sensitivity , and specificity of each of the individual codes and composite codes were also calculated . in order to account for the bias that may have resulted from our sampling method ( choosing from those who had cv coding and those who did not ) , we utilized the method described by weiner et al.20 to calculate sensitivity and specificity . students t - test and chi - square were used to determine differences in baseline variables , as appropriate . however the funding sources had no role in the study design ; in the collection , analysis and interpretation of data ; or in the writing of the report . the views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the u.s . this work was approved by the internal review board of the university of colorado and the veterans affairs medical center in denver , colorado . additionally , this project was approved by the scientific and ethics approval committee for the vara registry . of 862 individuals available for analysis , 229 ( 27 percent ) were coded for a cv - related event in the first position for icd-9 or in any position for icd-9-procedure or cpt ; 633 ( 73 percent ) had no cv - related event coding ( see figure 1 ) . of the cv - event coded cohort , the medical records of 107 of the intended 110 individuals were abstracted for acute cv - related coding ; of those without cv - event coding , the medical records of 60 individuals were abstracted for cv - related events ( for further details of those without cv - event codes , see supplemental table 1 ) . there were significant differences in age , gender , reported race / ethnicity , and ra disease duration between those coded for a cv - related event and those not coded for a cv - related event ( see table 1 ) . ra patients with a coded cv event were older , more often male , more likely to report caucasian race , and had a longer ra disease duration then those without a coded cv event . additionally , we examined the baseline differences between individuals at the three sites ( dallas , texas ; denver , colo . ; and omaha , neb . ) . significant differences were found in mean education , race / ethnicity , mean disease duration , and rf positivity ( see supplemental table 2 ) . there were significant differences in age at enrollment ( abstracted individuals 64.76 years versus 62.78 years in those not abstracted , p - value 0.041 ) and in rf positivity ( 92 percent versus 86 percent positive , p - value 0.041 ) . see supplemental table 3 for full details . the mean difference between an actual event and the cv - related code being assessed was 9.5 days ( sd 20.2 days ) . the ppv and its 95 percent confidence interval , npv , sensitivity and specificity of each individual code for both inpatient and outpatient records are presented in table 2 , while those of the inpatient records only are in table 3 and those of the outpatient records only are in table 4 . in general , there was a wide range of the ppv of the individual codes ranged ( from 0100% ) . after this initial ppv of the individual codes were determined , those with a ppv 50 percent were included in the composite coding algorithms . a summary of the composite codes for the total cohort , inpatient records only , and outpatient records only , may be found in table 5 . in comparing the composite codes between inpatient + outpatient records , inpatient only , and outpatient only , the inpatient only records had the highest ppv and specificity . however , the inpatient records in general had a lower sensitivity than that of either inpatient + outpatient or outpatient alone records . for example , the mi composite coding algorithm ( 410.x ) had a ppv of 0.87 for the inpatient + outpatient , 1.0 for the inpatient alone , and 0.75 for outpatient alone , while the sensitivity was 0.94 , 0.89 , and 0.93 , respectively . with regards to the any cv code composite , which consists of the mi composite , stroke composite , cabg composite , and pci composite , the inpatient + outpatient had a ppv of 0.88 , npv of 0.97 , sensitivity of 0.91 and specificity of 0.96 . the values for the inpatient and outpatient records , respectively , were the following : ppv 1.0 and 0.70 , npv 0.97 and 0.97 , sensitivity 0.87 and 0.88 , and specificity 1.0 and 0.90 . in general , the procedure codes ( icd-9 procedure and cpt ) exceeded the icd-9 diagnostic codes for ppv . using the administrative records of ra patients enrolled in the vara registry , we evaluated the accuracy of icd-9 , icd-9-procedure , and cpt codes for identifying acute cv events within a six - month window in inpatient , outpatient and inpatient + outpatient records . when the composite coding for any cv - related event is utilized for both inpatient and outpatient records , the ppv is 0.88 with an npv of 0.97.there were significant baseline differences between the event and no - event groups in age , gender , reported race / ethnicity , and average ra disease duration . this is not unexpected , as classic risk factors include age and gender,1 and longer ra disease duration has been associated with increased risk of cv events.2 as expected , the cv composite codes for the combined inpatient and outpatient records had a ppv between that of the inpatient and outpatient only records , as did sensitivity and specificity . qualitative review of the medical records revealed that the relatively low sensitivity for the composite cv code for inpatient records is likely due to the number of mi events not being captured by icd-9 codes in the va medical records ; frequently , this is because patients were evaluated and treated at outside facilities , and follow - up occurred outside of the six - month window . issue ( care received under two or more health care systems ) is likely more problematic for emergent conditions , such as mi . we attempted to evaluate this potential limitation by performing an outpatient and outpatient + inpatient records analysis , where a patient would likely report an event to that patient s primary care provider . stroke composite coding for outpatient records had a ppv of 0.55 , but a sensitivity of 1.0 . we hypothesize that the relatively low ppv is due to frequent assignment of stroke as either a working diagnosis during emergent events ( such as the emergency room visit , which does not create an inpatient record unless the patient is actually admitted ) or for follow up for neurologic sequelae of a stroke . for example , a patient presenting to the emergency room with altered mental status may be initially coded as stroke , but subsequent evaluations , including outpatient evaluations , may render a different diagnosis . composite coding for any cv event achieved acceptable levels for ppv for inpatient + outpatient ( 0.88 ) , inpatient ( 1.0 ) , and outpatient settings ( 0.70 ) . while certain circumstances may require the identification of specific types of events ( mi , stroke , cabg , pci ) as an outcome event , there are many situations that warrant the use of composite coding ( which would produce a higher event rate and allow researchers greater power in detecting differences in cv events between therapeutic approaches , for example ) . however , the specific types of records ( inpatient + outpatient , inpatient only or outpatient only ) should be determined by the researchers needs with regards to sensitivity and specificity . a number of investigators have evaluated the accuracy of administrative coding for cv conditions , including the following : mi;3,9,18,2124 stroke and transient ischemic attack;68,11,2528 cabg surgery;10,1214 and various procedures within the spectrum of pci , including percutaneous transluminal coronary angioplasty ( ptca ) and cardiac catheterization.10,1214 due to the wide range of differences in populations and methodologies used , these studies demonstrate an extraordinarily wide range of reported ppv for each individual icd-9 or cpt code . for example , the ppv for myocardial infarction icd-9 codes range from 2.5 to 100 percent,3 and there is a similarly broad range for incident stroke ( < 1 to 94 percent).8,11 furthermore , very little has been published regarding the sensitivity and specificity of these coding algorithms for the identification of cv disease ; and , of the studies that have reported the sensitivity and specificity , there is also a wide range . while some of these studies examine incident events68 most of these do not specify a specific window of allowable time for the event , making these results less useful for time - to - event analyses . also of importance , all previously mentioned articles rely exclusively upon data from hospital discharges , which may introduce a significant bias by neglecting to account for cv events documented in outpatient records . this may be particularly relevant to comprehensive health care systems or accountable care organizations , where a substantial proportion of patients live in rural locations . these patients may be managed acutely at a facility that is not within the health care system , and thereby avert detection in administrative data generated solely by the inpatient record . it is therefore vitally important in this setting to capture these events in the outpatient record , which may contain evidence of subacute care for the selected conditions delivered by the patients primary care physicians or other outpatient staff . depending upon the focus of the research , prevalent , incident , or acute coding algorithms may be the most appropriate for an individual researcher s use . we assessed the ppv , npv , sensitivity , and specificity of acute cv event coding within a specified window of time in inpatient , outpatient , and inpatient + outpatient settings to evaluate our particular codings for time - to - event and other analyses where timing is an essential element . administrative data are used for a variety of activities outside of their original billing - related purpose , including assessing quality of care , monitoring health care utilization , and performing health services research ( hsr).14 while these are all important applications , the value of administrative data for these uses depends upon the validity of the codes for the particular application . a number of factors may influence the validity of administrative data including the particular disease state under examination , whether prevalent versus incident disease is being assessed , whether the data are restricted to inpatient sources or whether outpatient data are incorporated , and the health care system in which the data are gathered ( a self - contained health care system versus fee - for - service insurance ) . as is the case with all studies , our investigation includes a number of strengths and limitations . notably , our study was conducted within the va using a well - defined ra population , which will allow application to other patient groups with ra . the va computerized patient record system ( cprs ) is an integrated systemwide emr that allows comprehensive capture of health care events for patients receiving care within the va system . however , these results may not be generalizable to other health care settings , and these coding algorithms will need to be validated in other systems . supporting the generalization or our results , however , is work demonstrating similar clinical characteristics for ra patients from our study cohort compared to more traditional female - predominant community - based cohorts.29 limitations include the fact that our study was performed in a cohort of subjects with ra . while clinicians or coders may exhibit a bias in assigning cv codes to ra patients when compared to non - ra populations , we believe that such a scenario is unlikely . as our subjects are registry participants , they may be systematically different from patients who do not participate in registries . additionally , we did not independently survey patients directly to determine whether they had experienced a cv event . such an approach might have identified more cv events ( and thus , diminished the observed sensitivity further ) ; however , patient surveys might have also resulted in substantially more misclassification of events ( e.g. , classification of angina as mi or of tias as stroke ) , when compared with clinician documentation , and resulted in a lower specificity . additionally , we chose to assess only the primary coding positions for both inpatient and outpatient records . we did not perform a sample size calculation , but rather chose our numbers to abstract a priori . these statistical choices may be improved upon when this study is repeated in other health care systems . also , it is important to note that because the va health care system pays for veterans care that is delivered outside the va system , policies are in place to reduce cost by aggressively seeking out veterans hospitalized outside the va and transferring them to va facilities . this likely improves the capture of non - va delivered care and , thus , is anticipated to improve the sensitivity of our approach . in conclusion , we report the ppv , npv , sensitivity , and specificity of cv - event coding algorithms in a well - defined ra population of u.s . our work has evaluated the proposed composite algorithms for identifying acute cv events in inpatient , outpatient , and inpatient + outpatient settings . the ability to detect incident cv events using reliable codes may further researchers efforts in health services research , guideline implementation , and health care utilization . these data will enable researchers to make a decision about whether the coding algorithms fit their requirements for use in their research . by providing ppv , npv , sensitivity , and specificity for different patient settings , our study provides investigators with reliable composite coding for mi , stroke , pci , and cabg in inpatient , outpatient , and inpatient + outpatient settings . future avenues of research include reproduction of our study in other health care settings , reproduction of our study in other patient populations , and evaluation of hybrid results of individuals with no cardiovascular related coding notes : tn= true negative ; mi= myocardial infarction ; cabg = coronary artery bypass graft ; pci = percutaneous coronary intervention . comparison of characteristics at the three sites contained in the cohort notes : sd = standard deviation ; age = age at the time of registry enrollment ; avg . disease duration= average rheumatoid arthritis disease duration at time of registry enrollment ; rf= rheumatoid factor ; anti - ccp= anti - citrullinated protein antibody ; rheum . nodules= rheumatoid nodules ; note : p - value represents test of differences between event and no event populations . characteristics abstracted individuals versus those not abstracted notes : sd = standard deviation ; age = age at the time of registry enrollment ; avg . disease duration= average rheumatoid arthritis disease duration at time of registry enrollment ; rf= rheumatoid factor ; anti - ccp= anti - citrullinated protein antibody ; rheum . nodules= rheumatoid nodules ; note : p - value represents test of differences between event and no event populations .

objective : to assess the accuracy of international classification of diseases , ninth revision , and current procedural terminology codes for identifying cardiovascular ( cv ) events ( myocardial infarction [ mi ] , stroke , coronary artery bypass graft [ cabg ] , and percutaneous coronary intervention [ pci ] ) in enrollees of the veterans affairs rheumatoid arthritis ( vara ) registry.design:we performed a validation study from vara enrollment until 6/1/2010 to compare the accuracy of cv events in those with and without cv - event coding in inpatient and outpatient records to evaluate for cv events + / 3 months of the coding . the positive predictive value ( ppv ) was calculated , and codes with a ppv 50% were included in a composite coding algorithm.results:we evaluated 107 individuals for 21 cv - event codes and 60 individuals without cv - event coding . the ppv varied between 0100% . composite coding algorithms ppv ranged from 70100%.conclusions : validation of these algorithms allows for identification of acute cv events with known accuracy . the sensitivity and ppv of coding algorithms for cabg and pci exceed that of stroke and mi .

primary care practices throughout the netherlands that were not involved in other diabetes care improvement programs were block randomized to intervention ( 26 practices ) or the control group ( 29 practices ) . the number of pcps working in each practice and the presence of a practice nurse before intervention were taken into account before randomization . the intervention , also described elsewhere ( 5 ) , consisted of 1 ) diabetes consultation hour run by a practice nurse , 2 ) a cdss that contained a diagnostic and treatment algorithm based on the dutch type 2 diabetes guidelines ( 6 ) and provided patient - specific treatment advice , 3 ) a recall system , and 4 ) feedback every 3 months regarding the percentage of patients meeting the treatment targets ( cessation of smoking , a1c < 7% , systolic blood pressure < 140 mmhg , total cholesterol < 4.5 mmol / l , ldl cholesterol < 2.5 mmol / l , and bmi < 27 kg / m ) on both the practice and the patient levels ( 6 ) . the pcps were advised that they should prescribe new medication and refer patients if necessary . the control group continued with the same diabetes care that they had received before entering the study , which means that diabetes care was provided by the pcp or by a practice nurse under pcp responsibility . the university medical center utrecht ethics committee approved the study , and patients provided written consent . from the 171,821 registered patients , all type 2 diabetic patients were identified . patients who had a short life expectancy , were unable to visit the primary care practice , or were receiving diabetes treatment from a medical specialist were excluded . initially , 3,979 patients were eligible ( 2,136 in the control group and 1,843 in the intervention group ) , but 548 subjects refused to participate ( 409 control and 139 intervention subjects ) , and an additional 40 ( 35 control and 5 intervention subjects ) failed to participate for unknown reasons ( for both groups , p < 0.05 ) . the final , mainly caucasian , study population consisted of 3,391 patients ( 1,692 control and 1,699 intervention ) . after 1 year , 2,841 patients ( 1,389 control and 1,452 intervention ) completed a follow - up examination ; 187 patients ( 115 control and 72 intervention ) refused to participate in the final measurements , and 13 others ( 12 control and 1 intervention ) failed to show for unknown reasons ( for both groups , p < 0.05 ) . the groups did not differ with regard to the number of patients who died , moved , became terminally ill , or were referred to a specialist . between march 2005 and august 2007 patients who did not show received one reminder . in the cdss , age , sex , ethnicity , blood pressure was measured according to a standard operating procedure . the 10-year coronary heart disease ( chd ) risk estimate , as established by the uk prospective diabetes study ( ukpds ) ( 7 ) , was calculated using the above - mentioned variables , excluding ldl cholesterol . secondary outcomes were the 1-year difference in the 10-year ukpds chd risk estimate and the percentage of patients that reached a1c 7% , systolic blood pressure 140 mmhg , total cholesterol 4.5 mmol / l , and ldl cholesterol 2.5 mmol / l ( 6 ) . we performed intention - to - treat analyses with baseline values carried forward in the case of missing values . to correct for clustering at the practice level , generalized linear models were used , and after clustering had been taken into account , a 0.3% difference in a1c and a 2% difference in ukpds chd risk could be detected with 90% power ( = 0.05 ) , with at least 1,080 patients in each treatment arm . there were more solo practices ( 58 vs. 50% ) and fewer duo practices ( 24 vs. 30% ) compared with national data ( 8) . the mean sd age ( 46.8 7.4 years ) of the participating pcps was comparable with the mean dutch pcp age ( 8) . baseline characteristics of the intervention and control groups were comparable , except for smoking status , history of cardiovascular disease , and hdl cholesterol levels ( table 1 ) . the difference in a1c change between the two groups was not significant . systolic and diastolic blood pressure and total and ldl cholesterol improved significantly more in the intervention group . as a result , after 1 year , significantly more patients in the intervention group reached the treatment targets , with 18.9% of the patients meting all treatment targets ( table 1 ) . the dcp is the first pragmatic diabetes care intervention using a cdss that improves patient outcome . as recommended by the national institute of clinical excellence , we calculated the 10-year ukpds chd risk estimate for all subjects and used this measurement as a determinant of clinical care . recently , the action in diabetes and vascular disease ( advance ) study showed that a1c reduction does not prevent chd ( 9 ) . this result indicates that we should focus on the patient 's total cardiovascular risk profile . our study showed no difference in a1c change between the two treatment arms , but the dcp led to improved diabetes care , which is shown by a 1.4% higher reduction in 10-year chd risk estimate in the intervention group . the cdss structures diabetes care , which may lead to improvements in the process of care ( 1 ) . in addition , the dcp added a practice nurse who acted as a case manager and provided periodic feedback . practices were self - selected , which may suggest a special interest of the pcp in improving diabetes care . this could be the reason why baseline values of a1c , blood pressure , and cholesterol were lower than those of most other dutch primary care diabetes studies ( 10 ) . because mean a1c at baseline was almost at the treatment target , there was little room for improvement . this could be explained by overly strict targets ( 11 ) , physicians inert in prescribing more medications ( 4 ) , or noncompliant patients ( 12 ) . whether the effects of the dcp will sustain

objective the diabetes care protocol combines task delegation ( a practice nurse ) , computerized decision support , and feedback every 3 months . we studied the effect of the diabetes care protocol on a1c and cardiovascular risk factors in type 2 diabetic patients in primary care.research design and methods in a cluster randomized trial , mean changes in cardiovascular risk factors between the intervention and control groups after 1 year were calculated by generalized linear models.resultsthroughout the netherlands , 26 intervention practices included 1,699 patients and 29 control practices 1,692 patients . the difference in a1c change was not significant , whereas total cholesterol , ldl cholesterol , and blood pressure improved significantly more in the intervention group . the 10-year coronary heart disease risk estimate of the uk prospective diabetes study improved 1.4% more in the intervention group.conclusionsdelegation of routine diabetes care to a practice nurse combined with computerized decision support and feedback did not improve a1c but reduced cardiovascular risk in type 2 diabetes patients .

a number of multiresistant bacterial pathogens inactivate antibiotics by producing znii - dependent -lactamases . we show that metal uptake leading to an active dinuclear enzyme in the periplasmic space of gram - negative bacteria is ensured by a cysteine residue , an unusual metal ligand in oxidizing environments . kinetic , structural and affinity data show that such znii - cys interaction is an adaptive trait tuning the metal binding affinity , thus enabling antibiotic resistance at restrictive znii concentrations .

innovative technologies , such as diode lasers , have provided considerable benefit to dental patients and dentists . in addition , the role of lasers in dentistry is well - established in both the conservative and surgical management of oral diseases . the diode laser is a semiconductor that uses solid - state elements , such as gallium , arsenide , aluminum , and indium , to change electrical energy into light energy . the light energy from the diode is greatly absorbed by the soft tissue and poorly absorbed by the teeth and bones . diode lasers are useful for oral soft tissue surgical procedures because their specific wavelength ( 810 - 980 nm ) is absorbed not only by water ( although less so than the carbon dioxide laser wavelength ) , but also by other chromophores , such as melanin , and in particular , oxyhemoglobin . moreover , the exclusive use of this laser by contact or at an extremely close distance avoids damage , due to beam escape, in an open field , which makes it much safer than other laser sources . in addition , diode lasers have the ability to cut the tissue to perform coagulation and hemostasis , and have a higher tissue ablation capacity and enough bleeding hemostatic properties compared to most laser systems . clinical experience suggests some advantages of the laser over scalpel surgical procedures on oral tissues . these advantages include greater precision , a relatively bloodless surgical and postsurgical course , sterilization of the surgical area , minimal swelling and scarring , coagulation , vaporization , cutting , minimal or no suturing , and less or no postsurgical pain . studies have shown that laser surgery is widely used for oral lesions , such as , simple soft tissue surgery ( frenectomy , epulis , gingival contouring plasty , etc . ) , vascular lesions ( hemangiomas , telangiectasias , etc . ) , pigmented lesions ( gingival pigmentations ) , white oral lesions ( oral leukoplakia , oral lichen planus , etc . ) , and low - level laser therapy ( lllt ) in vesiculobullous lesions ( mucous membrane pemphigoid ) . the purpose of this study is to determine the efficacy and safety of the 810-nm diode laser for treatment of oral soft tissue lesions or diseases , and to answer the question of whether the 810-nm diode laser is the best choice in oral soft tissue surgery . a total of 27 patients who had different benign oral lesions were treated with the 810-nm diode laser at the oral and maxillofacial surgery department , faculty of dentistry , ankara university , between the years 2009 and 2011 . an informed written consent form was obtained from all participating adults and from parents or legal guardians for minors or incapacitated adults ( 19 females and eight males , 10 smokers and 17 nonsmokers , 21 to 72 years of age ) in accordance with the declaration of helsinki . all patients had previously taken conventional medical treatment procedures ( local corticosteroid , beta carotene , vitamin e , fluocinonide , triamcinolone , lllt , etc . ) except the patients having epulis fissuratum or pathological frenulum . therefore , the diode laser treatment was selected as an alternative treatment option for these patients . patients with any systemic diseases were excluded from the study , and all operations were performed by the same surgical team . the diagnosis of all lesions or disorders was confirmed by histopathological examination , especially in patients who suffered from white , vesiculobullous , and pyogenic granuloma lesions . all treatments were performed with the patients under local anesthesia and on an outpatient basis . treatment was carried out using 12 different settings of the medart 426 diode laser system ( asah medico a / s , hvidovre , denmark ) . the laser output power ranged from 0 , 5 - 30 w , the pulse rate from 10 - 1000 msec in a pulsed mode , and the frequency from 0 , 3 - 100 hz . the laser could also be operated in a continous wave ( cw ) mode , as was done during the procedure . the target beam was generated by an aluminum , arsenide , and gallium laser ( 810 nm ) . the laser beam was delivered by ultrathin optical fibers of 400 - 1000 m , enabling it to be moved easily and quickly during surgical procedures . eighteen patients ( 10 epulis fissuratum [ figure 1a ] and eight labial frenectomy ) were treated with the diode laser . the laser had an output power of 5 - 10 w , high - power continuous wavelength , and a spot size of 2 mm . the surgical operation was performed using the excision method under local anesthesia [ figure 1b ] . ( a ) preoperative view of the epulis fissuratum lesion of a patient , ( b ) immediate postoperative view of the treated area , ( c ) view of the treated area two weeks after diode laser intervention five patients with histologically proven disorders , including two with leukoplakia [ figure 2a ] , two with oral lichen planus [ figure 3a ] , and one with oral mucous membrane pemphigoid , were selected for 810-nm diode laser treatment . they had been treated with different drugs ( fluocinonide , triamcinolone , lllt ) or a wait - and - see policy before the 810-nm diode laser evaporation . all patients , who had had recurring lesions or bad results with their previous treatment options , were prepared for laser surgery treatment . a defocused 810-nm diode laser was chosen for evaporation of the superficial mucosal lesions [ figure 2b , 3b ] . the laser had an output power of 10 - 15 w , high - power continuous wavelength and 2-mm spot size . ( a ) immediate preoperative view of the treated area of a leukoplakia patient , ( b ) immediate postoperative view of the treated area , ( c ) view of the treated area six weeks after diode laser intervention ( a ) preoperative view of a lichen planus lesion , ( b ) postoperative view of the treated lesion , ( c ) view of the healed lesion after six weeks laser treatment four patients with pyogenic granuloma , diagnosed clinically , were treated with a diode laser , with an output power of 10 - 15 w , high - power continuous wavelength , and 2-mm spot size . first , the lesions were excised with a focused 810-nm diode laser under local anesthesia and then the surgical material was sent for histopathological study . immediately , the surgical field was evaporated with a defocused 810 nm diode laser . in all the 27 patients treated , the areas surrounding the treated tissue were cooled after the laser surgical procedure . all the patients were given suitable postoperative care such as 0.2% chlorhexidine mouthwash , and paracetamol analgesics were prescribed . all patients were seen on a regular basis for follow - up : at one , two , and six weeks , and two months after treatment . eighteen patients ( 10 epulis fissuratum [ figure 1a ] and eight labial frenectomy ) were treated with the diode laser . the laser had an output power of 5 - 10 w , high - power continuous wavelength , and a spot size of 2 mm . the surgical operation was performed using the excision method under local anesthesia [ figure 1b ] . ( a ) preoperative view of the epulis fissuratum lesion of a patient , ( b ) immediate postoperative view of the treated area , ( c ) view of the treated area two weeks after diode laser intervention five patients with histologically proven disorders , including two with leukoplakia [ figure 2a ] , two with oral lichen planus [ figure 3a ] , and one with oral mucous membrane pemphigoid , were selected for 810-nm diode laser treatment . they had been treated with different drugs ( fluocinonide , triamcinolone , lllt ) or a wait - and - see policy before the 810-nm diode laser evaporation . all patients , who had had recurring lesions or bad results with their previous treatment options , were prepared for laser surgery treatment . a defocused 810-nm diode laser was chosen for evaporation of the superficial mucosal lesions [ figure 2b , 3b ] . the laser had an output power of 10 - 15 w , high - power continuous wavelength and 2-mm spot size . ( a ) immediate preoperative view of the treated area of a leukoplakia patient , ( b ) immediate postoperative view of the treated area , ( c ) view of the treated area six weeks after diode laser intervention ( a ) preoperative view of a lichen planus lesion , ( b ) postoperative view of the treated lesion , ( c ) view of the healed lesion after six weeks laser treatment four patients with pyogenic granuloma , diagnosed clinically , were treated with a diode laser , with an output power of 10 - 15 w , high - power continuous wavelength , and 2-mm spot size . first , the lesions were excised with a focused 810-nm diode laser under local anesthesia and then the surgical material was sent for histopathological study . immediately , the surgical field was evaporated with a defocused 810 nm diode laser . in all the 27 patients treated , the areas surrounding the treated tissue were cooled after the laser surgical procedure . all the patients were given suitable postoperative care such as 0.2% chlorhexidine mouthwash , and paracetamol analgesics were prescribed . all patients were seen on a regular basis for follow - up : at one , two , and six weeks , and two months after treatment . the study included 27 patients treated with 810-nm diode laser settings [ table 1 ] , including 10 with epulis fissuratum , eight with labial frenectomy , two with leukoplakia , two with oral lichen planus , one with mucous membrane pemphigoid lesion , and four with pyogenic granuloma . descriptive information on the 810-nm diode laser treatment at the two - week follow - up , the simple oral soft tissue surgery patients had healed without scarring or any other complications , such as hemorrhage [ figure 1c ] . at the six - week follow - up , the patients with oral white and vesiculobullous lesions had healed without scarring [ figure 2c , 3c ] . unfortunately , the patient with mucous membrane pemphigoid disorder had moderate bleeding intraoperatively and postoperatively , which was stopped by suturing the bleeding region . at the four - week follow - up , the patients with pyogenic granuloma lesions had healed without scarring or any other complications , such as hemorrhage . no charring or carbonization occurred during the procedure . in all cases , there were no incidents of infection in the days following the procedure . in our study , we have evaluated the effects of the 810-nm diode laser in the treatment of 27 patients with various benign oral soft tissue lesions . diode , neodymium , erbium , and co2 lasers are approved by the food and drug administration ( fda ) for use in oral surgery . diode lasers present a solid semiconductor as an active medium , by associating aluminum , gallium , and arsenate ( with wavelengths varying between 800 and 980 nm ) in the visible and invisible range of near infrared waves . as its wavelength is poorly absorbed by the hard dental tissue , the diode laser is safe and well indicated for soft oral tissue surgeries in regions near the dental structures and for cutting , vaporization , curettage , blood coagulation , and hemostasis in the oral region . , have reported success in the treatment of oral soft tissue lesions using a diode laser . in our study , an 810-nm diode laser was used surgically for safe elimination of soft oral tissue lesions ; no complications occurred in the surrounding soft tissue or hard tissue . we adopted the 810-nm diode laser for our patients due to its availability , the convenience of its application , the ability for large areas to be treated in a single application , and the possibility of precise control of laser fluence in all areas of the mouth . this pattern , especially of producing precise surgical incisions , was in accordance with those observed by genovese et al . and darcangelo et al . alongside all this , aras et al . compared diode laser with the er : yag laser revealed that er : yag laser was more comfortable because of the lower local - anesthesia requirement . soft tissue procedures cause postsurgical pain and discomfort when chewing , eating , breathing , and speaking . has reported that on evaluating the patients for pain during the first three hours after surgery , those in the er : yag laser group had a higher degree of pain than those in the diode laser group . kara has suggested that ne : yag laser frenulectomy provides better patient perception of success than that seen with conventional surgery . however , in our study , we have not encountered any reports of pain from the patients . according to the literature , the mainstay treatment for oral lichen planus is topical steroids . in oral lichen planus cases where topical approaches have failed , several studies have shown good results with this treatment , but side effects have also been reported . in our study the white lesions , especially oral lichen planus , were initially treated with the conventional therapy mentioned above . when we did not obtain a definite result from this treatment option , we decided to apply the 810-nm diode laser to our patients in order to obtain acceptable results . initially , clinicians treated oral leukoplakia with vitamin a , vitamin e , and beta - carotene . however , because of the toxicity of vitamin a and the unsatisfactory response to vitamin e and beta - carotene , the use of these drugs for the treatment of oral leukoplakia has been discontinued . the other treatment modalities for oral leukoplakia are scalpel excision or electrocautery and cryosurgery , for which there is a recurrence rate of approximately 33% . studies on the clinical usefulness of laser surgery in oral leukoplakia have shown that the laser surgery prevents not only recurrence and malignant transformation , but also postoperative dysfunction . in our study , we used 810-nm high - power laser surgery to treat these lesions and obtained excellent results . mucous membrane pemphigoid ( mmp ) is a rare chronic disorder of the mucosal tissue manifested largely by vesiculobullous lesions of the oral cavity and eyes . additionally , mmp most often involves the oral cavity followed by the conjunctiva and genitalia . the clinical manifestations of mucous membrane pemphigoid are varied , but the oral and conjunctival mucosae are most frequently involved . the condition is also commonly termed as cicatricial pemphigoid , but the term cicatricial later excluded patients without scarring . surgery has a diagnostic approach to the lesions , such as , stenosis and airway obstruction . whiteside et al . treated supraglottic airway stenosis with the co2 laser and had good results . first , our patient with an mmp lesion on the left side of her tongue had taken conventional therapy , such as , topical corticosteroids , tetramycin , lllt , and some immunosuppressive adjuvants ; unfortunately , these did not yield good results . we had a moderate bleeding complication intraoperatively and postoperatively , but the lesion healed completely within six weeks . the possible treatment methods are excision , curettage , cryotherapy , chemical and electric cauterization , and the use of lasers . surgical excision or electrocoagulation is considered the standard treatment for pyogenic granuloma . however , because of scarring , excision is a suboptimal form of therapy ; bleeding can also complicate the operation process , especially in huge granulomas or if the excision is close to the lesion . laser excision may be the ideal treatment option for these type of lesions . for our pyogenic granuloma patients , the use of lasers , especially diode lasers , in general dentistry is now an accepted treatment aid , with a wide range of applications in oral soft tissue surgery . we also see that the use of the 810-nm diode laser as the treatment of choice for oral soft tissue therapy is reliable because we obtained acceptable healing of the lesions with minimal adverse effect . thus , in oral soft tissue surgery , the use of 810-nm diode lasers may be the best choice .

objective : to evaluate the safety and efficacy of an 810-nm diode laser for treatment of benign oral soft tissue lesions.materials and methods : treatment with the 810-nm diode laser was applied to a group of eighteen patients with pathological frenulum and epulis fissuratum ; five patients with oral lichen planus , oral leukoplakia , and mucous membrane pemphigoid ; and four patients with pyogenic granuloma.results:although the conventional surgery wound heals in a fairly short time , in the present study , the simple oral soft tissue lesions healed within two weeks , the white and vesiculobullous lesions healed completely within six weeks , and the pyogenic granuloma lesions healed within four weeks . any complication was treated by using the 810-nm diode laser.conclusions:patient acceptance and satisfaction , without compromising health and function , have been found to be of a high degree in this present study . thus , we can say that the use of the 810-nm diode laser may indeed be the best choice in oral soft tissue surgery .

liver diseases other than autoimmune hepatitis ( aih ) can present with laboratory features of aih . alcoholic liver injury as well as drug - induced liver injury ( dili ) occasionally resemble chronic hepatitis . it has been reported that the activation of innate immune cells and the inflammatory cascade play a central role in the pathology of alcoholic liver disease . toll - like receptor 4 ( tlr4 ) expressed on innate immune cells and hepatocytes could recognize the gut - derived endotoxin and the tlr4-downstream could be activated through the interferon regulatory factor 3 ( irf3 ) . drugs and/or their metabolites and host immunological factors are rarely involved in idiosyncratic dili [ 4 , 5 ] and trigger a t cell - mediated cytotoxicity or b cell antibody response [ 5 , 6 ] . we report a woman with alcohol dependence who initially presented with atypical laboratory features resembling aih or dili . even with liver biopsy , it is difficult to diagnose certain patients . attention should be paid to the fact that alcoholic liver injury in some cases resembles aih or dili . a 50-year - old japanese woman was referred to chiba university hospital in april 2012 because of jaundice and abnormal liver tests . she took several drugs for her depression and hypertension and drank alcohol ( 90 g daily ) . one year before admission , she had had a history of dili by mianserin for her depression . laboratory findings were as follows : white blood cell count 7,100/l ( eosinophils 0.8% ) , platelets 155,000/l , prothrombin time 18% ( inr 2.36 ) , total protein 5.3 g / dl , total bilirubin 21.9 mg / dl ( direct bilirubin 15.0 mg / dl ) , alkaline phosphatase 313 iu / l , aspartate aminotransferase ( ast ) 314 iu / l , alanine aminotransferase ( alt ) 112 iu / l , and -glutamyl transpeptidase 190 serum immunoglobulin g of 1,695 mg / dl and immunoglobulin m of 116 mg / dl were normal , but antinuclear antibody ( ana ) was not positive ( 80 ) . virus serology was negative for hepatitis a , b , c and e viruses , cytomegalovirus and epstein - barr virus acute infection . computed tomography and abdominal ultrasound findings showed a non - atrophic liver with slightly ascites . we suspected a diagnosis of dili or aih , although her aih score was underscored [ 7 , 8 ] , and administered corticosteroids . iu / l ) , total bilirubin ( 3.4 mg / dl ) and prothrombin time ( 92% ) were improved and liver biopsy was performed to diagnose her disease ( fig . hepatic architecture was preserved without cirrhosis , and inflammation of the periportal area was obvious with centrilobular necrosis . there were mononuclear cells in the portal area , but marked plasma cell infiltration was not observed . after 1 month , she was discharged ( ast 22 iu / l , alt 56 iu / l , total bilirubin 1.8 mg / dl and prothrombin time 98% ) . two months later , her liver test had suddenly deteriorated ( ast 76 iu / l , alt 249 iu / l and -glutamyl transpeptidase 1,262 , we became aware of her alcohol consumption and she was diagnosed with alcohol dependence by a psychiatrist . we present a patient whose liver biopsy findings did not reveal any influence of alcohol and whose level of -glutamyl transpeptidase at administration was much lower than that after 2 months . unfortunately , we were not aware of her alcohol dependence while she was hospitalized . our patient had a previous history of dili , and liver biopsy showed chronic active hepatitis , but not cirrhosis . alcohol consumption is a common comorbid condition with other chronic liver diseases and may exacerbate liver injury in autoimmune liver diseases and chronic viral hepatitis . this synergism can result in increased hepatic inflammation and accelerated rates of fibrosis , although the influence of alcohol consumption in this patient with acute liver injury is unclear . alcohol consumption has been proposed as a risk factor for dili from medication , although there is still insufficient evidence to support this . we speculate that alcohol consumption might have worsened the background chronic liver injury of undetermined etiology . there is a recent report that deletion of sirtuin 1 , which is a nicotinamide adenine dinucleotide - dependent protein deacetylase that regulates hepatic lipid metabolism by modifying histones and transcription factors , promotes steatosis , inflammation and fibrosis in response to ethanol challenge . in alcohol - induced liver disease , the myd88-independent but irf3-mediated tlr4 signaling plays a role in alcohol - related liver inflammation and liver damage . gut - derived lipopolysaccharide , a ligand of tlr4 , plays an important role in triggering and maintaining the activation of kupffer cells in alcoholic hepatitis . innate immune signaling might be important in hepatic cell death . in the present case , the liver function tests , histology , female gender , positive ana and the remarkable resolution of disease on administration of corticosteroids all strongly point to this being aih ( acute - on - chronic exacerbation ) although her aih score was underscored [ 7 , 8 ] . liver histology of the present case was silent on the salient features of alcoholic hepatitis [ 13 , 14 ] : steatosis , marked perivenular and pericellular fibrosis , ballooning degeneration with neutrophilic inflammation , and mallory bodies in spite of the elevation of -glutamyl transpeptidase . carbohydrate - deficient transferrin , one of biological markers of alcoholism , might be useful . usually aih is diagnosed after excluding alcohol , hepatitis b and hepatitis c virus as a cause of liver injury [ 7 , 8 ] . this might be consistent with the fact that alcohol dependence initially presents with atypical laboratory features of aih or dili . in conclusion , it is important to remind oneself that alcohol dependence is not required for developing liver disease , since many patients with liver disease do not meet the criteria for alcohol dependence . in some cases , it is difficult to diagnose alcoholic liver injury even by liver biopsy . in any case , careful follow - up of the patient and consultation with a psychiatrist were useful in diagnosing alcohol dependence and related liver injury .

some patients with alcohol dependence may initially present with atypical laboratory and histological features resembling autoimmune hepatitis ( aih ) or drug - induced liver injury ( dili ) . even with liver biopsy , it may be difficult to diagnose certain patients with alcohol dependence . however , careful follow - up of our patient and consultations with the attending psychiatrist were successful in diagnosing alcohol dependence and its liver injury . the immune mechanisms of alcoholic liver diseases , aih and dili may be overlapping . certain patients are suffering from aih with flares on a background of alcohol abuse . certain patients with alcohol abuse may have a past history of dili . this might be consistent with the fact that alcohol dependence initially presents with atypical laboratory features of aih or dili . with careful observation , the clinician should remind himself that alcohol dependence is not always required for developing liver disease , since many patients with liver disease do not meet the criteria for alcohol dependence .

histoplasmosis caused by the dimorphic fungus histoplasma capsulatum ( hc ) is a granulomatous disease that primarily affects the lungs ; however , depending on the immune status of the host and the inhaled fungal load , the infection can progress to chronic pulmonary and disseminated diseases leading to death . . these yeasts may multiply inside macrophages , inducing cell death . however , when an appropriate immune response is triggered , alveolar macrophages are activated , killing intracellular hc and controlling the infection . host control and eradication of hc infection are mediated by a t helper type 1- ( th1- ) mediated cellular immune response , which is associated with production of il-12 , ifn- , and tnf- , and activation of macrophages , dendritic cells , and t helper lymphocytes . besides cytokines , previously , we demonstrated that leukotrienes ( lts ) , lipid mediators derived from arachidonic acid metabolism , play an important role in primary and secondary immune responses during histoplasmosis by increasing phagocytosis and killing by macrophages [ 57 ] . innate , sentinel dendritic cells , neutrophils , and mainly tissue macrophages are major producers of eicosanoids , as well as cytokines . considering the importance of macrophages for infection control , we can suggest that the enhancement of their effector activities might be critical and decisive to the fate of infections . that shows that treatment with erythropoietin ( epo ) activates the biological functions of macrophages . they observed that peritoneal macrophages and bone marrow - derived macrophages from epo - treated mice have increased phagocytic rates and nitric oxide production but a reduction in il-12 and il-10 . epo is a hormone that is classically known for regulating red blood cell production . in adults , epo is produced and secreted primarily by kidney tubular cells [ 12 , 13 ] . the nonerythroid effects of epo have been described [ 14 , 15 ] such as their role in immune system activation for controlling some infections . nishimura et al . showed that the absence of epo production decreases bone marrow erythropoiesis and aggravates anemia during trypanosoma brucei brucei infection . in addition , epo has been administered to patients with infectious diseases to treat pathogen - induced hemolytic anemia . during plasmodium berghei this phenomenon is likely due to the fact that epo ameliorated parasite - induced anemia , reduced tnf- and ifn- production , and decreased neuronal apoptosis , representing a potential therapeutic benefit in cerebral malaria . in contrast to that reported for protozoal infections , epo treatment in cases of salmonella typhimurium infection increased the mortality of mice and decreased the production of il-6 , tnf- , and il-1 in the liver . to the best of our knowledge , there are no reports evaluating the effect of epo pretreatment in fungal infections such as histoplasmosis . mason - garcia et al . demonstrated that erythrocytes in the presence of epo increase the production of ltb4 and 12-hydroxyeicosatetraenoic acid , and they suggested that this increase may be related to the activation of phospholipase a2 and c. however , nothing is known about the relationship between epo and lipid mediator production in fungal infections . the role of epo in pg synthesis or action has not been described , although some studies demonstrated the effects of pgs in epo production or action [ 2123 ] . considering that macrophages and lipid mediators are important for the control of hc infection and that epo might act on these two parameters , our aim was to investigate the effects of epo pretreatment on experimental histoplasmosis . we found that epo pretreatment increased the mortality of infected mice , the number of cells expressing cd80 and cd86 in the f4/80 population , and the production of inflammatory cytokines , chemokines , and ltb4 and upregulated late apoptosis . on the other hand , no alteration of fungal burden ( cfu ) was observed , and the number of mononuclear cells recruited to bronchoalveolar lavage fluid ( balf ) was reduced , as well as pge2 production . when extrapolated to humans , our study suggests that the use of epo for the treatment of anemia in patients with chronic renal disease might impair the control of fungal infections such as those caused by hc , leading to lung inflammation , tissue damage , and death . male , 8-week - old c57bl/6 mice were obtained from the faculdade de cincias farmacuticas de ribeiro preto , universidade de so paulo ( ribeiro preto , sp , brazil ) . uninfected mice , treated with epo and untreated , were kept at the animal facility of faculdade de cincias farmacuticas de ribeiro preto with a 12 h light / dark cycle and with water and food ad libitum . after hc infection , mice were kept in a biohazard facility in safety enclosures and under the same standard husbandry conditions used for uninfected mice . all procedures were performed following the guidelines of the animal care committee of universidade de so paulo ( protocol number 11.1.464.53.0 ) . recombinant human epo ( rhuepo ; hemax , from biosinttica - ach , brazil ) was diluted in phosphate - buffered saline ( pbs ) before use . the dose was chosen based on that used by lifshitz et al . and on a dose - response ( 45 , 50 , 100 , 150 , and 180 u of epo ) trial performed in our laboratory as pilot experiments ( data not shown ) . the dose of 45 u of epo in 100 l of pbs was selected and administered subcutaneously to each mouse three times per week for one week prior to hc infection . the hc isolate used in this study was obtained from a patient at the hospital das clnicas de ribeiro preto ( ribeiro preto , sp , brazil ) and maintained in our lab . the mycelial phase of hc was obtained as previously described . to obtain yeasts , mycelia were cultivated at 37c in bhi blood agar . samples were used when fungal viability was 90% , based on fluorescein diacetate and ethidium bromide exclusion assays , performed as previously described . then 100 l of pbs without or with a sublethal ( 5 10 yeasts / mouse ) or lethal ( 1 10 yeasts / mouse ) inoculum of viable hc cells was inoculated intratracheally ( i.t . ) . mice were euthanized 14 days after infection in a co2 chamber for lung , spleen , and bronchoalveolar fluid analyses . uninfected and untreated mice were euthanized on the same day as infected animals . in this study , we used the following 4 experimental groups of mice to evaluate the effect of epo pretreatment in hc infection : ( 1 ) untreated and uninfected mice ( control ) , ( 2 ) uninfected ( but inoculated i.t . with 100 l pbs ) mice treated with epo s.c . for one week ( epo group ) , ( 3 ) untreated ( but inoculated s.c . with 100 l of pbs ) mice infected i.t . with hc ( hc group ) , and ( 4 ) mice pretreated s.c . with epo for one week and then infected i.t . with hc ( epo + hc group ) . total cell counts in balf samples were achieved using a neubauer chamber , and differential cell counts were performed using cytospin preparations stained with panoptic ( laborclin , paran , brazil ) . two serial dilutions of tissue homogenates were made in pbs and 100 l of each diluted homogenate sample was placed on bhi - agar - blood medium and then incubated for 15 days at 37c and with 5% co2 . fungal colonies were counted manually and the fungal burden was expressed as the number of colony - forming units per gram of tissue ( cfu / g ) . lungs collected from mice 14 days after infection were fixed in 10% formalin , processed , and embedded in paraffin blocks , from which 5 m sections were cut and stained with hematoxylin and eosin ( h&e ) . stained tissue sections were analyzed with a leica dmr light microscope ( leica microsystems gmbh , wetzlar , germany ) equipped with a video camera ( leica microsystems ltd . images were analyzed using the leica application suite software ( leica microsystems image solutions , cambridge , uk ) , and the lung - lesion area ( lla ) was estimated by computerized planimetry analysis of the images using the cs3 photoshop software ( adobe , san jose , ca , usa ) and expressed as a percentage of the total lung tissue area using the formula ( lla / ta ) 100 , where ta is the total lung tissue area . to obtain tissue homogenates , lungs were removed from mice immediately after euthanasia and homogenized using a mixer homogenizer ( labortechnik , wasserburg am bodensee , germany ) in 2 ml of rpmi-1640 containing 10 m indomethacin and 1 mm edta . after centrifugation , supernatants were recovered , filtered , and acidified to ph 3.43.6 using 1 n hcl and applied slowly to a sep - pak c18 column ( waters , milford , ma , usa ) previously washed with 20 ml of 35% ethanol and 20 ml of water . after washing the columns with water , the lipids were eluted with 2 ml of absolute ethanol , dried under vacuum , and reconstituted in 100 l of methanol . samples were centrifuged to remove any indomethacin precipitate , and the supernatants were transferred to appropriate vials for determination of the concentration of pge2 and ltb4 by high - performance liquid chromatography - tandem mass spectrometry using an acquity uplc - ms system coupled to a xevo tq - s mass spectrometer ( waters ) . chromatographic separation was conducted using a supelco ascentis express c18 hplc column ( sigma - aldrich , st . louis , mo , eua ) with dimensions of 100 mm 3.0 mm , 2.7 m . a binary gradient system was employed in which phases a and b consisted of water / acetonitrile / acetic acid ( 70 : 30 : 0.02 , v / v ) and acetonitrile / isopropanol ( 70 : 30 , v / v ) , respectively . samples ( 10 l ) were eluted with a flow rate of 0.6 ml / min , with a linear gradient starting with 0% b , which was increased to 15% b at 2 min , 20% b at 5 min , 35% b at 8 min , 40% b at 11 min , 100% b at 15 min , 100% b at 18 min , and 0% b at 19 min and held there for 30 min . analyses were performed using multiple reaction monitoring ( mrm ) scan mode employing negative ionization . mrm transitions were 351.2 171 for pge2 and 335.1 195 for ltb4 . quantification was accomplished using calibration lines constructed with pge2 and ltb4 synthetic standards ( cayman chemical , ann arbor , mi , usa ) . data were collected and analyzed using the software mass lynx 4.0 ( waters ) . the concentrations of ifn- , il-6 , mcp-1 , and mip-1 in supernatants of lung homogenates were determined using commercially available elisa kits according to the manufacturer 's instructions ( r&d systems , minneapolis , mn , usa ) . cells from mouse balf samples were double - labeled for flow cytometric analysis as previously described , using combinations of the following fluorochrome - conjugated monoclonal antibodies : anti - f4/80-percp , anti - cd80-pe , anti - cd86-apc , and anti - mhc ii - fitc ( all from bd bioscience , san jose , ca , usa ) . samples were analyzed in a facscanto flow cytometer ( bd bioscience ) , and the percentage of cells double - positive for f4/80cd80 , f4/80/cd86 , and f4/80/mhc ii were calculated using the facsdiva software ( bd bioscience ) . balf cells from all groups were evaluated for apoptosis using an annexin v - fitc / propidium iodide ( pi ) apoptosis detection kit ( bd pharmingen ) , following the manufacturer 's instructions . balf cells were collected as mentioned previously , washed twice with pbs , centrifuged at 400 g for 15 min and suspended in annexin v binding buffer , and incubated with annexin v - fitc in total darkness for 15 min . a total of 10,000 events were acquired for each sample using the facscanto flow cytometer ( becton and dickinson , san jose , ca ) . a side - scatter gate was made to assess the apoptotic cells , and the results were evaluated by facsdiva software and expressed as a percentage of late apoptotic cells ( annexin / pi ) from total gated cells . one - way anova was used followed by newman - keuls multiple comparison test and student 's t - test to analyze differences between experimental groups . we observed that pretreatment with epo ( figure 1(a ) ) increased the mortality of sublethal or lethal hc - infected mice ( figures 1(b ) and 1(c ) ) . the survival of non infected mice was not altered by epo treatment following the same schedule ( data not shown ) . next , we investigate if increased mortality of infected epo pretreated mice was due to augmentation of fungal burden in the lungs and spleen . for this experiment , 14-day postinfection fungal burden in lung and spleen was determined by counting cfu . as seen in figure 2 , epo pretreatment did not alter the fungal burden in the organs , when compared to that observed in infected mice without epo treatment . these results suggest that the increased mortality observed in epo - treated mice was not due to increased fungal proliferation or dissemination . the effect of epo pretreatment on the cell populations in the bronchoalveolar space with or without infection was investigated . we observed a decrease in mononuclear cell counts ( figure 3(a ) ) without a change in neutrophil numbers ( figure 3(b ) ) in the balf of epo pretreated and hc - infected mice ( epo + hc ) , when compared to infected epo - untreated mice ( hc group ) . interestingly , epo treatment of non infected animals resulted exclusively in significant increases in mononuclear cells in the bronchoalveolar space ( figure 3(a ) ) . since epo pretreatment reduced the number of mononuclear cells in the balf of infected mice , we investigated whether this was due to increased apoptosis of mononuclear cells . the results demonstrate a decrease in the number of late apoptotic cells ( annexin / pi ) in balf from the hc group , when compared to control uninfected mice ( figure 3(b ) ) . however , epo pretreatment increased the number of late apoptotic cells present in balf ( epo + hc group ) , in comparison to the hc group . no differences were found in the number of early apoptotic or necrotic cells between epo pretreated mice and untreated controls ( data not shown ) . to understand why epo treatment increased the mortality of hc - infected mice without increasing fungal burden , we also investigated the degree of balf macrophage activation and lung inflammation . the expression of the activation markers cd80 , cd86 , and mhc ii was evaluated in f4/80 cells from balf . balf higher numbers of f4/80 cells expressing cd80 and cd86 molecules were found ( figures 4(a ) and 4(b ) ) , while the proportion of f4/80 cells expressing mhc ii was not affected ( figure 4(c ) ) , in comparison to only epo - treated mice . however , the expression of cd80 molecules was decreased in epo + hc and hc animals , when compared to uninfected control and to epo - treated animals ( figure 4(a ) ) . hc infection increased ifn- , il-6 , mip-1 , and mcp-1 production in lungs relative to those observed in uninfected and untreated mice . epo pretreatment further increased cytokine and chemokine concentrations in infected mice ( figure 5 ) . hc infection increased tnf- , kc ( il-8 ) , and rantes , but no further alterations were observed in the epo + hc group ( data not shown ) . because lipid mediators might contribute to lung inflammation , we evaluated ltb4 and pge2 production in the lungs of infected mice with or without epo pretreatment . in the epo + hc group , we observed decreased pge2 ( figure 6(a ) ) but increased ltb4 ( figure 6(b ) ) concentrations compared to that found in the hc group . infected mice treated or not with epo showed higher amounts of eicosanoids than uninfected animals , epo - treated or not ( figure 6 ) . as we observed increased inflammatory mediator concentrations , we assessed lung inflammation and parenchymal damage by histopathological analysis . corroborating our previous data , we observed that hc infection induced lung inflammation with severely compromised lung architecture ( figures 7(c ) and 7(d ) ) . epo pretreatment of infected mice clearly increased lung inflammation and damage , as seen by the expansion of llas ( figures 7(d ) and 7(e ) ) . we observed that epo pretreatment increased inflammatory cellular infiltration , mainly with mononuclear cells , and decreased alveolar structures in the lung parenchyma , suggesting that the impairment of respiratory capacity may be responsible for death of the mice . taken together , our results suggest that pretreatment with epo decreases the survival of infected mice due to excessive lung inflammation , which severely compromises lung architecture , impairing normal respiratory capacity . excessive inflammation in epo pretreated mice appears to be due to increased activation of macrophages that release higher amounts of inflammatory mediators than macrophages from infected mice in the absence of epo pretreatment . nonerythroid effects of epo have been described in several studies [ 11 , 13 , 14 , 18 ] . among their nonclassical effects , epo is known as a macrophage activating factor and seems to be involved in lipid mediator production . because of the importance of macrophages and lipid mediators for the control of fungal infection , the purpose of this study was to investigate the effect of epo pretreatment in the progression of lung infection induced by hc . we observed that epo treatment for one week before hc infection increased mice mortality without corresponding increases in fungal burden in the lungs or spleen , suggesting no correlation with fungal proliferation and/or dissemination . our analysis of different aspects of the innate immune response elicited by hc infection in epo pretreated mice revealed that increased mortality may result from excessive lung inflammation due to increased macrophage activation and the production of inflammatory mediators . our data are in agreement with the report of nairz et al . showing that 5 u / g epo augmented the mortality of mice infected with s. typhimurium as compared to infected mice in the absence of epo . increased mortality was accompanied with reduced no production and cytokines involved in controlling host resistance . the production of no is also important for the control of hc infection [ 3 , 28 ] . however , pretreatment with epo did not significantly alter no production ( data not shown ) , and fungal burden was not increased . however , mononuclear cell numbers in the bronchoalveolar space were decreased in epo + hc , as compared to the hc group . these results are surprising because mononuclear cells are the most efficient at triggering fungal death during hc infection . we also found a reduced number of apoptotic cells recovered from the balf of hc - infected mice . the pretreatment with epo partially increased apoptosis , which might explain the reduction in mononuclear cell numbers found in balf samples of epo pretreated and infected mice , compared to infected mice not pretreated with epo . the fact that the reduction in mononuclear cell numbers in epo pretreated mice did not result in increased fungal burden pretreatment with epo before hc infection increased the frequency of cd80 and cd86 ( but not mhc ii cells ) in the f4/80 macrophage population from balf samples . similarly , lifshitz and colleagues reported that the spleens of both c57bl/6 mice treated with rhuepo and tg6 mice ( which produce epo excessively ) had increased numbers of f4/80 cells coexpressing cd80 and mhc ii molecules . in a previous study using the same mouse model moreover , the pretreatment with epo also augmented the production of inflammatory mediators such as il-6 , ifn- , mcp-1 , mip-1 , and ltb4 in hc mice . although these mediators are important for the control of infection [ 5 , 3033 ] , their excessive production might be detrimental to the host by triggering excessive lung inflammation . disproportionate lung inflammation was confirmed by histopathological analysis in the epo + hc group ( figure 7 ) , which demonstrated increases in lla . our data are not in agreement with a previous study showing that epo pretreatment reduced il-6 , tnf- , and il-1 production in the liver during s. typhimurium infection . in addition to cytokines , the eicosanoids lt and pg also play a central role in controlling hc infection [ 58 ] . previous data from our laboratory demonstrated that pgs impair host control of hc infection and that pg synthesis inhibition by celecoxib led to yeast elimination and infection control . in the present study , we found increased pge2 concentration in hc - infected mice , but epo treatment before the infection reduced pge2 concentration , although no reduction in mortality was associated with this phenomenon . it is known that pge2 plays a role in apoptotic events . while pge2 induces apoptosis in some cell types , including resting immature and mature human lymphocytes and epithelial cells , inhibition of apoptosis by pge2 similarly , in the present report , there was a negative association between pge2 concentration and the number of apoptotic cells in balf samples . overall , our data strongly suggest that excessive inflammation caused by exacerbated activation of alveolar macrophages increases lung inflammation , tissue damage , and mortality in epo pretreated and infected mice . these results are consistent with a recent report from doitsh and colleagues demonstrating the role of excessive inflammation in maintaining cd4 t cell death during hiv infection . this phenomenon was related to cell death of nonpermissive cd4 t cells by pyroptosis , which is caused by excessive inflammation and also promotes further inflammation , maintaining the cycle of cell death and compromising the patient 's quality of life . in humans , the use of recombinant epo is very common in patients with chronic renal disease and/or those undergoing hemodialysis , because various forms of anemia are common in these cases . anemia , which impairs quality of life , occurs primarily due to deficiency in the production of epo by the kidney but can be exacerbated by iron deficiency , which contributes to the shortened lifespan of erythrocytes , among other complications . however , treatment is not based solely on epo administration ; antiproliferative cytokines and stimulation of iron uptake are used in combination with epo in attempts to reverse the anemia . it is important to highlight that although epo treatment is crucial for control of anemia in patients with chronic renal disease or those undergoing hemodialysis , our study focused on inappropriate immune responses that these patients might generate prior to a potential fungal infection acquired during the treatment period , which could contribute to their morbidity .

erythropoietin ( epo ) is a key hormone involved in red blood cell formation , but its effects on nonerythroid cells , such as macrophages , have not been described . macrophages are key cells in controlling histoplasmosis , a fungal infection caused by histoplasma capsulatum ( hc ) . considering that little is known about epo 's role during fungal infections and its capacity to activate macrophages , in this study we investigated the impact of epo pretreatment on the alveolar immune response during hc infection . the consequence of epo pretreatment on fungal infection was determined by evaluating animal survival , fungal burden , activation of bronchoalveolar macrophages , inflammatory mediator release , and lung inflammation . pretreatment with epo diminished mononuclear cell numbers , increased the recruitment of f4/80+/cd80 + and f4/80+/cd86 + cells to the bronchoalveolar space , induced higher production of ifn- , il-6 , mip-1 , mcp-1 , and ltb4 , reduced pge2 concentration , and did not affect fungal burden . as a consequence , we observed an increase in lung inflammation with extensive tissue damage that might account for augmented mouse mortality after infection . our results demonstrate for the first time that epo treatment has a deleterious impact on lung immune responses during fungal infection .

patellofemoral pain syndrome ( pfps ) is a common orthopedic disease , and often induces patella pain during climbing and long - distance running1 . the results of a previous study indicate that athletes have high risks of knee overuse2 . the quadriceps supplies the power of dynamic patellar movement , and the vastus medialis oblique ( vmo ) and vastus lateralis ( vl ) enable the patella to stabilize during tracking3 . a lack of muscle strength in the vmo decreases the medial muscular tension the patellar and causes the muscle imbalance to increase patellar maltracking4 . the ideal ratio of the vmo to vl is 1:1 , but many pfps patients have had a low ratio for both muscles in electromyography studies5 . some studies indicated that increasing the muscle strength of the vmo is a useful method of preventing pfps6 , 7 . some study results indicated that specific vmo exercise training can effectively prevent and reduce the incidence of pfps7 , 8 . the open kinetic chain is a movement in which the distal end of the extremity is not fixed to a relatively stable surface . in the closed kinetic chain , movement of a joint can not occur without causing predictable movements in the other joints of the extremity9 . a closed kinetic chain exercise for the knee joint can cause quadriceps and hamstring co - contraction to reduce displacement and improve joint stability , but this will increase the intra - articular pressure10 . this exercise can cause quadriceps contracture , and this is similar to the muscle activity recruitment occurring during weight - bearing in the gait11 . open kinetic chain exercise uses a lower amount of weight loading for pfps patients than closed kinetic chain exercise . the results of some studies show that closed kinetic chain exercise has a significant therapeutic effect for pfps patients11,12,13 . however , these studies often focused on the pain decrease and ignored discussing the effect of vmo muscle activity during the exercise training . sling exercise therapy ( set ) is a new training method for athletes and orthopedic patients utilized in recent years14 . because of its convenience and practicality , set has become more and more common in rehabilitation clinics and fitness training centers15 . however , studies about the effects of using set with open and closed kinetic chains on vmo muscle training in pfps patients are rare . we followed the theories about open and closed kinetic chain exercises to design two exercises using set and explore the vmo and vl muscle activity . to our knowledge , this is the first study to investigate this theme , and we think that the results of this study are important for pfps patients as well as physical therapists and athletic trainers providing therapeutic exercise . this study was a cross - over study and was approved by the institutional review board of china medical university and hospital . we informed all participants about the experimental process in detail and then obtained their informed consent before the study . we recruited healthy female college students and randomly assigned them to the two set exercises , comprising sling open chain knee extension ( socke ) exercise and sling closed chain knee extension ( sccke ) exercise ( fig . a redcord suspension system ( redcord trainer , record , staub , norway ) was used to train the quadriceps muscle by one athletic trainer . the two exercises were performed for 10 min , and 3 repetitions of maximum force were completed in this period . the socke exercise used set to suspend the knee and maintain knee flexion at 60 degrees . the sccke exercise used set to suspend the ankle and maintain the hip in full extension . we asked the individual to extend the knee to strengthen the concentric contraction of the quadriceps . these exercises were designed to improve the strength of the vmo for application in the field of sports medicine . scottsdale , az , usa ) was fixed to the lateral knee joint , which was the axis of the goniometer , and the stationary and movable arms were tied to the lateral midline of the thigh and shank . the range of motion of the knee was monitored , and the vmo and vl muscle activity was collected by electromyography by one examiner . socke ( a ) and sccke ( b ) exercises electromyography ( myotrace 400 , noraxon usa inc . , scottsdale , az , usa ) was used to calculate the maximal voluntary contraction ( mvc ) . the vl electrodes were placed on the line from the anterior superior iliac spine to the superolateral boarder of the patella and 10 cm from the patella . the vmo electrodes were placed at a distance of 4 cm from the superior side of the patella along a line inclined 50 that ran parallel to the line from the anterior superior iliac spine to the superolateral boarder of the patella16 . in the electromyography signal processing , the sampling frequency was set to 1,000 hz . we used a band - pass filter of 40 to 400 hz to reduce external noise and performed full - wave rectification . then , setting the root mean square at 100 ms smoothed the signal17 . finally , we set the amplitude normalization at 300 ms and normalized the different movement data by transforming mvc into mvc% with the following formula : mvc% = ( mean mvc / maximum force of the resistance test ) 100% , chicago , il , usa ) to compare the activations of the of vmo and vl . the wilcoxon test was used to compare the differences in the mvo% values of the vmo and vl between the sccke and socke exercises . the consistencies of activation of the vmo and vl for 3 repetitions of maximum contraction of one exercise within each individual were analyzed by using the intraclass correlation coefficient ( icc ) . seven healthy college students ( age = 21.3 0.6 ; weight = 51.3 6.3 kg ; height = 161.5 4.8 cm ) were recruited and performed 3 repetitions of maximum contraction for each exercise . the icc for activation of the vmo and vl for 3 repetitions of maximum contraction was 0.91 0.23 , and there were no significant differences in the socke and sccke exercises among the 3 repetitions of maximum contraction . table 1table 1.mvc results of the seven participantsnosocke exercisesccke exercisevmo ( % ) vl ( % ) vmo ( % ) vl ( % ) 198.9 2.484.4 2.379.9 3.363.2 1.3289.8 3.189.4 3.193.7 3.298.4 2.8360.6 3.586.3 4.278.6 2.671.2 2.3499.6 3.798.1 3.882.5 3.581.5 1.4573.3 3.289.9 2.782.2 3.386.2 3.3696.5 2.677.1 3.392.1 4.170.1 2.1785.4 1.478.2 2.990.7 3.274.4 3.2socke , sling open chain knee extension ; sccke , sling close chain knee extension exercise ; vmo , vastus medialis oblique ; vl , vastus lateralis shows that 5 participants ( nos . 1 , 2 , 4 , 6 , and 7 ) had higher mvc% values for the vmo than for the vl during the socke exercise . five participants ( nos . 1 , 3 , 4 , 6 , and 7 ) had higher mvc values for the vmo than for the vl during the sccke exercise . the mean mvc% values for the vmo ( 86.30 14.61 ) and vl ( 86.20 7.25 ) during the socke exercise were higher than those for the vmo ( 85.67 6.27 ) and vl ( 77.85 11.81 ) during the sccke exercise . no significant differences in mvc% values for the vl and vmo were found between the exercises ( p > 0.05 ) . however , the mvc% values for the vmo and vl during the socke exercise were higher than during the sccke exercise . the mvc% of the vl was higher than that of the vmo during both exercises . we also found that the ratio of the vmo to vl was 1.0 0.19 during the socke exercise and 1.11 0.15 during the sccke exercise . socke , sling open chain knee extension ; sccke , sling close chain knee extension exercise ; vmo , vastus medialis oblique ; vl , vastus lateralis this study compared the vmo and vl muscle activities during the sccke and socke exercises . our results showed that both exercises had a recruitment effect on the vmo muscle . in addition , the socke exercise had higher mvc% values for the vmo and vl than the sccke exercise . many studies have used closed kinetic chain and weight - bearing exercises , e.g. , double - leg squat and lunge exercise , to improve the vmo strength5 , 18 , 19 . their results showed that these exercises can significantly increase the activation of the vmo and vl . some studies showed that closed kinetic chain exercises can cause greater activation in the vmo than in the vl5 , 6 , 19 . however , combining closed kinetic chain exercise with weight - bearing exercise often provides a large increase in pulling strength , and increases the intra - articular pressure . we found that the using set in the sccke exercise can also cause greater activation in the vmo than in the vl . set can reduce the effect of body weight on the knee intra - articular pressure and can be used to train knee extension with non - weight bearing . the sccke exercise may be useful and helpful in training vmo activity for pfps patients . we also found the socke exercise to be more effective in activating the vmo than the sccke exercise . some studies reported that open kinetic chain exercise has little effect on ptps rehabilitation , because the activation of the vmo was not enough to improve patellar tracking20 , 21 . however , our study results showed that use of set in the socke exercise can increase the muscle activity of the vmo by more than that of the vl . we thought suspension of the knee by set would decrease the postural stability during the sccke exercise . this can challenge the stability of the trunk core muscles to facilitate knee extensor contracture . so , our study results showed that the socke exercise , which easily causes quadriceps muscle contraction , can be more effective in training the vmo than the sccke exercise . a previous electromyography study showed that the ideal ratio of the vmo to vl was 1:1 in knee extension exercise for the asymptomatic knee joint5 . in our study , the ratios of the vmo to vl in the socke and sccke exercises were over the ideal ratio of 1:1 . the socke exercise is more suitable training the vmo and vl of pfps patients than the sccke exercise because it has an approaching ideal vmo to vl ratio . this means that the signals detected for muscle fibers were the same for the socke and sccke exercises . we found that large muscle fibers were recruited for vmo activity during the socke exercise , and the ratio of the vmo to vl was 1.11:1 . previous studies showed high ratios of the vmo to vl at knee extension from 60 to 180 degrees during isokinetic open chain exercise21 , 22 . we used set to suspend the lower extremity and maintain extension of the knee from 60 to 180 degrees during the socke and sccke exercises . furthermore , our results showed that both exercises can recruit vmo activity , presenting ratios of the vmo to vl higher than 1:1 . so , both exercises can improve the vmo muscle contraction of pfsp patients , and the beneficial effect on vmo to vl ratio of the socke exercise is better than that of the sccke exercise . first , the healthy individuals in this study did not have pain and symptoms related to the patella and did not conform to the muscle activity of a ptps patient . second , the small sample size , 7 participants only , means that the findings can not be generalized to all ptps patients . so , we suggest that a larger sample of symptomatic ptps patients is required in a future study .

[ purpose ] the muscle strength of the quadriceps muscle is critical in patellofemoral pain syndrome . the quadriceps muscle supplies the power for dynamic patellar movement , and the vastus medialis oblique ( vmo ) and vastus lateralis ( vl ) enable the patella to stabilize during tracking . we followed the theories about open and closed kinetic chain exercises to design two exercises , sling open chain knee extension ( socke ) exercise and sling closed chain knee extension ( sccke ) exercise . the purpose of our study was to research the changes in quadriceps muscle activity during both exercises . [ methods ] electromyographic analysis was used to explore the different effects of the two exercises . the mvc% was calculated for the vmo and vl during exercise for analysis . [ results ] we found that the mean mvc% values of the vmo and vl during the socke exercise were higher than those during the sccke exercise . the ratio of the vmo to vl was 1.0 0.19 during the socke exercise and 1.11 0.15 during the sccke exercise . [ conclusions ] the socke exercise is targeted at quadriceps muscle training and has a recruitment effect on the vmo . the beneficial effect of the socke exercise is better than that of the sccke exercise .

image 1organismbacillus oleroniusstraindsm 9356sequencerillumina miseqdata formatassembledexperimental factorsgenome sequence of pure microbial cultureexperimental featuresgenome sequence followed by assembly and annotationconsentn / asample sourcele d'oleron , france the genus bacillus is a group of gram - positive , rod - shaped bacteria distributed extensively in the environment . their ubiquity in nature is because of their ability to produce endospores during adverse conditions . bacillus species includes pathogens of clinical significance , bacterial contaminants in food and as important industrial organisms producing various enzymes . bacillus oleronius is a non - motile endospore - forming bacterium which was originally isolated from the hindgut of the termite reticulitermes santonensis ( feytaud ) , where it plays a symbiotic role by aiding digestion . it is also found in the human skin parasitic mite demodex folliculorum , and is suspected to be related to the development of rosacea , a chronic inflammatory dermatological condition in humans . a school of thought is that demodex mites are vectors for bacteria including staphylococcus albus and microsporon canis , and of interest , b. oleronius that cause and exacerbate skin lesions . despite staining gram - negative , b. oleronius has gram - positive cell wall components shared amongst all bacillus species and thus closely related to other bacillus sp . that contaminate foods . although initially isolated from the hindgut of the termite and subsequently from mites , b. oleronius has been identified as a potential contaminant of milk and dairy products and has been sporadically identified and isolated from fodder , raw milk and milk processing equipment , , , . b. oleronius is associated with the bacillus firmus - lentus group , with its 16s rrna sequence 95.6% and 95.5% similar to that of b. lentus and b. firmus respectively . however , its closest phylogenetic neighbor is the highly heat resistant spore forming b. sporothermodurans , which may survive ultra - high temperature processing conditions during milk processing . albeit not as heat resistant as b. sporothermodurans , b. oleronius spores has been known to survive milk pasteurization , with spores isolated after 30 min heating at 100 c . in this study , the type strain rt10 ( dsm 9356 ) of b. oleronius , procured from the leibniz institute in germany was selected for whole genome sequencing . the principal reason is to enhance understanding of this bacterium in relation to other spore - forming bacillus species of importance to the dairy industry . overnight fresh culture of b. oleronius was inoculated into nutrient agar broth ( oxoid , uk ) and incubated at 37 c for 24 h. genomic dna was extracted using the zr bacterial dna miniprep kit ( zymo research , usa ) . dna extract was quantified using the qubit instrument and dsdna br assay kit ( life technologies , usa ) . multiplexed paired - end libraries were prepared using nextera xt dna sample preparation kit ( illumina , usa ) . genome sequencing was carried out on an illumina miseq system ( illumina , san diego , usa ) . the paired - end reads were checked for quality , trimmed and de novo assembled using the qiagen clc genomics workbench version 9 ( qiagen , netherlands ) . all resultant contigs were then submitted to genbank , where gene annotation was implemented using the ncbi prokaryotic genome annotation pipeline ( pgap ) . the annotation was further uploaded to rapid annotation using subsystem technology ( rast ) for subsystems - based annotation , , . the assembly contains 587 contig sequences of longer than 500 bp , covers 5,083,966 bp with g + c content of 35.00% , an n50 of 543,331 bp and a longest contig size of 90,648 bp . the total number of 5168 genes predicted by pgap includes 4899 protein coding genes , 130 pseudo genes , and 139 rna genes ( table 1 ) . the rast annotation assigned these genes into 462 subsystems , with maximum number of genes associated with amino acids and derivatives metabolism ( 14.84% ) , followed by carbohydrates ( 13.89% ) and protein metabolism subsystems ( 9.10% ) ( fig . 1distribution and counts of genes in cog categories for genome of bacillus oleronius strain dsm 9356.fig . oleronius dsm 9356 genome characteristics and resources.table 1s . nonamegenome characteristics and resources1ncbi bioproject idprjna3622822ncbi biosample idsamn062371563ncbi genome accession numbermtla000000004sequence typeillumina miseq5total number of reads2,305,9326read length3007overall coverage > 100 8estimated genome size5,083,966 bp9 g + c content ( % ) 35.0010genes ( total)516811protein coding genes489912trna coding genes11613rrna coding genes1814ncrna coding genes515pseudogenes130 distribution and counts of genes in cog categories for genome of bacillus oleronius strain dsm 9356 . the draft genome sequence of b. oleronius dsm 9356 has been deposited at ncbi under the bioproject number prjna362282 , biosample number samn06237156 and accession number mtla00000000 . the draft genome sequence of b. oleronius dsm 9356 has been deposited at ncbi under the bioproject number prjna362282 , biosample number samn06237156 and accession number mtla00000000 .

bacillus oleronius strain dsm 9356 isolated from the termite reticulitermes santonensis was sequenced to gain insights in relation to its closest phylogenetic neighbor bacillus sporothermodurans . the draft genome of strain dsm 9356 contains 5,083,966 bp with an estimated g + c content of 35% , 4899 protein - coding genes , 116 trnas and 18 rrnas . the rast annotation assigned these genes into 462 subsystems , with the maximum number of genes associated with amino acids and derivatives metabolism ( 14.84% ) , followed by carbohydrates ( 13.89% ) and protein metabolism subsystems ( 9.10% ) . the draft genome sequence and annotation has been deposited at ncbi under the accession number mtla00000000 .

the term neurodegeneration defines the progressive loss of function and structure of neurons that finally ends up with neuronal cell death . since neurodegeneration is a multifactorial and hence , a very complicated process , its exact mechanism still remains unknown , and its origin of onset is a type of chicken and egg situation . despite its multifaceted nature , it is known that over - activation of cysteine - protease calpains as a result of destabilization of calcium homeostasis is one of the main causative factors for neurodegeneration . there are number of neurodegenerative conditions such as amyloid beta ( a ) aggregation , metabolic alterations , and oxidative stress , which all cause destabilization of calcium homeostasis , resulting in the elevation of intracellular calcium levels , which in turn finally leads to pathological activation of calcium - sensitive cysteine - protease , calpain ( figure 1 ) . calpain is known to be one of the most effective proteins of which deregulation leads neurons into apoptosis through different pathways.14 calpains are heterodimeric proteins that are composed of a large 80 kda catalytic subunit and a small 30 kda regulatory subunit . domain v is the terminal domain and possesses glycine residues , while domain vi has five helix - loop - helix structural ef - hand calcium - binding motifs.5 the large 80 kda subunit is composed of four domains : i , ii , iii , and iv . domain i is the autolytic cleavage site , and domain ii has the cysteine protease activity and interacts with the substrates . domain iv contains e - helix - loop - f - helix motifs ( ef hands ) on which are calcium binding sites.6 calpain is localized in the cytosol in its inactive form in the absence of calcium . upon an increase in intracellular calcium level , calpain is translocated to the membrane , and there , it becomes activated with calcium and phospholipids . two subdomains of protease domain ii ( iia and iib ) are separated by structural constraints . in the first stage of activation , this structural constraint is released in the presence of calcium ions , which is a necessity to activate the calpain and to form the active catalytic site.7 calcium binds to ef - hand motifs in domains iii , iv , and vi , and this binding separates domain ii from domain iii . therefore , a 30 kda subunit dissociates from an 80 kda subunit at the end of the first stage . at the second stage , an active site on the protease domain ii is rearranged to be able to interact with its substrates.8 although calpain activity is strictly regulated , mainly by calcium , there is also an endogenous inhibitor of calpain , namely calpastatin , which regulates the activity of calpain.9 calpastatin reversibly binds to the active site and inhibits calpain only in the presence of calcium ions.10 activated calpain has a number of substrates such as growth factor receptors , cytoskeletal proteins , microtubule - associated proteins , and mitochondria in order to fulfill crucial roles in different cellular mechanisms such as progression of cell cycle,11 differentiation , apoptosis , long - term potentiation ( ltp ) , synaptic plasticity , and central nervous system ( cns ) development in neurons.12,13 ltp is a calcium - dependent regulator of memory development that results , in part , from remodeling of dendritic spines.14 activation of n - methyl - d - aspartate ( nmda ) receptors , which results in the activation of calpains and proteolysis of structural proteins such as spectrin,15 plays a crucial role in the induction of ltp.14 different studies also showed that calpain inhibition caused rapid axon retraction , attributing a role for calpain in axon maturation and maintenance during cns development.16,17 recent studies have indicated that calpains play significant roles in the apoptotic processes in the case of neurodegeneration.1,18,19 deregulation of calcium homeostasis results in calcium overload , and subsequent calpain over - activation forces neurons to ultimately undergo apoptosis for the reason that members of apoptotic machinery , such as b - cell lymphoma 2 ( bcl-2 ) family members of cell death regulators and nuclear transcription factors such as p53 and caspases are themselves direct substrates of calpains.20 several members of the bcl-2 family proteins that regulate programmed cell death21 are processed by calpains.22 putcha et al23 showed that bax ( a bcl-2 family member ) translocation from cytosol to mitochondria is a key event in neuronal apoptosis in the trophic factor - deprived sympathetic neuron model . the role of calpain in this process is to cleave bax into an 18-kda pro - apoptotic fragment that leads to cytochrome c release and subsequently , apoptosis.24 in addition to this , sedarous et al16 showed that inhibiting calpain by pharmacological calpain inhibitors ( mdl-28170 and pd 150606 ) significantly reduced p53 induction and subsequent cytochrome c release , and caspase-3 increase in embryonic cortical neurons , indicating that calpain is a key mediator of p53 induction and caspase - dependent apoptosis . in addition , experimental data suggest some neuroprotective effects for inhibitors of calpain and caspase in neurons.6,25 an increased body of evidence indicates an interaction between calpain and caspase proteolytic systems . caspases can also play a role in the degradation process of the specific endogenous calpain inhibitor , calpastatin , accelerating calpain activation . moreover , in an experiment using ultraviolet radiation to trigger apoptosis , it has been shown that calpain activity is required for caspase-3 activation.25,26 these findings obviously indicate that both calpain and caspase proteolytic systems are involved in the progression of neuronal death.27,28 since pathological calpain activation is one of the most important neurodegenerative factors causing activation of apoptotic machinery , it is crucial to develop effective and reliable approaches to prevent calpain - mediated apoptosis in degenerating neurons . an increasing number of studies demonstrated that there are many kinds of different stimuli that trigger pathological calpain activation.2931 thus , depending on the type of stimuli , there are numerous strategies developed by distinct research groups to inhibit apoptotic effects of calpain over - activity . although using calpain inhibitors is the most frequently applied strategy for the blockade of calpain - mediated apoptosis in neurodegeneration , considering its disadvantages , some other neuroprotective strategies are also being utilized . the most frequently used methods for this reason are over - expression or inhibition of certain proteins , especially those are involved in glutamate receptor signaling or using receptor antagonists , testing certain hormones and their receptors for their neuroprotective activities or designing competitive peptides to inhibit calpain s enzymatic activity are also utilized . glutamate is an important neurotransmitter of the cns that functions in many physiological cellular events through activating glutamate receptors.32,33 on the other hand , glutamate can be toxic for neurons in the case of excessive or prolonged exposure , which is known as glutamate neurotoxicity.34,35 glutamate neurotoxicity is known to be a major factor in a number of chronic neurodegenerative disorders such as amyotrophic lateral sclerosis and alzheimer s disease.36 it has been shown that abnormal ca influx through glutamate receptors is a part of glutamate neurotoxicity , which results in activation of certain enzymes such as calpain , leading to cleavage and degradation of proteins , membranes , and nucleic acids.37 although the underlying mechanisms of glutamate neurotoxicity are not completely known , the nmda receptor ( nmdar)-mediated ca overload and subsequent calpain activation have been indicated as strong candidates.38,39 there is a growing body of evidence indicating involvement of nmdars in calpain - mediated neuronal injury and neuronal death . in a study investigating the role of calpain in glutamate - induced retinal neuron injury , glutamate treatment has been shown to induce apoptosis by elevating ca influx and protein levels of calpain 2 and calpain - specific alpha - spectrin breakdown products ( sbdps).40 together with calpain induction , an increase in cyclin - dependent kinase 5 ( cdk5 ) and its co - activator p35 protein levels have been determined . under normal conditions , p35 is the partner for cdk5 , which is a non - mitotic neuron - specific kinase , and a cdk5/p35 complex is formed in important cellular events such as neuronal development and maturation.4143 however , in the case of neuronal calpain over - activation , calpain cleaves p35 into p25 and p10 fractions , as p35 is a substrate of calpain . however , p25 causes prolonged activation and mislocalization of cdk5.44 owing to prolonged activation of cdk5 by p25 , the calpain / p35p25/cdk5/nmdar signaling pathway is implicated in playing a role in neurological disorders by phosphorylation of nmdar subunit nr2a at the ser1232 site ( p - nr2as1232 ) , induction of the functional nmdarsexpression in the cell membrane , and subsequent glutamate - induced increase of ca.45,46 chen et al45 has utilized an nmdar antagonist , d-2-amino-5-phosphonovalerate and a non - nmdar antagonist , 6-cyano-7-nitroquinoxaline-2 , 3-dione to protect neurons against glutamate - induced increase of ca and as a result , elevation of calpain and its cleavage product sbdps have been shown to be attenuated . although different types of glutamate receptor antagonists , specifically those for nmdars , have been successfully utilized in both in vitro and in vivo studies , they have failed categorically in clinical trials , because completely blocking nmdars causes serious side effects , such as memory impairment , psychosis , and nausea.47 thus , there is a growing effort to search for alternative ways of inhibiting glutamate excitotoxicity in order to minimize these side effects . such an alternative strategy has been developed in an ischemic neuronal injury study.48 it has been previously reported that calpain - mediated cleavage of metabotropic glutamate receptor 1 ( mglur1 ) had an important role in excitotoxicity , and a transactivating regulatory protein ( tat)-mglur1 peptide , which was developed as an alternative to general receptor antagonists , was neuroprotective against this excito - toxicity.49 this peptide includes the calpain cleavage site of mglur1 and the peptide transduction domain of the tat of human immunodeficiency virus ( hiv ) . since over - activation of glutamate receptors is also widely recognized as an initiating factor in ischemic neuronal death , zhou et al48 tested this peptide in in vivo and in vitro neonatal hypoxia / ischemia ( h / i ) models for its neuroprotective effect . results have shown that the tat - mglur1 peptide is preventive against h / i - induced neuronal death in neonatal rats . the pathway of neuroprotection for the tat - mglur1 peptide is through blockade of calpain - mediated h / i - induced mglur1 degradation . however , calpain - mediated h / i - induced spectrin degradation is not affected by this peptide , suggesting that the neuroprotective effect of the peptide depends on inhibition of calpain - mediated mglur1 degradation , not on calpain inhibition . the results of this study indicated that tat - mglur1 peptide may be a strong alternative to prevent h / i - induced cytotoxic events , since it has the potential to cause milder side effects compared to those of glutamate receptor antagonists and calpain inhibitors . one of the important features of nmdar is its potential to form different subunit compositions that provide an advantage of having a number of receptor subtypes with different signaling and synaptic targeting characteristics . nmdars are composed of an essential glun1 subunit and different combinations of glun2 ( a d ) and glun3 ( a b ) subunits . in contrast with glun1/glun2 heteromers , which are well recognized by their high permeability to ca , glun3 subunits decrease nmdars ca permeability by at least tenfold.50,51 when this inhibitory glun3 subunit is involved in the subunit composition , it causes both reduction in ca influx via nmdar channels and alteration of synaptic targeting properties of the channel , indicating elimination of two neurodegenerative hallmarks of nmdars . a recent study , utilizing the neuroprotective potential of glun3a subunits against neurotoxin 3-nitropropionic acid ( 3-np)-induced striatal excitotoxic damage in glun3a over - expressing transgenic mice has demonstrated that mild over - expression of glun3a protected striatal neurons from excitotoxic damage of 3-np.52 results have also shown that glun3a - mediated neuroprotection is dose - dependent and potentially associated with inhibition of a ca - dependent protease , calpain , which is evident from the decrease in 3-np - induced cleavage of fodrin and striatal - enriched protein tyrosine phosphatase ( step ) by calpain as compared to controls . attempts at treating neurodegenerative diseases utilizing nmdar antagonists have far been unsatisfactory because it is difficult for most nmdars antagonists to efficiently cross the blood brain barrier ( bbb ) to reach their target.53,54 even if an inhibitor / antagonist can cross the bbb , direct inhibition of nmdars has major drawbacks in that glutamate receptors assume a role in numerous vital cellular processes , and blockade of them may result in failure of these cellular processes.5557 thus , prevention of the downstream processes of excess glutamate receptor induction such as calpain over - activation , which drives neurons into apoptosis , may present a broader range of opportunities for neuroprotection . calpain inhibition may provide advantages over glutamate receptor antagonists in that calpain is predominantly in its inactive proenzyme form under normal physiological conditions , and its significant activation occurs only under pathological conditions . although there is an intrinsic inhibitor of calpain named calpastatin , it has large active polypeptide fragments preventing it from crossing membrane barriers , which strongly reduces its therapeutic potential.58 by considering this problem , on the one hand , researchers have focused on designing exogenous small calpain inhibitor peptides , peptide - mimetics , or non - peptidic analogs that have good cell permeability and low toxicity ; on the other hand , researchers have been exploring novel strategies to overexpress or intrinsically up - regulate calpastatin.5962 a number of reversible or irreversible exogenous calpain inhibitors such as ak275,63 mdl-28170,64 pd150606,62,65 sja6017,66 a-705253,67,68 snj-1945,69 and calpeptin70 are frequently used in experimental studies ( table 1 ) . researchers continually work on enhancing stability , specificity , potency , and efficiency of the synthetic calpain inhibitors by developing third and even fourth generation calpain inhibitors with improved pharmacological profiles.7173 ak275 is one of the prototypic calpain inhibitors . in the very first exogenous calpain inhibitor experiments , ak275 was among the mostly preferred calpain inhibitors due to its selectivity , membrane permeability , solubility , and in vitro efficiency . bartus et al63 tested ak275 for the first time for its neuroprotective effect on cortical ischemic damage - associated focal ischemia in rats . over - activated calpain is one of the most potent participants of ischemic brain injury through different pathways such as hydrolysis of cytoskeletal and membrane proteins,74,75 and subsequent activation of certain enzymes like apoptotic caspase-3 , which causes neuronal death . for this reason , these researchers used the middle cerebral artery occlusion method to induce focal ischemia , and they applied ak275 through supracortical perfusion . results showed that abnormal calpain activity is an important factor for ischemia and that ak275 was a relatively reliable neuroprotectant for the treatment of focal brain ischemia . in addition to ak275 , calpeptin is another calpain inhibitor that may have an important function in protecting neurons against focal cerebral ischemia reperfusion injury . reperfusion injury experiments in rat models to prove the neuroprotective effect of calpeptin through a potential mechanism of caspase-3 inhibition . the results showed that calpeptin reduced neuronal apoptosis in the hippocampal ca1 section of focal - cerebral ischemia reperfusion - subjected rat brains by inhibiting caspase-3 expression . this caspase-3 inhibition may be due to inhibition of calpain , since it is well known that calpains can directly or indirectly modulate and activate p53 , which in turn causes caspase-3 activation.16 however , there is still need for further studies to reveal the exact mechanism of caspase-3 inhibition by calpeptin . mdl-28170 is another first generation calpain inhibitor which is membrane permeable and selective for calpain . it was used for the first time to prevent proteolysis of erythrocyte membrane - associated cytoskeletal proteins in rats.76 after that , brorson et al64 performed experiments using mdl-28170 to prevent glutamate receptor - mediated neurotoxicity , which is known to cause ca - dependent calpain over - activation . mdl-28170 was shown to successfully block proteolytic activity of calpain together with the advantage of limiting excitotoxicity even when applied 1 hour after calpain s toxic effects had been recorded . in addition , mdl-28170 has also been shown to be neuroprotective against both neuronal apoptosis occurring as a result of oxidative stress and subsequent calpain activation through intracellular ca - overload.77 in this study , using h2o2 to generate oxidative stress and using a23187 calcium ionophores to provide intracellular ca overload on neuron - like pc12 cells indicated significant increases both in calpain activity and mrna expression of pro - apoptotic bax gene . however , pretreatment of pc12 cells with mdl-28170 has been shown to prevent both calpain activity and an increase in mrna expression of pro - apoptotic bax gene , also suggesting a role for calpain in oxidative stress and ca influx - dependent neurodegeneration.77 a relatively less cytotoxic calpain inhibitor compared to other members of its class ( such as mdl-28170 , and calpain inhibitor i and ii ) , named sja6017 , has been studied in diffuse traumatic brain injury mice models.66 this was the first use of a calpain inhibitor in a diffuse traumatic brain injury model , and results have shown that sja6017 , when administered early ( 20 minutes post - injury ) , provided an improvement on functional outcome after 24 hours of injury , indicating a possible therapeutic role for calpain inhibitors in traumatic brain injuries . since calpain is well characterized as a part of the nmdar - mediated neurodegeneration , inhibition of cal - pain , instead of direct inhibition of nmdar activity , is a rational way to prevent neurodegeneration in order not to interfere with nmdar - mediated regulation of ltp and cognition . thus , nimmrich et al68 studied cholinergic neurons in disease - related animal models , which had excitotoxic lesions on cholinergic nucleus basalis magnocellularis of meynert ; these animal models also had a calpain inhibitor a-705253 to prevent excitotoxicity - induced neuronal decline without interfering with learning and memory processes . these researchers have shown that excitotoxicity seriously impeded novel object recognition ability , and that use of a-705253 calpain inhibitor prevented both this deficit and accompanying gliosis in a dose - dependent manner , while it did not inhibit ltp in hippocampal slices . granic et al67 then tested a-705253 in a rat animal model of a-induced neurodegeneration in which a-induced lesions of nucleus basalis resulted in a significant decrease in cholinergic neuron number . a-705253 has been shown to prevent cholinergic neurodegeneration together with accompanied neuroinflammatory response , in a dose - dependent manner.67 from the results of this study , it can be concluded that calpain inhibition may also be a useful strategy to prevent a-induced neurodegeneration . on the other hand , in order to unravel the mechanisms of degeneration occurring in spinal cord neurons in the case of parkinson s disease , snj-1945 , a cell - permeable calpain inhibitor , has been tested for its neuroprotective activity in differentiated sh - sy5y neuroblastoma cells that were exposed to two parkinsonian neurotoxicants , the toxic cation 1-methyl-4-phenylpyridinium ( mpp+ ) and rotenone to generate an experimental parkinsonism model.58 mpp+ and rotenone , which have been previously shown to elevate the intracellular free ca levels and induce calpain over - activation in vsc 4.1 cells.64,78 the results of the study showed that snj-1945 pre - treatment significantly protected cells in terms of viability and cellular morphology against mpp+ and rotenone exposure , indicating that calpain inhibition is the true strategy to protect neurons against parkinsonian neurotoxicants , and in this context , snj-1945 is a strong inhibitor of calpain.58 neuroprotective effect of snj-1945 has also been evaluated to reduce retinal cell death caused by retinal cell damage . researchers induced in vivo retinal damage in mice by injecting nmda intravitreally , and then they administered snj-1945.79 the in vitro part of the study was performed by inducing cell damage by using a 4-hour oxygen - glucose deprivation treatment followed by an 18-hour reoxygenation period in rgc-5 ( a rat retinal ganglion cell line ) cell culture . nmda injection has been shown to cause calpain activation in vivo and in vitro by immunochemically measuring its cleaved products , spectrin and p35 . the results of the study showed that snj-1945 significantly attenuated calpain activity , and reduced cell loss and also the number of positive cells for terminal deoxynucleotidyl transferase nick labeling in vivo and in vitro.79 these results not only indicate that calpain activation may be the key mechanism for retinal cell death , but also indicate that snj-1945 may be an effective neuroprotector in retinal diseases . despite their relatively efficient neuroprotective functions , there is a major limitation for the clinical use of available synthetic calpain inhibitors because of these inhibitors lack of specificity towards calpains among other cysteine proteases and other proteolytic enzymes and related risk for these calpain inhibitors of inhibiting cysteine proteases other than calpains . thus , developing alternative calpain inhibitors that have interaction with sites different from the catalytic site of calpain may provide enhanced specificity and potential for clinical use . alpha - mercaptoacrylate derivative pd150606 is one of these alternative kinds of calpain inhibitors , due to its relatively high specificity for calpains . it was described as a newly discovered class of calpain inhibitors by wang and yuen62 in 1994 , owing to its feature of uncompetitive inhibition with respect to substrate . since most calpain inhibitors are the peptides competing for the catalytic site of the protease , pd150606 differs from those inhibitors by its unique interaction with the calcium binding site of calpain rather than its catalytic site . the neuroprotective effect of pd150606 was shown for the first time by revealing its capability to inhibit hypoxic / hypoglycemic injury to cerebrocortical neurons and excitotoxic injury to purkinje cells.63 moreover , neuroprotection through pd150606 has also been tested in serum / potassium withdrawal - induced apoptosis of cerebellar granule cells.65 this research has demonstrated that 40 mm pd150606 prevented serum / potassium withdrawal - mediated apoptosis by inhibiting calpain , indicating a therapeutic role for this type of calpain inhibitor . although synthetic calpain inhibitors ( e64 , bda-410 , etc ) may also protect neurons in slow degenerative events such as alzheimer s disease80,81 and in ischemic and traumatic injuries , the range of neurodegenerative conditions in which calpain inhibitors could provide benefits still remains uncertain . most of the calpain inhibitor experiments up to now generally involved ischemic and traumatic injuries to the cns , and there are a few studies showing the efficiency of calpain inhibitors in slow - progressing neurodegenerative diseases.82 besides , calpain inhibitors , not having a complete specificity for calpain , can modulate other cysteine or serine proteases , which can have unknown effects on the progression of neurodegeneration.60 thus , developing strategies for inhibiting downstream apoptotic processes of calpain over - activation rather than direct inhibition of calpain may be of great importance to eliminate all of the disadvantages of using calpain inhibitors . even though there have been many attempts to develop effective calpain inhibitors , none of the calpain inhibitors has been approved for clinical use.83 this is probably due to the low specificity of existing calpain inhibitors . besides , combination of the nature and extent of enzyme inhibition is also important for effective inhibition of neuropathologic calpain activity . thus , there is still a need for more efficient pharmacological approaches with reduced side effects . considering these negative aspects , in our laboratory , we have been searching for non - synthetic inhibitor candidates for calpain - mediated apoptosis in neurodegeneration . a novel and alternative cell cycle regulator protein speedy / ringo is quite remarkable in this respect,84 due to its attributed additional function in preventing caspase - dependent apoptosis in mitotic u2os human osteosarcoma cells.85 as emphasized before , the calcium - activated proteolytic enzyme calpain is one of the key proteins that can directly or indirectly drive neurons into apoptosis . the indirect method is through cdk5 , a non - mitotic kinase , which is up - regulated through calpain over - activation , a process followed by a subsequent increase in p53 and active caspase-3 levels under neurodegenerative conditions.8688 the direct method is the up - regulation of p53 by calpain itself , since it has been demonstrated that p53 can be modulated and activated by calpains.16 speedy / ringo is an atypical cell cycle regulator , synthesized only in mitotic cells , which has been shown to have protective effects in mitotic cells against apoptosis by inhibiting caspase-3 activation in a p53-dependent manner.85,89 our aim was to reveal possible protective effects of speedy / ringo against calpain - induced caspase-3 activation in post mitotic neurons , which is crucial in terms of providing novel insights in preventing the caspase-3 activation cascade in neurodegeneration.84 for this purpose , rat primary hippocampal neurons have been transfected with speedy / ringo expression construct , and then intrinsic calpain has been over - activated via the calcium ionophore . as a result , we have shown that calpain over - activation leads to the up - regulation of p53 and a subsequent increase in active caspase-3 levels in rat primary hippocampal neurons , indicating activation of apoptotic machinery in neurons . this calpain - directed caspase-3 activation upon up - regulation of p53 is prevented by the expression of speedy / ringo in rat hippocampal neurons . furthermore , 4,6-diamidino-2-phenylindole ( dapi ) staining and terminal deoxynucleotidyl transferase dutp nick end labeling assays have been performed for detection of apoptosis . apoptotic analysis supported the finding that calpain over - activated neurons occurred in the early apoptotic stage , while calpain over - activated speedy / ringo - expressing neurons were not apoptotic.84 therefore , although its mechanism of action requires further elucidation , speedy / ringo may be a novel and alternative neuroprotectant , and it may act as a savior for neurons that are under apoptosis as a result of calpain activation due to caspase-3 activation . on the other hand , there are efforts to up - regulate the intrinsic calpain inhibitor , calpastatin , because it may overcome the specificity , efficiency , water solubility , and bbb - crossing problems that synthetic , exogenous calpain inhibitors have.60,90 taurine ( 2-aminoethanesulfonic acid ) , which is an organic acid widely distributed in mammalian tissues , has been utilized for this reason in an experimental study by sun and xu.91 taurine has a high cytoprotective potential due to its functions as a neurotransmitter , neuromodulator , modulator of intracellular calcium homeostasis , and anti - oxidant factor.9294 sun and xu91 have investigated the neuroprotective role of intravenously administered taurine through affecting calpain / calpastatin actions in a rat model of focal cerebral ischemia . results have indicated that taurine exerted dose - dependent neuroprotection by the mechanism of inhibiting the m - calpain- and caspase-3-mediated apoptotic neuronal death via increasing calpastatin synthesis.91 in order to use any substance as a neuroprotective agent , it is a prerequisite for that substance to have the ability to pass through the bbb . in this context , there is supporting evidence that taurine may pass through the bbb , and hence , may be used as a neuroprotective agent in brain ischemia ; for example , the demonstration of increased taurine levels in the brain after systemic administration of taurine indicates its ability to pass through the bbb.95,96 identification of the carrier - mediated transport of taurine from vascular space to the brain97 and indication of linearly increased taurine levels in rat brains after intraperitoneal injection also support the idea that taurine can cross the bbb.98 in addition to up - regulating endogenous calpastatin , over - expressing it using expression vectors to inhibit calpain activity is also noteworthy . in a recent study , this technique has been utilized to inhibit calpain , which is suspected to be responsible for generation of toxic ataxin 3 fragments and hence pathogenesis of the machado joseph disease , the most frequently found type of cerebellar ataxia.99 this study has produced adeno - associated viral vectors for overexpression of calpastatin and researchers have injected this viral construct into machado this study has demonstrated that calpain inhibition by over - expressed calpastatin significantly reduced the size and number of toxic ataxin 3 fragments and limited the progression of neurodegeneration in machado in contrast , calpastatin has been found to be markedly depleted in alzheimer s disease brains of patients which in turn would cause uncontrolled activation of calpain.95 based on this finding , rao et al100 have produced human calpastatin over - expressing hcast mice through an expression cassette containing thy-1.1 promoter to investigate the effects of calpastatin in kainic acid - induced neurotoxicity , which has been shown to be closely associated with alzheimer s disease.100 the results of this study have demonstrated that higher calpastatin levels inhibited kainic acid - induced cytoskeletal protein disruption , neurodegeneration , and accompanying reactive gliosis . although it is believed that calpain inhibition would have relatively less side effects,101,102 considering its inactive proenzyme form under normal physiological conditions , when necessary , upon high levels of calcium influx , calpain family members are activated and become crucially involved in multiple metabolic / regulatory pathways such as the cell cycle , differentiation , apoptosis , synaptic plasticity , and cns development.5,9 therefore , there still exist some limitations related to therapeutic use of calpain inhibitors , because multiple physiological functions of calpains might be disrupted by calpain inhibitors . thus , approaches other than using synthetic calpain inhibitors , which may be unpractical and insufficient , may be strong alternatives for the treatment of neurological injuries and neurodegenerative disorders . in this context , using non - synthetic calpain - mediated apoptosis inhibitors such as speedy / ringo may be more realistic and beneficial in order not to interfere with the vital physiological functions of calpains in the cellular environment . the female sex hormone estrogen has effects on the structure and function of the nervous system , such as supporting synaptic plasticity and preventing death of neurons , because brain regions related to sex differentiation have estrogen receptors . together with this function , estrogen is also a neuroprotective anti - oxidant that may interact with neuroprotective signaling pathways in neurons.103 there is a growing body of evidence demonstrating that hormone replacement therapy , which is defined as using different types of estrogen alone or together with progestins , is very efficacious against a number of neurodegenerative conditions , such as alzheimer s disease104,105 and parkinson s disease.106,107 by taking into consideration this neuroprotective role of estrogen , researchers have been studying to find out if preventing calpain - mediated neurodegeneration via estrogen hormone and its receptors would be a convenient and powerful approach . for this purpose , gamerdinger et al108 have analyzed estrogen and its receptors , er and er , with respect to their neuroprotective effect on over - expression of calpains under a massive attack of ca influx in er- and er-transfected human neuroblastoma sk - n - mc cells . the results of the study have revealed that calpain expression is inhibited by estrogen / er in a receptor subtype - specific fashion , since only er exerts this neuroprotective effect , not er. these data suggest an estrogen receptor subtype - specific neuroprotective action for calpain over - expressed degenerating neurons . in addition , the neuroprotective role of estrogen has recently been tested specifically against amyloid toxicity by using an alzheimer s disease rat model , which is produced by intra - hippocampal a140 injection.109 a140 treatment has been shown to cause at least two times more calpain activation in the cerebral cortex , suggesting a role for calpains in amyloid toxicity . however , introduction of estrogen has been demonstrated to reduce a140 peptide levels in rat brain , and it has also been demonstrated that rats on estrogen therapy have not developed a behavioral dysfunction . estrogen therapy has also been indicated to normalize a140-injected rats proteolytic systems , both in the hippocampus and cortex regions . this effect of estrogen may be due to selective estrogen - dependent calpain synthesis , which has been indicated in a study about estrogen treatment of spinal cord injury;110 estrogen may cause reduction in calpain s enzymatic activity , even under massive ca influx , indicating an enhanced tolerance against ca toxicity . since estrogen is a steroid hormone that has effects on multiple metabolic pathways , it is not easy to develop a single estrogen - based approach for the treatment of calpain - mediated neurodegeneration . however , it is still a strong therapeutic candidate as long as its mechanism of action is further analyzed . given the multifaceted nature of neurodegeneration , it does not seem to be easy to prevent onset or progression of neurodegeneration by clinically modulating a single process . however , while dissecting the underlying mechanisms of neurodegeneration , pathologic calpain activation has attracted great attention by many researchers for the reason that ca - activated calpains have been shown to be activated and to trigger apoptotic machinery through different pathways in degenerating neurons.14 the main reason for abnormal calpain activation is known to be the massive ca influx that is frequently seen under neurodegenerative conditions.9,111,112 glutamate neurotoxicity is one of the processes by which excessive ca influx is involved , and this process results in calpain over - activation , leading to neuronal apoptosis.37 thus , many researchers have focused on both revealing the exact mechanism of glutamate receptor - related calpain activation , and on developing neuroprotective strategies in the context of glutamate receptor - related calpain activation . for this purpose , they have mainly performed experiments aimed at direct blockade of receptor activity to prevent excessive ca influx ; they have used general receptor antagonists,45 alternative receptor blocking peptides,48 or over - expressing inhibitory subunits of the glutamate receptor,52 which reduces its activity . in contrast with its success in preventing calpain activation , direct blocking of glutamate receptors has too many drawbacks ; direct blocking can result in disruption of cellular and metabolic integrity , because glutamate receptor activity is vital for regulating neuronal processes such as synaptic plasticity and memory function.5557 from this point of view , rather than direct inhibition of glutamate receptor activity , studying of the inhibition of its downstream events , such as inhibiting calpain itself , sounds more realistic and beneficial if neuronal integrity is to be sustained . a number of synthetic peptide or non - peptide calpain inhibitors have been utilized for this purpose by different research groups . however , during these studies , many problems have arisen about specificity , efficiency , stability , and ability of these synthetic calpain inhibitors to cross the bbb.60 in order to overcome these problems , rather than complete inhibition of calpain , bbb - crossing competitive peptides or peptidomimetics , which inhibits active site of calpain or mimic certain substrates of calpain that are involved in neurodegenerative progress , can be designed to selectively prevent calpain s degradation activity on these substrates.113 in addition , for efficient and target - oriented delivery of these designed peptides to a desired location , a biocompatible delivery system can be used . examples of such delivery systems may be a fusion peptide , an antibody conjugate , an engineered virus , nanoparticles , or liposomes.114,115 on the other hand , apart from designing partial or complete calpain inhibitors , in order not to impede the essential functions of calpain , alternative approaches especially targeting downstream apoptotic events of calpain over - activation such as over - expression of anti - apoptotic speedy / ringo have successfully been tested in consideration of being beneficial against calpain - mediated apoptosis of neurons.84 lastly , as a less studied approach , steroid hormone estrogen has been examined for preventing pathologic calpain activation in neurons considering its well - known neuroprotective properties.108 results of these studies have shown that estrogen may be an effective neuroprotector against calpain - mediated neurodegeneration as long as some of its side effects are eliminated through a comprehensive understanding of its mechanism of action . taken together , studies reviewed here indicate the ever- growing picture of efficient , mechanism - based , target - oriented , feasible neuroprotective strategies against calpain - based neurodegeneration . taking into consideration all the negative aspects of available methods , it is essential to change direction to more specific , efficient , practical , less harmful , and most importantly , target - oriented novel strategies to overcome apoptotic effects of calpain activation in neurons . last , but not the least , not only calpain - mediated neurodegeneration , but also other candidate underlying mechanisms of neuronal degeneration , point toward the development of a number of neuroprotective strategies that still require further analysis before they can be introduced into clinical practice . although calpain over - activity is one of the most important and most potent underlying reasons for neurodegeneration , in order to remain realistic in our expectations , a multivisioned approach to developing therapeutic strategies for more than one degenerative mechanism is needed .

calpains are calcium - dependent proteolytic enzymes that have deleterious effects on neurons upon their pathological over - activation . according to the results of numerous studies to date , there is no doubt that abnormal calpain activation triggers activation and progression of apoptotic processes in neurodegeneration , leading to neuronal death . thus , it is very crucial to unravel all the aspects of calpain - mediated neurodegeneration in order to protect neurons through eliminating or at least minimizing its lethal effects . protecting neurons against calpain - activated apoptosis basically requires developing effective , reliable , and most importantly , therapeutically applicable approaches to succeed . from this aspect , the most significant studies focusing on preventing calpain - mediated neurodegeneration include blocking the n - methyl - d - aspartate ( nmda)-type glutamate receptor activities , which are closely related to calpain activation ; directly inhibiting calpain itself via intrinsic or synthetic calpain inhibitors , or inhibiting its downstream processes ; and utilizing the neuroprotectant steroid hormone estrogen and its receptors . in this review , the most remarkable neuroprotective strategies for calpain - mediated neurodegeneration are categorized and summarized with respect to their advantages and disadvantages over one another , in terms of their efficiency and applicability as a therapeutic regimen in the treatment of neurodegenerative diseases .

the steps of the procedure are shown schematically in fig . 1a . to reduce non - specific hydrodynamic breakage , dna samples were isolated from cells embedded in 0.5% low - melt agarose as described previously , , , , . about 6 million hek293 t cells in 2 ml of culture medium were pelleted by centrifugation at 2000 rpm in a minispin centrifuge ( eppendorf ) , resuspended in 0.3 ml of the same medium , gently mixed at 42 c with an equal volume of a solution of 1% low - melt agarose l ( lkb ) in pbs , and distributed on a mold containing 100-l wells . the agarose plugs were then placed in petri dishes with 5 ml of a solution containing 0.5 m edta ( ph 9.5 ) , 1% sodium lauroylsarcosine , and 12 mg of proteinase k per ml for 4048 h at 50 c , and stored at 4 c in the same solution for 3 months . each dna agarose plug usually contained about 15 g of dna , corresponding to about 1 million cells . to test the quality of isolated dna , fractionation in pulsed - field gels portions of the original agarose dna plugs ( 550 l ) containing 110 g of dna were used for electrophoresis without any restriction enzyme digestion . the dna samples were run in 0.8% agarose gels on a pulsaphor system ( lkb ) using a hexagonal electrode and switching times of 25 or 100 s. for elution of dna preparations , fractionation in a 0.8% agarose conventional mini - gel was performed . agarose plug was washed in 1 te three times ( for 15 min each ) , followed by washing ( three times ) in the same solution containing 17.4 g / ml phenylmethylsulfonyl fluoride ( pmsf ) in ethanol . after fractionation in the mini - gel , the ethidium bromide - stained dna band was excised and electroeluted inside a cellulose - membrane dialysis bag . after overnight dialysis without stirring against 1 l of 0.01 te at 4 c , the dna was concentrated with peg at 4 c . about 1.5 g of isolated dna ( see above ) was ligated with 70 ng of double - stranded oligonucleotide ( 25-bp long 5-phosphorylated 5 pcccctgcagtataaggagaattcggg 3 oligonucleotide annealed to a 26-bp long 5 biotinylated 5 bio - ccgaattctccttatactgcagggg 3 oligonucleotide ) in 150 l of a solution containing 0.1 m nacl , 50 mm tris hcl ( ph 7.4 ) , 8 mm mgcl2 , 9 mm 2-mercaptoethanol , 7 m atp , 7.5% peg , and 40 units of t4 dna ligase at 20 c for 16 h. after heating at 65 c for 10 min , the dna preparation was digested with sau3a enzyme to shorten the forum domain to the positions of the termini attached to the ligated oligonucleotide . the selection of such termini was performed in 0.5-ml eppendorf tubes using 300 l of a suspension containing streptavidin magnesphere paramagnetic particles , ( sa - pmp ; promega ) according to the manufacturer 's recommendations . after extensive washing with 0.5 ssc to remove dna fragments corresponding to the internal parts of forum domains , the forum termini ( ft ) dna preparation was eluted from the sa - pmp using digestion with ecori enzyme in a final volume of 50 l ( double - stranded ft ) . the ft were then ligated with 100 molar excess of double - stranded sau3a adaptor ( 5-phosphorylated 5 pgatcgtttgcggccgcttaagcttggg 3 oligonucleotide annealed to 5 cccaagcttaagcggccgcaaac 3 oligonucleotide ) . in some experiments , the dna preparation was eluted from the sa - pmp by incubation at 100 c for 3 min in 50 l of 0.01 te ( single - stranded ft ) . before heating , the ft preparation was ligated with a 100-fold molar excess of double - stranded sau3a adaptor in suspension with sa - pmp ( see above ) . both final dna samples ( double - stranded ft or single - stranded ft ) were used for pcr amplifications . pcr amplification ( 1520 cycles ) in 30 l of a solution containing 67 mm tris hcl ( ph 8.4 ) , 6 mm mgcl2 , 10 mm 2-mercaptoethanol , 16.6 mm ammonium sulfate , 6.7 m edta , 5 g / ml bsa , 1 mm dntps , 1 g of primer corresponding to sau3a adaptor ( 5 cccaagcttaagcggccgcaaac 3 ) , 1 g of primer corresponding to biotinylated oligonucleotide ( 5 ccgaattctccttatactgcagggg 3 ) , and 1 u of taq polymerase was performed using a mastercycler personal thermal cycler ( eppendorf ) . amplification conditions were 90 c for melting , 65 c for annealing , and 72 c for extension , for 1 min each . the final dna sample contained the amplified genome - wide preparation of dna fragments delimited by a base at a particular dsb and the nearest sau 3a site . libraries were prepared according to illumina 's instructions accompanying the dna sample kit ( part # 0801 - 0303 ) . briefly , dna was end - repaired using a combination of t4 dna polymerase , escherichia coli dna pol i large fragment ( klenow polymerase ) , and t4 polynucleotide kinase . the blunt , phosphorylated ends were treated with klenow fragment and datp to yield a protruding 3-a base for ligation of illumina 's adapters , which have a single t - base overhang at the 3 end . after adapter ligation , dna was pcr amplified with illumina primers for 15 cycles . library fragments of ~ 200400 bp and ~ 4001200 bp were isolated as bands from an agarose gel , and were sequenced on the genome analyzer iix and miseq , respectively , following the manufacturer 's protocols . the standard illumina analysis pipeline using their phix control software was used for base calling . at the first step of processing , quality control was performed using fastqc . next , reads were trimmed for raft primer sequences by use of cutadapt v. 1.3 . some options were common for both datasets :-- minimum - length = 30 --trimmed - only --quality - base = 33 --quality - cutoff = 3 -n 2 --minimum - length = 30 --trimmed - only --quality - base = 33 --quality - cutoff = 3 -n 2 the option " --trimmed - only " was used to remove from trimmed files all reads that did not have raft primers . this option setting ensures that after removal of primers the data set consists of reads of sufficient length to have contained raft primers before removal . the following options were applied to remove 5 ' attached raft primers from reads : g cccaagcttaagcggccgcaaac cutadapt was used in the paired - end mode for the paired - end illumina ga iix dataset and in the single - end mode for the single - end miseq dataset . at the next step , trimmed reads were mapped to hg19/grch37p10 in paired - end mode using bwa 0.7.5a and the mem algorithm , and by samtools 0.1.19 . final mappings were converted for further analysis into tables and formats , including bed and wig , using ad hoc perl scripts . first , all mappings that did not contain a sau3a recognition sequence ( gatc ) or contained two or more such sequences ( as a result of partial digestion ) were removed as erroneous . second , a coordinate for each dsb at the end opposite the sau3a sequence was calculated . finally , all mappings within 1 kb of each other were merged into groups , and the maximum coordinate and coverage value of the group replaced those of the individual mappings . the resulting sgr file contains the dsbs with one - nucleotide resolution and their coverage . to prepare the h3k4me3 mark dataset , the downloaded raw reads for rep1 , rep2 , and signal from encode accession wgencodeeh000953 were aligned to the same genome hg19/grch37p10 by use of bowtie v.1 with the following command line options : --best -m 1 --chunkmbs 1024 . peak calling was performed using the macs2 peak caller with the options callpeak --gsize hs to set the correct genome size . the steps of the procedure are shown schematically in fig . 1a . to reduce non - specific hydrodynamic breakage , dna samples were isolated from cells embedded in 0.5% low - melt agarose as described previously , , , , . about 6 million hek293 t cells in 2 ml of culture medium were pelleted by centrifugation at 2000 rpm in a minispin centrifuge ( eppendorf ) , resuspended in 0.3 ml of the same medium , gently mixed at 42 c with an equal volume of a solution of 1% low - melt agarose l ( lkb ) in pbs , and distributed on a mold containing 100-l wells . the agarose plugs were then placed in petri dishes with 5 ml of a solution containing 0.5 m edta ( ph 9.5 ) , 1% sodium lauroylsarcosine , and 12 mg of proteinase k per ml for 4048 h at 50 c , and stored at 4 c in the same solution for 3 months . each dna agarose plug usually contained about 15 g of dna , corresponding to about 1 million cells . to test the quality of isolated dna , fractionation in pulsed - field gels portions of the original agarose dna plugs ( 550 l ) containing 110 g of dna were used for electrophoresis without any restriction enzyme digestion . the dna samples were run in 0.8% agarose gels on a pulsaphor system ( lkb ) using a hexagonal electrode and switching times of 25 or 100 s. for elution of dna preparations , fractionation in a 0.8% agarose conventional mini - gel was performed . agarose plug was washed in 1 te three times ( for 15 min each ) , followed by washing ( three times ) in the same solution containing 17.4 g / ml phenylmethylsulfonyl fluoride ( pmsf ) in ethanol . after fractionation in the mini - gel , the ethidium bromide - stained dna band was excised and electroeluted inside a cellulose - membrane dialysis bag . after overnight dialysis without stirring against 1 l of 0.01 te at 4 c , the dna was concentrated with peg at 4 c . about 1.5 g of isolated dna ( see above ) was ligated with 70 ng of double - stranded oligonucleotide ( 25-bp long 5-phosphorylated 5 pcccctgcagtataaggagaattcggg 3 oligonucleotide annealed to a 26-bp long 5 biotinylated 5 bio - ccgaattctccttatactgcagggg 3 oligonucleotide ) in 150 l of a solution containing 0.1 m nacl , 50 mm tris hcl ( ph 7.4 ) , 8 mm mgcl2 , 9 mm 2-mercaptoethanol , 7 m atp , 7.5% peg , and 40 units of t4 dna ligase at 20 c for 16 h. after heating at 65 c for 10 min , the dna preparation was digested with sau3a enzyme to shorten the forum domain to the positions of the termini attached to the ligated oligonucleotide . the selection of such termini was performed in 0.5-ml eppendorf tubes using 300 l of a suspension containing streptavidin magnesphere paramagnetic particles , ( sa - pmp ; promega ) according to the manufacturer 's recommendations . after extensive washing with 0.5 ssc to remove dna fragments corresponding to the internal parts of forum domains , the forum termini ( ft ) dna preparation was eluted from the sa - pmp using digestion with ecori enzyme in a final volume of 50 l ( double - stranded ft ) . the ft were then ligated with 100 molar excess of double - stranded sau3a adaptor ( 5-phosphorylated 5 pgatcgtttgcggccgcttaagcttggg 3 oligonucleotide annealed to 5 cccaagcttaagcggccgcaaac 3 oligonucleotide ) . in some experiments , the dna preparation was eluted from the sa - pmp by incubation at 100 c for 3 min in 50 l of 0.01 te ( single - stranded ft ) . before heating , the ft preparation was ligated with a 100-fold molar excess of double - stranded sau3a adaptor in suspension with sa - pmp ( see above ) . both final dna samples ( double - stranded ft or single - stranded ft ) were used for pcr amplifications . pcr amplification ( 1520 cycles ) in 30 l of a solution containing 67 mm tris hcl ( ph 8.4 ) , 6 mm mgcl2 , 10 mm 2-mercaptoethanol , 16.6 mm ammonium sulfate , 6.7 m edta , 5 g / ml bsa , 1 mm dntps , 1 g of primer corresponding to sau3a adaptor ( 5 cccaagcttaagcggccgcaaac 3 ) , 1 g of primer corresponding to biotinylated oligonucleotide ( 5 ccgaattctccttatactgcagggg 3 ) , and 1 u of taq polymerase was performed using a mastercycler personal thermal cycler ( eppendorf ) . amplification conditions were 90 c for melting , 65 c for annealing , and 72 c for extension , for 1 min each . the final dna sample contained the amplified genome - wide preparation of dna fragments delimited by a base at a particular dsb and the nearest sau 3a site . libraries were prepared according to illumina 's instructions accompanying the dna sample kit ( part # 0801 - 0303 ) . briefly , dna was end - repaired using a combination of t4 dna polymerase , escherichia coli dna pol i large fragment ( klenow polymerase ) , and t4 polynucleotide kinase . the blunt , phosphorylated ends were treated with klenow fragment and datp to yield a protruding 3-a base for ligation of illumina 's adapters , which have a single t - base overhang at the 3 end . after adapter ligation , dna was pcr amplified with illumina primers for 15 cycles . library fragments of ~ 200400 bp and ~ 4001200 bp were isolated as bands from an agarose gel , and were sequenced on the genome analyzer iix and miseq , respectively , following the manufacturer 's protocols . the standard illumina analysis pipeline using their phix control software was used for base calling . at the first step of processing , next , reads were trimmed for raft primer sequences by use of cutadapt v. 1.3 . some options were common for both datasets :-- minimum - length = 30 --trimmed - only --quality - base = 33 --quality - cutoff = 3 -n 2 --minimum - length = 30 --trimmed - only --quality - base = 33 --quality - cutoff = 3 -n 2 the option " --trimmed - only " was used to remove from trimmed files all reads that did not have raft primers . this option setting ensures that after removal of primers the data set consists of reads of sufficient length to have contained raft primers before removal . the following options were applied to remove 5 ' attached raft primers from reads : g cccaagcttaagcggccgcaaac cutadapt was used in the paired - end mode for the paired - end illumina ga iix dataset and in the single - end mode for the single - end miseq dataset . at the next step , trimmed reads were mapped to hg19/grch37p10 in paired - end mode using bwa 0.7.5a and the mem algorithm , and by samtools 0.1.19 . final mappings were converted for further analysis into tables and formats , including bed and wig , using ad hoc perl scripts . first , all mappings that did not contain a sau3a recognition sequence ( gatc ) or contained two or more such sequences ( as a result of partial digestion ) were removed as erroneous . second , a coordinate for each dsb at the end opposite the sau3a sequence was calculated . finally , all mappings within 1 kb of each other were merged into groups , and the maximum coordinate and coverage value of the group replaced those of the individual mappings . the resulting sgr file contains the dsbs with one - nucleotide resolution and their coverage . to prepare the h3k4me3 mark dataset , the downloaded raw reads for rep1 , rep2 , and signal from encode accession wgencodeeh000953 were aligned to the same genome hg19/grch37p10 by use of bowtie v.1 with the following command line options : --best -m 1 --chunkmbs 1024 . peak calling was performed using the macs2 peak caller with the options callpeak --gsize hs to set the correct genome size . the raft procedure includes several steps in which very long dna molecules are manipulated in solution from the elution of dna domains to the ligation of biotinylated oligonucleotides ( steps 25 in fig . although only a gentle mixing of solution was performed , a random fragmentation of forum domains can not be excluded during these steps . nevertheless , our previous data strongly demonstrate that the level of this random hydrodynamic fragmentation of dna molecules in the conditions used is much lower than the non - random fragmentation detected at the hot spots of dsbs . the data on the distribution of hot spots of dsbs in the human genome could be used for the study of chromosomal breakage associated with regulation of gene expression and different genomic rearrangements ( translocations , inversions , and deletions ) . we studied the positional and ordering correlations between dsbs and h3k4me3 marks in the chromosomes of human hek293 t cells using genome track analyzer . the h3k4me3 mark is a well - known promoter - specific histone modification that is associated with transcription and active genes . this epigenetic mark selectively directs global tfiid recruitment to active genes , some of which are also p53 targets . the summary of correlations is shown in table 1 and demonstrates strong positional correlations between dsbs and h3k4me3 peak summits for all chromosomes of h293 t cells . interesting questions arise from the ordering correlations , which are significant only for chromosomes 2 , 3 , 18 , and 19 and show that in these chromosomes h3k4me3 peak summits often precede dsbs . in future work we plan to analyze the significant correlation pairs for these chromosomes in different genome browsers and automatic annotation systems to reveal the possible biological meaning of these correlations . the strong correlation between h3k4me3 marks and hot spots of dsbs has been described in human rdna units , suggesting an important role for dna breaks in actively transcribed genes . the bam file was used in locating hot spots of dsbs inside the tmprss2 and erg genes located on the minus strand of chr21 at a distance about 3 mb . these genes were selected because recurrent gene fusions between tmprss2 and ets family genes occur at high frequency in prostate cancer . we detected several regions in the tmprss2 and erg genes that are enriched with dsbs . deletion , rather than translocation , is reported to be the main mechanism for tmprss2-erg gene fusion ( 81 vs. 19% ) . it has been shown that the regions possessing hot spots of dsbs in human rdna genes often form contacts with other genomic regions also possessing hot spots of dsbs , and it has been suggested that this fact could explain the origin of robertsonian translocations . it is known that regions of the same chromosome make 3d contacts more often than between different chromosomes . tmprss2 and erg genes are located very close to each other on chr21 , providing a potential for contacts between their regions possessing dsb hot spots . currently , we are performing 4c ( circular chromosome conformation capture ) experiments in order to study genomic contacts between these genes , to uncover the possible mechanism of this and some other cancerogenic gene fusions . our data suggest that hot spots of dsbs are associated with various epigenetic mechanisms of gene regulation and with the formation of 3d chromosomal structures , both of which are conserved in different cell types , with dramatic consequences for genomic integrity should they go awry , . hence , data on the distribution of dsb hot spots in the human genome provide a new tool for studies of cancer genomics and genomic features associated with the regulation of gene expression .

hot spots of dna double - strand breaks ( dsbs ) are associated with coordinated expression of genes in chromosomal domains ( tchurikov et al . , 2011 [ 1 ] ; 2013 ) . these 50150-kb dna domains ( denoted forum domains ) can be visualized by separation of undigested chromosomal dna in pulsed - field agarose gels ( tchurikov et al . , 1988 ; 1992 ) and used for genome - wide mapping of the dsbs that produce them . recently , we described nine hot spots of dsbs in human rdna genes and observed that , in rdna units , the hot spots coincide with ctcf binding sites and h3k4me3 marks ( tchurikov et al . , 2014 ) , suggesting a role for dsbs in active transcription . here we have used illumina sequencing to map dsbs in chromosomes of human hek293 t cells , and describe in detail the experimental design and bioinformatics analysis of the data deposited in the gene expression omnibus with accession number gse53811 and associated with the study published in dna research ( kravatsky et al . , 2015 ) . our data indicate that h3k4me3 marks often coincide with hot spots of dsbs in hek293 t cells and that the mapping of these hot spots is important for cancer genomic studies .

the nasal septum is placed in the middle of the nasal cavity and it divides the nasal cavity and supports the external nose . it is an important structure for maintaining the facial skeletal structure , and it consists of the septal cartilage anteriorly , the perpendicular plate of the ethmoid bone and the vomer bone posteriorly . this structure comes in contact with other structures such as nasal bone , frontal bone , cribriform plate , maxillary bone , palatine bone and sphenoidal bone ( 1 ) . the septal cartilage starts to develop at the fetal stage and it continues to grow after birth . it reaches the size and structure of an adult by puberty , with some difference in the time frame between individuals ( 2 ) . it is known that the cartilage becomes denatured and undergoes degenerative changes , including decreasing cartilage cells , and it becomes calcified with age ( 3 ) . because it is important to thoroughly understand the developmental process of the nasal septum to safely carry out nasal septal procedures , many studies have been performed about the embryology and development of the nasal septum . however , most of these were studies based on autopsy or simple x - ray findings ( 4 - 7 ) . studies that are based on autopsies allow researchers to make realistic , concrete analysis . however , there are difficulties in generalizing the ideas from these autopsy studies as these studies did not have so many subjects . the studies conducted on the x - ray finding have limitations for providing specific information on the three - dimensional nasal septum structure . on the other hand , if the sagittal plane of magnetic resonance imaging ( mri ) is used , each structure could be better identified . therefore , a wide range of studies on the structure and development of nasal septum become possible with using mri . however , there have been no studies using the sagittal plane of mri for determining the structure and development of the nasal septum . we used the sagittal planes of mri to analyze the changes of the structures of the nasal bone and nasal septum , according to age and gender , so as to help physicians conduct safe nasal septal procedures . two hundred eighty patients , who had their entire septum well visualized on the midline sagittal view , were selected among the 3,904 patients who underwent brain mri from january , 2004 to december , 2006 . two subjects were analyzed from the patients from 1 year to 70 year of age , and this was done the same with each gender . signa excite ( ge medical system , twin speed : 1.5 tesla , huntley , il , usa ) and signa excite ( ge medical system , high definition : 1.5 tesla , huntley , il , usa ) magnetic resonance imaging machines were used . the t1 weighted sagittal plane images were used , which are the easiest method for observing the anatomic structure of the nasal septum . the repetition time ( tr ) was 500 - 600 msec , and the echo time ( te ) was 12 - 15 msec . pacs ( picture archiving and communication system : multivox , seoul , korea ) was used to measure and analyze the length and area of each part in the nasal bone and the nasal septum . the bony dorsal length was measured from the most posterior part of the bony nasofrontal angle to the rhinion ( fig . the cartilage dorsal length was measured as the length from the rhinion to the imaginary anterior septal angle . the imaginary anterior septal angle was defined as the point where the extensional line drawn from the anterior nasal spine to the caudal margin direction meets the nasal dorsum . the total dorsal length was measured from the most posterior part of the bony nasofrontal angle to the imaginary anterior septal angle ( fig . the length of the nasal septal cartilage ( sc)-nasal bone ( nb ) overlap was measured from the rhinion to the most posterior part of the plane in which the nasal septal cartilage and the nasal bone come into contact ( fig.1c ) . the total area of the nasal septum and the area of the nasal septal cartilage were measured , and we also measured the area of the nasal septal cartilage that excluded 1 cm each of the dorsal and caudal portions of the nasal septal cartilage ( fig . 1d - f ) . statistical analysis was performed using the statistical package for the social science , version 12.0 software system ( spss , inc . , chicago , il , usa ) , and independent t - tests were carried out for each age of the subjects . simple regression tests were used to determine the correlation ( pearson 's correlation coefficient ) between the length of the overlapped part of the nasal septum and nasal bone and each parameter . the bony dorsal length gradually increased after birth both in males and females up into their teens ( 16.92.7 mm ) and twenties ( 19.32.9 mm ) ( fig . the cartilaginous dorsal length increased after birth until their twenties ( 27.64.6 mm ) and it did not show significant changes later in adults , and no significant differences between the male and female were shown in each age group ( fig . 3 ) . the total dorsal length was also observed to grow until the twenties ; it did not show significant changes later in adults , and there were no significant gender differences ( fig . the length of the overlapped part of the nasal septal cartilage and the nasal bone increased in appearance until the twenties ( 9.93.2 mm ) and it later showed a significant decrease with no significant gender differences ( fig . 5 ) . the total nasal septal area was increased up to the twenties ( 2,323.5243.3 mm ) and it showed no significant change after the twenties . the area of the nasal septal cartilage was increased to the maximum by the twenties ( 889.4151.6 mm ) ( fig . the proportion of the area of the nasal septal cartilage to the total nasal septal area was shown to decrease as both the males and females got older ( fig . the area of nasal septal cartilage , with excluding 1 cm of the dorsal and caudal portion , was measured as 426.8 mm on average for the adults and the range was from 253 mm to 750 mm ( fig . 9 ) . the length of the overlapped portion between the nasal septum and the nasal bone showed a significant correlation with the total dorsal length , based on the adults in or beyond their twenties ( r=0.138 , p=0.049 ) , and this length also showed a significant correlation with the area of the nasal septal cartilage and the relative proportion of the nasal cartilage to the total nasal septum ( r=0.386 , p<0.001 and r=0.327 , p<0.001 , respectively ) ( fig . embryologically , the nasal septum consists of cartilaginous bone , and the ossification of the nasal bone and vomer is started at two ossification centers of the cartilaginous synchondroses . the ossification of the vomer occurs earlier than that of the nasal bone , and the two ossification centers are later united below the caudal part of the nasal septal cartilage ( 8 , 9 ) . most of the nasal septum of a young child consists of cartilage and this size increases until puberty as the ossification of the vomer , the nasal bone and the perpendicular plate of the ethmoid bone proceeds ( 10 , 11 ) . many studies focused on the changes of the nasal septal cartilage according to age have been performed . vetter et al . ( 3 ) reported that the anterior part of the nasal septal cartilage has a high cell density and it shows active proliferative ability , and the central part of the nasal septum shows the highest growth rate in childhood , with the cell density increasing until puberty , and the central part of the nasal septum shows a decreasing growth rate after puberty . ( 12 ) claimed that nasal plastic surgery should be performed after the age of 18 because the growth of the nose increases until the age of 18 and this does not show a significant difference beyond that age . ( 13 ) announced that females before 16 years and males before 17 years should not undergo nasal plastic surgery . in this study , the total dorsal length of the nasal septum and the cartilaginous dorsal length increased until the age of twenty and there was no significant difference in these lengths beyond the age of twenty . the growth rate of nasal bone was also increased up to the twenties and there were no significant differences in the older adults , except a little decrease in the sixties . godley et al . ( 7 ) carried out an anatomical study on the nose by using 60 adult cadavers and they reported that the cartilaginous dorsal length was 215 mm on average and this length was shorter in the females by approximately 54 mm . they also reported that the length of the dorsal part of the nasal septum that overlaps under the nasal bone with the perpendicular plate of ethmoid bone is 42 mm on average . in this study , which was based on radiological measurements , the cartilage dorsal length in adult was 264 mm on average and there were no significant differences in the dorsal length between the males and females . the overlapped part of the nasal septal cartilage and nasal bone was measured as 72 mm on average in the adults , and this seemed to gradually decrease with age . we observed some individual differences for the overlapped part according to the age , and these differences ranged from 3 mm up to 15 mm . takahashi et al . ( 14 ) demonstrated that the cartilage component occupies about 19% of the total nasal septum , however , miles et al . ( 6 ) reported that it is about 47.5% and the cartilage available for grafting , with excluding the l - strut , is about 420 mm . weiler et al . ( 15 ) reported , in the animal study using rat , that the size of the nasal septal cartilage increases after birth , it reaches a peak and it gradually decreases after , and that this was statistically significant for males . ( 10 ) , who analyzed the size of the nasal septal cartilage using 30 cadevers concluded the size of nasal septum after birth and of nasal septum throughout life . the size of the cartilage part of nasal septum gradually decreases as the bony part is increased . in this study , the total area of the nasal septum was 2,127 mm on average and the area did not show significant difference in the adults , whereas the area of the nasal septal cartilage was 692 mm on average and this area showed decreasing tendency with age . the relative proportion of the cartilage is approximately 32% of the total area of nasal septum in adults , and it was noted to decrease with age . in addition , it was noted that the maximal amount of nasal septal cartilage that can be used as graft material is about 427 mm , which was not different from the previous reports , and that this maximal amount of nasal septal cartilage decreased with age . the septal cartilage and upper lateral cartilage are engaged with each other below the nasal bone , and this is an important part that supports the entire cartilage structure of the nose , which attacks with the pyriform aperture dorsally , the nasal septum ventrally and the lower lateral cartilage caudally ( 16 ) . ( 18 ) explained that special care is needed when approaching this part during nasal plastic surgery or when performing bone dissection for removal of a hump . in this study , the keystone area , for which the dorsal part of the nasal septal cartilage lies upon the bottom of the nasal bone , was measured and studied by using two - dimensional mr images . this study showed that the length of the overlapped part between the nasal septum and the nasal bone had positive correlation not only with the absolute numerical value of the nasal septal cartilaginous area , but also with the relative proportion of the nasal septal cartilage . we speculate that the reduction of this length with age seems to be due to the decrease in the relative proportion of the nasal septal cartilage . this study has a limitation of excluding people with nasal septal deviation from the analysis because the subject of the study was set on the nasal septum in the median plane , as shown in two - dimensional planes . there were limitations for the analysis about the specific point when the growth of the nasal septum stops since the age range was divided into 10-year intervals . the area that the nasal bone and septal cartilage overlap also decreased , as the relative proportion and the area of the nasal septal cartilage got smaller . clinically , if this overlapped area is damaged when performing nasal septal surgery or nasal plastic surgery , then severe complication such as saddle nose can occur . much attention should be paid when treating older patients because of the risk of damaging their smaller overlapped nasal septal cartilage area .

objectivesthis study was designed to evaluate the normal development of the nasal septum in koreans using sagittal mri for the valuable clinical information on septal procedures.methodstwo hundred eighty patients who had their whole nasal septum visualized in the midline sagittal view were selected among the 3,904 patients with brain mri from january , 2004 to december , 2006 at dankook university hospital . the patients who had a history of nasal septal surgery or nasal trauma were excluded . following parameters are calculated and analyzed : lengths of bony and cartilage dorsum and septal cartilage - nasal bone overlap , total septal area , septal cartilage area and , the proportion of the cartilage area to septal area and the maximal harvestable cartilage for grafting were calculated using the pac program.resultsall the parameters were increased until adolescence . thereafter , bony dorsal length , cartilage dorsal length , total dorsal length , total septal area and maximal harvestable cartilage for grafting have not changed significantly with age , while sc - nb overlap length , septal cartilage area , and proportion of the cartilage area to the total septal area were significantly decreased with age . the sc - nb overlap length was positively correlated with the septal cartilage area and the proportion of the cartilage area to the total septal area.conclusionthe small septal cartilage area and its proportion to the total septal area were significantly correlated with a short overlap length of the septal cartilage under the nasal bone . septal procedures should be carefully performed in the elderly due to the risk of incurring saddle nose .

the parasitoid collecting sites used for the experiments were 1 ) the university of california cold canyon preserve located west of winters , ca ( 38 3027.40 n , 1220549.10 w ) , a natural habitat with historically low leafhopper densities ( population 1 ) ; 2 ) a low - input commercial vineyard in pope valley , ca ( 38 3902.62 n , 1222756.53 w ) with historically moderate leafhopper densities ( population 2 ) ; and 3 ) two adjacent high - input commercial vineyard blocks in napa valley , ca ( 38 2715.46 n , 122 2536.32 w ) with historically high leafhopper densities ( populations 3 and 4 ) . leafhopper density at each site was estimated by collecting a sample of 40 leaves from the middle third of each cane , randomly with respect to damage or leafhoppers present . one half of each leaf ( top and bottom ) was inspected under a stereomicroscope to count : 1 ) unemerged eggs without any sign of parasitism ; 2 ) unemerged eggs showing signs of parasitism ; 3 ) empty eggs from which a leafhopper had emerged ; and 4 ) empty eggs from which a parasitoid had emerged . leaves collected from each site and containing parasitized leafhopper eggs were held in cardboard emergence cages ( 12.7 cm in diameter and 24 cm in height ) fitted with a funnel at the top leading emerging wasps to a glass collecting vial supplied with a strip of honey - saturated filter paper . emerging female wasps were collected each morning between 07:00 and 10:00 h , allowed to mate with males from the same population , and then released singly into a rearing cage . rearing cages contained a single grapevine , vitis vinifera ( l. ) cv . chardonnay hosting either high or low densities of leafhoppers , erythroneura elegantula ( osborn ) . the vines were grown from dormant cuttings in a greenhouse free of pests and pesticides . leafhoppers were collected as adults from a high - density population in a small private vineyard in davis , ca ( 38 3258.86 n , 121 4648.03 w ) . we added either 10 adult leafhoppers per vine ( low density treatment ) or 30 adults per vine ( high density treatment ) 4 d prior to introducing a single mated female parasitoid per cage . the rearing cages were cylindrical , 12.7 cm in diameter and 33 cm in height , with fabric - covered vents on the side and top . ten days after introducing parasitoid females , vines were transferred into emergence cages to collect the progeny of the experimental parasitoids . emergence cages were of the same design as used in the original parasitoid collections but with the bottoms removed to allow them to be fitted over the potted grapevines . all parasitoids were sexed , and females were dissected under a stereomicroscope to establish egg load . all females were slide mounted , the length of a hind tibia measured with an ocular micrometer as an index of body size , and species identity confirmed using a phase - contrast microscope and antennal and wing morphology ( triapitsyn 1998 ) . to evaluate if the leafhopper egg density treatments were established successfully , we quantified egg densities in five half - leaves taken per cage for five replicates of each treatment . note that under our experimental design , the hypothesis of transgenerational phenotypic plasticity could be supported either by 1 ) an increase in fecundity of daughters whose mothers were exposed to higher host densities or 2 ) a decrease in fecundity of daughters whose mothers were exposed to lower host densities . for this reason , we suggest that our hypothesis can be tested with any starting parasitoid population . the experiment was analyzed as a mixed - model analysis of variance with jmp pro 11.0 ( sas institute 2013 ) , with random effects for population and family , and family nested within population ; hind tibia length as a covariate ; host density as a fixed effect ; and fecundity of daughters as the dependent variable . leafhopper host densities at the time leaves were collected to rear parasitoids were 0.048 0.008 ( n = 40 ) , 0.083 0.015 ( n = 40 ) , and 0.089 0.025 ( n = 40 ) eggs per cm of leaf surface for populations 1 , 2 , and 34 combined , respectively . host density treatments were established as desired in the laboratory : leaf samples revealed mean leafhopper egg densities of 0.053 0.021 ( n = 5 ) and 0.163 0.048 ( n = 5 ) eggs per cm of leaf surface for the low and high density cages , respectively , when the parasitoids were introduced . our experimental host density treatments fall comfortably within the range of leafhopper egg densities recorded in the field ( muphy et al . the host density treatments experienced by the mother parasitoids had no effect on the fecundity their daughters ( fig . 1 ; f = 0.09 , df = 14.9 , p = 0.77 ) . the slight , and nonsignificant , difference between the treatments was the opposite of what would be expected under adaptive plasticity : mothers experiencing lower host availability produced daughters with slightly higher fecundities ( 25.5 2.9 eggs ) than did mothers experiencing higher host availability ( 24.8 2.0 eggs ) . 1.mean ( 1 se ) fecundity of a. erythroneurae offspring produced by mothers given access to low ( 25.5 2.9 eggs , n = 9 ) versus high ( 24.8 2.0 eggs , n = 11 ) densities of hosts ( eggs of the western grape leafhopper , erythroneura elegantula ) . mean ( 1 se ) fecundity of a. erythroneurae offspring produced by mothers given access to low ( 25.5 2.9 eggs , n = 9 ) versus high ( 24.8 2.0 eggs , n = 11 ) densities of hosts ( eggs of the western grape leafhopper , erythroneura elegantula ) . significant variation in offspring fecundity was observed across families collected from the four source populations ( random effect covariance parameter estimate for family term : 40.9 20.6 [ se ] ; the 95% confidence interval [ 0.4281.3 ] did not overlap zero ; fig . too few parasitoids were collected from populations 1 and 2 to provide a formal test of differences between populations . 2.fecundity of individual a. erythroneurae females ( n = 122 ) reared from twenty different mothers collected from four populations . population 1 : wild vitis californica ( benth . ) site ; population 2 : low input agriculture site ; populations 3 and 4 : high input agriculture sites . fecundity of individual a. erythroneurae females ( n = 122 ) reared from twenty different mothers collected from four populations . population 1 : wild vitis californica ( benth . ) site ; population 2 : low input agriculture site ; populations 3 and 4 : high input agriculture sites . we found no evidence for transgenerational phenotypic plasticity in fecundity for a. erythroneurae parasitoids : mother parasitoids that experienced low versus high host densities when ovipositing did not produce daughters differing in mean fecundity . our experimental design also should have detected changes in fecundity triggered by daughter parasitoids having some mechanism of detecting host availability while they developed as pupae in a vitis leaf . thus , we see no evidence for phenotypic plasticity in fecundity for these solitary and strictly proovigenic parasitoids , whose egg maturation is completed prior to adult emergence . to our knowledge , this is the first test for transgenerational phenotypic plasticity in parasitoid fecundity that is not mediated by changes in clutch sizes produced per host by mother parasitoids ( a. erythroneurae is strictly solitary , with only one parasitoid offspring developing per host individual ) . our finding of significant between - family differences in mean fecundity suggests instead that the differences in fecundity documented by segoli and rosenheim ( 2013 ) for parasitoids collected from populations with varying host availability may have a genetic basis . transgenerational plasticity in parasitoids has been demonstrated to shape other important life history traits , including development time and the proliferation of clonal offspring in a polyembryonic wasp ( morag et al . thus , assessing its possible operation in a. erythroneurae was an important step in assessing different mechanisms that might underlie positive associations of parasitoid fecundity with local variation in host availability . future work will now focus on the hypothesis of local adaptation , which opens up the intriguing possibility that parasitoid fecundity could evolve as part of an eco - evolutionary feedback between host availability and parasitoid traits linked to the exploitation of host populations .

to improve biological pest control , we would like to understand the main factors that limit the reproductive success of key biological control agents , including parasitoid wasps . previous research with proovigenic parasitoids in the genus anagrus collected from multiple field sites revealed positive correlations between parasitoid fecundity and local host density . in this article , we test whether this variation in fecundity is a reflection of transgenerational phenotypic plasticity . we exposed females in laboratory microcosms to either low or high host densities and then quantified the fecundity of their adult daughters . mean fecundity of daughters did not differ across host density treatments , arguing against the operation of phenotypic plasticity . significant variation in mean fecundity across different families instead suggested the possibility that genetic variation may underlie the observed fecundity differences .

transposition of the great arteries ( tga ) occurs in 0.2 per 1000 live births , and comprises approximately 5% of all congenital heart defects . the condition is surgically repaired in the neonatal period by the arterial switch procedure ( aso ) . this involves transection of the proximal aorta above the aortic valve and moving the aorta posteriorly towards the left ventricle , along with dissection and relocation of the coronary arteries to the neoaorta . the pulmonary arteries are extensively dissected to minimise tension and the main pulmonary artery is transected and anastomosed to the right ventricular outflow . the original description of this by jatene has been largely replaced by slinging the pulmonary arteries in front of and straddling the ascending aorta as described by lecompte . this manoeuvre results in stretching of the pulmonary arteries which may result in pulmonary artery stenosis ( pas ) if there is inadequate dissection or not enough vessel length . pas is a recognised complication of this manoeuvre that occurs in up to 10% of cases . it has been speculated that this stenosis may be due to scar tissue formation at the anastomosis sites , inadequate somatic growth of the pulmonary artery and inadequate mobilization of both outflow tracts , with resultant tension at the anastomosis sites , as well as nonviable tissue at these sites ; it is likely to multi - factorial in many cases . surgical repair of such stenosis is possible but less invasive relief of obstruction is available by balloon angioplasty with or without stenting . ballooning alone also carries a higher failure rate than stenting so stenting is employed in tight stenosis , but the age at presentation plays a role . in this respect , pas either occurs soon after surgery and relatively rapidly or it can be a very slow process presenting later in life and associated with somatic growth . such procedures may involve negotiating tight bends in order to reach the site of stenosis . the passage of non - premounted stents may be problematic in such situations , especially with longer stents and tighter bends . care must also be taken in order to avoid inflating stents very proximally as these may impinge on the coronary arteries . if there is any suspicion of this , balloon interrogation should be performed prior to stenting . our patient presented in the early neonatal period with simple tga and underwent an uneventful aso . echocardiographic follow - up showed slowly progressive right ventricular outflow tract obstruction , as evidenced by tricuspid regurgitation gradients , but failed to elucidate the exact location of the obstruction . magnetic resonance imaging at fourteen years of age showed discrete narrowing of the right pulmonary artery ( rpa successive orthogonal views figures 1 and 2 ) with the narrowest orifice is 11x4 mm and vmax 1.1 m / s . there was also long segment narrowing of the left pulmonary artery ( lpa figures 3 and 4 ) with a minimal orifice diameter of 2.5 mm . and the main pulmonary artery was 9x18 mm successive orthogonal views obtained by magnetic resonance imaging using a balanced steady - state free precession pulse sequence , showing discrete narrowing of the right pulmonary artery ( arrowhead ) . orthogonal views obtained by magnetic resonance imaging showing narrowing long segment narrowing of the left pulmonary artery ( arrowhead ) . angiography in steep anteroposterior steep cranial as well as steep caudal ( 45 degrees ) angulations confirmed tight but discrete narrowing of the rpa origin and long segment narrowing of the proximal lpa ( figure 5 ) . after luminal measurements on fluoroscopic projections , an andrastent xxl 35 mm stent was hand - crimped on a 15 mm by 40 mm balt cristal balloon and an attempt was made to introduce this into the lpa through a 12f mullins sheath . however , when attempting to negotiate the stent and balloon over the apex of the lpa curve , the stent slipped back over the balloon . the balloon and stent were retrieved and the stent was recrimped , again by hand . the sheath was changed to 14f size but once again , the stent shifted on the balloon shaft . balloon and stent were once again retrieved and this time , after recrimping by hand , the stent was given a slight curve . after this manoeuvre , the stent traversed the bend without any problems and was deployed ( figures 6 and 7 ) . stent on balloon traversing a tight curve into the lpa stent on balloon traversing a tight curve . the stent was successfully deployed ( figure 8) with reduction of right heart pressures ( table 1 ) . pressures during the procedure ( mmhg ) rpa to mpa : 14/8 , mean 10 to 44/8 mean 20 lpa to mpa : 20/12 mean 15 to 44/8 mean 20 lv = left ventricle , rv = right ventricle , rpa = right pulmonary artery , lpa = left pulmonary artery we describe a case of bilateral branch pulmonary stenosis in an adolescent patient following the lecompte manoeuvre carried out in infancy for aso in a patient with tga . both stenoses were successfully treated percutaneously by balloon angioplasty and stenting , but the procedure was punctuated by challenges in negotiating a tight bend in the lpa . steep cranial or caudal angiograms should be obtained in order to decide on the best view to image and balloon / stent pulmonary artery stenosis . in the case of non - premounted stents , for the negotiation of tight curves within long sheaths , a slight curve of the stent on the balloon may be useful to negotiate a tight curve , particularly if the stent is on a small or low profile balloon . but any such manoeuvres must be done cautiously so as to avoid damaging the balloon . an additional benefit of introducing a slight bend on the stent after mounting and hand - crimping is that this increases the stability of the stent on the balloon . this technique assumes that other steps would have already been taken , such as , the use of a stiff wire parked distally in a pa branch and upsizing the sheath to augment the lumen .

simple transposition of the great arteries ( tga ) occurs in 0.2 per 1000 live births . the condition is surgically repaired in the neonatal period by the arterial switch procedure ( aso ) sometimes preceded by an atrial septostomy . the aso involves transecting the great arteries and relocating them to the appropriate ventriculo - arterial ( va ) connection with attachment of the disconnected coronary arteries to the aorta . in the process , the attachment of the pulmonary artery to the right ventricle involves the le compte manoeuvre and to achieve this the pulmonary arteries must be fully mobilised and sometimes the main pulmonary artery may require patch augmentation as well . nevertheless , pulmonary artery stenosis ( pas ) is one of the potential problems with the aso . however , with improved surgical techniques , this has dropped from around 15% in the 1980s to less than 3% . apart from surgical revision when pas occurs , there are interventional options which include angioplasty and/or stent insertion . the latter is preferred in small children and works well in around 60% but may require repeat procedures . in older patients or when angioplasty fails , stent insertion can be considered . these procedures may involve negotiating tight bends in order to reach the site of stenosis . the passage of non - premounted stents may be problematic in such situations , especially with longer stents and tighter bends as they tend to slip off balloon . we describe several techniques that may facilitate such interventions , and these were utilised in an adolescent patient who had had aso for tga in the neonatal period . these included manually giving the mounted stent a slight bend in order to help the balloon - stent assembly negotiate hairpin bends .

if the patient complains of leg symptoms / back pain , and there is a well - correlated lesion in lumbar spinal magnetic resonance ( mr ) imaging , the lesion is usually regarded as the problem . however , if the presentation of symptoms / signs is not typical , concurrent lesion in the other spine level , which is reported between 5% and 28% of patients with lumbar spine stenosis , is suspected , and it is important not to make a misdiagnosis2,4,8,9 ) . the monoparesis was caused by bleeding from probable vascular disorders such as cavernous malformation ( cm ) in the spinal cord , which should have been detected before the operation with a faithful physical examination and inquiry of the patient 's medical history . a 63-year - old man , who underwent decompressive laminectomy for spinal stenosis at l4 - 5 4 days previously in the other hospital , visited the emergency department with progressive weakness in his left leg and numbness below sensory level t7 on the right side and in the right leg . he had been cured of lung cancer with chemotherapy and conventional radiation therapy 10 years previously . unfortunately , detailed information about the irradiation was not available , but usual protocol of radiotherapy was 60 gy/30 fractions at that times . before the operation , he had complained of numbness and pain in the right leg . lumbar spine mr imaging showed spinal stenosis with spondylolisthesis at l4 - 5 . because the leg symptom was thought to be correlated with the lumbar stenosis the patient 's medical record showed that motor power was normal in the lower extremities . neither sensory nor deep tendon reflex abnormality was mentioned . although subjective numbness on right side trunk were present , further imaging studies were not performed for the cervical and thoracic spine . decompressive laminectomy was uneventful , but left leg monoparesis ( manual motor power grade ii / v ) suddenly developed 2 days after surgery . neurological examination demonstrated left leg weakness ( manual motor power grades : hip flexion , i / v ; knee extension , ii / v ; ankle dorsiflexion , ii / v ; ankle plantar flexion , ii / v ) , decreased sensation to light touch and pain below right sensory level t7 . whole spine mr imaging showed fatty marrow change from t1 to t8 , most likely due to previous irradiation ( fig . 1 ) . high signal intensity change in the t2-weighted mr image at t5 - 6 spinal cord showed normal diameter of the spinal cord without edema , and a sequela of previous irradiation was suspected ( fig . the t2-weighted mr image showed another high - signal lesion at t4 - 5 spinal cord surrounded by a low signal rim ( fig . 1 ) , and the t1-weighted mr image showed focal high signal intensity ( fig . the radiological interpretations were : suspicious bleeding , myelopathy , probable cm , and fatty marrow change from t1 to t8 . considering past medical history and fatty marrow change at the vertebrae which seemed to be the field of irradiation , vascular disorders such as cm were suspected as sequel of irradiation . the patient could walk 100 m without assistance 2 months later , but hypoesthesia persisted in the right side of the trunk and the leg . we reported a case of monoparesis after common lumbar decompressive laminectomy probably due to lesion at the thoracic spine . considering radiation induced change at thoracic vertebrae and spinal cord , many hemorrhagic vascular disorders may have similar radiological findings and without pathological examination and mr image before radiotherapy , the diagnosis was not conclusive18 ) . however , careful attention should have been paid to the history of irradiation to the spinal cord . infrequently , patients have suffered from unexpected neurological deterioration after common lumbar spinal surgery , and concurrent tandem lesions in other levels of the spine were among the causes2,4,8,18 ) . careful physical examination and imaging studies were recommended in suspected cases to prevent catastrophe2,4,8 ) . however , a false negative neurological examination was reported in 37% of patients with myelopathy , and suspicion with medical histories involving diseases such as cancer was also important6 ) . a cavernous malformationis is a blood vessel hamartoma that can affect any part of the central nervous system14,15,16,17,19 ) . these lesions are presumed to be congenital14,15,16,17 ) , but radiation - induced cms were reported after irradiation to the central nervous system , and the spinal cord was no exception11,13,14,17,18 ) . first , the cm may have present , but it was undetectable on neuroimaging study and enlarged due to vascular insult by radiation . radiation induced proliferation and dilation of the vascular endothelium with hyalinization and fibrinoid necrosis of blood vessel walls may induced cavernous malformation5,7,10 ) . four cases of de novo development of spinal cms following spinal axis radiation have been previously reported . the first report was published in 1999 by maraire et al.11 ) ; it involved a 22-year - old man who received 27 gy of craniospinal radiation and a 14.4 gy tumor boost after resection of suprasellar germinoma ( table 1 ) . the second case , reported by narayan and barrow13 ) in 2003 , involved a 17-year - old boy who underwent radiation therapy ( 30.6 gy to the whole brain , 54 gy tumor boost , and 27 gy to the entire spine ) after resection of posterior fossa medulloblastoma . the third case , reported by yoshino et al.18 ) in 2005 , involved a 16-year - old girl who underwent neuraxis radiotherapy ( 40 gy to the whole spine , 20 gy tumor boost ) after resection of pilocytic astrocytoma of the spinal cord . craniospinal radiation ( radiation dose was not describled ) to treat acute lymphocytic leukemia 19 years before . two cm13,18 ) occurred after irradiation to the spinal cord after surgery for spinal intramedullary tumor and the others11,12 ) occurred after craniospinal irradiation . pathological features of three cases11,13,18 ) demonstrated large , thin - walled blood vessels which were consistent with cm and vascular endothelial dilation and fibrinoid necrosis , which was typical of radiation - induced vascular change11,13,18 ) . radiotherapy induced cm at the spinal rootlets due to renal cell cancer ( radiation dose was not available ) , hodgkin 's disease ( 40 gy/20 fractions , 45 gy/40 fractions ) , testicular seminoma ( radiation dose was not available ) at 24 , 13 , 26 , and 47 years after radiotherapy , respectively3,10 ) . in the present case , although the result was obtained from brain , not cavernous malformation , but radiation - induced telangiectasia may present with varying amounts of hemorrhage and , occasionally , parenchymal hematomas , and may appear similar to cryptic vascular malformations on t2-weighted mr images5 ) . to prove radiation induced cm we need to verify the following conditions ; no lesion before radiation , latency period , the lesion within the radiation field and pathological examination1 ) . considering those criteria , there was not enough evidence to support the diagnosis of radiation induced cm in the present case . this case is suggestive that showed the importance of faithful neurological examination and history taking . a radiation induced lesion may be suspected if neurologic symptoms / signs are atypical and the spinal cord was previously irradiated even for patients with common lumbar degenerative disease .

occasionally , unexpected neurological deficits occur after lumbar spinal surgery . we report a case of monoparesis after lumbar decompressive surgery . a 63-year - old man , who had undergone decompression of l4 - 5 for spinal stenosis 4 days previously in the other hospital , visted the emergency department with progressive weakness in the left leg and hypoesthesia below sensory level t7 on the right side . he had been cured of lung cancer with chemotherapy and radiation therapy 10 years previously , but detailed information of radiotherapy was not available . whole spine magnetic resonance ( mr ) imaging showed fatty marrow change from t1 to t8 , most likely due to previous irradiation . the t2-weighted mr image showed a high - signal t4 - 5 spinal cord lesion surrounded by a low signal rim , and the t1-weighted mr image showed focal high signal intensity with focal enhancement . the radiological diagnosis was vascular disorders with suspicious bleeding . surgical removal was refused by the patient . with rehabilitation , the patient could walk independently without assistance 2 months later . considering radiation induced change at thoracic vertebrae , vascular disorders may be induced by irradiation . if the spinal cord was previously irradiated , radiation induced vascular disorders needs to be considered .

cardiovascular disease is a major cause of death worldwide , so people are advised to limit their intake of dietary cholesterol . ase can modulate cholesterol metabolism and decrease cholesterol levels in animals . in this study , the liver and ovary tissues of laying hens were collected and digital gene expression ( dge ) was performed to investigate the mechanisms underlying the cholesterol - lowering effect of ase . 150 hy - line brown hens at the age of 27 weeks were randomly divided into five groups ( five replicates were established per treatment , with 6 hens each replicate ) . hens in each treatment were randomly fed five different experimental diets : controls were fed a basal diet with 0 mg / kg ase , and the other four groups were fed otherwise identical diets supplemented with 60 mg / kg , 120 mg / kg , 240 mg / kg , and 480 mg / kg ase , respectively . the basal diet was designed to meet , or exceed , the national research council ( nrc ) nutrient requirements . the ingredients and calculated nutrient level of the basal diet are shown in table 1 . ( shijiazhuang , china ) as a standardized product that contained 61.64% saponins , 10.97% flavonoids , 8.12% polysaccharides , 7.11% moisture , and 12.16% unknown compounds . ase is extracted from medicago sativa l. the main active component is saponins . and ase played a positive role in lowering yolk cholesterol content , , . according to the cholesterol levels in yolk of feeding experiment , the control group and 120 mg / kg ase group were chosen for digital gene - expression profiling analysis ( table 2 ) . the liver and ovary tissues of 120 mg / kg ase group were isolated , frozen immediately in liquid nitrogen and stored at 80 c until rna extraction ( the liver from the control group ( li_ck ) , the liver from the 120 mg / kg ase group ( li_exp ) , the ovaries from the control group ( ov_ck ) , the ovaries from the 120 mg / kg ase group ( ov_exp ) ) . rna was assessed for quantity and quality using a nanodrop nd-1000 spectrophotometer and agilent 2100 bioanalyzer according to the manufacturer 's instructions . contaminating genomic dna in total rna samples were treated with dnase - i . 2 g of total rna ( 100 ng/l ) was pooled from three tissues within each group : li_ck , li_exp , ov_ck , ov_exp . four sequencing libraries were generated using neb next ultra directional rna library prep kit for illumina ( neb ) in accordance with the manufacturer 's recommendations . mrna was purified from total rna using poly - t oligo - attached magnetic beads ( life technologies , ca , usa ) . fragmentation was carried out using divalent captions under elevated temperature in neb proprietary fragmentation buffer . first strand cdna was synthesized using random oligonucleotides and m - mel reverse transcriptase ( rnase h- ) . second strand cdna synthesis was subsequently performed using dna polymerase i and rnase h. remaining overhangs were converted into blunt ends via exonuclease / polymerase activities and enzymes were removed . after adenylation of 3 ends of dna fragments , neb next adaptor oligonucleotides were ligated to cdna fragments . in order to select cdna fragments . dna fragments with ligated adaptor molecules on both ends were selectively enriched using neb universal pcr primer and index primer in a 10 cycles pcr reaction . products were purified and quantified using the agilent high sensitivity dna assay on the agilent bioanalyzer 2100 system . after the clustering of the index - coded samples , each library preparation was sequenced on an illumina hiseq 2000 platform and 50 bp single - end ( se ) reads were generated . for each dge library , clean tags were obtained by filtering out adaptor sequences and low - quality tags . then the clean tags were retained and mapped to the reference genome of gallus gallus at ftp://ftp.ensembl.org/pub/release-72/fasta/gallus_gallus/dna by tophat v1.4.0 . then reads per kilobase transcriptome per million mapped reads ( rpkm ) of each gene were calculated based on the length of the gene and reads count mapped to this gene . it is currently the most common method of for estimating levels of gene expression . in order to balance the number of false positives and false negatives a rpkm threshold value of 0.3 was established to determine whether or not given genes was expressed , as in several other studies , , . the q - values of 0.05 and log2 ( fold_change ) with no limitations were served as the threshold of significance for differential expression . here , 110 significantly expressed genes were identified in liver samples ; 66 were up - regulated and 44 were down - regulated . in ovary samples , 107 significantly expressed genes were obtained ; 63 were up - regulated and 44 were down - regulated ( table s1 ) . gene ontology ( go ) enrichment analysis of differentially expressed genes was implemented by the goseq r package . we used kobas software to test the statistical enrichment of differential expression genes in kegg pathways , . protein interaction networks of differentially expressed genes were acquired based on the information in string database ( http://string-db.org/ ) . cardiovascular disease is a major cause of death worldwide , so people are advised to limit their intake of dietary cholesterol . ase can modulate cholesterol metabolism and decrease cholesterol levels in animals . in this study , the liver and ovary tissues of laying hens were collected and digital gene expression ( dge ) was performed to investigate the mechanisms underlying the cholesterol - lowering effect of ase . 150 hy - line brown hens at the age of 27 weeks were randomly divided into five groups ( five replicates were established per treatment , with 6 hens each replicate ) . hens in each treatment were randomly fed five different experimental diets : controls were fed a basal diet with 0 mg / kg ase , and the other four groups were fed otherwise identical diets supplemented with 60 mg / kg , 120 mg / kg , 240 mg / kg , and 480 mg / kg ase , respectively . the basal diet was designed to meet , or exceed , the national research council ( nrc ) nutrient requirements . the ingredients and calculated nutrient level of the basal diet are shown in table 1 . ( shijiazhuang , china ) as a standardized product that contained 61.64% saponins , 10.97% flavonoids , 8.12% polysaccharides , 7.11% moisture , and 12.16% unknown compounds . ase is extracted from medicago sativa l. the main active component is saponins . and ase played a positive role in lowering yolk cholesterol content , , . according to the cholesterol levels in yolk of feeding experiment , the control group and 120 mg / kg ase group were chosen for digital gene - expression profiling analysis ( table 2 ) . the liver and ovary tissues of 120 mg / kg ase group were isolated , frozen immediately in liquid nitrogen and stored at 80 c until rna extraction ( the liver from the control group ( li_ck ) , the liver from the 120 mg / kg ase group ( li_exp ) , the ovaries from the control group ( ov_ck ) , the ovaries from the 120 mg / kg ase group ( ov_exp ) ) . rna was assessed for quantity and quality using a nanodrop nd-1000 spectrophotometer and agilent 2100 bioanalyzer according to the manufacturer 's instructions . 2 g of total rna ( 100 ng/l ) was pooled from three tissues within each group : li_ck , li_exp , ov_ck , ov_exp . four sequencing libraries were generated using neb next ultra directional rna library prep kit for illumina ( neb ) in accordance with the manufacturer 's recommendations . mrna was purified from total rna using poly - t oligo - attached magnetic beads ( life technologies , ca , usa ) . fragmentation was carried out using divalent captions under elevated temperature in neb proprietary fragmentation buffer . first strand cdna was synthesized using random oligonucleotides and m - mel reverse transcriptase ( rnase h- ) . second strand cdna synthesis was subsequently performed using dna polymerase i and rnase h. remaining overhangs were converted into blunt ends via exonuclease / polymerase activities and enzymes were removed . after adenylation of 3 ends of dna fragments , neb next adaptor oligonucleotides were ligated to cdna fragments . in order to select cdna fragments . dna fragments with ligated adaptor molecules on both ends were selectively enriched using neb universal pcr primer and index primer in a 10 cycles pcr reaction . products were purified and quantified using the agilent high sensitivity dna assay on the agilent bioanalyzer 2100 system . after the clustering of the index - coded samples , each library preparation was sequenced on an illumina hiseq 2000 platform and 50 bp single - end ( se ) for each dge library , clean tags were obtained by filtering out adaptor sequences and low - quality tags . then the clean tags were retained and mapped to the reference genome of gallus gallus at ftp://ftp.ensembl.org/pub/release-72/fasta/gallus_gallus/dna by tophat v1.4.0 . then reads per kilobase transcriptome per million mapped reads ( rpkm ) of each gene were calculated based on the length of the gene and reads count mapped to this gene . it is currently the most common method of for estimating levels of gene expression . in order to balance the number of false positives and false negatives a rpkm threshold value of 0.3 was established to determine whether or not given genes was expressed , as in several other studies , , . the q - values of 0.05 and log2 ( fold_change ) with no limitations were served as the threshold of significance for differential expression . here , 110 significantly expressed genes were identified in liver samples ; 66 were up - regulated and 44 were down - regulated . in ovary samples , 107 significantly expressed genes were obtained ; 63 were up - regulated and 44 were down - regulated ( table s1 ) . gene ontology ( go ) enrichment analysis of differentially expressed genes was implemented by the goseq r package . we used kobas software to test the statistical enrichment of differential expression genes in kegg pathways , . protein interaction networks of differentially expressed genes were acquired based on the information in string database ( http://string-db.org/ ) . based on the supplementing 120 mg / kg ase played a positive role in lowering yolk cholesterol content , the group with 120 mg / kg ase was used for digital gene - expression profiling analysis . in this study , some genes were found to be associated with the cholesterol metabolism . in order to shed more light on the functional roles of differentially expressed genes responsible for the cholesterol - lowering effect of ase , functional annotation was investigated using go classification and kegg pathway analysis between ov_ck and ov_exp samples . our results provided that ase were partially mediated by enhancement of cholesterol efflux in the liver and this reduced of cholesterol deposition in the ovary . the following are the supplementary data related to this article.table s1genes differentially expressed in the liver and ovary in ase and control groups.table s2kegg pathway classification of differentially expressed genes in the liver and the ovary .

cardiovascular disease is a major cause of death worldwide , so people are advised to limit their intake of dietary cholesterol [ 1 ] . egg consumption has been seriously reduced because of the high levels of cholesterol [ 2 ] . the objective of this study was to evaluate the cholesterol metabolism effects of alfalfa saponin extract ( ase ) in liver and ovary tissues using digital gene - expression ( dge ) profiling analysis . the liver and ovary tissues were isolated from laying hens fed with ase for rna sequencing . here , we provide detailed experimental methods and analysis pipeline in our study to identify digital gene expression of alfalfa saponin extract on laying hens and analysis pipeline published by singh and colleagues in the plos one [ 3 ] . the data generated in our work provide meaningful information for understanding the molecular mechanisms underlying the cholesterol - lowering effects of ase .

it has long been hypothesized that fishing can cause phenotypic changes in exploited fish populations ( miller 1957 ; borisov 1978 ; ricker 1981 ; law and grey 1989 ; stokes et al . in particular , fishing may lead to changes in life history traits , such as age and size at maturation , because these traits are especially sensitive to altered mortality schedules ( stearns 1992 ) . matching this expectation , substantial changes in age and size at maturation have been observed in many commercial fish stocks ( see reviews by miller 1957 ; policansky 1993 ; trippel 1995 ; rochet 1998 ; law 2000 ; stokes and law 2000 ; heino and godo 2002 ; hutchings and baum 2005 ; jorgensen et al . 2007 ; fenberg and roy 2008 ; heino and dieckmann 2008 ) . despite the apparent ubiquity of these trends , it has proven difficult to confirm that fishing per se is actually the cause because competing explanatory factors are hard to rule out in observational studies . in particular , long - term environmental trends ( e.g. , increasing sea surface temperatures ) have coincided with increasing exploitation in many fish stocks , and these environmental factors can influence maturation in fishes ( e.g. , cox and hinch 1997 ; shuter et al . although a few studies have formally considered some environmental factors , and ruled them out statistically ( ricker 1981 ; rijnsdorp 1993 ; barot et al . 2004 ) , many other studies have not rigorously accounted for them . moreover , some studies have concluded that the principal drivers of life history changes in particular fish stocks are environmental variables other than fishing pressure ( e.g. , cox and hinch 1997 ; cardinale and modin 1999 ) . we suggest that one way to inform the importance of fishing per se is to test , across multiple fish stocks , whether the rate of phenotypic change in life history traits is correlated with the intensity of exploitation . if fishing truly is a powerful and common driver of phenotypic change in nature , then lightly - fished stocks should exhibit little or no change in life history traits , whereas heavily - fished stocks should show rapid and dramatic changes . we surveyed the literature on commercial fish stocks to calculate rates of life history change . we then used this data set to ask ( i ) whether rates of phenotypic change in life history traits are correlated with the intensity of exploitation , and ( ii ) whether any such correlations are in the direction expected from theory . fishing may drive life history changes via at least two different mechanisms in addition to the immediate numerical effects of mortality ( nelson and soul 1987 ; stokes et al . 1993 ; smith 1994 ; trippel 1995 ; law 2000 ; heino and godo 2002 ) . for example , heavy fishing pressure often leads to drastic declines in population size ( hutchings and baum 2005 ) , which in turn can lead to the relaxation of intraspecific competition , and thus increase individual growth rates of the survivors ( policansky 1993 ) . as a result of these accelerated growth rates , fish in heavily exploited stocks can mature at younger ages , and can show associated changes in size at maturation ( heino and godo 2002 ; kuparinen and merila 2007 ) . second , fishing may induce evolutionary ( genetic ) changes in fish stocks by selecting against particular life histories . the potential for fisheries - induced evolution seems strong given that ( i ) almost all modern commercial fisheries are highly selective , especially with respect to age and size ( heino and godo 2002 ) ; ( ii ) fisheries sometimes remove as much as 50% of the individuals in populations annually ( stokes and law 2000 ) ; and ( iii ) life history traits in fish are at least moderately heritable ( h for age at maturation = 0.31 0.19 , mean from eight studies ; h for length at maturation = 0.30 0.21 , mean from 17 studies reviewed by law 2000 ) . furthermore , laboratory selection experiments using salmonids ( e.g. , gjedrem 1983 ) and atlantic silversides ( menida menida ) ( conover and munch 2002 ; conover et al . 2005 finally , rapid genetic divergence in life history traits driven by altered mortality schedules has also been documented in the wild for introduced populations of guppies ( poecilia reticulata ) ( reznick et al . 1990 ; reznick and ghalambor 2005 ) and mosquito fish ( gambusia affinis ) ( stearns 1983 ) . all of these results support the possibility of life history evolution on contemporary time scales in nature . the effects of these two mechanisms ( plasticity or genetic change ) can be jointly visualized using probabilistic maturation reaction norms ( pmrns ) . pmrns are functions that describe the probability that an immature individual of a given age and size will mature during a specified time interval ( heino et al . under this conceptual framework , plastic responses are visualized as shifts along the reaction norm mediated by changes in growth rate , whereas genetic changes are visualized as a shift in the position or shape of the maturation reaction norm itself . empirical studies are therefore increasingly examining changes in pmrns , instead of just size or age at 50% maturity , although the inferential strength of this method depends on accurately removing all of the important plastic effects ( if any ) . plastic and genetic changes can both occur in harvested populations , although they may operate on different time scales , at different rates , and even in different directions . in terms of genetic responses , life history theory generally predicts that fishing should select for earlier maturation at smaller sizes ( jorgensen et al . this expectation arises because most commercial fisheries are highly size - selective ; typically targeting individuals above a certain size threshold ( kuparinen and merila 2007 ) . this selectivity can result from the use of particular types of fishing gear , such as trawls , or specific regulations , such as minimum slot sizes ( jorgensen et al . 2009 ) . as a result , individuals that manage to reproduce at a small size / young age will often have higher fitness ( e.g. , ratner and lande 2001 ; ernande et al . 2004 ; de roos et al . 2006 ; andersen et al . 2007 ) . moreover , even fisheries that impose nonselective mortality can favor reduced age and size at maturation ( e.g. , law and grey 1989 ; de roos et al . one exception to the above prediction can occur when only sexually mature individuals are targeted , such as on spawning grounds . under this type of harvest regime , selection favours increased age at maturation , because fish that remain longer on the feeding grounds will grow larger , and thus have greater reproductive potential when they do mature and enter the spawning grounds where mortality is high ( law and grey 1989 ; heino 1998 ; ernande et al . 2007 ) , and the majority of fisheries likely select for reduced age and size at maturation . as introduced above , genetic responses to fisheries - induced selection can be accompanied by plastic shifts in life history traits , which can either reinforce , or mask , genetic trends . for age at maturation , plastic increases in growth rate in heavily - fished stocks should lead to earlier maturation ( policansky 1993 ; trippel 1995 ; law 2000 ; heino and godo 2002 ) , thus reinforcing the pattern expected from genetic change alone . for length at maturation , increased growth rates typically result in greater size at maturation ( abrams and rowe 1996 ) , thus potentially masking any genetic trend towards smaller size at maturation . however , models that incorporate both plastic and genetic effects still generally predict small increases in length at maturation when fishing mortality is low , but rapid decreases in length at maturation when fishing mortality is high ( de roos et al . overall , we predict that the dominant trend across commercial fish stocks should be a shift towards earlier maturation at smaller sizes . this pattern should manifest itself via negative rates of phenotypic change for length at 50% maturity , age at 50% maturity , and pmrns . furthermore , we predict that the rates of change in each of these life history traits across different fish stocks will be positively correlated with fishing mortality . fishing may drive life history changes via at least two different mechanisms in addition to the immediate numerical effects of mortality ( nelson and soul 1987 ; stokes et al . 1993 ; smith 1994 ; trippel 1995 ; law 2000 ; heino and godo 2002 ) . for example , heavy fishing pressure often leads to drastic declines in population size ( hutchings and baum 2005 ) , which in turn can lead to the relaxation of intraspecific competition , and thus increase individual growth rates of the survivors ( policansky 1993 ) . as a result of these accelerated growth rates , fish in heavily exploited stocks can mature at younger ages , and can show associated changes in size at maturation ( heino and godo 2002 ; kuparinen and merila 2007 ) . second , fishing may induce evolutionary ( genetic ) changes in fish stocks by selecting against particular life histories . the potential for fisheries - induced evolution seems strong given that ( i ) almost all modern commercial fisheries are highly selective , especially with respect to age and size ( heino and godo 2002 ) ; ( ii ) fisheries sometimes remove as much as 50% of the individuals in populations annually ( stokes and law 2000 ) ; and ( iii ) life history traits in fish are at least moderately heritable ( h for age at maturation = 0.31 0.19 , mean from eight studies ; h for length at maturation = 0.30 0.21 , mean from 17 studies reviewed by law 2000 ) . furthermore , laboratory selection experiments using salmonids ( e.g. , gjedrem 1983 ) and atlantic silversides ( menida menida ) ( conover and munch 2002 ; conover et al . finally , rapid genetic divergence in life history traits driven by altered mortality schedules has also been documented in the wild for introduced populations of guppies ( poecilia reticulata ) ( reznick et al . 1990 ; reznick and ghalambor 2005 ) and mosquito fish ( gambusia affinis ) ( stearns 1983 ) . all of these results support the possibility of life history evolution on contemporary time scales in nature . the effects of these two mechanisms ( plasticity or genetic change ) can be jointly visualized using probabilistic maturation reaction norms ( pmrns ) . pmrns are functions that describe the probability that an immature individual of a given age and size will mature during a specified time interval ( heino et al . plastic responses are visualized as shifts along the reaction norm mediated by changes in growth rate , whereas genetic changes are visualized as a shift in the position or shape of the maturation reaction norm itself . empirical studies are therefore increasingly examining changes in pmrns , instead of just size or age at 50% maturity , although the inferential strength of this method depends on accurately removing all of the important plastic effects ( if any ) . plastic and genetic changes can both occur in harvested populations , although they may operate on different time scales , at different rates , and even in different directions . in terms of genetic responses , life history theory generally predicts that fishing should select for earlier maturation at smaller sizes ( jorgensen et al . this expectation arises because most commercial fisheries are highly size - selective ; typically targeting individuals above a certain size threshold ( kuparinen and merila 2007 ) . this selectivity can result from the use of particular types of fishing gear , such as trawls , or specific regulations , such as minimum slot sizes ( jorgensen et al . individuals that manage to reproduce at a small size / young age will often have higher fitness ( e.g. , ratner and lande 2001 ; ernande et al . 2004 ; de roos et al . 2006 ; andersen et al . 2007 ) . moreover , even fisheries that impose nonselective mortality can favor reduced age and size at maturation ( e.g. , law and grey 1989 ; de roos et al . one exception to the above prediction can occur when only sexually mature individuals are targeted , such as on spawning grounds . under this type of harvest regime , selection favours increased age at maturation , because fish that remain longer on the feeding grounds will grow larger , and thus have greater reproductive potential when they do mature and enter the spawning grounds where mortality is high ( law and grey 1989 ; heino 1998 ; ernande et al . 2007 ) , and the majority of fisheries likely select for reduced age and size at maturation . as introduced above , genetic responses to fisheries - induced selection can be accompanied by plastic shifts in life history traits , which can either reinforce , or mask , genetic trends . for age at maturation , plastic increases in growth rate in heavily - fished stocks should lead to earlier maturation ( policansky 1993 ; trippel 1995 ; law 2000 ; heino and godo 2002 ) , thus reinforcing the pattern expected from genetic change alone . for length at maturation , increased growth rates typically result in greater size at maturation ( abrams and rowe 1996 ) , thus potentially masking any genetic trend towards smaller size at maturation . however , models that incorporate both plastic and genetic effects still generally predict small increases in length at maturation when fishing mortality is low , but rapid decreases in length at maturation when fishing mortality is high ( de roos et al . 2006 ) . overall , we predict that the dominant trend across commercial fish stocks should be a shift towards earlier maturation at smaller sizes . this pattern should manifest itself via negative rates of phenotypic change for length at 50% maturity , age at 50% maturity , and pmrns . furthermore , we predict that the rates of change in each of these life history traits across different fish stocks will be positively correlated with fishing mortality . we searched the peer - reviewed literature ( web of science ) for time series data on life history traits in commercial fish stocks . we did not place any restrictions on the date of publication or the source journal , and we used various permutations of the following search terms : change , trends , age at maturity , length at maturity , fish , maturation , life history , and fishing . we also included data from any relevant technical fisheries reports that were brought to our attention , particularly those published by the canadian department of fisheries and oceans ( dfo ) and the north atlantic fisheries organization ( nafo ) . for simplicity , we focused on commercial finfish species ( marine or freshwater ) . although we did not impose any a priori geographical restrictions , the majority of studies we found focused on temperate , marine systems from the north atlantic . because of the nature of our statistical analyses , we only included stocks for which both phenotypic time series and reliable fishing mortality statistics were available ( see below ) . this requirement greatly reduced the number of time series that we were ultimately able to analyze to 143 time series from 16 different studies . although this data set was certainly not exhaustive , inclusion or exclusion of studies was independent of the observed phenotypic changes , fishing intensities , and combinations thereof . phenotypic data were gathered for three different response variables : length at 50% maturity ( l50 ) , age at 50% maturity ( a50 ) , and midpoints of pmrns . l50 and a50 are derived from maturity ogives demographic functions that describe the probability of being mature at a given age or size , and which depend not only on maturation , but also on mortality and growth rates . in contrast , pmrns describe the maturation process after removing effects due to variation in growth rates and mortality , and thus disentangle some , but not all , of the potential environmental effects on maturation ( dieckmann and heino 2007 ) . pmrn mid - points represent the lengths at which the individuals of a given age have a 50% probability of maturing . studies reporting trends in length - at - age , weight - at - age , or mean age or size in commercial catches were not included ( with one exception ; see appendix a ) , because these data do not directly describe maturation , and they may vary with the selectivity of fishing gear . we chose not to collect data on growth rates , as these data were less widely reported , and because potential changes in growth in response to fishing are more difficult to predict and interpret . however , there is evidence that growth rates have declined in some commercial fish stocks ( jorgensen et al . 2007 ) . for each time series , we estimated the phenotypic value at the beginning ( z0 ) and end ( z1 ) of the study period , as well as the duration of the study ( t1t0 = t ) . to account for year - to - year fluctuations in time series , we estimated ( t0 , z0 ) and ( t1 , z1 ) as the mean of the first and last three data points in each time series , respectively . alternatively , if the authors had fit linear regressions to their time series , we used the ( x , y ) co - ordinates at the start and end of these regressions to estimate t0 and t1 and z0 and z1 . in some cases , the time series spanned a significant change in fishing intensity such as the imposition of a complete fishing moratorium . in these cases , we considered the pre- and postmoratorium periods separately , as in olsen et al . we did not include data in cases where t was less than the generation length of the species . the rate of phenotypic change for each trait was calculated in darwins , a common index for measuring rates of phenotypic change ( haldane 1948 ; gingerich 1993 ; hendry and kinnison 1999 ) . this index represents the proportional change per million years and is calculated as : an alternative phenotypic rate metric , the haldane , was not used because it required data ( i.e. , standard deviations in each time step ) that were not available for many time series . we did , however , consider one important element of haldanes by also considering elapsed time as the number of generations ( years divided by generation lengths ) . generation lengths were estimated from stock status reports , published data , and from consultation with fisheries scientists ( see appendices a these estimates generally reflect pre - exploitation conditions , and would likely change when age at maturation changes during exploitation . fishing intensity was estimated as the instantaneous rate of fishing mortality ( f , year ) . while data on fishing mortality are often scarce , f is a good proxy for the strength of selection imposed by a fishery , because it measures the proportion of a population that is removed by the fishery at a given time . unlike other more widely - reported metrics of fishing intensity ( e.g. , total landings , catch per unit effort ) , f is a per capita rate with no other dimensions than time , and therefore has the advantage of being readily comparable across studies . f values were rarely reported in the studies we examined , and so they were obtained from other sources , including stock status reports , online databases , and consultation with fisheries scientists ( see appendices a c for details ) . overall fishing mortality ( year ) for each stock was calculated as the average of yearly estimates of f over the time series ( or fraction thereof if some years were missing ) . in cases where age - specific f estimates were available , we used those for the age classes most heavily targeted by the fishery ( i.e. , the highest f values ) . given potential uncertainty in these estimates of f , we also assigned categorical levels of fishing intensity for each stock : low for f < 0.3 , med for 0.3 f < 0.6 , and high for f 0.6 . not all of the rate estimates in our initial data set were strictly independent : for example , some came from males and females in the same stock , from different age cohorts in the same stock , or from different studies of the same stock . we reduced this nonindependence by retaining only a single rate estimate for each life history trait for each stock , based on the criteria outlined below . although stocks are often arbitrary management units and may not always represent genetically distinct populations their use here is justified on several grounds : ( i ) life history traits often vary substantially among stocks ( e.g. , cosewic 2003 ; busby et al . 2007 ) , ( ii ) fishing intensity often varies among stocks ( appendices a c , this paper ) , ( iii ) data are typically reported on a stock by stock basis ( see for example department of fisheries and oceans canada stock status reports ) , and ( iv ) management decisions are usually made at the stock level ( e.g. , imposition of moratoria , see appendices a c , this paper ) . we experimented with reducing our data set in various ways ( e.g. , selecting the maximum or mean rate per stock , selecting one random entry per stock ) , but this did not influence our conclusions ( results not shown ) , so we settled on the following protocol . if time series were available for both sexes from a given stock , we included rate estimates for females only . if pmrn midpoints were reported for multiple ages from the same stock , we selected the time - series with the best resolution and smallest confidence intervals ( generally the middle of the range of ages reported ) . if two separate studies reported time series for the same stock , we included whichever series was the longest . if multiple rate estimates were calculated from the same time series ( e.g. , pre- and postmoratorium for the same stock ) , we excluded one at random . finally , even within these reduced stock - level data sets , one might argue for nonindependence of different stocks within a species . we therefore generated a further reduced data set that included only one rate estimate per species , selected at random from the stock - level data set . to test for the effects of fishing on the rate of phenotypic change in each trait ( l50 , a50 , pmrn ) we fit four separate general linear models in r ( v. 2.6.1 . the explanatory variables were fishing mortality ( continuous or categorical ) and time interval ( log10 years or number of elapsed generations ) , and the response variable was the magnitude of phenotypic change ( darwin numerator ) . we first fitted full models , but the interaction between fishing mortality and time was almost always non - significant . these models were fit separately for each trait and for the stock - level and species - level data sets . sample sizes were : 18 ( stocks ) and eight ( species ) for length at 50% maturity , 25 ( stocks ) and seven ( species ) for age at 50% maturity , and 11 ( stocks ) and four ( species ) for pmrns . we searched the peer - reviewed literature ( web of science ) for time series data on life history traits in commercial fish stocks . we did not place any restrictions on the date of publication or the source journal , and we used various permutations of the following search terms : change , trends , age at maturity , length at maturity , fish , maturation , life history , and fishing . we also included data from any relevant technical fisheries reports that were brought to our attention , particularly those published by the canadian department of fisheries and oceans ( dfo ) and the north atlantic fisheries organization ( nafo ) . for simplicity , we focused on commercial finfish species ( marine or freshwater ) . although we did not impose any a priori geographical restrictions , the majority of studies we found focused on temperate , marine systems from the north atlantic . because of the nature of our statistical analyses , we only included stocks for which both phenotypic time series and reliable fishing mortality statistics were available ( see below ) . this requirement greatly reduced the number of time series that we were ultimately able to analyze to 143 time series from 16 different studies . although this data set was certainly not exhaustive , inclusion or exclusion of studies was independent of the observed phenotypic changes , fishing intensities , and combinations thereof . phenotypic data were gathered for three different response variables : length at 50% maturity ( l50 ) , age at 50% maturity ( a50 ) , and midpoints of pmrns . l50 and a50 are derived from maturity ogives demographic functions that describe the probability of being mature at a given age or size , and which depend not only on maturation , but also on mortality and growth rates . in contrast , pmrns describe the maturation process after removing effects due to variation in growth rates and mortality , and thus disentangle some , but not all , of the potential environmental effects on maturation ( dieckmann and heino 2007 ) . pmrn mid - points represent the lengths at which the individuals of a given age have a 50% probability of maturing . studies reporting trends in length - at - age , weight - at - age , or mean age or size in commercial catches were not included ( with one exception ; see appendix a ) , because these data do not directly describe maturation , and they may vary with the selectivity of fishing gear . we chose not to collect data on growth rates , as these data were less widely reported , and because potential changes in growth in response to fishing are more difficult to predict and interpret . however , there is evidence that growth rates have declined in some commercial fish stocks ( jorgensen et al . 2007 ) . for each time series , we estimated the phenotypic value at the beginning ( z0 ) and end ( z1 ) of the study period , as well as the duration of the study ( t1t0 = t ) . to account for year - to - year fluctuations in time series , we estimated ( t0 , z0 ) and ( t1 , z1 ) as the mean of the first and last three data points in each time series , respectively . alternatively , if the authors had fit linear regressions to their time series , we used the ( x , y ) co - ordinates at the start and end of these regressions to estimate t0 and t1 and z0 and z1 . in some cases , the time series spanned a significant change in fishing intensity such as the imposition of a complete fishing moratorium . in these cases , we considered the pre- and postmoratorium periods separately , as in olsen et al . we did not include data in cases where t was less than the generation length of the species . the rate of phenotypic change for each trait was calculated in darwins , a common index for measuring rates of phenotypic change ( haldane 1948 ; gingerich 1993 ; hendry and kinnison 1999 ) . this index represents the proportional change per million years and is calculated as : an alternative phenotypic rate metric , the haldane , was not used because it required data ( i.e. , standard deviations in each time step ) that were not available for many time series . we did , however , consider one important element of haldanes by also considering elapsed time as the number of generations ( years divided by generation lengths ) . generation lengths were estimated from stock status reports , published data , and from consultation with fisheries scientists ( see appendices a these estimates generally reflect pre - exploitation conditions , and would likely change when age at maturation changes during exploitation . fishing intensity was estimated as the instantaneous rate of fishing mortality ( f , year ) . while data on fishing mortality are often scarce , f is a good proxy for the strength of selection imposed by a fishery , because it measures the proportion of a population that is removed by the fishery at a given time . unlike other more widely - reported metrics of fishing intensity ( e.g. , total landings , catch per unit effort ) , f is a per capita rate with no other dimensions than time , and therefore has the advantage of being readily comparable across studies . f values were rarely reported in the studies we examined , and so they were obtained from other sources , including stock status reports , online databases , and consultation with fisheries scientists ( see appendices a c for details ) . overall fishing mortality ( year ) for each stock was calculated as the average of yearly estimates of f over the time series ( or fraction thereof if some years were missing ) . in cases where age - specific f estimates were available , we used those for the age classes most heavily targeted by the fishery ( i.e. , the highest f values ) . given potential uncertainty in these estimates of f , we also assigned categorical levels of fishing intensity for each stock : low for f < 0.3 , med for 0.3 f < 0.6 , and high for f 0.6 . not all of the rate estimates in our initial data set were strictly independent : for example , some came from males and females in the same stock , from different age cohorts in the same stock , or from different studies of the same stock . we reduced this nonindependence by retaining only a single rate estimate for each life history trait for each stock , based on the criteria outlined below . although stocks are often arbitrary management units and may not always represent genetically distinct populations their use here is justified on several grounds : ( i ) life history traits often vary substantially among stocks ( e.g. , cosewic 2003 ; busby et al . 2007 ) , ( ii ) fishing intensity often varies among stocks ( appendices a c , this paper ) , ( iii ) data are typically reported on a stock by stock basis ( see for example department of fisheries and oceans canada stock status reports ) , and ( iv ) management decisions are usually made at the stock level ( e.g. , imposition of moratoria , see appendices a c , this paper ) . we experimented with reducing our data set in various ways ( e.g. , selecting the maximum or mean rate per stock , selecting one random entry per stock ) , but this did not influence our conclusions ( results not shown ) , so we settled on the following protocol . if time series were available for both sexes from a given stock , we included rate estimates for females only . if pmrn midpoints were reported for multiple ages from the same stock , we selected the time - series with the best resolution and smallest confidence intervals ( generally the middle of the range of ages reported ) . if two separate studies reported time series for the same stock , we included whichever series was the longest . if multiple rate estimates were calculated from the same time series ( e.g. , pre- and postmoratorium for the same stock ) , we excluded one at random . finally , even within these reduced stock - level data sets , one might argue for nonindependence of different stocks within a species . we therefore generated a further reduced data set that included only one rate estimate per species , selected at random from the stock - level data set . to test for the effects of fishing on the rate of phenotypic change in each trait ( l50 , a50 , pmrn ) we fit four separate general linear models in r ( v. 2.6.1 . the explanatory variables were fishing mortality ( continuous or categorical ) and time interval ( log10 years or number of elapsed generations ) , and the response variable was the magnitude of phenotypic change ( darwin numerator ) . we first fitted full models , but the interaction between fishing mortality and time was almost always non - significant . these models were fit separately for each trait and for the stock - level and species - level data sets . sample sizes were : 18 ( stocks ) and eight ( species ) for length at 50% maturity , 25 ( stocks ) and seven ( species ) for age at 50% maturity , and 11 ( stocks ) and four ( species ) for pmrns . rates of phenotypic change for length at 50% maturity in our stock - level analysis ( i.e. , after elimination of pseudoreplicated entries ) ranged from 24.8 10 to + 5.6 10 darwins ( mean sd = 10.6 9.6 ) . the vast majority ( 17 out of 18 ) of these rates were negative ( appendix a ) , significantly more than expected from chance ( fisher 's exact test , p = 0.007 ) . increasing fishing mortality ( f ) had a strong negative effect on length at 50% maturity , with the most heavily exploited stocks shifting most rapidly towards smaller sizes at 50% maturity ( figs 1a and 2a ) . this relationship was significant for the stock - level data sets , regardless of the fishing mortality metric ( continuous or categorical f ) , and the time metric ( years or generations ) ( table 1 ) . the direction of the trend remained the same for the species - level dataset ( fig . given comparable variances explained between the stock - level and species - level analyses , the reduced significance in the latter was probably the result of the smaller sample size and narrow range of f values ( table 1 ) . magnitude of phenotypic change in response to fishing mortality for length at 50% maturity ( a , b ) , age at 50% maturity ( c , d ) and mid - points of probabilistic maturation reaction norms ( pmrns ) ( e , f ) . the y axis shows residuals from a linear regression of darwin numerators ( [ ln(z1 ) ln(z0 ) ] ) over time ( log10 years ) ; i.e. , proportional phenotypic change after accounting for the effects of time . fishing mortality is the average of yearly estimates of fishing mortality for the time period over which the phenotypic change was measured . note that one data point ( f = 1.9 ) is not shown in panels c ( x = 0.19 , y = 0.10 ) and d ( x = 0.19 , y = 0.18 ) so as to match the scale in the other panels . trendlines were fit only in cases where fishing mortality was found to be significant ( p < 0.05 ) . rates of phenotypic change for stocks experiencing low ( f < 0.3 ) , medium ( 0.3 f < 0.6 ) and high ( f 0.6 ) levels of fishing mortality ( year ) . rates are expressed in darwins ( 10 ) and are plotted separately for length at maturity ( a ) , age at maturity ( b ) and midpoints of probabilistic maturation reaction norms ( pmrns ) ( c ) . the thick lines represent the median of each distribution , while the top and bottom of the boxes represent the 75th and 25th percentiles , respectively . the dashed error bars represent 1.5 times the interquartile range ( approximately 2 standard deviations ) . effect of fishing mortality on rates of change in length at 50% maturity ( l50 ) note : time is the duration of the time series measured in years ( log10-transformed ) ; and gen is the duration of the time series measured in generations for the species in question . partial is a measure of effect size and was calculated as sseffect/(sseffect + sserror ) . rates of phenotypic change for age at 50% maturity in our stock - level analysis ranged from 41.2 10 to + 12.6 10 darwins ( mean sd = 12.6 12.3 ) . the majority ( 23 out of 25 ) of these rates were negative ( appendix b ) , significantly more than expected from chance ( fisher 's exact test , p = 0.001 ) . in contrast to the pattern observed for length at 50% maturity , fishing mortality had much less of an effect on the rate of change in age at maturity among stocks ( figs 1c and 2b ) . fishing mortality explained almost none of the variation among rates when considered as a continuous variable ( table 2 ) and , although it was statistically significant when treated categorically ( table 2 ) , there was substantial overlap among categories ( fig . 1d ) , with no significant effect of fishing mortality regardless of the time metric used ( table 2 ) . effect of fishing mortality on rates of change in age at 50% maturity ( a50 ) note : in the species - level analysis , sample size was insufficient to fit models with fishing mortality as a categorical explanatory variable . for abbreviations and other conventions see note for table 1 . rates of phenotypic change for pmrns in our stock - level analysis ranged from 27.5 10 to + 7.3 10 darwins ( mean sd = 9.2 10.6 ) . the majority ( 8 out of 11 ) of these rates were negative ( appendix c ) , although this was not significantly more than expected from chance ( fisher 's exact test , p = 0.387 ) . increasing fishing mortality had a very strong negative effect on pmrns , with the most heavily exploited stocks showing the most rapid declines in prmn midpoints ( i.e. , shifts towards maturation at smaller size for a given age ) ( figs 1e and 2c ) . this relationship was significant for the stock - specific analyses , regardless of the fishing mortality metric ( continuous or categorical f ) , and the time metric ( years or generations ) ( table 3 ) . the direction of the trend remained the same for the species - level dataset ( fig . 1f ) , but there were insufficient data ( n = 4 ) to conduct any statistical tests . in contrast to the other traits , the interaction between fishing mortality and time was significant in some models ( table 3 ) . effect of fishing mortality on rates of change in probabilistic maturation reaction norm ( pmrn ) midpoints note : the sample size was insufficient to fit models at the species level . for abbreviations and other conventions , the rates of phenotypic change that we estimated for life history traits in commercial fish stocks are generally consistent with previous estimates ( jorgensen et al . 2007 ) , but are higher than many evolutionary rates previously reported in the literature for other traits , taxa , and contexts ( hendry and kinnison 1999 ; kinnison and hendry 2001 ; hendry et al . 2008 ) . this result supports the intuition that exploitation is a very strong selective force , probably outside the normal intensities of selection in most natural populations ( darimont et al . it is also true , however , that the particularly rapid rates of change observed here may be partly due to the relatively short duration of time series for harvested species ( 116 generations ) , given that rates of phenotypic change scale negatively with time interval ( gingerich 1993 , 2001 ; kinnison and hendry 2001 ) . one potential reason is that short studies may coincide with periods of intense selection that , in longer studies , would be averaged with periods of stasis and reversals ( grant and grant 2002 ) . it is important to acknowledge that the rapid rates of phenotypic change reported here could reflect plasticity rather than genetic change . this might be particularly likely for age and size at 50% maturity , both of which are known to show substantial plasticity ( stearns 1992 ) . pmrns , however , are meant to remove at least some of these plastic effects by accounting for variation due to changes in growth and mortality ( dieckmann and heino 2007 ) . thus , shifts in pmrns have been frequently interpreted as representing genetic , rather than plastic , changes in maturation schedules ( e.g. , olsen et al . 2004 ) , although such an inference must be made with caution ( dieckmann and heino 2007 ; kraak 2007 ) in our study , pmrns often changed as rapidly as age and size at 50% maturity , suggesting that plastic effects of growth are not the sole driver of changes in life history traits . how well do the observed trends fit theoretical predictions for fisheries - induced life history change ? for length at 50% maturity , we observed downward trends in the vast majority of stocks . this pattern is consistent with either genetic responses to fishing - induced selection ( ratner and lande 2001 ; ernande et al . 2007 ) , or with a combination of genetic and plastic effects ( de roos et al . 2006 ) . however , this pattern is the opposite of what we would generally expect from plastic effects alone in which case increases in size at maturation would have been most likely ( abrams and rowe 1996 ) . plastic decreases in size at maturation might be theoretically observed in species with positively - sloped pmrns ; however , almost all pmrns calculated to - date exhibit negative slopes ( heino and dieckmann 2008 ) . we also found that the intensity of fishing - induced mortality had a significant positive effect on the rate of change in length at 50% maturity , with the most heavily exploited populations experiencing the most precipitous declines in length . this correlation supports the hypothesis that fishing morality is a major driver of observed changes in size at 50% maturity across a diversity of commercial fish stocks . for age at 50% maturity , we also observed declining trends in the majority of stocks , which is consistent with either genetic or plastic responses to fishing ( heino and godo 2002 ; ernande et al . however , variation in the rate of decline among stocks was not significantly correlated with fishing mortality , suggesting that trends in age at 50% maturity may be influenced by variables not considered in the present study . for example , strong temporal trends in temperature , large changes in growth rate , or high levels of natural mortality may be the primary drivers of changes of age at 50% maturity in certain stocks . additionally , significant shifts in age at 50% maturity may have occurred earlier in the exploitation history of some stocks ( i.e. , before life history data were collected systematically ) , and further decreases may not be physiologically possible , despite continued high levels of mortality ( trippel 1995 ) . for pmrns , we observed declines through time for most stocks , in agreement with predicted genetic responses to size - selective harvesting ( ernande et al . increases in pmrn midpoints were typically observed only in postmoratorium situations ( e.g. , olsen et al . 2005 ) , or when fishing mortality was low ( e.g. , norwegian spring - spawning herring prior to 1955 ) . the rate of decline in pmrns was positively correlated with fishing mortality , and this relationship was stronger than for age and size at 50% maturity . overall , our results point to commercial fishing as a major driver of life history change ( at least for length at 50% maturity and pmrns ) . the above interpretations are subject to a number of caveats , stemming from the biases and uncertainties inherent in the data compiled for this review . first , our data set suffers from a strong geographical bias , with the majority of data coming from temperate , marine stocks from the northwest atlantic . as a result . it would be valuable to collect more data from other fish stocks , particularly from tropical and freshwater populations , which have been severely under - represented in this , and many earlier , reviews . second , the literature is dominated by work on only a few major commercial species , most notably atlantic cod , that have captured the public and scientific imagination because of the dramatic declines they suffered as a result of over - fishing . such stocks might exhibit anomalous or extreme trends , and thereby have an inordinate effect in combined analyses . on a related note , only a few fish families are represented in our data set , most notably the pleuronectidae , gadidae , and salmonidae . given that life history characteristics are often conserved within families , and that different life history characteristics influence vulnerability to fishing ( jennings et al . 1999 ) , the set of families that we considered are probably not representative of all possible responses to fishing . third , we used stocks defined for fisheries management as our units of replication and these stocks may not be demographically or evolutionarily independent . we attempted to address these last two issues by conducting analyses with reduced data sets that included only one rate estimate per species . statistical power was lacking in these analyses , but qualitative trends did not change , suggesting that any biases may not be too severe . these estimates came from a variety of different sources , showed considerable temporal variation , and were often available for only part of a given time series . another limitation was that some data sources reported f estimates averaged across the entire stock , whereas others reported it only for the most heavily exploited age classes . finally , the majority of f estimates do not include mortality due to by - catch from other fisheries , or discards at sea , which can be substantial ( jennings and kaiser 1998 ) . we attempted to minimize these uncertainties by also assigning categorical levels of fishing mortality to each stock . in all cases , trends were generally similar between the continuous and categorical analyses , suggesting that uncertainties about the magnitude of f may not be an overwhelming problem . finally , we recognize that such a broad - brush analysis of trends necessarily ignores a great deal of biological complexity . first , many stocks were exploited for centuries before any data on life history traits were collected . this past selection , which can not be assessed , has presumably shaped the starting point for evolution in response to recent fishing . this consideration also implies that postmoratorium rates must be interpreted with caution , as any phenotypic change during these periods may be influenced by the prior periods of intense exploitation . most notably , many stocks were at very low population densities at the time moratoria were imposed , and this could certainly have plastic effects on life history traits . however , we think that the inclusion of postmoratorium data is justifiable on several grounds . as mentioned above , even premoratorium estimates come after long periods of fishing pressure ( hundreds of years in the case of some cod stocks ) , for which we have little or no quantitative data . relying on these estimates in addition , excluding all postmoratorium estimates would dramatically reduce the range of f values for our analyses , because almost of all our low fishing mortality estimates come from postmoratorium contexts . of course it would be preferable to have long - term maturity data from unexploited or lightly exploited fish populations , but such data are scarce . a second important complication is that fishing alters marine ecosystems in complex and pervasive ways that extend far beyond the direct mortality imposed on the target stock ( jennings and kaiser 1998 ) . for example , fishing may change the densities of predators , prey , and competitors , which may alter growth rates and age - specific mortality and thus influence maturation schedules ( kuparinen and merila 2007 ) . notwithstanding these complexities , it is remarkable that simple models incorporating only recent , rough estimates of fishing intensity could explain 4793% of the variation in the magnitude of change among stocks ( for l50 and pmrns ) . keeping the above caveats in mind , we now return to the question that motivated this analysis : to what extent are the changes in life histories observed in commercial fish stocks driven by fishing ? we found significant correlations between fishing mortality and the rate of phenotypic change for two of the three traits examined , in a direction that was consistent with theoretical expectations for fisheries - induced evolution . these correlations explained a large portion of the variation in rates of change among populations , and were relatively robust to the types of metrics we used ( i.e. , years versus generations , f categorical or continuous ) , and the different assumptions underlying our analysis ( i.e. , stocks versus species as units of replication ) . although correlations can not prove a causal link between fishing and phenotypic change , they support this hypothesis and corroborate other types of evidence that fishing can cause evolutionary changes in natural populations , including mathematical models and simulations ( e.g. , ernande et al . 2004 ) , field - based estimates of fisheries - induced selection ( carlson et al . 2007 ) , and laboratory experiments ( e.g. , conover et al . 2005 ; conover and baumann 2009 ) . we conclude that the available evidence strongly points to exploitation as a major force driving life history change in commercial fish stocks . this reinforces the value of incorporating evolutionary thinking into fisheries management ( e.g. , law and grey 1989 ; dunlop et al .

age and size at maturation have declined dramatically in many commercial fish stocks over the past few decades changes that have been widely attributed to fishing pressure . we performed an analysis of such trends across multiple studies , to test for the consistency of life history changes under fishing , and for their association with the intensity of exploitation ( fishing mortality rate ) . we analyzed 143 time series from 37 commercial fish stocks , the majority of which originated from the north atlantic . rates of phenotypic change were calculated for two traditional maturation indices ( length and age at 50% maturity ) , as well as for probabilistic maturation reaction norms ( pmrns ) . we found that all three indices declined in heavily exploited populations , and at a rate that was strongly correlated with the intensity of fishing ( for length at 50% maturity and pmrns ) . these results support previous assertions that fishing pressure is playing a major role in the life history changes observed in commercial fish stocks . rates of change were as strong for pmrns as for age and size at 50% maturity , which is consistent with the hypothesis that fishing - induced phenotypic changes can sometimes have a genetic basis .

chronic diseases are associated with several psychosocial problems including quality of life , depression , anxiety and other psychological disorders . in the cases in which life conditions in some chronic diseases stigmatize the patient , the psychological impairment becomes more and more pronounced . chronic hepatitis c as one of the most commune causes of hepatic diseases is associated with a major psychosocial risk . the complications in the advanced stages of the hepatic diseases can be associated with a low level of the quality of life . the primary care of the doctors and psychiatrists is to identify the patients with psychiatric diseases which are exposed to the risk of virus b and c infection and observe them [ 1 , 2 ] . preexisting psychiatric disorders complicates the treatment of viral b and c chronic hepatitis , as the treatment of viral b and c chronic hepatitis can cause worst psychiatric disorders . below of being a mental marker of patients , measuring the quality of life should be capable of catching the differences between stages of a specific disease ( generic instruments allow for comparison between patients diagnosed with the target conditions and those without ) . the criteria of monitoring the quality of life are : sleepiness , fatigue , emotional function , anxiety , depression , vulnerability , lack of appetite , abdominal and systemic symptoms , joint pain / discomfort , loneliness , sexual problems and disfunctions . several studies showed the difference between the quality of life , which can differ in function of the severity of the disease , from asymptomatic to associated symptoms ( usually in cirrhosis and hepatocellular carcinoma ) . the stages of the disease affects the quality of life in function of the stage of disease ( table 1 ) . studies show that patients with chronic active hepatitis b have scores near compensated cirrhosis , but all have better scores than patients with cirrhosis and hepatocellular carcinoma . post - transplant patients showed an improvement of quality of life compared with patients with decompensated cirrhosis and hepatocellular carcinoma . summary of most important studies about the quality of life at patients with chronic hepatitis b most of the dates compare those with chronic hepatitis b with people who have chronic hepatitis c , nonalcoholic fat liver . scores had been higher at patients with chronic hepatitis b than patients with nonalcoholic fat liver . there been no differences between chronic hepatitis b and patients with chronic hepatitis c , although scores regarding the activity and questions about abdominal symptoms had been lower at patients with hcv than patients with hbv . the majority of the studies show that the quality of life at patients with hbv is better than patients with other liver diseases [ 3 ] . most of the researches show a low quality of life ( especially regarding psychiatric symptoms ) at patients with hcv compared to hbv . studies involving psychological disorders in hbv patients are generally limited to anxiety and depression . studies showed that psychiatric disorders ( mostly depression ) , anxiety and a poor global functioning were more common in chronic inactive hepatitis than healthy subjects . there was a higher rate of psychiatric disorders at patients diagnosed with chronic hepatitis b within 3 months . one of the areas with the fewest studies regarding psychosocial issues of patients with hbv refers to children . lai et all [ 4 ] in study above children found high levels of depression , anxiety and emotional instability at sick patients than healthy subjects . psychological disorders do nt affect only the child , but also have a negative impact above their parents . patients with severe psychiatric manifestations have generally a higher grade of poverty , fact that raises the risk of infection with hbv . the abuse of substances and high - risk behaviors are frequently associated with psychiatric manifestations [ 5 ] . hbv rate of prevalence among institutionalized patients with mentally handicap is 8% at more than 80% [ 6 , 7 ] . studies regarding stigma of hcv patients are one of the most deficient areas in the specialty literature . in a little study , 19 careers of hbv had been interviewed about their more important fears . while 60% of them expressed their worry of not infecting other people , patients with chronic virus c had a higher prevalence of psychiatric disorders , as well those with antiviral treatment [ 8 ] . at least 50% of the patients infected with virus c suffer of a psychiatric disease and the prevalence during life of psychosis , anxiety , impairment of quality of life is significantly higher than healthy subjects [ 6 , 9 - 11 ] . the prevalence of virus c infection among hospitalized patients in psychiatric hospitals is of 18% [ 7 ] . for some patients with preexisting mental illness ( psychosis , substance abuse , using and sharing same needles , intranasal drug abuse ) certainly raise the risk of infection with virus c [ 12 - 15 ] . for some other patients ( those with anxiety and those who have affective disorders ) the distinction between cause and effect is less clear [ 16 - 18 ] . despite the association between hcv and psychiatric diseases , these patients have a risk to develop hcv through risky sexual behaviors and intranasal drug abuse . the worry for hcv patients is challenging while most of psychotropic drugs are metabolized in liver and associated with a raised hepatotoxicity . patients with hcv and psychiatric diseases would nt answer as well as the difficulties to submit at the rigorous posttransplant regimen [ 16 - 18 ] . despite the evidence that hcv infected and preexisting or actual psychiatric diseases can safely be treated with antiviral medication , these patients are continuously excluded from ribavirin and interferon therapy because of neuropsychic side effects . [ 19 - 21 ] neuropsychiatrical side effects of ribavirin and interferon therapy appear in a proportion that varies between 24 and 49% of patients [ 9 , 22 ] . among them fatigue , depression , anxiety , hostility , maniacal symptoms , cognitive disorders , delirium , psychosis and suicidal ideation [ 20 , 23 , 24 ] . among more commune psychiatric side effects had been with systemic and gastrointestinal effects the most commune reason for giving up therapy . there is a general question if a patients who has chronic hepatitis c and has a personal history of psychiatric diseases , but which at actual moment does nt have psychiatric symptoms is able to do the antiviral treatment without interruption and with minimal psychiatric side effects . given the adverse effects of antiviral therapy depression is estimated that approximately 30 - 35% of patients treated with interferon will develop depression . most antidepressants are metabolized by the liver , which raises concerns regarding the potential hepatotoxicity [ 27 ] . however new antideprsiv approved desvenlafaxine could significantly improve risk due to metabolism in a very small proportion liver . prophylactic antidepressant should be taken into account but well monitored side effects ( nausea , vomiting , sexual dysfunction , paradoxical reactions ) . other examples of antidepressants that includes mirtazapine has a certainty that is associated with bone decalcification and agranulocytosis , which would place patients treated with interferon in a high risk for infection . quality of life is significantly impaired in patients with chronic hepatitis b , particularly in those with severe disease . c virus infection is a liver disease most likely affect the cns and is associated with increased proportion of neuropsychiatric disturbances . pre - existing psychiatric imbalances hcv complicates treatment as hcv treatment can cause exacerbation of psychiatric disorders . prevention of disease progression or treatment of early stage liver transplantation can improve quality of life . although some of antiviral drugs decrease the quality of life during treatment , it significantly improves after stopping it . patients in psychiatric institutions have increased risk of infection with virus b and c. immunization , continuous monitoring , educating those with psychiatric disturbances and their families and decrease length of stay may be potential benefits in reducing infection rates .

psychosocial issues and the quality of life are important components at the patients diagnosed with chronic hepatitis b and c. in function of the severity of the infection with virus b or the patients who already have cirrhosis , the treatment and psychosocial education should be improved because they have bigger problems . the frequency of psychosocial disorders seems to be raised at the patients diagnosed with chronic hepatitis b. factors as alcohol abuse and a low social support have a negatively impact above mental health of these patients . the prevalence rate of chronic hepatitis c infection at patients with severe mental illness can be nine times higher than in healthy population . usually patients with chronic hepatitis b have a quality of life and a mental health better than patients with chronic hepatitis c. patients with psychiatric affections ( especially institutionalized people ) have generally a higher risk of being infected with virus b in comparison with general population . patients with chronic hepatitis b and c suggest a higher grade of stigmatization from society . despite clinical challenges which treatment with interferon at patients with chronic hepatitis and comorbidities represents , recent studies indicate the fact that treatment can be administrated in safe conditions at patients with viral chronic hepatitis and psychiatric disorders .

epilepsy is one of the most prevalent neurological disorders which affects about 0.5- 1 percent of the world s population ( 1 ) . epilepsy is resulted from a recurrent spontaneously abnormal electrical discharge of a group of neurons in the brain and exhibits as seizure occurrence in the patients ( 2 ) . glutamate and -aminobutyric acid ( gaba ) are two important excitatory and inhibitory neurotransmitters in epilepsy ( 3 , 4 ) . in spite of the generally acceptable treatment of epilepsy by anticonvulsant drugs , about one - third of this population suffers from un - prevented neurological changes induced by epileptic seizures and also exhibit some accompanied side effects . the long time seizure - induced neuronal activity might result in neurological changes and finally is ended by neuronal death ( 5 ) . oxidative stress and free radicals production are of the most important mechanisms by which neurological disorders such as epileptic seizure occur ( 6 , 7 ) . nitric oxide ( no ) is known as a neurotransmitter in the brain that has shown paradoxical role in seizure modulation , as an inhibitor ( 8 - 10 ) and promoter ( 11 , 12 ) in different cases . the final product of lipid peroxidation is malondialdehyde ( mda ) , while mda level could be considered as an index of lipid peroxidation . increased level of mda , as an index of lipid peroxidation in the ptz mice may lead us to the conclusion that free fatty acids and free radicals are made from membrane phospholipid metabolism . superoxide dismutase ( sod ) is an intracellular antioxidant enzyme that catalyses converting the peroxidase to hydrogen peroxide ( h2o2 ) in order to protect the cell from superoxide radicals and oxidative stress . black mustard is a many - branched , aromatic , weedy annual plant , growing up to 4 meters in height . mustard seeds contain omega-3 fatty acids , essential oils , the minerals selenium , phosphorus , manganese , magnesium , iron , calcium , zinc , vitamins a , b - complex , and c , dietary fibre , protein , and phytonutrients ( 13 ) . in iranian traditional medicine , brassica nigra seed has been used as a sedate for neurotic pain and rheumatoid arthritis , treatment of the brain and lung edema , paralysis , migraine and epilepsy ( 14 ) . experimental reports have shown the antioxidant ( 15 ) , hypoglycemic ( 16 ) , anticancer ( 17 ) and antimicrobial effect ( 18 ) of brassica nigra seed . there are evidences implying the anti - epileptic effect of brassica nigra in iranian traditional medicine ( 14 ) . furthermore , the effect of this plant on oxidative stress and free radicals production is also reported ( 19 ) . therefore , in the present study we have investigated the antiepileptic effect of brassica nigra seed extract via assessing the anticonvulsant property of the plant . we have also tried to consider the antioxidant effect of the plant using mda , no and sod assessment using the kindling method . we have also compared the anticonvulsant and antioxidant effects of brassica nigra with valproate as a standard drug for epilepsy . in this experimental research , a total of 60 male albino mice weighing 20 - 25 g ( razi institute , iran ) were randomly divided into six experimental groups including : 1- control , 2- ptz - induced kindled , 3- positive control group which beside the ptz received valproate 100 mg /kg ( ip ( sigma , usa ) ) as an anti - convulsant drug , 4 , 5 and 6- treatment groups which beside the ptz received the brassica nigra seed extract in three doses of 75 , 150 and 300 mg / kg ; ip . ten mice were housed in each cage at temperature of 21 2c and 12 h light - dark cycling . all animals except the control groups ( group 1 ) were kindled by a total of 11 period injections of ptz ( 35 mg / kg ; ip ) . each administration was carried out every second day and in a period of 22 days . after an additional 30 min , the mice were observed for lethality before returning to the home cage . the challenge dose of 75 mg / kg ptz was injected to the kindled mice on 26th day ( the test day ) , which could produce convulsions ( tonic - clonic ) and lethality ( 5 ) . in the four treatment groups ( valproate and different doses of brassica nigra ) , ptz was administrated 30 min after the first treatment with valproate and different doses of brassica nigra . however , the exhibited phases of seizure ( 0 - 6 ) were observed and categorized using the following scale ( 19 ) for 30 min after the ptz injection . the scale introduces six phases as follows ; 0 : no response , 1 : ear and facial twitching , 2 : convulsive waves axially through the body , 3 : myoclonic body jerks , 4 : generalized clonic convulsions turn over into side position , 5 : generalized convulsions with tonic extension episode and status epilepticus , 6 : mortality . preparation of plant hydro - alcoholic extract the medicinal plant of brassica nigra seed was provided from the local market and was scientifically identified by the department of botany of shahed university . to prepare the hydro - alcoholic extract , using percolation method , 50 g of cleaned brassica nigra seed was crushed and mixed at ratio of 1 to 5 with ethanol 80% and kept for 24 h at room temperature . during this time , it was stirred several times . then , the solution was transferred to a percolator and then the deposit was separated using paper filter . the tab of the percolator released 2 - 3 drops of the solution per minute . after filtration , it was maintained in a water bath at 40c for 16 h to let the alcohol be evaporated from the filtered solution to reach a final concentration of 25% ( 20 ) . sample preparation and biochemical assays after the injection of the challenge dose of ptz and behavioral analysis , mice were decapitated . they were placed in freezer ( -30c ) , in a glass bottle ( less than 10 h ) . then , the brain pieces ( cutting the brain tissue using the scissors ) were homogenized using four times ice - cold tris - hcl buffer ( 50 mm , ph = 7.4 ) for two min at 5000 rpm . mda and no levels the supernatant solution was then extracted with a mixture of ethanol : chloroform ( a volume with ratio of 5 : 3 ) . after centrifugation at 5000g for 30 min , the clear upper layer ( the ethanol phase ) was taken and used for the evaluation of the sod activity . all experiments were carried out at + 4c ( 5 ) . the mda concentration ( thiobarbituric acid reactive substances , tbars ) in the supernatant was measured according to the following protocol . briefly , trichloroacetic acid and tbars reagent were added to the supernatant , then mixed and incubated at 100c for 80 min . after being cooled on ice , samples were centrifuged at 1000g for 20 min and the absorbance of the supernatant was read at 532 nm ( 21 ) . since no is a compound with a short half - life and is rapidly converted to the stable end products of nitrate ( no3- ) and nitrite ( no2- ) , the principle of the assay is the conversion of nitrate into nitrite by cadmium and followed by color development with griess reagent ( sulfanilamide and n - naphthyl ethylenediamine ) in acidic medium ( 22 ) . briefly , supernatant was incubated with xanthine and xanthine oxidase in potassium phosphate buffer ( ph = 7.8 , 37c ) for 40 min and nbt was added . the amount of protein that inhibited the nbt reduction to 50% of maximum was defined as 1 nitrite unit ( nu ) of sod activity ( 23 ) . comparison of seizure phase in different injection periods was carried out using repeated measurement of two - way analysis of variance ( anova ) . other data were subjected to one - way anova and its related post - hoc tests . effect of brassica nigra on the ptz - induced kindling intensity statistical analysis of results indicates that there are no significant differences among the experimental groups in seizure intensity until the 7th injection ( data is not shown ) . moreover , as it is shown in figure 1 , hydro - alcoholic extract of brassica nigra with 75 mg / kg dose at 7th injection and with 150 mg / kg at 9th and 11th injection were able to reduce the ptz - induced seizure significantly [ f ( 14 , 2 ) = 4.63 , p < 0.018 ] . however , valproate ( 100 mg / kg ) has reduced seizure intensity in all periods significantly . at the 12th injection , ( challenge dose ) , brassica nigra 75 mg / kg and valproate , had similar reducing effect on seizure intensity without any significant difference . p < 0.05 and * * p < 0.01 indicate significant differences as compared to ptz - kindled group . effect of brassica nigra on the ptz - induced kindling factors as could be seen in figure 2 , pretreatment of animals with different doses of brassica nigra do not have any significant effect on the duration time that the mice reach to phase 5 seizures . in addition , figure 3 indicates that only pretreatments of mice with brassica nigra 75 mg / kg and valproate 100 mg / kg are able to reduce the period that mice remain in phase 5 of seizure significantly [ f ( 4 , 45 ) = 1.44 , p < 0.02 ] . effect of valproate ( 100 mg / kg ) and three doses of brassica nigra ( 75 , 150 and 300 mg / kg ) on the latency of arriving to phase 5 of seizure . va and bn indicate valproate and brassica nigra respectively effect of valproate ( 100 mg / kg ) and three doses of brassica nigra ( 75 , 150 and 300 mg / k ) on the remaining time in the phase 5 . < 0.05 shows significant difference as compared to ptz - kindled group effect of brassica nigra on the biochemical indexes of oxidative stress and antioxidant table 1 indicates the brain levels of biochemical factor changes that are usually the indexes of oxidative stress in tissues , in kindled and non - kindled groups with or without pretreatment with valproate and brassica nigra extract . ptz - induced kindling has significantly increased the mda level in the brain tissue of kindled mice compared to the control group [ f ( 4 , 54 ) = 4.66 , p < 0.001 ] . however , the significant reductive effect of ptz on the sod level in the brain as compared to control mice was also observed . nonetheless , the no level in the brain of kindled mice compared to the control group was unchanged . the effect of valproate and three doses of brassica nigra on the no , mda and sod levels of brain tissue on the ptz - kindled mice brain levels of no , mda and sod are compared in six groups . in each group , n = 10 and bn indicates brassica nigra . * and # show significant differences as compared to control and ptz - kindled groups respectively ( p < 0.05 ) valproate administration was only able to significantly decrease the brain s no activity in the ptz - kindled mice compared to the control and non - treated ptz - kindled mice [ f ( 4 , 45 ) = 10.21 , 0.001 ] and did not change mda and sod level in the brain tissue . interestingly , in the pretreated group with 150 and 300 mg/ kg doses of brassica nigra , the no brain content had a significant increase compared to the other groups . the mda level of the brain tissue , only in brassica nigra treated group ( 300 mg / kg ) compared to the ptz - kindled mice , has been significantly decreased [ f ( 4 , 45 ) = 3.19 , p < 0.001 ] . finally , the effective dose of brassica nigra on the lowering of sod level of the brain compared to the control and ptz - kindled mice was 150 mg / kg . brassica nigra seed has been used as a treatment for epilepsy in iranian traditional medicine ( 13 ) . in the present research , it is observed that brassica nigra seed extract could reduce the intensity , improvement and duration time of ptz - induced seizure . our data analysis showed that the hydro - alcoholic brassica nigra extract in lower dose could significantly reduce the duration time that mice remain in phase 5 of seizure . experimental researches suggest the existence of flavonoids with antioxidant effects in the hydro - alcoholic brassica nigra seed ( 6 ) . so , vitamin a is able to prevent the kindling and convulsion , and is also able to inhibit the challenge of dose - induced tonic seizure ( 24 ) , one of the mechanism s actions of the plant that could be related to it . the important effect of free radicals is membrane lipid peroxidation and tissue injury by which results in cell membrane destruction and its dysfunction . normally , biological effects of free radicals in the body is controlled by a lot of antioxidants such as vitamins a , c and e , glutathione and also via anti - oxidant enzymes like glutathione reductase ( gr ) , glutathione peroxidase ( gp ) , sod and catalase ( 25 , 26 ) . generalized epilepsy is accompanied by reversible convulsing and can induce some species of reactive oxygen and superoxide in the brain ( 27 , 28 ) . since it is supposed that free radicals mediate the convulsion improvement , nowadays , searching for antiepileptic drugs with antioxidant and neuroprotective effects are of interests . however , some scientists suggest that only nmda receptor activation and no production , without glutamine synthetase inhibition are involved in the seizure ( 29 ) . using mice with amygdale convulsion , have shown the production of oxygen radicals following the seizure , and probably these radicals are involved in the seizure improvement and convulsion - induced neuronal death . they have also shown that seizure is able to increase lipid peroxidation in both hemispheres and cell death in all areas of hippocampus . interestingly , they have observed that during the seizure , antioxidants inhibit the lipid peroxidation in both hemispheres and cell death in the hippocampus ( 30 ) . it has been observed that antioxidants inhibit the ptz - induced seizure significantly and reduce the seizure - induced oxidative stress ( 5 ) . furthermore , in epileptic patients , the serum level of antioxidants is reduced and lipid peroxidation is increased and both effects are correctable with antiepileptic drugs ( 26 ) . in addition , possibly ptz is a starter of various processes such as membrane phosphorylation , proteolysis , and nuclease and consequently release of free fatty acids , diasylglycerols , eicosanoids , lipid peroxides and free radicals ( 31 ) . in the present study , significant increase of mda as an index of lipid peroxidation and meaningful reduction of antioxidant enzyme ( sod ) in the ptz - induced kindled group lead to the production of free radicals and existence of oxidative stress in the brain of the kindled mice . therefore , this research is in accordance with the theory that in the ptz - induced animals , the oxidative stress is possibly one of the parameters that participate in the pathophysiology of epilepsy . in the present study , 300 mg / kg of brassica nigra could decrease the mda level compared to the ptz mice , in a way that one can conclude probably the antioxidant effect of brassica nigra was able to decrease the oxidative injury , lipid peroxidation and mda reduction . possibly , the reductive effect of the brassica nigra extract on the seizure was resulted from the antioxidant property of the plant . sod is from antioxidant enzymes and catalyzes the conversion of superoxide to hydrogen peroxide and in this way , protects the cell against the superoxide and consequent oxidative stress . in the present study , it is observed that brassica nigra 150 mg / kg could increase the sod level compared to the ptz mice . this result can lead to the conclusion that probably brassica nigra seed with antioxidant effect and deletion of free radicals is able to preserve the antioxidant enzyme sod and consequently affect seizure intensity and duration . nowadays , no is known as an important neurotransmitter that in addition to various physiological duties , is also related to synaptic plasticity , neuronal excitability regulation , and epileptic activity ( 8 , 32 ) . oliveria et al . in 1997 have shown that nos inhibition in kindling model amplifies the 60 mg / kg ptz - induced seizure intensity , but has protective effect against 80 mg / kg ptz - induced tonic seizures ( 33 ) . so , they have concluded that the proconvulsant or anticonvulsant activity of nos and no inhibitors is dependent on the ptz dose and the seizure model . researchers have attributed the protective and inhibitory effect of nos on high doses of ptz to the contribution of no in the proconvulsant effect of limbic system ( 33 , 34 ) . using nnos mice ( lacking nnos gene ) and nnos ( neuronal no synthetase ) inhibitors , they have concluded that basic and enhanced levels , implies negative and positive modulatory effects respectively ( 34 ) . it is suggested that the anticonvulsant role of no is related to an implied feedback of no on the nmda receptor activation via different mechanisms ( 8) . however , no is known as a molecule that can easily react with o2.- radicals in the brain and reduce the oxidative stress - induced damage by eliminating free radicals ( 25 ) . controversial results make difficulties in predicting pro or anti - convulsant effect of no molecule . anyway , in the present research , the no level is decreased in ptz group compared to the control and is increased significantly in brassica nigra - treated group ( 300 mg / kg ) compared to the ptz mice . this indicates that probably brassica nigra seed extract can have suppressing effect on seizures via no synthesis mechanism activation . probably , the reduced level of no in ptz mice is resulted from free radicals production at seizure time , and its consumption due to its cleaning effect . besides , the enhanced level of no in the group treated with brassica nigra seed extract is due to its antioxidant effect by which it eliminates o2-radicals , and consequently prevents lipid peroxidation and oxidative stress - induced injury that result in the no level increment . in the chemical kindling model with ptz , which is identified by an increase in the seizure induction potential , furthermore , it has been reported that ptz is able to block the flow of chloride ionophore complex to the gaba receptor ( 38 ) . flumazenil , as an antagonist of benzodiazepine binding site of gabaa receptor increased the gaba - dependent chloride uptake in cultured cortical neurons ( 39 ) . using the flutamide ( androgen receptor antagonist ) and flumazenil ( benzodiazepine receptor ) in ptz - induced kindled mice , our results and these findings taken together , show that probably the anti - seizure effect of brassica nigra is via gabaergic system and benzodiazepine receptor . in conclusion , the present research indicates that hydro - alcoholic brassica nigra extract have anti - seizure effect on ptz - induced kindling in mice . in addition , since the experimental epilepsy is mediated by oxidative stress and free radicals , it could be suggested that brassica nigra is able to prevent seizures by an antioxidant mechanism . however , the involvement of gaba receptor agonists in the brassica nigra anti - seizure effect should not be ruled out .

considering the high rate of epilepsy today , with respect to the insufficiency of the available therapies , new strategies and methods are recommended for medical treatment of epileptic patients . therefore , the present study experimentally investigated the anticonvulsant effect of a herbal medicine candidate brassica nigra , by using kindling method . sixty male mice were randomly selected and divided into six experimental groups ( n = 10 ) including : 1-control , 2-pentylentetrazole ( ptz)-kindled mice , 3-positive control group received valproate ( 100 mg / kg ) as anti - convulsant drug , 4 - 5 and 6 received brassica nigra seed extract in three doses ( 75 , 150 and 300 mg / kg ; ip ) . all groups except for the control ones were kindled by 11 period injections of ptz ( 35 mg / kg ; ip ) . in the 12th injection , all groups except for the control group were tested for ptz challenge dose ( 75 mg / kg ) . however , the exhibited phases of seizure ( 0 - 6 ) were observed and noted till 30 min after the ptz injection . at last , the brains of all the mice were removed and then malondialdehyde ( mda ) , superoxide dismutase ( sod ) and nitric oxide ( no ) levels of the brain tissues were determined . statistical analysis of the data shows that the seed extract could reduce the intensity , improvement and duration of seizure . in addition , the brassica nigra extract increased the sod and no levels and decreased the mda level in the brain tissues . attained results show that the extract of brassica nigra seed can be used in grand mal seizure treatment . moreover , the antiepileptic effect of this extract is probably caused by its antioxidant properties and acts via enzyme activity mechanism .

cataract surgery is one of the most commonly performed ophthalmic procedures in the world . in modern ophthalmology , phacoemulsification with intraocular lens ( iol ) implantation is becoming more of a refractive procedure with patients having higher expectations and seeking spectacle independence . despite advances in iol formulas and surgical and biometric techniques , common causes of undesired refractive error include inaccuracies in preoperative measurements or biometric calculations,13 variations in lens formulas,4,5 incorrect selection of iol power,6 manufacturing precision , or postoperative positional changes.1,6 established surgical techniques to rectify refractive error after iol implantation are excimer laser surgery,718 astigmatic keratectomy,19 iol exchange,1923 and an implantation of a piggyback iol.1923 laser in situ keratomileusis ( lasik ) or photorefractive keratectomy ( prk ) were found to be the most accurate and safest surgical options for pseudophakic ametropia,1923 with other techniques ( iol exchange or secondary iol ) being used mainly in the cases of higher ametropia or in eyes where excimer laser ablation is contraindicated . in patients with multifocal iols , postoperative refractive error can possibly exacerbate problems inherent to these lens designs , such as loss of contrast sensitivity or photic phenomena , resulting in higher postoperative dissatisfaction.24,25 some reports also suggest that surgical correction of refractive error in eyes with multifocal iol might be technically more difficult.16,17 this is due to difficulties in estimating refractive error caused by increased depth of focus and split of light to several foci in multifocal designs,16,17 as well as problems associated with measuring refractive error by automated devices26 or estimation of higher - order aberration with hartmann shack aberrometers if wavefront - guided ( wfg ) treatment is intended.27,28 to date , only a few reports have been published on clinical outcomes of lasik / prk in pseudophakic patients implanted with multifocal iols,11,1317 with small numbers of patients involved . the aim of this study was to present predictability , safety , and efficacy of excimer laser correction for undesired ametropia in pseudophakic patients implanted with multifocal iols in a large cohort of patients . a retrospective data review was performed to identify patients who had excimer laser retreatment for residual ametropia following the primary cataract / refractive lens exchange with an implantation of a multifocal iol between december 2013 and june 2015 . the study was deemed exempt from full review by the committee of human research at the university of california , san francisco , because it used only the retrospective , de - identified patient data . written informed consent to undergo primary and enhancement procedure was obtained from all patients . criteria for retreatment were that patients were unhappy with some aspect of their vision ( either blurred distant and/or near vision or visual symptoms ) that was related to their residual refractive error and an in - clinic demonstration using trial frames improved their symptoms . if there was any doubt about eligibility for enhancement , patients underwent a contact lens or spectacle trial for several days to demonstrate whether the gain in near / distance vision would warrant a surgical procedure . in addition , they had to have a stable manifest refraction of no more than a 0.50 d change in either sphere or cylinder , documented over a minimum of 3 months . only patients with a follow - up of 1 month or more post - enhancement were included in this study . exclusion criteria for enhancement were active ophthalmic diseases , presence of posterior capsular opacification , abnormal corneal shape , concurrent medications , or medical conditions that could impair healing and a calculated residual stromal bed < 250 m . ophthalmic examination prior to the initial procedure included manifest and cycloplegic refraction , uncorrected distance visual acuity ( udva ) , corrected distance visual acuity ( cdva ) , uncorrected near visual acuity ( unva ) , slitlamp evaluation , dilated fundoscopy , autorefraction and tonometry ( tonoref ii ; nidek co. ltd . , gamagori , japan ) , corneal topography ( pentacam ; oculus , inc . , wetzlar , germany ) , endothelial cell count ( sp-2000p specular microscope ; topcon europe bv , newbury , uk ) , biometry ( iolmaster ; carl zeiss meditec ag , jena , germany ) , and retinal optical coherence tomography ( cirrus 4000 oct ; carl zeiss meditec ag ) . visual acuity was measured at distance using a snellen visual acuity chart and close - up using a logarithmic near visual acuity chart ( the early treatment diabetic retinopathy study ) at 40 cm . near visual acuity biometry ( iolmaster ) was used for lens calculation and surgeon preference determined the choice of iol formula . generally , haigis or holladay ii formulas were used in most cases , and other formulas ( such as hoffer - q and srk / t ) were considered in eyes with extreme axial lengths . the same examinations as prior to initial procedure were carried out prior to enhancement , excluding cycloplegic refraction and biometry . all patients were advised to return for follow - up at 1 day , 4 days ( surface ablation only ) , 1 week , 1 month , and 3 months and thereafter as required . manifest refraction , udva , unva , cdva , and slitlamp examinations were performed postoperatively . it was self - administered by the patient using a password protected and secure computer terminal in an isolated area of the clinic . the questionnaire responses were stored in the secured optical express central database , which is compliant with iso 27001 for information security management systems . the questionnaire was derived from the joint lasik study task force.29 patients were asked to rate their satisfaction with visual acuity as well as difficulty with night vision phenomena and various tasks that require close - up or distance vision ( figure 1 ) . the last available questionnaire prior to enhancement and the last available questionnaire post - enhancement were used for analysis . all intraocular surgeries were performed under sub - tenon s anesthetic block with a mild sedation . a toric iol was used in patients with preoperative corneal cylinder > 1.50 d ( 94 eyes ) . after phacoemulsification , a foldable multifocal iol was inserted into the capsular bag through a 2.75 mm corneal incision . postoperatively , patients were instructed to instill one drop of levofloxacin 0.5% ( oftaquix ) four times daily for 2 weeks and one drop of dexamethasone 0.1% ( maxidex ) four times daily for 2 weeks . lasik eyes had corneal flaps created by a femtosecond laser ( ifs ; abbott medical optics inc . the diameter of the femtosecond flaps ranged from 8.0 mm to 9.2 mm , and the programmed depth ranged from 100 m to 120 m . standard postoperative treatment was administered to all patients consisting of topical levofloxacin 0.5% and topical prednisolone acetate 1% four times a day for 1 week , and preservative - free artificial tear drops . in eyes having surface ablation , the eye was topically anesthetized and a 9 mm well was placed on the cornea and filled with 20% ethanol . following a 3040-second application , the alcohol was drained with a surgical spear and the eye was irrigated with a balanced salt solution . the epithelium was removed with a blunt spatula , the programmed treatment was applied , and a bandage contact lens was placed on the eye and left in place until the cornea was re - epithelialized . postoperative medication consisted of topical levofloxacin 0.5% , four times a day for 1 week , and 4 weeks of a tapering dose of topical fluorometholone ophthalmic solution 0.1% in the following sequence : four times a day for 1 week , three times a day for 1 week , two times a day for 1 week , and once a day for 1 week . excimer laser enhancements were performed with a conventional ablation profile using visx star s4 ir excimer laser ( abbott medical optics inc . ) . treatment was based on patient s manifest refraction , and nomogram adjustment was applied based on the previous experience with conventional ablations on primary laser vision correction ( lvc ) cases . for myopic treatments , the optical zone ( oz ) diameter was 6.5 mm ; for myopic astigmatism , the major axis of the elliptical oz was 6.5 mm with a minor axis as small as 5.0 mm depending on the amount of myopia and astigmatism . the transition zone was 8 mm , unless there was < 1.0 d of myopia . where clinical parameters ( such as corneal pachymetry and topography ) did not allow creation of lasik flap , prk was performed ( 8.8% of eyes ) . when normality condition could be assumed , paired student s t - test was used to compare preoperative and postoperative data . when parametric analysis was not possible , wilcoxon rank sum test was applied in place of paired t - test . to compare independent groups of patients , unpaired t - test or mann whitney u - test all the data were analyzed using microsoft office excel 2007 program ( microsoft corporation , redmond , wa , usa ) and statistica ( statsoft inc . all intraocular surgeries were performed under sub - tenon s anesthetic block with a mild sedation . a toric iol was used in patients with preoperative corneal cylinder > 1.50 d ( 94 eyes ) . after phacoemulsification , a foldable multifocal iol was inserted into the capsular bag through a 2.75 mm corneal incision . postoperatively , patients were instructed to instill one drop of levofloxacin 0.5% ( oftaquix ) four times daily for 2 weeks and one drop of dexamethasone 0.1% ( maxidex ) four times daily for 2 weeks . lasik eyes had corneal flaps created by a femtosecond laser ( ifs ; abbott medical optics inc . , santa ana , ca , usa ) . the diameter of the femtosecond flaps ranged from 8.0 mm to 9.2 mm , and the programmed depth ranged from 100 m to 120 m . standard postoperative treatment was administered to all patients consisting of topical levofloxacin 0.5% and topical prednisolone acetate 1% four times a day for 1 week , and preservative - free artificial tear drops . in eyes having surface ablation , the eye was topically anesthetized and a 9 mm well was placed on the cornea and filled with 20% ethanol . following a 3040-second application , the alcohol was drained with a surgical spear and the eye was irrigated with a balanced salt solution . the epithelium was removed with a blunt spatula , the programmed treatment was applied , and a bandage contact lens was placed on the eye and left in place until the cornea was re - epithelialized . postoperative medication consisted of topical levofloxacin 0.5% , four times a day for 1 week , and 4 weeks of a tapering dose of topical fluorometholone ophthalmic solution 0.1% in the following sequence : four times a day for 1 week , three times a day for 1 week , two times a day for 1 week , and once a day for 1 week . excimer laser enhancements were performed with a conventional ablation profile using visx star s4 ir excimer laser ( abbott medical optics inc . ) . treatment was based on patient s manifest refraction , and nomogram adjustment was applied based on the previous experience with conventional ablations on primary laser vision correction ( lvc ) cases . for myopic treatments , the optical zone ( oz ) diameter was 6.5 mm ; for myopic astigmatism , the major axis of the elliptical oz was 6.5 mm with a minor axis as small as 5.0 mm depending on the amount of myopia and astigmatism . the transition zone was 8 mm , unless there was < 1.0 d of myopia . lasik was the preferred enhancement procedure , which was performed on 91.2% of eyes . where clinical parameters ( such as corneal pachymetry and topography ) did not allow creation of lasik flap , prk was performed ( 8.8% of eyes ) . when normality condition could be assumed , paired student s t - test was used to compare preoperative and postoperative data . when parametric analysis was not possible , wilcoxon rank sum test was applied in place of paired t - test . to compare independent groups of patients , unpaired t - test or mann whitney u - test all the data were analyzed using microsoft office excel 2007 program ( microsoft corporation , redmond , wa , usa ) and statistica ( statsoft inc . , the mean age at the time of enhancement was 57.47.2 years ( range : 3476 years ) . of all patients , 266 ( 46.2% ) were females , and 310 ( 53.8% ) were males . table 1 presents the lens designs implanted during primary cataract / refractive lens exchange procedure . a majority of eyes ( 98.7% ) the initial data ( prior to intraocular procedure ) and pre- and post - enhancement data are summarized in table 2 . figure 2 shows the distribution of pre- and post - enhancement manifest spherical equivalent ( mse ) . prior to laser enhancement , 41.2% of eyes were within 0.50 d and 84.7% were within 1.00 d of mse . post - enhancement , the percentage of eyes within 0.50 d and 1.00 d of emmetropia was 90.4% and 99.5% , respectively . the reduction in the mean refractive sphere and cylinder was statistically significant ( table 2 ) . a linear regression of attempted pre - enhancement vs achieved post - enhancement mse correction ( figure 3 ) had a slope of 1.06 and intercept of 0.002 . figure 4 depicts the cumulative monocular udva prior to primary procedure , prior to enhancement , and post - enhancement . prior to enhancement , 2.3% of patients had udva 20/16 or better and 7.8% 20/20 or better . these were mostly eyes with low amount of hyperopia or mixed astigmatism , where the small amount of refractive error affected the multifocal performance of the iol . the percentage of eyes achieving 20/16 or 20/20 or better udva post - enhancement was 39.4% and 74.9% , respectively . binocularly , 44.1% of patients had udva 20/20 or better prior to enhancement and 92.0% post - enhancement . the percentage of patients achieving monocular unva 20/40 ( j5 ) or better was 61.3% prior to enhancement and 76.6% post - enhancement . binocularly , 75.8% and 86.6% of patients achieved 20/40 unva pre- and post - enhancement , respectively . figure 6 shows the comparison of pre - enhancement cdva to post - enhancement cdva . the mean change in cdva was not statistically significant ( table 2 ) . the loss of two lines of cdva at the last available follow - up was recorded in 1.4% of eyes ( eleven eyes ) . ocular surface dryness / superficial punctate keratitis , one eye age - related macular changes that were not present at the time of enhancement , one eye traumatic eye injury not related to the enhancement resulting in prolonged corneal edema . table 3 shows the pre- and post - enhancement clinical outcomes stratified by the type of pre - enhancement refractive error . prior to enhancement , 32.5% of eyes had myopia or myopic astigmatism , 49.2% of eyes were hyperopic or had hyperopic astigmatism and 18.3% had mixed astigmatism . all the three categories had significant reduction in sphere , cylinder , and mse ( table 3 ) . the percentage of patients achieving post - enhancement udva 20/20 or better was 71.1% in the myopic group , 76.4% in the hyperopic group , and 77.4% in the mixed astigmatism group . there was no statistically significant difference in pre - enhancement myopic or hyperopic sphere ( myopic sphere : prk , 0.720.41 d ; lasik , 0.730.49 d ; p=0.85 ; hyperopic sphere : prk , + 0.850.49 d ; lasik , + 0.820.47 d ; p=0.68 ) . eyes in the prk group had higher pre - enhancement cylinder ( prk , 1.030.58 d ; lasik , 0.860.55 d ; p=0.02 ) and worse pre - enhancement cdva ( prk , 0.020.08 logmar [ logarithm of the minimum angle of resolution ] ; lasik , 0.040.06 logmar ; p=0.02 ) . there was no statistically significant difference in postoperative sphere ( prk , + 0.180.32 d ; lasik , + 0.140.35 d ; p=0.49 ) , cylinder ( prk , 0.300.36 d ; lasik , 0.290.32 d ; p=0.81 ) , udva ( prk , 0.00.10 logmar ; lasik , 0.00.10 logmar ; p=0.99 ) , or cdva ( prk , 0.030.07 logmar ; lasik , 0.040.07 logmar ; p=0.53 ) between the two groups . when evaluating difficulties with post - enhancement dry eye symptoms ( measured on scale 1= no difficulty to 7= severe difficulty ; figure 1 ) , the mean score for prk was 2.21.4 and for lasik 2.21.5 ( p=0.89 ) . there was also no statistically significant difference in post - enhancement visual symptoms between the two groups ( mean score : starburst : prk 2.21.4 , lasik 2.11.5 , p=0.64 ; glare : prk 1.91.3 , lasik 2.31.6 ; p=0.17 , halo : prk 1.81.3 , lasik 2.21.6 , p=0.19 ; ghosting : prk 1.81.4 , lasik 1.91.5 , p=0.45 ) . ninety - four eyes in the study had a toric multifocal iol implanted during their primary iol procedure . the mean refractive cylinder in this subgroup of patients was 1.250.74 d prior to enhancement , and reduced to 0.230.30 d ( p<0.01 ) . there was no statistically significant difference in the residual post - enhancement refractive cylinder in eyes that had a toric iol and non - toric iol ( mean post - lvc cylinder in patients with non - toric iol : 0.300.33 d , p=0.06 ) . out of all eyes that had lasik enhancements ( n=713 ) , two eyes ( 0.3% ) developed peripheral epithelial ingrowth that stabilized and did not affect visual acuity or refraction . mild diffused lamellar keratitis was observed in 33 eyes ( 4.6% ) , and except for a temporary increase in topical steroid dosing , all resolved without additional intervention . one eye had more severe diffused lamellar keratitis ( grade 3 ) and was successfully treated with increased topical steroids . there was one case of flap striae affecting the patient s cdva / quality of vision and a surgical intervention was required . following a successful flap lift , the patient s cdva returned to the preoperative level . in one eye without inflammation or pain , a presumed sterile corneal infiltrate was observed on the first postoperative day . the patient was maintained on his / her broad - spectrum topical antibiotic , and the topical steroid was increased in frequency . the infiltrate resolved within the first 5 postoperative days . in the group of eyes with surface ablation ( n=69 ) , two eyes ( 2.9% ) developed mild haze , which cleared within the first 6 postoperative months . there was one case of delayed epithelial healing that required management with therapeutic contact lenses for 1 month and , finally , resolved without any consequence . one patient suffered from recurrent erosion syndrome following the bilateral prk , which was also managed with therapeutic contact lenses and intense ocular surface lubrication . of all patients , 81.3% ( n=468 ) completed both pre- and post - enhancement questionnaires . the mean patient satisfaction ( measured on the scale 15 ; figure 1 ) changed from 2.31.1 before enhancement to 1.91.0 ( p<0.01 ) post - enhancement . the percentage of patients being dissatisfied / very dissatisfied with their vision decreased from 17.0% prior to enhancement to 9.5% post - enhancement ( p<0.01 ) . figure 7 shows the proportion of patients experiencing severe difficulty with night vision phenomena and severe difficulty with tasks requiring distance or near vision . there was a slight improvement in all symptoms from pre- to post - enhancement level , although most of the percentage differences were not statistically significant ( figure 7 ) . the mean change in visual phenomena scores from pre- to post - lvc enhancement ( all measured on a scale between 1= no difficulty to 7= severe difficulty ) indicated a slight but statistically significant improvement in all symptoms . the mean score for starburst improved from 2.41.7 before lvc enhancement to 2.11.5 ( p=0.01 ) post - enhancement , and the change in other optical side effects was as follows : glare : 2.51.6 to 2.21.6 , p=0.03 ; halo : 2.41.7 to 2.11.6 , p=0.03 ; ghosting / double vision : 2.21.7 to 1.91.5 , p<0.01 . the mean score for dry eye symptoms changed from 2.01.4 prior to enhancement to 2.21.5 post - enhancement ( p=0.06 ) . out of all clinical parameters , the final satisfaction with visual acuity was most strongly correlated with postoperative monocular udva ( r=0.19 , p<0.01 ) . post - lvc enhancement satisfaction was also strongly correlated with all the other questionnaire items . for example , the correlation between satisfaction and difficulty with glare post - enhancement was r=0.55 ( p<0.01 ) , and the same applied to all the other ocular side effects ( starburst , glare , ghosting / double vision ) . it was the final post - lvc enhancement glare ( or any other visual phenomena ) that affected patient satisfaction more than the change between pre- and post - lvc enhancement glare , which was still significantly correlated with patient satisfaction , but the correlation was weaker ( r=0.26 , p<0.01 ) . despite the pre - enhancement demonstration of visual symptoms in trial frames or with contact lens / spectacle trial , some patients may have believed that the enhancement would further improve their quality - of - vision issues . it is likely that many of these persistent symptoms were inherent to the multifocal iol . the satisfaction was also affected by the difficulty performing tasks such as night driving ( r=0.54 , p<0.01 ) , close - up activities ( r=0.53 , p<0.01 ) , and difficulty with outdoor activities ( r=0.44 , p<0.01 ) . there was a strong relationship between patient satisfaction and the difficulty with dry eye symptoms at the last follow - up ( r=0.50 , p<0.01 ) . there was no statistically significant difference in pre - enhancement myopic or hyperopic sphere ( myopic sphere : prk , 0.720.41 d ; lasik , 0.730.49 d ; p=0.85 ; hyperopic sphere : prk , + 0.850.49 d ; lasik , + 0.820.47 d ; p=0.68 ) . eyes in the prk group had higher pre - enhancement cylinder ( prk , 1.030.58 d ; lasik , 0.860.55 d ; p=0.02 ) and worse pre - enhancement cdva ( prk , 0.020.08 logmar [ logarithm of the minimum angle of resolution ] ; lasik , 0.040.06 logmar ; p=0.02 ) . there was no statistically significant difference in postoperative sphere ( prk , + 0.180.32 d ; lasik , + 0.140.35 d ; p=0.49 ) , cylinder ( prk , 0.300.36 d ; lasik , 0.290.32 d ; p=0.81 ) , udva ( prk , 0.00.10 logmar ; lasik , 0.00.10 logmar ; p=0.99 ) , or cdva ( prk , 0.030.07 logmar ; lasik , 0.040.07 logmar ; p=0.53 ) between the two groups . when evaluating difficulties with post - enhancement dry eye symptoms ( measured on scale 1= no difficulty to 7= severe difficulty ; figure 1 ) , the mean score for prk was 2.21.4 and for lasik 2.21.5 ( p=0.89 ) . there was also no statistically significant difference in post - enhancement visual symptoms between the two groups ( mean score : starburst : prk 2.21.4 , lasik 2.11.5 , p=0.64 ; glare : prk 1.91.3 , lasik 2.31.6 ; p=0.17 , halo : prk 1.81.3 , lasik 2.21.6 , p=0.19 ; ghosting : prk 1.81.4 , lasik 1.91.5 , p=0.45 ) . ninety - four eyes in the study had a toric multifocal iol implanted during their primary iol procedure . the mean refractive cylinder in this subgroup of patients was 1.250.74 d prior to enhancement , and reduced to 0.230.30 d ( p<0.01 ) . there was no statistically significant difference in the residual post - enhancement refractive cylinder in eyes that had a toric iol and non - toric iol ( mean post - lvc cylinder in patients with non - toric iol : 0.300.33 d , p=0.06 ) . out of all eyes that had lasik enhancements ( n=713 ) , two eyes ( 0.3% ) developed peripheral epithelial ingrowth that stabilized and did not affect visual acuity or refraction . mild diffused lamellar keratitis was observed in 33 eyes ( 4.6% ) , and except for a temporary increase in topical steroid dosing , all resolved without additional intervention . one eye had more severe diffused lamellar keratitis ( grade 3 ) and was successfully treated with increased topical steroids . there was one case of flap striae affecting the patient s cdva / quality of vision and a surgical intervention was required . following a successful flap lift , the patient s cdva returned to the preoperative level . in one eye without inflammation or pain , the patient was maintained on his / her broad - spectrum topical antibiotic , and the topical steroid was increased in frequency . the infiltrate resolved within the first 5 postoperative days . in the group of eyes with surface ablation ( n=69 ) , two eyes ( 2.9% ) developed mild haze , which cleared within the first 6 postoperative months . there was one case of delayed epithelial healing that required management with therapeutic contact lenses for 1 month and , finally , resolved without any consequence . one patient suffered from recurrent erosion syndrome following the bilateral prk , which was also managed with therapeutic contact lenses and intense ocular surface lubrication . of all patients , 81.3% ( n=468 ) completed both pre- and post - enhancement questionnaires . the mean patient satisfaction ( measured on the scale 15 ; figure 1 ) changed from 2.31.1 before enhancement to 1.91.0 ( p<0.01 ) post - enhancement . the percentage of patients being dissatisfied / very dissatisfied with their vision decreased from 17.0% prior to enhancement to 9.5% post - enhancement ( p<0.01 ) . figure 7 shows the proportion of patients experiencing severe difficulty with night vision phenomena and severe difficulty with tasks requiring distance or near vision . there was a slight improvement in all symptoms from pre- to post - enhancement level , although most of the percentage differences were not statistically significant ( figure 7 ) . the mean change in visual phenomena scores from pre- to post - lvc enhancement ( all measured on a scale between 1= no difficulty to 7= severe difficulty ) indicated a slight but statistically significant improvement in all symptoms . the mean score for starburst improved from 2.41.7 before lvc enhancement to 2.11.5 ( p=0.01 ) post - enhancement , and the change in other optical side effects was as follows : glare : 2.51.6 to 2.21.6 , p=0.03 ; halo : 2.41.7 to 2.11.6 , p=0.03 ; ghosting / double vision : 2.21.7 to 1.91.5 , p<0.01 . the mean score for dry eye symptoms changed from 2.01.4 prior to enhancement to 2.21.5 post - enhancement ( p=0.06 ) . out of all clinical parameters , the final satisfaction with visual acuity was most strongly correlated with postoperative monocular udva ( r=0.19 , p<0.01 ) . post - lvc enhancement satisfaction was also strongly correlated with all the other questionnaire items . for example , the correlation between satisfaction and difficulty with glare post - enhancement was r=0.55 ( p<0.01 ) , and the same applied to all the other ocular side effects ( starburst , glare , ghosting / double vision ) . it was the final post - lvc enhancement glare ( or any other visual phenomena ) that affected patient satisfaction more than the change between pre- and post - lvc enhancement glare , which was still significantly correlated with patient satisfaction , but the correlation was weaker ( r=0.26 , p<0.01 ) . despite the pre - enhancement demonstration of visual symptoms in trial frames or with contact lens / spectacle trial , some patients may have believed that the enhancement would further improve their quality - of - vision issues . it is likely that many of these persistent symptoms were inherent to the multifocal iol . the satisfaction was also affected by the difficulty performing tasks such as night driving ( r=0.54 , p<0.01 ) , close - up activities ( r=0.53 , p<0.01 ) , and difficulty with outdoor activities ( r=0.44 , p<0.01 ) . there was a strong relationship between patient satisfaction and the difficulty with dry eye symptoms at the last follow - up ( r=0.50 , p<0.01 ) . despite the use of accurate lens calculation formulas , optimization of a - constants , use of customized toric lenses , and improvements in micro - incision intraocular surgery , undesired postoperative refractive error is still an issue.1923 in a recent retrospective review of > 17,000 cataract procedures,30 emmetropia ( defined as spherical equivalent within 0.50 d with < 1.00 d of astigmatism ) was finally achieved only in 55% of eyes . in our previous series of 9,366 eyes implanted with a nonrotational symmetric lens,31 which is the iol primarily used in the majority of eyes in this study , although this refractive predictability was good , it also indicates that a not - insignificant fraction of these patients would require additional procedures to achieve emmetropia . variables routinely measured before cataract surgery ( such as age , refraction , axial length , and intraocular pressure ) can not accurately predict the need for lvc enhancement.32 in the current study , 90.4% of eyes were within 0.50 d and 99.5% within 1.00 d of emmetropia following the laser enhancement . only 7.8% of patients had udva 20/20 or better prior to enhancement , and this number increased to 74.9% post - enhancement . the mean cdva was almost the same before ( 0.040.06 logmar ) and after enhancement ( 0.040.07 logmar ) . the symptoms of photic phenomena slightly reduced following the enhancement ( figure 7 ) , but they did not completely disappear , which is expected with the use of multifocal lens designs . there are several reasons why results of laser enhancements in pseudophakic patients could be different from primary laser patients . patients with cataract are typically 2 to 3 decades older , and older age can be associated with lower predictability and efficacy of excimer ablation,33,34 as well as higher susceptibility to tear - film deficiency.35 the pseudophakic patients typically have one or two corneal incisions , as well as additional incisions to correct astigmatism . it is possible that these incisions could be slightly distorted during suction required for the creation of lasik flap and affect postoperative outcomes.12,23 despite these potential concerns , excellent outcomes have been reported with the use of excimer laser ablation in pseudophakic patients,718 and it remains the preferred surgical option for unintended ametropia.1923 predictability was confirmed in many studies in patients with monofocal iol;710,12,16 however , to our knowledge , only a few studies have been previously published on the use of excimer laser surgery in patients with a multifocal lens.11,1317 in the first report , leccisotti11 presented outcomes of 18 eyes that had prk for residual refractive error after the implantation of array sa40n refractive iol ( amo , inc . , santa ana , ca , usa ) . postoperative spherical equivalent was slightly hyperopic ( + 0.33 d ) , with the mean postoperative udva of 0.8 ( = 0.1 logmar ) . the study concluded that prk significantly improved visual acuity , but it had limited effect on halos associated with array lens and some patients eventually required an exchange to a monofocal lens . alfonso et al14 presented outcomes of 53 eyes that underwent lasik for residual ametropia after the implantation of acrysof restor ( alcon , inc . , hnenberg , switzerland ) apodized diffractive iol , using conventional ablation profile and femtosecond laser for flap creation . excellent outcomes were achieved in terms of predictability : all eyes were within 1.00 d and 96.2% were within 0.50 d of the desired refraction . this is slightly superior to our outcomes , but a much lower range of refractions was treated in this study ( pre - enhancement mse : 2.00 d to + 1.00 d , compared to the mse of the current study , ranging between 3.38 d and + 2.25 d ) . six months after lasik , the mean udva was 0.830.20 ( 0.08 logmar ) . in 2008 , jendritza et al13 published outcomes of 27 pseudophakic eyes treated with wfg lasik . twenty - four eyes had a diffractive multifocal iol ( tecnis ; abbott medical optics inc . , or restor ) , and four eyes had a refractive iol ( rezoom ; abbott medical optics inc . ) . the conclusion was that wfg lasik could successfully treat residual ametropia in patients with multifocal lens implant , but it did not improve higher - order aberrations . the authors advised against the use of wfg ablation in patients with refractive iols , as the measurements with wavefront sensors may not be reliable . wfg lasik in eyes with diffractive iols did not affect multifocality of the lens , and the patients retained about the same corrected near and distance visual acuity following the excimer laser ablation . on the other hand , in the group of patients with a refractive iol , there was one - line loss of distance corrected and two lines loss of near corrected visual acuity . the majority of patients in our study ( 98.7% ) had an iol with refractive design , and there was no loss in the mean cdva . muftuoglu et al15 retrospectively studied 85 eyes with apodized diffractive iol , which were enhanced for residual myopia , hyperopia , or mixed astigmatism . pre - enhancement spherical equivalent ranged between 2.58 d and + 1.63 d and postoperatively , 96% and 99% of eyes were within 0.50 d and 1.00 d of targeted refraction . postoperative mean udva was 0.050.08 logmar , and there was no difference in achieved udva between eyes with preoperative myopia , hyperopia , and mixed astigmatism . this is comparable to our outcomes , where udva was very close to 20/20 ( 0.0 logmar ) in all the three subcategories ( table 3 ) . piero et al16 compared the outcomes of lasik for pseudophakic ametropia in eyes with monofocal iol and multifocal iol with 50 eyes in each group . of all eyes , 84% were within 0.50 d of emmetropia in the monofocal group and 70% in the multifocal group , and this difference was not statistically significant . postoperative udva was similar in both the groups ( 0.150.18 logmar in the multifocal group , 0.140.11 logmar in the monofocal group ) . when examining eyes with multifocal iol , no difference in postoperative outcomes was found between eyes with diffractive and refractive lenses . the authors further divided the eyes with multifocal iols into those that had hyperopic and those with myopic pre - enhancement refractive error , and they concluded that eyes with hyperopic error had less predictable outcomes . eyes with preoperative hyperopia had tendency for undercorrection , and there was a higher variability in postoperative mse ( + 0.320.72 d ) , whereas eyes with preoperative myopia were slightly overcorrected , but the standard deviation of postoperative mse was lower ( + 0.280.45 d ) . this was attributed to the difficulties in estimating refractive error in patients with multifocal iol caused by the presence of several foci , which can result in artifacts in subjective refraction due to several refractive options providing similar visual quality . they recommended the use of the midpoint refraction of the clear vision interval provided by the depth of field of the iol to avoid postoperative problems of predictability . in contrast , albarrn - diego et al17 describe difficulty in treating pseudophakic patients with multifocal iol with myopic residual refraction . however , this study is only a case report of three eyes with unexpected hyperopic surprise . the findings of our study did not confirm those of piero et al.16 eyes with preoperative myopia or hyperopia had both predictable outcomes with the mean postoperative spherical equivalent close to emmetropia , with very similar standard deviation ( myopic eyes : + 0.040.36 d , n=254 ; hyperopic eyes : 0.030.33 , n=385 ) . the percentage of eyes within 0.50 d of targeted refraction was actually slightly higher in patients with preoperative hyperopia ( 91.7% ) than those with myopic error ( 87.5% ; table 3 ) , although this difference was not statistically significant . patients with slight hyperopic error are mostly affected by inability to see well close - up , but they often have very good distance visual acuity . thus , contact lens or spectacle trial is often necessary to ensure that the patient will benefit from enhancement and that postoperative overall visual acuity will not be affected . the refraction determined from this trial is then used in preoperative planning . in this study , lasik was the preferred choice for the enhancement of pseudophakic ametropia , which was performed in 91.3% of eyes . although the group of patients with surface ablation was much smaller , there was no statistically significant difference in any of the postoperative clinical parameters between the two techniques . lasik is a popular choice due to its fast and relatively pain - free recovery ; however , there could be an increased risk of dry eye symptoms with the creation of the corneal flap , especially in a group of older patients who are more prone to tear - film abnormalities . despite this concern , we did not find a statistically significant difference in postoperative dry eye symptoms between the two techniques . surface ablation might require longer re - epithelialization period due to the age - related decrease in the healing response,36,37 which was observed only in one case in this study . on the other hand , the creation of lasik flap temporarily increases intraocular pressure , which could result in complications associated with posterior segment of the eye,38 such as retinal detachment , macular hemorrhage , damage to optic nerve , and visual field defect . we did not see any vitreoretinal complications related to lasik in our cohort of 724 eyes . however , our patients were younger ( 57.47.2 years ) than a typical population of patients undergoing cataract surgery . in addition , patients with posterior segment pathology were excluded during preoperative screening as this would be a contraindication to multifocal iol implantation . although there were a large number of patients involved , the study was retrospective and there was a variation in the time between the enhancement and the last postoperative visit . thus , we could not calculate the stability of outcomes over the period of time . despite this , findings of this study confirmed that excimer ablation can be safely and successfully utilized in eyes with multifocal lens and undesired pseudophakic ametropia . due to the age of this population , patients should be carefully counseled for possible increase in dry eye symptoms , and some of the undesired optical side effects might remain despite the successful correction of refractive error .

purposethe aim of this study was to assess visual and refractive outcomes of laser vision correction ( lvc ) to correct residual refraction after multifocal intraocular lens ( iol ) implantation.patients and methodsin this retrospective study , 782 eyes that underwent lvc to correct unintended ametropia after multifocal iol implantation were evaluated . of all multifocal lenses implanted during primary procedure , 98.7% were refractive and 1.3% had a diffractive design . all eyes were treated with visx star s4 ir excimer laser using a convectional ablation profile . refractive outcomes , visual acuities , patient satisfaction , and quality of life were evaluated at the last available visit.resultsthe mean time between enhancement and last visit was 6.34.4 months . manifest spherical equivalent changed from 0.020.83 d ( 3.38 d to + 2.25 d ) pre - enhancement to 0.000.34 d ( 1.38 d to + 1.25 d ) post - enhancement . at the last follow - up , the percentage of eyes within 0.50 d and 1.00 d of emmetropia was 90.4% and 99.5% , respectively . of all eyes , 74.9% achieved monocular uncorrected distance visual acuity 20/20 or better . the mean corrected distance visual acuity remained the same before ( 0.040.06 logmar [ logarithm of the minimum angle of resolution ] ) and after lvc procedure ( 0.040.07 logmar ; p=0.70 ) . there was a slight improvement in visual phenomena ( starburst , halo , glare , ghosting / double vision ) following the enhancement . no sight - threatening complications related to lvc occurred in this study.conclusionlvc in pseudophakic patients with multifocal iol was safe , effective , and predictable in a large cohort of patients .

type 2 diabetes mellitus ( t2 dm ) is an endocrine disease characterized by impaired insulin excretion by the pancreas and insulin resistance of body tissues . chronic hyperglycemia leads to micro- and macrovascular complications in patients with t2 dm ; diabetic retinopathy ( dr ) is the most common and the specific microangiopathy . diabetes duration , dyslipidemia , genetic factors , obesity , hypertension , smoking , proteinuria , and hypermetropic refractive changes may all play a role in development of dr development . abnormal insulin activation in patients with t2 dm may increase platelet activation and precipitate microvascular complications . some authors have emphasized the importance of platelet dysfunction in macrovascular ( cardiovascular disease [ cvd ] , stroke , and peripheral artery disease [ pad ] ) and microvascular ( nephropathy , neuropathy , and retinopathy ) complications , which lead to increased morbidity and mortality in t2 dm [ 4 , 5 ] . various parameters reflect the condition of platelets , including platelet count , plateletcrit , and mean platelet indices ( mpi ) ( mean platelet volume [ mpv ] , platelet distribution width [ pdw ] , and platelet large cell ratio [ plcr ] ) . it is a marker that indicates subclinical platelet activation and may be increased in some vascular conditions such as myocardial infarction ( mi ) , coronary artery disease ( cad ) , cerebral ischemia , and pad . other platelet markers such as pdw , plcr , and plateletcrit ( pct ) , which reflect platelet morphology , are also important in vascular events such as atherosclerosis and thrombosis . pdw gives an indication of the distribution of platelet size , plcr indicates the ratio of younger platelet group that has the largest volume , and pct gives the total mass of platelets . studies have been published on the relationship between dr stages and mpv , pdw , and pct values , which give information about platelet morphology and function alteration [ 12 , 13 ] . however , to our knowledge , this is the first study investigating all of the morphologic parameters that reflect subclinical platelet activity ( mpv , pdw , plcr , pct , and platelet count ) between dr stages . the clinical research was performed between march 2015 and january 2016 at the department of ophthalmology , kocaeli derince research and training hospital . patients who were diagnosed as any type of dr in department of ophthalmology and diabetic patients who were referred from the department of internal medicine were enrolled in this study . the hc group consisted of healthy individuals with no history of systemic or ocular diseases , who had undergone routine ophthalmic examination . all participants underwent full ophthalmologic examination , and retinopathy status was assessed by fundus photography , fluorescein angiography , and optical coherence tomography . all of the study procedures were conducted in accordance with the declaration of helsinki , and informed consent was obtained from all of the participants after approval from the institutional review board . the study protocol was approved by the ethics committee of kocaeli university school of medicine . , was applied . according to this formula , ( n = 1 + 2c(s / d ) ) where n was the sample size , c was the equation constant with 0.8 power and two sided = 0.05 , s was the standard deviation of platelet parameters ( mpv , pdw , plcr , platelet count , and pct ) in people , and d was the effect size . therefore , we designed a 1 : 1 case - control study with groups of 85 . we prospectively evaluated platelet parameters ( mpv , pdw , plcr , pct , and platelet count ) in complete blood sample in 262 ( 138 females , 124 males ) patients with t2 dm and 85 ( 46 females , 39 males ) age- and sex - matched adult healthy controls ( hcs ) ( group 1 ) . patients with t2 dm were separated into three groups based on the findings of the clinical ocular examination : 88 with no dr ( ndr ) ( group 2 ) , 88 with nonproliferative dr ( npdr ) ( group 3 ) , and 86 with proliferative dr ( pdr ) ( group 4 ) . exclusion criteria were presence of uncontrolled hypertension , anemia ( hematocrit below 38% ) , any cardiovascular disease , stroke , or chronic renal failure ; treatment with anticoagulant ; or history of alcohol consumption . complete blood count samples were taken from each participant and drawn into vacutainer tubes containing 0.04 ml of the 7.5% k3 salt of edta and were analyzed within 90 minutes after sampling with a commercially available analyzer ( sysmex 1800 i automated cell counter , japan ) . differences between the four groups for platelet parameters were evaluated using one - way anova , where applicable . all statistical analyses were performed using spss for windows 18.0 software ( spss inc . , logistic regression analysis was used to assess the associations between platelet parameters and dr stage . the average age of the patients was 63.39 8.52 years in group 1 , 60.87 8.31 in group 2 , 62.72 7.19 in group 3 , and 61.66 7.91 in group 4 . there was no difference in age or sex of the patients between the four groups ( p > 0.05 ) ( table 1 ) . mpv was 7.42 0.68 fl in group 1 , 7.84 0.76 fl in group 2 , 7.90 0.85 fl in group 3 , and 8.31 0.76 fl in group 4 ( table 1 ) . the blood samples showed a marked elevation in mpv levels in the groups with t2 dm compared with controls : group 2 , p = 0.036 ; group 3 , p = 0.016 ; and group 4 , p < 0.01 . patients with dr had higher mpv levels among dr stages , but the difference was significant only between group 2 and group 4 ( p = 0.036 ) . mean pdw values were 12.19 1.36% in group 1 , 13.02 1.29% in group 2 , 13.49 1.18% in group 3 , and 13.77 1.26% in group 4 ( table 1 ) . there was a significant difference in pdw levels between the diabetic groups and hcs ( group 2 , p = 0.003 ; group 3 , p < 0.001 ; and group 4 p < 0.001 ) . patients with dr had higher pdw levels among dr stages , but the difference was significant only between group 2 and group 4 ( p = 0.006 ) . mean plcr values were 28.59 2.28% in group 1 , 30.45 2.19% in group 2 , 31.31 2.15% in group 3 , and 31.71 2.16% in group 4 ( table 1 ) . there was a significant difference in plcr levels between the diabetic groups and hcs ( group 2 , p = 0.019 ; group 3 , p < 0.001 ; and group 4 , p the dr groups had higher plcr levels among dr stages , but the difference was not statistically significant ( p = 0.993 between groups 2 and 3 , p = 0.264 between groups 2 and 4 , and p = 0.833 between groups 3 and 4 ) . mean pct values were 0.27 0.05 in group 1 , 0.26 0.06 in group 2 , 0.25 0.04 in group 3 , and 0.24 0.03 in group 4 ( table 1 ) . there was no statistically significant difference in pct values between groups ( p < 0.05 ) . mean platelet count was 253.76 50.87 10/l in group 1 , 253.86 60.87 10/l in group 2 , 254.77 72.87 10/l in group 3 , and 264.96 64.44 10/l in group 4 . there was no statistically significant difference in platelet counts between groups ( p < 0.05 ) . according to logistic regression analysis , there was a 0.91-fold increase in the risk of retinopathy development ( or : 0.913 ; p = 0.07 ) and a 1.14-fold increase in the risk of proliferative dr ( or : 1.148 ; p = 0.06 ) as the mpv value increased . there was a 3.10-fold increase in the risk of retinopathy development ( or : 3.106 ; p = 0.002 ) and a 1.90-fold increase in the risk of proliferative dr ( or : 1.908 ; p = 0.005 ) as the pdw value increased . there was a 0.67-fold increase in the risk of retinopathy development ( or : 0.676 ; p = 0.07 ) and a 0.64-fold increase in the risk of proliferative dr ( or : 0.645 ; p = 0.06 ) as the plcr value increased . dm is a multisystemic disease that affects the eyes , kidneys , and peripheral nerves and leads to micro- and macroangiopathy through chronic hyperglycemia . dr is the main cause of retinal vascular disease , which causes blindness between the third and sixth decades of life . studies have revealed the relationship between increased platelet aggregation and vascular complications of dm [ 17 , 18 ] . the main problem in diabetic platelets is hypersensitivity against the excretions , causing their activation . platelet aggregates were seen in the retinal capillaries of diabetic rats in some histological studies [ 21 , 22 ] . high mpv levels indicate the presence of many large platelets , which are newer , denser , and more active . cad , oad , and cerebral ischemia were all found to be associated with elevated mpv in previous studies . reported that mpv values were significantly higher in patients with t2 dm compared with controls , whereas there was no significant difference between patients who had background retinopathy and those who developed retinopathy later . ayhan tuzcu et al . found a correlation between mpv values and dr stage in their study with 192 individuals . stated that mpv was significantly higher in patients with proliferative dr and proposed that mpv is a risk factor for retinal neovascularization . by contrast , aydinli et al . advocated that there was no association between mpv and vascular complications in t2 dm . in the current study , we found a statistically significant difference in mpv values between patients with t2 dm and hcs . when we compared the different diabetic patient groups , we detected a significant difference between patients with pdr and without dr . pdw is also a specific marker of platelet activation and increases in heterogeneity of platelet volume distribution . reported that mpv and pdw were elevated together in platelet activation but emphasized that pdw is a more specific marker . in the study of rechcski et al . , pdw was found to be an independent risk factor for cardiac mortality and for the occurrence of either death , recurrent mi , or need for another revascularization procedure . found that pdw was significantly increased in patients with t2 dm and reported that it was higher in patients who developed microvascular complications . found higher pdw levels in patients with diabetes compared with hcs , but this was not statistically significant . in our study , pdw values of diabetic patient groups increased according to retinopathy stage ; there was a significant increase in patients who developed pdr compared with patients who had no dr . logistic regression analysis was performed to assess the effect of pdw values on the risk of developing dr , showing a 3.10-fold increase in the risk of retinopathy development with increased pdw values ( or : 3.106 ; p = 0.002 ) and a 1.90-fold increase in the risk of proliferative dr as pdw value increased ( or : 1.908 ; p = 0.005 ) . plcr is another marker related to platelet volume , and it is an indicator of the largest platelet fraction . an increase in plcr usually occurs together with an increase in the number of newly produced platelets , which are the largest platelet type . plcr is usually correlated with mpv , but it is more sensitive to the increase in platelet size . babu and basu showed that plcr is inversely proportional to platelet count and directly related to mpv and pdw . an increase in plcr may indicate the presence of platelet aggregates , microerythrocytes , and giant platelets . plcr can serve as useful prognostic factors for long - term mortality in patients after acute mi . advocated that pdw and plcr are prognostic factors after mi and suggested that they could be better than other markers , particularly mpv . the interrelationships between plcr and t2 dm microvascular complications were previously studied by jindal et al . , who found that diabetic patients in general , and patients with diabetes and microvascular complications in particular , have higher values of plcr but the difference was not significant . plcr values were statistically significantly higher in the diabetic groups compared with the hc group . plcr levels also correlated with the degree of retinopathy , but these differences were not statistically significant . it is a quantitative test for abnormalities in platelet count and calculated as platelet count mpv/10 . found a significant increase in pct values in patients with t2 dm compared with their control group ; however , they did not report a difference between patients with t2 dm with or without microvascular complications . showed that there was no significant difference in pct level between their diabetic groups and healthy individuals , and there was also no significant difference in mean pct values between dr stages . similar conflicting results also exist between platelet count and t2 dm ; while some studies report no relationship [ 25 , 31 , 32 ] , others report a positive correlation [ 33 , 34 ] . in our study , no significant difference in pct values or platelet count between the diabetic and hc groups was detected . some publications have suggested that proliferative retinopathy is associated with cad events ; therefore , retinopathy and cad may have similar pathophysiological backgrounds [ 35 , 36 ] . platelet parameters provide information on the severity of dr and they may also contain tips which are related to the cad . mpv is also referred to frequently in the pathogenesis , treatment , and prognosis of cvd and also appears to be a useful marker in other systemic and vascular diseases [ 7 , 37 ] . the role of pdw in systemic disease and specifically in patients with cvd and acute coronary events is yet to be explored . according to some publications pdw is becoming a more valuable marker compared with other platelet markers [ 10 , 26 ] . similarly , in our study pdw was found to be the highest independent risk factor compared with the other platelet parameters . because this parameter is generated by only a few machines ( with the sysmex analyzer being one ) , few studies are available about the importance of plcr in systemic and vascular diseases . studies with larger patient groups are required to clarify its function in patients with t2 dm and its role in potential vascular complications . all these indices can be measured by an inexpensive and readily available routine blood count . mpi values may be important in diagnosing and treating patients with dr , and it is possible that higher mpi values will be correlated with the rates of cardiovascular disease and cardiac mortality in these patients . our study supports the fact that platelet function ( mpi ) is altered even in patients with dr potentially contributing to increased cardiovascular risk later on . in our study , mpi ( mpv , pdw , and plcr ) values were significantly higher in patients with t2 dm compared with healthy individuals . values were statistically significantly higher in patients who developed proliferative dr compared with patients without retinopathy . a statistically significant difference in pct and platelet was not observed between the diabetic and hc groups . based on our results , an increase in mpi values reflects an increased risk for pdr stage .

purpose . to investigate whether platelet morphology or function is altered in patients with diabetic retinopathy ( dr ) . methods . this prospective study enrolled 85 healthy controls ( hcs ) ( group 1 ) and 262 patients with type 2 diabetes mellitus ( t2 dm ) . patients were subclassified into three groups according to ocular findings : no dr ( group 2 ; n = 88 ) ; nonproliferative dr ( group 3 ; n = 88 ) , and proliferative dr ( group 4 ; n = 86 ) . mean platelet volume ( mpv ) , platelet distribution width ( pdw ) , platelet large cell ratio ( plcr ) , plateletcrit ( pct ) values , and platelet count were measured in the studied groups . results . mpv , pdw , and plcr levels were significantly altered in groups 24 compared with hcs ( p < 0.05 , p < 0.05 , p < 0.05 ) . compared with group 2 , both dr groups had higher mpv and pdw levels , with a significant difference between groups 2 and 4 for both mpv ( p = 0.036 ) and pdw ( p = 0.006 ) . plcr correlated with retinopathy stage , but no significant difference was found between the dr groups . platelet count and pct values were not significantly different between the groups ( p > 0.05 ) . conclusion . our findings suggest an association between mean platelet indices ( mpi ) ( i.e. , mpv , pdw , and plcr ) and dr stage . therefore , mpi could be a beneficial prognostic marker of dr in patients with t2 dm .

severe trauma and major surgery frequently result in the development of inflammatory complications , including systemic inflammatory response syndrome , sepsis , and septic shock with subsequent splanchnic hypoperfusion . tissue injury after ischemia is primarily caused by reduced oxygen supply and the injury may be worsened during reperfusion phase via formation of reactive oxygen radicals which can promote cell destruction and bowel necrosis through peroxidation of membrane lipid with subsequent increase in the intestinal mucosal level of malondialdehyde ( mda ) . in addition , membrane protein oxidation induces early increase in mucosal protein carbonyl ( pc ) level owing to formation of reactive chlorinating species capable of oxidizing protein . moreover , the oxidative stress inhibit the endogenous antioxidant system which includes glutathione peroxidase and superoxide dismutase . intestinal fatty acid binding protein ( i - fabp ) , a cytosolic protein distributes in the small bowel mucosa from the duodenum to the distal segment of the ileum , and its serum level elevates rapidly at the early stages of small bowel ischemia . hence , it is considered as an early biomarker for intestinal disease , ischemia , and damage . ischemia stimulates localized infiltration of activated neutrophils which is manifested by significant increase in the mucosal level of myloperoxidase enzyme activity ( mpo ) . research in this field has identified key molecular and signaling players that could modulate the tissue injury during this disease process . however , further elucidation of the molecular mechanisms should provide the rationale to identify much needed novel therapeutic options to ameliorate organ damage . atrial natriuretic peptide ( anp ) besides its role in regulating volume homeostasis , has a protective effect during ischemia - reperfusion injury ( iri ) in many tissues . based on the multiple protective effects of nps , it was approved for use in the united states in 2001 by the food and drug administration for acute decompensated heart failure . a previous study demonstrated protective effect of np in iri in heart , lung , brain , and liver owing to the extracardiac expression for np receptors . hence , in this work , we attempted to evaluate the role of anp in attenuating intestinal injury in septic shock . this is a prospective observer - blinded study carried out in surgical intensive care unit ( icu ) from march 2013 to april 2014 on 40 patients in septic shock after approval by hospital ethical committee . patients in septic shock according to the american college of chest physicians / society of critical care medicine definitions , which include strongly suspected infection or positive blood culture plus persistent hypotension despite adequate fluid resuscitation requiring vasopressors to maintain mean arterial blood pressure ( map ) 65 mmhg , and lactate 2 mmol / lpatients were treated with antibiotics according to blood culture and sensitivity testpatients on vasopressor noradrenaline intravenous ( iv ) infusion ( 0.050.2 micg / kg / min)map 65 mmhg . patients in septic shock according to the american college of chest physicians / society of critical care medicine definitions , which include strongly suspected infection or positive blood culture plus persistent hypotension despite adequate fluid resuscitation requiring vasopressors to maintain mean arterial blood pressure ( map ) 65 mmhg , and lactate 2 mmol / l patients were treated with antibiotics according to blood culture and sensitivity test patients on vasopressor noradrenaline intravenous ( iv ) infusion ( 0.050.2 micg / kg / min ) patients on noradrenaline dose > 0.2 micg / kg / minpatients with electrolyte imbalancepatients with one or more organ failurepatients with renal dysfunctionpatients with valvular stenosis , restrictive or obstructive cardiomyopathy , constrictive pericarditis , and pericardial tamponade . patients on noradrenaline dose > 0.2 micg / kg / min patients with electrolyte imbalance patients with one or more organ failure patients with renal dysfunction patients with valvular stenosis , restrictive or obstructive cardiomyopathy , constrictive pericarditis , and pericardial tamponade . the group assignment numbers were sealed in an envelope and kept by the study supervisor . after admission to icu , a written informed consent was obtained from the nearest relatives . group ( e ) : patients received anp ( natrecor scios inc . , titusville , nj 08560 , austria ) ) supplied from sigma company in form of 1.5 mg vial added to 250 ml plastic iv bag ( 1 ml = 6 mg ) given at 2 mcg / kg iv bolus over 1 min followed by 0.01 mcg / kg / min for 24 h ( the dose approved for acute decompensated heart failure ) . patients were monitored for mean arterial pressure ( map ) ( mmhg ) , heart rate ( hr ) ( b / min ) , central venous pressure ( cvp ) ( cmh2o ) , temperature , urine output ( uop ) , total fluid balance and routine investigation ( complete blood count , arterial blood gases , serum electrolytes , liver , and kidney function ) . the primary outcome measurements were blood marker of intestinal hypoperfusion in the form of the following laboratory data which was measured before the start of anp infusion , 6 h , 12 h and 24 h after the start of infusion . blood sample ( 5 ml ) was centrifuged and serum was used to measure : i - fabp as a marker of intestinal ischemia using ( elisa ) measured by spectrophotometer at wavelength 450 nmmda measured by spectrophotometer at wavelength 535 nmpc level measured by spectrophotometer at wavelength 366 nmmpo measured by spectrophotometer at wavelength 655 nmglutathione peroxidase activity ( gpa ) ( lipid peroxide scavenger ) was expressed as nanomols of reduced nicotinamide adenine dinucleotide phosphate oxidized to nadh measured by spectrophotometer at wavelength 340 nm . i - fabp as a marker of intestinal ischemia using ( elisa ) measured by spectrophotometer at wavelength 450 nm mda measured by spectrophotometer at wavelength 535 nm pc level measured by spectrophotometer at wavelength 366 nm mpo measured by spectrophotometer at wavelength 655 nm glutathione peroxidase activity ( gpa ) ( lipid peroxide scavenger ) was expressed as nanomols of reduced nicotinamide adenine dinucleotide phosphate oxidized to nadh measured by spectrophotometer at wavelength 340 nm . the secondary outcome measurements were the duration of noradrenaline infusion ( days ) , duration of icu stay , hospital mortality rate and complications related to anp such as hypotension , headache , nausea , and back pain.(if developed during time of the study , the drug infusion was stopped , and complication was recorded ) . sample size of forty patients was calculated for 90% power , = 0.05 , = 0.1 , and anticipated effect size = 0.40 using sample size software ( g * power version 3.00.10 , germany ) . analytic statistics was performed on ibm compatible computer using spss 11.5 ( spss inc . , data compared using unpaired student 's t - test ; p < 0.05 was considered as statistically significant . patients in septic shock according to the american college of chest physicians / society of critical care medicine definitions , which include strongly suspected infection or positive blood culture plus persistent hypotension despite adequate fluid resuscitation requiring vasopressors to maintain mean arterial blood pressure ( map ) 65 mmhg , and lactate 2 mmol / lpatients were treated with antibiotics according to blood culture and sensitivity testpatients on vasopressor noradrenaline intravenous ( iv ) infusion ( 0.050.2 micg / kg / min)map 65 mmhg . patients in septic shock according to the american college of chest physicians / society of critical care medicine definitions , which include strongly suspected infection or positive blood culture plus persistent hypotension despite adequate fluid resuscitation requiring vasopressors to maintain mean arterial blood pressure ( map ) 65 mmhg , and lactate 2 mmol / l patients were treated with antibiotics according to blood culture and sensitivity test patients on vasopressor noradrenaline intravenous ( iv ) infusion ( 0.050.2 micg / kg / min ) patients on noradrenaline dose > 0.2 micg / kg / minpatients with electrolyte imbalancepatients with one or more organ failurepatients with renal dysfunctionpatients with valvular stenosis , restrictive or obstructive cardiomyopathy , constrictive pericarditis , and pericardial tamponade . patients on noradrenaline dose > 0.2 micg / kg / min patients with electrolyte imbalance patients with one or more organ failure patients with renal dysfunction patients with valvular stenosis , restrictive or obstructive cardiomyopathy , constrictive pericarditis , and pericardial tamponade . the group assignment numbers were sealed in an envelope and kept by the study supervisor . after admission to icu , a written informed consent was obtained from the nearest relatives . group ( e ) : patients received anp ( natrecor scios inc . , titusville , nj 08560 , austria ) ) supplied from sigma company in form of 1.5 mg vial added to 250 ml plastic iv bag ( 1 ml = 6 mg ) given at 2 mcg / kg iv bolus over 1 min followed by 0.01 mcg / kg / min for 24 h ( the dose approved for acute decompensated heart failure ) . patients were monitored for mean arterial pressure ( map ) ( mmhg ) , heart rate ( hr ) ( b / min ) , central venous pressure ( cvp ) ( cmh2o ) , temperature , urine output ( uop ) , total fluid balance and routine investigation ( complete blood count , arterial blood gases , serum electrolytes , liver , and kidney function ) . the primary outcome measurements were blood marker of intestinal hypoperfusion in the form of the following laboratory data which was measured before the start of anp infusion , 6 h , 12 h and 24 h after the start of infusion . blood sample ( 5 ml ) was centrifuged and serum was used to measure : i - fabp as a marker of intestinal ischemia using ( elisa ) measured by spectrophotometer at wavelength 450 nmmda measured by spectrophotometer at wavelength 535 nmpc level measured by spectrophotometer at wavelength 366 nmmpo measured by spectrophotometer at wavelength 655 nmglutathione peroxidase activity ( gpa ) ( lipid peroxide scavenger ) was expressed as nanomols of reduced nicotinamide adenine dinucleotide phosphate oxidized to nadh measured by spectrophotometer at wavelength 340 nm . i - fabp as a marker of intestinal ischemia using ( elisa ) measured by spectrophotometer at wavelength 450 nm mda measured by spectrophotometer at wavelength 535 nm pc level measured by spectrophotometer at wavelength 366 nm mpo measured by spectrophotometer at wavelength 655 nm glutathione peroxidase activity ( gpa ) ( lipid peroxide scavenger ) was expressed as nanomols of reduced nicotinamide adenine dinucleotide phosphate oxidized to nadh measured by spectrophotometer at wavelength 340 nm . the secondary outcome measurements were the duration of noradrenaline infusion ( days ) , duration of icu stay , hospital mortality rate and complications related to anp such as hypotension , headache , nausea , and back pain.(if developed during time of the study , the drug infusion was stopped , and complication was recorded ) . sample size of forty patients was calculated for 90% power , = 0.05 , = 0.1 , and anticipated effect size = 0.40 using sample size software ( g * power version 3.00.10 , germany ) . analytic statistics was performed on ibm compatible computer using spss 11.5 ( spss inc . , all results presented in the form of mean standard deviation . data compared using unpaired student 's t - test ; p < 0.05 was considered as statistically significant . figure 1 shows the flow diagram for this study , in which 52 patients were assessed for eligibility and 40 adult patients were included in the study . the trial flow diagram , including enrollment , intervention allocation , and analysis there was no significant difference between both groups as regard patients characteristics and disease severity [ table 1 ] . patients characteristics in comparison with group c , group a showed a significant decrease ( p < 0.05 ) in serum level of mpo , mda , pc , and i - fabp , with a significant increase ( p < 0.05 ) in serum level of gpa 6 h , 12 h and 24 h after the start of anp infusion [ table 2 ] . there was significant decrease ( p < 0.05 ) in mean duration of noradrenaline infusion , the length of icu stay and hospital mortality rate in group a in comparison with group c [ table 3 ] . in group a , after 12 h of anp infusion , there was significant decrease ( p < 0.05 ) in map , cvp , hr , and serum sodium with a significant increase ( p < 0.05 ) in uop [ table 4 ] . seven patients had map < 65 mmhg but respond to volume resuscitation with insignificant change in noradrenaline dose , three patients serum sodium was 125130 tissue perfusion following a period of hypoperfusion represents complex series of events during shock . in this study , before administration of anp , there was a significant increase in serum level of mda , pc , mpo , and i - fabp with a significant decrease in serum gp activity . these results could be attributed to the generation of reactive oxygen species which are generated when oxygen is reintroduced to xanthine oxidase enzyme activation during ischemia . these potentially toxic reactive oxygen species can promote cell destruction and bowel necrosis through peroxidation of membrane lipid and protein manifested in this study by a significant increase in serum level of mda and pc . the intestine is one of the richest sources in the body of xanthine oxidase enzyme that exhibit a gradient of increasing activity which play a major role in the generation of toxic superoxide radicals . under normal condition xanthine dehydrogenase form is predominant but during ischemia xanthine dehydrogenase will be oxidized into xanthine oxidase which becomes a major source for toxic superoxide radical production once the tissues are reperfused and oxygen is reintroduced . moreover , the reactive oxygen species could stimulate localized infiltration of activated neutrophils which is demonstrated in this work by a significant increase in serum level of mpo which in turn is a potential source for further release of reactive oxygen radicals with enhanced secretion of proinflammatory cytokines by mast cells and macrophages . the inflammatory cytokines leading to the formation of reactive chlorinating species capable of oxidizing protein with a subsequent increase in plasma pc level . this work demonstrated that iv infusion of anp for 24 h recorded a significant decrease in serum level of mda , pc , mpo , and ifap with a significant increase in serum gp activity in comparison with control group . anp and its receptors were demonstrated to be expressed in diverse tissues beside the cardiovascular and renal system . many previous studies recorded significant antioxidant and anti - inflammatory effects of anp in many tissues . some studies reported anp could attenuate activation of nuclear factor kappa b in macrophages . the reduced binding activity of this redox - sensitive transcription factor was accompanied by diminished m rna expression of tumor necrotizing factor alpha chemokines and cell adhesion molecules thereby reducing leukocytes infiltration manifested in this study by a significant decrease in the serum level of mpo . fabp is one of the intracellular proteins that plays important role in transportation and metabolism of long - chain fatty acids which is rapidly released into circulation just after small intestinal mucosal injury . alexandra et al . stated that anp could inhibit the activity of inducible no synthetase ( inos ) in macrophages via destabilization of inos mrna and this protective effects of anp are mediated by cyclic guanosine monophosphate . inhibition of no formation will inhibit the reactive oxygen species and hence inhibit lipid and protein peroxidation manifested in this work by a significant decrease in serum level of mda and pc . in addition , this study shows that anp induces significant increase in serum gp activity . chujo et al . stated that anp could stimulate the antioxidant defense system or may induce antioxidant activity by itself . revealed that administration of anp infusion at the onset of lung reperfusion attenuates reperfusion injury . moriyama et al . found that anp attenuates ischemia - reperfusion in induced renal failure through inhibition of inflammatory mediators , mpo and neutrophils activation when given as iv infusion for 2 h after reperfusion injury . yamada et al . found the protective effect to anp in hepatic ischemia - reperfusion . in cases of septic shock , concomitant administration of anp with noradrenaline may have a protective effect against intestinal injury through a decrease in the level of intestinal hypoperfusion markers owing to its anti - inflammatory and antioxidant effect .

background and aims : the aim of this study is to assess the effect of atrial natriuretic peptide ( anp ) on intestinal ischemia - reperfusion injury in septic shock.material and methods : a prospective randomized controlled , observer - blinded study was carried out in surgical intensive care unit ( icu ) , university hospital . forty adult patients in septic shock were randomly divided into two groups , control group ( group c ) received normal saline and anp group ( group a ) patients received anp in the form of 1.5 mg vial added to 250 ml solvent in plastic bag ( 1 ml = 6 micg ) given at 2 mcg / kg intravenous bolus over 1 min followed by 0.01 mcg / kg / min for 24 h. the primary outcome measurements were blood marker of intestinal hypoperfusion in form of intestinal fatty acid binding protein ( i - fabp ) , malondialdehyde ( mda ) , myloperoxidase enzyme activity ( mpo ) , protein carbonyl ( pc ) , and glutathione peroxidase activity ( gpa ) measured before start of anp infusion , 6 h , 12 h , and 24 h after start of infusion . the secondary outcome measurements were the duration of noradrenaline infusion , duration of icu stay , hospital mortality rate , and complications related to anp.results:in comparison with group c , group a showed a significant decrease ( p < 0.05 ) in serum level of mpo , mda , pc , and i - fabp , with a significant increase ( p < 0.05 ) in serum level of gpa , 6 h , 12 h , and 24 h after the start of anp infusion . there was significant decrease ( p < 0.05 ) in mean duration of noradrenaline infusion , the length of icu stay and mortality rate in group a in comparison with group c. in group a , seven patients had mean arterial blood pressure < 65 mmhg but respond to volume resuscitation , three patients serum sodium was 125130 mmol / l.conclusion : in cases of septic shock , concomitant administration of anp with noradrenaline may have a protective effect against intestinal injury through a decrease in the level of intestinal hypoperfusion owing to its anti - inflammatory and antioxidant effect .

new drugs for treating eye diseases have been developed over the past decade and are very unique for each eye diseases , such as , glaucoma , cataracts , and age - related macular degeneration ( amd ) . it is estimated that 1.6 million adults in the usa over the age of 50 and above suffer from age - related macular degeneration and about 200,000 cases are diagnosed annually . drugs currently utilized for amd are delivered via repeated intravitreal injections of the drug into the eye . risks of repeated intravitreal injections can include intraocular infections ( endophthalmitis ) , intraocular hemorrhage , and retinal detachment . also , reducing the frequency of dosing will clearly benefit the patient by reducing the need for risky intravitreal injections and improving the pharmacokinetics of the drug in the eye . eye disease in the posterior segment includes two different forms of amd , such as , dry and wet . approximately 90% of patients with amd have the dry form shown in small yellow and white deposits form made of proteins and waste products . wet adm is caused by abnormal blood vessels grow out of the retina followed by rapid vision loss . however , these amd diseases limit drug delivery in the retina region to eye drops [ 2 , 3 ] . the drug using a needle with syringe can be injected , but it barely provides the right amount of dose and over doses may cause more severe problems , such as , swelling , fatigue , and damage photoreceptor molecules . developing an implantable drug delivery device will help reduce the costs and risks associated with frequent injections and facilitate delivering the drug in a controlled manner and in the required amounts and improve therapeutic efficacy and safety of drugs . ocular diseases , such as , glaucoma , age - related macular degeneration ( amd ) , diabetic retinopathy , and retinitis pigmentosa require drug management in order to prevent blindness . these incurable diseases require lifelong treatment through orally administered medications , intraocular injections , and biodegradable implants . drug delivery to ocular tissue is very difficult due to area and size limitations in the eye . there are currently at least three major categories of ocular drug delivery systems as discussed in : biodegradable or nonbiodegradable , atypical implantable pump systems , and implantable pump systems . implantable pump systems dispense drugs from an internal reservoir and have the advantage of providing control over drug delivery rate and volume . several types of implantable pumps , such as , infusion pumps , osmotic pumps , and peristaltic pumps have been developed and used successfully for applications , such as , insulin delivery but have been found to be unsuitable for ocular drug delivery due to space limitations . typical implantable pump systems ( hydrogel systems infused with drug swell via intake of biological fluids for example ) minimize the drug volume required for treatment and provide targeted delivery at a constant rate . however surgical procedures are required to implant and replace these devices , which may in turn contribute to additional side effects . vitrasert and retisert distributed by bausch and lomb are commercially available examples of nonbiodegradable systems . in these systems , the drug is released as a polymer matrix infused with drug dissolves or the drug is distributed from a nonbiodegradable reservoir . the disadvantages with these systems include in vivo polymer degradation ( and the drug can not be refilled ) and the fact that drug delivery is dependent upon a limited volume available within the polymer . recently , a review of barriers to posterior eye drug delivery and the challenges and opportunities were discussed by thrimawithana et al . . table 1 summarizes various drugs , their diffusion coefficients , the average dosage , and the frequency to treat amd diseases . fick 's second law of diffusion can be used to describe the transport of drug into the eye using microchannels . as the drug delivery device is implanted in the vitreous body of the eye , usually the diffusion depends on the local concentration rate between a drug reservoir and an aqueous humor . the fick 's second law of diffusion model can predict the diffusion time of the local tissue concentration in the eye following a variety of microchannel geometries for ocular implantable delivery . the diffusion coefficient of drugs may vary based on the chemical properties and internal structure as well as the molecular weight of the drug . several micro electro - mechanical system ( mems ) devices , such as , microreservoirs and micropumps have been fabricated to address the spatial constraints posed by ocular drug delivery [ 1 , 4 ] . microreservoirs offer maximum control of drug delivery but can not be refilled or reused , thereby ruling out suitability for treating chronic eye conditions . peristaltic micropumps provide targeted drug delivery through active pumping but require considerable space to achieve a desired volume of flow per minute . to overcome these limitations , lo et al . recently developed a first generation prototype polymer mems delivery device with a refillable drug reservoir for treating ocular diseases . in addition to the refillable drug reservoir , the device consists of a transcleral cannula , check valve , and suture tabs . the device requires surgical implantation underneath the conjunctiva and the specified dose of medication is dispensed from the device when the reservoir is mechanically activated by the patient 's finger . this device has several advantages when compared to existing systems including the following : the device is refillable , requires only a single surgical intervention , and is suitable for treating chronic ocular conditions ; it is compact and fits within the dimensions imposed by the ocular orbit ( < 2 mm thick ) . however , the device requires patient 's intervention in dispersion of the drug . in order to cater to multiple scenarios in terms of amount of drug delivery and constraints , alternate mems devices nano-/microchannel - based drug delivery technologies represent an unprecedented opportunity to realize radically new devices that would exploit the novel features of the nanochannels , in which chip contains that drug reservoir with dose , provide unique performance in terms of diffusion and kinetics over existing technologies for drug delivery applications . this study focuses on the design , simulation , and development of an implantable ocular drug delivery device . a novel design concept consisting of micro / nanochannels embedded between top and bottom covers with a drug reservoir made from pdms material was developed . several simulations were carried out with different microchannel configurations in order to see the feasibility for ocular drug delivery applications . finally , a prototype illustrating the three components of the drug delivery design is presented . as shown in figure 1 , the drug is stored in a reservoir at one end of the device . microchannels are coated with hydrophilic coatings so that the drug from the reservoir diffuses through the channels at specified / designed rate into the eye eliminating the need for any controlled actuation . hydrogels ( miragel , mira inc , waltham , mass ) , consisting of poly ( methyl acrylate - co-2-hydroxyethyl acrylyte ) are used as means to passively induce the drug delivery into the microchannels so that the drug diffuses freely through the channels and reaches the outlet for delivery . the microchannel component with inlet / outlet reservoirs will be enclosed in a pdms case whose base is rounded to match the curvature of the eye globe . the device is attached securely to the sclera of the eye with fine 100 or 90 nylon sutures . ideally , the device would be surgically , transclerally implanted in the vitreous space with an external thin curved spherical surface flange that would be nearly flush with the sclera and sutured in place ( see figure 2 ) . the design requirements for the proposed drug delivery device are as follows : target overall volume is less than 280 mm ; diffusion rate is less than 0.07 nl / min ; target diffusion time period will be around 1 to 2 years ; kinetics : reliable diffusion coefficient of drugs through the microchannels ; implantable : eliminate repeated injections for effective treatment ; actuation : sustained release drug delivery methods . target overall volume is less than 280 mm ; diffusion rate is less than 0.07 nl / min ; target diffusion time period will be around 1 to 2 years ; kinetics : reliable diffusion coefficient of drugs through the microchannels ; implantable : eliminate repeated injections for effective treatment ; actuation : sustained release drug delivery methods . to illustrate the targeted volume and rate of the drug delivery device , the following section provides the details of calculations . it has been assumed that drug - contained deionized water will be transported through the microchannel from a reservoir . the corticosteroid fluocinolone acetonide has low solubility , so that solution was made by dissolving 59 mg of c24h30f2o6 in deionized water of 50 l ( concentration in the device 1.18 mg/l ) . we also assume that the concentration of drugs in the water within the reservoir is around 1.18 mg / cm and zero concentration within the retina region of the eye . using this value of concentration , we can estimate the flux density of drug - contained water transport into the retina region by molecular diffusion . however , in this study , we assume that the diffusion coefficient for typical eye drug , which is the corticosteroid fluocinolone acetonide in the deionized water , is equal to 2.3 10 cm / s . the concentration of drug in the reservoir is very large in comparison to the concentration in the retina region . to calculate the flux density , we use fick 's law ( 1 ) , assuming that the gradient of concentration with length is linear over the microchannels path . the diffusive flux will be from the reservoir to the eye , from a high concentration to a lower concentration . fick 's first law , which relates the diffusive flux to the concentration and is given as , ( 1)j=d x , where , j is the diffusion flux ( g / cms ) , d is the diffusion coefficient or diffusivity in dimension of cm / s , and is the concentration of drugs in the reservoir . using , the above values , we get ( 2)j= 2.3 107 cm2/s(1.18 g / cm3/0.8 cm)= 3.39 107 g/(cm2)s . ignoring the diffusion direction , we calculate the flux density of 3.39 10 g / cm s and it can be used to calculate the total mass flux of drug into the eye using ( 3 ) given below . for example , if the straight microchannel has an inlet area of 0.0005 cm with 12 separate pathways , then the total flux into the eye is ( 3)mtotal = ja , where , a is a section area at the inlet . using the above values , we get ( 4)mtotal=3.39107 g / cm2s0.0005 cm260 s / minute=1.02108 g / min 1.04104 l / min or 2.58 mg / month ( total 12 microchannels ) . as per our specification , the drug delivery device contains drug of 6 mg in the deionized water , it can be continuously used for around 11 to 12 months without refilling injection . in order to illustrate the proof - of - concept , the overall dimensions of microchannels were within a range of 1.5 ~ 8.0 mm in length , had a depth of 5 to 100 m and a width may vary based on the geometry of microchannels ( 50 ~ 500 m ) as shown in figure 3 . the length of microchannels depends on the geometry of diffusion channels . after the surface modification , such as , oxygen plasma , the channels will provide various diffusion rates in conjunction with the drug 's diffusion coefficient . the injection cannula ( needle gauge # 25 or 32 ) on the outlet of the device routes the drugs into the targeted region . in order to understand the design characteristics of the microchannels , we developed a coarse - grained representation of the microchannel geometry through computational fluid dynamic analysis and optimization . specifically , the role of the microchannel geometry in passive free diffusion that molecules can pass freely through the microchannel follow concentration gradients is investigated and discussed . finite element ( fe ) analysis using ansys - multiphysics module was used to perform the design simulations . six different microchannel geometries are developed to simulate the thermal diffusivity as shown in figure 4 . drug concentration at the reservoir is assumed to be 1 kmol / m and drug concentration at the outlet to be 0 kmol / m . drug diffusivity is assumed to be the same as the synthetic corticosteroid fluocinolone acetonide in deionized ( di ) water ( 2.3 10 cm / s ) . simulation results of drug diffusion within the microchannels as a function of time and drug diffusion from the drug reservoir revealed high - concentration areas to zero drug concentration areas in the vitreous body . since drug consumption occurred at the blood vessel of a vitreous body which can be approximated as zero , the amount of diffusion may vary with the concentration gradient , however , a constant drug release rate can be obtained . master molds for both upper and bottom layers of the reservoir are made of acura 50 plastic ( 3d system corp . ) and constructed from 3d stereolithography process using 3d viper sla system ( 3d system corp . ) . pdms is mixed silicone elastomeric base and a curing agent with a 10 : 1 ratio ( sylgard 184 , dow corning ) and poured into the master molds . the pdms is degassed in a vacuum machine for 20 minutes ( durable medical equipment inc . , richmond , va ) and cured at room temperature for 24 hours or 80c for 2 hours . the microchannel geometries will be formed using soft lithography on the 4 silicon wafers after baking at 1000c for at least 10 hours to get at least 1 m thickness of an oxides layer . the wafers are vapor coated with hexamethyldisilazane ( hmds ) adhesion promoter . after the mask is completed , photoresist ( az eci # 3012 , az electronic materials , branchburg , nj , usa ) is poured on the wafer around 2.5 ml and spin coated at 4000 rpm for 30 s ( expected thickness less than 0.8 m layer ) , and then the wafer is baked at 90c for 1 minute . after exposure , native oxide is removed with a 20% koh solution dip at 80c for 2 hours and 5 hours so that 100 and 250 m etch depths for the microchannels will be achieved . the final step of the wafer fabrication is to remove the oxide by using a boe etch . lastly , the assorted microchannels will be assembled to the pdms reservoir and sealed using the o2 plasma etching processes in accordance with 600 mtorr pressure and 20 w power for 35 s. the length and width of the straight microchannel for this simulation are 8 mm and 500 m . in this simulation , we assumed that the drug diffuses from the drug reservoir , as we discussed using fick 's law , which states that molecules will diffuse out to an area of low concentration from an area of high concentration through microchannels . the movement of drug across a micro channels in a manner driven solely by the concentration gradient . the results of drug diffusion at 50 seconds through various microchannel configurations considered are shown in figure 6 . it is interesting to note that different microchannel configurations will give rise to different diffusion rates . the drug diffusion rate as a function of time for various microchannel configurations is presented in figure 7 . it can be seen from figure 7 that each of the microchannel configurations exhibits different diffusion characteristics in terms of drug diffusion rates . initially , there is a drastic increase and after a certain time , the diffusion rate is almost constant . if the drug was to be delivered at a constant rate over a one - hour period then the inlet flux of straight microchannel would be 6.25 10 kmol / s . over the first second , there is a rapid increase of diffusion rates up to approximately 1.24 10 kmol / s and then a more gradual increase to approximately 6 10 kmol / s after 105 seconds . overall , each of the microchannel configurations can deliver the drug at different diffusion rates . based on the results obtained through various microchannel configurations , the designs : osmotic i and osmotic ii best satisfied the diffusion rate specifications ( less than 0.07 nl / min ) for the developed ocular drug delivery device . these results are presented in figure 8 . in order to demonstrate the diffusion through the entire device , an analysis is carried out using the osmotic ii microchannel and the reservoir for 1500 seconds using ansys software . it is assumed that the thermal conductivity and specific heat and density were set as 0.6 wmk , 4181.3 j / kgk , and 850 kg / m , respectively , for simulating the diffusion through the device . the temperature of 120c at the inlet ( center top of reservoir ) and 37.5c at the outlet is applied for this simulation . it can be seen from figure 9 that a very slow diffusion occurs most likely at the narrow channel paths . this demonstrates that the developed microdevice is capable of delivering the drug through the osmotic ii microchannel configuration . the results obtained from the simulations confirmed that the microchannels have the potential to be used as a drug delivery system depending on desired flow rates and drug concentrations . the proposed device can produce a constant delivery rate , which is favorable to the treatment of eye disease . diffusion rates can be customized to obtain effective levels by varying height , width , and length of microchannels . the overall fabricated device is shown in figure 10 . currently , the functionality of the device is being explored and will be tested in future . the design involves development of an implantable device with micro-/nanochannels with top and bottom covers . based on the results obtained , channel design of osmotic i and ii satisfied the diffusion rates required for ocular drug delivery . in addition to design simulations , the top and bottom covers were fabricated from pdms through replica - molding techniques . currently , the device is being tested for its functionality and diffusion characteristics . however , there are significant challenges related to achieving reliable and sustainable integration , bonding , diffusion of the drug into channels , and controllability . the test evaluation will be performed measuring the change in ph of a neutral solution using a strong citric acid ; it can be diffused out through the device .

ocular diseases , such as , glaucoma , age - related macular degeneration ( amd ) , diabetic retinopathy , and retinitis pigmentosa require drug management in order to prevent blindness and affecting million of adults in usa and worldwide . there is an increasing need to develop devices for drug delivery to address ocular diseases . this study focuses on the design , simulation , and development of an implantable ocular drug delivery device consisting of micro-/nanochannels embedded between top and bottom covers with a drug reservoir made from polydimethylsiloxane ( pdms ) which is silicon - based organic and biodegradable polymer . several simulations were carried out with six different micro - channel configurations in order to see the feasibility for ocular drug delivery applications . based on the results obtained , channel design of osmotic i and osmotic ii satisfied the diffusion rates required for ocular drug delivery . finally , a prototype illustrating the three components of the drug delivery design is presented . in the future , the device will be tested for its functionality and diffusion characteristics .

medicine retailers play an important role , especially at the community level , in developing countries . medicine vendors in these medicine outlets fill the gap created by inadequate skilled health professionals ( pharmacists ) required for the procurement , storage , and distribution of medicine . in nigeria , patent medicine stores are registered by licensed patent medicine vendors ( pmvs ) , while community pharmacies are registered by licensed pharmacists . pmvs are individuals with no formal pharmacy training , and are only authorized to sell over - the - counter medicines and are usually licensed to practice by the pharmacists council of nigeria ; however , no such licensing exists for medicine vendors in community pharmacies . the law does not specify the minimum educational level for medicine vendors ; however , the minimum educational level for pmvs in nigeria is the primary level of education . in nigeria , medicine vendors are required , by law , to sell medicines in their original package . pmvs are not allowed , by law , to alter a medicine package or sell a portion of medicines from their package as this constitutes dispensing . however , in response to their clients demand , they are often seen dispensing medicines from their original package or dispensing generic medicines like paracetamol from their tins . however , some medicine vendors in nigeria are trained as pharmacy technicians with basic knowledge of dispensing , to serve as assistants to registered pharmacists . appropriate self - medication requires the individual to accurately recognize symptoms , set therapeutic objectives , select the appropriate medicine to be used for his medical condition , and determine the appropriate dosage and dosage schedule , taking into account his medical history , contraindication , and possible side effects of the medicine . nalini , evaluated self - medication practice among government doctors in india , and concluded that the prevalence of self - medication practice among doctors is high . baros et al . , demonstrated that self - medication was a frequent practice among nurses working in public hospitals in brazil , and bamgboye et al . , showed that self - medication among workers involved in medical care services in tertiary hospitals in nigeria is high . no study has looked at self - medication practice and medicine knowledge among medicine sellers in nigeria . this study was therefore aimed at evaluating self - medication practice and medicine knowledge among medicine vendors , to determine if a relationship exists between the two . there are about 119 registered community pharmacy outlets and several patent medicine stores in jos - north and jos - south local government areas , where this research was carried out . the first stage involved stratifying jos into two , based on the two local governments that made up jos . each local government was further stratified into two according to the place of practice ( community pharmacy and patent medicine store ) of the respondents , and these formed the primary sampling unit . the primary sampling unit ( psu ) was obtained by a convenience method and the medicine vendors in the psu who consented to participate in the study were included in the study . a total of 129 psus , consisting of 55 community pharmacies and 74 patent medicine stores were used for this study . the participants responded to questions on demography , self - medication practices within the last month , reasons for self - medication , and sources of medicine information . a knowledge assessment test was also administered to test the respondents over - the - counter medicine knowledge on key areas of medicine indication , drug administration , brand and generic names of medicines , contraindication , interaction , and side effects of medicines . the study was approved by the ethical committee of the faculty of pharmaceutical sciences , university of jos , jos , nigeria . approval to carry out the study in each psu was obtained from the managers of the psus . verbal informed consent was sought from each participant before administering the questionnaire and the participants were told that participation in the study was voluntary and information obtained would be anonymous and confidential . data were entered into the statistical package for social sciences ( spss ) version 16.0 ( spss inc , chicago il ) to generate descriptive statistics . relationships between variables were tested using the chi - square test . the knowledge assessment test was graded and the score was converted into percentages . a percentage score of less than 50% was considered inadequate , while a score of between 50 to 69% was considered marginal , and a score of 70 to 100% was considered adequate . there are about 119 registered community pharmacy outlets and several patent medicine stores in jos - north and jos - south local government areas , where this research was carried out . the first stage involved stratifying jos into two , based on the two local governments that made up jos . each local government was further stratified into two according to the place of practice ( community pharmacy and patent medicine store ) of the respondents , and these formed the primary sampling unit . the primary sampling unit ( psu ) was obtained by a convenience method and the medicine vendors in the psu who consented to participate in the study were included in the study . a total of 129 psus , consisting of 55 community pharmacies and 74 patent medicine stores were used for this study . the participants responded to questions on demography , self - medication practices within the last month , reasons for self - medication , and sources of medicine information . a knowledge assessment test was also administered to test the respondents over - the - counter medicine knowledge on key areas of medicine indication , drug administration , brand and generic names of medicines , contraindication , interaction , and side effects of medicines . the study was approved by the ethical committee of the faculty of pharmaceutical sciences , university of jos , jos , nigeria . approval to carry out the study in each psu verbal informed consent was sought from each participant before administering the questionnaire and the participants were told that participation in the study was voluntary and information obtained would be anonymous and confidential . data were entered into the statistical package for social sciences ( spss ) version 16.0 ( spss inc , chicago il ) to generate descriptive statistics . relationships between variables were tested using the chi - square test . the knowledge assessment test was graded and the score was converted into percentages . a percentage score of less than 50% was considered inadequate , while a score of between 50 to 69% was considered marginal , and a score of 70 to 100% was considered adequate . a total of 310 questionnaires were distributed to medicine vendors , but 236 of them were returned , representing a response rate of 76.1% . about 62% of the respondents were between the ages of 21 and 30 years and most respondents ( 65.7% ) had post secondary education with 28% of the respondents holding a certificate in health sciences [ table 1 ] . there was a significant association between the respondents place of practice with their highest educational level attained and holding a health certificate , as 67.7% of those with tertiary education and 71.2% of those holding a health certificate worked in community pharmacies . demographic characteristics of the respondents self - medication was found to be common ( 75.4% ) among medicine vendors and it was not found to be associated ( p>0.05 ) with any of the demographic characteristics of the respondents [ table 2 ] . prevalence of self - medication practice among respondents the classes of medicines commonly used by respondents for self - medication were analgesics ( 31.4% ) , anti - malarials ( 22.6% ) , multivitamins ( 17.7% ) , and antibiotics ( 11.25% ) [ table 3 ] . classes of medicine used by respondents for self - medication the common reasons given by the respondents for engaging in self - medication were that their illness was mild ( 31.7% ) , they were knowledgeable about the treatment of their conditions ( 23.5% ) , and self - medication saved time ( 17.6% ) . other reasons given by the respondents were that self - medication saved money ( 12.2% ) and that they had taken the same medicine in the past ( 10.9% ) common sources of medicine information among respondents were from the health professionals ( 47.3% ) , medicine leaflets ( 23.3% ) , and the media ( 19.1% ) . other sources of medicine information among respondents were from friends and relatives ( 7.6% ) and the internet ( 0.8% ) . the knowledge assessment test revealed that 32.6% of the respondents had inadequate knowledge , 33.1% had marginal knowledge , and 34.3% had adequate knowledge . there was no significant ( p>0.05 ) relationship between self - medication practice among respondents and their medicine knowledge . however , the medicine knowledge scores were found to be significantly ( p<0.05 ) associated with holding a certificate in health sciences , the years of experience , and place of practice [ table 4 ] . the demographic data gathered from this study showed that a number of medicine vendors had post secondary education with about 28% of them holding a certificate in various fields of health sciences . those holding a certificate in health sciences reported being trained as nurses , community health workers , and pharmacy technicians . although the policy does not consider being trained in these fields as a prerequisite to being a medicine vendor , having trained staff at the community level is of public health significance , as these staff work in premises that are cited within local communities and they can advice the public on the safe use of medicines , minor ailments , and healthy lifestyles . this rate is similar to a finding in a tertiary hospital in nigeria , wherein , the prevalence of self - medication among workers was 73% . although the prevalence of self - medication in this study was not associated with any of the demographic characteristics studied , the high prevalence of self - medication among medicine vendors may be as a result of their perceived medicine knowledge ( as most of them reported that they were knowledgeable about the medicine they used for self - medication ) and increased access to medicines , as these factors have been shown to promote self - medication . the high use of analgesics for self - medication confirms the findings that analgesics are the commonly used over - the - counter medicines for self - medication , while the high use of anti - malarial drugs correlates with the high incidence of malaria in nigeria . multivitamins were common because people take them as supplements for promoting health , preventing illness ; to boost the immune system , for prevention of stress , and to supplement regular nutrition . self - medication with antibiotics is of public health concern because inappropriate use of antibiotics has been reported even among health workers . inappropriate use of antibiotics results in antibiotic resistance , which is a major problem worldwide especially in developing countries . medicine information from friends / relatives , media , and the internet are unreliable sources of medicine information and people can easily be misinformed by these sources . a number of the studied population relies on these sources of information , hence , the need for these vendors to be enlightened on the appropriate and reliable sources of medicine information . the study demonstrated poor medicine knowledge among the respondents ; only 34.3% of the respondents had adequate knowledge as per the medicine knowledge assessment test . medicine knowledge was found to be associated ( p<0.05 ) with holding a certificate in health sciences , years of experience , and place of practice . vendors with a health certificate , those with more years of experience , and those working in a community pharmacy had more adequate knowledge . the higher medicine knowledge level achieved by medicine vendors in community pharmacies , rather than those in patent medicine stores , could be a result of the high percentage of trained medicine vendors working in community pharmacies or might have to do with their working environment and experience , as vendors working in community pharmacies had more exposure with respect to the types of medicines they handled and they also worked under the supervision of a trained pharmacist , who could be contributing to their medicine knowledge . a few limitations exist with this study ; the study population may not be representative of the medicine vendors found in jos ; and the study was based on retrospective information obtained from the vendors and this is prone to recall error and bias in reporting . a few limitations exist with this study ; the study population may not be representative of the medicine vendors found in jos ; and the study was based on retrospective information obtained from the vendors and this is prone to recall error and bias in reporting . the present study demonstrated that self - medication practice was high and inadequate medicine knowledge existed among respondents . hence , there is a need for education on appropriate self - medication practice among medicine vendors .

background : medicine vendors fill the gap created by inadequate skilled professionals required for medicine procurement , storage , and distribution in developing countries.aim:to evaluate self - medication practice and medicine knowledge among medicine vendors and to determine if a relationship exists between both.materials and methods : a descriptive , cross - sectional study was conducted , using a pretested questionnaire on 236 medicine vendors in jos , nigeria , sampled through a two - stage stratified design . data collected were analyzed using the statistical package for social sciences version 16 , and the chi - square test was used to determine the association between variables.results:self-medication was common ( 75.4% ) among respondents and was not associated ( p>0.05 ) with any of the demographic characteristics studied . the classes of medicines commonly used by respondents for self - medication were analgesics ( 31.4% ) , anti - malarials ( 22.6% ) , multivitamins ( 17.7% ) , and antibiotics ( 11.25% ) . a knowledge assessment test revealed that only 34.3% of the respondents had adequate knowledge . there was no significant ( p>0.05 ) relationship between self - medication practice and medicine knowledge , among the respondents . however , the medicine knowledge scores were significantly ( p<0.05 ) associated with holding a certificate in health sciences , years of experience , and the place of practice of the medicine vendors.conclusion:the present study demonstrated that self - medication practice was high and inadequate medicine knowledge existed among respondents .

ectopic thyroid is an uncommon embryological abnormality characterized by the presence of thyroid tissue in a site other than its usual pretracheal location [ 1 , 2 ] . of all ectopic thyroids 90% are found to be lingual it is a rare congenital anomaly appearing with prevalence of 1 : 100.000 . patients with lingual thyroid tissue usually present with symptoms such as dysphagia , choking , hemorrhage , and dyspnea and occasionally life - threatening airway obstruction . in addition , malignant transformation of the lingual thyroid has been reported , albeit rarely . for patients with obstructive symptoms , lingual thyroid is estimated to occur in 0.2 per cent of normal children , being more common in females and on the left side of the thyroid gland . in this case report , a child of eight years old came to our department with dysphagia and foreign body sensation in the throat . after examination , the patient presented a solid , spherical mass with 2 cm of diameter , covered with intact mucosa , located at the base of the tongue . examination of neck did not reveal cervical lymphadenopathy , the past medical history of the patient was insignificant , and she was not currently taking any medication . thyroid scintigraphy with tc and ultrasound were performed , which confirmed oval mass in the base of tongue , suggestive of ectopic thyroid tissue ( figures 1 and 2 ) . laryngoscopy was performed and showed the presence of a mass at the base of the tongue with displacement of the uvula . thyroid hormone tests showed elevated thyroid - stimulating hormone ( tsh = 10.79 ( 0.44.2 ) ) concentrations and normal t4 concentrations ( t4 = 0.81 ( 0.611.48 ) ) . from the clinical and imaging findings four months later a new evaluation was performed and there were no changes in the tsh levels . so , other exams were requested ( anti - tpo antibodies 4 < 3.0 ( normal value < 9.0 ) ; anti - thyroglobulin antibodies = 0.3 ( normal value < 4.1 ) ) to rule out the association between hashimoto 's thyroiditis and ectopic thyroid ; however these tests showed normal values . we observed no changes in the tsh after four months , showed in table 1 . then the dose was increased again for 75 mcg / day . after four months the patient remains with thyroid hormone tests in normal range , and there are clinical improvement of the symptoms of dysphagia and foreign body sensation in the throat . upon examination , there were no finds of a solid mass located at the base of the tongue . therefore a surgical excision of the mass was not required ; thus a follow - up protocol was established . clinically , lingual thyroid is presented as a mass at the base of the tongue , pink and firm . the most important diagnostic tool is thyroid scan with technetium tc-99 m sodium ; computerized tomography and magnetic resonance imaging may help in defining the extension and location of the ectopic thyroid gland . in this case report the diagnostic was obtained with aid of thyroid scintigraphy , ultrasound , and laryngoscopy . literature search showed only a few cases of lingual thyroid with hyperthyroidism and all of them were treated with a combination of antithyroid drugs , steroids , and surgery . the case presents a subclinical hypothyroidism that receives treatment of regularization of tsh levels with levothyroxine sodium successfully . the clinical presentation of lingual thyroid could be classified into two groups according to the appearance of the symptoms . in the first group of infants and young children , whose lingual thyroid is detected via routine screening may suffer from failure to thrive and mental retardation ; or even severe respiratory distress and requiring emergency care [ 8 , 9 ] . other cases may present with onset of slowly progressing dysphagia and symptoms of oropharyngeal obstruction before or during puberty . this occurs as a response to the increased demand for thyroid hormone in these hypermetabolic states . treatment of a lingual thyroid depends on the sex and age of the patient as well as on the severity of the symptoms and the associated ulceration and hemorrhage . patients with mild symptoms can be treated successfully by medical suppression , as presented in this paper . the treatment with lt4 could result in partial involution of lingual thyroid volume . benign or malignant neoplastic changes were sometimes described in ectopic thyroid tissue . concerning these considerations , patients who are symptomatic need different therapeutic approaches depending on the localization of the gland , type of symptoms , malignancy of symptoms , and functional status . the suppressive drug therapy based on suppressive dose of levothyroxine depends on the ectopic gland size . this kind of approach is a good option for patients who are not suitable for surgery and have only obstructive symptoms . the patients who present a slow and progressive enlargement of the mass can receive this treatment and postpone the surgical procedure . drug therapy based on levothyroxine has been suggested to prevent malignant transformation of the ectopic gland and to prevent developing hypothyroidism . in our case report the time required to regularize the levels of tsh was longer than usual ; it could be related to time required to schedule new appointments and performed blood tests , once the patient was assisted in a public institution with a great workload in most hospitals and places . patients complaining of dysphagia should be evaluated to check whether there is a presence of ectopic thyroid . the presence of mass or abnormality in this area could be a sign of lingual thyroid ectopic , once this is the most affected region , and this place is the embryological origin of the thyroid . this case report highlights the management of this condition with drug therapy based on suppressive dose of levothyroxine in a child .

lingual thyroid gland is a rare clinical entity . the presence of an ectopic thyroid gland located at the base of the tongue may be presented with symptoms like dysphagia , dysphonia , and upper airway obstruction . we are introducing a case of an 8-year - old girl who had lingual thyroid that presented dysphagia and foreign body sensation in the throat . the diagnostic was reached with clinical examination , thyroid scintigraphy with tc99 m and ultrasound . a laryngoscopy was performed which confirmed a spherical mass at base of tongue . investigation should include thyroid function tests . in this case we observed subclinical hypothyroidism . there are different types of surgical approaches for the treatment of this condition ; however , the treatment with levothyroxine sodium allowed the stabilization of tsh levels and clinical improvement of symptoms in a follow - up of 2 years .

traumatic brain injury ( tbi ) is a major public health problem , accounting for a high morbidity and mortality rates in children and youth . the national center for injury prevention and control estimates that over 510,000 traumatic brain injury ( tbi ) cases occur annually in children 014 years of age in the united states . ten percent of tbi is classified as severe and associated with a mortality rate of approximately 50% . while cardiac dysfunction after brain death has been described in a variety of brain injury paradigms , it has not been well described after tbi in the pediatric population , although the pathophysiology may be similar . as most neurologic causes of brain death in the pediatric population tend to occur after trauma , patients with severe tbi and brain death make a large proportion of pediatric patients presenting for possible organ donation . to understand the association between tbi - induced brain death and cardiac function , we examined the incidence of cardiac dysfunction among severe pediatric tbi patients , both with and without a diagnosis of brain death . we conducted a retrospective cohort study among pediatric patients with severe tbi , both with and without a diagnosis of brain death , who underwent echocardiography during the first 2 weeks after tbi between 2003 and 2011 . we compared cardiac dysfunction among patients who were diagnosed with brain death , as compared with patients who were not diagnosed with brain death . the study was performed at harborview medical center ( hmc ) , a 413-bed county medical center in seattle , wa , affiliated with the university of washington and is the only level 1 trauma center in the northwest region of the united states . pediatric severe tbi [ defined as admission glasgow coma scale ( gcs ) 8 or head abbreviated injury scale ( ais ) > 3 ] patients who underwent echocardiography during the first 2 weeks after tbi between 2003 and 2011 were examined . eligibility criteria included : ( 1 ) age < 18 years ; ( 2 ) discharge diagnosis of tbi ( icd 9 codes 800 - 801.9 , 803 - 804.9 or 850 - 854.1 ) , ( 3 ) presence of transthoracic or transesophageal echocardiogram report in the medical record within 14 days of tbi admission , and ( 4 ) no documentation of cardiac arrest or cardiopulmonary resuscitation . data sources were the hmc trauma registry , hmc billing data , and hmc electronic medical records . a list of eligible patients were identified from the hmc trauma registry ( n = 1,413 ) and linked to the hmc billing data source to yield a list of patients with severe tbi who underwent echocardiograms ( n = 47 ) . fifteen patients were excluded because 1 ) echocardiogram coding was inaccurate ( n = 4 ) , and 2 ) echocardiograms were performed beyond 14 days of tbi admission ( n = 11 ) . during the study period , patients were resuscitated according to institutional practice . for severe tbi patients , the 2003 infant , children and adolescents brain trauma foundation guidelines ( 14 ) were followed including : intracranial pressure monitoring , maintaining intracranial pressure ( intracranial pressure ) < 20 mmhg , maintaining a minimum cerebral perfusion pressure ( cpp ) 4050 mm hg , avoiding prophylactic severe hyperventilation to a paco2 < 30 mm hg , keeping sao2 > 90% , and using cooling / warming blankets or intravascular cooling devices when necessary for maintenance of normothermia . practices involving requests for echocardiogram were not standardized during the time of this study and echocardiograms were requested at the discretion of the primary attending intensivist . the main outcome measured was the presence of cardiac dysfunction based on echocardiogram findings , defined as the presence or absence of abnormal echocardiogram findings ( ejection fraction [ ef ] < 50% or regional wall motion abnormality [ rwma ] grades 1 = normal , 2 = mild hypokinesis , 3 = severe hypokinesis , 4 = akinesis and 5 = dyskinesis ) . statistical analyses were performed by statistical package for the social sciences software ( spss19.0 , chicago , illinois ) and stata 12.0 ( college station , texas ) software . descriptive statistics were used to describe clinical characteristics , echocardiogram findings , computed tomography scan defined tbi lesions , and mortality ; data are presented as median and standard deviation for non - parametric variables . a student 's t - test was used to examine differences between normally distributed continuous variables , a mann - whitney u test was used to test for differences in these non - parametric continuous data , and chi - squared analysis was used to test for differences between categorical variables . univariate logistic regression was performed to calculate odds ratios and 95% confidence intervals . due to multiple testing , a significant p value of 0.025 was set , using a bonferroni correction . the study was performed at harborview medical center ( hmc ) , a 413-bed county medical center in seattle , wa , affiliated with the university of washington and is the only level 1 trauma center in the northwest region of the united states . pediatric severe tbi [ defined as admission glasgow coma scale ( gcs ) 8 or head abbreviated injury scale ( ais ) > 3 ] patients who underwent echocardiography during the first 2 weeks after tbi between 2003 and 2011 were examined . eligibility criteria included : ( 1 ) age < 18 years ; ( 2 ) discharge diagnosis of tbi ( icd 9 codes 800 - 801.9 , 803 - 804.9 or 850 - 854.1 ) , ( 3 ) presence of transthoracic or transesophageal echocardiogram report in the medical record within 14 days of tbi admission , and ( 4 ) no documentation of cardiac arrest or cardiopulmonary resuscitation . data sources were the hmc trauma registry , hmc billing data , and hmc electronic medical records . a list of eligible patients were identified from the hmc trauma registry ( n = 1,413 ) and linked to the hmc billing data source to yield a list of patients with severe tbi who underwent echocardiograms ( n = 47 ) . fifteen patients were excluded because 1 ) echocardiogram coding was inaccurate ( n = 4 ) , and 2 ) echocardiograms were performed beyond 14 days of tbi admission ( n = 11 ) . during the study period , patients were resuscitated according to institutional practice . for severe tbi patients , the 2003 infant , children and adolescents brain trauma foundation guidelines ( 14 ) were followed including : intracranial pressure monitoring , maintaining intracranial pressure ( intracranial pressure ) < 20 mmhg , maintaining a minimum cerebral perfusion pressure ( cpp ) 4050 mm hg , avoiding prophylactic severe hyperventilation to a paco2 < 30 mm hg , keeping sao2 > 90% , and using cooling / warming blankets or intravascular cooling devices when necessary for maintenance of normothermia . practices involving requests for echocardiogram were not standardized during the time of this study and echocardiograms were requested at the discretion of the primary attending intensivist . the main outcome measured was the presence of cardiac dysfunction based on echocardiogram findings , defined as the presence or absence of abnormal echocardiogram findings ( ejection fraction [ ef ] < 50% or regional wall motion abnormality [ rwma ] grades 1 = normal , 2 = mild hypokinesis , 3 = severe hypokinesis , 4 = akinesis and 5 = dyskinesis ) . statistical analyses were performed by statistical package for the social sciences software ( spss19.0 , chicago , illinois ) and stata 12.0 ( college station , texas ) software . descriptive statistics were used to describe clinical characteristics , echocardiogram findings , computed tomography scan defined tbi lesions , and mortality ; data are presented as median and standard deviation for non - parametric variables . a student 's t - test was used to examine differences between normally distributed continuous variables , a mann - whitney u test was used to test for differences in these non - parametric continuous data , and chi - squared analysis was used to test for differences between categorical variables . univariate logistic regression was performed to calculate odds ratios and 95% confidence intervals . due to multiple testing , a significant p value of 0.025 was set , using a bonferroni correction . thirty - two children with severe tbi underwent echocardiography within 14 days of admission ; 10 ( 31.2% ) of these patients were declared brain dead secondary to their primary neurologic event , and none of the patients suffered a cardiac arrest and subsequent cardiopulmonary resuscitation . patients had a median of age of 16 years and were predominantly male ( 75% ) . differences were not seen in clinical characteristics between severe tbi patients who were and were not declared brain dead [ table 1 ] . clinical characteristics and early echocardiogram findings of pediatric patients with severe traumatic brain injury ( n=22 ) and brain death patients ( n=10 ) in the group of severe pediatric tbi without brain death , associated injuries included : orthopedic ( 14 ; 63.6% ) , chest ( 12 ; 54.5% ) , abdominal ( 6 ; 27.3% ) , and isolated tbi ( 6 ; 27.3% ) . in the group of severe pediatric tbi with brain death , associated injuries included : orthopedic ( 2 ; 20% ) , chest ( 3 ; 30% ) , abdominal ( 2 ; 20% ) , and isolated tbi ( 5 ; 50% ) . no significant difference was observed in the proportion of severe pediatric tbi patients with or without brain death who had chest injuries ( p = 0.21 ) . in all , 32 ( 2.3% ) of 1,413 pediatric tbi patients underwent echocardiogram evaluation . median time to echocardiogram for the entire cohort of 32 patients was 3 days ( 113 days ) after tbi . of the 32 patients , 28 ( 87.5% ) patients had a transthoracic echocardiogram ( tte ) performed , 2 ( 6.3% ) patients underwent only transesophageal echocardiography ( tee ) , and 2 ( 6.3% ) patients underwent tee in addition to tte . table 2 shows the early echocardiogram findings from severe pediatric tbi patients with and without brain death . four ( 40% ) of 10 pediatric tbi patients who were brain dead had low ef ( 39 + 10% ) and rwma ( 1 mild hypokinesis , 2 severe hypokinesis and 1 akinesis ) . one ( 4.5% ) of 22 severe pediatric tbi patients without brain death had a low ef . three ( 13.6% ) of 22 severe pediatric tbi patients without brain death had rwma ( 2 mild hypokinesis and 1 severe hypokinesis ) . patients with severe tbi and brain death had a higher incidence of low ef than non - brain dead patients ( or 14 , 95% ci 1.31150.02 , p = 0.024 ) but no higher incidence of rwma ( or 4.2 , 95% ci 0.7324.44 , p = 0.165 ) . early echocardiogram findings in final sample of severe pediatric traumatic brain injury without brain death ( n=22 ) and tbi with brain death ( n=10 ) thirty - two children with severe tbi underwent echocardiography within 14 days of admission ; 10 ( 31.2% ) of these patients were declared brain dead secondary to their primary neurologic event , and none of the patients suffered a cardiac arrest and subsequent cardiopulmonary resuscitation . patients had a median of age of 16 years and were predominantly male ( 75% ) . differences were not seen in clinical characteristics between severe tbi patients who were and were not declared brain dead [ table 1 ] . clinical characteristics and early echocardiogram findings of pediatric patients with severe traumatic brain injury ( n=22 ) and brain death patients ( n=10 ) in the group of severe pediatric tbi without brain death , associated injuries included : orthopedic ( 14 ; 63.6% ) , chest ( 12 ; 54.5% ) , abdominal ( 6 ; 27.3% ) , and isolated tbi ( 6 ; 27.3% ) . in the group of severe pediatric tbi with brain death , associated injuries included : orthopedic ( 2 ; 20% ) , chest ( 3 ; 30% ) , abdominal ( 2 ; 20% ) , and isolated tbi ( 5 ; 50% ) . no significant difference was observed in the proportion of severe pediatric tbi patients with or without brain death who had chest injuries ( p = 0.21 ) . in all , 32 ( 2.3% ) of 1,413 pediatric tbi patients underwent echocardiogram evaluation . median time to echocardiogram for the entire cohort of 32 patients was 3 days ( 113 days ) after tbi . of the 32 patients , 28 ( 87.5% ) patients had a transthoracic echocardiogram ( tte ) performed , 2 ( 6.3% ) patients underwent only transesophageal echocardiography ( tee ) , and 2 ( 6.3% ) patients underwent tee in addition to tte . table 2 shows the early echocardiogram findings from severe pediatric tbi patients with and without brain death . four ( 40% ) of 10 pediatric tbi patients who were brain dead had low ef ( 39 + 10% ) and rwma ( 1 mild hypokinesis , 2 severe hypokinesis and 1 akinesis ) . one ( 4.5% ) of 22 severe pediatric tbi patients without brain death had a low ef . three ( 13.6% ) of 22 severe pediatric tbi patients without brain death had rwma ( 2 mild hypokinesis and 1 severe hypokinesis ) . patients with severe tbi and brain death had a higher incidence of low ef than non - brain dead patients ( or 14 , 95% ci 1.31150.02 , p = 0.024 ) but no higher incidence of rwma ( or 4.2 , 95% ci 0.7324.44 , p = 0.165 ) . early echocardiogram findings in final sample of severe pediatric traumatic brain injury without brain death ( n=22 ) and tbi with brain death ( n=10 ) the main finding of our study is that cardiac dysfunction was more common in pediatric patients with tbi - induced brain death as compared to tbi patients without brain death . this finding may have clinical significance for the pediatric cardiac donor population , especially because the incidence of cardiac dysfunction in this population is similar to the adult brain death population , where echocardiographic abnormalities are found in as many as 42% of patients with brain death , causing the loss of potential heart donors for transplantation . physiologic stress is hypothesized as a mechanism for cardiac dysfunction , including in brain dead patients . our findings show that low ef occurred frequently in this sample of severe pediatric tbi patients , and our incidence is consistent with the range of 9%30% , which reported in adults with subarachnoid hemorrhage , and 42% reported after brain death . this similarity suggests that the observed cardiac dysfunction in this sample represents stress related cardiomyopathy , a phenomenon which has never been reported in pediatric tbi but is well recognized in adult neurological conditions . myocardial dysfunction after brain death has not only been reported as common , but may also represent a common pathway of an end - stage cardiac injury , which may have multiple etiologies , including tbi . three main theories explaining the pathogenesis of brain injury induced cardiac dysfunction include multi - vessel coronary spasm causing ischemia , microvascular dysfunction , and the catecholamine hypothesis . catecholamine - induced cardiac injury is one theory explaining the underlying cause of cardiac dysfunction in adult tbi patients ; plasma catecholamine concentrations ( noradrenaline , adrenaline and 3-methoxy-4-hydroxy - phenylethylene glycol ) are elevated one day after tbi . using sah as the best - studied model of cardiac dysfunction after neurologic injury , we observe that adult patients with sah have an increase in plasma norepinephrine within 48 hours after the hemorrhage , which persists during the first week and normalizes by six months . potential pediatric heart donors have been shown previously to have a high prevalence of left ventricular systolic and diastolic dysfunction . the ventricular dysfunction is likely to be worse in younger patients because of a greater density of -adrenergic receptors within myocardium in the setting of a sudden increase in sympathetic activity . interestingly , a longer interval between brain death and donor cardiectomy improved rejection - free survival in the recipient , presumably secondary to hemodynamic optimization of the donor prior to transplant . first , this was a single - center retrospective study , and the sample size is small . however , this was a preliminary study to determine initial impressions of the incidence of cardiac dysfunction in pediatric tbi . second , echocardiogram timing and utilization were driven by clinical care needs and not by study design , thus creating a possible selection bias toward patients with a greater degree of hemodynamic compromise ; furthermore , because pre - injury echocardiograms were not collected , the possibility of reverse causality can not be fully excluded . third , the majority of patients in our study were adolescents , and the generalizability of our findings to a younger patient population may be limited . however , despite the study limitations , our study documents cardiac dysfunction , which has the potential to impact both hemodynamic management of the pediatric tbi patient with brain death and transplantation outcomes from donor pediatric tbi patients with brain death . in summary , to our knowledge , our preliminary study is the first to document an effect of severe tbi on cardiac function by echocardiogram early after injury in children . brain death , representing the most severe form of tbi , demonstrated the highest rate of cardiac dysfunction . because of the small sample size and limitations on generalizability , hopefully our data can open the door for larger prospective studies evaluating cardiac dysfunction after pediatric tbi with and without brain death .

background : cardiac dysfunction after brain death has been described in a variety of brain injury paradigms but is not well understood after severe pediatric traumatic brain injury ( tbi ) . cardiac dysfunction may have implications for organ donation in this patient population.materials and methods : we conducted a retrospective cohort study of pediatric patients with severe tbi , both with and without a diagnosis of brain death , who underwent echocardiography during the first 2 weeks after tbi , between the period of 20032011 . we examined cardiac dysfunction in patients with and without a diagnosis of brain death.results:in all , 32 ( 2.3% ) of 1,413 severe pediatric tbi patients underwent echocardiogram evaluation . most patients had head abbreviated injury score 5 ( range 26 ) and subdural hematoma ( 34.4% ) . ten patients with tbi had brain death compared with 22 severe tbi patients who did not have brain death . four ( 40% ) of 10 pediatric tbi patients with brain death had a low ejection fraction ( ef ) compared with 1 ( 4.5% ) of 22 pediatric tbi patients without brain death who had low ef ( or = 14 , p = 0.024).conclusions : the incidence of cardiac dysfunction is higher among pediatric severe tbi patients with a diagnosis of brain death , as compared to patients without brain death . this finding may have implications for cardiac organ donation from this population and deserves further study .

the cystic fibrosis transmembrane conductance regulator ( cftr ) is a phosphorylation and nucleotide regulated chloride ( cl ) channel . the incidence of cystic fibrosis ( cf ) ( mim # 219700 ) is very varies in different areas for example in australia is 1:248 000 live births . however , the incidence of cf in many studies was reported 1:2500 live births and 0.4 in carriers . in us cf disease caused by mutations in cftr gene that localized on the long arm of chromosome number 7 ( 7q31 , 2 ) . this gene has 27 exon and expressed protein by this gene had a 1480 amino acid , which encodes a cyclic adenosine monophosphate - dependent cl channel that is found at the apical membrane of epithelial cells , including respiratory epithelia and submucosal glands , exocrine pancreas , liver , sweat ducts , and the reproductive tract . cftr is a member of the adenosine triphosphate - binding cassette membrane transporter superfamily that includes proteins such as the multiple drug resistance protein and bacterial periplasmic permeases . a 6-month - old girl with weakness and meconium ileus referred to the pediatric clinic in ilam , in the west of iran [ figure 1 ] . patient 's skin was dark , and their parents stated that the child had a weight loss . there are no evidence for edema , hypoproteinemia , liver and gallbladder problems , rectal prolapsed or finger clubbing [ table 2 ] . geographic place of patient resident symptoms and clinical property of patient the pediatric specialist requested sweet test . by using macroduct method ( wescor , logan , ut ) , the concentration of ( cl ) and sodium in patient 's sweat was 130 - 135 mmol / l and 125 - 128 mmol / l , respectively . a sample of 4 ml blood was taken for desoxyribonucleic acid ( dna ) extraction . all samples were sequenced in both the forward and reverse direction using the same primers used in the polymerase chain reaction ( pcr ) reactions . the conditions used for amplification were as follows : 5 min at 94c ; 30 cycles of 30 s at 94c followed by 30 s at annealing temperature , and 30 s at 72c ; and a final extension step for 5 min at 72c . the primers used in this study were synthesized by operon ( metabion , germany ) . the sequencing reactions were vacuums purified with the qiaquick pcr purification kit and analyzed on an abi 3130 gene analyzer ( applied biosystems , usa ) . the sequences were compared with the wild - type cftr nucleotide sequence using dna sequencing analysis v5.2 software ( applied biosystems , usa ) . after comprehensive genetic analysis of exon 10 , there was a mutation at the position c. 1499g > c to be heterozygous ( gly > ala ) [ figures 2 and 3 , table 3 ] . the child has also a f508 mutation as a heterozygous [ figure 4 and table 3 ] . the heterozygote c.1499 mutation ( g > c ) . the sequence resulted from exon 10 sequencing which heterozygote cd500 mutation has created in 1499 place identification of mutation in exon 10 of cystic fibrosis transmembrane conductance regulator gene list of mutations in patient the sequence resulted from sequencing process which f508 mutation is seen as a heterozygote type . in this sequence , disruption in arrangement of the peaks demonstrates that one of the transmitted hereditary genes from parents to the child had been deficit the investigation to better recognition of cftr mutations not only is useful in clinical recognition of cf , but it further strengthens the available genetical knowledge . these findings are also beneficent in pre - marriage consultant protocols , and they lead to easier carrier identification . the results in this report and other related papers indicate a high rate of heterogeneity in cftr mutations . up to 2012 , more than 1800 ( cystic fibrosis mutation database , http://www.genet.sickkids.on.ca / cftr/. ) mutations in the cftr gene were detected and reported in caucasian or- non - caucasian . the type and distribution of mutations varies widely between different countries and/or ethnic groups . according to the geographical and ethnic origin of patients , many of these mutations are rare , and some of them have been reported in only one family . a patient with a sign of cf with consanguine parents ( degree of 3 ) in ilam province ( west of iran ) she was a 6 month baby and found with acute respiratory disorders symptoms , growth retardation and abnormal stool . the mutation of c. 1499g > c is being reported for the first time in this case report . the patient had symptoms of meconium ileus and gastrointestinal obstruction and was found to be conveying heterozygote f508 mutation . the sign of liver disorders , hypoproteinemia , rectal prolapse and mellitus diabetes were not found in the patient . in middle east populations , there is no complete information about the cf disease and limited studies have been conducted . hence , it seems that ethnic and tribal relationships in these areas have led to frequent native mutations . for prevention of increase cf cases , many of preventive programs like establishment of screening tests for diagnostic of carrier statues must be done special in developing countries like iran . a patient with a sign of cf with consanguine parents ( degree of 3 ) in ilam province ( west of iran ) [ figure 1 ] has been studied . she was a 6 month baby and found with acute respiratory disorders symptoms , growth retardation and abnormal stool . the mutation of c. 1499g > c is being reported for the first time in this case report . the patient had symptoms of meconium ileus and gastrointestinal obstruction and was found to be conveying heterozygote f508 mutation . the sign of liver disorders , hypoproteinemia , rectal prolapse and mellitus diabetes were not found in the patient . in middle east populations , there is no complete information about the cf disease and limited studies have been conducted . hence , it seems that ethnic and tribal relationships in these areas have led to frequent native mutations . for prevention of increase cf cases , many of preventive programs like establishment of screening tests for diagnostic of carrier statues must be done special in developing countries like iran . a patient with a sign of cf with consanguine parents ( degree of 3 ) in ilam province ( west of iran ) [ figure 1 ] has been studied . she was a 6 month baby and found with acute respiratory disorders symptoms , growth retardation and abnormal stool . the mutation of c. 1499g > c is being reported for the first time in this case report . the patient had symptoms of meconium ileus and gastrointestinal obstruction and was found to be conveying heterozygote f508 mutation . the sign of liver disorders , hypoproteinemia , rectal prolapse and mellitus diabetes were not found in the patient . in middle east populations , there is no complete information about the cf disease and limited studies have been conducted . hence , it seems that ethnic and tribal relationships in these areas have led to frequent native mutations . for prevention of increase cf cases , many of preventive programs like establishment of screening tests for diagnostic of carrier statues must be done special in developing countries like iran .

so far , more than 1800 mutations identified in the cystic fibrosis transmembrane conductance regulator ( cftr ) gene . in this case report , we presented first report of c. 1499g > c mutation in a 6-month - old girl with cystic fibrosis ( cf ) diagnosis . a 6-month - old girl with weakness and meconium ileus referred to the pediatric clinic in ilam , in the west of iran . patient 's skin was dark and suffered from bronchiectasis . the sweat test was performed , and the concentration of chloride and sodium in patient 's sweat was 130 - 135 mmol / l and 125 - 128 mmol / l , respectively . the exon 10 mutation analysis of a cf patient was performed . cftr mutation analysis revealed the identification of 2 mutations in patient , the mutations were p.f508del ( f508 ) and c. 1499g > c ( cd500 ) , respectively . the mutation c. 1499g > c ( cd500 ) were found for the first time in the world . assessing this mutation in future study and genetic investigation is recommended .

mbius syndrome is a heterogeneous congenital disorder that is linked to bilateral palsies of the cranial nerves vi and vii , resulting in congenital facial paralysis sometimes associated with impaired ocular abduction . we present the case of a 44-year - old woman with mbius syndrome and inferior recurrent keratitis secondary to scleral show in both eyes . we decided to use a cartilage graft from the ear in the inferior eyelid to avoid eyelid retraction and scleral show . their treatment must be individualized , depending on their age , clinical examination and symptoms . mbius syndrome is a heterogeneous congenital disorder that is linked to bilateral palsies of the cranial nerves vi and vii [ 1 , 2 ] . from an ophthalmological perspective , these patients present symmetrical facial diplegia with variable patterns of horizontal gaze paresis and facial paralysis [ 3 , 4 ] , which affect palpebral dynamics in adult life . we present the case of a 44-year - old woman with mbius syndrome and congenital craniofacial cleft . during her childhood , she had undergone multiple surgeries in the departments of traumatology , plastic surgery and maxillofacial surgery . the patient was being monitored due to her eye problems derived from the facial paralysis . four years ago , she had undergone a permanent partial bilateral tarsorrhaphy that improved her eyelid closure problems . however , at the last checkups , the lower eyelids had retracted again , and she presented recurrent lower keratitis caused by a scleral exposure of 12 mm in both eyes , in spite of a good bell 's phenomenon and an intensive treatment with lubricants ( fig . 1 ) . we decided to introduce a graft for the lower eyelid in order to alleviate the retraction and reduce the scleral exposure . in view of the fact that the patient had undergone surgery of the palatal area on several occasions due to her craniofacial malformations ( fig . we performed an incision at the end of the lower conjunctival sac , we separated the retractor muscles from the tarsal plate , and we disinserted the capsulopalpebral ligament , so that the tarsal plate was left free and completely exposed . a 6 20 mm section of auricular cartilage was extracted and its upper border was sewn into the lower edge of the lower tarsal plate with 7 - 0 vicryl . the lower edge of the graft was sutured to the retractors , and the incision in the conjunctival sac was also sutured with 7 - 0 vicryl . two weeks after the procedure , we observed that the lower eyelid retraction had improved notably . the patient did not present scleral exposure , and the eyelid closure had also improved ( fig . , the patient presented recurrent type iii keratitis despite intensive treatment with lubricants ; however , after the graft had been inserted , we only observed type i keratitis sporadically and always when she had forgotten to apply tear supplements . mbius syndrome is a rare complex of malformations which was first described by von graefe in 1880 . its etiology is unknown , although some components have been described , either genetic , vascular or related with the intake of some drugs like misoprostol during pregnancy . it is characterized by bilateral congenital paralysis of the cranial nerve pairs vi and vii , and it is accompanied by symmetrical facial diplegia with variable patterns of horizontal gaze paresis . facial paralysis and lagophthalmos have been documented in as many as 80% of cases with mbius syndrome . during childhood , the phenomena derived from facial paralysis are not very noticeable . lagophthalmos or other alterations of the eyelid dynamics are rare before 20 years of age , but facial amimia of the patients is common in the patients , especially in expressions like crying or smiles . in patients younger than 7 years of age , a certain function of the facial muscles can be achieved with the transference of the temporal muscle or the latissimus dorsi , or the gracilis muscle , which can be reinnervated with the mandibular branch of the trigeminal nerve or with a ramification of the spinal nerve [ 6 , 7 , 8 ] . however , patients are usually admitted as adults with no previous treatment , when the effects of facial paralysis are more evident . the main reasons for consultation are problems caused by the appearance of exposure keratitis due to poor eyelid closure during the night . in this case , the main therapeutic measure is a medical approach with lubricants and hygienic measures in order to prevent the evaporation of tears . when the alterations in the eyelid dynamics increase , patients suffer a retraction of the lower eyelid with scleral exposure . this aggravates the recurrence and severity of keratitis , which in some cases causes extremely severe corneal ulcers that increase the risk of neurotrophy and can lead to corneal perforation and endophthalmitis . it is also common to see ectropion of the lower eyelid and eyebrow ptosis . in these cases , surgery is required , and the approach will depend on the severity of the facial paralysis , the exploration of the dynamic and static condition of the eyelids and the symptoms of the patient . static approaches narrow the opening of the eyelid either horizontally ( tarsorrhaphy ) or vertically ( elevation of the lower eyelid via desinsertion of the retractors , placing of spacers and/or a facial lifting ) . dynamic approaches improve the eyelid closure , and the most common procedure is a gold weight implant . to transfer a muscle , either vascularized or nonvascularized , such as the platysma muscle , towards the free edge of the eyelid and to combine it with a gold weight implant the authors report no conflicts of interest . the authors alone are responsible for the content and writing of the paper .

introductionmbius syndrome is a heterogeneous congenital disorder that is linked to bilateral palsies of the cranial nerves vi and vii , resulting in congenital facial paralysis sometimes associated with impaired ocular abduction.case reportwe present the case of a 44-year - old woman with mbius syndrome and inferior recurrent keratitis secondary to scleral show in both eyes . we decided to use a cartilage graft from the ear in the inferior eyelid to avoid eyelid retraction and scleral show.discussionpatients with mbius syndrome have a severe dysfunction of their facial mimic . their treatment must be individualized , depending on their age , clinical examination and symptoms .

the media was prepared using 10% fbs ( 16140 - 071- gibco ) in dmem / f-12 ( 1x ) , liquid 1:1 ( 10565 - 042- invitrogen ) with 0.6 % agar solution purified , granulated ( 1.01614.1000- emd ) . for the preparation of media 5ml of fbs ( 16140 - 071- gibco ) was added to 45ml of dmem / f-12 ( 1x ) , liquid 1:1 ( 10565 - 042- invitrogen ) making final volume of 50ml . the agar solution was cool down to 37c temperature by keeping it into the water bath . we used equal volume of media from step 1.2 and 1.3 and prepared stock solution . the ratio of 0.6 % agar ( 0.5ml ) and 10% fbs - d - mem / f-12 media ( 0.5ml ) was 1:1 , making the total volume of 1 ml in each well of 6 well plates . glioblastoma multiforme ( gbm ) tissues were received immediately after the surgery from department of pathology . the tissue was transferred to patri - plate using forceps carefully and washed with ice - cold 5ml 1xpbs for 3 times . serial sections of the specimens were cut from the dissected samples to create tumor blocks ( approximate 10 mm in diameter ) with a surgical blade ( feather-2976#10 ) and forcep ( fisher scientific ) . tumor blocks were then injected with either dmso ( 5% ) or tmz ( 2.5 nm ) and incubated for 16 hours at 37 c in humidified air containing 5% co2 . ( 5% ) or tmz was injected 3 times in tumor blocks at different places for consistent treatment . the drug was injected using insulin syringe 1ml 0.37x12.7 mm 28g1/2 ( comfort point-26027 ) . after washing the blocks were fixed with 10ml of 10 % v / v formalin for 24 hours ( ricca chemical company ) in 50 ml tubes ( basix-5539802 ) and processed for paraffin embedded 4m sections . the formalin fixed section was placed in beaker on the stir plate with " no heat " and processed through the following solutions for 30 minutes in each solution . 30% ethanol , 50% ethanol , 75% ethanol , 80% ethanol , 95% ethanol , 100% ethanol , and xylene . the tissue was blotted on paper towels and placed in melted paraffin ( 58 to 60 c , fisher paraplast paraffin ) for 30 minutes . the tissue was embedded in molds using surgipath blue ribbon paraffin , embedded tissue was cooled to room temperature . xylene for 3x5 minutes ( 3 times for 5 minutes ) , 100% ethanol for 3x5 minutes , 95% ethanol for 1x5 minutes , 70% ethanol for 1x5 minutes , rinse in running tap water for 3 minutes . antigen retrieval with heat induced epitope retrieval immerse slides in 1x citrate buffer ( thermo scientific - ap9003 - 500 ) diluted in distilled water in a glass beaker . boil for 15 minutes on a hot plate ( make sure sections don"t fall off the slide ) . cool the solution for 30 minutes at room temperature . rinse the slides with 1x pbs for 3x5 minutes . inhibition of internal peroxide immerse the slides in methanol ( fisher scientific - a-452 - 4 ) with 0.3% hydrogen peroxide ( fisher scientific- bp2633 - 500- diluted in distilled water ) in a glass beaker for 15 minutes . rinse in 1x pbs for 3x5 minutes . blocking cover the tissues with 10% normal goat serum . transfer the slides in a humid box and incubate for 1 hour at room temperature . primary antibody sections were incubated overnight at 4c with primary antibody at following concentrations : human specific caspase-3 ( 1:1,000 , r&d systems , af835 ) , ki67 ( 1:1 , dako , 15626 ) diluted with 1x pbs . secondary antibody reaction cover the tissues with 2 - 3 drops of hrp labeled secondary antibody ( envision systems ) . detection develop for the chromogen dab kit ( vector laboratories - sk-4100 ) for detection of primary antibody following the manufacturer 's protocol . counter staining immerse the slides with hematoxylin ( 10 - 15 seconds , make sure don"t overrun dab signal ) . dehydration immerse the slides in following order , 70% ethanol for 5 minutes , 95% ethanol for 5 minutes , 100 % ethanol for 3x5 minutes , xylene for 3x5 minutes . cover slip the slides with permount mounting reagent ( fisher scientific - sp-15 - 100 ) . images were taken using olympus fluorescence microscope ( dp-72).3.11 ) . in all experiments , specific labeling the patients underwent the first surgery without prior chemotherapies including temozolomide ( tmz ) . the t1-weighted image of mri with gadolinium enhancement in figure-1a demonstrated an enhanced tumor in the right frontal lobe before surgery . the surgical tissues were sent to the department of pathology for the clinical diagnosis purpose , and the remaining specimens were processed to the tissue procurement under the approved institutional review board ( irb ) protocol at the ohio state university medical center . hematoxylin and eosin ( h&e ) staining demonstrated the presence of necrosis , pseudopallisading cells , and microvascular proliferation ( figure 1-c ) . of note , tumor explants following incubation without tmz up to 48 hours maintained the cytoarchitecture of gbm .in contrast , with incubation for 72 hours or longer , we noticed the increased number of condensed nuclei in the specimens , suggesting some of the tumor cells starting to undergo apoptosis . as a result , tumor tissues following longer incubation contained patchy regions that lost tumor cells , which were preferentially observed at and around necrotic areas ( data not shown ) . in order to investigate if these tumor explant samples can reliably be utilized for the purpose of drug efficacy test , we tested the effect of tmz treatment a recent study indicated that intratumoral injection of tmz has more potent effect on growth control of malignant glioma than that of the systemic administration of this drug . following incubation for 16 hours , these explants were embedded with paraffin and serial sections were prepared for staining . immunohistochemistry of tmz - treated gbm tissues demonstrated a substantial reduction of ki-67-positive tumor cells in comparison with the control samples . in turn , immunohistochemistry with an apoptosis marker , caspase-3 , did not yield any significant difference between tmz - treated glioma specimens and the control samples ( figure-3 ) . treatment effect with tmz was further characterized by combining this explant assay together with either in vitro culture or flow cytometry . specifically , we sought to determine the effect on stem cell - like tumor cells ( scltc ) . therefore , following intratumoral treatment with tmz , we performed sphere forming assay and flow cytometry of these tumor explants . the tmz - treated specimens were dissociated into individual single cells and these single cells were seeded in a low density ( 1 cell per microliter or lower ) in 96-well plates in serum - free medium supplemented with bfgf and egf . in the control group , given that sphere formation is a property of scltc , alterations in the number of tumor spheres by a drug treatment indicates the effect on scltc . similarly , flow cytometry of the dissociated tumor explants with a cell surface marker , cd133 , was carried out to verify the effect on scltc . if scltc in heterogeneous tumor cells in gbm are selectively eradicated by a drug treatment , one might predict that flow cytometry should detect decrease of the cd133-positive fraction by treatment ( data not shown ) . figure-2b shows dissection of tumor block into 10 mm small pieces with the help surgical blade . immunohistochemistry of gbm tissue blocks injected with dmso or tmz with activated caspase-3 and ki67 . the media was prepared using 10% fbs ( 16140 - 071- gibco ) in dmem / f-12 ( 1x ) , liquid 1:1 ( 10565 - 042- invitrogen ) with 0.6 % agar solution purified , granulated ( 1.01614.1000- emd ) . for the preparation of media 5ml of fbs ( 16140 - 071- gibco ) was added to 45ml of dmem / f-12 ( 1x ) , liquid 1:1 ( 10565 - 042- invitrogen ) making final volume of 50ml . the agar solution was cool down to 37c temperature by keeping it into the water bath . we used equal volume of media from step 1.2 and 1.3 and prepared stock solution . the ratio of 0.6 % agar ( 0.5ml ) and 10% fbs - d - mem / f-12 media ( 0.5ml ) was 1:1 , making the total volume of 1 ml in each well of 6 well plates . glioblastoma multiforme ( gbm ) tissues were received immediately after the surgery from department of pathology . the tissue was transferred to patri - plate using forceps carefully and washed with ice - cold 5ml 1xpbs for 3 times . serial sections of the specimens were cut from the dissected samples to create tumor blocks ( approximate 10 mm in diameter ) with a surgical blade ( feather-2976#10 ) and forcep ( fisher scientific ) . tumor blocks were then injected with either dmso ( 5% ) or tmz ( 2.5 nm ) and incubated for 16 hours at 37 c in humidified air containing 5% co2 . 0.1ml of dmso ( 5% ) or tmz was injected 3 times in tumor blocks at different places for consistent treatment . the drug was injected using insulin syringe 1ml 0.37x12.7 mm 28g1/2 ( comfort point-26027 ) . after washing the blocks were fixed with 10ml of 10 % v / v formalin for 24 hours ( ricca chemical company ) in 50 ml tubes ( basix-5539802 ) and processed for paraffin embedded 4m sections . the formalin fixed section was placed in beaker on the stir plate with " no heat " and processed through the following solutions for 30 minutes in each solution . 30% ethanol , 50% ethanol , 75% ethanol , 80% ethanol , 95% ethanol , 100% ethanol , and xylene . the tissue was blotted on paper towels and placed in melted paraffin ( 58 to 60 c , fisher paraplast paraffin ) for 30 minutes . the tissue was embedded in molds using surgipath blue ribbon paraffin , embedded tissue was cooled to room temperature . xylene for 3x5 minutes ( 3 times for 5 minutes ) , 100% ethanol for 3x5 minutes , 95% ethanol for 1x5 minutes , 70% ethanol for 1x5 minutes , rinse in running tap water for 3 minutes . antigen retrieval with heat induced epitope retrieval immerse slides in 1x citrate buffer ( thermo scientific - ap9003 - 500 ) diluted in distilled water in a glass beaker . boil for 15 minutes on a hot plate ( make sure sections don"t fall off the slide ) . cool the solution for 30 minutes at room temperature . rinse the slides with 1x pbs for 3x5 minutes . inhibition of internal peroxide immerse the slides in methanol ( fisher scientific - a-452 - 4 ) with 0.3% hydrogen peroxide ( fisher scientific- bp2633 - 500- diluted in distilled water ) in a glass beaker for 15 minutes . rinse in 1x pbs for 3x5 minutes . blocking cover the tissues with 10% normal goat serum . transfer the slides in a humid box and incubate for 1 hour at room temperature . primary antibody sections were incubated overnight at 4c with primary antibody at following concentrations : human specific caspase-3 ( 1:1,000 , r&d systems , af835 ) , ki67 ( 1:1 , dako , 15626 ) diluted with 1x pbs . secondary antibody reaction cover the tissues with 2 - 3 drops of hrp labeled secondary antibody ( envision systems ) . put slides in a humid box and incubate for 1 hour at room temperature . detection develop for the chromogen dab kit ( vector laboratories - sk-4100 ) for detection of primary antibody following the manufacturer 's protocol . counter staining immerse the slides with hematoxylin ( 10 - 15 seconds , make sure don"t overrun dab signal ) dehydration immerse the slides in following order , 70% ethanol for 5 minutes , 95% ethanol for 5 minutes , 100 % ethanol for 3x5 minutes , xylene for 3x5 minutes . cover slip the slides with permount mounting reagent ( fisher scientific - sp-15 - 100 ) . images were taken using olympus fluorescence microscope ( dp-72).3.11 ) . in all experiments , specific labeling the patients underwent the first surgery without prior chemotherapies including temozolomide ( tmz ) . the t1-weighted image of mri with gadolinium enhancement in figure-1a demonstrated an enhanced tumor in the right frontal lobe before surgery . the surgical tissues were sent to the department of pathology for the clinical diagnosis purpose , and the remaining specimens were processed to the tissue procurement under the approved institutional review board ( irb ) protocol at the ohio state university medical center . hematoxylin and eosin ( h&e ) staining demonstrated the presence of necrosis , pseudopallisading cells , and microvascular proliferation ( figure 1-c ) . thus , these tumors were histopathologically diagnosed as gbm . of note , tumor explants following incubation without tmz up to 48 hours maintained the cytoarchitecture of gbm .in contrast , with incubation for 72 hours or longer , we noticed the increased number of condensed nuclei in the specimens , suggesting some of the tumor cells starting to undergo apoptosis . as a result , tumor tissues following longer incubation contained patchy regions that lost tumor cells , which were preferentially observed at and around necrotic areas ( data not shown ) . in order to investigate if these tumor explant samples can reliably be utilized for the purpose of drug efficacy test , we tested the effect of tmz treatment a recent study indicated that intratumoral injection of tmz has more potent effect on growth control of malignant glioma than that of the systemic administration of this drug . following incubation for 16 hours , these explants were embedded with paraffin and serial sections were prepared for staining . immunohistochemistry of tmz - treated gbm tissues demonstrated a substantial reduction of ki-67-positive tumor cells in comparison with the control samples . in turn , immunohistochemistry with an apoptosis marker , caspase-3 , did not yield any significant difference between tmz - treated glioma specimens and the control samples ( figure-3 ) . treatment effect with tmz was further characterized by combining this explant assay together with either in vitro culture or flow cytometry . specifically , we sought to determine the effect on stem cell - like tumor cells ( scltc ) . therefore , following intratumoral treatment with tmz , we performed sphere forming assay and flow cytometry of these tumor explants . the tmz - treated specimens were dissociated into individual single cells and these single cells were seeded in a low density ( 1 cell per microliter or lower ) in 96-well plates in serum - free medium supplemented with bfgf and egf . in the control group , given that sphere formation is a property of scltc , alterations in the number of tumor spheres by a drug treatment indicates the effect on scltc . similarly , flow cytometry of the dissociated tumor explants with a cell surface marker , cd133 , was carried out to verify the effect on scltc . if scltc in heterogeneous tumor cells in gbm are selectively eradicated by a drug treatment , one might predict that flow cytometry should detect decrease of the cd133-positive fraction by treatment ( data not shown ) . figure-2b shows dissection of tumor block into 10 mm small pieces with the help surgical blade . immunohistochemistry of gbm tissue blocks injected with dmso or tmz with activated caspase-3 and ki67 . there is a gap between the drug efficacy in the current pre - clinical experimental models and the efficacy in patients . more specifically , in the brain tumor research field , novel and reliable methods are required that would help filling the existing gap between the drug evaluation with the current methods and the efficacy in patients . the described assay in this study is an additional asset , if not a solution , to facilitate filling the discrepancy of the experimental data and outcome of affected patients . in vitro cell cultures preferentially expand certain tumor cell types regardless of the culture conditions , and represent only a subpopulation of the entire tumor . in addition , genetic and phenotypic transformation of the artificial cell expansion occurs and is inevitable with the long - term cell cultures . one of the major hurdles to treat malignant glioma is the heterogeneity of tumor cells , both within a single tumor and between tumor samples . therefore , selective enrichment of a certain population of tumor cells , regardless of one type or another , may not be an appropriate means to determine efficacy of any chemotherapeutic agents on heterogeneous tumor cells . any animal model of brain tumors derived from a subpopulation of tumor cells shed lights on drug efficacy on a subpopulation , but not the entire tumor . several limitations , on the other hand , still remain in this tumor explant method . malignant tumor cells are considered to acquire resistance to most , if not all , therapies over time , initial control of the short - term tumor cell growth may not be reflected by the long - term patient prognosis , as was recently reported with an anti - angiogenic agent , bevacizumab . thus , improvement of the protocol for this explant assay to preserve tissues for a longer period is required with further investigations . another question is a specific effect of a tested drug on scltc in the tumor . given that scltc are relatively resistant to the current therapies for malignant glioma , identification of novel anti - cancer drugs that potently eradicate scltc we currently seek to combine this explant assay with the subsequent in vitro experiments , such as neurosphere forming assay ( data not shown ) . we expect to learn more about the effects , if any , of a drug candidate on scltc through these combined assays . lastly , using small blocks of tumor samples may not reflect the entire tumor cell populations , as is the case with the conventional tumor cell lines or primary cultures from surgical specimens . these issues aside , preserving tumor stroma , including the vascular niche , is helpful in characterizing the environmental factors for tumor cells . therefore , this assay may have potential to evaluate any therapeutic strategies under a more physiologically relevant condition . here , we established an assay for drug efficacy testing with explants of surgical specimens of gbm . in fact , with the tested surgical specimens , we found that a direct injection of tmz reduces proliferation in the gbm samples . this tissue explant method is theoretically applicable to other solid cancers and provides asset to determine drug efficacy on a patient 's tumor , which will hopefully help us avoiding another failure in clinical trials for cancers .

the current therapies for malignant glioma have only palliative effect . for therapeutic development , one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients . thus , novel and reliable methods for evaluating drug efficacy are warranted in pre - clinical phase . in vitro culture of tumor tissues , including cell lines , has substantial phenotypic , genetic , and epigenetic alterations of cancer cells caused by artificial environment of cell culture , which may not reflect the biology of original tumors in situ . xenograft models with the immunodeficient mice also have limitations , i.e. , the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment . here , we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects . to validate this method , data with the current first - line chemotherapeutic agent , temozolomide ( tmz ) , are described . we used the freshly - removed surgical specimen of malignant glioma for our experiments . we performed intratumoral injection of tmz or other drug candidates , followed by incubation and analysis on surgical specimens . here , we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti - cancer therapies so that we may be able to overcome , at least , some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient 's tumor . this method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment .

expansion of the mid - palatal suture is an important part of the clinician s armamentarium in the correction of malocclusions . this procedure increases the posterior dentition width rapidly , which is followed by active bone formation in the mid - palatal suture.1,2 rapid maxillary expansion ( rme ) appliances show the best examples of true orthopaedics in that changes are produced primarily in the underlying structures and therefore are found to be more stable.35 however , clinical and histological studies have shown that relapse , microtrauma of the temporomandibular joint , microfractures at the mid - palatal suture and especially external root resorption are observed in rme treatment.6,7 the relapse phenomenon after rme is complex since the regulation of bone metabolism and stresses generated on mid - palatal and circum - maxillary sutures depend on many factors . although the reason for early relapse is not fully understood , velocity and quality of bone formation in the mid - palatal suture during and after expansion may affect the post - treatment relapse.1 it would be potentially beneficial therefore to accelerate bone formation in mid - palatal suture after expansion to prevent relapse of arch width and to shorten the retention period.1,2 the vitamin d metabolism plays an essential role in calcium and bone homeostasis.8 yamamoto9 reported that administration of 2-b-(3-hydroxypropoxy)-1a,25-dihydroxyvitamin d3 ( ed-71 ) , an analog of synthetic vitamin d3 , increased the bone mineral content at the lengthened callus . ed-71 lowers bone resorption without reducing bone formation in ovariectomized rats and in prednizolone - treated rats.10 in the rabbit model , distraction osteogenesis with ed-71 increases callus volume during early period after the completion of lengthening , resulting in thick cortical bone formation.11 the effect of ed-71 on bone metabolism in the sutures after rme , however , remains to be unclear . there have been few studies that attempted to change in bone regeneration capacity in mid - palatal suture during maxillary expansion . sawada and shimizu2 investigated the expression of transforming growth factor-1 ( tgf-1 ) in rme of the mid - palatal suture to evaluate its synergistic effects on bone formation and found that application of tgf-1 during early stage was essential to attain the most effective bone formation . saito and shimizu1 evaluated the effects of low - power laser irradiation on bone regeneration during expansion of a mid - palatal suture in rats and suggested laser therapy may have therapeutic benefit in inhibiting relapse and shortening the retention period through acceleration of bone regeneration . in a recent study , uysal et al12 evaluated the effects of dietary boron on bone formation in response to expansion of mid - palatal suture during different retention periods in rabbits and concluded that boron has positive effects on early phase of bone regeneration in mid - palatal suture and may be beneficial in routine maxillary expansion procedures . the aim of this experimental study was to evaluate the effects of ed-71 , a new active vitamin d analog , on bone regeneration in response to expansion of mid - palatal suture , in rats . these effects were evaluated with quantitative bone histomorphometric examination . for the purposes of this study , the null hypothesis assumed that ed-71 has stimulating effects on bone formation during expansion of mid - palatal suture , in rats . sixteen male , 5060 days old wistar rats weighing 162.5315.25 gram were used in this study . all animals were housed in polycarbonate cages in a 12-hour light / dark environment at the constant temperature of 23c and fed a standard pellet diet ( expanded pellets , stepfield , witham , essex , uk ) with tap water ad libitum . the experimental protocol was approved by university of erciyes , regional animal research ethics committee and carried out in the hakan cetinsaya experimental and clinical research centre . animals were randomly separated into two groups ( control and experimental ) of eight rats each . expansion appliance comprised of helical springs that fabricated from 0.014-inch , stainless - steel wires . the force was measured with a gauge ( 30 grams ) , and the springs were not reactivated during the 5-day expansion period . appliances were attached to maxillary incisors of all animals under anaesthesia ( xylasine+ketamine combination , 0.5 ml / kg and 1 ml / kg intramuscular , respectively ) . a hole was drilled in both incisors at the level of the lingual gingival papilla and springs were inserted into the holes , buccally ( figure 1 ) . helical springs were removed and a piece of rectangular retaining - wire was inserted into the holes between two incisors for retention . tooth separation was maintained during the retention phase . the distance between the mesial corners of the maxillary incisors was measured at the beginning and on the fifth - day of the expansion with a digital calliper ( msi - viking gage , south carolina , usa ) . occlusal radiographs were taken at three stages : at the beginning , end of expansion and at the end of the retention periods . ed-71 [ 1,25-dihydroxy-2-(3-hydroxypropoxy ) vitamin d3 ] obtained from chugai pharmaceutical co. , ltd ( tokyo , japan ) , were dissolved in propylene glycol . in this histomorphometric study , experimental group was treated with single dose of ed-71 ( 0.8 g / kg body weight ) and eight control animals received vehicle solution . twenty - four hours after expansion , one dose 10 l ed-71 or vehicle solutions was injected into the mid - palatal suture with a micro - syringe ( hamilton injection syringe , hamilton company , nevada , usa ) . the rats were monitored during the experiment , and all animals were weighed at the beginning , after expansion and after retention periods . however , deep mucosal infection / dehiscence was observed in one animal in experimental and superficial infection was observed in one animal in control group and both animals excluded from the study groups . after retention , the rats were sacrificed with an overdose of ketamine / xylazine combination and their pre - maxillae were dissected and placed in bottles contains 10% formalin . during decalcification , after fixation , the retaining wires were removed , and the pre - maxillae were decalcified with 5% formic acid for 3-days . the decalcified pre - maxillae were fixed again in the same manner and sectioned . the section was cut perpendicular to the sagittal plane and was determined by two points , one at the alveolar crest and the other 4 mm apically . histological sections were stained with haematoxylin - eosin prior to optical microscope examination ( figures 2 and 3 ) . bone histomorphometric measurement was performed 200 m under the surface of the osseous palate facing the oral cavity because bone formation of the surface area was sometimes irregular and not suitable for quantitative measurement . measurements were based on observations of the sections under a microscope and calculated using an image analysis program ( figure 4 ) . for this purpose a microscope and digital camera system ( olympus cx41/dp25 research system , olympus corp . japan ) and computer assisted image analysis software ( analysis 2.1 , soft - imaging software gmbh , mnster , germany ) were used for histomorphometric evaluation . histomorphometric evaluation was performed in a blinded analysis by two experienced authors and results were an average of the counts . the associated analyzed parameters were mineralized area ( md.ar , m ) , fibrosis area ( fb . ar , m ) , mineralized area / fibrosis area ( md.ar/fb.ar , % ) and bone area ( b.ar , m ) within the pre - maxillary suture , taken according to previous descriptions reported by parfitt et al.13 sections were stained with tgf-2 receptor by immunohistochemical method and the number of osteoblast ( ob.n ) which reflecting the new bone forming activity was counted . all data were analyzed with the statistical package for social sciences , 13.0 ( spss for windows , spss inc , chicago , illinois , usa ) . descriptive statistics are given as mean , standard deviation , standard error , minimum and maximum . the group differences were studied by the mann - whitney - u test ( with the bonferroni correction ) . sixteen male , 5060 days old wistar rats weighing 162.5315.25 gram were used in this study . all animals were housed in polycarbonate cages in a 12-hour light / dark environment at the constant temperature of 23c and fed a standard pellet diet ( expanded pellets , stepfield , witham , essex , uk ) with tap water ad libitum . the experimental protocol was approved by university of erciyes , regional animal research ethics committee and carried out in the hakan cetinsaya experimental and clinical research centre . animals were randomly separated into two groups ( control and experimental ) of eight rats each . expansion appliance comprised of helical springs that fabricated from 0.014-inch , stainless - steel wires . the force was measured with a gauge ( 30 grams ) , and the springs were not reactivated during the 5-day expansion period . appliances were attached to maxillary incisors of all animals under anaesthesia ( xylasine+ketamine combination , 0.5 ml / kg and 1 ml / kg intramuscular , respectively ) . a hole was drilled in both incisors at the level of the lingual gingival papilla and springs were inserted into the holes , buccally ( figure 1 ) . helical springs were removed and a piece of rectangular retaining - wire was inserted into the holes between two incisors for retention . tooth separation was maintained during the retention phase . the distance between the mesial corners of the maxillary incisors was measured at the beginning and on the fifth - day of the expansion with a digital calliper ( msi - viking gage , south carolina , usa ) . occlusal radiographs were taken at three stages : at the beginning , end of expansion and at the end of the retention periods . ed-71 [ 1,25-dihydroxy-2-(3-hydroxypropoxy ) vitamin d3 ] obtained from chugai pharmaceutical co. , ltd ( tokyo , japan ) , were dissolved in propylene glycol . in this histomorphometric study , experimental group was treated with single dose of ed-71 ( 0.8 g / kg body weight ) and eight control animals received vehicle solution . twenty - four hours after expansion , one dose 10 l ed-71 or vehicle solutions was injected into the mid - palatal suture with a micro - syringe ( hamilton injection syringe , hamilton company , nevada , usa ) . the rats were monitored during the experiment , and all animals were weighed at the beginning , after expansion and after retention periods . however , deep mucosal infection / dehiscence was observed in one animal in experimental and superficial infection was observed in one animal in control group and both animals excluded from the study groups . after retention , the rats were sacrificed with an overdose of ketamine / xylazine combination and their pre - maxillae were dissected and placed in bottles contains 10% formalin . during decalcification , after fixation , the retaining wires were removed , and the pre - maxillae were decalcified with 5% formic acid for 3-days . the decalcified pre - maxillae were fixed again in the same manner and sectioned . the section was cut perpendicular to the sagittal plane and was determined by two points , one at the alveolar crest and the other 4 mm apically . histological sections were stained with haematoxylin - eosin prior to optical microscope examination ( figures 2 and 3 ) . bone histomorphometric measurement was performed 200 m under the surface of the osseous palate facing the oral cavity because bone formation of the surface area was sometimes irregular and not suitable for quantitative measurement . measurements were based on observations of the sections under a microscope and calculated using an image analysis program ( figure 4 ) . for this purpose a microscope and digital camera system ( olympus cx41/dp25 research system , olympus corp . japan ) and computer assisted image analysis software ( analysis 2.1 , soft - imaging software gmbh , mnster , germany ) were used for histomorphometric evaluation . histomorphometric evaluation was performed in a blinded analysis by two experienced authors and results were an average of the counts . the associated analyzed parameters were mineralized area ( md.ar , m ) , fibrosis area ( fb . ar , m ) , mineralized area / fibrosis area ( md.ar/fb.ar , % ) and bone area ( b.ar , m ) within the pre - maxillary suture , taken according to previous descriptions reported by parfitt et al.13 sections were stained with tgf-2 receptor by immunohistochemical method and the number of osteoblast ( ob.n ) which reflecting the new bone forming activity was counted . all data were analyzed with the statistical package for social sciences , 13.0 ( spss for windows , spss inc , chicago , illinois , usa ) . descriptive statistics are given as mean , standard deviation , standard error , minimum and maximum . the group differences were studied by the mann - whitney - u test ( with the bonferroni correction ) . there was no evidence of diarrhea or other gastrointestinal symptoms in any of the animals . the body weight of the one rat in experimental group decreased during the expansion period , but subsequently recovered . no statistically significant changes in body weight were observed between groups during expansion and retention periods ( table 1 ) . the biometric analysis for the amount of expansion was done by image analysis software at the most anterior part of the pre - maxilla on histological sections . suture width measurements from histological sections showed that the mid - palatal suture was expanded following application of an activated helical loop ( figure 2 ) , with the lateral part of the maxillary bone tipped towards the skull base . also it was determined in the frontal sections that oral side of the suture was expanded more widely than the nasal side . the results indicated that the mean amount of expansion was less in ed-71 group than the control ( 178.1019.31 m and 186.2921.06 m , respectively ) . the md.ar ( p<.001 ) , fb.ar ( p<.001 ) , md.ar/fb.ar ( p<.001 ) and b.ar ( p<.01 ) measurements showed statistically significant differences ( table 3 ) . for all investigated histomorphometric parameters ed-71 group showed more positive results than the control related to the new bone formation . the descriptive statistics and statistical comparison of groups for n.ob measurement were presented in table 4 . ed-71 group with a mean of 24.556.47 osteoblast showed statistically higher n.ob then the control group ( mean of n.ob : 12.825.81 osteoblast ) . thus , according to formative changes in all histomorphometric parameters , the null hypothesis of this study was failed to be rejected . for fracture healing or distraction osteogenesis protocols , various experimental studies have been carried out to shorten the healing period , and most of these involve bone stimulation . demineralized bone matrix,14 autologous marrow cells,15 and cultured periosteal cells16 have been transplanted into the lengthened area , during distraction osteogenesis procedures . in addition , many experiments have been carried out to stimulate the callus mechanically , such as micro movements applied in the direction axial to the callus,17 static compression for shortening the callus after callotasis,15 as well as electrical stimulation18 and electromagnetic stimulation.19 however , yet , most of these methods have not been applied to sutural region during maxillary expansion procedures . palatal expansion is referred through a multi - factorial adaptive response within the mid - palatal suture . mechanical expansion results in distortion of the sutural structure , inducing a biologic chain of events leading to osseous modelling that allows the suture to restore itself to its original architecture.20 the clinical results are increasing of maxillary skeletal and dentoalveolar widths . in the present study , the stimulatory effect of ed-71 administration on bone regeneration in the mid - palatal suture in response to expansion was investigated by using a histomorphometric method . this method is a reliable technique that is frequently used in quantitative evaluation of bone remodelling in vivo.21 in the current study , locally administered ed-71 and its effect on bone regeneration was evaluated in response to maxillary expansion . there are some limitations to evaluate the effects of systemically administered pharmacological agents or nutrients on bone formation . to minimize adverse effects systemically , and to support bone formation in definite time interval and area , thus , we administered the ed-71 to the centre of the investigated area to evaluate the pure effects of the product in that region . the nature of the effects of force on rate of bone mineralization can be undertaken by experimental studies on animals . while monkey and cat have maxillary sutures similar in most aspects to that of man and have been used in maxillary expansion experiments , the ideal animals with which to obtain a clear picture of bone and suture changes under stress are the rabbit and the rat.22 thus in the present study , according to the ethical considerations , the smallest animal model was chosen to test new material in bone modelling . burstone and shafer23 reported that the normal pre - maxillary suture in young rats , measured approximately 20 to 60 m in thickness and found that expansion of the suture by the rubber wedges over a period of 5-days resulted in an opening the suture to an average width of 377104 m . pre - maxillary suture was opened by helical springs in this study and occlusal radiographs of the rats snout showed wide separation of pre - maxillary bones after 5-days of expansion and the suture width measurements were found in range between 157.26 m and 209.98 m . the amount of expansion in all groups was similar and showed no statistically significant differences . as suture width measurements were less in ed-71 group , it indicates new forming bone along the medial margins of bone segments . synthetic vitamin d3 is widely administered in osteoporosis , and effectively controls reduction of bone volume by its bone resorption inhibitive and bone formation stimulative action . recently , analogs of vitamin d3 have been studied , and it was found that ed-71 markedly accelerates bone formation.24 using ed-71 in animal tests with histomorphometric evaluation , tsurukami et al24 found by histomorphometric evaluation that , in ovariectomized rats , ed-71 not only inhibited bone resorption but also increased parameters of bone formation , such as osteoid surface and mineral apposition rate . in addition , tsubota et al16 reported that , upon administration of ed-71 to rats receiving steroids , reduction in bone volume was prevented . to investigate the effect of ed-71 on bone formation , we evaluated the effects of single injection of ed-71 or vehicle solutions in the expanding mid - palatal suture , during maxillary expansion procedures in the rats , histomorphometrically . hou et al25 indicated that the expansion of suture in rat resulted in ossification of the suture and loss of the normal suture layered structure of fibrous tissue sandwiched between cartilage - covered palatal bone plates . when ed-71 was given to rats 24 hours after starting the expansion procedure , bone formation in the suture was markedly stimulated , md.ar was increased and fb.ar was decreased . these data revealed that the mean md.ar was lower and mean fb.ar was higher than in animals which had taken no ed-71 during the study period . increased in md.ar and decreased in fb.ar measurements also indicated new bone formation in ed-71 administration groups . okuda et al26 reported that bone marrow cells from ed-71-treated mice contained more osteogenic progenitor cells than vehicle - treated mice , indicating that ed-71 treatment enhanced the osteogenic commitment of bone marrow cells . similar to the findings of okuda et al,26 we found that bone matrix remodelling was enhanced , with the peak levels of total bone area elevated following ed-71 administration . the enhancement of bone area and osteoblast number in ed-71-treated animals suggested that this compound promotes bone formation through augmentation of the proliferation and/or differentiation of osteoblasts . it may have a synergetic effect on bone formation in response to mechanical and biological stimuli . ed-71 may be useful for a wide range of applications , including the treatment of osteoporosis and the enhancement of bone formation , as might be helpful during distraction osteogenesis , fractured bone repair or in the reconstruction of bone defects . these findings suggest that locally administered ed-71 can stimulate bone regeneration in an orthopedically expanded mid - palatal suture , during expansion and retention periods .

objectivesthe aim of this experimental study was to evaluate the effects of ed-71 , a new active vitamin d analog , on bone regeneration in response to expansion of the mid - palatal suture , in rats , histomorphometrically.methodssixteen male 5060 days old wistar rats were separated into two equal groups ( control and experimental ) . both groups were subjected to expansion , and 30 grams of force was applied to the maxillary incisors with a helical - spring . experimental group was treated with single - dose ed-71 ( 0.8 g / kg body weight ) in the mid - palatal suture locally and eight control animals received vehicle solution . bone regeneration in the mid - palatal suture was evaluated by bone histomorphometric method and mineralized area ( md.ar ) , fibrosis area ( fb.ar ) , mineralized area / fibrosis area ( md.ar/fb.ar ) , bone area ( b.ar ) and osteoblast number ( n.ob ) parameters were evaluated . mann whitney - u test was used for statistical evaluation at p<.05 level.resultsstatistical analysis showed significant differences between groups for all investigated histomorphometric parameters . md.ar ( p<.001 ) , md.ar/fb.ar ( p<.001 ) , b.ar ( p<.01 ) and n.ob ( p<.001 ) parameters were significantly increased and fb.ar ( p<.001 ) measurement was significantly decreased in experimental group . ed-71 group with a mean of 24.556.47 showed statistically higher n.ob than the control group ( mean n.ob : 12.825.81).conclusionsed-71 has positive effects on early phase of bone regeneration in the mid - palatal suture in response to expansion and may be beneficial in routine maxillary expansion procedures .

clinically , it is typified by swelling , pain , and reduced function in affected joints . uncontrolled ra causes disability and reduces quality of life , placing a high disease burden on affected populations.1 disease - modifying antirheumatic drugs ( dmards ) and biologic drugs , including tumour necrosis factor inhibitors , can reduce disease activity and improve disability . however , despite a host of new immune - mediated therapies available to treat ra , significant numbers of patients exist who continue to experience pain , despite the use of dmards.2 the uk - based national institute for health and care excellence ( nice ) guidance has outlined best practice,1 and several international guidelines for ra care exist to guide treatment.3 current interventions achieve remission in 30% of patients but leave many , ie , 50%60% , with ongoing disease activity in the uk alone.4 an increasing challenge in ra management is to optimize disease remission and treatment of pain in a significant number of patients who report ongoing pain despite treatment with often expensive disease - modifying drugs . in a recent uk - based study of 1,189 people with ra , after 1 year of treatment with disease - modifying drugs , the level of pain reporting remained high.5 mcwilliams et al5 showed that there was no significant change in reported pain levels despite the use of disease - modifying drugs . such observations , now also from other groups,6,7 have led to the formulation of the hypothesis that people with ra have a heightened pain experience very early on in their disease . it is possible that people with early ra may have multiple components of pain , including neuropathic and sensitization elements . by sensitization we mean a process of heightened pain perception derived from hypersensitivity to stimuli by sustained activation of peripheral nociceptors , eg , in the arthritic joint.8,9 merskey8 defined pain as an emotional experience with an unpleasant sensation that is accompanied by an actual or potential damage or injury to tissue . pain is a persisting symptom in people with ra , and up to 70% would like to see improvements in pain compared with other symptoms of ra.2,10,11 in this article , we propose methods by which pain assessment in the clinic can assist to establish the nature of pain phenotypes in ra . a number of groups have recently reported the use of the paindetect questionnaire12 as a quantitative tool for measuring noninflammatory , neuropathic , or sensitization elements of pain . the paindetect questionnaire has already been investigated in distinct groups of people with musculoskeletal pain , including fibromyalgia,13 back pain,14 and osteoarthritis.15 all of the studies described have reported neuropathic / sensitization features of pain in the musculoskeletal conditions described , including in people who were already being treated with analgesic drugs . however , to our knowledge , no reports on the use of the paindetect questionnaire in pain reporting in ra have been published to date . we used the published version of the paindetect questionnaire , which was developed by freynhagen et al,12 and used it for the first time in people with ra to assess pain characteristics in this autoimmune condition . our work has found that ra pain is likely to be a multimodal entity with features of inflammation , neuropathic pain , and sensitization . we propose that wider use of paindetect in the clinical setting of arthritis clinics may assist in identifying neuropathic or sensitization pain features in people with ra to help optimize their future pain management . we conducted a study of 100 participants ( 32 males and 68 females ) with confirmed ra based on american college of rheumatology / european league against rheumatism criteria3 from a tertiary care rheumatology center at st george s hospital , london , uk . a patient reporting tool was formulated , consisting of 24 questions , and was used to evaluate the effectiveness of pain management and monitoring . pain analysis tools , including the visual analog scale ( vas ) ( 0100 mm ) and the paindetect questionnaire , were included to evaluate pain intensity , duration , and nature in the group . additional demographic data and disease activity score in 28 joints ( das28 ) for ra duration , use of dmards , and demographic data were also collected . full ethical approval for this study was provided by the london surrey borders ethics committee ( rec no 115212 ) . they were eligible to participate in the study if their treatment had been stable for the previous 3 months and they did not require corticosteroid therapy . the vas is a widely used tool in pain assessment for ra and forms part of the pain reporting tool in das28.16,17 to date , additional tools such as the paindetect questionnaire , which is now increasingly used to assess neuropathic elements of pain , have not been reported widely in assessments of people with ra . to investigate the value of pain assessment tools in addition to vas , we explored the use of the paindetect questionnaire in a group of people with established ra . we set parameters for vas and paindetect based on published reporting for subsets of pain . previous studies have described vas measurements for pain as mild ( 030 mm ) , moderate ( 3153 mm ) , and severe ( 54100 mm).18 with respect to pain - detect , scores suggesting likely neuropathic pain are 19 , probable neuropathic pain 1318 , and unlikely neuropathic pain 12 . the paindetect questionnaire was validated and developed in germany.12 it is a patient - based questionnaire that could be used by specialists as well as nonspecialists . it consists of nine items of which seven are weighted sensory descriptor items and two items relate to the spatial ( radiating ) and temporal characteristics of the individual pain pattern . from the answers , an overall score is generated ranging from 1 to 38 . according to the researchers who developed the paindetect questionnaire,12 a score of 12 indicates that a neuropathic component is unlikely ( with a true negative sensitivity of < 15% ) , a score of 19 suggests that the pain is likely to have a neuropathic component ( with a true positive sensitivity of > 90% ) , and a score of 1318 indicates that there is a slight chance that there is a neuropathic component . this tool for assessing neuropathic pain when compared with clinical diagnosis has 85% sensitivity and 80% specificity.12 pain reporting data were also compared with recorded das28 scores in our patient group . the simplified das28 is a modified derivative of the original das , assessing 28 designated joints from the 44 joints in original reports.19,20 this tool is generally used by the physician or specialist nurse to assess the number of swollen and tender joints out of the 28 . it also takes account of the erythrocyte sedimentation rate ( esr ) or the c - reactive protein ( crp ) . the esr and crp can both be used to measure the degree of inflammation in the serum , with usually one of the readings , either esr or crp , being used to calculate the das28 for the patient according to the formula given here . in addition , the vas component of the das28 is usually the only pain assessment tool used in many clinical settings for ra care . once the scores are calculated , they are then used in a mathematical formula to produce the overall disease activity score : das=0.56tjc+0.28sjc+0.70ln(esr)+0.014gh where tjc = tender joint count , sjc = swollen joint count , esr = erythrocyte sedimentation rate , and gh = general health . values of das28 > 5.1 indicate high disease activity , 3.25.1 means moderate disease activity , 2.63.2 means low disease activity , and a score of < 2.6 means the patient is in remission.21 all data were analyzed using graphpad prism 7 software . variables following a normal distribution were assessed as mean standard deviation . when comparing three or more groups , one - way analysis of variance was used . nonparametric data were presented as descriptive statistics ( median , interquartile range ) and analyzed using the chi - square test . the categorical variables were recorded as numbers ( n ) and percentages ( % ) and were compared using the chi - square test if they had three or more categories . variables following a normal distribution were assessed as mean standard deviation . when comparing three or more groups , one - way analysis of variance was used . nonparametric data were presented as descriptive statistics ( median , interquartile range ) and analyzed using the chi - square test . the categorical variables were recorded as numbers ( n ) and percentages ( % ) and were compared using the chi - square test if they had three or more categories . the results from our study demonstrate that participants with ra reported relatively high pain levels , despite the widespread use of disease - modifying drugs in this group of patients ( tables 2 and 3 ) . the majority of participants with ra , namely 54% , reported severe pain on the vas , which identifies people with a vas of 54100 mm as having the highest severity of pain . all participants evaluated had been stable on dmard therapy for at least 3 months prior to completing the study and had not required a change in their treatment or addition of corticosteroid therapy during that time . the majority of participants were being treated with disease - modifying drugs , including the commonest agent , methotrexate ( 82% ) . table 2 shows a summary of the dmard agents being taken by the group as a whole , with the majority being on methotrexate and combination therapy in the form of additional oral dmards , eg , methotrexate , sulfasalazine , hydroxychloroquine , penicillamine , or ciclosporin . additionally , all the subjects who were on biologic drugs and therefore by definition had more severe disease reported pain in the severe pain group . that is , of 39 participants who were taking one of the biologic agents infliximab , abatacept , or tocilizumab , 76.9% reported severe pain . our data suggest that initial treatment may not have been efficient in achieving disease control measured by das28 score , leading to the decision to use combination dmard therapy . the use of pain - relieving medications was also greatest in the severe pain group , which had the highest use of analgesic medications including paracetamol , ibuprofen , and other pain - relieving agents . the paindetect questionnaire was used to evaluate for a possible neuropathic element of pain among the ra group . figure 1 shows that 33% of participants reported possible or likely neuropathic pain by paindetect . additionally , there was a clear positive trend between vas scores and paindetect scores , suggesting that the high level of pain reported on the vas also correlated with high neuropathic pain scores ( r=0.757 ) . one of the components of the paindetect questionnaire is the body map , where subjects can mark body areas in which they felt pain . the majority of participants with probable neuropathic pain or likely neuropathic pain reported pain around their knees , ankles , thighs , lower back , wrists , and shoulders on body mapping , suggesting widespread pain in keeping with likely sensitization in our ra group . analysis of sex differences in pain reporting did not identify any significant differences between men and women ( figure 2 ) . when asked about their current pain compared with worst ever pain in their joints , a significant number of participants reported an improvement in their pain reporting at current levels ( figure 2b ) , suggesting that the majority of subjects had experienced higher pain levels previously . we also assessed the group for evidence of obesity by body mass index ( bmi ) , since higher levels of obesity are known to correlate with higher pain reporting.22 we found that the reporting of pain by vas increased according to subgroupings for subjects who were overweight and obese ( figure 3 ) . bmi group and the overweight or obese bmi group were statistically significant ( p<0.05 ) . when participants were grouped according to those on oral dmards alone ( ie , methotrexate , sulfasalazine , hydroxychloroquine , penicillamine ) , biologic drugs , and dmards ( ie , methotrexate and infliximab or tocilizumab or abatacept ) , the mean vas for the oral dmard group was 52.021.3 mm ( figure 4a ) . for the biologic and oral dmard group , the mean vas was 68.218.5 mm , suggesting that participants on combination oral and biologic dmard therapies had the highest vas pain scores reported compared with participants not on any dmard therapy ( p0.0001 ) . in comparison , 14% of the total group were not on any dmard therapy , with a mean vas of 30.419.5 mm . the results of the paindetect questionnaire divided subjects into three groups : unlikely neuropathic pain , possible neuropathic pain , and likely neuropathic pain . we found that the likely neuropathic pain group also reported the highest pain levels on vas , with a mean vas of 90.14.3 mm ( figure 4b ) , which was statistically significant compared with the unlikely neuropathic pain group ( p0.01 ) . interestingly , participants in the possible neuropathic pain group also demonstrated high vas reporting , with a mean vas of 77.810.3 mm , which was statistically significant compared with the unlikely neuropathic pain group ( p0.0001 ) . our results suggest that people with ra reporting high pain levels by vas in the clinic could have neuropathic elements to their pain measured by tools such as paindetect . we have found that high levels of pain reporting exist in ra in people with fairly well - controlled disease activity measured by das28 . the disconnect observed between pain reporting and disease activity shows that specific components of pain in ra exist that may not be fully captured by the vas . our data from the paindetect score showed that a large proportion of subjects with ra demonstrated likely or probable neuropathic pain features . neuropathic pain classically refers to pain that is arising from the central nervous system , and it could be , as demonstrated by our study , that people with ra are sensitized to pain very early on in their disease . we have shown that despite the use of disease - modifying agents for ra , including both oral and biologic dmards , according to nice guidelines , pain reporting remains high . our data suggest that there may be a noninflammatory or neuropathic component that is mediating certain components of pain perception in ra . in the group we studied , most subjects were on oral dmard therapy , with the majority on at least methotrexate ( 82% ) plus additional disease - modifying drugs . of note , although the majority of subjects were prescribed oral dmards , 54% reported high pain levels despite use of oral and biologic therapies . the use of analgesic medications in the moderate and severe pain groups was the highest . clinically , ra is identified with synovitis , which classically corresponds with inflammation - driven pain . studies have shown that inflammation of the synovium leads to an acceleration of prostaglandin and bradykinin production , which leads to the activation of thin unmyelinated sensory nerves ( c fibers ) in the synovium.23 inflammation is pivotal in the overall perception of pain in ra . however , several studies , including the data herein , suggest that despite sufficient suppression of inflammation , there is still persistent pain even with the treatment of anti - inflammatory drugs , indicating that other factors mediating pain perception are involved in ra.24 it has been proposed that after a period of time , arthritic joints expand their total receptive field to the surrounding area of normal noninflamed tissue . the enhanced response to stimulation of joints that are inflamed could be mediated by peripheral sensitization.25 additional increased pain responses to noninflamed tissue could be generated in the spinal cord , leading to central sensitization . central sensitization is when normal inputs begin to produce aberrant feedback due to the excitation of the neurons in the central nervous system.26 recent work in ra has shown that features such as anxiety , low mood , and depression have an impact on clinical pain reporting in ra and can influence brain activation in frontal regions , measured by brain functional neuroimaging studies.27 more recently , rech et al28 showed that people with ra treated with biologic therapies could have early improvements in brain pain sensitization , observed by brain functional imaging using blood oxygen level - dependent signals , within days of treatment with biologic agents such as certolizumab pergol , well before an improved clinical response in the joints was observed . from our results , we have identified that people with ra prescribed a combination of biologics and dmards had an overall high mean vas score compared with the other groups , despite das28 scores suggesting good control of disease activity . this finding further suggests that there may be additional factors involved in ra pain processing other than inflammation alone . biologics are powerful and expensive treatments usually given to patients with high das28 ( > 5.1 ) . a reduction in das28 of 1.2 points or more is considered as guidance for response criteria to biologic therapies and also considered as a benchmark for continuing treatment.29 dmards are a group of drugs that are unrelated and used in the treatment of ra to slow down the disease progression , limiting joint damage and improving function.30 a window of opportunity has emerged in ra treatment where earlier treatment with dmards equates with improved long - term outcomes.31,32 dmards help primarily to reduce inflammation and likely inflammatory pain . together with agents such as nonsteroidal anti - inflammatory drugs , they help to limit and control the symptoms of ra . additional analgesic agents that were reported to be of high usage in our study included paracetamol and codeine ( can also be given in combination , known as co - codamol).33 apart from central and peripheral sensitization , pain in ra can be further exacerbated by comorbidities , including obesity , fibromyalgia , diabetes mellitus , smoking , and secondary osteoarthritis.5 psychological and physiological factors also contribute to the perception of pain , which is seen in patients with ra who also suffer from depression and anxiety.34 clinicians could evaluate people with ra using paindetect in addition to current tools being used to assess disease activity in the future . if they showed evidence of likely neuropathic pain , it could be addressed potentially with a trial of analgesics targeted at neuropathic pain . interestingly , of the participants in our study who had severe pain , approximately 30% had been prescribed amitriptyline . potential limitations of our study are that numbers of patients were relatively small with 100 participants recruited . however , this was a feasibility study to test the practicality of using the vas and paindetect in a clinical setting where many parameters are already being used to assess patients , including das28 , blood tests , and clinical examinations . we asked participants to complete the questionnaire , which took an additional 1015 minutes of their appointment , and patients reported that it was user - friendly and practical within a clinical setting . a further limitation is that we did not evaluate mood disorders in our study group . there is extensive literature ( reviewed by dougados et al35 ) to suggest that the emotional component of pain interferes with the efficacy of treatment . future work is aimed at increasing the sample size , collecting data on mood disorders , and assessing test in summary , the data from our study suggest that people with ra have higher pain sensitivity , with a smaller but significant number demonstrating pain sensitization at both joint and nonjoint sites using the paindetect and vas questionnaires . our data concur with those of mcwilliams et al,5 who reported high vas scores in people with ra despite active treatment and das28 scores suggesting good control of disease activity . we suggest that the paindetect score could be a useful tool in the clinic as a measure of the sensitization element to ra pain . our findings suggest that implementation of multimodal pain assessment tools such as the vas and paindetect questionnaires in the arthritis clinic setting could open new avenues in the assessment and treatment of ra pain in the future .

rheumatoid arthritis ( ra ) is an inflammatory autoimmune condition typified by systemic inflammation targeted toward synovial joints . inhibition of proinflammatory networks by disease - modifying antirheumatic drugs , eg , methotrexate and biologic therapies , including tumor necrosis factor- inhibitors , often leads to suppression of disease activity observed at the clinical level . however , despite the era of widespread use of disease - modifying treatments , there remain significant groups of patients who continue to experience pain . our study formulated a pain assessment tool in the arthritis clinic to assess feasibility of measurements including the visual analog scale ( vas ) and paindetect to assess multimodal features of pain in people with established ra ( n=100 ) . clinical measures of disease activity ( disease activity score in 28 joints [ das28 ] ) were also recorded . our data showed that despite the majority of subjects on at least one disease - modifying agent , the majority of patients reported severe pain ( 54% ) by vas , despite well - controlled clinical disease , with mean das28 2.070.9 . using the paindetect questionnaire , 67% of patients had unlikely neuropathic pain . a significant proportion of subjects ( 28% ) had possible neuropathic pain and 5% had features of likely neuropathic pain by paindetect scoring . we found a positive correlation between vas and paindetect ( r2=0.757 ) . of note , the group who had likely or probable neuropathic pain also showed significantly increased pain reporting by vas ( p<0.01 ) . subjects who were clinically obese ( body mass index > 30 ) also had statistically higher proportions of pain reporting ( vas 89.00.7 mm ) compared with subjects who had a normal body mass index ( vas 45.221.8 mm ) , p<0.05 . our findings suggest that multimodal features of pain perception exist in ra , including neuropathic and sensitization elements , perhaps explaining why a subgroup of people with ra continue to experience ongoing pain , despite their apparent suppression of inflammation .

international exchange and training in the field of public health is organized by the world and european association of public health and their working groups established during the past 20 years since these associations exist . formation of these working groups followed a stream of demand for their content depending on the acute public - health problems ( 1 ) . most of them are focused on diseases and conditions that are widespread in the field of public health , such as cardiovascular diseases , malignant diseases , aids , mental health or other contemporary illness . the schools of public health are founded within these working groups based in some european cities and within their university centers . generally , the content of these workshops cover ( 1 , 2 , 3 ) : analysis of public health and management ; monitoring and control of work in the field of public health ; public health aspects of medical information and their international comparability ; rapid response and action plans ; exploration of the most important parameters for the current public health problems . within this are implemented appropriate studies in the field of public health and made strategies for development of : assistance in decision making ; assistance in the development of information and communication technology ; presentation of the results at the scientific meetings . these results and experiences are presented at the annual eupha conference in some of the european cities and are organized on regular basis or as continuous supervision by experts , and program coordinators who are responsible for organizing workshops and help their quality organization from distance . eupha and whpa have developed an excellent international network and well designed 24-hours active websites with the possibility of international communication between public health experts and also very respectable journals of which is among the most prominent european journal of public health which is continuously published for 15 years and is indexed in current contents , medline and other databases . great attention is paid to the promotion of young researchers in the field of public health especially within countries in transition . in this domain international public health through eupha whpa emphasis is focused on the following activities : the formulation of appropriate research structures and quality implementation in practice with specific goals and activities for individual countries of the european union , southeast europe and beyond ; definition of national structures and policies of public health in the countries of southeast europe and the european union where the emphasis is placed on the development of national strategies and the internal relationships between different health care systems in european countries , also with emphasis on the identification of european mechanisms to enhance cooperation and exchange ; collection of basic data from which are made strategic plans and implement public health policy of the projects in the countries of southeast europe and the european union , with application of best practice models ; simulations of already implemented and evaluated research projects with quality scales in european union and countries in transition ; assistance of public health experts from european union whose practical experiences are approved and confirmed by the world health organization and at the national level by experts in public health in countries in transition , assessment of the adequacy and validity of the application of these research projects in the field of public health in the structures of public health organizations , the civil sector in public health in countries in transition and spreading of these results by publication through local media and relevant publications which are available to health planners . eupha and whpa , both scientific and professional associations are organized within the broad frame work program of activities with detailed objectives and quality created information system and database in order to : carry out effective preventive public health actions for mass communicable and noncommunicable diseases ; organizing the optimal public health system applicable for users of health care at different levels ( visible structures , policies and results ) ; assess is the health care policy comparable with the european union but also within services at the local level ; measure the results of the public health sector through the development of registries for specific diseases for their ongoing monitoring ( malignant diseases , cardiovascular , etc . ) be able to create a policy of health based on evidence and facts , as cohrane cooperation in clinical medicine and relate it to those in the field of public health . public health research is dependent on the political , organizational , structural and financial factors , and they are all part of the application of public health in member countries of the european union and more recently of the same concept in the southeastern europe ( very often used model is the danish one from 1980 which results and experiences are of so high quality and efficient that is used as a model in several countries of the european union in its policies and development strategies to improve public health and health systems at local and national level ) . the literature often quotes the experience of public health and very respectable usa center for disease control ( cdc ) . this research center and its experts were with their models of research in the field of public health example and role model to many countries in europe to implement similar ones in their practice ( 1 ) . eupha , european public health associations is a non - profit organization founded in 1992 and registered in the netherlands ( http//www.nivel.nl / eupha/ ) . eupha objectives are to promote and direct the health research and training in europe . besides these their goal is to improve communication between researchers , practitioners and politicians in a field related to public health in europe . the founders are the national health care organizations from 22 countries in europe , and many major research groups were active in the early eu research programs . eupha publishes a journal , organize annual international conferences and supports the five sections that work in certain specialized research areas . besides this it transfers knowledge and development policy onto public health units of the european committee . epha , european public health alliance , is an association of over 60 volunteer organizations founded in 1994 ( www.epha.org/ ) and includes a volunteer assistance to research of many diseases , such as the european link for heart disease , santa for mental illness , european league for cancer , evropka program for the prevention of injuries and infectious diseases . it publish every two - months a journal , electronic and personal possibility to exchange information , and provides the scientific and political support to the european parliament for health . it is funded directly by member states and additionally from the european committee ( 2 , 3 ) . aspher , association of public health schools in the european region , it is a non - profit association registered in france ( www.ensp.fr/aspher/ ) . aspher is main independent organization in europe with a commitment to streamline public health through training of health professionals and researchers . established in 1966 aspher has more than 60 organizations that are members of the eu and the world health organization . this organization provides a formal system between colleagues made to improve the quality of health professionals training , in addition to the annual conferences held . it is financed by member organizations . authorized by open social institutions for the development of schools of public health in central and eastern europe as well as to improve the communication relationship between health care professionals . enhpa , european community of health promotion agencies represents national health agencies of 21 european countries that are associated with their ministries of health ( www.eurohealthnet.org ) . formed in 1996 to help promote health priorities of the european committee . technical secretary is in the iupe / euro office in the dutch institution for health promotion and disease prevention . enhpa is a non - profit network and was founded by the european committee and the action ministries of health . enhpa recently took a major role in the development of european research on health disparities , and developing administrative support for national public health researchers , a common exchange policy and research development . we identified 45 clinical studies , in addition to many individual level , such as control of various treatments of patients rather than studies at a population level for the prevention of disease and the organization . it has the support of public health researchers , including research , such as standards of health informations and promotional projects of basic diseases . annual meetings of european public health associations have shown constant growth of public health care quality and collaboration between researchers across the europe and were partly supported by eu funding . the existing common infrastructure in addition offers a framework for development of public health and applicative research . eupha build such cooperation in order to find out how far it can be the exchange of interventions between countries as well as at the national and local level . in order to improve the health of people in europe , it is necessary to move research from individual level to programs of disease prevention and control . joint management and exploitation of knowledge collaboration between researchers , citizens groups and national research management is the key to success in the development of health care in europe . differences in health care are increasing as well as the cost and quality of health services . historically governments are not interested in the differences , but they are for reduction of the increasing costs of health care . with the combination of the four categories of health and illness we get the model of modern public health : individual treatment of disease ; collective treatment of disease ; collective management of health and individual health management . public health debates and reforms of health systems are focused on the following issues : access to health services ; coordination of care ; ethical aspects of health care ; strengthening the coalition building ; community capacity to cope with problems in the community ; capacity for organizational learning ; capacity for certain health organizations that address health problems ; quality of program planning ; sustainability of the program .

summarypublic health is a broad field that touches several different medical disciplines such as epidemiology , public health informatics , health statistics , environmental protection ( ecology ) , sociology , health promotion , disease prevention and organization of health care for nosological groups of the population . in europe and the world today is developed a fairly extensive network of scientific and professional institutions engaged in research projects , studies , organization of scientific conferences and publishing scientific and professional articles from the field of public health . their goal is to promote scientific advances in the field of public health through the exchange of experiences of public - health professionals , training , demonstration of appropriate research models , simulation and application in medical practice .

zebrafish exhibits many qualities that are essential in a good genetic model : relatively small size for vertebrates , high fecundity , reasonably short generation time , low maintenance costs , amenability to large - scale saturating mutagenesis screening , availability of mutants , genetic maps , and a sequenced genome . however , the lack of some classical tools in genome manipulation has been a serious drawback . in recent years , several transposable elements from heterologous hosts ( tc3 , mariner , tol2 , sleeping beauty , frog prince , and ac / ds ) have been successfully applied in zebrafish [ 1 - 6 ] . all of these ' cut - and - paste ' transposon vectors are designed to operate as two component systems : a non - autonomous element that inserts into the genome of the host and a transposase that catalyzes transposition of the non - autonomous element . a non - autonomous element can carry a cargo inserted between the two end sequences that are required in cis for transposition . the transposase is usually supplied in the form of mrna by microinjection into fertilized eggs to induce transposition of the non - autonomous element . the ease of microinjection in fish is advantageous ; the injected transposase mrna is degraded some hours after injection , and new insertions of the non - autonomous element in the genome remain immobilized . the choice of many transposon vectors provides zebrafish biologists with a plethora of possibilities , but it also raises the issue of which to use and when . the tc3 element from caenorrhabditis elegans and the mariner ( mos1 ) element from drosophila mauritiana however , since the initial reports , no further developments using these transposons have been described in the literature . two transposable elements of fish origin , tol2 from medaka ( a member of the hat [ hobo / ac / tam3 ] superfamily ) and sleeping beauty ( a synthetic member of the tc1/mariner superfamily ) , have both become widely used in the past few years ( table 1 ) [ 7 - 12 ] . recently , a transposable system from plants has been added to this arsenal . the modified maize ac / ds elements are effective transformation vectors and produce high genomic ds transposition and re - transposition rates in zebrafish ( table 1 ) . reported performance of various transposable elements in zebrafish the combined size of 5 ' and 3 ' cis sequences of the element . the size of the nontransposon dna fragment confined between the 5 ' and 3 ' cis sequences ( excluding the letter ) . all transposon - mediated transgenesis data were produced in an essentially similar experimental setup . in each case the transposon dna and the transposase rna were co - injected into fertilized zebrafish eggs . germline transgenesis was calculated as the percentage of founders that produced green fluorescent protein ( gfp)-positive offspring among the total number of founders screened ( shown in brackets ) . except for a few cases , indicates that no reporter gene was used ; transgenic offspring was identified using polymerase chain reaction ( pcr ) . '' indicates that the number included those expressing gfp reporter and those that were pcr - positive but did not express detectable amounts of gfp reporter . in these reports the transposase rna was injected into fertilized eggs of a transgenic fish carrying a single transposon insert in its genome . the genomic re - transposition rates were calculated as the number of founders that produced offspring with novel enhancer trap patterns distinct from the pattern of the founder . , it appears that sleeping beauty produces lower transgenesis rates and is more sensitive to size of cargo fragments compared with tol2 and ac / ds ( table 1 ) . the tol2 and ac / ds systems produce the highest transgenesis rates reported to date , and they are capable of carrying large cargo fragments without significant reduction in activity . both systems can generate multiple insertions that are advantageous for insertional mutagenesis . on the other hand , working with multiple insertion lines and maintaining them can be cumbersome . furthermore , highly active transposons can undergo multiple excision - insertion events , leaving behind the untagged footprints and genomic rearrangements . it is surprising that despite a huge phylogenetic distance between hosts , the ac / ds system performs as well in zebrafish as in plants . moreover , transposition activity of ac / ds in fish is comparable to that of the fish - derived member of the hat superfamily tol2 ( table 1 ) . the ac / ds system has been utilized in many plant species , yeast , and now in vertebrates , which is the widest host range demonstrated for any transposon . these facts strongly suggest that members of the hat superfamily are probably the most versatile cut - and - paste transposons , and can be broadly utilized in genetic manipulation of various organisms . this makes hat elements the best candidates for introduction into new species that do not yet have established transgenesis and insertional mutagenesis techniques . different insertion preferences produced by different transposable elements can allow better distribution of insertions throughout the genome . the absence of cross - activation between different transposons permits independent sequential use of several elements within the same animal and can be useful for various applications . for example , one can envision using one transposon to generate transgenic reporter lines for any given cell type of interest , and these lines can subsequently be mutagenized using a different transposon to create a bank of mutations affecting that particular cell type . the simplicity and the ease of use , and the ability to carry complex cargo fragments , combined with high cargo capacity , makes transposons superior to retroviral vectors for fish transgenesis . vertebrate genes contain disproportionately large introns compared with exons . therefore , most random insertions would not directly disrupt coding sequences . in some cases such insertions can be mutagenic by affecting the regulatory sequences , causing mis - expression , or they may result in aberrant splicing or premature termination of transcription . however , such events often cause hypomorphic mutations , and the null alleles are assumed to occur rarely . several approaches have been proposed to select for insertions within genes and to enhance the chances of disrupting gene function . a classic gene trap vector ( sometimes referred to as 5 ' gene trap ) harbors a splice acceptor site upstream of a promoterless reporter . when inserted into an intron of a transcribed gene it can cause abnormal fusion transcripts and expression of the reporter gene under regulation of the promoter of the gene into which it is inserted . because gene trap vectors target introns , they are effective in species with large introns such as vertebrates . however , the rate of alternative splicing through the splice acceptor sequence of the gene trap cassette can be insufficient to prevent fully the synthesis of the original transcript , resulting in generation of hypomorphic alleles or no detectable effect on gene function . multiple splice acceptor sequences and multiple transcription terminator sequences can be introduced into gene trap vectors to improve their mutagenicity and tagging efficiency . another drawback of 5 ' gene trap is that only insertions in transcriptionally active genes are selected . genes that are expressed at low levels or are not expressed during the stages observed in the screen remain undetected even if a tagged gene is successfully inactivated by the gene trap . to circumvent this , polya trap vectors have been designed to tag genes regardless of their transcription status . a basic polya trap vector ( sometimes termed 3 ' gene trap ) generally harbors a promoter driving the transcription of a reporter gene , which has no polya signal but a downstream 3 ' splice donor sequence . such reporters normally produce unstable transcripts , which after successful splicing are polyadenylated using polya signals of the tagged gene . this strategy combines the mutagenic capacity of 5 ' gene trap with the high trapping capacity of polya trap vectors . the ' gene - breaking ' transposon vector developed by sivasubbu and coworkers harbors a 5 ' mutagenicity cassette and a 3 ' polya trap cassette in the same transposon vector . the 5 ' mutagenicity cassette produces strong termination of transcription by combining an effective splice acceptor sequence with a polya signal of ocean pout antifreeze protein . to date , only a few examples of successful gene inactivation using gene trap vectors have been reported in zebrafish . unbiased systematic studies using a statistically significant number of tagging events are required to estimate the actual mutagenic ability of the various gene trap designs and to compare it with the mutagenicity of transposon insertions carrying simple reporters or enhancer traps . transposon vectors can also be designed to screen for gain - of - function mutations by random activation and/or over - expression of genes . in these screens , usually referred to as ' activation tagging ' or ' modular mis - expression ' , transposons are equipped with enhancer or promoter sequences that can induce transcription of the endogenous gene adjacent to the insert . several laboratories have begun testing these tools in zebrafish , but there are no published reports yet . if a transposon insertion residing inside an intron is found to be nonmutagenic , then such a transposon can potentially be re - mobilized in order to produce more insertions in the same gene or to generate flanking deletions . various transposons preferentially re - transpose to nearby chromosomal sites ( for example ac / ds in plants , p - element in drosophila , and sleeping beauty in mouse ) . several elegant approaches utilize this ability of transposons to inactivate closely linked genes ( figure 1 ) . ( a ) regional mutagenesis using gene trap elements ( for simplicity , only a 5 ' gene trap is illustrated ) . here a ' jumper ' element carrying a selection marker ' a ' ( for example , green fluorescent protein under regulation of a constitutive promoter ) is inserted between an open reading frame of a marker gene ' b ' ( for example , red fluorescent protein ) and a suitable promoter . when the jumper element is excised , the expression of the ' b ' is switched on . animals carrying an empty donor site and retaining the jumper element can be analyzed by polymerase chain reaction ( not shown ) . ( c ) regional mutagenesis from a launch pad combined with site - specific recombination system ( cre / lox in this case ) . the system is a modification of the method shown in part b but the carrier and jumper elements both carry loxp sites . after local re - transposition of the jumper element the region between the loxp sites is deleted or inverted according to the orientation of loxp sites using cre recombinase . ( d ) selecting flanking deletions using flanking marker recombination ( from the method of p induced male recombination in drosophila ; see text for references ) . this approach requires two closely linked markers ( a and b ) around the donor site . the transposon is usually retained at one side of the deletion . this approach can detect the same events as the methods shown in parts c and d but no additional markers are required . the animals that harbor deletion events can be identified by loss of one flanking marker , whereas retention of the other marker shows presence of the donor site . because not all offspring from a founder will harbor re - transpositions , it is beneficial to have a selection scheme for the transposition events . one simple approach is to use a genomic insertion of a gene trap transposon as a donor , and select for gene trap events ( figure 1a ) . a similar selection principle was successfully utilized in the mouse using the sleeping beauty ( sb ) transposon by keng and coworkers . they used two unique donor sites containing multiple concatemeric copies of the sb element , a feature that ensured high re - transposition rates . the reporter gene carried by the sb element in these donor sites was silenced , and its expression was activated when re - transpositions caused gene trap events . the authors demonstrated high efficiency of this approach for region - specific saturation mutagenesis , producing insertions in all genes within a 4 megabase region surrounding the donor site . the selection scheme shown in figure 1a can only detect transposition events that cause detectable reporter gene expression . a more effective strategy for selecting re - transposition events uses a combination of two selection markers : one to detect the excision events and another to retain the re - transposed element ( figure 1b ) . a ' carrier ' element harbors a ' jumper ' element and an excision marker . the ' carrier ' is used to generate insertions in the genome that are subsequently used as the donor sites for transposition of the ' jumper ' element . in plant genetics , such donor sites , usually called ' launch pads ' , have successfully been used to saturate specific regions of the genome with insertions . because zebrafish exons are small , the mutagenesis efficiency of this approach is expected to be low . arming the jumper element with an additional ' another possible approach combines the local re - transposition from a ' launch pad ' with a site - specific recombination system such as cre / lox or flp / frt ( flipase recombination enzyme / flipase recognition target ) in order to produce flanking deletions or inversions ( figure 1c ) . in this method , the carrier and the jumper elements both harbor loxp sites . after the local re - transposition of the jumper element , a fragment between the loxp sites is deleted or reversed according to the relative orientation of the loxp sites . in plants this approach was demonstrated using a combination of the cre / lox system , t - dna as the carrier , and ds element as the jumper [ 33 - 35 ] . in zebrafish , there are at least three heterologous transposon systems that can be used in various combinations with each other . tol2 and ac / ds can both be used as carrier elements because of their large insert capacity . upon mobilization , a transposable element can generate deletions , inversions , and other rearrangements around the donor site . when two insertions of the same transposon are found at different sites on the same chromosome , deletions and inversions of the region between the two insertions can be recovered . two trans - heterozygous insertions ( located at different positions on homologous chromosomes ) have also been used to generate deletions in drosophila . p - element insertions are frequently used as starting - points to generate flanking deletions in drosophila by screening for male recombination events [ 39 - 41 ] or ' imprecise excision ' events . the former strategy ( figure 1d ) generally relies on the lack of male meiotic recombination in drosophila . in this approach , recombination between two flanking markers in the male germline occurs only when the p - element undergoes a transposition process called ' hybrid element insertion ' , which causes frequent deletions . in zebrafish , meiotic recombination in males is also suppressed relative to that of female meiosis , especially near centromeres , but the rate of suppression is unlikely to be sufficient for effective application of this strategy . however , it is not known whether similar recombination events occur in fish and what is the frequency of long deletions . when a transposable element is excised , the double - strand break generated during the excision is sometimes repaired imprecisely , generating flanking deletions or other rearrangements . although , the actual mechanism of ' imprecise excision ' is unclear , it should be distinguished from the ' hybrid element insertion ' , which leaves the transposon at the deletion site ( figure 1d ) . because large deletions ( several kilobases ) are expected to occur at low rates , this strategy may be feasible only when the insert is found very close to an exon of the target gene or when there is a convenient selection marker , which can be used to detect deletion events . a compound element consists of a carrier element that harbors a ' deleter ' element inside , flanked by a pair of visual markers ( for instance , green fluorescent protein [ gfp ] and red fluorescent protein [ rfp ] ) . during primary screening , the genomic insertions of the compound element are produced using the transposase for the carrier element . if the insert is found close to the gene of interest or if it lands in an intron causing no effect on gene function or hypomorphic mutation , then such insertion can be used as a starting point to produce flanking deletions by activating the deleter element . in this scheme , there is no need for additional genetic markers ; deletions on either side of the original insertion can easily be identified by the absence of one of the flanking markers , whereas the other marker shows the presence of the donor site . a similar selection scheme with a single flanking marker was utilized in plants using the ds transposon as a deleter . the strategies for generating flanking deletions illustrated in figure 1c , f both have advantages and disadvantages . the strategy shown in figure 1c requires two steps ( re - mobilization and excision ) to produce deletions , whereas the simpler strategy shown in figure 1f needs only one step , thereby reducing the experimental time for at least one generation . on the other hand , the strategy shown in figure 1c provides a platform for inducible and tissue / cell - specific gene inactivation . until now there have been no reliable data published on the distribution preference of transposable elements in fish therefore , to evaluate the feasibility of the approaches described above , it is important to determine the level of preference to insert to linked positions and to determine the frequency and extent of the flanking deletions . saturating the genome with random insertions ( obtaining at least one effective hit in every gene ) requires generation of a very large number of insertions , significantly larger than the total number of genes in the genome . it is problematic to generate , analyze , maintain , and maximize use of such huge collections . in the classical mutagenesis screens , this problem is addressed in two steps : animals are heavily mutagenized with highly active mutagens such as enu ( n - ethyl - n - nitrosourea ) or retroviruses , so that every founder carries multiple mutations ; and such collections are screened for the visible phenotypes , and only the mutant strains are retained and analyzed [ 49 - 51 ] . the main disadvantage of the phenotype - based mutant screening ( forward genetics ) is that only a relatively small number of genes are targeted , and mutations that do not cause obvious phenotypes are ignored or discarded . in the post - genome era , the most common problem is gaining insight into the function of genes that have been identified by sequencing but for which mutant information is lacking ( reverse genetics ) . therefore , reverse genetics approaches to mutant screening must operate with a complete collection of random mutations representing all genes , regardless of the phenotypic manifestation . until recently , only one reverse genetic approach has been implemented in zebrafish to obtain mutations in the gene of interest . this approach utilizes tilling ( targeting induced local lesions in genomes ) and re - sequencing of enu - mutagenized fish for target - selected screening of point mutations in the gene of interest . although this approach is very powerful , considerable screening efforts are required each time to obtain mutations in every new gene . furthermore , this method of mutagenesis generates predominantly missense mutations , which may or may not result in phenotypes . an alternative approach to reverse genetic mutagenesis screening was recently pioneered by znomics , inc . ( portland , or , usa ) , who have produced a large - scale library of retroviral insertions in zebrafish . the company has been identifying the locations of retroviral insertions from cryopreserved sperm samples containing , on average , 25 insertions per sample by sequencing the flanking dna adjacent to the insertions . with 100,000 insertions already identified , znomics hopes to map a total of half a million insertions . thus , their bank of retroviral insertions promises to be a valuable resource for the zebrafish research community . however , the actual efficiency of the znomics screen and the utility of the strains remain to be determined . despite the recent vector improvements , the frequency of transposon insertions still remains lower than that of retroviral insertions ( table 1 ) . it is therefore important to determine whether transposon vectors have the potential to complement the retroviral projects in the field of large - scale insertional mutagenesis . one way to increase the number of transposon insertions per germline is to use the donor lines carrying multiple inserts for re - transposition . the distribution of retroviral insertions ( or any other known insertional mutagen ) is not absolutely random . thus , a single insertional mutagen ( retrovirus ) would be inefficient in hitting genes at ' cold spots ' , but instead it will produce in vast excess insertions in ' hot spots ' when trying to achieve genome - wide saturation . the presence of hot and cold spots narrows the limits for efficient use of a single insertional mutagen in saturation mutagenesis screens . in order to achieve saturation more productively , it is worthwhile to launch several parallel screens of smaller scale using different insertional mutagens , rather than using a single agent alone . in this respect , having an arsenal of several transposable elements is an advantage . because vertebrate genomes contain only a small proportion of coding sequences , the majority of retroviral insertions ( or other random insertions ) are found either in the intergenic regions or inside introns . in case of retroviral mutagenesis , the only way to increase the number of exon hits is by significantly increasing the number of insertions . unlike retroviral insertions , transposons can be re - mobilized in order to obtain exon insertions or to generate flanking deletions ( figure 1 ) . re - transposition also offers a unique approach to reducing the size of insertion libraries - the ' launch pad ' strategy . a relatively small collection of the launch pads ( figure 1 ) regularly distributed throughout the genome must be generated and deposited in a stock center . then , even a moderate sized laboratory should be able to use a launch pad that is closest to their gene of interest for region - specific saturation mutagenesis to generate insertions in the target gene . although thousands of transgenic fish lines can be created , stored , and recorded in a database in individual laboratories , the long - term maintenance of such collections and wide public distribution of the materials by the same laboratories appear problematic . because large - scale projects in the field of functional genomics promise vast resources for the scientific community , the necessary funding can usually be obtained . in contrast , it is difficult to raise the additional funds necessary to further maintain and distribute the transgenic lines , and therefore the produced materials and data often remain largely unutilized and eventually wasted . other sensitive issues such as authorship , sharing of intellectual property rights , and oppressive material transfer agreements , among other factors , are common and can significantly hinder progress . operating on a cost - recovery basis may present a solution , but recovery from the cryopreserved stocks , quality control measures such as prevention of material loss , and insert confirmation can significantly increase running costs . recently , a private company ( znomics , inc . ) produced a large collection of retroviral insertion strains , which are available for purchase on a per strain basis . however , it is early to speculate whether demand for the transgenic zebrafish lines will be large enough to extend this model to projects of smaller scale . the distribution problem is unlikely to be resolved without a dedicated stock center that can take on these responsibilities . the success of large - scale functional genomics projects in other model organisms was achieved largely because of centralization . for example , arabidopsis stock centers maintain hundreds of thousands of genotypes produced by insertional mutagenesis projects [ 57 - 60 ] , and large - scale collections of transposon insertions in drosophila were also made available through stock centers . the existing zebrafish stock center will need to scale - up its capacity in order to accommodate many thousands of new samples that might be generated very soon by several laboratories . at the present time , there appears to be some variability from laboratory to laboratory in cataloging transposon lines . because various transposon screens are designed for different purposes ( mutagenesis , enhancer and gene trapping , driver lines , and so on ) , it is important to develop a set of standard requirements for the lines that are deposited at the stock center . these requirements should define the alternative descriptions to be provided with each line that would allow a searchable database to be established . at the same time , these requirements should not deter the community from depositing their collections into the stock center . arguably the simplest way to characterize this information can be used to map the insertions along the identified genome sequence ( znomics model ) . alternatively , flanking sequences can be used to build - up the blast dataset , which would allow checking for the availability of insertions inside any dna fragment using the blast search program . it is much more difficult to produce comprehensive expression pattern information , and to systematize and organize this information into a searchable database . high - throughput reverse genetics screening using transposable elements can generate a huge resource for zebrafish research , but the actual benefit of large - scale projects will depend on the subsequent utilization of this resource . this article has been published as part of genome biology volume 8 , supplement 1 , 2007 : transposons in vertebrate functional genomics .

the recent introduction of several transposable elements in zebrafish opens new frontiers for genetic manipulation in this important vertebrate model . this review discusses transposable elements as mutagenesis tools for fish functional genomics . we review various mutagenesis strategies that were previously applied in other genetic models , such as drosophila , arabidopsis , and mouse , that may be beneficial if applied in fish . we also discuss the forthcoming challenges of high - throughput functional genomics in fish .

organisms develop mechanisms to adapt to their changing environment ( tinbergen 1963 ; seligman 1970 ; mayr 1974 ) . they use exquisitely designed sensory systems to receive environmental cues and respond appropriately , favoring cues that aid survival and reproduction , while avoiding those that indicate danger ( hollis 1982 ; domjan 2005 ) . these complex and often energetically costly sensory systems ( e.g. , olfactory , gustatory , visual ) are typically coupled with downstream machinery ( e.g. , cognitive , hormonal , motor output ) in order to take full advantage of environmental changes ( ames and li et al . perhaps the most crucial of these functions is the ability to recognize a threat : an increase in temperature , a poisonous fruit , or the scent of a predator . individuals that fail to detect and avoid threats are likely to experience injury or death ; thus , the machinery governing threat response must be precise ( plutchik 1980 ) . fear and anxiety are complex behaviors that represent responses to environmental threats . these two behaviors differ in that fear is a response to a real or clearly identifiable threat and functions to remove the individual from a harmful situation ( belzung and philippot 2007 ) . in contrast , anxiety is a contrived or exaggerated fear response ( chaffey et al . this important distinction has led most psychologists and evolutionary biologists to regard fear as an appropriate and adaptive response ( barlow and durand 2011 ) , and anxiety as psychologically unhealthy and often associated with unwarranted physiological stress across multiple organ systems ( kessler et al . 2005 ) . in this review , we first briefly summarize the history and biology of anxiety - related disorders ; we then describe how invertebrates , with their powerful genetic tools and relatively simple neurocircuitry , can contribute to our understanding of anxiety - related ailments . of course , an inherent challenge with the use of an invertebrate model system to analyze a human emotional behavior is that organisms such as fruit flies and nematodes do not possess the full suite of characters that comprise human emotion ( tulving 2001 ) . most notably , invertebrates are missing a sophisticated sense of self - awareness , termed autonoetic consciousness . this heightened sense of self facilitates the full experience of human behavior by mentally placing an individual in the past , the future and in possible alternative situations as the threatening stimulus is processed ( tulving 1987 ; gardiner 2001 ; lou et al . there is currently no compelling evidence that any non - hominids are in possession of this cognitive ability ( tulving 2001 ; cloninger 2009 ) . however , if an emotional behavior is reduced down to its individual components , one finds that certain components of the behavior are accessible in simple model systems . for example , psychologists largely agree that anxiety is composed of three steps : ( 1 ) perception and appraisal of a threatening stimulus , ( 2 ) cognitive and physiological processing of the stimulus , and ( 3 ) specific behavioral output ( including the fight or flight response ) ( arnold 1960 ; scherer 1984 ; lowe and ziemke 2011 ) . in the final section of this review , we describe recent work in the nematode , caenorhabditis elegans , which lends to a circuit level understanding of how a threatening stimulus is sensed , and studies in the fruit fly , drosophila melanogaster , which describes how a behavioral response to a threat ( fighting ) is generated . the greeks may have been the first civilization to formally recognize anxiety - related disorders as a medical condition ( gabriel 1987 ; hunt 1988 ) . in 332 bc , vapors emanating from the heart and brain could induce a hysteric or especially nervous condition ( stone 1997 ) . panic is derived from the greek god , pan , who elicits irrational fear when aroused from his sleep ( stone 1997 ) . the earliest recorded attempts to ameliorate anxiety symptoms used sedatives and electrical stimulation . in the first century ad a hundred years later , claudius galen , a prominent roman physician / philosopher ( 131201 ad ) , applied shocks from electric eels to remedy anxiety - related ailments with modest success ( stillings 1975 ) . medical advances during the nineteenth century allowed anatomists to correlate specific lesions in brain - damaged patients to a cognitive or emotional deficiency , a process known as functional localization ( kandel et al . 2000 ) . in 1861 , pierre paul broca used this technique to correctly link the capacity to articulate speech with a confined portion of the cortex , the inferior frontal gyrus , now termed broca s area ( broca 1861 ) . in 1937 , james papez , a cornell university anatomist , proposed an influential theory of how emotional behaviors are generated and , most importantly , identified a neural pathway driving these behaviors , the papez circuit ( papez 1995 ) . papez suggested that emotions are invoked via neural pathways beginning with sensory information and integrated hypothalamal and cortical stimulation ( ledoux 1996 ) . subsequent neuroanatomical findings have modified this circuit ; however , the broader concept of integrated neural pathways underpinning emotional behavior remains intact ( bear 2006 ) . our current understanding of the neurobiology of fear and anxiety centers largely on three interrelated systems : the amygdala , the hpa ( hypothalamic pituitary adrenal gland ) axis , and neuromodulators ( e.g. , serotonin ) ( fig . 1 ) . each of these systems will be briefly discussed ; however , this review will largely focus on the role of serotonin in modulating these behaviors.fig . a fearful reaction occurs in response to a definite threat : a real attacker wielding a knife . an anxious response occurs in response to an imprecise or unknown threat : a dark shadow reminiscent of an attacker ( kaplan et al . b physiological processing of threatening stimulus . distressing visual information enters the retina , is processed by visual circuitry , and activates the hypothalamus ( shi and davis 2001 ; davis 2006 ) . once engaged , the hypothalamus initiates the hpa axis by releasing crf ( corticotrophin - releasing factor ) . in response , the pituitary releases acth ( adrenocorticotrophin - releasing hormone ) into the bloodstream , which prompts the adrenal gland to release glucocorticoids ( ex . the glucocorticoids bind receptors on the hypothalamus and pituitary , preventing further release of crf and acth ( mathew et al . amygdala processing ( purple arrows ) : visual data captured by the retina is first processed by the thalamus , which in turn innervates the amygdala ( green regions ) . the amygdala integrates memory information from the hippocampus and context and autonoetic consciousness from the cortex ( ledoux 2000 ; zald 2003 ; phelps and ledoux 2005 ) ( e.g. , orbitofrontal cortex , anterior cingulate cortex ) . additionally , the amygdala reciprocally innervates various brain regions including locus coeruleus , bnst ( bed nucleus of the stria terminalis ) , anterior insula , and hypothalamus ( paulus and stein 2006 ; mathew et al . serotonergic circuit ( red arrows ) : serotonin is released from pre - synaptic neurons within the dorsal and median raphe nuclei on the midline of the brainstem . serotonergic neurons innervate regions of the cerebellum , thalamus , hippocampus , hypothalamus , basal ganglia , frontal cortex , and amygdala ( kandel et al . 2000 ) neurobiology of mammalian fear / anxiety response . a fearful reaction occurs in response to a definite threat : a real attacker wielding a knife . an anxious response occurs in response to an imprecise or unknown threat : a dark shadow reminiscent of an attacker ( kaplan et al . distressing visual information enters the retina , is processed by visual circuitry , and activates the hypothalamus ( shi and davis 2001 ; davis 2006 ) . once engaged , the hypothalamus initiates the hpa axis by releasing crf ( corticotrophin - releasing factor ) . in response , the pituitary releases acth ( adrenocorticotrophin - releasing hormone ) into the bloodstream , which prompts the adrenal gland to release glucocorticoids ( ex . the glucocorticoids bind receptors on the hypothalamus and pituitary , preventing further release of crf and acth ( mathew et al . amygdala processing ( purple arrows ) : visual data captured by the retina is first processed by the thalamus , which in turn innervates the amygdala ( green regions ) . the amygdala integrates memory information from the hippocampus and context and autonoetic consciousness from the cortex ( ledoux 2000 ; zald 2003 ; phelps and ledoux 2005 ) ( e.g. , orbitofrontal cortex , anterior cingulate cortex ) . additionally , the amygdala reciprocally innervates various brain regions including locus coeruleus , bnst ( bed nucleus of the stria terminalis ) , anterior insula , and hypothalamus ( paulus and stein 2006 ; mathew et al . serotonergic circuit ( red arrows ) : serotonin is released from pre - synaptic neurons within the dorsal and median raphe nuclei on the midline of the brainstem . serotonergic neurons innervate regions of the cerebellum , thalamus , hippocampus , hypothalamus , basal ganglia , frontal cortex , and amygdala ( kandel et al . 2000 ) information encoding a threatening stimulus enters an individual s brain , undergoes context - dependent processing through a sensory neural circuit , and is eventually relayed to the hypothalamus ( shi and davis 2001 ; davis 2006 ) . once activated , the hypothalamus engages the hpa axis : a hormonal feedback system between two brain regions ( hypothalamus and pituitary ) and the adrenal gland ( a small organ on the top of the kidneys ) ( fig . the hpa axis regulates the bodily reaction to stress and constitutes an important facet of fearful or anxious behavior ( jacobson 2005 ) . the neurochemical component of fearful / anxious hpa signaling begins when stressful stimuli prompt the hypothalamus to release the neuropeptide crf ( corticotrophin - releasing factor ) . crf release triggers the proximally located pituitary to release acth ( adrenocorticotrophin - releasing hormone ) into the bloodstream ( jacobson 2005 ) . the adrenal cortex detects the increase of acth in the bloodstream and , in turn , triggers the adrenal gland to release glucocorticoids , including cortisol . a negative feedback loop is completed when glucocorticoids bind the glucocorticoid receptors on the hypothalamus and pituitary , thereby suppressing further release of crf and acth ( mathew et al . abnormalities in these neurochemical hpa interactions are implicated in multiple stress - related disorders , including anxiety ( laue et al . the amygdala , an almond shaped cluster of nuclei within the medial temporal lobes of complex vertebrate brains , is also critical for generating emotional fear and anxiety behaviors ( kandel et al . the amygdala receives inputs from the sensory systems ( e.g. , auditory , visual ) via the thalamus and integrates these stimuli with cortical processing ( context , memory , and conscious self - regulation ) before generating efferent signals that contribute to the threat response ( fig . correlative experiments linking functional imaging in the brain with fear conditioning emphasize the key role played by the amygdala in fear and anxiety behaviors . fear conditioning , or pavlovian fear conditioning , is a form of learning in which a neutral stimulus ( conditioned stimulus ) elicits fearful or anxious behavior after association with an aversive event ( unconditioned stimulus ) ( pavlov 1927 ) . ledoux and others surmise these two stimuli ( conditioned and unconditioned ) are integrated within the amygdala circuitry via synaptic plasticity and the hebbian processes ( johansen et al . , pharmaceutical attempts to treat anxiety disorders by modulating neurotransmitter activity have had mixed success . in 1904 , a barbiturate that soon became the drug of choice in private nervous clinics ( shorter 1997 ) . barbiturates act mostly by enhancing gaba signaling by binding gaba receptors on target neurons ( nemeroff 2003 ) . despite its addictive side effect , veronal s initial success led to the development of chemically similar anxiolytics ( anxiety - reducing compounds ) . throughout the past century , chemists have produced various classes of anxiolytics ( e.g. , beta - blockers , tricyclic anti - depressants , benzodiazepines ) in an attempt to improve efficacy while minimizing detrimental side effects ( barlow and durand 2011 ) . a recent meta - analysis of multiple classes of drug treatments for general anxiety disorder determined that fluoxetine ( prozac ) , a selective serotonin reuptake inhibitor ( ssri ) , elicits the highest patient response to treatment ( baldwin et al . ssris block the reuptake of serotonin into the presynaptic nerve terminals , thereby increasing the synaptic concentration of serotonin ( fig . 2000 ) . of patients suffering from anxiety , 63 % report amelioration of symptoms when treated with prozac ( baldwin et al . this high efficacy has made prozac the primary , first - line treatment for anxiety disorders ( koen and stein 2011).fig . there are 2 mammalian tph isoforms : tph-1 and tph-2 ( walther al . 2003 ) . the vmat vesicular monoamine transporter pumps 5-ht from the cytoplasm into either small synaptic vesicles or dense core vesicles ( liu and edwards 1997 ) . there are 2 mammalian vmat proteins : vmat1 , found in neuroendocrine cells , and vmat2 , expressed in all cns serotonergic neurons ( erickson et al . 5-ht1 , 5-ht2 , 5-ht4 , 5-ht5 , 5-ht6 , 5-ht7 are gpcr g - protein - couple receptors ( hartig 1997 ) . 5-ht3 is a ligand - gated sodium channel with no orthologue in the invertebrates ( hanna et al . there are 2 forms : mao a and mao b ( bach et al . 1988 ) . serotonin ( 5-ht ) is synthesized by 2 tryptophan hydroxylase homologues : dtrhn ( hydroxylates tyryptophan ) and dtphu ( hydroxylates both tryptophan and phenylalanine ) ( neckameyer and white 1992 ; neckameyer et al . 5-ht is packaged into vesicles with dvmat , homologous to mammalian vmat1 and vmat2 ( greer et al . 2005 ) . 4 classes of serotonin receptors have been identified : 5-ht7dro is functionally similar to 5ht7 ( becnel et al . 5-ht is removed from the synaptic cleft by dsert , homologous to mammalian sert ( demchyshyn et al . 5-ht is packaged with vmat , homologous to mammalian vmat1 and vmat2 ( duerr et al . four are g - protein coupled receptors ( gpcr ) : ser-4 resembles 5ht1 ( olde and mccombie 1997 ) , ser-5 has only sequence similarity to 5-ht6 ( hapiak et al . 2009 ) , ser-1 , a 5ht2-like receptor ( hamdan et al . 1999 ) and ser-7 , homologous to 5-ht7 mammalian receptor ( hobson et al . mod-1 is a 5-ht gated chloride channel not found in mammals ( ranganathan et al . 5-ht is removed from the synaptic cleft by mod-5 , a serotonin reuptake transporter ( cooper et al . once removed from the cleft , 5ht is degraded by an enzyme homologous to monoamine oxidase ( weyler 1992 ) conservation of serotonin pathway machinery . a mammalian serotonin pathway . there are 2 mammalian tph isoforms : tph-1 and tph-2 ( walther al . 2003 ) . the vmat vesicular monoamine transporter pumps 5-ht from the cytoplasm into either small synaptic vesicles or dense core vesicles ( liu and edwards 1997 ) . there are 2 mammalian vmat proteins : vmat1 , found in neuroendocrine cells , and vmat2 , expressed in all cns serotonergic neurons ( erickson et al . 5-ht1 , 5-ht2 , 5-ht4 , 5-ht5 , 5-ht6 , 5-ht7 are gpcr g - protein - couple receptors ( hartig 1997 ) . 5-ht3 is a ligand - gated sodium channel with no orthologue in the invertebrates ( hanna et al . there are 2 forms : mao a and mao b ( bach et al . 1988 ) . serotonin ( 5-ht ) is synthesized by 2 tryptophan hydroxylase homologues : dtrhn ( hydroxylates tyryptophan ) and dtphu ( hydroxylates both tryptophan and phenylalanine ) ( neckameyer and white 1992 ; neckameyer et al . 5-ht is packaged into vesicles with dvmat , homologous to mammalian vmat1 and vmat2 ( greer et al . 2005 ) . 4 classes of serotonin receptors have been identified : 5-ht7dro is functionally similar to 5ht7 ( becnel et al . 5-ht is removed from the synaptic cleft by dsert , homologous to mammalian sert ( demchyshyn et al . 5-ht is packaged with vmat , homologous to mammalian vmat1 and vmat2 ( duerr et al . four are g - protein coupled receptors ( gpcr ) : ser-4 resembles 5ht1 ( olde and mccombie 1997 ) , ser-5 has only sequence similarity to 5-ht6 ( hapiak et al . 2009 ) , ser-1 , a 5ht2-like receptor ( hamdan et al . 1999 ) and ser-7 , homologous to 5-ht7 mammalian receptor ( hobson et al . mod-1 is a 5-ht gated chloride channel not found in mammals ( ranganathan et al . 5-ht is removed from the synaptic cleft by mod-5 , a serotonin reuptake transporter ( cooper et al . once removed from the cleft , 5ht is degraded by an enzyme homologous to monoamine oxidase ( weyler 1992 ) the high success rate of ssris strongly suggests a biological link between anxiety and the serotonin pathway ; however , the details of this link remain unclear . serotonin ( 5ht ) , a monoamine neurotransmitter , is utilized by most of the animal kingdom as a neural circuit modulator ( hen 1993 ) . in mammals , serotonin modulates a wide range of behaviors , including pain perception , sleep , aggression , feeding , and mood ( hen 1993 ; weiger 1997 ; carre - pierrat et al . serotonergic neurons originate from the dorsal and median raphe nuclei in the brain stem and project into multiple forebrain and limbic structures ( e.g. , amygdala , thalamus , hypothalamus , hippocampus , and frontal cortex ) , thereby constituting a serotonin circuit ( fig . fear and anxiety stimuli selectively activate serotonergic neurons in the dorsal raphe nucleus , which then project into the amygdala and the hypothalamic portion of the hpa axis ( goddard and charney 1997 ; lowry 2002 ; bauman and amaral 2005 ; spannuth et al . one model to explain ssri efficacy reasons that serotonin can suppress the hyperactivation of the amygdala ( harmer et al . while ssris might have multiple neural circuit targets , their treatment successes emphasize a crucial role for serotonin in modulating human anxiety - related behavior . the molecular machinery governing serotonin signaling across neurons is well characterized and also well conserved among vertebrates and invertebrates ( fig . 2 ) . serotonin is synthesized from tryptophan by tryptophan hydroxylase in the cytoplasm of the presynaptic serotonergic neuron . these vesicles fuse with the cell membrane and the stored neurotransmitter is released into the synaptic cleft and bound by serotonin receptors on the surface of postsynaptic cells . the signal is terminated when unbound serotonin is reabsorbed back into the presynaptic cell by reuptake transporters , thereby limiting the spread of 5ht concentration . finally , a catabolic enzyme , monoamine oxidase , metabolizes 5ht to non - active aldehyde derivatives ( horvitz et al . 1982 ; kandel et al . 2000 ; chase and koelle 2007 ) . discrepancies in the activity of the serotonin pathway correlate with anxious behaviors . combining functional imaging with genomic analysis , researchers have associated a short allele of the promoter ( 5-httlpr ) for the human serotonin transporter gene ( slc6a4 ) with anxiety - related personality traits , for example , fear condition - ability ( hariri et al . individuals carrying one or two copies of the truncated version of slc6a4 exhibit reduced serotonin signaling and , interestingly , greater amygdala activity . consistent with these findings , high - anxiety subjects carrying the short allele of the serotonin transporter promoter , 5-httlpr , exhibit elevated amygdala activity during an anxiety - inducing public speaking task compared to carriers of the long allele ( furmark et al . these studies suggest 5ht can suppress the activity in the amygdala that is relevant to anxiety . while these studies implicate a strong link between serotonergic circuits and fear and anxiety behavior , less is known about how serotonin modulates neural circuits to affect these behaviors ( baldwin et al . 2011 ; koen and stein 2011 ) . treatments that deplete serotonin levels have produced both anxiety - reducing and anxiety - inducing behavior ( handley 1995 ) . this paradox is further illustrated by the fact that long - term exposure to ssris reduces anxiety , while acute exposure ( first 2 weeks of treatment ) is often accompanied with anxiety - inducing behavior in both humans and animal models ( gordon and hen 2004 ; grillon et al . furthermore , it should be noted that the significance of ssri efficacy , in regard to its antidepressant benefits , is currently being debated . irving kirsch and colleagues performed a meta - analysis of published and unpublished data ( obtained from the fda via the freedom of information act ) analyzing 35 placebo - controlled trials of four popular ssri drugs ( fluoxetine , venlafaxine , nefazodone , paroxetine ) . the study concluded that , compared with placebo , these new - generation ssri antidepressants do not produce clinically significant improvements in patients with mild or moderate depression , but do show significant effects only in severely depressed patients ( kirsch et al . it remains unclear what biological variation generates this multitude of behavioral responses to pharmacological intervention . taken together , these ssri - related incongruities emphasize our incomplete understanding of the complexities of serotonin signaling as it pertains to cognitive behaviors . while functional imaging and genetic analysis of humans have provided a neuroanatomical map of serotonin - modulated behaviors , attempts to elucidate a circuit level understanding of anxious behavior have only been marginally successful due to the cellular complexity of the mammalian brain . this is understandable ; the human brain consists of approximately 100 billion interconnected neurons , which are largely inaccessible for analysis ( kandel et al . fortunately , the serotonin machinery is largely conserved throughout the animal kingdom ( fig . 2 ) , facilitating the study of this intricate pathway in biological systems that are more tractable than the mammalian brain . ideally , such a model should be capable of executing sophisticated behaviors that are modulated by serotonin signaling . small invertebrate model systems such as d. melanogaster and c. elegans are exceptionally suited to this task ( kaletta and hengartner 2006 ; iliadi 2009 ; pungaliya et al . 2009 ; dimitriadi and hart 2010 ; kullyev et al . 2010 ) . the nematode , c. elegans , with just 302 neurons connected by identified chemical and electrical synapses ( white et al . 1986 ) is ideally suited to unravel conserved serotonin signaling pathways driving whole animal behavior . in the worm , serotonin is made from 6 neuronal types ( hsn , nsm , vc4 , vc5 , adf , and cp1 - 6 ) by a tryptophan hydroxylase ( tph-1 ) with strong homology to mammalian tph-1 ( sze and victor et al . 2 ) , a c. elegans vmat packages serotonin into vesicles ( duerr et al . 1999 ) . also , c. elegans neurons express at least five different serotonin receptors : ser-4 ( 5-ht1-like ) , ser-1 ( 5-ht2-like ) , ser-5 ( 5-ht6-like ) , ser-7 ( 5-ht7-like ) , and mod-1 ( a 5-ht gated cl- channel not found in mammals ) ( olde and mccombie 1997 ; hamdan et al . 1999 ; ranganathan et al . 2000 ; hobson et al . 2003 ; hapiak et al . moreover , mod-5 , the worm homologue of mammalian sert , acts to remove serotonin from synapses ( ranganathan et al . interestingly , fluoxetine , a commonly used ssri that blocks mammalian sert , can also block mod-5 ( c. elegans sert homologue ) , among other targets in the worm ( kullyev et al . 2010 ) . in c. elegans , serotonin signaling plays a significant role in mediating avoidance to the repellent odorant , octanol ( segalat et al . as previously mentioned , the perception and appraisal of a stressful or repellent stimulus is the first component of anxious or fearful behavior in humans . before an organism can determine an appropriate behavioral output , the threatening stimulus must be accurately processed via the sensory system . as described below , elegant studies in c. elegans , which seek to unravel the mechanisms of avoidance behavior , provide insights into serotonin - mediated sensory activity . the studies reveal specific interactions of different serotonin receptors acting in concert with peptidergic and additional monoaminergic pathways ( chao et al . the komunieki laboratory has utilized sensory - mediated avoidance behavior , driven by a well - defined neuronal circuit , to dissect the pathways governing serotonin - mediated stimulus appraisal ( chalfie et al . . the group has identified cell - specific roles for serotonin receptors facilitating avoidance to 30 % octanol , a c. elegans repellent . to administer the repellent , a hair is taped to a toothpick , dipped in 30 % octanol , and placed in front of a worm exhibiting forward sinusoidal locomotion ( harris et al . using mutant analysis and cell - specific rnai knockdown , harris and colleagues determined that mod-1 , the serotonin gated cl- channel , is necessary in the aib interneuron to properly receive sensory input and that ser-1 , the g - protein - coupled serotonin receptor , is required in the ria / ring motor neuron to properly innervate head muscle . upon determining the repellent was sensed by the ash amphid sensory neuron but not finding a known serotonin receptor in ash , the group performed an rnai knockdown of previously uncharacterized c. elegans biogenic amine receptors and identified ser-5 to be essential for the serotonergic modulation of aversive responses to 30 % octanol ( harris et al . ser-5 is a novel serotonin receptor that , based on sequence similarity and conserved functional motifs , is most closely related to mammalian 5-ht6 receptors . together , these findings identify multiple points within a neurological circuit from which serotonin can exert control on the response to an aversive agent . additional reports from this group have described how peptide signaling and octopamine signaling integrate with this serotonin circuit to provide a dynamic system for modulating sensory - mediated locomotion ( harris et al . harris and colleagues found octopamine signaling inhibits the serotonin sensitized ash neuron from initiating aversive behavior . octopamine is an invertebrate biogenic amine that is , structurally , closely related to norepinephrine ( roeder et al . 2003 ) . the group found that octopamine exerts this negative control in the ash neuron via three alpha - adrenergic - like octopamine receptors : octr-1 , ser-3 , and ser-6 ( mills et al . interestingly , this group also identified a peptidergic pathway that exerts a positive effect on ash activity . cell - specific rnai knockdown determined that c. elegans aversive response requires a suite of peptides encoded by the neuropeptide , nlp-3 . nlp-3 signals via the receptor npr-17 and acts in coordination with a suite of g alpha genes that overlap with octopamine signaling ( harris et al . to summarize , these studies describe in impressive detail the manner that a sensory circuit is modulated by multiple monoaminergic and peptidergic signaling pathways . one of the key elements of fear / anxiety behavior in the vertebrate is the ability to appropriately respond to an external threat . one aspect of a threat response , aggression , particularly in the context of a threat from a con - specific , has been modeled in a simple invertebrate , drosophila . the fly brain is composed of multiple cell clusters , many of which include varicosities containing serotonin . a complete account of serotonin positive neurons was first mapped in drosophila ( valles and white 1988 ) using immunocytochemical techniques that were originally used to identify serotonin circuits in non - drosophila insects ( e.g. , grasshoppers , cockroaches ) ( bishop and oshea 1983 ; nassel and klemm 1983 ; taghert and goodman 1984 ) . though the numbers of drosophila serotonin positive neurons identified represent a small fraction of the total neuronal population , their arborization pattern indicates a crucial role for serotonin . the larval serotonin ( 5-ht ) pattern consists of 84 neurons distributed in clusters of one to five neurons labeled sp1 , sp2 , ip , lp1 , lp2 , se1 , se2 , se3 , t1 , t2 , t3 , a1 - 7 , and a8 ( valles and white 1988 ) . further , these clusters are arranged across the two brain hemispheres and the projections fasciculate around the central commissure and innervate the contralateral brain hemisphere ( valles and white 1988 ; lundell and hirsh 1994 ) . in the periphery , 5-ht positive neurons project to the pharynx , proventriculus , the midgut , and the ring gland ( larval endocrine organ ) . in the adult , two new 5-ht clusters ( lp2a and lp2b ) are added and these are located in the brain near the medulla neuropil . 5-ht is also found in the ellipsoid body of the central complex , all neuropil regions of the optic ganglia , the thoracic ganglia , and the fused abdominal ganglia ( monastirioti 1999 ) . interestingly , in drosophila and likely other animals , 5-ht is found in a number of neurons that can not synthesize 5-ht ( valles and white 1988 ) . this result suggests that serotonin re - uptake machinery is expressed in non - serotonin producing cells allowing this key neuromodulator to influence additional circuits ( circuits not identified by 5-ht immunoreactivity ) . 2b ) , serotonin is synthesized by two tryptophan hydroxylases and packaged into vesicles by dvmat ( neckameyer and white 1992 ; greer et al . these receptors are homologous to mammalian receptors in regard to sequence and downstream activity . 5-ht1adro and 5-ht1bdro are homologues of mammalian 5ht1 and are also positively coupled to adenylate cyclase ( witz et al . 5-ht7dro is functionally similar to vertebrate 5-ht7 and upon activation increases camp signaling ( saudou et al . finally , a serotonin transporter , dsert , has been identified that has strong sequence and functional homology to the mammalian sert . this transporter functions to remove serotonin from the synaptic cleft and is highly sensitive to cocaine and anti - depressants ( corey , quick et al . this conserved signaling machinery and powerful genetics make drosophila a suitable model to dissect serotonin - mediated behavior . as mentioned , we focus our discussion to one such behavior , a fighting response to an environmental stressor . drosophila exhibits aggressive behavior ( jacobs 1978 ) , which is ethologically ( skrzipek et al . 1979 ; lee and hall 2000 ) and evolutionarily well established ( boake and hoikkala 1995 ; boake et al . 1998 ) . male flies under appropriate conditions will occupy a food patch and defend it against other males ( hoffman and cacoyianni 1989 ; hoffman and cacoyianni 1990 ) . this observation has been transformed into a behavioral assay where pairs of 5- to 7-day - old male flies are placed together in a small chamber ( 1.6 cm diameter , 1.1 cm height ) . a careful filming of these encounters found unambiguous offensive fighting elements including wing threat , lunging , holding , tussling , and boxing ( dierick and greenspan 2007 ) . interestingly , serotonin pharmacology has been found to play an important role in mediating this aggressive fighting behavior . flies exposed to 5-htp , a serotonin precursor , experience a maximum increase in serotonin ( 5-ht ) expression 3 days after treatment , while mtp , an inhibitor , blocked 5-ht signaling 4 days after exposure . elevating 5-ht pharmacologically increases aggression in flies , while silencing the serotonergic circuits makes the flies behaviorally unresponsive to the drugs ( dierick and greenspan 2007 ) . this study was further extended by testing the acute modulation of 5-ht neurotransmission ( alekseyenko et al . expression of temperature - sensitive dtrpa1 channels allowed acute activation of serotonergic neurons , which caused flies to escalate fights faster and at higher intensity . in contrast , disruption of 5-ht signaling interferes with the male s ability to fight and create a dominance relationship ( alekseyenko et al . behavioral traits , such as aggression , are biologically complex , and therefore , establishing a connection between the drosophila aggression phenotype and its underlying genotype often necessitates an understanding of how specific genes orchestrate the development and physiology of the neural circuits that govern the behavioral action ( siwicki and kravitz 2009 ) . as mentioned above , drosophila aggression is a male - specific phenotype , which suggests aggression is controlled by a sexually dimorphic neural circuit . ongoing work from the kravitz laboratory explores how genetic interactions in certain neurons specify the development of these sexually dimorphic neural circuits . fruitless ( fru ) , a zinc - finger - like transcription factor , is a part of the sex determination hierarchy of genes in drosophila that control both morphological and behavioral sexual dimorphisms ( siwicki and kravitz 2009 ) . transcripts of fru undergo complex sex - specific splicing early in drosophila development , ultimately yielding a male - specific fru isoform , fru , and a female - specific fru isoform , fru . the expression of fru is necessary and sufficient to specify the neural systems that generate the male patterns of aggressive behavior ( demir and dickson 2005 ; vrontou et al . interestingly , a distinctive group of large serotonergic neurons at the posterior tip of the abdominal ganglion ( sabg neurons ) express multiple versions of the fru isoform ( lee and hall 2001 ; lee et al . the presence of multiple fru isoforms complicates analysis , but the implication is that different combinations of fru gene products specify particular components of the neural circuitry concerned with sexually dimorphic behaviors ; then , once developed , this circuit can be modulated by serotonergic transmission to effect aggression ( siwicki and kravitz 2009 ) . of course , further cell - specific experiments are needed to define serotonin s contribution to the neural circuits that drive aggression . we expect the identity of these serotonergic neurons coupled with genetic marking tools ( e.g. marcm ) should reveal additional modulators of the aggression circuitry , including but not limited to peptidergic or other monoaminergic pathways . together , these studies highlight the importance of serotonin signaling and position d. melanogaster as a model to study the role of serotonin in cognitive processes relevant to the mammalian brain . many of these processes are contingent on the unique neuroarchitecture of complex human cognition and physiology . as stated , one could never accurately model the entire panoply of a human emotional behavior , like anxiety , in an organism that lacks human - like emotional and cerebral capacity . however , we speculate that it is possible to reduce a human emotional behavior down to its component parts and analyze the biological machinery governing individual components using a simpler genetic model . of course , this analysis is only relevant if the biological machinery in question is conserved between humans and the model organism . three interrelated processes help govern human anxiety : the hpa axis , amygdala processing , and serotonin neuromodulation . neuroanatomical ensembles ( hpa axis and amygdala ) are vertebrate specific and , therefore , are not candidates for analysis using invertebrate model organisms . however , serotonergic transmission is remarkably well conserved between d. melanogaster , c. elegans , and h. sapiens ( fig . 2 ) , despite approximately 550 million years of independent evolutionary trajectories ( valentine et al . this conservation allows the possibility of utilizing nematodes and flies , with their tractable nervous systems , to explore the specifics of serotonin circuitry as it relates to complex behavioral outputs . serotonin signaling certainly modulates affective disorders ( i.e. , anxiety ) in mammals and , as described , also modulates aversive behavior and aggression in invertebrate model organisms . aversion and aggression do not serve as complete models of anxiety , but rather represent components of this complex human behavior . by observing aversion and aggression in model systems while simultaneously perturbing the genetic / cellular components of the serotonin pathway , one can begin to resolve the complexity of this neurotransmitter s actions and its intricate interactions with additional signaling pathways . this level of analysis is currently unavailable to mammalian systems , and results from the invertebrate models have the potential to inform the broader neuroscience community . treating anxiety disorders with selective serotonin reuptake inhibitors has demonstrated efficacy ; however , the adverse side effects associated with ssris and their habit of acute anxiety - induction suggest that ssris have multiple effects on neurological circuits . a circuit level comprehension of serotonin neuromodulation will greatly enhance our ability to understand and treat anxiety - related disorders . as discussed , serotonin circuits in c. elegans are controlled at multiple points by cell - specific receptors and further modulated by octopamine and neuropeptide signaling pathways ( harris et al . 2009 , 2010 ) . perhaps similar circuit level interactions are governing serotonin signaling in the mammalian amygdala , as it pertains to anxiety - related behaviors . the amygdala , specifically the lateral amygdala , has been identified as a key anatomical brain region where circuits involved in the processing of threatening stimulus are housed ( ledoux 2000 ; kim and jung 2006 ) . in all mammalian species studied , the lateral amygdala exhibit exceptionally dense serotonergic innervation , largely originating from the dorsal raphe nucleus ( smith and porrino 2008 ; bonn et al . it is possible that particular serotonin receptors acting in specific neurons can modulate the manner conditioned and unconditioned stimulus are processed ( i.e. , coupled ) within the serotonergic circuits in the amygdala . intricate interactions between peptidergic ( e.g. , neuropeptide y ) and monoaminergic systems ( e.g. , dopamine , norepinephrine ) may couple with serotonin signaling ( via distinct serotonin receptors ) to provide a more dynamic and sophisticated response to environmental stressors . as circuit level details emerge from invertebrate model systems , candidate interactions are identified , which can then be explored within serotonergic circuits of the lateral amygdala in mammalian systems . we propose that further circuit level analysis of serotonin - mediated behaviors in invertebrate systems will increase our comprehension of serotonin - influenced fearful and anxiety - related behaviors in humans , such as ptsd , phobias , and panic attacks .

fear , a reaction to a threatening situation , is a broadly adaptive feature crucial to the survival and reproductive fitness of individual organisms . by contrast , anxiety is an inappropriate behavioral response often to a perceived , not real , threat . functional imaging , biochemical analysis , and lesion studies with humans have identified the hpa axis and the amygdala as key neuroanatomical regions driving both fear and anxiety . abnormalities in these biological systems lead to misregulated fear and anxiety behaviors such as panic attacks and post - traumatic stress disorders . these behaviors are often treated by increasing serotonin levels at synapses , suggesting a role for serotonin signaling in ameliorating both fear and anxiety . interestingly , serotonin signaling is highly conserved between mammals and invertebrates . we propose that genetically tractable invertebrate models organisms , such as drosophila melanogaster and caenorhabditis elegans , are ideally suited to unravel the complexity of the serotonin signaling pathways . these model systems possess well - defined neuroanatomies and robust serotonin - mediated behavior and should reveal insights into how serotonin can modulate human cognitive functions .

obesity is recognized as a major cause of morbidity and mortality and obesity rates have shown dramatic increases in both adults and school - aged populations over the last 30 years . the rate of obese adolescents aged 12 - 19 in the united states increased from 5% in 1980 to nearly 18% in 2010 . adolescence is a critical stage for psychological and physical development since any excessive weight gained in this stage may be easily transferred to adulthood and may affect the quality of life . 80% of obese adolescents become obese adults with various health problems and long - term risk of diseases related to their obese state . summer break represents about 23% of a typical american school year and is the largest consecutive period of out - of - school time . during the summer break , the decline in physical fitness and weight gain in adolescents are results of physical inactivity as well as increased food intake . previous research shows that the increase in body mass index ( bmi ) of children during the summer is roughly twice as fast as that in the school year . during the summer break , children tend to live a sedentary lifestyle while having an unhealthy calorie intake with larger portion sizes , fewer nutrients , and more energy dense foods than the foods they eat at school . it has been suggested that schools employ an effective approach in improving the level of physical activity and the diet of their students during the school year , therefore , out - of school time , especially long summer breaks , may be more problematic for weight management . compared to the school year when children are influenced by both academic and non - academic environments , students spend their breaks solely in a non - academic environment . consequently , their physical activity and diet may be affected by the socio - economic status of their family during the summer break . in underprivileged groups such as racial and/or ethnic minorities , those living in poverty and those with low levels of education compared to non - hispanic whites , african - american and hispanic children showed a higher prevalence of obesity due to more obesity promoting behaviors such as skipping breakfast , low intake of fruits and vegetables , and physical inactivity . family income had a larger effect for hispanic children than for non - hispanic whites and african - americans . obesity and physical levels of fitness are believed to affect adolescents psychological outcomes , resulting in low self - esteem and depression [ 15 - 17 ] . higher self - esteem can provide adolescents with a buffer against the impact of negative influences and help them choose better physical health and health - related behaviors such as regular exercise and healthy diet patterns . self - body image satisfaction is also known to be central to emotional well - being , which has a strong influence on eating habits and regular physical activity . several groups have reported success in the loss or prevention of weight gain during the summer break by conducting summer camp studies that incorporate physical activity and diet interventions ; however , no summer intervention studies have been conducted in underprivileged communities . most summer intervention studies targeted children who were socio - economically able to attend camps . children from unprivileged community may have less access to out - of school activities such as sports and summer camps . to our knowledge , none of the studies investigated the relationship between psychosocial outcomes and changes in body composition or physical fitness over the summer break depending on whether the students attended a structured and extended summer school program . therefore , we hypothesized that a structured and extended school program during the summer may reduce weight gain and loss of physical fitness in underprivileged adolescents by dampening the effects of the out - of - school environment and psychosocial outcomes , thus , reducing the prevalence of obesity in this population . the primary purpose of this study was to determine the effects of psychosocial outcomes and a five week summer school participation , designed to increase physical activity and reduce dietary intake , on changes in body composition and physical fitness in underprivileged high school students . participants were recruited from the freshman and sophomore student populations ( ages 15 - 17 yrs ) in a high school located in an underprivileged hispanic area in southern texas . in this high school , all freshman students participate in a compulsory 5 week summer school program in their first summer break . therefore , freshman participants were considered summer school attendants while sophomore participants were summer school non - attendants . the number of students in each grade was about 100 students . excluding students who had known chronic diseases , were taking medication that could alter metabolic , cardiovascular or immune function , had musculoskeletal limitations , or were not willing to volunteer for this study , a total of 145 high school students performed the pre test , but only 138 students ( 70 summer school attendants vs. 68 non - attendants ; 71 males vs. 67 females ) completed both pre and post data collection . seven students that could not participate in the post test due to sickness ( three students ) , missing school ( three students ) , and changing schools ( one student ) were excluded from data analyses . the experimental protocol [ tamiu irb 2011 - 04 - 05 ] was approved by the institutional review board of texas a&m international university , and participants provided written informed consent . the summer school program was held five days a week during regular school hours ( 08:00 - 16:00 ) for 5 weeks . this was exactly the same schedule as the regular school days , with an hour of physical activity every day . during the physical activity period , students conducted aerobic exercises , resistive exercises with body weight ( push - up , sit - up , etc ) and a variety of games . since the high school was located in an underprivileged community , all summer school attendants were provided with breakfast ( 250 - 400 kcal ) and lunch ( 700 - 900 kcal ) . for both the summer school attendants and non - attendants , all variables were measured and assessed before ( pre : last two weeks of school year in may ) and after ( post : first two weeks of new school year in august ) the summer break . height and weight were measured to the nearest 0.1 cm and 0.1 kg , respectively , with the participants wearing indoor clothes without shoes . percent body fat ( % fat ) was estimated using the data obtained with a skin fold caliper ( beta technology incorporated , cambridge , md ) . the thickness of the skin was measured and used to calculate percent body fat based on previous research . for this study , the males were assessed in the following three areas : chest , abdominals , and front thigh . for the females , the following areas were assessed for percent body fat : the triceps , supra - spinale , and the front thigh . the skin fold measurements and equations used for this study were as follows : percent fat = ( ( 4.95/bd)-4.5)100 bd : body density several other fitness tests such as the queens college step test , push - ups , and the sit - and - reach test were used to assess cardiovascular function , muscular strength and endurance , and flexibility , respectively . the queens college step test was used to estimate cardiovascular function ( vo2max ) and is known as a valid and reliable measure for adolescents [ 23 - 25 ] . chatterjee and colleagues reported that estimated vo2max from the queens college step test using a standardized procedure was highly correlated with actual vo2max in male and female participants whose mean height were 166 cm and 157 cm , respectively ( r = .95 ) . mean heights for male and female students in this study were 172 and 156 cm , respectively , therefore , the queens college step test was conducted without height and speed adjustment . each participant stepped up and down on the 41.3 cm height platform , at a rate of 22 steps per minute for females and 24 steps per minute for males , for a total of 3 minutes . the speed of the step exercise was set up using a metronome at 96 and 88 beats per minute for male and female students , respectively . the participants immediately stopped upon completion of the test , and their heart rates were counted for 15 seconds from 5 - 20 seconds of recovery . a prediction of vo2max was calculated using the following formula ; male : vo2max ( ml / kg / min ) = 111.33 - 0.42 heart rate ( bpm ) female : vo2max ( ml / kg / min ) = 65.81 - 0.1847 heart rate ( bpm ) the push - up test was performed according to the procedures outlined in the fitnessgram reference guide . the sit - and - reach test was performed using a commercially available metal sit - and - reach box . participants sat on the floor in the long - sitting position with knees straight and with the soles of their feet against the box and performed the test according to acsm guidelines . a survey with an approximate time of 20 minutes the survey captured socio - economic data including sex , level of parents education , living status , caregiver status , annual house hold income , and frequency of fast food consumption . a self - esteem scale by rosenberg and the self - body shape questionnaire developed by copper , taylor , cooper , & fairburn were used in this study to estimate psychological data such as self - esteem and self - body shape image . power calculations were conducted to assess the overall change in body composition within the group using results from smith et al . . the results indicated that 54 participants in each group were needed to detect a significant increase in body composition during the summer break with 90% power at = .05 . two - way analysis of variance ( anova ) with repeated measures were used to analyze changes in body composition and levels of physical fitness during the summer break as well as the effects of socio - economic status on changes in body composition and levels of physical fitness . pearson correlation analysis was performed to establish a relation between psychosocial outcomes and changes in physical fitness during the summer break . participants were recruited from the freshman and sophomore student populations ( ages 15 - 17 yrs ) in a high school located in an underprivileged hispanic area in southern texas . in this high school , all freshman students participate in a compulsory 5 week summer school program in their first summer break . therefore , freshman participants were considered summer school attendants while sophomore participants were summer school non - attendants . the number of students in each grade was about 100 students . excluding students who had known chronic diseases , were taking medication that could alter metabolic , cardiovascular or immune function , had musculoskeletal limitations , or were not willing to volunteer for this study , a total of 145 high school students performed the pre test , but only 138 students ( 70 summer school attendants vs. 68 non - attendants ; 71 males vs. 67 females ) completed both pre and post data collection . seven students that could not participate in the post test due to sickness ( three students ) , missing school ( three students ) , and changing schools ( one student ) were excluded from data analyses . the experimental protocol [ tamiu irb 2011 - 04 - 05 ] was approved by the institutional review board of texas a&m international university , and participants provided written informed consent . the summer school program was held five days a week during regular school hours ( 08:00 - 16:00 ) for 5 weeks . this was exactly the same schedule as the regular school days , with an hour of physical activity every day . during the physical activity period , students conducted aerobic exercises , resistive exercises with body weight ( push - up , sit - up , etc ) and a variety of games . since the high school was located in an underprivileged community , all summer school attendants were provided with breakfast ( 250 - 400 kcal ) and lunch ( 700 - 900 kcal ) . for both the summer school attendants and non - attendants , all variables were measured and assessed before ( pre : last two weeks of school year in may ) and after ( post : first two weeks of new school year in august ) the summer break . height and weight were measured to the nearest 0.1 cm and 0.1 kg , respectively , with the participants wearing indoor clothes without shoes . percent body fat ( % fat ) was estimated using the data obtained with a skin fold caliper ( beta technology incorporated , cambridge , md ) . the thickness of the skin was measured and used to calculate percent body fat based on previous research . for this study , the males were assessed in the following three areas : chest , abdominals , and front thigh . for the females , the following areas were assessed for percent body fat : the triceps , supra - spinale , and the front thigh . the skin fold measurements and equations used for this study were as follows : percent fat = ( ( 4.95/bd)-4.5)100 bd : body density several other fitness tests such as the queens college step test , push - ups , and the sit - and - reach test were used to assess cardiovascular function , muscular strength and endurance , and flexibility , respectively . the queens college step test was used to estimate cardiovascular function ( vo2max ) and is known as a valid and reliable measure for adolescents [ 23 - 25 ] . chatterjee and colleagues reported that estimated vo2max from the queens college step test using a standardized procedure was highly correlated with actual vo2max in male and female participants whose mean height were 166 cm and 157 cm , respectively ( r = .95 ) . mean heights for male and female students in this study were 172 and 156 cm , respectively , therefore , the queens college step test was conducted without height and speed adjustment . each participant stepped up and down on the 41.3 cm height platform , at a rate of 22 steps per minute for females and 24 steps per minute for males , for a total of 3 minutes . the speed of the step exercise was set up using a metronome at 96 and 88 beats per minute for male and female students , respectively . the participants immediately stopped upon completion of the test , and their heart rates were counted for 15 seconds from 5 - 20 seconds of recovery . a prediction of vo2max was calculated using the following formula ; male : vo2max ( ml / kg / min ) = 111.33 - 0.42 heart rate ( bpm ) female : vo2max ( ml / kg / min ) = 65.81 - 0.1847 heart rate ( bpm ) the push - up test was performed according to the procedures outlined in the fitnessgram reference guide . the sit - and - reach test was performed using a commercially available metal sit - and - reach box . participants sat on the floor in the long - sitting position with knees straight and with the soles of their feet against the box and performed the test according to acsm guidelines . a survey with an approximate time of 20 minutes the survey captured socio - economic data including sex , level of parents education , living status , caregiver status , annual house hold income , and frequency of fast food consumption . a self - esteem scale by rosenberg and the self - body shape questionnaire developed by copper , taylor , cooper , & fairburn were used in this study to estimate psychological data such as self - esteem and self - body shape image . power calculations were conducted to assess the overall change in body composition within the group using results from smith et al . . the results indicated that 54 participants in each group were needed to detect a significant increase in body composition during the summer break with 90% power at = .05 . two - way analysis of variance ( anova ) with repeated measures were used to analyze changes in body composition and levels of physical fitness during the summer break as well as the effects of socio - economic status on changes in body composition and levels of physical fitness . pearson correlation analysis was performed to establish a relation between psychosocial outcomes and changes in physical fitness during the summer break . table 1 . shows changes in body composition and levels of physical fitness in summer school attendants and non - attendants over the summer break . summer school attendants showed no significant changes in any of the variables during the summer break . in contrast , summer school non - attendants gained body weight ( 63.6 1.9 kg 65.6 1.9 , p = .003 ) and % body fat ( 24.4 0.7 % 26.17 1.2 , p = .014 ) without significant changes in height during the summer break . estimated cardiovascular fitness ( vo2max ) via the step test also decreased significantly ( 44.3 1.3 ml / kg / min 42.7 1.5 , p = .018 ) . the push - up and sit - and - reach results did not change significantly in both groups over the summer break . effects of socio - economic status on changes in body composition and levels of physical fitness over the summer break are provided in table 2 , 3 and 4 . the numbers for body composition and physical fitness variables reflect the changes during the summer obtained by subtracting values before the summer from those after the summer . each group was further divided into two or three sub - groups depending on socio - economic status and changes in body composition . school attendants , body composition and physical fitness were not affected by socio - economic status during the summer break . in contrast , body composition and physical fitness in summer school non - attendants were influenced by their socio - economic status . summer school non - attendants who had parents that were educated beyond the university level ( p < .05 ) , lived with both parents ( p = .0001 ) , had day time adult caregivers during the summer break ( p = .015 ) , had higher house hold income ( p = .002 ) , or consumed fast - food less than once a week ( p = .0001 ) gained less body fat . a lesser decrease in push - up ability was found in summer school non - attendants who lived with both parents ( p = .012 ) and had fast - food less than once a week ( p = .024 ) . non - attendants who lived with both parents ( p = .0001 ) , had higher house hold income ( p = .014 ) and fast - food consumption less than once a week ( p = .0001 ) showed a smaller decrease in sit - and - reach score compared to their counterparts . the correlations between psychological factors and changes in body composition as well as the levels of physical fitness are demonstrated in table 5 . no psychological factors were correlated with changes in body composition and levels of physical fitness in summer school attendants . in summer school non - attendants , self - esteem was significantly correlated with changes in body composition ( r = -0.542 , p = 0.0001 ) , and sit - and - reach score ( r = 0.399 , p = 0.001 ) . self - body image ( level of dissatisfaction ) was negatively correlated with changes in body composition ( r = 0.370 , p = 0.002 ) , and sit - and - reach score ( r = -0.337 , p = 0.004 ) , indicating that relatively high self - esteem and self - body satisfaction were closely associated with less weight gain . to our knowledge , this is the first study to investigate the combined effects of a structured summer school program and psychosocial outcomes on changes in body composition and physical fitness in hispanic adolescents during the summer break . the primary purpose of this study was to examine whether a student s participation in a summer school program can prevent an increase in their body composition and a decrease in their physical fitness levels . furthermore , the secondary purpose of this study was to investigate whether such changes are associated with socio - economic and psychological status in underprivileged hispanic high school students . to achieve these purposes , we examined changes in body composition and physical fitness during the students summer break and also conducted a survey with the students and their parents to determine their psychosocial outcomes . the summer break represents approximately one quarter of a typical school year and is the largest consecutive out - of - school time . there are many habits and lifestyle changes that may develop during this time , all of which can affect the student s body weight and physical fitness . moreno et al . investigated changes in weight over the school year and the summer break for 5 years and found that the most weight gain occurred during the summer break . an increase in % body fat during this period was more than double the rate during the school year . the results of these two investigations indicate the importance of the summer break for weight management . the results of this study show that students that did not attend summer school experienced significant increases in body weight and % body fat in addition to a decrease in estimated vo2max . for these students , 3 months of summer break seemed to have negative effects on body composition and physical fitness . however , summer school attendants effectively maintained their body composition and levels of physical fitness during the summer break . none of the variables changed significantly for these students during the summer break , with the exception of estimated vo2max , which increased significantly . studies that employed either community based [ 4 , 8 ] or school based intervention found that participants improved or maintained their body composition and physical fitness . in contrast , studies that observed changes in body composition and physical fitness without any intervention reported accelerated weight gain during the summer break [ 5 - 7,17 ] . the reasons for the more rapid increases in body weight and % body fat and the decrease in physical fitness levels in adolescents during the summer break are not clear . potential mechanisms for the accelerated summer weight gain observed and the loss in physical fitness are suggested to be at least partially due to physical inactivity , easy access to unhealthy food , unstructured schedules , and increased sedentary behaviors . even though none of the studies including our investigation directly investigated any of the potential mechanisms above , the combined results from studies with and without summer intervention revealed that the school environment has a protective effect against accelerated weight gain and a decline in physical fitness . the national school lunch program provides students with most of their daily intake of nutrient - dense fruits and vegetables , and a school meal includes less sugar and soda than an out of school meal . schools also offer students the opportunity for physical education , sports , and extracurricular activities . in the present study , summer school attendants participated in regular school programs for five weeks between 8:00am-4:00pm from monday to friday , with an hour of physical activities every day . , it appears that a structured summer school program provides healthy food and the opportunity for physical activity just as provided in the school year and may promote healthy lifestyle habits in students year - round . adolescents who attend summer school have more opportunities to be physically active , which could be the reason for the increase in physical fitness during the summer break . meanwhile , summer school non - attendants are often limited to inactivity at home . in this respect , the results of this study correspond with previous findings that adolescent weight gain during the summer break may be due to physical inactivity . it is known that vo2max decreases by 4 - 20% with physical inactivity of two weeks or more . therefore , physical inactivity might be one of the causes for the vo2max decrease in summer school non - attendants in this study . summer school attendants effectively maintained their body composition and level of physical fitness during the summer break , which indicates the possibility of cancelling out disadvantages of the summer break . previous research investigated the association of the prevalence rate of obesity in adolescents with socio - economic status such as annual house hold income , sex , race and ethnicity . the studies reported that the rates of obesity were greater in african american and hispanic adolescents than in their white counterparts . the higher rate of obesity in minority adolescents is known to be related to socioeconomic status in complex ways . we found in this study that socio - economic status did not have an influence on changes in body composition and levels of physical fitness in summer school attendants . we believe this was partly due to the 5 week summer school program , which extended the regular program offered during the school year and included physical education as well as a breakfast and lunch program . another finding in this study was that the influence of socio - economic status was observed in changes in body composition and levels of physical fitness in summer school non - attendants . summer school non - attendants that had higher levels of parental education , daytime caregivers , and were living with both parents showed less of an increase in % body fat . also , adolescents who regularly experienced parental care and participated in summer school had a significantly lower risk of gaining weight and reducing the level of physical fitness during the summer break . similar results were reported by mahoney and parente , who suggested that parental supervision can protect against poor developmental outcomes in lifestyle behavior especially during out - of - school periods . this study found that adolescents with lower house hold income were more likely to gain a greater amount of body fat during the summer break if they did not attend summer school . these results are similar to previous research performed during the school year [ 36 - 38 ] . reported significantly higher levels of physical activity and lower levels of energy intake in adolescents with higher socio - economic status compared to their counterparts . children from low socio - economic backgrounds may have less access to out - of - school activities such as sports , extracurricular activities , and summer camps during the summer break [ 4 , 39 ] , which can prevent weight gain during the out - of - school season . the frequency of fast - food consumption is one of the most important factors affecting weight gain during the summer break . summer school non - attendants who had 0 - 1 fast - food meals per week showed significantly lower increase in % body fat as well as lower decreases in muscular strength and flexibility compared to the summer school non - attendants who had fast food more than 2 - 3 times per week or more than 4 times per week . overall , socio - economic status did not have any effects on changes in body composition and physical fitness for the adolescents who were actively engaged in a summer program such as sports , summer camp or a structured summer school program . however , it did have a faster and negative effect on adolescents with a sedentary lifestyle during the summer break . to our knowledge , several studies have reported a relationship between obesity and psychological outcomes , but no study has investigated the relationship between psychological outcome and changes in body composition or physical fitness over the summer break . we observed changes in body composition and physical fitness through the summer break and found that they were significantly associated with psychological outcomes such as self - esteem and body satisfaction when the adolescent did not attend a summer school program . psychological status such as self - esteem and body satisfaction can be key factors that may motivate students to maintain physical fitness and control their diets . in a review of the literature on self - esteem and obesity based on cross - sectional studies , french et al . consistently found that obese adolescents had relatively lower levels of self - esteem in comparison with healthy - weighed counterparts . strauss reported that the relationship between obesity and self - esteem in white and hispanic students was significantly greater than in african - american students and suggested significant social consequences of reduced self - esteem such as elevated loneliness , sadness and nervousness that may contribute to the increased prevalence rate of obesity in the hispanic population . participating in summer camps had health benefits and significantly increased self - esteem . body satisfaction is central to the emotional well - being associated with self - esteem and is known to affect eating habits such as binge eating or overeating [ 21 , 43 ] . conducted a five year longitudinal study to investigate associations between body satisfaction and health behavior and found that adolescents with lower body satisfaction employed more unhealthy weight control behaviors and binge eating and were physically less active . quick et al . suggested that adolescents with body dissatisfaction attempt dieting to lose weight . however , they are more likely to gain weight due to their use of restrictive and unhealthy weight control behaviors . . carried out a study using a 12 month behavioral weight management program and found that eating behavior was positively predicted by investment in body image change , indicating that a high level of body satisfaction may be related to healthy eating behaviors and may prevent weight gain . the results of this study correspond with previous studies in which body dissatisfaction was highly correlated with an increase in % body fat for summer school non - attendants but had no influence on changes in body composition in summer school attendants . participants were recruited from a high school located in an underprivileged hispanic society because the school was an exemplary school that required participation in a 5 week summer school program at the end of the freshman year . therefore , volunteers from freshman and sophomore classes were automatically considered summer school attendants and non - attendants , respectively . although the results of this study demonstrate an association between psychosocial outcomes and changes in body composition as well as levels of physical fitness , the causal relationship remains speculative . occupation was one of the variables in socio - economic status that was not investigated in this study . in reference to house hold income , even the highest category of house hold income in this study was in the average house hold income range in the overall united states . lastly , this study was conducted over the summer break without any observations during the school year . since changes in body composition and levels of physical fitness during the school year were not examined , it is not clear whether the changes we recorded in this study were due to the summer break . in the united states , the typical length of the summer break is about 23% of the calendar year and it is the largest consecutive period of non - school time . how adolescents spend their summer break has important implications for their well - being . results of this study revealed that a long summer break may increase summer weight gain and decrease physical fitness in underprivileged adolescents , indicating that a structured summer school program may minimize the negative effects of non - school environments with regard to psychosocial aspects . during the summer break , adolescents experience tremendous variations in non - school environments . on the other hand , during the school year , all students are in relatively similar environments , all of which tend to dampen variability . in our findings , summer school attendance dampens the effects of psychosocial outcomes on changes in body composition and levels of physical fitness similar to the regular school year . only for summer school non - attendants did a significant increase in body composition and decrease in physical fitness levels occur and these were affected by psychosocial outcomes . this observation indicates the importance of summer school programs on the management of weight and physical fitness during the summer break in school aged adolescents , especially in underprivileged students who are more susceptible to psychosocial outcomes . these findings support the idea that schools provide healthier environments , whereas the environments are downgraded during the summer break for underprivileged adolescents . additionally , the home environment including the parents level of education , living and caregiver status , and fast - food consumption may influence the health habits of these adolescents during the summer break . psychological status , such as self - esteem , and concerns regarding body shapes may be determinant factors for the management of body composition during the summer break .

[ purpose]evidence suggests that adolescents gain more weight during the summer break than they do during the school year , and that participation in the summer school program is beneficial in maintaining their healthy lifestyle . it is known that obesity and physical fitness in adolescents can be affected by their socio - economic and psychological status , especially during a long school break . the purpose of this study was to examine the effects of summer school participation and psychosocial outcomes on changes in body composition and physical fitness in underprivileged adolescents during the summer break.[methods]body composition and physical fitness in 138 underprivileged adolescents were measured at the beginning and end of the summer break . a survey on socio - economic and psychological status was conducted at the beginning of the summer break . two - way repeated measures anova and tukey post hoc tests were used for data analysis . pearson correlation analysis was performed to establish a relation between psychological outcomes and changes in body composition and physical fitness during the summer break.[results]significant increases in body weight ( p = .003 ) and % body fat ( p = .014 ) as well as a decrease in vo2max ( p = .018 ) were found in summer school non - attendants during the summer whereas no significant changes were found in summer school attendants . summer school non - attendants with lower psychosocial outcomes had a greater decline in physical fitness and weight gain ; however , summer school attendants were not affected by psychosocial outcomes . the summer school program effectively prevented summer weight gain among underprivileged adolescents due to the structured environment , restricted food access , and scheduled time for exercise in addition to minimizing the effects of their psychosocial outcomes.[conclusion]results indicated that summer school non - attendants may require comprehensive intervention for psychosocial outcomes and nutritional education to maintain body weight and physical fitness levels during the summer break .

the peak incidence occurs in pre - school children , between 1 to 5 years of age . few as they are , there are still more reports on the tumor in children [ 13 ] . what can be defined as extrarenal wilms tumor extrarenal nephroblastoma was first reported in 1961 by moyson et al . , who described a chest tumor in which the histological image resembled nephroblastoma . about 300 cases of the disease have been reported around the world since then . in all of them , the tumor was located in the retroperitoneal space , excluding an intrarenal location [ 2 , 3 , 5 ] . the second most common location is the area of the pelvis minor , in particular the inguinal canal , the female reproductive organs ( vagina , uterus ) , the round ligament of the uterus , the ovaries and the testicles [ 6 , 7 , 911 ] . nephroblastoma was also found in the sacro - coccal area in children . from the embryological point of view , extrarenal nephroblastoma is frequently reported to be located in the vicinity of the fetal urogenital ridge and mesonephron , which might account for the location of the tumor in the organs mentioned above [ 12 , 13 ] . the retroperineal location is reported to be typical of males [ 4 , 9 , 11 , 12 ] , whereas the inguinal region is believed to predominate in females [ 6 , 7 , 14 , 15 ] . a case of extremely rare extrarenal nephroblastoma within the isthmus of the horseshoe kidney histology of the tumor revealed an association with teratoma and three cell types which typically form nephroblastoma : blastemal , stromal and epithelial [ 8 , 9 ] . although recent studies maintain that extrarenal wilms tumor most commonly occurs in children , according to multicentre research analyzing the data of 34 patients by cooppes et al . , the patients age varied from 2 months to 56 years . the occurrence of the tumor in men and women was comparable in the group of 4 adults and 3 teenagers . the remaining 27 patients were children below 10 , predominantly between 1 to 4 years of age . according to becwith et al . , nephroblastoma in a typical location can be classified as having either favourable or unfavourable histology . in addition , one more category has been singled out by siop [ society of pediatric oncology ] and nwts ( national wilms tumor study ) pathologists , comprising intermediate risk tumors . the tumor is believed to arise from mesonephric remnants or metanephretic tissue [ 8 , 9 , 13 , 15 , 18 , 19 ] . alternatively , it is assumed to originate in a teratoma , but this conception has not yet been confirmed . however , mature teratoma was recognized in a teratoid wilms tumor found in the horseshoe kidney , even though other forms such as blastematous elements with abortive glomeruli , tubules and immature chondromatoid elements predominated . it was the presence of these three phases blastemal , epithelial and stomal that justified the diagnosis of a teratoid extrarenal wilms tumor [ 4 , 8 , 12 , 20 , 21 ] . it is claimed that extrarenal wilms tumor develops from mesonephric or pronephric remnants and its tissue is more primitive than the tissue of intrarenal nephroblastoma . according to kaptur et al . , between the 6 and 7 week of fetal development , a nexus between metanephric blastema and a ureteral bud is formed . another theory points to the role of the mesonephric duct in the pathogenesis of extrarenal nephroblastoma . male urogenital structures such as the epididymis , vas deferens and seminal vesicles , as well as the female gartner 's duct , differentiate from the mesonephric duct . the gartner 's duct disappears by the 4 month of gestation . yet , as was observed , it could remain beyond that time as juxtagonadal mesonephric gromeruli , which might be the cause of nephroblastoma in genitalia . on the other hand , identified the wt1 gene in 25% of patients with extrarenal nephroblastoma and suggested that the gene mutation might cause transformations of extrarenal mesonephric or pronephric splinters in wilms tumor . electron microscopy of wilms tumor showed protein elements , vimentin filaments , as well as cytokeratin and epithelial antigen , which might suggest a relation between stroma , blastema and epithelium , also observed in intrarenal nephrobastoma . the most common presentation is nonspecific abdominal pain and , as in the case of intrarenal nephroblastoma , a palpable mass ( figure 1 ) . pelvic and retroperitoneal location is associated with gastrointestinal disorders , cachexia , haematuria , gynaecological haemorrhage and in few patients even raised temperature . the clinical presentation depends mainly on the tumor size [ 3 , 4 , 12 , 2224 ] . ct and mri are used to detect tumors in the retroperiteneal space , inguinal , sacro - coccal and scrotal area . however , extrarenal nephroblastoma does not show characteristic radiological features . the treatment of extrarenal nephroblastoma , based on the guidelines for the management of intrarenal nephroblastoma , comprises of surgery , chemotherapy and radiotherapy . the commonly shared opinion is that the surgical removal of the tumor together with the organ it originates from and adjacent lymph nodes is the most important element of the therapy [ 3 , 4 , 9 , 13 ] . tumors in the retroperiteneal space , inguinal or sacro - coccal area must be radically resected . surgery is the treatment of choice and should be as radical as it is for intrarenal nephroblastoma . the whole area of resection should be carefully inspected and the enlarged lymph nodes from the area must be removed . chemotherapy as adjuvant therapy was reported to have been used in all studies [ 12 , 17 ] coopies at al . stage i amounted to 30% , ii to 10% , iii to 57% and iv to 3% . in all patients , two - year asymptomatic survival postoperative treatment depends on the stage of the tumor and its histological result . both factors ( stage and histology ) it seems controversial to refer to the tnm classification in cases of tumors unrelated to the organ . blastemal type , diffuse anaplasia , clear cell sarcoma , and rhabdoid tumor are all high risk tumors . mesoblastic nephroma , cystic partially differentiated nephroblastoma and complete necrotic nephroblastoma belong to the low risk group . as in all reports on extrarenal nephroblastoma , the tnm classification was referred to and the types of treatment , as well as the results , were be compared . stage 1 organ - confined tumor , resected completely , no injury to a pseudocyst , clear surgical margin . stage 2 regional tumor , biopsied before removal or invading blood vessels ; resected completely , clear surgical margin . stage 3 tumor not completely resected , regional lymph node metastasis present , tumor spread to the abdominal organs and peritoneum . , chemotherapy regimens recommended by siop or nwts have been introduced according to the stage and histological image of the tumor . in poland , the chemotherapy regimen recently recommended by siop is used . in the case of the boy with extrarenal nephroblastoma reported by the authors , a three - agent therapy [ actinomycin , vinkristine and doxorubicin ] , after that , the patient was treated with 20 gy radiation . according to both nwts and siop pathologists , diffuse anaplastic changes generally predict a poor outcome and full recovery is achieved in 30% of cases [ 16 , 17 ] . what also confers poor prognosis is a tumor mass above 250 mg , volume above 400 cm3 and lymph nodes metastases . extrarenal nephroblastoma was first reported in 1961 by moyson et al . , who described a chest tumor in which the histological image resembled nephroblastoma . about 300 cases of the disease have been reported around the world since then . in all of them , the tumor was located in the retroperitoneal space , excluding an intrarenal location [ 2 , 3 , 5 ] . the second most common location is the area of the pelvis minor , in particular the inguinal canal , the female reproductive organs ( vagina , uterus ) , the round ligament of the uterus , the ovaries and the testicles [ 6 , 7 , 911 ] . nephroblastoma was also found in the sacro - coccal area in children . from the embryological point of view , extrarenal nephroblastoma is frequently reported to be located in the vicinity of the fetal urogenital ridge and mesonephron , which might account for the location of the tumor in the organs mentioned above [ 12 , 13 ] . the retroperineal location is reported to be typical of males [ 4 , 9 , 11 , 12 ] , whereas the inguinal region is believed to predominate in females [ 6 , 7 , 14 , 15 ] . a case of extremely rare extrarenal nephroblastoma within the isthmus of the horseshoe kidney was described as well . histology of the tumor revealed an association with teratoma and three cell types which typically form nephroblastoma : blastemal , stromal and epithelial [ 8 , 9 ] . although recent studies maintain that extrarenal wilms tumor most commonly occurs in children , according to multicentre research analyzing the data of 34 patients by cooppes et al . , the patients age varied from 2 months to 56 years . the occurrence of the tumor in men and women was comparable in the group of 4 adults and 3 teenagers . the remaining 27 patients were children below 10 , predominantly between 1 to 4 years of age . according to becwith et al . , nephroblastoma in a typical location can be classified as having either favourable or unfavourable histology . in addition , one more category has been singled out by siop [ society of pediatric oncology ] and nwts ( national wilms tumor study ) pathologists , comprising intermediate risk tumors . the tumor is believed to arise from mesonephric remnants or metanephretic tissue [ 8 , 9 , 13 , 15 , 18 , 19 ] . alternatively , it is assumed to originate in a teratoma , but this conception has not yet been confirmed . however , mature teratoma was recognized in a teratoid wilms tumor found in the horseshoe kidney , even though other forms such as blastematous elements with abortive glomeruli , tubules and immature chondromatoid elements predominated . it was the presence of these three phases blastemal , epithelial and stomal that justified the diagnosis of a teratoid extrarenal wilms tumor [ 4 , 8 , 12 , 20 , 21 ] . it is claimed that extrarenal wilms tumor develops from mesonephric or pronephric remnants and its tissue is more primitive than the tissue of intrarenal nephroblastoma . according to kaptur et al . , between the 6 and 7 week of fetal development , a nexus between metanephric blastema and a ureteral bud is formed . another theory points to the role of the mesonephric duct in the pathogenesis of extrarenal nephroblastoma . male urogenital structures such as the epididymis , vas deferens and seminal vesicles , as well as the female gartner 's duct , differentiate from the mesonephric duct . the gartner 's duct disappears by the 4 month of gestation . yet , as was observed , it could remain beyond that time as juxtagonadal mesonephric gromeruli , which might be the cause of nephroblastoma in genitalia . on the other hand , roberts et al . identified the wt1 gene in 25% of patients with extrarenal nephroblastoma and suggested that the gene mutation might cause transformations of extrarenal mesonephric or pronephric splinters in wilms tumor . electron microscopy of wilms tumor showed protein elements , vimentin filaments , as well as cytokeratin and epithelial antigen , which might suggest a relation between stroma , blastema and epithelium , also observed in intrarenal nephrobastoma . the most common presentation is nonspecific abdominal pain and , as in the case of intrarenal nephroblastoma , a palpable mass ( figure 1 ) . pelvic and retroperitoneal location is associated with gastrointestinal disorders , cachexia , haematuria , gynaecological haemorrhage and in few patients even raised temperature . the clinical presentation depends mainly on the tumor size [ 3 , 4 , 12 , 2224 ] . ct and mri are used to detect tumors in the retroperiteneal space , inguinal , sacro - coccal and scrotal area . however , extrarenal nephroblastoma does not show characteristic radiological features . the treatment of extrarenal nephroblastoma , based on the guidelines for the management of intrarenal nephroblastoma , comprises of surgery , chemotherapy and radiotherapy . the commonly shared opinion is that the surgical removal of the tumor together with the organ it originates from and adjacent lymph nodes is the most important element of the therapy [ 3 , 4 , 9 , 13 ] . tumors in the retroperiteneal space , inguinal or sacro - coccal area must be radically resected . surgery is the treatment of choice and should be as radical as it is for intrarenal nephroblastoma . the whole area of resection should be carefully inspected and the enlarged lymph nodes from the area must be removed . chemotherapy as adjuvant therapy was reported to have been used in all studies [ 12 , 17 ] coopies at al . stage i amounted to 30% , ii to 10% , iii to 57% and iv to 3% . in all patients , two - year asymptomatic survival postoperative treatment depends on the stage of the tumor and its histological result . both factors ( stage and histology ) it seems controversial to refer to the tnm classification in cases of tumors unrelated to the organ . blastemal type , diffuse anaplasia , clear cell sarcoma , and rhabdoid tumor are all high risk tumors . mesoblastic nephroma , cystic partially differentiated nephroblastoma and complete necrotic nephroblastoma belong to the low risk group . as in all reports on extrarenal nephroblastoma , the tnm classification was referred to and the types of treatment , as well as the results , were be compared . stage 1 organ - confined tumor , resected completely , no injury to a pseudocyst , clear surgical margin . stage 2 regional tumor , biopsied before removal or invading blood vessels ; resected completely , clear surgical margin . stage 3 tumor not completely resected , regional lymph node metastasis present , tumor spread to the abdominal organs and peritoneum . , chemotherapy regimens recommended by siop or nwts have been introduced according to the stage and histological image of the tumor . in poland , the chemotherapy regimen recently recommended by siop is used . in the case of the boy with extrarenal nephroblastoma reported by the authors , a three - agent therapy [ actinomycin , vinkristine and doxorubicin ] , after that , the patient was treated with 20 gy radiation . according to both nwts and siop pathologists , diffuse anaplastic changes generally predict a poor outcome and full recovery is achieved in 30% of cases [ 16 , 17 ] . what also confers poor prognosis is a tumor mass above 250 mg , volume above 400 cm3 and lymph nodes metastases . the occurrence of the extrarenal nephroblastoma is exceedingly rare . in absence of typical clinical presentation or conclusive imaging tests , the recommended management of the extrarenal nephroblastoma is similar to the treatment used for intrarenal nephroblastoma .

introductionnephroblastoma is one of the most common solid tumours in children . the occurrence of extrarenal nephroblastoma is exceedingly rare . what can be defined as extrarenal wilms tumor must satisfy the following criteria : histologically confirmed nephroblastoma and extrarenal location.material and methodscurrent data on extrarenal nephroblastoma based on a selective review of the literature.resultsthe retroperineal location is reported to be typical in males , whereas the inguinal region is believed to predominate in females . there are no characteristic manifestations of extrarenal nephroblastoma . the symptoms depend on the location and stage upon diagnosis . us , ct and mri are used to detect tumours in the retroperiteneal space , inguinal , sacro - coccal and scrotal area . however , extrarenal nephroblastoma does not show characteristic radiological features.conclusionsin absence of typical clinical presentation or conclusive imaging tests , the diagnosis is based on histology after the resection of the tumor . the recommended management of the extrarenal nephroblastoma is similar to the treatment of intrarenal nephroblastoma .

the human resources crisis created by personnel shortages is currently the greatest impediment to generalizing proven strategies for improving maternal and perinatal mortality [ 14 ] . in mali , referral - evacuation systems ( ress ) are one strategy in the fight against maternal and perinatal mortality , where the implementation has had to cope with shortages of staff , particularly of midwives in rural settings . an res looks after the upgrading of emergency obstetric services , the financial coverage of patients by community cost - sharing schemes , and the transfer of patients , as needed , to better equipped facilities . it closely links peripheral healthcare structures ( community health centres ( chcs ) ) ) , district health centres ( dhcs ) , and regional hospitals . it is organized around the district , the basic functional unit of mali 's healthcare system . its interventions are concentrated on the intrapartum period , during which most maternal deaths occur . it includes a functional referral and allows for simultaneous action on the demand and supply sides . indeed , the res ensures rapid management of obstetric complications in mali 's context , which is characterized by low rates of institutional delivery , especially in rural settings . in 2006 , the rates of facility - based deliveries were 45% in mali , 34% in kayes , and 90.3% in bamako , the national capital . ress have been implemented in all of them , with considerable variation in the levels of inter- and intraregional functionality . in kayes region , where this study was conducted , the seven districts ' ress were set up between 2002 and 2005 . they are supposed to facilitate normal deliveries in the chcs and the referral of complicated cases to the dhcs . for early screening and referral of obstetric complications , whose occurrence is most often unpredictable , all deliveries should be carefully monitored by staff qualified for deliveries essentially either midwives or other comparably skilled professionals [ 9 , 10 ] . thus , in kayes region , counting one midwife per chc , 223 midwives would be required to implement the ress , in addition to those at the dhcs and the regional hospital . however , in 2005 , there were only seven midwives at the chc level , and , in 2006 and 2009 , only eleven . in the face of these shortages of qualified personnel , one that took immediate effect was to extend obstetric services coverage in chcs by using matrons . they are much less qualified than midwives but are more easily trained , recruited and , especially , retained in rural areas because they come most often from the local communities that train and employ them . midwives complete three years of study after their baccalaureate degree , whereas matrons undergo a few months of internship and some theoretical training in a maternity unit . the content of the matrons ' training varies depending on the maternity unit they attend . moreover , no particular level of schooling is required for this training , although most matrons have at least completed primary school . in 2005 , kayes region had 196 matrons in chcs and 222 in 2006 . another strategy was to train obstetric nurses , who are less qualified than midwives but more than matrons . they receive three years of theoretical and practical training in competencies similar to midwifery skills , after a basic teaching diploma ( six years of primary and four years of secondary schooling ) . in the chcs of kayes region , there were eight in 2005 and 14 in 2006 . despite these compromises in staff qualification at the peripheral levels , the implementation of ress doubled the number of women who had access to obstetric care and reduced by half their risk of dying from obstetric complications . these results were made possible through the combined action of all components of the res . in addition to the shortages of midwives , the numbers and qualifications of staff in the chcs ' healthcare teams vary considerably . in kayes region , as everywhere in mali , chc management falls under the responsibility of local communities and is conducted through community health associations ( asaco ) that can take the initiative in staff recruitment and remuneration [ 6 , 14 ] . kayes region is the largest source of malian emigrants to france , and the asacos enjoy the financial support of malian citizens from kayes who are living abroad . healthcare projects are sometimes funded as much as 80% by emigrants from the kayes region , which represents 30% of the combined total contributions to the region 's development . thus , while some chcs might have only three professionals a nurse , a matron and a pharmacy manager in addition , around 10% of the chcs in the kayes region have physicians . in chcs where there is a physician , that person is automatically the centre 's manager : the physician - in - charge ( mdecin chef de poste , or mcp ) . otherwise , it is the most qualified staff , often the nurse , who is the manager : the nurse - in - charge ( infirmier chef de poste , or icp ) . it showed that the joint mother - newborn survival is significantly influenced in the kayes maternal referral system by combined effects of the skill configuration of chc personnel and distance traveled . thus , women referred from a chc where there was a physician were six times more likely to survive from an obstetric complication than were those transferred from a chc without a physician , based on comparable morbidity and controlling for distance travelled and other cofactors . pregnancies complications were not managed at the chc , but , at the dhc , the protective role of physicians ' presence in chcs was therefore attributed to a better management of complicated cases before their evacuation [ 18 , 19 ] , or to earlier screening of women potentially at risk of obstetric complications , or to the beneficial effects of ambulance transport . despite those hypotheses , a more rigorous understanding is needed of the mechanisms that link the management of pregnancies , the qualification of staff , and the organization of services at the first level of care provision , especially at a time when shortages of qualified personnel lead to the adoption of task - delegation strategies [ 2023 ] . we conducted a multiple - case study in the districts of kayes and diema . using purposive and stratified sampling , we selected , in two stages , 25 chcs from the districts ' total of 67 chcs as units of analysis . first , we selected 13 chcs headed by physicians ( mcps ) , taking into account the number of healthcare personnel and the distance between each chc and the dhc , to maximize variability in the care environment . then , we selected 12 other chcs headed by nurses ( icps ) ; these were comparable to the mcp - managed chcs in every respect except for the qualification of the manager in charge . we carried out semistructured interviews with the personnel involved in maternal care : matrons , nurses , obstetric nurses , midwives , and physicians . in each chc we visited , participants were invited to freely elaborate about the organization of maternal services in their centre and about what they did to improve the outcome of care for the women . this enabled us to develop a picture of the centre 's functioning and to validate it with the staff . through nonparticipant observations , we were able to observe professional interactions in the teams and to verify whether the organizational modalities corresponded to what was stated in the interviews . the interviews were carried out by the first author and a sociologist from the region . on average , we interviewed icps ( n = 10 ) ( two icps were absent from their posts and one interview with an icp was discarded because of technical problems with the recording ) , nurses ( n = 3 ) , matrons ( n = 23 ) , midwives ( n = 5 ) , obstetric nurses ( n = 4 ) , and mcps ( n = 11 ) ( one mcp had just been named to his post and was not interviewed ) . in all the coding and analysis of the transcribed material were done using qsr international 's nvivo 8 software . we developed the coding plan from a list of codes inspired by the literature on human resources in healthcare services and on quality of services . the list was combined with an open coding to allow the emergence of new themes . comparisons between mcps and icps , reported in this study , allowed us to identify points in common as well as differences . this project received ethical approval from the research centre of the university of montreal hospital centre , from mali 's national department of health , and from the kayes regional department of health . in accordance with local practices , verbal informed consent was obtained from the asaco managers and health personnel for the interviews as well as for their recording . to maintain respondents ' anonymity , extracts from the interviews variations in the care environment include how work is organized , the level of technology , the skill and numbers of staff in the chc , and the creation of a competitive environment . in the 13 chcs with icps , the distribution of tasks for the clinical management of pregnant women followed a homogeneous model , while those with mcps showed a variety of profiles . the icps looked after general consultations , oversaw the proper functioning of the chc , and delegated responsibility for maternity activities to the matrons . the matrons organized prenatal consultations and did the deliveries ; they sought technical advice from the icps for complicated cases . the following excerpt illustrates this work organization , which was nearly uniform in all chcs headed by nurses : when there is no complication , the matron is in charge , but under my supervision . ( icp_10 ) . in some chcs managed by icps , this division of tasks , which was sometimes associated with a physical separation between the maternity unit and other buildings , led to the perception that the chc was made up of two distinct entities : the first matron is our boss and the icp is the boss of everyone she ( the matron ) is the person that the women listen to and are influenced by the most . ( matron_44 ) . analysis of the organization of work in chcs with mcps shows three models of service organization . the first model is comparable to chcs managed by icps and was found in five chcs with mcps . matrons managed deliveries and only called on the mcp in serious cases . despite only being directly involved in a selective and limited way with the serious cases , the mcps did systematic telephone followup of patients with the matrons . in the second model , seen in three chcs , the mcp 's involvement with pregnant women was more frequent and involved both simple and complicated cases . this model was characterized by two complementary measures : the systematic examination of women by at least two members of the personnel , and the organization of weekly staff meetings and presentations where the week 's difficult cases were discussed . two of these mcps said they implemented these measures to improve the knowledge of their team members and to provide patients with appropriate management of complicated cases . we divide the work ; the nurse looks after the prenatal consultation , and in real time , if a woman arrives , the matron can look after her . ( even ) if she is very busy , the nurse sees the woman , systematically , even if she has to let the matron continue . the third model of work distribution seen in three mcps was characterized by their very strong involvement in the management of pregnant women . they conducted the first examinations of many women and sometimes did deliveries , even for simple cases . often at night i do not wake the matron , i do the deliveries myself . ( mcp_48 ) . in a context in which chcs are managed autonomously by the asacos , the possibility of having mcps with obstetric skills was accompanied by the purchase of ultrasound equipment in three of the chcs that had physicians . in addition , with the support of some kayes emigrants , two chcs were fitted with operating suites and were in negotiations with the regional department of health to obtain authorization for interventions . expertise in the use of ultrasound was one of the recruitment criteria for staff in these facilities and was helpful for the early diagnosis of certain pathologies , as this physician explained . when a patient arrives who has had no prenatal visits , i quickly do an ultrasound , and this lets me know if it is a case of twins , or of placenta praevia , and we can make decisions quickly . while no icps envisioned raising the level of technology in their centres since according to them , the complicated cases should be referred to the dhcs the mcps were preparing development plans for their chcs . in these development plans , the mcps envisioned raising the levels of emergency obstetric care ( emoc ) competency in their teams , setting up a functional laboratory in addition to ultrasound , installing internet connections , promoting the use of solar energy , and , in the longer term , creating functional operating suites . i would like to see it transformed into a referral health centre ( dhc ) someday , since , as you can see for yourself , it 's far to refer a patient ; god knows what could happen . we already have ultrasound ; i 'm looking into how we can also set up a lab so i can do the initial analyses here . six mcps had a particular interest in obstetrics which led them to acquire skills in emergency obstetric and neonatal care ( emonc ) . three of them had done a thesis in a gynaecology - obstetric service , and the other three had undergone supplementary training in emonc or in obstetric ultrasound , most often at their own expense . all of them were more deeply involved ( model 2 or 3 ) in the management of pregnant women . when i go to france , i use my vacations to do applied training sessions with my colleagues , but it 's really a personal choice . ( mcp_23 ) . aside from training offered by the regional department of health to upgrade their emoc skills , either they did not satisfy the conditions required for acceptance into these training programs , or they were unwilling to pay for the training themselves . none of the icps we interviewed had recently been able to update their emoc skills . these training sessions competed with several others ; also , they were held in the regional capital , such that the icps would have to travel . staff who went for training were expected to brief the rest of the personnel . so the icps , knowing that the matrons would brief them and the other staff on the maternal care training they received , and wishing to limit their own absences from their posts , preferred instead to attend training sessions on hiv - aids and on policies , standards , and procedures . in addition to the acquisition of additional skills , the presence of mcps in chcs was associated with staff recruitment . nursing students preferred doing training internships in chcs where there was an mcp in order to learn more . in addition , with the support of the mcp , these trainees were able to negotiate a contract as volunteers at the end of their studies . this ability to attract personnel changed the staffing levels in the chcs , the workloads , and the combination of skills available for maternal services . however , they asserted that they preferred these centres , not because of the presence of a physician , but because they were high - volume centres , so they would not risk losing their skills . some midwives regretted never having had the opportunity in the chcs to practise even the simplest emergency procedures . the presence of midwives and obstetric nurses in the team improved the combination of skills available for maternal care ; however , having female staff whose families did not live in the chc 's village complicated human resources management because of absences due to family reasons , as illustrated by the following excerpt . but the only problem is the instability of the on ( obstetric nurse ) , who spends one week here and three weeks in bamako ( nurse_36 in an mcp - managed chc ) . the presence of physicians in the chcs of kayes region created a competitive services environment that could , directly or indirectly , provide incentives for professionals ' performance . since , as you know , in this district there are a lot of physicians , there 's competition ; if you 're not competent , the villagers will go elsewhere for their care . the mcps reported benchmarking practices . to improve their performance , they compared themselves against best practices in healthcare in the region . on their own initiative , some arranged informally to take introductory courses on ultrasound from other colleagues in the region . we even went to k with the members of the asaco , to exchange ideas and experiences in the context of advancing the chc . because k is a chc that does a lot . the mcps paid particular attention to relations with the professionals of the dhcs and with the patients . the icps see interactions between the chcs and the dhcs as administrative relations that should be maintained but should not influence the management of referred patients . ( icp_1 ) and the decider ( icp_24 ) ; it ensures the proper functioning of the chcs through the supply of vaccines and other materials and it receives quarterly reports from the health information system . ( icp_1 ) only one mcp stated that relations between the two levels of care had no influence at all on the management of pregnant women . all the other mcps considered it crucial to maintain good relations with the dhc to ensure better management of referred patients . some mcps nurtured relationships with the higher level of care that were sometimes already preferential . our connections with the referral centre ( dhc ) are excellent because , as i said , these are colleagues , friends ; they 're civil servants like me , and we have to work together to get results with regard to the women who are evacuated , generally they give us feedback when i evacuate someone to , where i have good connections , and i 'm in contact with everyone , so then i just make a phone call to get whoever is on call to refer my patient , who is quickly taken on . the advantages to patients of privileged relations with the higher level of care were confirmed by a midwife who had previously worked in a dhc . when you arrive with a referral letter , they take care of you faster , they do not just leave you hanging . ( midwife_32 ) . according to the mcps , establishing a relationship of trust with the population made it possible to mobilize all the resources of the village and facilitated women 's acceptance of medical recommendations , such as transfers to dhcs . unlike the icps , who did not mention the importance of this relational aspect , the mcps reported that they made an effort to gain people 's confidence . if the population is not informed and aware , we wo n't have good results i've gained people 's confidence , we understand each other , and when i say something , they do it the variations identified in the mcps ' care environment were sometimes deliberate and actively induced changes in the way work was organized , the level of technology , and the qualifications of the personnel and sometimes they arose passively and unintentionally . analysis of the functioning of chcs shows that mcps , depending on their interests in obstetrics , had varying levels of involvement in the management of deliveries . on the other hand , in chcs with icps , the organization of work was uniform and conformed to the official model provided in the res . more frequent , direct involvement of a physician in the management of pregnant women improved the combination of skills applied in maternal services . it is true that people perform better in contexts that correspond to their personal preferences , and , as this study shows , the mcps with the greatest involvement were those who had supplementary training in obstetrics . still , an interest in obstetrics alone does not explain the development of diverse models . in fact , even when they organized the work like the icps did , leaving matrons in charge of deliveries , the mcps put in place systems such as systematic phone call followup and management of patients by several professionals , to ensure complicated cases were properly managed . in this way , they were not restricted to the traditional care model of the res used by nurses . also , in the present study , the icps spoke only about the administrative aspects of their relations with the dhc , while mcps nurtured their relations with those working at the higher levels with whom they established personal contacts to accelerate the handover of their patients . a quick management of obstetric complications is a factor in improving the quality of obstetric care [ 26 , 27 ] ; nevertheless , further studies are needed to document better the consequences for patient care of the relations between the two levels of care . the mcps also developed relationships of trust with patients , while the icps did not mention this . yet , in the case of a referral system , social interactions with patients are recognized as being beneficial to patients . although giving preferential treatment to patients referred by a physician might be criticized from an ethical standpoint , in the context of a network where final outcomes depend on a succession of prior actions , an interdependent team collaboration with information exchange between the different levels is more effective than working independently , and even more effective than consultation referral . in addition to going beyond the traditional model of care and developing the relational and interpersonal aspects of care , the presence of mcps can raise the level of technology in chcs and create a competitive environment that helps to improve performance . in a context of staff shortages where the general trend is to use less - qualified staff , the tasks , roles and responsibilities of qualified staff that are usually delegated are technical ones , such as anesthesia , cervical smears , first - trimester abortions , or surgical interventions . however , these acts are usually carried out in the district level centers . at the peripheral level , often the responsibilities of the healthcare personnel , beyond technical interventions , include relational activities such as accompaniment and social support . it would seem appropriate , therefore , to wonder whether the tasks to be delegated , especially in the health centres and in a referral system , should not also include relational and interpersonal skills . moreover , unintentional mechanisms that improve performance , such as the capacity to attract personnel and especially to create a competitive environment , should be identified and maintained . the presence of mcps with an interest in obstetrics may improve the quality of care , but it does not resolve the problem of shortages of staff qualified to assist during women 's deliveries . in fact , as this study shows , the difficulties of retaining midwives and obstetric nurses in rural areas continue , while filling these positions would have a long - term positive impact on maternal care [ 11 , 33 , 34 ] . also , the environmental variations produced by physician managers veer away from the logic underlying the res . however , the present study shows that , with management autonomy and support from expatriates , some chcs have acquired increasingly qualified staff and sophisticated equipment , including operating wards . with no governmental regulation of what acts are to be done in chcs , mcps with obstetric competencies could take over the management of complicated cases at the most peripheral levels . however , this displacement would not be cost - effective and the number of interventions would most certainly be too small to maintain skills . through the study , we were able to explore relations between the qualifications of the staff heading up the chcs , the organization of care , and the management of pregnant and child birthing women 's care in rural settings . while its generalizability to other contexts is limited , this study provides some understanding , in the specific context of kayes region , of the complexity and diversity of care organization in chcs . kayes region ( districts of kayes and diema ) is a specific context in which innovations in service provision can be studied . significant levels of emigration and the flow of resources back to the emigrants ' communities of origin have allowed these communities to acquire a service offer that exceeds national standards . a significant proportion of first - line health centres in rural settings have a physician . this profoundly changes the organization of care , because physicians bring a development model to their chcs that is centred on clinical practices and closely linked to their personal and professional development . when nurses are in charge of first - line health centres in rural areas , their approach is more administrative ; they tend to adopt a more hierarchical than clinical perspective in their relations with the higher district level and a work organization model in which they delegate obstetric responsibilities to matrons . in the referral - evacuation system of kayes region , the presence of physician managers in the chcs created more opportunities to improve patient care outcomes . an analysis of the chcs ' models of care organization reveals organizational , relational , and interpersonal mechanisms that can improve their performance . still , this should be an ad hoc and temporary strategy , as the problem of shortages of staff qualified for delivery particularly midwives and obstetric nurses continues , and there is a real risk that the referral system 's operating principles would be modified in this context of autonomous management of chcs .

in mali , a poor sub - saharan country , maternity referral systems were implemented to combat the still - high rates of maternal mortality . this qualitative study was aimed at understanding the relationships between the qualification of staff in community health centres , the organization of services , and the management of pregnant women in the maternity referral system in kayes , a rural region of mali . physicians who managed chcs actively or passively modified work organization , the level of technology , their obstetric skills , and staffing . they also created a competitive environment and developed relationships of trust with patients and with the district health centre . these findings are helpful in orienting decision - making for better personnel management .

the incidence of congenital dysplasia of the hip ( cdh ) is known to be higher in china than in the european and american countries2 . ultimately , cdh can cause osteoarthritis and other joint destruction , and lead to loss of the joint function2 . it is known that one of the main functions of the artificial joint is to overcome the anatomic deformity of the hip1 , 3 . studying the anatomic parameters of cdh before and after total hip replacement is of great scientific and clinical value . lindgren et al . had measured 47 cases of hip dysplasia after total hip replacement , analyzing the differences between the femoral eccentricity , hip abductor arm , height and hip rotation center , and the leg lengths3 . dastane m et al . had measured femoral offset to investigate the guiding role of the total hip replacement1 . according to the radiographic images , the difference in the femoral eccentricity between the preoperative arthritic hip and the contralateral normal hip was 12 to 21 mm ( average , 1.5 mm ) , while the postoperative femoral offset difference value between the two sides was 1.46.4 mm2 . the study did not present any preoperative and postoperative data on the hip femoral offset , length of the femoral neck , and femoral head height . normal femoral offset , hip abductor muscle of the arm , the soft tissue tension , and abductor muscle strength are important parameters for the improvement of the gait and lower limb movement . cdh is more complex than single femoral head necrosis or osteoarthritis surgery ; thus , the postoperative anatomic parameters directly affected the quality of surgery and the long - term follow - up . the objective of this study was to investigate the relevant preoperative and postoperative anatomic parameters , and evaluate the surgical corrective effect , while providing a reference for future improvement of the surgical method . location : the people s liberation army ( pla ) general hospital ( 301 hospital , beijing ) and the people s hospital of lishui city ( zhejiang province ) , china . patients : the inclusion criteria were : ( a ) i iii congenital dysplasia of the hip and patients with unilateral total hip replacement for the first time ; ( b ) patients treated with class iii and class iv ceramic prosthesis ; ( c ) preoperative and postoperative pelvic radiography . all the experimental designs and processes were approved by the ethics committee of the pla general hospital ( 301 hospital ) and the people s hospital of lishui city . according to the developmental congenital dysplasia of the hip ( ddh ) classification , we grouped the cases as follows : ( 1 ) type i : femoral head translocation<50% of the femoral head height , or femoral head translocation<10% of the pelvic height ; ( 2 ) type ii : femoral head translocation<5075% of the femoral head height , and femoral head translocation<1015% of the pelvic height ; ( 3 ) type iii : femoral head translocation<75100% of the femoral head height , or femoral head translocation<1520% of the pelvic height . materials used for the measurements : for this study , to estimate the size of the prosthesis we used concentric circles made on paper cards . by using photoshop cs 53 ( adobe , usa ) , we drew various concentric circles with a minimum radius of 0.5 mm ( minimum radius value was 0.5 mm and this was increased to 0.5 mm ) . the concentric circles were printed on h2so4 paper and a 6 cm 6 cm square card was prepared . the concentric circles card was placed on the screen to determine the center of the femoral head . in order to choose the appropriate size of the hip prosthesis , when the magnified prosthetic template matched the values evidenced by the radiographic images , we estimated the size of the acetabular cup and that of the femoral handle . methods : the elected surgical method was the lateral approach . to start , the joint capsule was cut and removed , followed by anterior dislocation of the hip joint . a space of 4 cm was left between the femoral neck and the small tuberositas ; the femur length was maintained at 1015 mm , while the femoral head was removed . the prosthesis was placed into the reshaped socket , and two holes were drilled into the greater trochanter of the femur . further , the medius gluteus was stitched on the rotator tendon of the femur , and further the integrity of the hip joint lateral tissue was reconstructed . the following preoperative and postoperative parameters of the hip were analyzed on the radiograms using the ps software ( adobe photoshop cs5 , adobe systems inc . ) . ( 1 ) the center of the femoral head : h2so4 paper was used to determine the center of the femoral head on its both sides . ( 2 ) femoral neck length ( fnl ) : the distance between the center of the femoral head and the central and femoral neck long axes . ( 3 ) the height of the femoral head ( hfh ) : the distance between the center of the femoral head and the connection to the sciatic nerve branch . ( 4 ) the longitudinal plane of gluteus medius ( lgm ) : the distance between the center of the femoral head or the center of the femoral head of the prosthesis to the big tuberosity of the femur . ( 5 ) postoperative and preoperative harris hip score . ( 6 ) femoral eccentricity ( fe ) : the distance of the center of femoral head to femoral long axis . correlation analysis : the correlation between the femoral eccentricity , length of the femoral neck , femoral head height , and hip abductor muscle arm , and the hip motion with harris hip score were compared . the spss 13.0 ( spss , usa ) package was used to analyze the statistical significance of the results , which were expressed as mean + standard deviation ( sd ) . t - test was used to compare the results obtained before and after the surgical operation and the difference was statistical different for p<0.05 . furthermore , logistic regression analysis was used when analyzing the relationships between the parameters . this study included 78 cases , of which 50 were males and 28 were females . according to the ddh classification , 28 cases were type i , 26 cases were type ii , and 24 cases were type iii . the mean weight of the patients was 55.1 23.9 kg , and the mean height was 150 20.5 cm . for all the patients the chosen prosthetic components were betacup acetabular cup ( waldemar link , germany ) , corail shank ( johnson & johnson , depuy synthes , product number 3l92509 - 15 ) . additionally , the collodiaphyseal angle was 135. from each patient preoperative and postoperative radiograms were collected . additionally , a circular metal mark ( 5 cm in diameter ) was drawn at a distance of 10 cm from the small tuberositas to correct the magnification of the radiologic image . when analyzing the types of prosthesis , in 90% of the cases betacup acetabular cup and corail head were used during surgery . additionally , the diameter for all the four ceramic femoral heads was of 36 mm . because of economic considerations or acetabulum - related conditions , for 10% of the cases an interface low - density polyethylene hinge and a metal head ( 28 mm in diameter ) were used . table 1table 1.preoperative and postoperative values of the hip parameters on total type ( x sd)mmpreoperative postoperativefemoral eccentricity33.3 8.4 * 39.1 7.1the height of the femoral head59.5 8.8 * 68.9 11.0femoral neck length50.9 10.8 * 61.5 10.4longitudinal of gluteus medius54.3 9.6 * 64.7 10.1*there are significant differences between the preoperative and postoperative values . presents the preoperative and postoperative hip parameters for the total type of congenital dysplasia of the hip . table 2table 2.preoperative and postoperative values of the hip parameters on different type ( x sd)mmpreoperative postoperativetype ife3435.1 7.2 * 38.9 5.7hfh59.8 10.1 * 71.3 10.1fnl57.0 12.0 * 67.2 10.4lgm56.8 12.0 * 66.7 10.2type iife33.9 6.6 * 39.6 9.6hfh60.6 6.1 * 68.9 9.0fnl49.6 8.5 * 61.2 9.5lgm54.3 7.1 * 65.0 11.6type iiife30.2 10.4 * 38.7 5.3hfh57.9 10.1 * 65.8 14.1fnl45.6 9.3 * 55.8 8.7lgm51.8 7.7 * 62.7 8.8fe : femoral eccentricity ; hfh : height of the femoral head ; fnl : femoral neck length ; lgm : longitudinal of gluteus medius . presents the preoperative and postoperative hip parameters based on the three types of congenital dysplasia of the hip . fe : femoral eccentricity ; hfh : height of the femoral head ; fnl : femoral neck length ; lgm : longitudinal of gluteus medius . the range of hip motion ( anterior and posterior stretch , interior and external rotation extension ) during the preoperative assessment was of 60.5 8.2 ( minimum=0 , maximum=136 ) , while during the postoperative assessment it was of 168.2 8.1 ( minimum=130 , maximum=230 ) . the results evidenced a relationship between the range of hip motion and the femoral offset ( r=0.419 , p<0.001 ) . the preoperative and postoperative harris hip scores were 32.4 4.6 points ( minimum - maximum : 862 points ) and 93.6 5.2 points ( minimum - maximum : 83100 points ) . the analysis evidences a significant difference of t : 0.471 , p : 0.0001 . the goal of total hip replacement is to influence the hip biomechanics reconstruction process4 . the degree of improvement of the biomechanics of the hip joint function was directly related to the total hip arthroplasty and the time period since using the prosthesis5,6,7 . among the four parameters studied ( eccentricity , femoral head height , length of the femoral neck , and abductor muscle arm of the hip ) , the eccentricity parameter was the most important . according to the results , eccentricity had a positive relationship with the other three parameters . when the eccentricity value was too small , it would reduce the distance between the femur end and the pelvis ; it would reduce the range of the hip motion and the hip muscle relaxation ; and it would increase the risk of collision and joint dislocation . alternatively , if the eccentricity value was high , the acetabulum interface friction would increase the risk of plastic deformation and lead to dissolution . for patients with ddh , the acetabulum hypoplasia would directly affect the eccentricity , therefore , for all the three types of hip replacement the acetabulum and the center of rotation would influence hip arthritis development . furthermore , patients affected by necrosis of the femoral head evidenced a change in eccentricity . some studies had shown that the change of the rotation center could cause the change of the eccentricity8,9,10,11 . to reconstruct the femoral eccentricity we used one specific method . when the femoral handle length increased , the lower limb length would increase , thus causing sciatic nerve palsy and low back pain . compared with the opposite hip side that did not undergo surgery , the rebuilt eccentricity increased less than 4 mm . for our study , 80% of the patients evidenced a large diameter of the femoral head ( 36 mm ) , which increased the eccentricity without increasing the lower limb length . additionally , when choosing the small collodiaphyseal angle of the femoral prosthesis handle , this would increase the eccentricity , and would maintain the length of the body . furthermore , it will increase the rotation torque of the femoral neck and the risk of a broken neck . as for the artificial total hip replacement , future consideration should include rebuilding the physiological anatomy as soon as possible . the reconstruction of the femoral eccentricity , femoral head height , and hip muscle arm would improve hip stability8 , 12,13,14,15,16 , reduce the prosthetic hip wear , avoid postoperative hip muscle weakness , claudication , and improve the prosthesis lifetime . careful preoperative plan and the use of the appropriate anatomy of the prosthesis handle are very important .

[ purpose ] to study preoperative and postoperative hip circumference data of various types of congenital dysplasia of the hip treated with total hip replacement , including the femoral offset , femoral neck length , height , and hip abductor arm parameters . [ subjects and methods ] this study included seventy - eight cases of congenital dysplasia of the hip ( i iii type ) . furthermore , four parameters were measured , including the preoperative and postoperative femoral offset . statistical data analysis was performed using the spss 13.0 software . [ results ] the femoral offset was 33.3 8.4 mm ( preoperative ) and 39.1 7.1 mm ( postoperative ) . the femoral head height was 59.5 8.7 mm ( preoperative ) and 68.8 11.0 mm ( postoperative ) . the femoral neck length was 50.8 10.8 mm ( preoperative ) and 61.5 10.4 mm ( postoperative ) . the hip abductor arm was 54.3 9.6 mm ( preoperative ) 64.7 10.1 mm ( postoperative ) . the preoperative and postoperative parameters showed statistical differences . furthermore , no significant differences were evidenced when comparing the postoperative hip parameters with the normal data parameters . [ conclusion ] total hip replacement on congenital dysplasia of the hip could lead to the rebuilt of an almost normal physiological anatomy for each hip case ( type i iii ) .

many stem cell research studies require insertion of genes or genetic sequences in human pluripotent stem cells ( hpscs ) in a way that ensures their expression is predictable , reliable and sustained over time . random insertion by retroviral vectors or plasmid transfection usually results in unpredictable expression subject to variegation and silencing . bacterial artificial chromosome transgenesis can overcome these issues in some cases , but it is an inefficient and cumbersome process . furthermore , the majority of gene therapy applications currently in the clinic or in advanced preclinical development rely on gene addition and , while in situ gene correction may prove feasible and practical for some genetic diseases in the future , gene addition will always be the approach of choice for a number of gene therapy applications . with recent advances in technologies for targeted gene insertion ( gene targeting ) , it is becoming increasingly feasible to introduce a transgene into a predetermined genomic site . the advent of crispr as a genome - editing tool , in particular , dramatically reduced the complexity of gene targeting making it more accessible to the wider scientific community . however , these advances have not been paralleled by advances in the identification and validation of appropriate genomic sites . very few sites have so far been explored for their ability to accommodate newly introduced transgenes . the adeno - associated virus site 1 ( aavs1 ) in chromosome 19 is the site most commonly used today for gene insertion . it was originally recovered as a natural site of integration of wild - type aav in cultured human cell lines and gained popularity due to , on one hand , its ability to afford robust expression of inserted reporter transgenes in various cell types , including hpscs ( embryonic stem cells and induced pluripotent stem cells ( ipscs ) ) and , on the other hand , the development of efficient commercially available tools for its targeting . however , more systematic recent studies have revealed that the aavs1 locus is prone to silencing in some cell lineages . furthermore , a number of applications require dual transgene or reporter expression or transgene insertion in hpsc lines where the aavs1 locus is already occupied by other transgenic sequences , for example by an inducible genome editing enzyme . in the latter example , recently generated hpsc lines harboring a doxycycline - inducible cas9 ( icas9 ) inserted in one allele of the aavs1 locus ( and the reverse tetracycline transactivator , in the other allele ) could be further exploited to mediate high - efficiency crispr - cas9-facilitated insertion of a transgene in a genomic locus provided one was known and available . two other candidate loci for gene insertion that have been investigated in past studies , the chemokine ( c - c motif ) receptor 5 ( ccr5 ) gene a coreceptor of wild - type hiv-1and the human ortholog of the mouse rosa26 locus , lack robust expression in most cell types including hpscs and hence have not found use by the scientific community . a more recently described intergenic locus , h11 , located on chromosome 22 , can support expression of multiple transgenes in hpscs and appears to be a promising candidate worthy of further evaluation . at this stage , the identification of additional candidate loci is therefore warranted if we were to deliver to the research community genomic sites that can support reliable expression and can be used alternatively or in combination to the aavs1 locus . we previously used lentiviral integration to isolate genomic sites in human ipscs and examine their location with respect to known annotated genomic elements with the goal to predict their potential for insertional oncogenesis . however , in this previous study , we did not assess transgene expression across many different sites or the likelihood that a given site will afford sufficient expression based on its position relative to endogenous genes . here , we use a lentiviral screen to analyze the capacity for transgene expression in a large number of genomic sites in human ipscs . we select a specific site on chromosome 15 affording robust expression and establish an ipsc line with a preexisting cassette inserted therein for efficient and versatile gene insertion through recombinase - mediated cassette exchange ( rmce ) . in order to isolate ipsc clones with single vector integrations , which could subsequently be re - used through rmce , we modified our previously described lentiviral vector tns9.3/fng , encoding -globin in an erythroid - specific manner , as well as a selection cassette consisting of a neomycin resistance gene and egfp driven by the constitutive human phosphoglycerate kinase promoter . in the derivative tns9.3/eng vector we use in this study , the hpck - egfp cassette was flanked by two heterospecific loxp sites a wild - type loxp and a lox2272 site in inverse orientation towards each other ( figure 1a , b ) . this cassette can be exchanged by rmce with a cassette harboring the same configuration of two loxp sites , as heterospecific loxp sites can not recombine with each other but can recombine with homologous sites . the placement of the two loxp sites in inverse orientation relative to each other further reduces the probability of recombination events between them ( which would result in cassette excision ) . we transduced the tns9.3/eng vector into the ipsc line thal5.10-cre8 , derived from a patient with -thalassemia and devoid of permanently integrated transgenes . after transduction at a range of multiplicity of infection , the groups of cells more likely to be enriched in single vector copy clones ( transduction efficiency 1030% , as assessed by % egfp cells ) were single - cell subcloned . after selection , 202 neomycin - resistant clones were picked . sixty of them were found to be single - copy by quantitative polymerase chain reaction ( qpcr ) ( figure 1c ) . of these 40 were confirmed by southern blotting to be single clones with single vector integrations ( figure 1d ) , 27 of which could be mapped to the genome ( supplementary table s1 ) . the 40 single copy clones were grown for 34 weeks after the end of antibiotic selection to allow sufficient time for egfp expression to reach steady state . analysis of egfp expression by flow cytometry showed that of the 40 single copy clones , 25 were predominantly ( more than 50% of cells ) egfp and 15 were predominantly egfp ( supplementary figure s1 ) . we analyzed the location of the integration sites in the 27 mapped clones with respect to the four core criteria : ( i ) inside a gene transcription unit ; ( ii ) at a distance of less than 50 kb from a gene 5 ' end ( transcriptional start site ) ; ( iii ) at a distance of less than 300 kb from a gene associated with cancer in humans or model organisms and ( iv ) at a distance of less than 300 kb from a microrna gene ( supplementary table s1 ) . two of the 27 clones ( clones 72 and 99 ) met the four core criteria in agreement with our previous estimates regarding the frequency of lentiviral integration sites genome - wide that are located in safe harbor sites by these criteria . we thus next asked whether sites that can support expression are less likely to meet each of the four criteria . by comparing all 40 egfp and egfp clones against the criteria , we observed that sites supporting sustained expression are more likely to reside inside transcription units and in proximity to genes , including cancer genes ( figure 2 ) . in contrast , sites wherein transgenes silence are randomly distributed with regards to these criteria . on the other hand , proximity to mirna genes was not predictive of the probability of expression , unlike the other three criteria . these results show that transgene insertion sites that support expression at least those accessible to lentiviral integration are enriched in genomic sites that are located inside or in close proximity to endogenous genes . we selected clone a3 ( hereafter referred to as lentiviral - ips - a3 or lips - a3 ) harboring an integration site in chromosome 15 ( hereafter referred to as lips - a3 site , lips - a3s ) , on the basis of its sustained egfp expression over more than 20 passages and its distance from cancer - related genes ( figure 3a , supplementary table s1 ) . the lips - a3s site is within the 3 ' utr region of the dennd4a gene . we did not detect any perturbation of expression of the dennd4a gene or of any of five other coding genes located within 300 kb on either side of the integration site ( figure 3b ) . in order to test whether lips - a3s could support expression of different transgenes we used rmce to exchange the egfp reporter in the lips - a3 line . as a donor for cassette exchange , we constructed an integrase - deficient lentiviral vector ( idlv ) carrying a cassette consisting of the puromycin resistance and the mcerulean genes driven by human phosphoglycerate kinase flanked by loxp and lox2272 sites ( figure 4a ) . this was codelivered with a second integrase - deficient lentiviral vector expressing cre recombinase together with mcherry ( supplementary figure s2 ) . after transduction with both vectors at high efficiency ( over 85% ) , and selection with puromycin , 33 resistant clones were picked . they were shown to express mcerulean and to have lost egfp expression ( figure 4c ) . cassette exchange was further tested and confirmed by multiple pcr primer sets ( figure 4b , d ) and by southern blotting ( figure 4e ) . cre recombinase expression or dna could not be detected by flow cytometry or qpcr , respectively ( supplementary figure s2d , e ) . the cells maintained all features of pluripotency , expressed pluripotent cell markers and could form teratomas comprising tissues of all three embryonic germ layers ( figure 5a , b ) and were karyotypically normal ( figure 5c ) . these results show that the lips - a3 line can be used to efficiently and rapidly introduce transgenes at a known genomic site that can maintain robust and long - term expression . in order to isolate ipsc clones with single vector integrations , which could subsequently be re - used through rmce , we modified our previously described lentiviral vector tns9.3/fng , encoding -globin in an erythroid - specific manner , as well as a selection cassette consisting of a neomycin resistance gene and egfp driven by the constitutive human phosphoglycerate kinase promoter . in the derivative tns9.3/eng vector we use in this study , the hpck - egfp cassette was flanked by two heterospecific loxp sites a wild - type loxp and a lox2272 site in inverse orientation towards each other ( figure 1a , b ) . this cassette can be exchanged by rmce with a cassette harboring the same configuration of two loxp sites , as heterospecific loxp sites can not recombine with each other but can recombine with homologous sites . the placement of the two loxp sites in inverse orientation relative to each other further reduces the probability of recombination events between them ( which would result in cassette excision ) . we transduced the tns9.3/eng vector into the ipsc line thal5.10-cre8 , derived from a patient with -thalassemia and devoid of permanently integrated transgenes . after transduction at a range of multiplicity of infection , the groups of cells more likely to be enriched in single vector copy clones ( transduction efficiency 1030% , as assessed by % egfp cells ) were single - cell subcloned . after selection , 202 neomycin - resistant clones were picked . sixty of them were found to be single - copy by quantitative polymerase chain reaction ( qpcr ) ( figure 1c ) . of these 40 were confirmed by southern blotting to be single clones with single vector integrations ( figure 1d ) , 27 of which could be mapped to the genome ( supplementary table s1 ) . the 40 single copy clones were grown for 34 weeks after the end of antibiotic selection to allow sufficient time for egfp expression to reach steady state . analysis of egfp expression by flow cytometry showed that of the 40 single copy clones , 25 were predominantly ( more than 50% of cells ) egfp and 15 were predominantly egfp ( supplementary figure s1 ) . we analyzed the location of the integration sites in the 27 mapped clones with respect to the four core criteria : ( i ) inside a gene transcription unit ; ( ii ) at a distance of less than 50 kb from a gene 5 ' end ( transcriptional start site ) ; ( iii ) at a distance of less than 300 kb from a gene associated with cancer in humans or model organisms and ( iv ) at a distance of less than 300 kb from a microrna gene ( supplementary table s1 ) . two of the 27 clones ( clones 72 and 99 ) met the four core criteria in agreement with our previous estimates regarding the frequency of lentiviral integration sites genome - wide that are located in we thus next asked whether sites that can support expression are less likely to meet each of the four criteria . by comparing all 40 egfp and egfp clones against the criteria , we observed that sites supporting sustained expression are more likely to reside inside transcription units and in proximity to genes , including cancer genes ( figure 2 ) . in contrast , sites wherein transgenes silence are randomly distributed with regards to these criteria . on the other hand , proximity to mirna genes was not predictive of the probability of expression , unlike the other three criteria . these results show that transgene insertion sites that support expression at least those accessible to lentiviral integration are enriched in genomic sites that are located inside or in close proximity to endogenous genes . we selected clone a3 ( hereafter referred to as lentiviral - ips - a3 or lips - a3 ) harboring an integration site in chromosome 15 ( hereafter referred to as lips - a3 site , lips - a3s ) , on the basis of its sustained egfp expression over more than 20 passages and its distance from cancer - related genes ( figure 3a , supplementary table s1 ) . the lips - a3s site is within the 3 ' utr region of the dennd4a gene . we did not detect any perturbation of expression of the dennd4a gene or of any of five other coding genes located within 300 kb on either side of the integration site ( figure 3b ) . in order to test whether lips - a3s could support expression of different transgenes we used rmce to exchange the egfp reporter in the lips - a3 line . as a donor for cassette exchange , we constructed an integrase - deficient lentiviral vector ( idlv ) carrying a cassette consisting of the puromycin resistance and the mcerulean genes driven by human phosphoglycerate kinase flanked by loxp and lox2272 sites ( figure 4a ) . this was codelivered with a second integrase - deficient lentiviral vector expressing cre recombinase together with mcherry ( supplementary figure s2 ) . after transduction with both vectors at high efficiency ( over 85% ) , and selection with puromycin , 33 resistant clones were picked . they were shown to express mcerulean and to have lost egfp expression ( figure 4c ) . cassette exchange was further tested and confirmed by multiple pcr primer sets ( figure 4b , d ) and by southern blotting ( figure 4e ) . cre recombinase expression or dna could not be detected by flow cytometry or qpcr , respectively ( supplementary figure s2d , e ) . the cells maintained all features of pluripotency , expressed pluripotent cell markers and could form teratomas comprising tissues of all three embryonic germ layers ( figure 5a , b ) and were karyotypically normal ( figure 5c ) . these results show that the lips - a3 line can be used to efficiently and rapidly introduce transgenes at a known genomic site that can maintain robust and long - term expression . the mouse rosa26 locus , a locus widely used for transgenesis in the mouse , was discovered through a retroviral gene trap screen . because lentiviral vectors preferentially integrate in transcriptionally active genomic sites , here we used a lentiviral screen to capture genomic sites enriched in actively transcribed sites . as expected from this , over half of the sites we retrieved ( 25 out of 40 ) expressed egfp ( supplementary table s1 and supplementary figure s1 ) . the locus control region ( lcr ) elements ( dnase - i - hypersensitive sites ) from the beta - globin locus contained in the tns9.3/eng vector that we used for this screen may have further contributed to preserving an open chromatin configuration and active transcription in the neighboring human phosphoglycerate kinase - driven reporter gene . of the 15 sites that did not support egfp expression , approximately half were silenced early on , whereas the other half contained mostly egfp cells shortly after transduction , but showed gradual silencing over several days . these kinetics are consistent with two distinct silencing events , also described by others , one early and rapid and one late that extinguishes transgene expression over a prolonged period of time . in some of the clones that showed late gradual silencing , but not in others , egfp cells could be sorted or picked under a fluorescent microscope and maintained stably expressing egfp thereafter . while lentiviral integrations are enriched in transcriptionally active genes , they are at the same time more likely to reside in the vicinity of endogenous genes and hence perturb their expression . we previously found that less than 20% of lentiviral integrations genome - wide meet our core safe harbor criteria . in agreement with this , the present set of sites contained 2 out of 27 sites that met the criteria , none of which expressed egfp . furthermore , we found an overall inverse correlation between probability of transgene expression and location outside of or far from coding ( but not noncoding ) genes ( figure 2 ) . in other words , genomic sites inside or in proximity to coding genes were enriched in egfp compared to egfpclones . in agreement with our previous estimates , our experimental scheme enabled us to retrieve approximately 1 transgene - expressing clone for which clonality , single integration and the location of the latter in the genome could be thoroughly established for every 10 clones initially picked ( 20 out of 202 in the current study , figure 1c ) . the process of rmce provides the possibility of deriving a variety of ipsc lines expressing transgenes of choice from a single well - characterized paternal line . although transgene expression in differentiated cells of various lineages needs to be further tested , the lips - a3 line described here can potentially be of use in a broad range of applications . these include constitutive or inducible knockdown or overexpression of genes for functional genetics studies , ectopic expression of genes for reprogramming or lineage conversion , insertion of reporters for lineage tracing studies and introduction of therapeutic transgenes for preclinical gene therapy studies . rmce multiplexing using different recombination systems ( cre / loxp and flp / frt ) can further enable more complex serial rmce - based modifications at the lips - a3s locus . rmce is at least as easy and user - accessible as endonuclease - based gene targeting , as it only requires expression of cre recombinase and construction of a compatible donor , similarly to the requirement for expression of the nuclease and delivery of a donor dna in a nuclease - based approach . a potential advantage of rmce over gene targeting mediated by site - specific nucleases is that off - target effects can be avoided and the need to test for them is eliminated . the broader impact of this work lies in the identification of a genomic site the lips - a3s locus on chromosome 15for reliable transgene expression . with gene targeting tools becoming increasingly accessible to the wider scientific community the need for predetermined sites that can support reliable transgene expression is becoming an urgent need . to the best of our knowledge , this is the only genomic site other than aavs1 that has been shown to support robust and long - term expression of multiple transgenes in hpscs . the future development of genome editing tools specific for this site could expand the applications and facilitate the generation of hpsc lines with multiple reporters . these may include any type of site - specific endonuclease : zinc - finger nucleases , meganucleases , transcription activator - like effector nucleases or the crispr - cas9 system . transgene expression afforded by the lips - a3s locus in multiple lineages , cell types , and developmental stages can easily be assessed using the existing lips - a3 line with in vitro differentiation and/or teratoma formation assays . finally , while this site does not meet our previously proposed safe harbor criteria , it is far from known cancer genes and its safety features , including the dysregulation of endogenous genes in the neighborhood of the site by integrated transgenes , can be tested in the future . site may open possibilities for its use in human cell and gene therapy relying on gene addition . while we did not find evidence for perturbation of expression of nearby endogenous genes by our vector integrated in the lipsc - a3s site also , integration of diverse expression cassettes with different constitutive or tissue - specific promoters and regulatory elements can impact the expression of transgenes from this site , as well as the interaction of the integrated cassette with neighboring genetic elements and will thus need to be tested on a case - by - case basis . in conclusion , we report here a human ipsc line with a preinserted cassette that can easily be exchanged with any transgene of choice by rmce , obviating the need to test for off - target effects , as well as a new locus of the human genome that can support reliable transgene expression . human ipscs were cultured on a feeder layer of mitomycin c - treated mouse embryonic fibroblasts ( globalstem , rockville , md ) or in feeder - free conditions and passaged with dispase or accutase as previously described . the tns9.3/eng vector was derived from tns9.3/fng by substituting the 3 ' loxp site with a lox2272 site in inverse orientation relative to the loxp site . lentiviral vector packaging was performed by triple co - transfection of the plasmid dna encoding the vector , pucmd.g and pcmvr8.91 into 293 t cells , as described . for packaging of the integrase - deficient lentiviral vector encoding the cre recombinase , undifferentiated ipscs were dissociated with accutase , and stained with alexa fluor 647-conjugated anti - tra-181 or anti - tra-160 or anti - ssea3 or anti - ssea4 ( bd biosciences , san jose , ca ) . data were acquired in a lsrii cytometer ( bd biosciences ) and analyzed with the flowjo software ( tree star , ashland , or ) . formation assays , ipscs were suspended in he s medium containing 10 mol / l of the rho - associated kinase ( rock ) inhibitor y-27632 ( tocris , bristol , uk ) . approximately 2 million cells were injected intramuscularly into nod - scid il2rg - null ( nsg ) mice ( jackson laboratory , bar harbor , me ) . five to six weeks later , the tumors were surgically dissected and fixed in 4% formaldehyde . all animal experiments were conducted in accordance with protocols approved by the university of washington institutional animal care and use committee and following national institutes of health guidelines for animal welfare . forty - eight hours after transduction , the cells were harvested with accutase and vigorously triturated into single cells . an aliquot of the cells was used for analysis of egfp expression by flow cytometry . transduced cells with gene transfer < 30% ( as estimated by the percentage of egfp cells ) were replated at a density of 1,500 cells / cm on a layer of neo - resistant mitomycin c treated mouse embryonic fibroblasts ( globalstem ) . g418 ( invitrogen , carlsbad , ca ) was added at a concentration of 12.5 g / ml between days 5 and 9 after transduction . approximately 20 days post - transduction , neo - resistant colonies were manually picked and replated into six - well dishes on mitomycin c - treated mouse embryonic fibroblasts . one week later , ~100200 cells from each clone were manually picked and lysed as described . measurement of vector copy number was performed with multiplex qpcr using a standard curve , as described . for southern blot analysis , 510 g of genomic dna was digested with ecori and a radiolabeled fragment spanning the egfp coding region was used as probe , as described . integration site analysis . tns9.3/eng vector integration sites in single - copy clones were mapped by inverse pcr ( ipcr ) , as described . integration sites were judged to be authentic if the sequences were adjacent to vector ltr ends and had a unique hit when aligned to the draft human genome ( university of california santa cruz , ucsc hg19 ) . genomic annotations were also obtained from ucsc hg19 genome browser and mapped against the integration sites . the integration sites of clones a3 , a29 , 72 and 99 were confirmed by integration - specific pcr using an ltr universal forward primer and reverse primers corresponding to the specific genomic sequence adjacent to the vector integration . reverse transcription was performed with superscript iii ( life technologies ) and qpcr was performed with the ssofast evagreen supermix ( bio - rad , hercules , ca ) using primers dpp8-f : gct tgg tcc atc cta cta gat cg , dpp8-r : tag tgg cgt cac aga atc agg , ptplad1-f : ctt aga cct tgt gaa acc aga g , ptplad1-r : ctg ctt tgt gag tct ctc cca , vwa9-f : ctg ctt tgt gag tct ctc cca , vwa9-r : gtt gat ttt gag tgg cta ggg , slsc24a1-f : tca agg gag atc aga agg aga at , slsc24a1-r : gcg tgg att tca ccc tca tct t , dennd4a - f : cca cac acg ttc tgc aaa gtc , dennd4a - r : agg aac aga ttc cgg tag tga , rab11a - f : caa caa gaa gca tcc agg ttg a , rab11a - r : gca cct aca gct cca cga taa t , actin - f : tga agt gtg acg tgg aca tc , actin - r : gga gga gca atg atc ttg at . reactions were carried out in triplicate in a 7500 fast real - time pcr system ( applied biosystems , foster city , ca ) . the donor plm - epc vector was derived from vector plm - mcerulean - p2a - cmyc after replacing the cmyc cdna with the puromycin resistance gene and inserting loxp sites . the vector was packaged as an integrase - deficient lentiviral vector , as described above . for southern blot analyses , two radiolabeled fragments corresponding to the neomycin resistance and the puromycin resistance gene sequences were used as probes . , cells were assayed on a bd fortessa and data were analyzed with flowjo software ( tree star ) . for qpcr , gdna was isolated with the dneasy blood and tissue kit ( qiagen , hilden , germany ) . qpcr was performed with the ssofast evagreen supermix ( bio - rad ) using primers cre - f : tta cgg cgc taa gga tga ct and cre - r : tgc atg atc tcc ggt att ga . reactions were carried out in triplicate in a 7500 fast real - time pcr system ( applied biosystems ) .

transgenesis of human pluripotent stem cells ( hpscs ) can enable and empower a variety of studies in stem cell research , including lineage tracing and functional genetics studies . while in recent years much progress has been made in the development of tools for gene targeting , little attention has been given to the identification of sites in the human genome where transgenes can be inserted and reliably expressed . in order to find human genomic sites capable of supporting long - term and high - level transgene expression in hpscs , we performed a lentiviral screen in human induced pluripotent stem cells ( ipscs ) . we isolated 40 ipsc clones each harboring a single vector copy and characterized the level of transgene expression afforded by each unique integration site . we selected one clone , lips - a3 with an integration site in chromosome 15 maintaining robust expression without silencing and demonstrate that different transgenes can be inserted therein rapidly and efficiently through recombinase - mediated cassette exchange ( rmce ) . the lips - a3 line can greatly facilitate the insertion of reporter and other genes in hpscs . targeting transgenes in the lips - a3s genomic locus can find broad applications in stem cell research and possibly cell and gene therapy .

the study design of the advance study is reported in detail elsewhere ( 24 ) . in brief , 11,140 patients with type 2 diabetes , aged at least 55 years at study entry and with at least one other cardiovascular risk factor , underwent factorial randomization to 1 ) the fixed combination of perindopril and indapamide ( 4 mg/1.25 mg ) or matching placebo and 2 ) intensive ( gliclazide mr - based , to an hba1c target of 6.5% ) or standard ( usual care ) glucose control . the advance trial enrolled subjects from 20 countries in asia , australasia , europe , and north america . a total of 11,126 patients with hdl - c measurements at baseline were included in these analyses . from this population , 1,602 patients were included in the advance retinal measurement ( adrem ) study , which involved serial retinal photography ( 1,241 had assessable images ) . study design and the methods used in adrem are published in detail elsewhere ( 25 ) . approval for the study was obtained from each center s institutional ethics committee , and all participants provided written informed consent . at baseline , venous blood was taken for a fasting lipid profile , hba1c , and creatinine . in addition , urine samples were collected at baseline , 24 months , 48 months , and at the end of the trial for determination of the urinary albumin - to - creatinine ratio ( acr ) . hdl - c level was repeated at 24 and 48 months or at the end of the study . all samples were analyzed by local laboratories , and all assessments could be repeated at physician discretion . participants in adrem had retinal stereoscopic photographs taken after the advance trial randomization visit and at the final visit of the blood pressure arm of the trial . all images were graded centrally according to the early treatment of diabetic retinopathy study classification as modified in the uk prospective diabetes study ( 26 ) . the main outcomes for this analysis were microvascular events , defined as a composite of total renal and retinal events . total renal events were defined as the development of new microalbuminuria ( urinary acr 30300 g / mg ) , new macroalbuminuria ( urinary acr of > 300 g / mg ) , a doubling of creatinine to at least 200 mol / l , the need for renal replacement therapy , or death as a result of renal disease . total retinal events were defined as the development of proliferative retinopathy ( new blood vessels or fibrous proliferations on the disc or elsewhere , vitreous hemorrhage , or preretinal hemorrhage ) , macular edema , diabetes - related blindness , or the use of retinal photocoagulation therapy . participants could record more than one event relating to renal and retinal disease , but only the first event was analyzed for total microvascular events . differences in variables at baseline between subgroups of the study population were tested using the student t test , the mann - whitney test , or the test , as appropriate . participants were censored at their date of death or , for those still alive at the end of follow - up , the date of their last visit . the regression dilution bias in hdl - c was assessed using a linear mixed model , with hdl - c during follow - up as the outcome and baseline hdl - c as the predictor . to calculate the correction factor , hdl - c measurements after microvascular events were excluded ( 27 ) . the risks of events associated with baseline hdl - c level were estimated using cox proportional hazards models , with adjustment for potential confounding baseline covariates including age ( continuous ) , sex ( male / female ) , ethnicity ( white / asian / other ) , treatment groups ( standard vs. intensive glucose control and placebo vs. fixed - dose blood pressure lowering treatment ) , history of microvascular disease ( yes / no ) , smoking status ( current / previous / never ) , current alcohol consumption ( yes / no ) , hba1c ( continuous ) , bmi ( continuous ) , systolic blood pressure ( continuous ) , diabetes duration ( continuous ) , total cholesterol ( continuous ) , creatinine ( continuous ) , and statin use ( yes / no ) . the selection of variables was based on identifying all measured clinical variables of known or suspected prognostic importance for the outcomes of interest . the assumption of the proportional hazards was checked graphically using the log cumulative hazard plot for all variables included in the cox model . the primary analyses compared patients in the highest with those in the lowest third ( or tertile group ) of hdl - c . additional sensitivity analyses were performed comparing the risks in those in the lowest with those in the highest fourths ( or quartile groups ) , excluding patients with microvascular disease , renal disease , or retinal disease at baseline and examining subgroups defined by age , sex , ethnicity , systolic blood pressure , bmi , and age . all p values were calculated from two - tailed tests of statistical significance with a type i error rate of 5% . all analyses were performed using sas version 9.1 ( sas institute , cary , nc ) . at baseline , venous blood was taken for a fasting lipid profile , hba1c , and creatinine . in addition , urine samples were collected at baseline , 24 months , 48 months , and at the end of the trial for determination of the urinary albumin - to - creatinine ratio ( acr ) . hdl - c level was repeated at 24 and 48 months or at the end of the study . all samples were analyzed by local laboratories , and all assessments could be repeated at physician discretion . participants in adrem had retinal stereoscopic photographs taken after the advance trial randomization visit and at the final visit of the blood pressure arm of the trial . all images were graded centrally according to the early treatment of diabetic retinopathy study classification as modified in the uk prospective diabetes study ( 26 ) . the main outcomes for this analysis were microvascular events , defined as a composite of total renal and retinal events . total renal events were defined as the development of new microalbuminuria ( urinary acr 30300 g / mg ) , new macroalbuminuria ( urinary acr of > 300 g / mg ) , a doubling of creatinine to at least 200 mol / l , the need for renal replacement therapy , or death as a result of renal disease . total retinal events were defined as the development of proliferative retinopathy ( new blood vessels or fibrous proliferations on the disc or elsewhere , vitreous hemorrhage , or preretinal hemorrhage ) , macular edema , diabetes - related blindness , or the use of retinal photocoagulation therapy . participants could record more than one event relating to renal and retinal disease , but only the first event was analyzed for total microvascular events . differences in variables at baseline between subgroups of the study population were tested using the student t test , the mann - whitney test , or the test , as appropriate . participants were censored at their date of death or , for those still alive at the end of follow - up , the date of their last visit . the regression dilution bias in hdl - c was assessed using a linear mixed model , with hdl - c during follow - up as the outcome and baseline hdl - c as the predictor . to calculate the correction factor , hdl - c measurements after microvascular events were excluded ( 27 ) . the risks of events associated with baseline hdl - c level were estimated using cox proportional hazards models , with adjustment for potential confounding baseline covariates including age ( continuous ) , sex ( male / female ) , ethnicity ( white / asian / other ) , treatment groups ( standard vs. intensive glucose control and placebo vs. fixed - dose blood pressure lowering treatment ) , history of microvascular disease ( yes / no ) , smoking status ( current / previous / never ) , current alcohol consumption ( yes / no ) , hba1c ( continuous ) , bmi ( continuous ) , systolic blood pressure ( continuous ) , diabetes duration ( continuous ) , total cholesterol ( continuous ) , creatinine ( continuous ) , and statin use ( yes / no ) . the selection of variables was based on identifying all measured clinical variables of known or suspected prognostic importance for the outcomes of interest . the assumption of the proportional hazards was checked graphically using the log cumulative hazard plot for all variables included in the cox model . the primary analyses compared patients in the highest with those in the lowest third ( or tertile group ) of hdl - c . additional sensitivity analyses were performed comparing the risks in those in the lowest with those in the highest fourths ( or quartile groups ) , excluding patients with microvascular disease , renal disease , or retinal disease at baseline and examining subgroups defined by age , sex , ethnicity , systolic blood pressure , bmi , and age . all p values were calculated from two - tailed tests of statistical significance with a type i error rate of 5% . all analyses were performed using sas version 9.1 ( sas institute , cary , nc ) . the advance study enrolled patients with type 2 diabetes at high risk for macrovascular events . at baseline , 10% ( n = 1,155 ) had evidence of microvascular disease . during a median follow - up period of 5 years , 32% ( n = 3,585 ) of participants developed new or worsening microvascular disease . almost one - third ( n = 3,161 [ 28% ] ) developed a renal event : microalbuminuria was the most frequent event , followed by macroalbuminuria , doubling of creatinine to at least 200 mol / l , renal - related death , and the need for renal replacement therapy ( 25 , 3.4 , 1.2 , 0.3 , and 0.2% , respectively ) . a retinal event occurred in 6% ( n = 680 ) : the need for laser therapy was the most frequent event , followed by macular edema , proliferative retinopathy , and diabetes - related blindness ( 3.5 , 3.2 , 2.6 , and 0.4% , respectively ) . table 1 shows the distribution of variables by thirds of baseline hdl - c level . compared with the lowest third of hdl - c , those subjects in the highest third were less likely to be male ( 44.8 vs. 70.8% ) or taking a statin ( 24.9 vs. 31.1% ) and more likely to have lower bmi ( 27.7 vs. 29.1 kg / m ) , lower serum creatinine ( 82.5 vs. 91.0 mol / l ) , and to have never smoked ( 65.3 vs. 49.6% ) . low hdl - c also was associated with higher triglycerides and lower total cholesterol and ldl cholesterol . age , diabetes duration , systolic blood pressure , hba1c , ethnicity , and nonstatin lipid - lowering medication use all were statistically significantly different between thirds ; however , the absolute differences were very small . the relative differences between hdl groups and the baseline variables of statin use , nonstatin lipid - lowering medication use , systolic blood pressure , and hba1c remained stable over the course of study follow - up ( supplementary table 1 ) . baseline variables of study subjects by thirds of serum hdl - c table 2 shows the association , both adjusted and unadjusted , between baseline thirds of hdl - c and the risk of developing new or worsening microvascular disease . compared with the highest third of hdl - c , those in the lowest third had an 11% higher risk of a microvascular event ( unadjusted hazard ratio [ hr ] 1.11 [ 95% ci 1.021.20 ] , p = 0.01 ) . following multivariable adjustment and taking into account regression dilution , the risk was 17% higher ( 1.17 [ 1.061.28 ] , p = 0.001 ) . this finding was driven by an adjusted 19% higher risk of a renal event ( 1.19 [ 1.081.32 ] , p = 0.0005 ) . there was a similar significantly higher risk of developing new microalbuminuria and macroalbuminuria ( 1.14 [ 1.031.27 ] , p = 0.01 , and 1.42 [ 1.071.87 ] , p = 0.01 , respectively ) . furthermore , we observed that those patients in the lowest third of hdl - c were more likely to maintain or progress to a worse category of urinary acr over time , compared with those in the middle and highest third of hdl - c ( 66.8 vs. 62.0 and 58.8% , respectively ) ( supplementary table 2 ) . association between baseline thirds of hdl - c ( tertile [ t ] 1 , t2 , and t3 ) and subsequent development of microvascular disease the direction of the effect was similar , however not statistically significant following adjustment , to the other renal outcomes of doubling of creatinine to at least 200 mol / l and renal - related death . in contrast to the other renal outcomes , the association with the need for renal replacement therapy ( the end point with the smallest number of events ) was nonlinear . there was no association between baseline thirds of hdl - c and the development of retinopathy or any specific type of retinal event . furthermore , there was no association between baseline hdl - c and a wide range of predefined retinal outcomes from the adrem substudy involving 1,241 participants who underwent assessable serial retinal photography , which included progression by the early treatment of diabetic retinopathy study classification and the development of the individual signs of retinopathy ( data not shown ) . sensitivity analyses by fourths ( or quartile groups ) of hdl - c revealed similar findings , namely an inverse relationship between hdl - c level and total microvascular events that was driven primarily by renal events , with no relationship found between hdl - c and retinal events . sensitivity analyses excluding patients with baseline microvascular disease also were similar to the main analysis . when stratified by subgroups defined by age , sex , ethnicity , systolic blood pressure , bmi , or hba1c , there was no evidence of heterogeneity in the association . this study is the largest prospective analysis specifically addressing hdl - c level and risk of microvascular disease in patients with type 2 diabetes . the main finding is that lower baseline hdl - c level is a significant and independent predictor of the development and progression of diabetic nephropathy . compared with patients in the highest third , those in the lowest third of baseline hdl - c had a 19% higher risk of nephropathy , after adjustment for a wide range of potential confounders and accounting for regression dilution . in contrast , there was no association between baseline hdl - c and the risk of diabetic retinopathy . these findings suggest that differences exist in the pathophysiology between the two types of microvascular disease . our findings provide the strongest evidence to date for a role of hdl - c in the development and progression of diabetic nephropathy in patients with type 2 diabetes . several small prospective studies have shown that low hdl - c predicts progression of microalbuminuria ( 810 ) ; however , others have not identified such an association ( 28,29 ) . the largest previous study evaluating this question included 2,193 patients with type 2 diabetes and normal renal function at baseline . in this study , each 0.26 mmol / l higher level of hdl was associated with a 24% lower risk of developing stage 3 chronic kidney disease ( 11 ) . this was not a prespecified end point of the advance study ; however , we are the first to show an inverse relationship between baseline hdl - c and total renal microvascular events . we observed no significant association between hdl - c and a whole spectrum of retinal complications . although some non - hdl lipid fractions previously have been reported to be associated with the severity and progression of retinopathy in patients with type 2 diabetes , no study has shown an association with hdl - c ( 16,20 ) , and our study is concordant with this literature . our findings may be explained by the nature of the retinal events examined as the most frequent retinal end point was laser photocoagulation . because this procedural event is likely to be driven by health service availability and local usual practice in addition to biological risk in a multicenter , international study such as this , its inclusion may have diluted any causal link , if any such association existed . however , we also found no evidence of an association with any of the specific end points examined in the small proportion of advance trial participants who underwent serial retinal photography , a more sensitive and objective measure of retinal disease . the fenofibrate intervention in end point lowering in diabetes trial showed that fenofibrate reduced the development of renal dysfunction and the need for retinal laser therapy ( 22,30 ) . in support of our findings of an association between hdl - c and renal events but not retinal events , fenofibrate seemed to be more protective against the estimated glomerular filtration rate loss in those with baseline dyslipidemia ( defined as low hdl - c and high triglycerides ) ( 30 ) , whereas the retinal benefit was independent of any lipid fraction ( 22 ) . in much the same way , the more recent action to control cardiovascular risk in diabetes eye study showed a benefit of fenofibrate added to statin therapy on the progression of diabetic retinopathy , without an appreciable change in hdl - c level ( hdl - c at 1 year 1.03 mmol / l in the fenofibrate group compared with 1.01 mmol / l in the placebo group ) ( 23 ) . the salient conclusion from these two studies of fenofibrate therapy is that treatment reduced both microvascular complications in type 2 diabetes . this effect , at least in part , may be related to hdl - c level for renal but not retinal complications . on the horizon , there is the promising cholesterylester transfer protein inhibitor , anacetrapib , which increases hdl - c by 138% ( 31 ) . our findings suggest that investigating the effect of hdl - c modulating agents on the development of diabetic nephropathy is warranted . the pathogenesis of diabetic microvascular disease is complex and involves the interplay of endothelial dysfunction , advanced end - glycation products , oxidative stress , and the abnormal production of cytokines and growth factors ( 32,33 ) . the pathobiology of diabetic nephropathy and retinopathy is heterogeneous , with no single characteristic lesion ( 33,34 ) . although retinopathy and nephropathy can coexist in the same patient , the association is less clear in type 2 rather than type 1 diabetes ( 35 ) , perhaps an indication of differing pathophysiology . hdl - c itself is a complex of several lipoproteins , cholesterol , and triglycerides and has several postulated mechanisms of vascular action , notably reverse cholesterol transport as well as anti - inflammatory and antioxidant properties ( 36 ) . furthermore , there is emerging evidence that hdl - c improves glycemic control by modulating glucose uptake into skeletal muscle ( 37 ) and protects against islet -cell dysfunction ( 38 ) . although the exact putative mechanism for protection is unknown , it is clear that hdl - c exerts beneficial effects on many of the pathways known to be detrimental to vascular and kidney biology , but how this could be different in retinopathy is unclear . following the development of microalbuminuria , 2040% of patients with type 2 diabetes will progress to overt nephropathy ( 1 ) . many randomized trials have shown a clear benefit for the treatment of this asymptomatic condition ; thus , early detection is critical ( 39 ) . international guidelines recommend screening at the time of diagnosis of diabetes and thereafter annually for patients with normoalbuminuria ( 1 ) . the use of hdl - c level , along with other established risk factors , may help identify patients at high risk of development and progression of nephropathy and therefore warrant more frequent testing . the strengths of this study are its large sample size , with an ethnically diverse population and rigorous collection of data . this allowed precise estimation of the independent effect of hdl - c on the development and progression of predefined diabetic microvascular disease outcomes . our study also has a number of limitations . because it is a post hoc observational analysis of data from a randomized controlled trial , the results should be interpreted as hypothesis generating . despite the size of this study , there were very few events for some of the end points such as the need for renal replacement therapy and renal death , which limited our ability to provide accurate estimates of the associations for these outcomes . although the difference was small ( < 2.7% ) , adherence to the study ace inhibitor ( perindopril ) was slightly lower in the lowest hdl - c third compared with the middle and highest third ( 77.1 , 79.2 , and 79.8% , respectively ) . in addition , it is possible that not all subtypes of hdl - c have the same relationship with microvascular outcomes . however , we were unable to measure subtype and function of hdl - c in order to explore these relationships in our study . in conclusion , in a large population of patients with type 2 diabetes and after adjustment for a wide variety of confounders , low hdl - c level was shown to be an independent risk factor for the development and progression of diabetic nephropathy . additional research is needed to explore the possible benefits of therapies that increase hdl - c in patients with type 2 diabetes .

objectivealthough low hdl cholesterol ( hdl - c ) is an established risk factor for atherosclerosis , data on hdl - c and the risk of microvascular disease are limited . we tested the association between hdl - c and microvascular disease in a cohort of patients with type 2 diabetes.research design and methodsa total of 11,140 patients with type 2 diabetes and at least one additional vascular risk factor were followed a median of 5 years . cox proportional hazards models were used to assess the association between baseline hdl - c and the development of new or worsening microvascular disease , defined prospectively as a composite of renal and retinal events.resultsthe mean baseline hdl - c level was 1.3 mmol / l ( sd 0.45 mmol / l [ range 0.14.0 ] ) . during follow - up , 32% of patients developed new or worsening microvascular disease , with 28% experiencing a renal event and 6% a retinal event . compared with patients in the highest third , those in the lowest third had a 17% higher risk of microvascular disease ( adjusted hazard ratio 1.17 [ 95% ci 1.061.28 ] , p = 0.001 ) after adjustment for potential confounders and regression dilution . this was driven by a 19% higher risk of renal events ( 1.19 [ 1.081.32 ] , p = 0.0005 ) . there was no association between thirds of hdl - c and retinal events ( 1.01 [ 0.821.25 ] , p = 0.9).conclusionsin patients with type 2 diabetes , hdl - c level is an independent risk factor for the development of microvascular disease affecting the kidney but not the retina .

sudden death has been recognized as an important public health problem for over four centuries . in the 18th century , pope clement xi became concerned with the increase in occurrence of sudden death in rome , and charged his papal physician geofredo lancisi to investigate its cause to no avail . through the centuries the instantaneous nature of death has provided both a scientific and spiritual challenge to investigators to understand its mechanism . despite the interest and intense investigation , neither lancisi nor subsequent investigators have advanced our understanding of either the physiologic triggers of the event , or the nature of the event itself . there have been numerous electrocardiographic recordings of the event , but they have provided little insight into its mechanism . cardiac arrest is largely expressed as ventricular fibrillation , but in approximately 20% of patients the event is due to asystole . with the development of emergency medical care systems , relatively large populations of resuscitated cardiac arrest became available for both retrospective and prospective study . these studies indicate that cardiac arrest usually occurs in individuals who are known to have cardiovascular disease and who have left ventricular dysfunction . although sudden death can occur as the first expression of coronary heart disease , and usually as an acute myocardial infarction , even in these individuals prior evidence of left ventricular damage or dysfunction is present . in addition , many individuals with nonischemic left ventricular dysfunction also experience sudden cardiac arrest . investigators have used animal models and single - cell preparations in an attempt to understand the mechanism of ventricular fibrillation in order to prevent its occurrence . all of these attempts have been frustrated by the instantaneous and transient nature of the physiologic phenomena that precipitate cardiac arrest . for a large part of the past half century , a great deal of research was focused on the suppression of ambient ventricular beats as a surrogate marker of the efficacy of antiarrhythmic drugs . the number of pharmacologic blind alleys that were entered are too numerous to recount , but it is clear that no agent directed at the suppression of ventricular premature beats has been found to be either safe or effective . the most recent investigations with amiodarone carried out in europe and canada failed to demonstrate any convincing evidence for its use for the prevention of sudden death , although the concomitant use of -blockers with amiodarone appeared to offer some advantage . with unusual prescience and technologic skill , mirowski realized that antiarrhythmic therapy was a blind alley and began the development of the automatic internal defibrillator almost 40 years ago , and reported its first successful use in humans in 1980 . that device is refined to such a degree that it is now easily implantable ; it is small in size , but still large in cost . its proven efficacy has spawned a large industry of both physicians and biotech companies that are directed at the expanded clinical use of the device . although the american heart association and the american college of cardiology have reported implantation guidelines , common clinical practice has led to a significant deviation from those guidelines . the ease of implantation and the fear of recurrent sudden death have encouraged both the patient and physician to ' creep - up ' on the guidelines . there is little argument that implantation of the icd in patients with recurrent ventricular fibrillation or symptomatic ventricular tachyarrhythmias in the absence of acute myocardial infarction or ischemia is beneficial . even in this regard , however , the definition has been vague and has provided a window through which any syncopal episode , with little more than the presence of complex ventricular ectopy , has become fair game for the implantation of the icd in patients with left ventricular dysfunction . many of the patients who receive an icd would have been better been treated with revascularization or -blocker therapy . it is essential to understand this practice reality as we embark on the following discussion . recently , patients with heart failure have become a potential target population for implantation of an icd . it is estimated that there are almost 5 million individuals with heart failure in the usa . initially , the device was considered as a bridge to transplantation , because the mortality rate , often sudden death , in patients waiting for a donor heart was high . it soon became clear , however , that most of the patients who were dying on the transplant waiting list were dying of rhythm disturbances as a complication of worsening heart failure . as electrophysiologists became aware of the natural history of heart failure , they realized that sudden death was actually a greater problem in patients with nyha class ii and iii than in those with advanced heart failure . recent clinical trials have emphasized that sudden death represents almost two - thirds of the mortality in mild - to - moderate heart failure . heart failure therapy for most of the past decade has been based primarily on angiotensin - converting enzyme ( ace ) inhibitors , which had little effect on arrhythmic death . the striking decrease in sudden death in the recent -blocker trials has provided , for the first time , a pharmacologic approach to sudden death . these observations could have been assumed from studies in post - myocardial infarction patients with congestive heart failure , in whom propanolol was shown to have a profound effect on sudden death . the recent trials have demonstrated an decrease in total mortality in addition to a decrease in sudden death by 40 - 50% , without any adverse effects and excellent tolerance . therapy with -blockers has provided an entirely new approach to both the treatment of heart failure and the prevention of sudden death . the reduction in sudden death mortality was observed across a broad spectrum of subgroups studied in the metoprolol cr / xl randomized intervention trial in congestive heart failure ( merit - hf ) , the largest -blocker mortality trial , regardless of ejection fraction or heart failure etiology . not only do these drugs decrease the incidence of sudden death , but they also have an important effect on total cardiovascular mortality and death due to worsening heart failure . treatment with carvedilol , bisoprolol and metoprolol in the three major randomized clinical trials decreased the total mortality by approximately 35% and sudden death by 45% . more recently , patients in nyha class iii - iv who were treated with spironolactone experienced a 29% decrease in incidence of sudden death , in addition to a 31% decrease in cardiovascular deaths . it is possible that a combination of these drugs could make an even larger impact on sudden death prevention . the annual cardiovascular mortality rate in patients with heart failure treated with ace inhibitors and -blockers is now approximately 7% in nyha class ii and iii patients . although sudden death remains an important problem , to which a device could appropriately be applied , this solution would pertain to less than 5% of the heart failure patients treated with -blockers . it can be estimated that , if the icd was implanted in 100 patients treated with ace inhibitors and -blockers , sudden death could be prevented in five individuals over the next year . this is presuming that the aid would prevent all of the deaths due to ventricular fibrillation or asystole and that none of the sudden deaths were due to acute progression of the primary disease . because many of these individuals have ischemic heart disease , it can be presumed that some will experience further loss of ventricular function as a result of myocardial infarction . asystole is also assumed to be successfully treated by the icd , because most of the devices have pacing capability . the estimated benefit that could be achieved by icd is rather small and would be obtained at a considerable cost . this cost is significant in the face of the almost 5 million individuals in the usa with heart failure . little has been said about the risk or side effects of the device when left in place for years or the discomfort of inappropriate discharge . it is clear , however , that icd implantation and its safety have improved during the past few years . the device that mirowski developed over 40 years ago has moved far beyond his vision at that time . the appropriate role for the icd a number of major trials are now underway to examine the benefit of these devices in heart failure patients . although these trials fall short in their design , because they do not mandate -blocker therapy , they will expand our understanding of the usefulness of icd in heart failure . perhaps a more appropriate research direction would be to try to identify those persons within the large population of heart failure patients who would benefit the most from implantation of an icd . unfortunately , the easiest answer to the question of who should have the device implanted is to implant it into everyone . this appears to be the most expedient if not efficient approach to the problem , and the one that appears to hold sway in today 's clinical practice . i would submit that the benefit of the icd must be judged in terms of both the socioeconomic and medical costs . in the interval period , it is clear that the addition of -blocker therapy to the treatment of the broad spectrum of patients with heart failure has provided important protection from sudden death and death due to progressive heart failure . it is my view that the icd will add little additional benefit to the treatment of patients with mild - to - moderate heart failure .

recent clinical trials indicate that approximately two - thirds of patients in new york heart association ( nyha ) class ii and iii , who comprise almost 90% of patients with heart failure , die suddenly . patients in nyha class iv usually die of progressive heart failure . implantation of implantable cardioverters defibrillators ( icds ) in this population would represent a huge logistic problem and economic expense . clinical trials have recently demonstrated that -blocker therapy with carvedilol , bisoprolol , and toprol xl decrease the sudden death rate by almost 50% , in addition to impacting significantly on death due to worsening heart failure . this medical approach is beneficial to all patients , and should be our major therapy . however , it is reasonable to attempt to identify that subpopulations of heart failure patients who could benefit from an icd .

allogeneic stem cell transplantation ( allo - sct ) is a curative therapy for different life - threatening malignant and nonmalignant hematologic disorders . in hematologic malignancies , the therapeutic efficacy of allo - sct is due to the graft - versus - tumor ( gvt ) effect . however , the beneficial effect of gvt is counterbalance by the immunological recognition and destruction of cells and tissues of the recipient by the immune effectors of the donor , termed graft - versus - host disease ( gvhd ) . gvhd remains a major source of morbidity and mortality following allo - sct , which limits the use of this treatment in a broader spectrum of patients . it is therefore essential to improve the management of gvhd , in both forms , acute and chronic . this requires a better understanding of the pathophysiology of gvhd in order to identify new therapeutic target and develop new immunosuppressive drugs . furthermore , early diagnosis of gvhd , particularly of acute gvhd , is often difficult because of the nonspecific nature of the associated symptoms and of the numerous differential diagnoses . these issues may lead to delay of the initiation of corticosteroids , which may have dramatic consequences for patients . consequently several teams develop laboratory test to predict the risk of developing gvhd or responsiveness to treatment . indeed the development of biomarkers in the allo - sct setting is crucial , because the early identification of patients at high risk for gvhd has important therapeutic consequences , including more stringent monitoring and intensified prophylaxis of gvhd . beside biological biomarkers , it is essential to develop new tools to aid in early diagnosis of gvhd and particularly of gastrointestinal ( gi ) acute gvhd , whose diagnosis can be particularly difficult . in recent years many progress has been made in medical imaging test and endoscopic techniques , and the interest of these different techniques in the diagnosis of gi acute gvhd has been evaluated in several studies . the aim of this work is , after recalling the clinical aspect and the current practice for the diagnosis of gi acute gvhd , to review the available evidence on these medical imaging test and endoscopic features for the diagnosis of gi acute gvhd ; data on biomarkers have been extensively reviewed by paczesny et al . historically , gvhd was clinically divided as acute and chronic , according to the time of onset . acute gvhd was defined as arising within 100 days after allo - sct , whereas chronic gvhd occurs after 100 days after allo - sct ( with or without preceding acute gvhd ) . however , this distinction was not always so clear - cut , in particular after reduced - intensity conditioning , an increasingly used regimen [ 4 , 5 ] . this led the national institute of health ( nih ) to a new classification , including 2 new entities , late onset acute gvhd and overlap syndrome . late onset acute gvhd is defined as gvhd occurring after day 100 , with clinical features of acute gvhd and no feature of chronic gvhd ; the overlap syndrome is defined as gvhd with features of both acute and chronic gvhd . acute gvhd is a clinicopathological syndrome involving mostly three organs : the skin , the gastrointestinal tract , and the liver . severity of acute gvhd is assessed by the severity of involvement of these three target organs , according to the classification described by glucksberg et al . and modified in 1995 , to include upper intestinal symptoms within the definition of acute gvhd and drop the use of the clinical performance score . including late onset acute gvhd , up to 80% of patients will experience grades ii iv acute gvhd after allo - sct with a match related or unrelated donor . patients presented a typical maculopapular rash , pruritic , palmoplantar impairment is frequent at diagnosis , and the rash can spread to the whole body sparing the scalp . skin detachment may occur in the most severe cases . liver involvement of acute gvhd is first suspected on the biological test , patients have an elevation of serum bilirubin , and then patients presented icterus when bilirubin reached 3050 mol / l . the diagnosis is often difficult as there are many other causes of liver dysfunction after allo - sct , such as viral infection , iron overload , venoocclusive disease , sepsis , or toxic drug effects . a definitive diagnosis could be made by the examination of a liver biopsy ; however , this is rarely performed due to the highly invasive character of the procedure . anorexia or vomiting , alone or associated with diarrhea , is also considered as gi acute gvhd symptom , according to the revised glucksberg classification . diarrhea in acute gvhd is secretory and usually voluminous , often reaching more than 2 l per day . in the most severe cases , unfortunately , these symptoms are nonspecific and encountered in numerous differential diagnoses frequently observed after allo - sct such as infection with clostridium difficile colitis , viral infection ( mainly cytomegalovirus ( cmv ) ) , drug toxicity , or neutropenic enterocolitis . in the setting of umbilical cord blood allo - sct these symptoms may also correspond to the cord colitis syndrome , related to an infection by a newly described bacteria , bradyrhizobium enterica . the diagnosis of gi acute gvhd is based upon the analysis of clinical and laboratory criteria in the appropriate clinical context after excluding other causes . thus bacteriological , virological , and parasitological stool culture , a search for clostridium difficile toxin in stool , and virus dna screening in plasma ( cytomegalovirus ( cmv ) , adenovirus ( adv ) , etc . ) are usually performed in order to exclude other differential diagnoses . the gold standard in the diagnosis of gi acute gvhd is upper and lower gi endoscopy with histological validation . indeed gi acute gvhd is characterized by a patchy distribution of the lesions , which can either affect a short segment of the small bowel or the entire digestive tract [ 1721 ] . given that esophagogastroduodenoscopy ( egd ) and colonoscopy only explore a small segment of the small bowel , their diagnostic yield is limited . also one should bear in mind that endoscopic abnormalities are usually found in a minority of cases ( 1632% ) and are usually nonspecific . consequently , additional biopsies are necessary , despite the lack of specificity of apoptotic bodies , the main histological hallmark , especially in the early phase after allo - sct . finally endoscopic examinations and biopsies are relatively invasive in such fragile patients , often at high risk of bleeding in case of thrombocytopenia . in the last decade , several imaging modalities have been developed to offer extensive and noninvasive examination of the entire small bowel . from the beginning of allo - sct development , attempts have been made to use medical imaging to help diagnose gi acute gvhd . in 1988 , plain abdominal radiography has been evaluated ; 95% of patients with gi acute gvhd presented abnormal radiography with separation of bowel loop indicative of wall thickening , air fluid level , and small bowel dilatation ; however , these features are not specific . consequently several studies have evaluated the contribution of computed tomography ( ct scan ) for the diagnosis of gi acute gvhd ; the features most often found with ct scan are bowel wall thickening , abnormal mucosal enhancement , and bowel dilatation [ 21 , 2527 ] . indeed , similar radiologic finding is seen after allo - sct in many other complications , such as infection with clostridium difficile colitis , viral infection ( mainly cytomegalovirus ( cmv ) ) , or neutropenic enterocolitis [ 21 , 25 ] . however , some features may help differentiate gi acute gvhd from other gi complications of allo - sct . regarding bowel wall thickening , in gi acute gvhd , the thickening is generally moderate , whereas more severe thickening rather suggests clostridium difficile or cmv colitis and neutropenic enterocolitis . furthermore , in gi acute gvhd , bowel wall thickening involved both small and large intestine in almost all cases ; we can thus exclude clostridium difficile colitis which affects only the large intestine . neutropenic enterocolitis also involves both small and large intestine ; however , right colonic or caecum involvement present in 75100% of neutropenic enterocolitis [ 25 , 28 ] is uncommon in gi acute gvhd [ 2527 ] and a discontinuous distribution of bowel involvement is more frequent in gi acute gvhd . abnormal mucosal enhancement after administration of intravenous contrast material has been reported in up to 89% of patients with gi acute gvhd [ 2527 ] and seems to be more common than in other gi complications after allo - sct . some author reported the use of negative oral contrast material , leading to a lower rate of visualization of mucosal enhancement . furthermore , gi symptoms of gi acute gvhd often prevent the use of oral contrast material . regarding other radiological findings seen in gi acute gvhd ; the incidence of bowel dilatation varies in an important way according to the studies , from 23 to 86% [ 2527 ] . however , this remains more frequent than in other gi disorders after allo - sct or in neutropenic patients . mesenteric infiltration , ascites , or blood vessel abnormality ( engorgement of vasa recta ) is also frequently observed after gi acute gvhd [ 2527 ] and could help for the diagnosis . results confirm that ct scan morphology of gi acute gvhd is independent of the delay between gi acute gvhd onset and the time of execution of the ct scan . the interest of this study lies on the development of a severity ct scan score based on 6 criteria to grade gi acute gvhd . thus ct scan lacks specificity for the diagnosis of gi acute gvhd ; however , with an experienced user , it can provide valuable assistance for the diagnosis of gi acute gvhd . , one must pay attention to the nephrotoxicity of intravenous iodine contrast material , since renal function impairment is frequent after allo - sct . furthermore , a ct scan score that correlates with clinical grading could be performed and provide valuable information regarding the evaluation of gi acute gvhd severity . the use of magnetic resonance imaging ( mri ) was reported only in a few cases [ 30 , 31 ] . mri findings are comparable to the ct scan features ; in particular a bowel wall thickening associated with abnormal mucosal enhancement with gadolinium is reported . as for ct scan these features are not specific and do not permit discriminating gi acute gvhd from other etiologies . thus mri should not be performed for the diagnosis of gi acute gvhd ; however , when mri is performed after allo - sct for another indication , these features should make us consider the diagnosis of gi acute gvhd . findings with ultrasonography ( us ) are comparable to those of ct scan . author reported bowel wall thickening and bowel dilatation [ 3134 ] . blood vessel abnormalities are also described using color doppler imaging [ 33 , 34 ] . this study included 52 patients with gi symptoms after allo - sct , 15 patients were lost to followup , 17 patients develop gi acute gvhd , and in 20 patients no gi acute gvhd was diagnosed ( 4 with chemo / radiotoxicity , one with escherichia coli sepsis , and 15 without a specific diagnosis ) . us detects bowel wall thickening or bowel dilatation in 16/17 patients with gi acute gvhd . the sensitivity and specificity were , respectively , 94 and 95% in this study . however , one may question the control group , for gi acute gvhd diagnosis , and the interest of complementary investigation is to discriminate with infectious complication and neutropenic enterocolitis , whereas only 1 patient presented infectious gi complication in the control group . thus , despite this study , us lacks specificity for gi acute gvhd diagnosis and we do not recommend its use in this setting . on the other hand , recent progress has been made , with the development of contrast - enhanced ultrasound ( ceus ) . it is a real - time microvascular imaging technique , which use has been possible thanks to the development of novel echocontrast - enhancing agent . these echocontrast - enhancing agents are gas - filled microbubbles , administered intravenously to the systemic circulation . ceus has been used in active crohn 's disease , in which neovascularization of the small bowel wall has been described . considering the role of neovascularization in the early stages of gi acute gvhd recently described , several teams evaluated the use of ceus for the diagnosis of gi acute gvhd . after standard us , an ultrasound contrast agent is administered i.v . and ceus is performed on the intestine . in 2011 , schreyer at al . reported that in contrast to crohn 's disease patients and healthy volunteers , patients with gi acute gvhd showed transmural penetration of microbubbles into the bowel lumen . out of 17 patients with biopsy proven gi acute gvhd , 14 showed penetration of the iv applied microbubbles into the bowel lumen , whereas in patients with viral or bacterial infection of the gi tract , no transmural penetration of the microbubbles was observed . the sensitivity , specificity , npv , and ppv were , respectively , 82 , 100 , 81 , and 100% in this study . the second study compares ceus finding in 14 patients with lower gi acute gvhd , 16 patients with only upper gi acute gvhd , and 4 patients with neutropenic enterocolitis . transmural penetration of microbubbles into the bowel lumen was observed in all patients with lower gi acute gvhd and in one patient with neutropenic enterocolitis and was not reported in patients with only upper gi acute gvhd . furthermore , in patients with lower gi acute gvhd , ceus normalized after response to treatment , whereas , in corticosteroid resistant patients , it remained unchanged . overall , ceus appears to be a sensitive method for assessment of gi acute gvhd , with a very high specificity from 75% to 100% ; however , there were very few patients in the control group , 3 in one , and in the other 4 ( only patients with neutropenic enterocolitis were clinically relevant ) . finally , ceus is a noninvasive promising tool for assessment and monitoring of gi acute gvhd , restricted to patients with lower gi acute gvhd . furthermore , it requires specific device and a highly trained physician able to perform the procedure , not available in many hospitals . f - fluorodeoxyglucose positron emission tomography ( fdg - pet / ct ) is widely used in haematological malignancies and solid tumours [ 42 , 43 ] . more recently fdg - pet / ct has been found to be useful in detecting inflamed areas throughout the entire intestinal tract in inflammatory bowel diseases . furthermore , two preliminary case reports suggest that fdg - pet / ct is appropriate to assess the exact localization of gi acute gvhd and to evaluate treatment response [ 45 , 46 ] . these observations have led to the evaluation of fdg - pet / ct for the diagnosis of gi acute gvhd in prospective studies . stelljes et al . demonstrated in 2008 that gi acute gvhd was visualized by fdg - pet / ct in a murine model and translated these results in a cohort of 30 patients with diarrhoea and suspected lower gi acute gvhd . no increased fdg was detected in 13 patients without histologic evidence of gi acute gvhd , whereas 14 of 17 patients with biopsies proven gi acute gvhd showed significant fdg uptake in the gut . the se , sp , npv , and ppv were , respectively , 82 , 100 , 81 , and 100% . a secondary fdg - pet / ct was performed in some patients and a significant decrease of fdg uptake was observed in patients ' responder to gi acute gvhd therapy . recently we published the results of a prospective study evaluating the predictive value of fdg - pet / ct for early diagnosis of gi acute gvhd in 42 patients . fdg - pet / ct was systematically performed at a median of 28 days ( range : 2438 ) after allo - sct and gi acute gvhd onset was monitored within 4 weeks after completion of fdg - pet / ct . among the 10 patients who presented gi acute gvhd , 9 had a positive fdg - pet / ct ( figure 1 ) . regarding the 32 patients without gi acute gvhd , only 5 patients had a positive fdg - pet / ct . the se , sp , npv , and ppv were 81 , 90 , 96 , and 60% , respectively . fdg - pet / ct appears to be a noninvasive , sensitive , and very specific biomarker for gi acute gvhd diagnosis in patients with diarrhoea and may help to monitor the response to corticosteroids [ 45 , 47 ] . furthermore fdg - pet / ct can detect inflammatory activity of the gi tract associated with subclinical gi acute gvhd before clinical symptoms onset [ 46 , 48 ] in contrast to ct scan , mri scan , or us , where radiological features occur later . finally , data remains limited so far and larger prospective studies seem indispensable before using fdg - pet / ct in routine in this setting . the development of new pet tracers targeting apoptosis , one of the histological hallmarks of gi - agvhd , is very promising . wireless video - capsule endoscopy ( wce ) is a sensitive , noninvasive diagnostic tool for the exploration of the small intestine . wce is routinely used for the diagnosis of anemia and occult bleeding and crohn 's disease and recognition of intestinal tumors . given that gi acute gvhd is characterized by a patchy distribution of lesions , which can either affect a short segment or involve the whole gastrointestinal tract , wce that explores the whole small intestine is a seductive approach for gi acute gvhd diagnosis . several studies evaluated wce in adults [ 20 , 5154 ] and children [ 55 , 56 ] with gi acute gvhd symptoms . these studies highlight the heterogeneity of the involvement of the small bowel ; in some patients wce shows lesion of the whole small bowel , whereas in others lesions are discontinuous , sparing some area of the small bowel . regarding lesions observed , author reported edema , erythema , erosion , ulceration , and bleeding ( figure 2 ) . a delayed gastric transit time was also reported [ 52 , 54 ] . it should be noted that intestinal stenosis contraindicated wce . fortunately intestinal stenosis is exceptional and occurs in patients with a long history of refractory gi acute gvhd . we highlight that no retention of the capsule in the small bowel was reported in the setting of gi acute gvhd . evaluated wce in 10 patients with suspected gi acute gvhd and results were compared with egd and duodenal biopsies . five patients had a normal wce examination ; egd and duodenal biopsies were also normal in those five patients . four patients were successfully treated symptomatically and one patient died from toxoplasmosis . regarding the five remaining patients , wce disclosed gi acute gvhd lesion , whereas egd was considered as normal in 2 of them and duodenal biopsy in one of them . gi symptoms improved in all of these patients after adjustments of their immunosuppressive treatment . the contribution of wce for the adaptation of immunosuppressive therapy in patients with suspected gi acute gvhd and the apparently high npv in this study is very appealing . neumann et al . also investigated the role of wce in 14 patients with clinical symptoms of gi acute gvhd . only 13 patients could be evaluated , given that the capsule remained in the stomach and was removed endoscopically in one patient . in all 7 of 13 patients with histologically confirmed gi acute gvhd , wce reveals typical signs of gi acute gvhd , whereas egd reveals sign of gvhd in only 4 patients . in all 6 remaining patients , egd , histology , and wce were normal but in one patient wce showed erosive enteritis . here to , the npv of wce is very high , with better sensitivity than egd . however , biopsies can not be performed during wce , and despite the apparently high npv in these studies , they are based on furthermore , wce probably lacks specificity given that erosion and ulceration were reported on wce performed in patients with cmv diseases after allo - sct [ 52 , 57 ] . overall , wce realization can be useful in patients with gi acute gvhd symptoms and normal egd . furthermore , a visual grading of gi acute gvhd lesion has been performed in several studies [ 20 , 51 ] according to the brand criteria as follows : grade 0 , normal ; grade i , focal erythema ; grade ii , moderate or diffuse erythema , nodularity ; grade iii , erosion and or vulnerable mucosa ; and grade iv , ulceration , denuded mucosa , and bleeding . it could be useful to evaluate the severity of gi acute gvhd and manage immunosuppressive treatment . finally , one limit of wce for the investigation of gi acute gvhd is the bowel preparation required . indeed , despite it being noninvasive , patients had a 12-hour overnight fast and drank 1 liter of a polyethylene glycol - electrolyte ( peg ) solution 2 hours prior to swallowing the wce . the absorption of the peg may be difficult in frail patients with upper gastrointestinal symptoms and limits wce use in this setting . regarding new procedure in endoscopic examination , another interesting method is confocal laser endomicroscopy , a high - resolution imaging modality , allowing access to in vivo histology at the subcellular level during ongoing endoscopy . confocal laser endomicroscopy , either using an endoscope - based ( ecle ) or a probe - based technology ( pcle ) , aims to decrease the number of standard biopsies and their associated risk , by providing real time in situ microscopy . cle requires the iv injection of a fluorescent contrast agent , fluorescein , allowing vasculature and cellular architecture to be better appreciated . in addition acriflavine could be administered topically providing staining of cell nuclei , not stained by fluorescein . established cle applications include the diagnosis of barrett 's oesophagus , gastric intestinal metaplasia coeliac disease , and microscopic colitis . apoptotic bodies on histological lesion is one of the hallmarks of gi acute gvhd and the ecle equivalent of an apoptotic cell is 100% nuclear staining with topical acriflavine . examined endomicroscopy features and conventional histology on targeted biopsies of sigmoid and rectum in 35 patients with acute diarrhea after allo - sct . in 16 patients , ecle and histology showed no evidence of gi acute gvhd . in 19 cases , there was evidence of gi acute gvhd on conventional histology . in 14 of these 19 cases , the diagnosis of gi acute gvhd could be already performed by ecle during the procedure . endomicroscopy showed single cell apoptosis within the crypt epithelium in histologically proven grade 1 gi acute gvhd . other features seen at endomicroscopy for grades 24 include apoptosis of entire crypts , focal destruction of crypt , and capillary leakage of fluorescein ; in severe case ( grade 4 gi acute gvhd ) , near complete destruction of the colonic crypt with flat mucosa was observed . in the 30 controls ( 15 infectious colitis and 15 ulcerative colitis ) although this study is limited to lower gi acute gvhd , data suggest that cle can be used to diagnose upper gi acute gvhd . thus hundorfean et al . reported the diagnostic of gastric acute gvhd utilization of fluorescein guided cle . more recently our team evaluated wce and pcle to predict the risk of gi acute gvhd in early stage asymptomatic patients . 15 patients were prospectively examined with a wce and duodenal and colorectal pcle and underwent standard biopsies between day 21 and day 28 following allo - sct independently of the presence or absence of digestive symptoms . in the current study pcle was used after iv injection of fluoresceine and we were not able to study the nuclei . however , our study focused on the vascular compartment and dynamic changes , since the role of neovascularization in the early stages of gi acute gvhd has been recently highlighted . furthermore , acriflavine is not approved for use in france and in many other countries . during follow - up period pcle accurately identified 7 out of 8 patients who developed acute gvhd during followup ( figure 3 ) . pcle presents mild anomaly in 2 patients who did not develop any sign of acute gvhd . the se , sp , npv , and ppv of pcle to predict the onset of acute gvhd were 87 , 71 , 77 , and 83% , respectively . overall , cle allows rapid diagnostic of gi acute gvhd with high accuracy while performing endoscopy . reported a sp of 100% and discriminated very accurately gi acute gvhd from infectious complications . once gi acute gvhd has been diagnosed in vivo , unnecessary biopsy could be avoided , in these frequently thrombocytopenic and at high risk of bleeding patients . furthermore , pcle allows very early detection of lesions suggestive of gi acute gvhd before the onset of symptoms , which probably reflect the global alloreactivity in the body . this confirmation is indispensable before being able to recommend the use of this technique in routine . furthermore , it requires specific device and experienced users , which are not yet available in all centers . achievements have been made regarding the development of new tools to assess the diagnosis of gi acute gvhd . advantages , limitations , future developments , and recommendations regarding the use of each technique are summarized in table 1 . the contribution of conventional imaging techniques , ct scan , mri scan , and us , is limited . their use is not recommended in clinical practice even if in some situations they could be valuable . data regarding the new imaging technique , fdg - pet / ct and more particularly ceus , are more promising , even if further prospective studies are warranted to validate their use in clinical practice . however , starting today , their use may be considered in some patients where the diagnosis of gi acute gvhd could not be achieved and to evaluate the response to corticosteroids . regarding wce its use should be considered in patients with gi acute gvhd symptoms and normal upper and lower endoscopy or in case of contraindication in order to explore the whole small bowel . finally , in situ histology using cle is probably the future , to avoid biopsies in thrombopenic patients and make an immediate diagnosis , enabling the clinician to begin corticosteroids without delay .

allogeneic stem cell transplantation ( allo - sct ) is a curative therapy for different life - threatening malignant and nonmalignant hematologic disorders . graft - versus - host disease ( gvhd ) remains a major source of morbidity and mortality following allo - sct , which limits the use of this treatment in a broader spectrum of patients . early diagnostic of gvhd is essential to initiate treatment as soon as possible . unfortunately , the diagnosis of gvhd may be difficult to establish , because of the nonspecific nature of the associated symptoms and of the numerous differential diagnosis . this is particularly true regarding gastrointestinal ( gi ) acute gvhd . in the recent years many progress has been made in medical imaging test and endoscopic techniques . the interest of these different techniques in the diagnosis of gi acute gvhd has been evaluated in several studies . with this background we review the contributions , limitations , and future prospect of these techniques in the diagnosis of gi acute gvhd .

the antibiotic squalamine forms a lyotropic liquid crystal at very low concentrations in water ( 0.3 - 3.5% w / v ) , which remains stable over a wide range of temperature ( 1 - 40 c ) and ph ( 4 - 8 ) . squalamine is positively charged , and comparison of the alignment of ubiquitin relative to 36 previously reported alignment conditions shows that it differs substantially from most of these , but is closest to liquid crystalline cetyl pyridinium bromide . high precision residual dipolar couplings ( rdcs ) measured for the backbone 1h-15n , 15n-13c , 1h-13c , and 13c-13c one - bond interactions in the squalamine medium fit well to the static structural model previously derived from nmr data . inclusion into the structure refinement procedure of these rdcs , together with 1h-15n and 1h-13c rdcs newly measured in pf1 , results in improved agreement between alignment - induced changes in 13c chemical shift , 3jhnh values , and 13c-13c rdcs and corresponding values predicted by the structure , thereby validating the high quality of the single - conformer structural model . this result indicates that fitting of a single model to experimental data provides a better description of the average conformation than does averaging over previously reported nmr - derived ensemble representations . the latter can capture dynamic aspects of a protein , thus making the two representations valuable complements to one another .

recently , many new antiepileptic drugs ( aeds ) have been developed , which has led to additional options for the care of patients with epilepsy . new aeds are needed because there are still patients with epilepsy that is refractory to established aeds . furthermore , there is a need for new aeds without pharmacokinetic drug interactions , drugs with different mechanism of action to allow synergistic combination therapy , and drugs with fewer adverse events ( including idiosyncratic , teratogenic , and cognitive ones ) compared with old aeds . therefore , it has become more important to gain understanding of the efficacy , mechanisms of action , and adverse - event profiles of new aeds and drug interactions between these drugs , which play a key role in rational and effective combination therapy . the national institute for health and care excellence ( nice ) guidelines also point out that the aim of treatment is to abolish seizures completely , while also keeping the side effects of treatment to a minimum so that patients can lead a life that is as normal as possible.1 it is well known that more than 30% of patients continue to have seizures despite pharmacological treatment . in other words , up to 70% of patients seizures can be well controlled by aeds,2 although some patients experience fluctuating courses between remission and seizure recurrence.3,4 the probability of seizure freedom in patients with newly diagnosed epilepsy declines rapidly after the failure of the first monotherapy . therefore , the selection of the initial aed depends on the characteristics and frequency of aed adverse events to a greater extent . if efficacy is not very different between aeds , the propensity and nature of adverse events are the determining factors in the choice of aed . although the good efficacy of the initial monotherapy is well known , the relative efficacy of new aeds over old aeds has not been established . new aed approvals are achieved by add - on trials of patients with refractory epilepsy . this system can not represent all patients in real clinical practice , especially patients who need initial monotherapy.5 only a few studies have been conducted to evaluate this issue . the sanad trial was designed in an unblinded , randomized fashion to evaluate the effectiveness of carbamazepine , gabapentin , lamotrigine , oxcarbazepine , or topiramate in the treatment of partial epilepsy.6 the comparison parameters were time to treatment failure , which was defined as the discontinuation of randomized drugs because of either inadequate seizure control or intolerable side effects . the retention rate encompassed the efficacy , tolerability , and quality of life ( fig . 1 ) . in summary , regarding time to treatment failure , lamotrigine was significantly better than carbamazepine , gabapentin , and topiramate , and exhibited a nonsignificant advantage compared with oxcarbazepine . regarding time to 1-year remission , carbamazepine was significantly better than gabapentin , but exhibited a nonsignificant advantage against lamotrigine , topiramate , and oxcarbazepine . in other words , even though some new aeds yielded a better retention rate , they failed to show better efficacy than old aeds . in another sanad trial , the effectiveness of valproate , lamotrigine , or topiramate to treat generalized and unclassified epilepsy was evaluated.7 regarding time to treatment failure , valproate was significantly better than topiramate , but there was no significant difference between valproate and lamotrigine . valproate was significantly better than lamotrigine in time to 1-year remission , but there was no significant difference between valproate and topiramate . therefore , for the treatment of generalized or unclassified epilepsy , new aeds also failed to show better efficacy than old aeds . one randomized double - blinded trial was performed to compare the efficacy of levetiracetam and controlled - release carbamazepine ( carbamazepine cr ) in patients with newly diagnosed epilepsy.8 the 6-month seizure - freedom rate was almost the same for both drugs : 73.1% for levetiracetam and 72.8% for carbamazepine . fewer patients on levetiracetam ( 14.4% ) discontinued treatment because of adverse events compared with carbamazepine cr ( 19.2% ) , although this difference did not reach statistical significance . recently , the komet study was completed to compare the efficacy of carbamazepine cr and extended - release sodium valproate ( val - proate er ) , as well as levetiracetam in patients with newly diagnosed epilepsy.9 this was a two - parallel - group , stratified trial . if the physician thought that valproate er was the best treatment , the patients were randomized into treatment with valproate er or levetiracetam . when the physician regarded carbamazepine cr as the best choice , the patients in this stratum were randomized into levetiracetam or car - bamazepine cr arms . the conclusion of this trial was simple : levetiracetam was not superior to either valproate er or carbamazepine cr regarding the time to treatment withdrawal or time to first seizure . if the efficacy of new aeds is not superior to that of old aeds , what is the meaning of developing and using new aeds ? patients with symptomatic or cryptogenic epilepsy are more likely to exhibit continuation of seizures.10 combination therapy or epilepsy surgery can be considered for these patients . when combining aeds , many important factors should be considered , including efficacy , adverse events , spectrum , pharmacokinetic drug interactions , pharmacodynamic interactions , titration speed , and speed of action onset of aeds . pharmacodynamics is the mechanism of action of aeds and the manner in which aeds exert their effect on the target organ for the generation of programmed action . aeds can exert their antiepileptic effect via the inhibition of excitatory neurotransmitter systems or the enhancement of inhibitory neurotransmitter systems . there are many different mechanisms of action for various aeds.11 moreover , some aeds have multiple mechanisms . in excitatory neurotransmitter systems , phenytoin , carbamazepine , valproate , lamotrigine , felbamate , topiramate , and zonisamide involve voltage - gated sodium channels ( fig . 2 ) . lacosamide acts on voltage - gated sodium channels and is unique in the slow inactivation of sodium channels . levetiracetam has a unique mechanism , as it binds to the sv2a protein of synaptic vesicles , which functions in synaptic modulation . topiramate and perampanel may have an effect on kainate receptors and on the ampa receptor , respectively . 3 ) , barbiturate and benzodiazepine bind directly to the gaba receptor , which hyperpolarizes cell membranes by opening chloride channels . tiagabin inhibits gaba reuptake in the presynaptic membrane , which leads to the accumulation of gaba in the synaptic cleft . the same effect can be achieved by vigabatrin , by binding irreversibly to gaba transaminase ( which metabolizes gaba ) . when combining drugs , in theory , the combination of aeds with different mechanisms may have a higher chance of synergistic effects.12 a synergistic effect implies that the effect of the combination of two drugs is greater than just the simple sum of the effects of the individual drugs.13 an animal experiment showed that some drug combinations might exhibit synergism.12 for example , oxcarbazepine plus levetiracetam or lamotrigine plus valproate may have advantages in this sense . some combinations of aeds sometimes exacerbate adverse events . the combination of two sodium - channel blockers can increase neurotoxic adverse events , such as dizziness , diplopia , or ataxia . therefore , understanding the pharmacodynamics of aeds is important to design effective and safe combinations of aeds . new aeds have unique and multiple mechanisms of actions , which can allow the development of various effective and rational combinations . pharmacokinetic interactions represent the mutual effect of two combined drugs on the absorption , distribution , metabolism , and excretion of drugs . when combining aeds , the absence of pharmacokinetic drug interactions is easier to handle.14 several new aeds do not involve cytochrome p-450 enzyme systems , or do so to a lesser extent ( fig . furthermore , in patients who are taking other drugs to treat comorbid diseases , the absence of drug interactions is important . as mentioned above , a 50% responder rate is usually used to assess the efficacy of a test drug for the approval of new aeds . however , the goal of epilepsy treatment is to abolish seizure with minimization of adverse events there are various outcome measures for aeds , including percent seizure reduction , responder rate , time to first seizure , presence of adverse events , retention rate , and assessment of quality of life ( qol).5 the treatment goal should be relevant to real - world settings and should include comprehensive measures of efficacy , tolerability , and qol based on reliable and valid assessment tools . clinical effectiveness encompasses all these aspects , and the retention rate is very similar to this concept ( fig . for example , the qol of patients is dependent on seizure freedom , adverse events of aeds , and presence of anxiety or depression.5,15 qol improvement occurs primarily among patients who achieve complete seizure freedom . there is no measurable impact of the degree of reduction in seizure frequency on qol . adverse events of aeds and the presence of anxiety / depression are other important factors that affect qol . therefore , even though new aeds can not achieve seizure freedom , if they have fewer adverse events and play a role in reducing anxiety or depression , they would contribute to the improvement of the qol of patients . although the efficacy of new aeds does not exceed that of old aeds , on an individual basis , there should be differences in efficacy and in the appearance of adverse events among different drugs . for example , if the efficacy of drugs a and b is similar and one patient does not respond to drug a , this does not necessarily mean that the patient does not respond to drug b ( fig . if many drugs are available , the number of therapeutic tools is increased and there is a higher chance of treating patients successfully . furthermore , the development of a new drug enables a greater number of drug combinations . although the effect of valproate is excellent on generalized epilepsies , its use is limited in many patients because of its side effects , such as increased incidence of congenital malformations , low iq of offspring , and polycystic ovaries.16,17 in that sense , the development of new aeds with broad - spectrum efficacy is also important . the increased incidence of congenital malformations after aed treatment is a serious issue for the treatment of childbearing women . several nationwide pregnancy registries have been developed to clarify this issue.1821 to date , some new aeds have been proven to be relatively safe during pregnancy . the long - term effects of old aeds are another issue with this type of drug . old aeds , especially those with enzyme - inducing effects , have a negative impact on bone metabolism.22 the use of old aeds is associated with reduced bone density and increased risk of fracture . long - term treatment with old aeds has been associated with metabolic changes that lead to an increased risk of atherosclerosis in patients with epilepsy.23 patients who were receiving long - term monotherapy with carbamazepine , phenytoin , or valproate exhibited altered circulatory markers of vascular risk , which was significantly associated with the duration of epilepsy . it was documented that treatment with new aeds , such as levetiracetam , oxcarbazepine , and top - iramate , increased the level of low - density lipoprotein , homocysteine , and apolipoprotein b.24 patients with epilepsy , especially elderly ones , exhibit a high frequency of concomitant diseases , such as vascular , cardiac , and cognitive disorders . new aeds without drug interactions are suitable for the treatment of patients with epilepsy who have concomitant diseases . even though the efficacy of new aeds is not stronger than that of old aeds , there are advantages in using new aeds and essential needs for their development . there are still many patients who are refractory to established aeds . when combining aeds to treat these patients , new aeds have advantages . they have unique or different mechanisms of action that enable the design of possible synergistic combinations . an absence of response to a certain aed does not necessarily mean an absence of response to other aeds . if many new aeds are developed , the number of therapeutic weapons available to obtain this individual response is increased . many new aeds with fewer adverse events , including the induction of congenital malformations , have been developed . other concerns about the long - term effect of established aeds , such as bone health and the development of atherosclerosis , may be alleviated by the use of new aeds .

achieving complete seizure remission without adverse events is the goal of epilepsy treatment . recently , many new antiepileptic drugs ( aeds ) have been developed . even though the efficacy of new aeds is not stronger than that of old aeds , there are advantages in using new aeds . they have unique or different mechanisms of action that enable the creation of possible synergistic combinations . they usually exhibit fewer or no pharmacokinetic drug interactions . furthermore , the response to aeds varies individually . a similar efficacy does not imply a similar response from all patients . many new aeds have fewer adverse events , including induction of congenital malformations . other concerns about the long - term effects of established aeds , such as bone health and development of atherosclerosis , may be alleviated by the use of new aeds . new aeds are needed to achieve better care of patients with epilepsy .

femoroacetabular impingement ( fai ) is an anatomic and functional condition that results in a mechanical conflict in the hips , producing pathological contact between the acetabulum and the femoral head neck junction . some authors have suggested a relationship between fai deformity and the subsequent development of osteoarthritis [ 1 , 2 ] . three types of fai have been described : pincer , cam and mixed . pincer fai is characterized by focal or general over - coverage of the femoral head by the acetabulum , while cam - type fai is characterized by the presence of an aspherical portion of the femoral head neck junction . the mixed type is diagnosed when both types of impingements are found [ 3 , 4 ] . the alpha angle is a radiological measure that has been proposed for the diagnosis and evaluation of surgical treatment in cam type fai ( figs . 1 and 2 ) . the normal value of the angle is controversial ; even more so is the value to be considered pathological . first described in 2002 , notzli et al . suggested that the pathological value was greater than 50. in this study , the alpha angle was measured using magnetic resonance imaging ( mri ) . tannast et al . , in a review published in 2007 , proposed the same cut - off value , but with computed tomography ( ct ) . it was measured in an oblique axial ct reconstruction of the femoral neck , at the anterolateral region . 2 . shows the measurement of an angle alpha in symptomatic patients who underwent surgery . it was measured in an oblique axial ct reconstruction of the femoral neck , at the anterolateral region . it was measured in an oblique axial ct reconstruction of the femoral neck , at the anterolateral region . it was measured in an oblique axial ct reconstruction of the femoral neck , at the anterolateral region . in 2005 , proposed a cut - off value of 50. the study compares alpha angle , measured in ct , between symptomatic and asymptomatic individuals . five years later , the same author published an article , in which , the average alpha angle , measured in mri , was 50.15 in asymptomatic population . allen et al . in 2009 , using radiographs , and kang et al . in 2010 using ct , reported that angle values of more than 55.5 and 55 , respectively , have to be considered pathological ; only asymptomatic volunteers were included in both studies [ 5 , 10 ] . in an earlier performed study by the current group , we found that a cut - off value of 50 using ct would characterize 28% of the asymptomatic population as pathological . lastly pollard et al . , in 2010 using radiographs , established that the average normal value of the alpha angle is 47.5 , with a 95% confidence interval of 4649. the initial studies that set pathological cut - off values between 50 and 55 for the alpha angle have been invalidated by later studies performed on healthy populations , in which greater values for the alpha angle have been found in asymptomatic volunteers . in addition , those initials studies lacked the appropriate statistical methodology to determine cut - off values for diagnostic tests [ 13 , 14 ] . the aim of this study was to compare measurements of the alpha angle in asymptomatic volunteers and patients who had undergone surgery for symptomatic cam - type fai and determine a diagnostic cut - off value using a regression model and roc curve . a case control type diagnostic test study was designed and approved by our institution s ethics review board . all participants provided informed consent . cases were defined as patients who had undergone arthroscopy surgery for symptomatic fai in our institution between 2011 and 2012 . controls were defined as patients with no history of hip pain who underwent a ct scan for a non - joint or bone - related reason . patients that met inclusion criteria for cases were those who presented hip pain referred to the inguinal region , a positive flexion adduction internal rotation ( fadir ) test on physical examination and a positive lidocaine test , and subsequently underwent surgical treatment via hip arthroscopy . for the lidocaine test , a radiologist , who specializes in the musculoskeletal area , injected 3 cc of 5% lidocaine directly into the hip under an ultrasound guide . a fadir test was performed before and after the injection , and was considered positive when the patient subjectively experienced a 50% pain decrease in a visual analogue scale . in addition , inclusion in the study required the presence of an intraoperative cam type fai pathomorphology and osteochondroplasty procedure performed . exclusion criteria were previous hip surgeries , history of hip dysplasia and patients in whom surgical procedures were performed only in the acetabulum during arthroscopy . a retrospective review of clinical records was conducted using the above inclusion and exclusion criteria to identify cases for this study . the control population consisted of individuals who consulted in our institution for a non - joint or bone - related reason , and who required an abdominal and pelvic ct for diagnosis . prior to enrolment , volunteers completed a questionnaire , which included asking for current or past history of hip - related pain or hip surgery . information was reprocessed to multi - planar reconstructions of 3 mm . in both cohorts , the alpha angle was measured in an oblique axial ct reconstruction of the femoral neck , in the anterolateral region at 1:30 . in this location , the neck was divided into thirds , the measurement was taken in the middle third . only the affected hip was measured in cases , while controls underwent measurement of both hips , with the average of the left and right hip recorded . measurements were performed in both groups by the chief musculoskeletal radiology ( j.d . ) of our institution . figures 1 and 2 show two examples of the femoral alpha angle measurement performed in the study . descriptive analysis was first conducted , and average and standard deviations of the alpha angle of both groups were reported . in the second step , a logistic regression model was estimated , in which the presence of fai ( case / control ) was used as the dependent variable and the alpha angle measurement as the independent variable . a hosmer lemeshow goodness - of - fit test was done to test logistic regression assumptions ( this is considered appropriate if p > 0.15 ) . the receiver operating characteristics ( roc ) curve was calculated to determine the discrimination capacity . the area under the curve was interpreted according to hosmer and lemeshow recommendations ( table i ) . finally , probabilities associated with each cut - off value of the alpha angle were calculated , with the value that maximized sensitivity and specificity selected as the diagnostic cut - off value [ 13 , 15 ] . a significance level of 0.05 was established and 95% confidence interval was reported . all analyses were performed using stata v11.2 ( statacorp lp , college station , tx , usa ) . table i. shows discrimination ability of roc curve according to the value of area under the curve based on hosmer and lemeshow area under roc curvediscrimination0.500.60luck0.610.70low0.710.80acceptable0.810.90very good0.911excellent shows discrimination ability of roc curve according to the value of area under the curve based on hosmer and lemeshow thirty - eight patients ( 82.6% ) that underwent surgery for fai between 2011 and 2012 at our institution were included ; all of them underwent hip arthroscopy . eight patients were excluded due to the presence of pincer - type only fai without any evidence of cam - type pathomorphology . the average age was 36.1 years ( 11.8 ) among cases and 36.8 years ( 14.4 ) among controls , gender and age were summarize in table ii . the average wiberg angle was 38 ( 7.2 ) among controls and 39 ( 4.1 ) among cases . a non - parametric median test was performed showing no difference ( p = 0.30 ) , and a logistic regression was estimated showing a non - significant odds ratio 0.98 [ 0.921.04 ] . table ii . shows descriptive analysis of age and gender by groupscasecontrolsp ( test)male21/38 ( 55.26%)41/101 ( 40.59%)female17/38 ( 44.74%)60/101 ( 59.41%)0.13 ( fisher exact)age ( years)36.12 ( 11.82)36.82 ( 14.43)0.95 ( wilcoxon unpaired)male age ( years)30.20 ( 11.78)36.50 ( 13.18)0.10 ( wilcoxon unpaired)female ( years)40.79 ( 09.78)37.29 ( 16.24)0.17 ( wilcoxon unpaired ) shows descriptive analysis of age and gender by groups the average alpha angle was 66.8 ( 12.2 ) among cases and 47.8 ( 5.3 ) among controls . a logistic regression model was estimated and an odds ratio of 1.28 [ 1.181.39 ] was obtained , meaning that , as the angle alpha increases , the risk of being symptomatic increases , this was statistically significant . an alpha angle of 57 maximized sensitivity ( 92% ) and specificity ( 95% ) , and correctly classified 94% of patients in this study . table iii shows the sensitivity and specificity estimated by the regression model at 50 , 57 , 60 and 65 degrees of alpha angle . the area under the curve was 0.96 , which is excellent based on hosmer and lemeshow . table iii . shows the sensitivity and specificity estimated by the logistic regression model at different cut - off values of femoral alpha anglecut off valuesensivity ( % ) specificity ( % ) > 509774>579295>607595>654896 shows roc curve obtained after the estimation of the logistic regression model . the area under the curve was 0.96 , which is excellent based on hosmer and lemeshow . shows the sensitivity and specificity estimated by the logistic regression model at different cut - off values of femoral alpha angle the alpha angle has been frequently cited as a measure of cam - type fai . however , there are controversies in the contemporary literature regarding the ability of this angle to discriminate between asymptomatic and symptomatic individuals , and even more controversy regarding the cut - off value that defines cam - type fai . in a study published in 2002 , notzli et al . included 39 patients with hip symptoms , positive physical exam and positive mri for cam - type fai and compared those patients with 40 controls . however , this study only showed that both groups had different average values for the alpha angle , with the authors suggesting that an angle > 50 should be used as the cut - off value . in 2005 , beaul et al . published a retrospective study that included 30 patients who had undergone surgery for cam - type fai and 12 healthy individuals the alpha angle was measured using ct and the selected cut - off value was 50.5 because that gave 100% specificity to the sample . it should be noted that there was only one control every three cases , and the methodology for choosing the cut - off value was based on a descriptive analysis without an estimated model such as logistic regression . in a retrospective review of 2803 anteroposterior radiographs , gosvig et al . suggested a pathological value of 83 for men and 57 for women . however , the medical reasons to request the radiographs were not mentioned in the article ; as the radiographies were retrospectively reviewed , no clinical elements were used for inclusion or exclusion criteria and some patients who were included had osteoarthritis . in addition , the use of the standard deviation is not the most accurate method to determine a diagnostic cut - off value . allen et al . , in a study that included 113 individuals , found that an alpha angle of 60 has an odds ratio of 2.59 of having hip pain . nonetheless , the odds ratio of this study was calculated respecting the contralateral hip of the same individual ; in others words , the contralateral asymptomatic hip was used as the control group . theoretically , this assumption is wrong for morphological analysis ; it can not be assumed that the left and right hips provide independent observations , taking into account the fact that both have the same genetic basis , makes findings difficult to interpret . sutter et al . performed a study that included 53 asymptomatic individuals and 53 patients with symptomatic cam - type fai . they measured the alpha angle in radially reformatted mri images , assessing the contour of the head neck junction . they found that a diagnostic cut - off value of 60 in the anterolateral region had a sensitivity of 7276% and specificity of 7380% . this finding is similar to that reported in this study , stating that the cut - off value is greater than the 5055 suggested by earlier studies . however , measurement of the alpha angle in radians , as performed by sutter et al . , is mostly used for investigative purpose and not in clinical practice . in addition , sutter et al . used a control / case ratio of only 1 ; in contrast , we used three times more controls than cases to determinate the cut off value . finally , to discriminate patients with pincer - type fai from those with cam or mixed type fai , sutter et al . used an alpha angle of < 55 as an exclusion criteria for patients ( cases ) with symptomatic fai . in this study , this must be taken into account as a potential source of bias , as sutter et al . study is that the anterolateral region is the area with better discrimination ( the same region where we perform our single measurement ) , nevertheless the cut - off value can be overestimated as they exclude patients based on the alpha angle measurement . a strength of this study is that the recruitment of healthy individuals ( controls ) was prospective , measuring the alpha angle with ct scanning with no unnecessary radiation . furthermore , we recruited a case / control ratio of 3:1 , which , we believe , gives the study greater reliability . as the proportion of female / male was different between groups we conducted a fisher exact test to determine if this difference was significant . a p value of 0.13 was obtained , meaning that it was not statically significant ( table ii ) . secondly , in an earlier performed study by the current group , alpha angle measurement in healthy people was not associated with gender , weight , height or age . another important strength is that the cases presented symptoms , physical examination and an intraarticular lidocaine test consistent with cam - type fai . finally , the use of a logistic regression model is a valid methodology to obtain diagnostic cut - off points for a pathology [ 13 , 14 ] . another strength of the study is that no difference in wiberg angle was found between both cohorts , so pincer has little influence in the hip pain of the case groups . the lack of analysis of inter or intraobserver agreement is an important issue in this study . the same author ( j.d . ) , who is the chief of musculoskeletal radiology at our institution , performed all measurements for both cohorts . taking this into consideration , we can say that the measurements were performed by a highly trained observer , and if any measurement error exists , it is a systematic error . moreover , beyond the radiology report of the measurement , he did not participate in the clinic s decision whether the patients should or should not need hip arthroscopy . it must be noted that in our institution , patients who undergo hip arthroscopy have a special sheet in their clinic record that states symptom location , physical examination findings , ct measurements and arthroscopy findings . however , as patients were included retrospectively , we can not assess how the angle measured influenced the indications for the procedure and the indication for osteochondroplasty . another weakness is that control volunteers were recruited without a physical examination of the hip . fai is a dynamic condition , asymptomatic individuals could have a high alpha angle , but no pain , only because the patient does not place cam pathomorphology in risk with an activity that use extreme hip range of motion [ 19 , 20 ] . on the other hand , the measurement on ct scan was only a single measurement and multiple measurements along the femoral head neck junction may add further diagnostic ability . the alpha angle value measured in an oblique ct reconstruction of the femoral neck has a high discriminating capacity for the diagnosis of symptomatic cam - type fai . if a patient complains of hip pain and an alpha angle of 57 is found in ct , strongly suggest that cam impingement is causing the pain .

the normal value of alpha angle is controversial . the aim of this study was to compare the alpha angle in asymptomatic volunteers versus patients who had undergone surgery for symptomatic cam - type femoroacetabular impingement ( fai ) and determine a diagnostic cut - off value for symptomatic cam impingement . this is a diagnostic test study . cases were defined as those patients who had undergone surgery for symptomatic cam or mixed type fai . controls were defined as asymptomatic volunteers , with no history of hip pain who had undergone a computed tomography ( ct ) scan of the abdomen and pelvis for a non - joint or bone - related reason . in both groups , the alpha angle was measured in an oblique axial ct reconstruction of the femoral neck . a logistic regression model was first estimated and a receiver operating characteristics ( roc ) curve was then calculated . the diagnostic cut - off value selected was the one that maximizes sensitivity and specificity . data were analysed from 38 consecutive cases of cam or mixed fai and 101 controls . the average alpha angle was 67(12 ) among cases and 48(5 ) among controls . an odds ratio of 1.28 [ 1.181.39 ] was obtained . a roc curve of 0.96 [ 0.930.99 ] was calculated , and using an alpha angle of 57 as the diagnostic cut - off value , provided a sensitivity of 92% and a specificity of 95% . if a patient complains of hip pain and an alpha angle of 57 is found in ct , strongly suggest that cam impingement is causing the pain .

pioneer ( nct 01185314 ) was a prospective , multinational , epidemiological study of egfr mutation status in patients from asia with newly diagnosed advanced ( stage iiib or iv ) nsclc of adenocarcinoma histology . secondary objectives were to investigate the correlation between egfr mutation status and demographic and clinical factors ; to investigate the attrition rates of egfr mutation testing ; and to investigate the correlation of egfr mutation status between histology and cytology for patients who provided both samples . eligible patients were aged 20 years or older , with histological / cytological confirmed advanced ( stage iiib / iv ) , treatment - nave , adenocarcinoma nsclc . data collected included date of the first diagnosis of nsclc , histological type , american joint committee on cancer disease stage , and number of organs with metastases . availability of tumor samples ( biopsy , surgical specimen , or cytology ) was an inclusion criterion in the study . the study was conducted in accordance with the declaration of helsinki , the international conference on harmonisation guidelines for good clinical practice , applicable regulatory requirements , and astrazeneca s policy on bioethics , and was approved by the ethics committees of all study centers . all patients provided written informed consent before the initiation of data collection and sample testing . acquisition , preparation , and processing of tumor material were performed in line with routine clinical practice at participating hospital laboratories . tumor egfr mutation status was determined by analyzing dna extracted from formalin - fixed , paraffin - embedded archival tumor tissue ( using validated methods previously published by fukuoka et al . ) or from cytology samples ( including fine - needle aspirates and bronchial washings ) . samples underwent central , histopathological review to ensure that they were adequate for use and where appropriate , hematoxylin and eosin - stained tissue was classified by suitably qualified pathologists according to the most recent world health organization classification . samples considered suitable for downstream biomarker analysis were progressed to biomarker analysis ( on the basis of quality , sample source , and tumor content [ > 100 tumor cells ] ) . all samples were tested by using an amplification refractory mutation system ( arms)-based egfr mutation detection kit ( scorpion arms ivd2 ; qiagen , crawley , united kingdom ) . the arms ivd2 kit is able to detect 29 mutations : three in exon 18 ( g719a , g719s , and g719c ; the kit was unable to distinguish between these subtypes , which are referred to as g719x hereafter ) , 19 deletions in exon 19 , two mutations in exon 20 ( s768i , t790 m ) , three insertions in exon 20 , and two mutations in exon 21 ( l858r , l861q ) . the egfr mutation status of each patient s tumor was assessed from the individual status of all egfr mutation types and recorded as one of the following : positive ( mutation detected for at least one of the mutation types assayed ) , negative ( no mutation detected in any of the mutation types assayed ) , or undetermined / unknown ( a positive or negative result could not be determined as per laboratory assessment [ assay fail , insufficient dna , fail because of assay criteria , or no / insufficient sample ] ) . the overall distribution of egfr mutation status ( primary end point ) was summarized as the number of patients in the per - protocol ( pp ) population ( all patients who did not significantly deviate from the study protocol ) classified in each of the three mutation status categories ( positive , negative , or undetermined / unknown ) . percentages of patients in the positive and negative groups were calculated with corresponding 95% confidence intervals ( cis ) using the wilson score method , both overall and for demographic / clinical subgroups , including country / region , sex , ethnic group , smoking status , smoking pack - years , disease stage , and histology type . the specific egfr mutations detected by the arms kit were only summarized , with no formal statistical comparison performed . frequency of egfr mutation was compared between demographic and clinical subgroups with the use of /fisher s exact test , with no correction made for multiple testing . to best predict egfr mutation frequency , factors with p less than 0.05 in the univariate analysis were further analyzed by multivariate logistic regression ( at 1% significance level because of the large data set ) . to investigate any correlation of egfr mutation status between histology and cytology for patients who provided both types of samples , the probability ( and 95% cis ) of agreement between sample types sample size was calculated to obtain an accurate estimate of the proportion of patients with egfr mutation - positive tumors . assuming a percentage of 40% , more than 1047 samples were required to ensure a 95% ci of less than 3% ( wilson score method ) . taking into account patients with tumors of undetermined status , an overall sample size of 1270 was chosen . pioneer ( nct 01185314 ) was a prospective , multinational , epidemiological study of egfr mutation status in patients from asia with newly diagnosed advanced ( stage iiib or iv ) nsclc of adenocarcinoma histology . secondary objectives were to investigate the correlation between egfr mutation status and demographic and clinical factors ; to investigate the attrition rates of egfr mutation testing ; and to investigate the correlation of egfr mutation status between histology and cytology for patients who provided both samples . eligible patients were aged 20 years or older , with histological / cytological confirmed advanced ( stage iiib / iv ) , treatment - nave , adenocarcinoma nsclc . data collected included date of the first diagnosis of nsclc , histological type , american joint committee on cancer disease stage , and number of organs with metastases . availability of tumor samples ( biopsy , surgical specimen , or cytology ) was an inclusion criterion in the study . the study was conducted in accordance with the declaration of helsinki , the international conference on harmonisation guidelines for good clinical practice , applicable regulatory requirements , and astrazeneca s policy on bioethics , and was approved by the ethics committees of all study centers . all patients provided written informed consent before the initiation of data collection and sample testing . acquisition , preparation , and processing of tumor material were performed in line with routine clinical practice at participating hospital laboratories . tumor egfr mutation status was determined by analyzing dna extracted from formalin - fixed , paraffin - embedded archival tumor tissue ( using validated methods previously published by fukuoka et al . ) or from cytology samples ( including fine - needle aspirates and bronchial washings ) . samples underwent central , histopathological review to ensure that they were adequate for use and where appropriate , hematoxylin and eosin - stained tissue was classified by suitably qualified pathologists according to the most recent world health organization classification . samples considered suitable for downstream biomarker analysis were progressed to biomarker analysis ( on the basis of quality , sample source , and tumor content [ > 100 tumor cells ] ) . all samples were tested by using an amplification refractory mutation system ( arms)-based egfr mutation detection kit ( scorpion arms ivd2 ; qiagen , crawley , united kingdom ) . the arms ivd2 kit is able to detect 29 mutations : three in exon 18 ( g719a , g719s , and g719c ; the kit was unable to distinguish between these subtypes , which are referred to as g719x hereafter ) , 19 deletions in exon 19 , two mutations in exon 20 ( s768i , t790 m ) , three insertions in exon 20 , and two mutations in exon 21 ( l858r , l861q ) . the egfr mutation status of each patient s tumor was assessed from the individual status of all egfr mutation types and recorded as one of the following : positive ( mutation detected for at least one of the mutation types assayed ) , negative ( no mutation detected in any of the mutation types assayed ) , or undetermined / unknown ( a positive or negative result could not be determined as per laboratory assessment [ assay fail , insufficient dna , fail because of assay criteria , or no / insufficient sample ] ) . acquisition , preparation , and processing of tumor material were performed in line with routine clinical practice at participating hospital laboratories . tumor egfr mutation status was determined by analyzing dna extracted from formalin - fixed , paraffin - embedded archival tumor tissue ( using validated methods previously published by fukuoka et al . ) or from cytology samples ( including fine - needle aspirates and bronchial washings ) . samples underwent central , histopathological review to ensure that they were adequate for use and where appropriate , hematoxylin and eosin - stained tissue was classified by suitably qualified pathologists according to the most recent world health organization classification . samples considered suitable for downstream biomarker analysis were progressed to biomarker analysis ( on the basis of quality , sample source , and tumor content [ > 100 tumor cells ] ) . all samples were tested by using an amplification refractory mutation system ( arms)-based egfr mutation detection kit ( scorpion arms ivd2 ; qiagen , crawley , united kingdom ) . the arms ivd2 kit is able to detect 29 mutations : three in exon 18 ( g719a , g719s , and g719c ; the kit was unable to distinguish between these subtypes , which are referred to as g719x hereafter ) , 19 deletions in exon 19 , two mutations in exon 20 ( s768i , t790 m ) , three insertions in exon 20 , and two mutations in exon 21 ( l858r , l861q ) . the egfr mutation status of each patient s tumor was assessed from the individual status of all egfr mutation types and recorded as one of the following : positive ( mutation detected for at least one of the mutation types assayed ) , negative ( no mutation detected in any of the mutation types assayed ) , or undetermined / unknown ( a positive or negative result could not be determined as per laboratory assessment [ assay fail , insufficient dna , fail because of assay criteria , or no / insufficient sample ] ) . the overall distribution of egfr mutation status ( primary end point ) was summarized as the number of patients in the per - protocol ( pp ) population ( all patients who did not significantly deviate from the study protocol ) classified in each of the three mutation status categories ( positive , negative , or undetermined / unknown ) . percentages of patients in the positive and negative groups were calculated with corresponding 95% confidence intervals ( cis ) using the wilson score method , both overall and for demographic / clinical subgroups , including country / region , sex , ethnic group , smoking status , smoking pack - years , disease stage , and histology type . the specific egfr mutations detected by the arms kit were only summarized , with no formal statistical comparison performed . frequency of egfr mutation was compared between demographic and clinical subgroups with the use of /fisher s exact test , with no correction made for multiple testing . to best predict egfr mutation frequency , factors with p less than 0.05 in the univariate analysis were further analyzed by multivariate logistic regression ( at 1% significance level because of the large data set ) . to investigate any correlation of egfr mutation status between histology and cytology for patients who provided both types of samples , the probability ( and 95% cis ) of agreement between sample types sample size was calculated to obtain an accurate estimate of the proportion of patients with egfr mutation - positive tumors . assuming a percentage of 40% , more than 1047 samples were required to ensure a 95% ci of less than 3% ( wilson score method ) . taking into account patients with tumors of undetermined status , an overall sample size of 1270 was chosen . from september 29 , 2010 to july 31 , 2011 , 1510 patients were enrolled from 51 investigational sites in seven asian countries / regions ( china mainland , hong kong , india , philippines , taiwan , thailand , and vietnam ) ( fig . 1482 had no important protocol deviations , had samples available for mutation analysis , and were included in the pp population . of note , three patients were less than 20 years old , deviating from the inclusion criteria that patients should be 20 years or older ; however , this was considered a minor deviation and these patients were included in the pp population . two patients had major protocol deviations : one patient did not have histologically or cytologically confirmed nsclc , and one patient had received previous treatment for nsclc . three patients were < 20 years old and therefore violated the study inclusion criteria . this deviation was considered to be minor and these patients were subsequently included in the pp population . four patients had tumor samples that were not tested for egfr mutations : two biopsy samples were lost after pathological reading , one sample did not have a clot for preparing a tissue block , and the dna concentration of one block was insufficient for testing . egfr , epidermal growth factor receptor ; nsclc , non small - cell lung cancer ; pp , per - protocol population . overall demography / clinical characteristics for the pp population are summarized in table 1 ; 43.4% ( 643 of 1482 ) were female , median age was 60 years ( range , 1794 years ) , and 52.6% ( 779 of 1482 ) were never - smokers . nearly three - quarters of patients ( 73.8% [ 1093 of 1482 ] ) were of chinese ethnicity . key demographic and clinical characteristics ( pp population ) of 1486 patients with no important protocol deviations , tumor samples from four were not tested : two biopsy samples were lost after pathological reading , one sample did not have a clot for preparing a tissue block , and the dna concentration of one block was insufficient for testing . of the remaining 1482 samples tested , 169 ( 11.4% ) egfr mutation analysis was successful in samples from 1450 of 1482 patients ( 97.8% ; 95% ci , 97.0%98.5% ) ; 32 of 1482 ( 2.2% ; 95% ci , 1.5%3.0% ) were undetermined ( unknown ) , of which eight were cytology samples . among the 1450 evaluable samples , 746 ( 51.4% ; 95% ci , 48.954.0 ) were egfr mutation positive and 704 ( 48.6% ; 95% ci , 46.051.1 ) were egfr mutation negative . of patients with a known time interval between physicians requesting and obtaining a test result ( n = 1475 ) , the mean ( sd ) time interval for reporting the test was 17.6 ( 13.3 ) days ( median , 15.0 ; range , 1148 days ) ; for the majority of patients ( 1168 ; 79.2% ) , the time interval was less than 21 days . tumor egfr mutation frequency for patients in demographic / clinical subgroups is presented in table 2 . factors with a statistically significant association with egfr mutation status ( or fisher s exact test ) were country , sex , ethnicity , smoking status , smoking pack - years ( all p < 0.001 ) , disease stage ( p = 0.009 ) , and histology type ( p = 0.016 ) , and are briefly summarized below . caution is advised when interpreting results of these univariate analyses because individual demographic / clinical factors may be influenced by others and therefore may not represent a true effect of that variable : egfr mutation frequency for demographic and clinical characteristic subgroups ( pp population ) country / region egfr mutation frequency was highest in patients from vietnam ( 64.2% [ 77 of 120 ] ) and lowest in patients from india ( 22.2% [ 16 of 72 ] ) ( other countries 47.2% [ 76 of 161 ] to 62.1% [ 108 of 174 ] ) . sex egfr mutation frequency was significantly higher in females ( 61.1% [ 384 of 628 ] ) than males ( 44.0% [ 362 of 822 ] ) . egfr mutation frequency was highest for those of kinh ( vietnamese ) ethnicity ( 64.2% [ 77 of 120 ] ) and lowest for those of indian ethnicity ( 21.9% [ 16 of 73 ] ) . egfr mutation frequency was highest among never - smokers ( 60.7% [ 462 of 761 ] ) compared with ex - smokers ( 43.2% [ 130 of 301 ] ) , occasional smokers ( 51.6% [ 33 of 64 ] ) , or regular smokers ( 37.3% [ 121 of 324 ] ) . smoking pack - years egfr mutation frequency was highest among never - smokers ( 60.7% ) and decreased as pack - year number increased ( > 010 pack - years : 57.9% ; > 50 pack - years : 31.4% ) . a similar trend was observed by sex : males ( 010 pack - years : 55.9% [ 161 of 288 ] , > 1030 pack - years : 46.6% [ 123 of 264 ] , and > 30 pack - years : 28.2% [ 74 of 262 ] ) ; females ( 010 pack - years : 62.5% [ 371 of 594 ] , > 1030 pack - years : 37.5% [ 9 of 24 ] , and > 30 pack - years : 40.0% [ 4 of 10 ] ) . egfr mutation frequency was significantly higher among patients with stage iv disease ( 53.5% [ 612 of 1144 ] ) compared with iiib ( 43.2% [ 117 of 271 ] ) or other stage ( 48.6% [ 17 of 35 ] ) . egfr mutation frequency was significantly higher among patients with adenocarcinoma not otherwise specified histology ( 52.2% [ 718 of 1376 ] ) compared with adenocarcinoma bronchoalveolar histology ( 37.8% [ 28 of 74 ] ) . factors not significantly correlating with egfr mutation frequency were age ( p = 0.565 ) , time from diagnosis ( p = 0.612 ) , existence of malignant pleural effusion ( p = 0.265 ) , primary tumor stage ( tumor staging t1-tx ; p = 0.454 ) , regional lymph node involvement ( p = 0.075 ) , and tumor grade ( p = 0.369 ) . multivariate logistic regression ( with 1% significance level ) identified ethnicity ( p < 0.001 ) and smoking pack - years ( p < 0.001 ) to be independent predictive factors for egfr mutation status ( table 3 ) . when stratified by smoking status or pack - years , sex was no longer found to be significant ( fig . multivariate logistic regression analysis for egfr mutation status ( pp population ) combined effect of sex and ( a ) smoking status and ( b ) pack - years on frequency of egfr mutation ( pp population ) . a , p values for logistic regression model . smoking history definitions : never - smoker ( patients who had never smoked cigarettes in their lifetime ) ; ex - smoker ( patients who had previously smoked but no longer smoked ) ; occasional smoker ( patients who smoked , but not every day ) ; regular smoker ( patients who smoked every day ) . individual mutation types , including multiple mutations , are summarized in table 4 . of the 1450 evaluable samples , 671 ( 46.3% ) harbored activating ( sensitizing ) mutations alone , 42 ( 2.9% ) had resistance mutations alone , and 33 ( 2.3% ) had a combination of activating and resistance mutations . the most common mutations detected were deletion in exon 19 ( deletion alone 22.1% [ 321 of 1450 ] ; alone and in combination with others 24.3% [ 352 of 1450 ] ) and l858r point mutation in exon 21 ( l858r alone 20.9% [ 303 of 1450 ] ; alone and in combination with others 22.9% [ 332 of 1450 ] ) . tumors from 21 patients ( 1.4% ) harbored t790 m resistance mutations , of which five ( 0.3% ) had t790 m alone . summary of individual egfr mutation types ( including multiple mutations ) in total , 23 patients ( 1.5% of pp population ) provided both histology and cytology samples for mutation analysis . the egfr mutation status ( and specific mutations found ) of matched histology and cytology samples were concordant for 21 patients ( positive n = 13 , negative n = 8 ; 91.3% concordance [ wilson score 95% ci , 73.2%97.6% ; coefficient 0.817 ] ) . matched samples from two patients were discordant ( one patient s samples were positive for histology and negative for cytology , with the other patient having a reverse of this result ) . from september 29 , 2010 to july 31 , 2011 , 1510 patients were enrolled from 51 investigational sites in seven asian countries / regions ( china mainland , hong kong , india , philippines , taiwan , thailand , and vietnam ) ( fig . 1482 had no important protocol deviations , had samples available for mutation analysis , and were included in the pp population . of note , three patients were less than 20 years old , deviating from the inclusion criteria that patients should be 20 years or older ; however , this was considered a minor deviation and these patients were included in the pp population . two patients had major protocol deviations : one patient did not have histologically or cytologically confirmed nsclc , and one patient had received previous treatment for nsclc . three patients were < 20 years old and therefore violated the study inclusion criteria . this deviation was considered to be minor and these patients were subsequently included in the pp population . four patients had tumor samples that were not tested for egfr mutations : two biopsy samples were lost after pathological reading , one sample did not have a clot for preparing a tissue block , and the dna concentration of one block was insufficient for testing . egfr , epidermal growth factor receptor ; nsclc , non small - cell lung cancer ; pp , per - protocol population . overall demography / clinical characteristics for the pp population are summarized in table 1 ; 43.4% ( 643 of 1482 ) were female , median age was 60 years ( range , 1794 years ) , and 52.6% ( 779 of 1482 ) were never - smokers . nearly three - quarters of patients ( 73.8% [ 1093 of 1482 ] ) were of chinese ethnicity . key demographic and clinical characteristics ( pp population ) of 1486 patients with no important protocol deviations , tumor samples from four were not tested : two biopsy samples were lost after pathological reading , one sample did not have a clot for preparing a tissue block , and the dna concentration of one block was insufficient for testing . of the remaining 1482 samples tested , 169 ( 11.4% ) were cytology samples . in the overall pp population , egfr mutation analysis was successful in samples from 1450 of 1482 patients ( 97.8% ; 95% ci , 97.0%98.5% ) ; 32 of 1482 ( 2.2% ; 95% ci , 1.5%3.0% ) were undetermined ( unknown ) , of which eight were cytology samples . among the 1450 evaluable samples , 746 ( 51.4% ; 95% ci , 48.954.0 ) were egfr mutation positive and 704 ( 48.6% ; 95% ci , 46.051.1 ) were egfr mutation negative . of patients with a known time interval between physicians requesting and obtaining a test result ( n = 1475 ) , the mean ( sd ) time interval for reporting the test was 17.6 ( 13.3 ) days ( median , 15.0 ; range , 1148 days ) ; for the majority of patients ( 1168 ; 79.2% ) , the time interval was less than 21 days . tumor egfr mutation frequency for patients in demographic / clinical subgroups is presented in table 2 . factors with a statistically significant association with egfr mutation status ( or fisher s exact test ) were country , sex , ethnicity , smoking status , smoking pack - years ( all p < 0.001 ) , disease stage ( p = 0.009 ) , and histology type ( p = 0.016 ) , and are briefly summarized below . caution is advised when interpreting results of these univariate analyses because individual demographic / clinical factors may be influenced by others and therefore may not represent a true effect of that variable : egfr mutation frequency for demographic and clinical characteristic subgroups ( pp population ) country / region egfr mutation frequency was highest in patients from vietnam ( 64.2% [ 77 of 120 ] ) and lowest in patients from india ( 22.2% [ 16 of 72 ] ) ( other countries 47.2% [ 76 of 161 ] to 62.1% [ 108 of 174 ] ) . sex egfr mutation frequency was significantly higher in females ( 61.1% [ 384 of 628 ] ) than males ( 44.0% [ 362 of 822 ] ) . ethnic group egfr mutation frequency was highest for those of kinh ( vietnamese ) ethnicity ( 64.2% [ 77 of 120 ] ) and lowest for those of indian ethnicity ( 21.9% [ 16 of 73 ] ) . egfr mutation frequency was highest among never - smokers ( 60.7% [ 462 of 761 ] ) compared with ex - smokers ( 43.2% [ 130 of 301 ] ) , occasional smokers ( 51.6% [ 33 of 64 ] ) , or regular smokers ( 37.3% [ 121 of 324 ] ) . smoking pack - years egfr mutation frequency was highest among never - smokers ( 60.7% ) and decreased as pack - year number increased ( > 010 pack - years : 57.9% ; > 50 pack - years : 31.4% ) . a similar trend was observed by sex : males ( 010 pack - years : 55.9% [ 161 of 288 ] , > 1030 pack - years : 46.6% [ 123 of 264 ] , and > 30 pack - years : 28.2% [ 74 of 262 ] ) ; females ( 010 pack - years : 62.5% [ 371 of 594 ] , > 1030 pack - years : 37.5% [ 9 of 24 ] , and > 30 pack - years : 40.0% [ 4 of 10 ] ) egfr mutation frequency was significantly higher among patients with stage iv disease ( 53.5% [ 612 of 1144 ] ) compared with iiib ( 43.2% [ 117 of 271 ] ) or other stage ( 48.6% [ 17 of 35 ] ) . egfr mutation frequency was significantly higher among patients with adenocarcinoma not otherwise specified histology ( 52.2% [ 718 of 1376 ] ) compared with adenocarcinoma bronchoalveolar histology ( 37.8% [ 28 of 74 ] ) . factors not significantly correlating with egfr mutation frequency were age ( p = 0.565 ) , time from diagnosis ( p = 0.612 ) , existence of malignant pleural effusion ( p = 0.265 ) , primary tumor stage ( tumor staging t1-tx ; p = 0.454 ) , regional lymph node involvement ( p = 0.075 ) , and tumor grade ( p = 0.369 ) . multivariate logistic regression ( with 1% significance level ) identified ethnicity ( p < 0.001 ) and smoking pack - years ( p < 0.001 ) to be independent predictive factors for egfr mutation status ( table 3 ) . when stratified by smoking status or pack - years , sex was no longer found to be significant ( fig . multivariate logistic regression analysis for egfr mutation status ( pp population ) combined effect of sex and ( a ) smoking status and ( b ) pack - years on frequency of egfr mutation ( pp population ) . a , p values for logistic regression model . smoking history definitions : never - smoker ( patients who had never smoked cigarettes in their lifetime ) ; ex - smoker ( patients who had previously smoked but no longer smoked ) ; occasional smoker ( patients who smoked , but not every day ) ; regular smoker ( patients who smoked every day ) . individual mutation types , including multiple mutations , are summarized in table 4 . of the 1450 evaluable samples , 671 ( 46.3% ) harbored activating ( sensitizing ) mutations alone , 42 ( 2.9% ) had resistance mutations alone , and 33 ( 2.3% ) had a combination of activating and resistance mutations . the most common mutations detected were deletion in exon 19 ( deletion alone 22.1% [ 321 of 1450 ] ; alone and in combination with others 24.3% [ 352 of 1450 ] ) and l858r point mutation in exon 21 ( l858r alone 20.9% [ 303 of 1450 ] ; alone and in combination with others 22.9% [ 332 of 1450 ] ) . tumors from 21 patients ( 1.4% ) harbored t790 m resistance mutations , of which five ( 0.3% ) had t790 m alone . of 1486 patients with no important protocol deviations , tumor samples from four were not tested : two biopsy samples were lost after pathological reading , one sample did not have a clot for preparing a tissue block , and the dna concentration of one block was insufficient for testing . of the remaining 1482 samples tested , 169 ( 11.4% ) were cytology samples . in the overall pp population , egfr mutation analysis was successful in samples from 1450 of 1482 patients ( 97.8% ; 95% ci , 97.0%98.5% ) ; 32 of 1482 ( 2.2% ; 95% ci , 1.5%3.0% ) were undetermined ( unknown ) , of which eight were cytology samples . among the 1450 evaluable samples , 746 ( 51.4% ; 95% ci , 48.954.0 ) were egfr mutation positive and 704 ( 48.6% ; 95% ci , 46.051.1 ) were egfr mutation negative . of patients with a known time interval between physicians requesting and obtaining a test result ( n = 1475 ) , the mean ( sd ) time interval for reporting the test was 17.6 ( 13.3 ) days ( median , 15.0 ; range , 1148 days ) ; for the majority of patients ( 1168 ; 79.2% ) , the time interval was less than 21 days . tumor egfr mutation frequency for patients in demographic / clinical subgroups is presented in table 2 . factors with a statistically significant association with egfr mutation status ( or fisher s exact test ) were country , sex , ethnicity , smoking status , smoking pack - years ( all p < 0.001 ) , disease stage ( p = 0.009 ) , and histology type ( p = 0.016 ) , and are briefly summarized below . caution is advised when interpreting results of these univariate analyses because individual demographic / clinical factors may be influenced by others and therefore may not represent a true effect of that variable : egfr mutation frequency for demographic and clinical characteristic subgroups ( pp population ) country / region egfr mutation frequency was highest in patients from vietnam ( 64.2% [ 77 of 120 ] ) and lowest in patients from india ( 22.2% [ 16 of 72 ] ) ( other countries 47.2% [ 76 of 161 ] to 62.1% [ 108 of 174 ] ) . sex egfr mutation frequency was significantly higher in females ( 61.1% [ 384 of 628 ] ) than males ( 44.0% [ 362 of 822 ] ) . ethnic group egfr mutation frequency was highest for those of kinh ( vietnamese ) ethnicity ( 64.2% [ 77 of 120 ] ) and lowest for those of indian ethnicity ( 21.9% [ 16 of 73 ] ) . egfr mutation frequency was highest among never - smokers ( 60.7% [ 462 of 761 ] ) compared with ex - smokers ( 43.2% [ 130 of 301 ] ) , occasional smokers ( 51.6% [ 33 of 64 ] ) , or regular smokers ( 37.3% [ 121 of 324 ] ) . smoking pack - years egfr mutation frequency was highest among never - smokers ( 60.7% ) and decreased as pack - year number increased ( > 010 pack - years : 57.9% ; > 50 pack - years : 31.4% ) . a similar trend was observed by sex : males ( 010 pack - years : 55.9% [ 161 of 288 ] , > 1030 pack - years : 46.6% [ 123 of 264 ] , and > 30 pack - years : 28.2% [ 74 of 262 ] ) ; females ( 010 pack - years : 62.5% [ 371 of 594 ] , > 1030 pack - years : 37.5% [ 9 of 24 ] , and > 30 pack - years : 40.0% [ 4 of 10 ] ) egfr mutation frequency was significantly higher among patients with stage iv disease ( 53.5% [ 612 of 1144 ] ) compared with iiib ( 43.2% [ 117 of 271 ] ) or other stage ( 48.6% [ 17 of 35 ] ) . egfr mutation frequency was significantly higher among patients with adenocarcinoma not otherwise specified histology ( 52.2% [ 718 of 1376 ] ) compared with adenocarcinoma bronchoalveolar histology ( 37.8% [ 28 of 74 ] ) . factors not significantly correlating with egfr mutation frequency were age ( p = 0.565 ) , time from diagnosis ( p = 0.612 ) , existence of malignant pleural effusion ( p = 0.265 ) , primary tumor stage ( tumor staging t1-tx ; p = 0.454 ) , regional lymph node involvement ( p = 0.075 ) , and tumor grade ( p = 0.369 ) . multivariate logistic regression ( with 1% significance level ) identified ethnicity ( p < 0.001 ) and smoking pack - years ( p < 0.001 ) to be independent predictive factors for egfr mutation status ( table 3 ) . when stratified by smoking status or pack - years , sex was no longer found to be significant ( fig . multivariate logistic regression analysis for egfr mutation status ( pp population ) combined effect of sex and ( a ) smoking status and ( b ) pack - years on frequency of egfr mutation ( pp population ) . never - smoker ( patients who had never smoked cigarettes in their lifetime ) ; ex - smoker ( patients who had previously smoked but no longer smoked ) ; occasional smoker ( patients who smoked , but not every day ) ; regular smoker ( patients who smoked every day ) . individual mutation types , including multiple mutations , are summarized in table 4 . of the 1450 evaluable samples , 671 ( 46.3% ) harbored activating ( sensitizing ) mutations alone , 42 ( 2.9% ) had resistance mutations alone , and 33 ( 2.3% ) had a combination of activating and resistance mutations . the most common mutations detected were deletion in exon 19 ( deletion alone 22.1% [ 321 of 1450 ] ; alone and in combination with others 24.3% [ 352 of 1450 ] ) and l858r point mutation in exon 21 ( l858r alone 20.9% [ 303 of 1450 ] ; alone and in combination with others 22.9% [ 332 of 1450 ] ) . tumors from 21 patients ( 1.4% ) harbored t790 m resistance mutations , of which five ( 0.3% ) had t790 m alone . in total , 23 patients ( 1.5% of pp population ) provided both histology and cytology samples for mutation analysis . the egfr mutation status ( and specific mutations found ) of matched histology and cytology samples were concordant for 21 patients ( positive n = 13 , negative n = 8 ; 91.3% concordance [ wilson score 95% ci , 73.2%97.6% ; coefficient 0.817 ] ) . matched samples from two patients were discordant ( one patient s samples were positive for histology and negative for cytology , with the other patient having a reverse of this result ) . in pioneer , the first epidemiological study of egfr mutation frequency across asian countries / regions , approximately half of the unselected patients with adenocarcinoma nsclc had tumors that harbored egfr mutations . frequency of tumor mutation was significantly lower in indian patients compared with other countries , and at 22.2% , was more comparable with the frequency for a broad white population ( approximately 20% ) than to the east asian countries / regions in the study ( approximately 4764% ) , or the approximately 30% and approximately 36% reported in japanese and korean patients , respectively . interestingly , a higher tumor mutation frequency of 44% was reported in a similarly sized ( n = 75 ) adenocarcinoma population of indian patients in a study by sahoo et al . , using scorpion arms polymerase chain reaction . however , only 90 of 220 tumor samples could be histologically subclassified , which may have resulted in potential bias . in contrast , the large data set and consistent use of mutation test methodology in pioneer permitted comprehensive and reliable subgroup analysis . in pioneer , a patient s ethnicity and smoking status / pack - years were independent predictive factors for tumor egfr mutation status . of note , there was no association between sex and tumor egfr mutation status when results were stratified by smoking status . generally , female sex , adenocarcinoma histology , never - smoking status , and asian ethnicity are considered the most important factors associated with egfr mutation and response to egfr - tkis . in pioneer , univariate analyses showed that country , sex ( subsequently negated when stratified by smoking status ) , ethnic group , smoking status , pack - years , disease stage , and adenocarcinoma histology type all had a statistically significant association with egfr mutation status ( note that our study was ongoing before the publication of the new adenocarcinoma classification system ) . the highest frequency of egfr mutation was among female never - smokers , in agreement with previous studies . however , tumor egfr mutations can be found in patients with clinical characteristics other than female sex , adenocarcinoma histology , never - smoking status , or asian ethnicity . indeed , the frequency of egfr mutation in tumors from asian males in pioneer was 44% ( arms ) , in sharp contrast to the 8.2% reported in a comparable european male population ( dna sequencing ) . similarly , frequency of egfr mutation in asian heavy - smokers was approximately 30% in pioneer , much higher than the 5.8% observed in european heavy - smokers . thus , physicians should not discount these other populations from egfr mutation testing on the basis of clinical characteristics . in a large , retrospective u.s . study of 2142 patients with stage i to iv nsclc , egfr mutations in tumors from former / current smokers represented 40% of all mutations detected ( direct sequencing ; 201 of 503 ; 95% ci , 36%44% ) , and those from men represented 31% ( 157 of 503 ; 95% ci , 27%35% ) ; if only female never - smokers had been tested , 57% of mutation - positive tumors would have remained undetected . in pioneer , more than 50% of patients with egfr mutation - positive tumors came from subpopulations other than female never - smokers . these data highlight that egfr mutation testing is warranted even in males and smokers , particularly in asian populations , and emphasize that egfr mutation status can only be confirmed by performing egfr mutation testing . egfr - tki efficacy in non - asian patients with egfr mutation - positive nsclc was demonstrated in the phase iii european tarceva versus chemotherapy ( eurtac ) study . this study of 173 european white patients with egfr mutation - positive tumors ( dna sequencing ) reported significantly longer progression - free survival ( pfs ) with first - line erlotinib ( n = 86 ; 9.7 months ) compared with chemotherapy ( n = 87 ; 5.2 months ) ( hazard ratio [ hr ] , 0.37 ; 95% ci , 0.250.54 ; p < 0.0001 ) , with benefit in both female ( n = 126 ; pfs hr , 0.35 ; 95% ci , 0.220.55 ) and male subgroups ( n = 47 ; pfs hr , 0.38 ; 95% ci , 0.170.84 ) . these data further strengthen the rationale for routine assessment of tumor egfr mutations in all patients ( where possible ) before initiation of nsclc therapy . indeed , recent molecular testing guidelines copublished by three societies recommended the use of egfr mutation testing to guide patient selection for therapy with an egfr inhibitor , in all patients with advanced - stage adenocarcinoma , regardless of sex , race , smoking history , or other clinical risk factors , and to prioritize egfr testing over other molecular predictive tests . in pioneer , the success rate ( known positive or negative result ) of egfr mutation analysis was very high at 97.8% , and only 2.2% of patients had tumor samples for which mutation status could not be determined . this high success rate is very encouraging as it indicates that even though the acquisition , preparation , and processing of tumor material varied between test centers / participating laboratories because of differences in routine clinical practice , the quality of most samples was such that mutation testing was successful and a definite result obtained . the high success rate also indicates that the in vitro diagnostic mutation arms kit used throughout the study was suitable for a range of samples where the collection method was not standardized , highlighting its suitability for adoption / routine use at local test centers . indeed , previous studies have found arms to be successful at detecting egfr mutations in a variety of sample types , including those of cytological origin , with a reported increase in detection of egfr mutations with arms when compared with direct sequencing . although the success rate of mutation testing in pioneer was very high , the median time interval between requesting and reporting a result was 15.3 days ; not ideal from a clinical perspective . however , egfr mutation testing was not performed routinely in some participating countries , and performing tests solely for the experimental purposes of our study will have exacerbated this time interval . commercial test centers routinely performing tests are currently reporting turnaround times of 8 to 10 days ; however , even this may not be satisfactory , given that patients need access to the most appropriate treatment as quickly as possible . research into more rapid mutation identification , such as allele - specific testing , may help to reduce this waiting period and provide more rapid access to appropriate personalized therapies . mutation test results were concordant for the majority of patients in pioneer who provided matched histology and cytology samples , indicating that cytology samples could be considered for mutation testing if tumor biopsy samples are not available . however , the small number of samples ( n = 23 ) limits the interpretation of these findings , although mutation test methodology studies are providing more conclusive evidence which corroborates these data . there is currently a need to generate data for a pan - asian guideline for the management of nsclc . such a guideline has been difficult to establish owing to differences in ethnicity and medical care across asian countries / regions , in addition to longer drug approval times compared with the european union and united states . lack of standardization in testing methodology has also reduced the feasibility of large - scale testing . however , the consensus from a recent meeting to discuss egfr mutation testing in east asia recommended testing all recently diagnosed patients with nonsquamous nsclc ( as is current practice in some centers and community practices ) , and patients with squamous nsclc with clinical features associated with higher prevalence of egfr mutations . tissue acquisition and pre - test sample evaluation were also considered important steps to increase sensitivity / specificity , and thus help standardize mutation test methodology . the substantial body of data generated by pioneer therefore has valuable clinical implications for the treatment of advanced nsclc across asia , and indicates that large - scale testing across countries is feasible , can be standardized , and can result in a high analysis success rate . further multinational studies are required to help establish guidelines and realize these recommendations . in summary , the observed frequency of tumor egfr mutation in demographic and clinical subgroups of asian patients in pioneer suggests that egfr mutation testing should be considered for all patients with stage iiib / iv adenocarcinoma nsclc in an asian population . such an approach should help ensure the optimal identification and treatment of patients whose tumors harbor egfr mutations . the authors thank sarah lewis , from complete medical communications , who provided medical writing support funded by astrazeneca .

introduction : pioneer ( nct01185314 ) was a prospective , multinational , epidemiological study of epidermal growth factor receptor ( egfr ) mutations in patients from asia with newly diagnosed advanced lung adenocarcinoma.methods:eligible patients ( aged 20 years ) had untreated stage iiib / iv adenocarcinoma . the egfr mutation status ( primary end point : positive , negative , or undetermined ) of tumor samples ( biopsy , surgical specimen , or cytology ) was determined ( scorpion amplification refractory mutation system ) . egfr mutation frequency was calculated and compared between demographic and clinical subgroups.results:of 1482 patients from seven asian regions , 43.4% of patients were female , median age was 60 years ( range , 1794 ) , and 52.6% of patients were never - smokers . egfr mutation status was evaluable in tumors from 1450 patients ( 97.8% ) ( 746 [ 51.4% ] positive ; 704 [ 48.6% ] negative ) . country , sex , ethnicity , smoking status , pack - years ( all p < 0.001 ) , disease stage ( p = 0.009 ) , and histology type ( p = 0.016 ) correlated significantly with egfr mutation frequency . mutation frequency was 61.1% in females , 44.0% in males ; lower in patients from india ( 22.2% ) compared with other areas ( 47.2%64.2% ) ; highest among never - smokers ( 60.7% ) ; and decreased as pack - year number increased ( > 010 pack - years , 57.9% ; > 50 pack - years , 31.4% ) ( similar trend by sex ) . ethnic group ( p < 0.001 ) and pack - years ( p < 0.001 ) had statistically significant associations with mutation frequency ( multivariate analysis ) ; sex was not significant when adjusted for smoking status.conclusion:pioneer is the first prospective study to confirm high egfr mutation frequency ( 51.4% overall ) in tumors from asian patients with adenocarcinoma . the observed high mutation frequency in demographic / clinical subgroups compared with white populations suggests that mutation testing should be considered for all patients with stage iiib / iv adenocarcinoma , even males and regular smokers , among asian populations .

it has been used widely in the assessment of executive functions , neuropsychological functions , and motor cognitive processing speed ( mcps ) . mcps varies with number of possible valid stimulus , type , order and intensity of stimulus , arousal , age , gender , physical fitness , hand dominance , practice and error , fatigue , fasting , distraction , alcohol , finger tremor , stress , drugs , intelligence , learning disorder , brain injury , illness , personality type , and accuracy in hearing and vision . lesser mcps accelerates the achievements in various fields such as games , studies , fine arts , martial arts , and defense . by identifying the person 's mcps , we can predict the reacting abilities in the above - mentioned situations . in case of children , this guides us to diagnose the children with sustained rt and its origin . however , high cost and professional guidance in estimating rt make this unavailable for the schoolchildren . although mobile - based android applications are available for estimating rt , the restricted usage of mobiles at schools makes this as a tough task . hence , there is a need to validate a simple gadget frequently used in schools such as ruler . the participant / athlete was asked to perform rdm by sitting on a chair of appropriate height with his / her dominant forearm resting on a flat horizontal table surface . the ruler was dangled erect by an examiner / assistant such that the other end of the ruler was aligned with the top of the participant / athlete open hand and he / she was instructed to catch the ruler quickly once it was dropped by the examiner / assistant . then , the distance traveled by the ruler was converted into time by the formula , d = vt + at . however , the instrument has ceiling effect , and in case of children , it might have a major effect . to minimize this effect , we have proposed a simple method to estimate mcps by a ruler dropped at least a meter distance from the ground . a stainless steel meter ruler is used in the study to estimate the rt in children . in our pilot study , we evaluated the reliability and validity of rt of 12 schoolchildren aged between 6 and 10 years using rdm . we performed a small modification in the procedure that the ruler was suspended vertically such that 5 cm below the child 's open hand , and the distance the ruler traveled from the initial point was recorded . , we used an android - based mobile application known as criterion - referenced reaction speed . we found good intrarater reliability ( 0.81 ) and moderate - to - good degree of validity ( 0.54 ) . this study was aimed to derive a prediction equation to estimate mcps among the primary schoolchildren by rdm . a total of 204 children participated in this cross - sectional study with prior permission from the principal of participating school and also from the parents / legal guardians by cluster sampling technique . the ethical clearance was obtained from the university research ethics committee of maharishi markandeshwar university , mullana , ambala , haryana , india ( mmu / iec/445 ) . the study was performed according to ground rules settled by the declaration of helsinki ( revised 2013 ) . the assent from children and written consent from their parents / legal guardians were collected prior to the study . children with confirmed history of neurological , orthopedic , and metabolic disorders , upper extremity injury or surgery , complaints of weakness or pain , and any other mediwcal , surgical , and psychological conditions were excluded from the study . all anthropometric measurements were taken before the beginning of the study in a sports laboratory . body weight was measured in children with minimum clothing by a calibrated weighing scale ( equinox br-9015 , analog weighing scale ) , and height was measured in centimeters , without shoes and socks , with a standard height measuring flat metallic piece over a mounting measuring tape ( freemans mrl 2 m 16 mm , metal wired tape ) on the wall with feet about 2530 cm apart and they were recorded to the nearest 1 kg and 1 mm , respectively . body mass index ( bmi ) was calculated by dividing the weight ( kg ) by the square of the height ( m ) . to measure mcps by rdm , the children were asked to sit with their dominant side elbow flexed at 90 with mid - pronated , and the forearm rested over a flat of the table surface such that the hand was placed outside the table . ruler was hung down vertically by the investigator such that its lower edge was aligned across 5 cm between the web space ( i.e. , thumb and index finger ) of the children 's hand . children were instructed to grasp the ruler quickly as it was liberated by examiner as shown in figure 1 . measuring motor cognitive processing speed by the ruler drop method note : figure 1 has appeared in our previous publication in , aranha vp , joshi r , samuel aj , sharma k. catch the moving ruler and estimate reaction time in children . then , this distance was converted into mcps using the following formula , mcps = 2d / g , where d is the distance traveled by the ruler and g is the acceleration due to gravity , a constant ( 9.8 m / s ) . three trials were taken , and then , mean of this was used for the analysis of normative value . statistical analysis of the collected data was performed using the statistical package for social sciences ( spss , version 20.0 inc . , chicago , il , usa ) software for windows 7 ultimate edition . as the data follow normal distribution , descriptive statistics were expressed in terms of mean standard deviation ( sd ) and 95% confidential interval ( 95% ci ) . demographic character differences between male and female were explored by the statistical test of significance , independent t - test . mean rt was reported in mean sd and 95% ci with range . to identify the effect of independent variables such as gender , age , height , weight , and bmi over the depending variable , multiple linear regression was used . multiple linear regression analysis was performed to identify the role of the independent variable such as age , height , weight , and bmi for estimating the rt . by stepwise exclusion , prediction equation for rt in the age group between 6 and 12 years was made . a total of 204 children participated in this cross - sectional study with prior permission from the principal of participating school and also from the parents / legal guardians by cluster sampling technique . the ethical clearance was obtained from the university research ethics committee of maharishi markandeshwar university , mullana , ambala , haryana , india ( mmu / iec/445 ) . the study was performed according to ground rules settled by the declaration of helsinki ( revised 2013 ) . the assent from children and written consent from their parents / legal guardians were collected prior to the study . children with confirmed history of neurological , orthopedic , and metabolic disorders , upper extremity injury or surgery , complaints of weakness or pain , and any other mediwcal , surgical , and psychological conditions were excluded from the study . all anthropometric measurements were taken before the beginning of the study in a sports laboratory . body weight was measured in children with minimum clothing by a calibrated weighing scale ( equinox br-9015 , analog weighing scale ) , and height was measured in centimeters , without shoes and socks , with a standard height measuring flat metallic piece over a mounting measuring tape ( freemans mrl 2 m 16 mm , metal wired tape ) on the wall with feet about 2530 cm apart and they were recorded to the nearest 1 kg and 1 mm , respectively . body mass index ( bmi ) was calculated by dividing the weight ( kg ) by the square of the height ( m ) . to measure mcps by rdm , the children were asked to sit with their dominant side elbow flexed at 90 with mid - pronated , and the forearm rested over a flat of the table surface such that the hand was placed outside the table . ruler was hung down vertically by the investigator such that its lower edge was aligned across 5 cm between the web space ( i.e. , thumb and index finger ) of the children 's hand . children were instructed to grasp the ruler quickly as it was liberated by examiner as shown in figure 1 . measuring motor cognitive processing speed by the ruler drop method note : figure 1 has appeared in our previous publication in , aranha vp , joshi r , samuel aj , sharma k. catch the moving ruler and estimate reaction time in children . then , this distance was converted into mcps using the following formula , mcps = 2d / g , where d is the distance traveled by the ruler and g is the acceleration due to gravity , a constant ( 9.8 m / s ) . three trials were taken , and then , mean of this was used for the analysis of normative value . statistical analysis of the collected data was performed using the statistical package for social sciences ( spss , version 20.0 inc . , chicago , il , usa ) software for windows 7 ultimate edition . as the data follow normal distribution , descriptive statistics were expressed in terms of mean standard deviation ( sd ) and 95% confidential interval ( 95% ci ) . demographic character differences between male and female were explored by the statistical test of significance , independent t - test . mean rt was reported in mean sd and 95% ci with range . to identify the effect of independent variables such as gender , age , height , weight , and bmi over the depending variable multiple linear regression analysis was performed to identify the role of the independent variable such as age , height , weight , and bmi for estimating the rt . by stepwise exclusion , prediction equation for rt in the age group between 6 and 12 years was made . a total of 269 children with a mean age of 9.1 1.9 years ( range of 612 years ) were selected for the study . of these 269 children although the recruited sample composed of unequal gender distribution with 56.9% of female ( 116 ) and 43.1% of male ( 88 ) , there exist no statistical differences among their demographic parameters ( p > 0.05 ) , which is shown in table 1 . mean mcps of the entire sample , i.e. 230.01 ms 26.5 sd ( 95% ci : 226.4233.7 ms ) ranged from 162.9 to 321.6 ms . the effect of multiple independent variables which include age , gender , height , weight , and bmi over the dependent variable mcps was derived from the multiple regression . the overall prediction of mcps by the above independent variables is r = 0.43 with the variability of r = 0.19 and adjusted r = 0.17 . the f - ratio in the anova test demonstrates that the overall regression model is a good fit for the data . here , the independent variables statistically significantly predict the dependent variable , f ( 5 , 198 ) = 9.078 , p < 0.001 . hence , the regression model is a good fit of the data . by stepwise regression analysis , the nonstatistically significant independent variables were excluded from the analysis , and regression analysis was performed by including the independent variable , age of the children , which is only statistically significant ( p < 0.001 ) . demographic characteristics of the primary schoolchildren recruited motor cognitive processing speed ( ms ) among individual age group expressed in mean ( standard deviation ) the predictability of mcps by age is 41.3% ( r = 0.413 ) with a variability of 17.1% ( r = 0.171 and adjusted r = 0.166 ) as shown in table 2 . the derived regression equation is as follows : regression analysis of variables to determine the influence of independent variable , age on motor cognitive processing speed mcps ( ms ) = 279.625 ( 5.495 age [ in years ] ) . for example , while estimating mcps of 8 years , 6-month - old child , substitute 8.5 for age in the above formula . the preliminary aim of this study was to establish the prediction equation for mcps by rdm among primary schoolchildren aged between 6 and 12 years . these years , also known as mid - childhood , where they make a move toward adulthood , demonstrate the major shift in their cognitive abilities . as we know rt is the time lapse between the application of stimulus and task completion , the mcps of an individual can be quantified by calculating rt . rdm is simple and has good inter - rater reliability and it is a good moderate validity method to assess the simple rt in school - going children . the apparatus used here is a universal ruler made by steel with 1 m length , which is easily available in commercial market in a feasible cost . the comprised battery methods which are used to assess rt will not have this upper limit . newton 's second law of motion states that the applied force and weight of moving object determine its acceleration ( a = f / m ) . however , for an object falling from the height , its own body weight will act as the force ( f = w ) at constant air resistance . the weight of object is the product of mass and gravity ( w = m g ) . thus , gravitational force is proportional to its mass and decides the acceleration ( a = mg / m ) . thereby , newton proves the assumptions of galileo given approximately four century before ( a = g ) . galileo dropped two cannon balls of same size ( made up of different material such as wood and iron , respectively ) from the top of pisa tower , and he observed that both balls reach earth at the same time . he reported that the distance covered by the object depends on the square of the time and velocity increased as the ball moves down , which suggests that the mass and dimension of the object do not affect the acceleration of the object . thus , it is clear that the size , shape , and material of the ruler will not alter the result . from a total of 269 children , we recruited 223 children depending on the selection criteria . among them , 15 children were absent to the school on the day of data collection . although there exists an unequal gender distribution ( boys 88 and girls 116 ) , they are matched in their age ( p = 0.12 ) , height ( p = 0.24 ) , weight ( p = 0.27 ) , and bmi ( 0.82 ) . practice trials were performed before the actual trial , and these trial data were excluded from the analysis . here , we demonstrated mcps of 204 children is 230.01 ms , and it varies from 162.9 to 321.6 ms . the considerable variations that are found in the distribution of mcps is possibly due to person 's individuality , complexity of task , and items in the given task . the mcps for children between 8 and 10 years measured by anxiety related information - processing biases method lies close to our findings . the highly trained children can exhibit significantly shorter rt than other children of similar age group . proved this by comparing the mcps of international level qualified taekwondo ( a form of martial art ) players ( 0.19 0.03 s ) of 1016 years with the nontrained children ( 0.22 0.02 s ) of the same age . another study of comparing mcps between circus artist and nontrained adults of the same age group shows significant differences in their mcps . our study report also exhibits these age - related differences in mean mcps among different age . the rt and age are inversely related to each other . approximately up to an age of 50 years , the mean mcps will decrease with progression of age . this reduction in mcps with increasing age may be due to distracted attention , ignorance , declining cognitive ability , and motor response . the development of human brain according to heterochronicity principle may be another possible reason for the age - related mcps differences . this is also supported by the regression analysis in identifying the factors affecting mcps . by considering the prediction equation , mcps ( ms ) = 279.625 ( 5.495 age ) , rt can be estimated in the children aged between 6 and 12 years . the mcps difference among individual age group between 6 and 12 years is relatively constant ; it is also supported by rueda 's study on the development of attention network among 610-year - old children . in our experience during testing and data collection , the rdm was easy to perform and individual child responded with nearly homogeneous eagerness and competitiveness . the children found that dropping ruler is intrinsically interesting , and hence , they appeared to be motivated to give excellent outcome . second , other variables which could affect mcps such as intelligent quotient , sociodemographics , and child behavior were not assessed . third , not analyzing the muscle and nerve parameter such as muscle cross - section area , muscle strength , pinch grip , and nerve conduction velocity , which is beyond the scope of this study . nevertheless , this is the first study to derive the prediction equation for mcps among the primary schoolchildren by the simple method , rdm . further , the study can be extended over the other age groups and in children with special needs such as children with cerebral palsy and down syndrome . the study has high clinical implication that mcps could be estimated by knowing only the age of the primary schoolchildren with the help of prediction equation . this greatly reduces the time in estimating rt / mcps through any known objective method . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .

objectives : motor cognitive processing speed ( mcps ) is often reported in terms of reaction time . in spite of being a significant indicator of function , behavior , and performance , mcps is rarely used in clinics and schools to identify kids with slowed motor cognitive processing . the reason behind this is the lack of availability of convenient formula to estimate mcps . thereby , the aim of this study is to estimate the mcps in the primary schoolchildren.materials and methods : two hundred and four primary schoolchildren , aged 612 years , were recruited by the cluster sampling method for this cross - sectional study . mcps was estimated by the ruler drop method ( rdm ) . by this method , a metallic stainless steel ruler was suspended vertically such that 5 cm graduation of the lower was aligned between the web space of the child 's hand , and the child was asked to catch the moving ruler as quickly as possible , once released from the examiner 's hand . distance the ruler traveled was recorded and converted into time , which is the mcps . multiple regression analysis of variables was performed to determine the influence of independent variables on mcps.results:mean mcps of the entire sample of 204 primary schoolchildren is 230.01 ms 26.5 standard deviation ( 95% confidence interval ; 226.4233.7 ms ) that ranged from 162.9 to 321.6 ms . by stepwise regression analysis , we derived the regression equation , mcps ( ms ) = 279.6255.495 age , with 41.3% ( r = 0.413 ) predictability and 17.1% ( r2 = 0.171 and adjusted r2 = 0.166 ) variability.conclusion:mcps prediction formula through rdm in the primary schoolchildren has been established .

pancreatic cancer is a well - known cause of morbidity and mortality world - wide . pancreatic cancer has one of the lowest at 5-year survival rates of all cancers.1 this low survival rate is mainly due to the late presentation of patients with pancreatic cancer and limited treatment modalities for advanced disease ; the average survival time after diagnosis is only 6 months.2 therefore , early and precise diagnosis is very important for improving the results of surgery . surgical resection remains the only potentially curative treatment for pancreatic adenocarcinoma and is associated with momentous periprocedural morbidity and mortality.3 accurate pre - operative diagnosis is critical to avoid unnecessary laparotomy in those with benign disease . furthermore precise pre - operative staging to those with malignant lesions is vital , because only 10% of patients have the chance of surgical cure at the time of diagnosis . correct staging of patients with pancreatic malignancy will allow accurate identification of those who may benefit from surgery.4 the ability to obtain high quality images and perform fine needle aspiration ( fna ) has led endoscopic ultrasound ( eus ) to become the recommended procedure for diagnosis and staging of pancreatic tumours with a low rate of complications ( < 2%).4 previous studies aimed to evaluate eus - fna in diagnosis of solid pancreatic lesions reported a range of diagnostic accuracy between 62% and 96%.56 ultrasonography ( us ) guided percutaneous fna biopsy is a well - established method for obtaining tissue for cytological examination since the 1970s . us - fna of the liver and pancreas has been shown to be an accurate method for the cytological diagnosis of malignancy ; the diagnostic yield has been reported to be from 84% to 95%.7 moreover , us - fna has very low rate of complications if contraindications are followed.8 to reach the highest accuracy of diagnosis , us - fna must be performed by well - experienced sonographers and cytopathologists.9 controversy has arisen about whether the percutaneous approach with computed tomography ( ct)/us - fna or eus - fna is the preferred method to obtain diagnostic tissue.1011 after being approved by the local scientific ethical committee and obtaining written informed consent from all participants , this multicenter prospective study was conducted on 197 consecutive patients presented with pancreatic head masses based on ct , magnetic resonance imaging ( mri ) and/or eus confirmation from september 2008 to january 2013 . according to accessibility and feasibility , they were sub - classified into two main groups : group 1 included 125 patients , underwent percutaneous us - fna . inclusion criteria for enrolment were accessibility of the tumor , platelet count > 50 10 and prothombin concentration > 50% . local xylocaine 2% was given in most patients and deep sedation by propofol was given in few irritable patients . eus examination was done using a pentax eg-3830ut machine connected to a hitachi machine eub-7500 , japan . fna was done using 19 and 22-g echotip needles ( cook endoscopy , winston - salem , nc ) . on - site cytopathology was done in only 15 patients , as it was nt available until recently and this small group only benefited from such maneuver . a final diagnosis was based on definitive cytopathology , surgical pathology and clinical follow - up for more than 18 months . data were statistically described in terms of frequencies ( number of cases ) and percentages . accuracy was represented using the terms sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and overall accuracy . all statistical calculations were performed using computer programs statistical package for the social science ( spss inc . , chicago , il , usa ) version 15 for microsoft windows . a final diagnosis was based on definitive cytopathology , surgical pathology and clinical follow - up for more than 18 months . data were statistically described in terms of frequencies ( number of cases ) and percentages . accuracy was represented using the terms sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and overall accuracy . all statistical calculations were performed using computer programs statistical package for the social science ( spss inc . , chicago , il , usa ) version 15 for microsoft windows . a final diagnosis was based on definitive cytopathology , surgical pathology and clinical follow - up for more than 18 months . data were statistically described in terms of frequencies ( number of cases ) and percentages . accuracy was represented using the terms sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and overall accuracy . all statistical calculations were performed using computer programs statistical package for the social science ( spss inc . , chicago , il , usa ) version 15 for microsoft windows . a total of 197 patients were included in the study ; of these , 152 were males and 45 females . patient characteristics and final diagnosis of groups 1 and 2 are summarized in ( tab . patient 's characteristics and final diagnosis of both groups out of 125 diagnosed by us - fna , 83 were malignant pancreatic head masses , 71 cases were true positive and four cases were false positive . 42 cases were benign , 38 cases were true negative and 12 cases were false negative , with sensitivity of 85.54% , specificity of 90.48% , ppv of 94.67% , npv of 76% and accuracy of 87.20% ( tab . effect of the number of passes on the accuracy of both us - fna and eus - fna in diagnosing pancreatic cancer out of 72 cases diagnosed by eus - fna , 50 were malignant pancreatic head masses , 42 cases were true positive and no cases showed false positive results . 22 cases were benign , 22 cases were true negative and eight cases were false negative , with sensitivity of 84% , specificity of 100% , ppv of 100% , npv of 73.33% and overall accuracy of 88.89% ( tab . sensitivity , specificity , ppv , npv and accuracy of us - fna / eus - fna in diagnosing pancreatic cancer within the eus - fna group , the sensitivity was 81.82% in those cases where no on - site cytology was available , while the sensitivity reached 92.85% when on - site cytology was available . no on - site cytopathology was available for any of the us - fna group . effect of the presence of on - site cytology on the accuracy of eus - fna in diagnosing pancreatic cancer the sensitivity was 60% in those cases where one pass was used while the sensitivity reached 90.91% when three passes was used . the overall accuracy increased from 76.47% with one pass to 93.75% as shown in tab 2 . complications occurred in eight patients ( 4.06% ) , one of the 72 patients underwent eus - fna ( 1.38% ) and 7 of the 125 patients underwent us - fna ( 5.6% ) . the patient who underwent eus - fna had severe epigastric pain due to acute pancreatitis requiring hospitalization for 3 days with improvement . 3 of patients underwent us - fna had severe epigastric pains that responded to non - steroidal anti - inflammatory drugs within 1 - 3 days without hospitalization . three patients had seeding of malignant cells in the peritoneal cavity in two cases and in the subcutaneous tissue at the biopsy site in one case 1 year after radical surgical excision of the pancreatic head mass . the last case has severe infection in the form of pancreatic abscess requiring surgical debridement and drainage . out of 125 diagnosed by us - fna , 83 were malignant pancreatic head masses , 71 cases were true positive and four cases were false positive . 42 cases were benign , 38 cases were true negative and 12 cases were false negative , with sensitivity of 85.54% , specificity of 90.48% , ppv of 94.67% , npv of 76% and accuracy of 87.20% ( tab . effect of the number of passes on the accuracy of both us - fna and eus - fna in diagnosing pancreatic cancer out of 72 cases diagnosed by eus - fna , 50 were malignant pancreatic head masses , 42 cases were true positive and no cases showed false positive results . 22 cases were benign , 22 cases were true negative and eight cases were false negative , with sensitivity of 84% , specificity of 100% , ppv of 100% , npv of 73.33% and overall accuracy of 88.89% ( tab . 3 ) . sensitivity , specificity , ppv , npv and accuracy of us - fna / eus - fna in diagnosing pancreatic cancer within the eus - fna group , the sensitivity was 81.82% in those cases where no on - site cytology was available , while the sensitivity reached 92.85% when on - site cytology was available . no on - site cytopathology was available for any of the us - fna group . effect of the presence of on - site cytology on the accuracy of eus - fna in diagnosing pancreatic cancer the sensitivity was 60% in those cases where one pass was used while the sensitivity reached 90.91% when three passes was used . the overall accuracy increased from 76.47% with one pass to 93.75% as shown in tab 2 . complications occurred in eight patients ( 4.06% ) , one of the 72 patients underwent eus - fna ( 1.38% ) and 7 of the 125 patients underwent us - fna ( 5.6% ) . the patient who underwent eus - fna had severe epigastric pain due to acute pancreatitis requiring hospitalization for 3 days with improvement . 3 of patients underwent us - fna had severe epigastric pains that responded to non - steroidal anti - inflammatory drugs within 1 - 3 days without hospitalization . three patients had seeding of malignant cells in the peritoneal cavity in two cases and in the subcutaneous tissue at the biopsy site in one case 1 year after radical surgical excision of the pancreatic head mass . the last case has severe infection in the form of pancreatic abscess requiring surgical debridement and drainage . out of 125 diagnosed by us - fna , 83 were malignant pancreatic head masses , 71 cases were true positive and four cases were false positive . 42 cases were benign , 38 cases were true negative and 12 cases were false negative , with sensitivity of 85.54% , specificity of 90.48% , ppv of 94.67% , npv of 76% and accuracy of 87.20% ( tab . effect of the number of passes on the accuracy of both us - fna and eus - fna in diagnosing pancreatic cancer out of 72 cases diagnosed by eus - fna , 50 were malignant pancreatic head masses , 42 cases were true positive and no cases showed false positive results . 22 cases were benign , 22 cases were true negative and eight cases were false negative , with sensitivity of 84% , specificity of 100% , ppv of 100% , npv of 73.33% and overall accuracy of 88.89% ( tab . 3 ) . sensitivity , specificity , ppv , npv and accuracy of us - fna / eus - fna in diagnosing pancreatic cancer within the eus - fna group , the sensitivity was 81.82% in those cases where no on - site cytology was available , while the sensitivity reached 92.85% when on - site cytology was available . no on - site cytopathology was available for any of the us - fna group . effect of the presence of on - site cytology on the accuracy of eus - fna in diagnosing pancreatic cancer the sensitivity was 60% in those cases where one pass was used while the sensitivity reached 90.91% when three passes was used . the overall accuracy increased from 76.47% with one pass to 93.75% as shown in tab 2 . complications occurred in eight patients ( 4.06% ) , one of the 72 patients underwent eus - fna ( 1.38% ) and 7 of the 125 patients underwent us - fna ( 5.6% ) . the patient who underwent eus - fna had severe epigastric pain due to acute pancreatitis requiring hospitalization for 3 days with improvement . 3 of patients underwent us - fna had severe epigastric pains that responded to non - steroidal anti - inflammatory drugs within 1 - 3 days without hospitalization . three patients had seeding of malignant cells in the peritoneal cavity in two cases and in the subcutaneous tissue at the biopsy site in one case 1 year after radical surgical excision of the pancreatic head mass . the last case has severe infection in the form of pancreatic abscess requiring surgical debridement and drainage . sometimes the ordinary imaging tools like ct and mri do nt provide a paved way for definite diagnosis and a necessity for cytopathological diagnosis is mandatory to define the protocol of therapy . us - fna and eus - fna has been established during the last decades as a diagnostic tool for many hepatobiliary and pancreatic malignancies . in 2012 , hewitt et al.4 pooled 4984 patients in his wide meta - analysis research and demonstrated that eus - fna has a sensitivity of 85% , specificity of 98% , ppv of 99% and a npv of 72% in diagnosis of solid pancreatic masses . these results are very similar to the results of the current study that showed a sensitivity of 84% , specificity of 100% , ppv of 100% and npv of 73% . the results of the current study showed that us - fna and eus - fna of pancreatic masses have a nearly similar diagnostic accuracy ( 87.20% and 88.89% respectively ) . in 2011 , dumonceau et al . published the guidelines of the european society of gastrointestinal endoscopy regarding the clinical impact of eus - guided sampling in gastroenterology and reported that eus - fna seems to present a higher diagnostic accuracy than us - fna.12 eloubeidi et al.13 studied major complications in a total of 355 consecutive patients with a solid pancreatic mass underwent eus - fna . examples of complications included acute pancreatitis , severe pain after the procedure , fever and surgical debridement for necrosis . only seven patients ( 1.97% ) required hospitalization ( range : 1 - 16 days ) . they reported no haemorrhage , perforation or death . in the present study , complications occurred in eight patients ( 4.1% ) . only 1 of the 72 patients underwent eus - fna ( 1.38% ) had acute pancreatitis requiring hospital admission . 7 of the 125 patients underwent us - fna ( 5.6% ) had complications , three patients ( 2.4% ) had tumor seeding , 3 ( 2.4% ) had severe epigastric pains and 1 ( 0.8% ) had a pancreatic abscess . this was also the conclusion of hewitt et al.4 who demonstrated that the observed complication rate of eus - fna was also low , at 1%-2% , with complications occurring more commonly when eus - fna was performed on cystic lesions than on solid lesions . examples of complications include bleeding , infection , self - limiting pancreatitis , and tumour seeding ; however , there are similar risks for ct - guided biopsy . all masses underwent us - fna were larger than 20 mm , because lesions less than 20 mm were not easily visible or accessible for us - fna , while eus - fna could be done in masses as small as 9 mm and more . in the developing country like egypt , us - fna is much cheaper than eus - fna ( 170 vs. 750 i.e. , about one - fifth of the cost ) , with nearly similar diagnostic accuracy ( 87.2% vs. 88.89% ) , so it is more cost - effective . however , the main drawbacks of us - fna that : it needs larger tumour sizes , not all tumours were accessible or even visible by such modality and it had a higher incidence of complications . ( 1 ) eus - fna has nearly similar diagnostic accuracy as us - fna of pancreatic masses with a lower complication rate . eus - fna of pancreatic masses should be the first - line procedure , but since us - fna is much cheaper and nearly as accurate , may be considered an acceptable alternative when eus is not available , but in rather larger masses ( i.e. , > 20 mm ) and more complications rate . ( 2 ) on - site cytopathological examination increases the sensitivity and accuracy of the procedure . however our limitations were that such maneuver became available only recently . further studies with application of on - site cytology on a larger number of patients are needed to yield better results .

objective : pancreatic carcinoma is one of the leading cancer morbidity and mortality world - wide . controversy has arisen about whether the percutaneous approach with computed tomography / ultrasonography - guidance fine needle aspiration ( us - fna ) or endoscopic ultrasound - guided fine needle aspiration ( eus - fna ) is the preferred method to obtain diagnostic tissue . our purpose of this study is to compare between the diagnostic accuracy of eus - fna and percutaneous us - fna in diagnosis of pancreatic cancer.patients and methods : a total of 197 patients with pancreatic masses were included in the study , 125 patients underwent us - fna ( group 1 ) and 72 patients underwent eus - fna ( group 2).results : eus - fna has nearly the same accuracy ( 88.9% ) as us - fna ( 87.2% ) in diagnosis of pancreatic cancer . the sensitivity , specificity , positive predictive value and negative predictive value for eus - fna was 84% , 100% , 100% , 73.3% respectively . it was 85.5% , 90.4% , 94.7% , 76% respectively for us - fna . eus - fna had a lower complication rate ( 1.38% ) than us - fna ( 5.6%).conclusion : eus - fna has nearly the same accuracy as us - fna of pancreatic masses with a lower complication rate .

lips are a central part of facial aesthetics due to their color , surface texture , and shape . well defined and full lips represent attractiveness and beauty . loss of volume and elasticity of subcutaneous soft tissue , retraction of lip red , bone resorption , loss of teeth , smoking , and ultraviolet exposure contribute to perioral aging and loss of attractiveness . furthermore , lips are exposed to irritants and environmental factors that challenge the barrier function of their thin horny layer . the lips are conceived as signs of aging , loss of attractiveness , and fragility.1 lips are part of the aesthetic unit that involves the mouth and the perioral tissue . aging of this area is characterized by perioral fine lines , marionette lines , and flattening of the cupid bow . the philtrum becomes longer and ill defined , indirectly contributing to a thinner upper lip.2 furthermore , the dynamics of lip movement change with age . people older than 50 years of age lose their ability of a high smile.3 skin and lip care may help to preserve a youthful and healthy appearance.4,5 dentistry plays a significant role in restoring perioral and mouth aesthetics , not only in elderly people.6 restorative techniques of the perioral soft tissue will further enhance the aesthetic outcome of aesthetic restorative dentistry.7 in our review , we will focus on minimally invasive procedures such as dermal filler injection and botulinum toxin a application in women 50 years of age and older . all procedures need a detailed knowledge of anatomy , pharmacology , and technical skills to be safe for patients . the usual medical background is dermatology or plastic surgery . although the legal situation can vary between countries , attendance of certified courses and workshops by medical societies the mandible , maxillary bone , and teeth represent the hard tissue structures that will shape the perioral area and the mouth ( figure 1 ) . maxillary retrusion of the maxilla is evident in both fully dentated patients and those with loss of teeth . deepening of the maxilla supports a posterior positioning of the upper lip and the nasolabial fold.8 three dimensional computed tomography ( ct ) demonstrated that mandibular height and length decrease with age.9 the perioral muscles are arranged in several layers . the orbicularis oris muscle interlaces with perioral muscles , influencing the dynamic actions during speech , smiling , and whistling . on the apical area , fibers of the levator labii superioris insert into the upper lip and the oral sphincter . laterally , the levator anguli oris and both zygomaticus muscles insert into the upper lip . the risorius muscle and depressor anguli oris insert into the corner of the mouth . the lower lip is inserted by the depressor labii inferioris and mentalis , with mentalis also inserting into chin dermis . both interlace with platysma fibers . perioral muscles have a different nerve supply from the zygomatic and buccal branches of the facial nerve ( zygomaticus , levator anguli oris , levator labii superioris , and part of orbicularis oris ) , and marginal branches of the facial nerve ( part of orbicularis oris , depressor anguli oris , mentalis).10 superficial fat compartments are arranged in the perioral area . the jowl compartment above the mandible and the nasolabial compartment can influence the lateral upper lip and the corners of the mouth.11 facial fat is divided into deep and superficial planes . the perioral fat is characterized by a deep department beneath mentalis and orbicularis oris muscles different from small lobuled superficial fat of the lips . the wet dry transition zone of the lips defines the most anterior border of the submuscular deep fat tissue.12 cadaveric studies13 of perioral soft tissue demonstrated a lipomatous area without defined ligamentous attachment . the perioral mound with a scaffold of fine trabecular fibrouse separations defines a separate fat pad compartment . with aging it becomes lipodystrophic and ptotic.13 deep fat depots have been defined under the melomental ( marionette ) line . since these depots are surrounded by retaining ligaments ( condensations of fibrous tissue ) , this may affect volumizing procedures.14 the philtrum is characterized by a fat compartmentation by delicate membranes related to its particular vascular anatomy.15 sensory innervation of the perioral skin arises from infraorbital nerve branches ( upper lip and philtrum ) , the buccal nerve ( corners of the mouth ) , and the mental nerve ( lower lip and chin).10 there is no doubt about the need for validated grading scales to measure effects of aging and objective evaluations of therapeutic trials . a validated 5-point photonumeric lip scale , the lip fullness scale ( lfs ) has been developed by carutthers et al.16 separate grading for upper and lower lips is recommended . the scale ratings are : very thin ( 0 ) , thin ( 1 ) , moderately thick ( 2 ) , thick ( 3 ) , and full ( 4).16 the same group of international experts developed a validated grading scale for melomental folds ( marionette lines ) , the marionette line grading scale.17 the 5-point photonumeric scale differentiates no visible folds ( 0 ) , visible fold with slight indentation ( 1 ) , moderately deep folds not visible when skin is stretched ( 2 ) , very long and deep folds ( 3 ) , and extremely long and deep folds ( 4).17 a limitation of both grading systems is that they do not address dynamic movements of the area . there are more grading systems for the perioral area available but a detailed discussion is not the focus of this review.15,16 dermal fillers can be subdivided into temporary and permanent fillers . in this article , we will focus on temporary fillers based on crosslinked hyaluronic acid ( ha ) fillers . this filler type consists of biphasic ( particulate ) and monophasic fillers ( gel only).17 the injection technique is dependent on the filler , area to inject , and the physician and patient preference . to reduce injection and filling related pain , topical anesthetics or nerve blocks the injections should be done with a slow motion to reduce pain , bruising , and risk of irregularities . cold compresses before and after the procedure increase comfort and reduce swelling and tenderness . for lip augmentation the ha filler is often placed near the border of the wet and dry lip . bleeding , nodule formation , and temporary edema are the most frequent adverse effects.18 overcorrections and nodules can easily be corrected by injection of hyaluronidase , a big advantage compared to other filler materials.19 nonaminal stabilized ha ( [ nasha ] restylane , q - med ab , uppsala , sweden ) represents a particulate filler type . restylane perlane ( q - med ) consists of 10,000 particles / ml . juvederm ( allergan inc , irvine , ca , usa ) , belotero ( merz pharma gmbh and co , frankfurt , germany ) , glythone ( pierre fabre , france and merz pharma gmbh and co ) , or teosyal ( teoxane sa , geneva ) are monophasic fillers . fillers also differ by ha concentration , crosslinking technology , and rheological properties.18,20 in an open trial,21 50 female subjects were treated with a monophasic ha filler ( juverderm ultra , allergan inc ) for lip enhancement . the mean injected volume was 1.6 ml . more than 90% of investigators and subjects were satisfied with the results . at week 12 , 71% of subjects had an improvement of 1 grade on the lfs . twelve weeks later , lip improvement was still present for 56% of subjects.21 in a multicenter european trial,22 60 subjects were treated with the ha filler juvederm vobella ( allergan ) . , incorporation of short chain and long chain ha is used to enhance crosslinking without increasing stiffness . the juvederm vobella filler has an elastic modulus , g , of about 160 pa , which is lower when compared to other fillers like juvederm ultra or restylane . the improvement of 1 grade in the lfs was reported for 98.3% of subjects at 3 months and 48.3% of subjects at 12 months.22 in two studies using biphasic , nasha restylane ( q - med ) covering 180 subjects and 1,446 subjects , subject satisfaction after 6 months was 70% and 50.8% , respectively.23,24 medical history is important in the sense of allergies , bleeding disorders , neurologic or autoimmune diseases , immunodeficiencies , or iatrogenic immunosuppression . body dysmorphic disorders are not limited to younger patients . such subjects will be inappropriate for filler injections . medications and over the counter drugs should be recorded.25 the facial mimics are studied in rest and in motion . with increasing age facial asymmetries the mirror and camera are the most important tools to define with the patient what should be modified to obtain a better appearance . we included females older than 49 years of age ( n = 57 ) with a mean age of 59.2 7.9 years ( age range 50 to 84 years ) . we used only monophasic ha dermal fillers ( belotero , glytone , or juvederm ) with 27 or 30 gauge needles . botulinum toxin a ( vistabel , allergan ; bocouture , merz ) was diluted in physiological sodium chloride solution according to the manufacturer s recommendation . the median amount of botulinum toxin a units ( botox units ) in the perioral area was 12 8 units . the position of the upper lip is modified by the nasolabial fold and the cheek fat pads . a minor lift of the upper lip with an improved show of the wet roll is possible by liquid lift of the cheeks ( figure 2 ) . augmentation by filler along the vermillion border ( white roll ) attenuates the convexity of this area and can improve perioral fine wrinkling ( figure 3 ) . soft filler with a lower stiffness is preferred even if the durability is more limited . stiffer fillers would impair the delicate motion of the lips when speaking , singing , or smiling . although injections of small amounts of botulinum toxin a are used to improve perioral lines in younger and middle aged females , we would not recommend this method for elderly subjects . intrinsic and extrinsic aging eventually causes lines in rest that can not be corrected by botulinum toxin . loss of volume of the upper lips is often associated with flattening of the vermillion border and partial loss of cupid s bow . subdermal injection of filler along the philtrum columns by threading technique improves not only the three dimensional appearance of the philtrum but cupid s bow as well ( figures 35 ) . deep fat injection of the upper lip into the wet roll is a method to volumize and to reshape . in case of advanced bone resorption or dental problems and loose connective tissue , advanced volumizing may lead to an undeserved ptosis ( duckbill look ) and loss of fine lip motion . in such cases , plastic surgery and dental restoration the downward turn of the corners of the mouth can be seen in subjects of various ages . whereas in younger individuals the strength of the platysma and depressor anguli oris is responsible and can be corrected by botulinum toxin injections , this is rarely the case in elderly women.26,27 ptosis , loss of elasticity , repetitive facial expression , gravity , and extrinsic aging contribute to the downward turn which eventually results in melomental folds . their negative appearance may be further increased by loss of volume in the lateral parts of lower lip . when the corner of the mouth is stretched laterally deep fat augmentation by filler injection into the wet roll will volumize and reshape the lower lips and elevate the corners of the mouth . this will also reduce the depth of the melomental fold . in case of grade 3 of melolabial folds , serial punctures or threading along the fold will be necessary to obtain a good outcome . only in patients with a strong platysma , small amounts of botulinum toxin a injections into the anterior part of the platysma and close to the mandibular insertion point of the depressor anguli oris will contribute to a lift of the corner of the mouth.26 injections need to be placed superficially because of the particular muscular anatomy.28 the chin crease can be improved by deep filler placement . in cases of poppy chin , the procedure may be combined with injections of 58 units of botulinum toxin a ( botox units ) . the satisfaction with perioral rejuvenation in elderly females was high ; 96.5% of the females were satisfied or highly satisfied . facial aging is accompanied by perioral hard and soft tissue changes leading to wrinkles , folds , and loss of contour and volume of the lips . this becomes more pronounced in elderly subjects . with individually tailored filler injection techniques , possibly in conjunction with low dose botulinum toxin ,

lips and the perioral area are of outstanding importance in youthful appearance , attractiveness , and beauty . in contrast to younger and middle aged females , there is only scant published data on minimally invasive procedures to restore and revitalize lips and perioral soft tissue in elderly females . in this review we report the signs of aging in this particular region and the underlying anatomy . we review studies on lip restoration in younger females and present our techniques for elderly women . with an individually tailored approach , elderly females benefit from minimally invasive techniques .

a 5-year - old girl presented to our clinic ( kashani hospital , isfahan , iran , 2011 ) with a history of 4-month pain around her right knee . there was a moderate tenderness in the proximal of tibia in palpation without any warmth or change in color of the skin in the suspicious area . the child was not febrile and there was no sign or symptom of systemic illness . hematological investigations ( include leukocyte count , cell profile , erythrocyte sedimentation rate , calcium , phosphorus and alkaline phosphatase ) were normal . before referring to our clinic , she had been visited 3 months ago by an orthopedic surgeon , and a plain radiography had been requested . at that time , the plain radiography of the right knee demonstrated a small well - circumscribed eccentric lesion in the proximal medial metaphyseal region of the tibia without disruption of the cortex ( figure 1 ) , and the patient had been treated conservatively with suspicious of fibrous cortical defect . first presentation with small eccentric lucent lesion 3 months later she was referred to our clinic with a new plain radiography , which demonstrated a large well - defined centric lytic lesion with involvement of both medial and lateral portion of proximal tibial metaphysis , without disruption of cortices and physis ( figure 2 ) . there was no evidence of fallen leaf sign or obvious sclerotic border . because of rapid progression of the lytic lesion , a magnetic resonance imaging ( mri ) was requested . mri reported an expansile centric , septated cystic lesion in metadiaphyseal part of the right tibia without fracture or secondary changes in muscular component ( figure 3 ) . rapid expansion of lesion after three months sagittal view shows expansile centric , septated cystic lesion the patient was admitted to orthopedics ward for open biopsy . macroscopically , there was blood accompanied by epithelial lining without straw fluid or infective discharge . histological examination revealed highly vascular thin membranous lining composed primarily of epithelium - like cells and giant cells accompanied with hemosiderin deposition ( figure 4 ) . histological examination shows highly vascular lining with giant cells and hemosiderin deposition after confirmation of the diagnosis , the patient underwent curettage and grafted with bone substitute ( figure 5 ) . the risk of progressive valgus deformity discussed with patient 's parents thoroughly before proceeding with surgery and they were justified to be followed and evaluated postoperatively . at 3 months follow - up , the patient was asymptomatic and clinical examination was normal . unicameral bone cysts ( ubc ) are benign , fluid - filled lesions which occur predominantly in children with average age of 9 - 11 years . the male - to - female ratio is 2 - 2.5 to 1.1 94% of ubcs occur in the proximal humerus and proximal femur , with the proximal humerus being affected 2 - 3 times more frequently than the proximal femur.2 the remaining 6% occur in other bones including the calcaneus ( 2% ) , ilium ( 2% ) , talus , tibia , fibula , metatarsals , ischium , pubic rami , sacrum , vertebral bodies , forearm , and craniofacial bones.3 the exact incidence is difficult to determine as many lesions are silent prior to fracture.4 although ubcs were first recognized by virchow in 1876 , the exact etiology remain unknown , however a number of etiologies have been proposed to explain ubc , such as venous obstruction , blocked drainage of interstitial fluid , invagination of synovial membrane , and traumatic hematoma with cystic resorption.567 pain in the affected region may be present , but in approximately 70% of cases the first and only symptom is a pathological fracture following a trivial trauma.8 the radiological features on plain x - rays include a centrally located , expansile lesion of the metaphysic . cortical thinning without disruption is seen.9 fallen fragment sign is virtually pathognomonic of a multiloculated bone cyst.39 the growth plate moves away from the cyst as the child grows . this explains why two - thirds of these lesions are not in contact with the growth plate when discovered in children older than 10 years.9 in mri , prolonged t1 and t2 relaxation times suggest a cyst , although t1 shortening may reflect proteinaceous content resulting in signal which is higher than water.10 the most characteristic histopathologic finding is the thin membranous lining of the cyst , composed primarily of flattened to plump epithelium - like cells ; the lining may also possess osteoclast - type giant cells , cholesterol cells and fat cells . hemosiderin , fibrin , calcification , and reactive bone may be seen in focal areas of the cyst.11 there is no consensus or official guideline for when and how to treat ubcs.12 as ahn et al.13 stated age , site , size , degree of loculation and cortical erosion , the stage of the cyst and whether it is active or not are all factors to be considered . there is a risk of recurrence in cysts which are lying within 2 centimeter of growth plate.14 progressive tibia valga seem to be relatively common in patients younger than 10 years who have had curettage of the proximal tibial metaphysis.15 the major differential diagnoses include aneurysmal bone cysts , monostotic fibrous dysplasia , and atypical eosinophilic granuloma.16 all of these lesions may be radiolucent . however , features typically associated with these lesions usually help differentiate them from simple bone cysts . findings appear somewhat interesting but unusual characteristics of this case were female gender with low age ( 5 years ) , being painful although there was no cortical disruption in plain x - ray and mri , rapid progression of the lytic lesion ( in three months ) , and the site of lesion which unfortunately is accompanied by relative risk of tibia valga .

unicameral bone cysts ( ubcs ) are benign , osteolytic lesions which are often asymptomatic and are commonly seen in the proximal of humerus and femur . the average age at diagnosis is 9 - 11 years and there is a male preponderance with a male - to - female ratio of approximately 2 - 2.5 to 1 . we describe a case of 5-year - old girl who presented to orthopedic clinic with a 4-month history of painful limping . plain radiography of the right knee demonstrated a well - defined lytic lesion in the proximal of the tibia . open biopsy and then curettage and bone grafting with bone- substitute was performed . the diagnosis of this condition requires a high index of suspicion . this report demonstrates that all ubcs do not have the same clinical patterns and with adequate attention good results can be achieved .

the first is the evolution of a foetally derived placenta which enables the foetus to influence its own destiny , whilst placing a considerable burden in terms of time and energy investment on an expectant mother . hormonal cues provided by the developing foetus influence maternal neural function , increase maternal food consumption to compensate for increased energetic costs , suppress oestrus and sexual behaviours [ 2 , 3 ] , decide the timing of parturition , and prime the maternal hypothalamus to initiate maternal care [ 5 , 6 ] . one unique feature of placentation is that there are three matrilineal generations and hence genomes interacting in one organism at the same time ( mother , foetus , and postmeiotic oocytes ) which provide a mechanism by which a mother can transmit adaptive or predictive information concerning potential future environmental conditions to the developing foetal brain . it also provides a mechanism to ensure intergenerational coadaptation of the maternal hypothalamus , foetal brain , and placenta . this ensures that a foetus whose hypothalamus has coadaptively engaged with its placenta and maternal hypothalamus to provide optimal transfer of energetic resources during development will when mature have a hypothalamus that will be able to efficiently transfer energetic resources to its own offspring [ 711 ] . placentation also hands disproportionate control of epigenetic inheritance to the matriline as epigenetic markers undergo global erasure and reprogramming in the developing zygote [ 1214 ] . the evolution of placentation is also accompanied by the evolution of monoallelic gene expression or genomic imprinting , where gene expression is silenced depending on the parent of origin . genomic imprinting is absent in egg laying mammals and only around 6 imprinted genes have been detected in a range of marsupial species ( who possess a choriovitelline placenta ) ; this is in contrast to eutherian mammals where around 150 imprinted genes have been described [ 15 , 16 ] . original theories of the evolution of imprinted genes are derived from the hypothesis that paternally and maternally expressed genes act antagonistically within a developing offspring to promote differential interests with regard to nutrient transfer , but this is difficult to reconcile with the observation that the erasure and reprogramming of epigenetic markers and imprints is under matrilineal control [ 1214 ] . hypotheses that explain the evolution of imprinted genes have been derived that include kinship , sexual antagonism , and varying models of maternal - offspring and placental hypothalamic coadaptation [ 711 , 19 ] . the huge expansion of the neocortex especially in primates has facilitated a shift in the complexity of social interactions and in primates complex social cues replace changes in hormonal states to promote maternal and sexual responses . parturition is not required to initiate maternal behaviour which is therefore displayed towards a range of offspring in a social group and sexual interactions may occur out of oestrus and are used to reinforce social attachment . the most important phases of neocortical development and socialisation occur during periods of maternal attachment , among stable groups of females that extend over more than one generation . there are often complex social hierarchies within these groups and social rank is often inherited matrilineally from the mother . the developing mammalian brain can demonstrate remarkable plastic and adaptive responses many of which serve to adapt the brain to an environment that it will encounter when mature . this may account for the ease in which mammals have undergone adaptive radiation into a wide range of environmental niches , where evolution of the brain and neuronal phenotype has paralleled adaptations in physical structure . in this review we focus on the importance of a number of mechanisms that contribute to this and emphasise the importance of the matriline in these mechanistic processes . we also suggest that failure in these processes can result in the induction of deleterious neural and peripheral phenotypes that can not be viewed as traditionally pathological but are phenotypes which are adaptive to the environmental conditions with which an individual or its mother interacted during early development . subsequent shifts in environmental conditions during an organism 's later development may leave it with a poorly environmentally adaptive phenotype and a propensity for health problems . the hypothesis of predictive adaptive responses or plasticity ( par ) was originally developed at a whole organism level to provide a mechanistic framework by which a developing organism can attempt to predict and modify its developmental phenotype to accurately match the environment that it will encounter when mature . it also incorporated a range of ideas that attempted to explain why early life experiences are associated with an increased risk of disease in later life , especially in contemporary human societies [ 2123 ] . this is likely to be a more reliable proposition in animal species with short intergenerational times ( mice 4 - 5 weeks ; cf . humans 1315 years ) , with the exception of humans where technological advances and cultural transmission have produced extended environmental stability . secondly , an organism which has during development successfully predicted its adult environment will benefit from increases in both survival and darwinian fitness . thirdly , successful predictive responses are primarily advantageous to the organism until reproductive age , and for reproduction itself , postreproductive beneficial or deleterious effects on an organism 's health do not affect darwinian fitness . and finally this predictive mechanism may incorporate a fatal evolutionary flaw as a mismatch between an environmental prediction and subsequent reality may lead to a poorly adaptive phenotype and health problems [ 2022 ] . a range of predictive adaptive responses involving the brain have been described in a number of mammalian species . one of the simplest and earliest related to the observation that vole pups ( microtus pennsylvanicus ) born in late summer and early autumn have much thicker coats than those born in the spring . the proximal cue for this is a reduction in day length , which is transmitted to the developing vole in utero via melatonin signalling originating in the mother [ 24 , 25 ] . it is a tautological prediction that reduced day length precedes winter and this adaptation enables autumn born voles to survive winter better than they would with thinner coats . generally these predictive adaptive responses are advantageous to survival but in dynamically changing environments especially when intergenerational times are extended , predictive adaptive responses may have contingent or dichotomous properties which may lead to a mismatch between phenotype and environment . one of the most studied examples is the relationship between maternal nutrition and offspring metabolism . a range of epidemiological observations has established that maternal nutrition developmentally programmes the metabolic , behavioural , and developmental phenotype of their offspring . well - nourished mothers have offspring who are adapted to affluent environments ; offspring born to mothers with restricted nutrition are adapted to impoverished environments . children born with low birthweights ( which is thought to reflect impoverished intrauterine nutritional supply ) enter puberty early [ 2629 ] and have a preference for high fat foods [ 30 , 31 ] and higher satiation thresholds and a smaller stature , phenotypical characteristics that are adaptive in a life of impoverished circumstances . the proof of principle of this adaptive response has been characterised in a range of animal species , as direct human evidence is scant and incorporates a range of confounders derived from adult socioeconomic status . anecdotal evidence obtained from concentration and prisoner - of - war camps suggests that physically large individuals died first whilst at least some small individuals survived . and in a famine afflicted ethiopian population a high birthweight was associated with a 9-fold greater risk of rickets . harvests in finland failed and poor people suffered greatly in terms of both adult and infant mortality . a key prediction of the predictive adaptive response hypothesis should be that poor individuals who were born in impoverished years should be more resistant to the effects of the famine than those born in more affluent years ; however this was not the case and poor people who were born in more affluent years were less affected than those born in impoverished years . gluckman and hanson argued that the poor physical and economic conditions of those born in impoverished years may have persisted throughout life and therefore their less resilient responses did not represent a predictive adaptive response to the 1887 - 8 famine . the predictive adaptive response hypothesis has been used to explain why variations in the early interuterine and neonatal environment exert profound effects on health and disease in later life . a mismatch between an environmental prediction made during early development and subsequent reality may mean that an organism may have a poorly adapted phenotype to its adult environment which may predispose it to health problems . babies reared in affluent environments enjoy a reduced risk of a range of diseases as adults including diabetes [ 3739 ] ; however babies born in impoverished but who move to more affluent environments demonstrate increased rates of metabolic and cardiovascular diseases in later life [ 40 , 41 ] . placentation is universally thought to be an iconic feature of mammalian evolution but this view is incomplete and based on observations of extant mammalian species . for much of early mammalian evolution the majority of mammal species despite possessing a suite of iconic mammalian features including hair , thermoregulation , lactation , and a highly developed neocortex laid eggs . the subsequent evolution of placentation ensured that during gestation there are three matrilineal generations , present in one organism at one time ( mother , foetus , and postmeiotic oocytes ) which provides a unique template by which a mother can transmit environmental information to her developing foetus and her potential grandchildren . the placenta is produced by the developing foetus and provides a mechanism by which it can attempt to control the physiology and neural function of its mother to its own advantage . progesterone secreted by the placenta suppresses sexual activity [ 2 , 3 ] ; its metabolite allopregnanolone increases maternal food intake , exerts a neuroprotective influence , and promotes hippocampal neurogenesis in both foetus and mother . placental hormones also prime the oxytocin system in anticipation of parturition where oxytocin release is required to support uterine contractions , milk ejection , and maternal behaviour in the mother [ 5 , 6 ] and to promote solicitation from and attachment to the mother in the offspring , this acts to coordinate the evolutionary appropriate behavioural responses in both mother and infant . one potential caveat of placentation means that the mother has to contend with the potential immune response generated by an invasion of semiallogenic foetal trophoblast cells . this has facilitated the evolution of a range of coadaptive responses ; the foetal placenta has adapted to avoid the maternal immune system but maternal natural killer cells have also adapted to activate growth and development of the placenta , by promoting angiogenesis , vascular remodelling , and trophoblast invasion of the uterus . the developing foetus begins to exert considerable leverage on the neural function of its maternal hypothalamus during early placental development at the same time as it develops a hypothalamus of its own [ 711 , 47 , 48 ] . this intimate transgenerational interaction of two separate genomes and hypothalami can provide an efficient mechanism to promote both information transfer about environmental cues and coadaptive function across generations . this model of development has been termed hypothalamic / placental coadaptation and states that a developing foetus whose placenta and hypothalamus are coadapted to regulate optimal resource transfer from the mother will develop an adult hypothalamus that will be able to efficiently transfer resources to its own offspring in utero via placentation or postnatally via lactation and efficient maternal care [ 711 , 47 , 48 ] . genomic imprinting refers to a form of monoallelic gene expression whereby the expression of an allele depends on its parental origin . the fact that genomic imprinting is absent in egg laying mammals and the fact that there are only 6 or so recorded imprinted genes in marsupials but around 150 imprinted genes in eutherian mammals have driven the development of over 14 interesting theories that explain their existence [ 14 , 48 ] . a number of these theories may not be mutually exclusive . in the context of this review two similar hypotheses involving transgenerational coadaptation and information transfer wolf and hager , 2006 , suggested that monoallelically maternally expressed genes facilitate coadaptation of interacting maternal and offspring traits during development , which acts to increase offspring fitness . one prediction of this is that increasingly biased patterns of monoallelic maternal gene expression would increase the overall fitness of any subsequent generation . keverne argued that imprinted genes facilitate the coadaptation of the developing foetal hypothalamus and placenta with the hypothalamus of the mother [ 711 , 47 , 48 ] . this would also increase overall fitness of offspring by promoting the transgenerational formation of a hypothalamic centred network that acts to adapt and stabilise optimal energetic and nutrient transfer between an expectant mother and the developing foetal brain and placenta . the hypothalamic neural circuitry responsible for the extraction of maternal energetic resources displays a remarkable overlap with the hypothalamic circuitry responsible for the provision of resources to offspring . this ensures that a brain adapted to extract optimal energetic resources when developing will be predisposed to provide optimal energetic resources to its own young . many imprinted genes are coexpressed in the developing hypothalamus and placenta , and a range of studies using mice with an inactivated paternally expressed gene 3 ( peg3 ) has provided a range of evidence that supports the role of imprinted genes in the development of coadapted responses between the hypothalamus and placenta . the peg3 gene can be inactivated in the maternal hypothalamus but not the placenta or hypothalamus of offspring or be functional in the maternal hypothalamus but inactivated in the hypothalamus or placenta of offspring . both of these paradigms produce remarkably similar deficits in hypothalamic cell number , maternal behaviour , and impaired suckling , which leads to impaired growth , onset of puberty , and reproduction [ 48 , 49 ] . disruption of this hypothalamic - placental response with 24 h of maternal food deprivation induces a significant decrease in peg3 expression in the placenta and other gene expression changes that are induced resemble those when the peg3 gene is inactivated and include the disruption of genes expression linked to autophagy and ribosomal turnover . in the foetal hypothalamus peg3 expression these results suggest that the foetus attempts to control its own destiny during periods of acute starvation by a short term sacrifice of its placenta which preserves resources required for neuronal development [ 48 , 50 ] . imprinted genes are found in clusters around heritable imprint control regions ( icrs ) , which are thought to be important for the regulation of monoallelic gene expression and disruption to these can be associated with prader - willi and angelman 's syndrome . imprinted control regions are highly conserved and predominately matrilineal and have important roles for embryo survival and maternal / foetal interactions during development [ 52 , 53 ] . they are also thought to have important roles in the allocation of matrilineal time and energy , which is an important evolutionary consideration given that often 90% of mammalian adult female life is committed to pregnancy , lactation , and maternal care . the development of the brain is by definition a complex epigenetic process ; neural systems are designed to respond to environmental changes as the interactions of over a trillion synaptic connections both are activity dependent and can be subjected to epigenetic modification . this occurs throughout the life of an individual but at key stages of development , epigenetic modification of neural function is under disproportionate control of the matriline . the erasure and reprogramming of the epigenetic landscape in the developing oocyte , interaction of the developing brain and placenta with environmental cues , the ability of a developing brain to accurately assess present conditions and accurately anticipate the future , and programming of the postpartum brain via efficient maternal care and lactation are all disproportionately matrilineal processes . no two brains are ever alike and even monozygotic twins exhibit increasing differences in behaviour and predisposition to psychiatric disorders as they age [ 54 , 55 ] which are thought to be associated with subtle epigenetic influences resulting from minor variations in developmental environments . immediately before birth , coordinated epigenetic and transcriptional remodelling begins to facilitate the brains adaptation to an extrauterine environment . at birth the foetus is disconnected from its own placenta and supply of placental hormones controlled by its own genome , including allopregnanolone , which facilitates a rapid shift in gaba function [ 46 , 47 ] . these processes enable rapid postnatal synaptogenesis and neocortical expansion which allows the integration of activity dependent neuronal gene expression and development with salient environmental and developmental cues . in humans this remodelling extends from just before birth to the first year of life [ 56 , 57 ] . activity dependent neuronal gene expression , remodelling , and maturation during development are critical for the proper development of neural circuits . in the visual , auditory , and attentional systems , disruption of this process results in a delay in the maturation of the systems supporting these functions and lifelong cognitive deficits [ 58 , 59 ] . this underappreciated process has important implications for neonatal medicine , where interventions during this critical transformative process may induce lifelong deficits in neuronal function [ 60 , 61 ] . a range of intergenerational based matrilineal epigenetic influences on neural function and behaviour have been described mainly in the context of maternal behaviour and stress responses [ 6264 ] . rodents reared by mothers who display poor quality maternal care acquire heritable epigenetic changes to their oestrogen and oxytocin receptor systems and then in turn display poor quality maternal care to their own offspring [ 62 , 63 ] . although both developmental and intergenerational epigenetic modifications are under disproportionate control of the matriline , paternal transgenerational influences in neural function are also important . male mice conditioned to an aversive stimulus paired with an odour ( acetophenone ) which activates a specific odorant receptor ( olfr151 ) have offspring and grandoffspring that demonstrate enhanced biological sensitivity to acetophenone and increases in olfr151 activity . bisulfite sequencing of sperm dna in acetophenone conditioned fathers and their offspring demonstrated cpg hypomethylation in the olfr151 gene . this paternal transgenerational mechanism is adaptive in that it allows potential predator avoidance cues to be transferred quickly through a population . other examples of paternal epigenetic influence may not be adaptive and there is evidence that paternal age , nutrition , substance abuse , and stress may predict mental and physical health in offspring [ 6568 ] . the exact molecular mechanisms by which paternal epigenetic influences can be transmitted across generations are poorly understood but are thought to require the active cooperation of the matriline , as dna methylation marks are globally erased and reprogrammed in the developing zygote following fertilisation [ 1214 ] . mothers may also modify paternal influences on neural development by dynamically adjusting their reproductive investment in response to the qualities of their mate . female mice that mate with males housed in a socially enriched environment exhibit increased levels of maternal care towards their offspring of pup nursing and licking towards their offspring , which are associated with increases in expression of brain derived neurotrophic factor in the maternal hypothalamus . the evolution of paternally derived intergenerational effects on offspring fitness and phenotype may suggest one further mechanism by which adaptive traits can be passed rapidly through an expanding population . beneficial genetic variations and epigenetic modifications of monoallelically paternally expressed genes will spread far more rapidly through a population than those of monoallelically expressed maternally expressed genes . this is because mammals typically have fewer ancestral fathers than mothers as in males freed from the energetic burden of placentation reproductive success is based on competing to impregnate as many females as possible . this means that many low status males fail to breed and yet high status males often have large numbers of offspring . in populations that are moving into novel environments , who then subsequently undergo a rapid expansion this reproductive asymmetry coupled with the evolution of paternally expressed ( and hence maternally silenced ) gene expression provides an elegant mechanism to rapidly distribute traits that are adaptive to neural function . adaptive gene variants and epigenetic modifications of paternally expressed genes will spread extremely rapidly in such rapidly expanding population , especially in circumstances where there are few ancestral or founding fathers . there are animal examples of this gender asymmetry of ancestral influences but one rare but illustrative human example of this process relates to the amazing fecundity of genghis khan who was thought to have sired over 1,500 children . there are now over 16 million men in asia who can trace their y chromosome lineage to an extremely small number of reproductively successful men living in the 12 and 13th centuries . in circumstances where an organism 's early life environment provides accurate predictive cues to the environmental conditions prevailing when adult , the ability of the developing foetal and neonatal mammalian brain to adjust its development to optimally match its function to the requirements of its adult environment confers profound advantages in terms of survival and reproductive fitness . one evolutionary development that elegantly facilitates this process in eutherian mammals is the development of a foetally derived placenta , by which the foetus can attempt to control its own destiny and hormonally regulate the maternal hypothalamus to leverage maternal resources for its own benefit . this is a unique situation that requires the interaction and coadaptation of three matrilineal generations and hence genomes interacting in one organism at the same time ( mother , foetus , and postmeiotic oocytes ) . this provides a template by which a mother can transmit adaptive information concerning present and potential future environmental conditions to the developing foetal brain over an extended period . when combined with the evolution of imprinted genes and disproportionate maternal control over the mechanisms of epigenetic inheritance , this provides a mechanism to ensure intergenerational coadaptation of the maternal hypothalamus , developing brain , and placenta , integrate potential maternal and paternal interests , and promote the optimal allocation of maternal resources and transfer of matrilineal cultural influences . in circumstances either where the early environment provides inaccurate cues to the environmental conditions prevailing when adult due to rapid environmental change or when disruptions to normal neural development occur , the mismatch between the environmental predictions made during early development and subsequent reality may mean that an organism may have a poorly adapted phenotype to its adult environment which may predispose it to health problems . an appreciation of these underlying evolutionary salient processes may provide a novel perspective on the casual mechanisms of a range of health problems . the concept of a brain that is not pathological in the classical sense but it is simply mismatched to its environment has been most extensively studied in the context of ancestral and early developmental nutrition [ 2123 , 2628 , 3135 , 40 , 41 ] . however , this concept can be extended to provide insights into the development of a range of alternative neural phenotypes . these include psychopathy whose defining characteristics of impulsivity , recklessness , lack of empathy , and a predisposition to violence are adaptive in extreme conflict driven environments but catastrophically maladaptive in normal circumstances [ 70 , 71 ] . examination of the adaptive potential of a range of neural and cognitive deficits in the context of evolutionary derived foetocentric brain and placental development , epigenetics and environmental adaptation may provide novel insights into the development and potential treatment of a range of health , neurological , and cognitive disorders .

the evolution of intrauterine development , vivipary , and placentation in eutherian mammals has introduced new possibilities and constraints in the regulation of neural plasticity and development which promote neural function that is adaptive to the environment that a developing brain is likely to encounter in the future . a range of evolutionary adaptations associated with placentation transfers disproportionate control of this process to the matriline , a period unique in mammalian development in that there are three matrilineal genomes interacting in the same organism at the same time ( maternal , foetal , and postmeiotic oocytes ) . the interactions between the maternal and developing foetal hypothalamus and placenta can provide a template by which a mother can transmit potentially adaptive information concerning potential future environmental conditions to the developing brain . in conjunction with genomic imprinting , it also provides a template to integrate epigenetic information from both maternal and paternal lineages . placentation also hands ultimate control of genomic imprinting and intergenerational epigenetic information transfer to the matriline as epigenetic markers undergo erasure and reprogramming in the developing oocyte . these developments , in conjunction with an expanded neocortex , provide a unique evolutionary template by which matrilineal transfer of maternal care , resources , and culture can be used to promote brain development and infant survival .

mri of the breast has been applied clinically for more than 20 years , and in western countries methods of using , it has been established in the form of guidelines [ 1 , 2 ] . the core applications of mri of the breast lie in detecting , diagnosing , and evaluating the efficacy of treatment of breast cancer [ 1 , 2 ] . today , however , besides the information on tumor morphology and blood flow obtained by contrast - enhanced mri , it has become possible to obtain a variety of function images and molecular information , and pet and mr spectroscopies are representative studies that are capable of actually being used clinically . choline is considered an important metabolite in proton mr spectroscopy in the mammary gland area . cholines are substances that have attracted interest in regard to every organ and disease , and because they are precursors of the phospholipids that compose cell membranes , increases in choline signals are thought to reflect increased membrane synthesis . in the mammary gland area , choline shows a promise of enabling differentiation between benign and malignant tumors and of serving as an indicator of tumor activity and viability . clinically , the attention has been focused on differentiation between benign and malignant tumors and prediction of the efficacy of chemotherapy . the markers that are useful in breast diseases are centered at 3.2 ppm and are generally referred to as the choline peak . however , myo - inositol , taurine , and so forth are included in the choline peak in addition to such compounds as choline ( cho ) , phosphocholine ( pc ) , and glycerophosphocholine ( gpc ) , which are parts of the membrane lipid metabolic pathways , and because of the closeness of the ranges of their chemical shifts , they are observed as a single peak when measurements are made in vivo . at the cellular level , gpc is higher than pc in normal breast tissue , and since a marked increase in pc and decrease in gpc have been demonstrated as a result of carcinogenesis , the main component in breast cancer is pc . moreover , it has been reported that if we examine minute chemical shifts even with a 1.5 t mr unit , it is possible to separate and observe a 3.22 - 3.23 ppm ( pc ) peak and a 3.27 - 3.28 ppm peak ( gpc / taurine / myo - inositol ) , and that they are useful in differentiating between benign and malignant tumors . the lactose peak is characteristic of the lactation period , and choline has also been found to be detected at 3.2 ppm during that period . the choline observed in the lactating breast matches the choline ( 3.27 ppm ) observed in benign diseases [ 4 , 6 ] . in contrast to the central nervous system area , because of the presence of normal tissue , fat , and so forth , in the mammary glands , the magnetic fields in the voxels tend to be nonhomogeneous . thus , when analyzing the spectra of tumors , it is important to set the voxels so that tissue outside the tumor is excluded . however , because there is a tradeoff between voxel size and acquisition time , a certain voxel size is unavoidable in order to obtain a high signal - to - noise ratio ( snr ) . assuming the addition of mr spectroscopy before or after approximately 20 to 30 minutes contrast - enhanced mri , we think being able to perform mr spectroscopy in approximately 10 minutes would be ideal , and a minimum voxel size of about 15 mm 15 mm 15 mm would therefore appear to be appropriate for lesions with a diameter of 1 cm or greater . in contrast to the central nervous system , manual shimming as well as autoshimming is essential to make the magnetic fields of the voxels homogeneous . with manual shimming , we use full width at half - maximum ( fwhm ) or t2 * for reference , and 2030 hz is the approximate target for fwhm . when the tumor is large , chemical shift imaging ( csi ) is sometimes even used as a means of assessing the internal nonhomogeneity of the tumor ( figure 1 ) . however , because multivoxelization is performed by using the point spread function by phase encoding , the same as for mri , contamination from adjacent voxels occurs . because of this defect , single - voxel spectroscopy is now the standard method . a spin - echo type , point - resolved spectroscopy sequence ( press ) is generally used in order to obtain single - voxel spectroscopy data . when selecting echo time ( te ) of proton mr spectroscopy , the decision as to whether to use a short te or long te involves making a tradeoff between signal intensity and signal contrast . a high snr is obtained with short te whereas long te is superior in terms of ability to separate the contrast signal from the fat signal . with long te ( 135270 ms ) , the signal intensity decreases , but the fact that the fat signal also decreases means an improvement in ability to detect the choline signal . application of mr spectroscopy to the whole body has recently increased greatly , and many methods of fat suppression have been developed . band - selective inversion with gradient dephasing ( basing ) is one of the latest fat suppression techniques and makes it possible to suppress any chemical shift spectrum signal desired by incorporating an additional pulse into the press sequence . the pulses used for suppression , such as chemical - shift selective ( chess ) excitation pulses , are positioned in the form of prepulses immediately before the excitation pulses whereas with basing the pulses for suppression are positioned within the body of the sequence itself . basing enables suppression of the peak of any chemical shift desired , and it has been applied to peak editing , and so forth . it is preferable for the water peak to persist to some extent in order to be able to use it as a standard to compute the chemical shift of a metabolite . while it is possible to perform water and fat suppression simultaneously by basing , we use chess for water suppression at our hospital because of its strong suppressive effect . it is possible to set the intensity of the residual water peak by setting the waiting time after applying the chess pulse . we actually set the waiting time after applying the chess pulse at 200 ms . two methods are available for quantification when performed by comparison with known concentrations : the internal reference method and the external reference method . in the internal reference method , the water signals in measurement voxels are used to quantify the target metabolites , whereas in the external reference method , a phantom is set up close to the measurement site , and the concentration in the volume in the phantom is used as the standard . we use the external reference method to perform quantification at our hospital . with the internal reference method , the water content of the voxels changes during chemotherapy , and that is a potential pitfall with using it . the greatest disadvantage of the external reference method is the need to perform an additional independent measurement as an external reference , and it also requires correction for the partial volume effect and separate calibration experiments . furthermore , since the t1 and t2 values of choline used were the values cited , there is the possibility of errors being introduced between these values and the actual values in the patients . in addition , because of patient movements , accurate correction is difficult even with frequency correction . however , quantification is essential in order to compare changes in the amount of choline for the patients treated with neoadjuvant chemotherapy . we believe that quantification is very useful and requires dedicated multidisciplinary team including radiologists , medical physicists , physicists , and chemists . at our hospital , we perform proton mr spectroscopy with a 1.5 t unit ( magnetom avanto ; siemens ag healthcare sector , erlangen , germany ) and circularly polarized ( cp ) and four - channel phased - array breast coil ( breast matrix coil ; siemens ag healthcare sector , erlangen , germany ) . the parameters used for mr spectroscopy are tr / te = 1620/270 ; voxel size = 15 15 15 mm ; acquisitions = 256 ; spectral width = 1,000 hz ; data points = 1,024 ; and the time of acquisition is 7 minutes . a cylindrical bottle phantom ( syngo grace external phantom ; siemens ag healthcare sector , erlangen , germany ) 4.0 cm high and 2.5 cm in diameter was inserted behind the breast coil and fixed in position . the phantom was filled with 1.25 g of niso46h2o per 1000 g of h2o . proton mr spectroscopy of the phantom was performed immediately after the mr spectroscopy examination of the breast lesion . the voxel size was 7 7 15 mm , and the acquisition time was 4 seconds . the following formula was used to calculate the scaling factor : ( 1)scaling factor = ( 106mwh2o)(nh2oncho ) (ft1h2oft1cho ) (ft2h2oft2cho)(voxelh2ovoxelcho)(coilsensh2ocoilsenscho),ft1=1exp ( trt1 ) , ft2=exp ( tet2 ) , where ncho and nh2o are the numbers of h nuclei of choline molecules and water molecules , respectively . the scaling factor can be converted to molar concentration by correcting for the number of h nuclei per molecule and the molecular weight of the solvent ( mwh2o ) . the ft1 and ft2 relaxation factors were corrected by using the equation for relaxation times . t1 was calculated from the images obtained with two different trs according to the spin - echo sequence , with te maintained constant , and t2 was calculated from the images obtained with 16 different tes with tr maintained constant . the mean t1 was 375 ms , and the mean t2 was 270 ms . ( t1cho = 1513 ms , t2cho = 269 ms ) were adopted as the t1 and t2 values for choline in vivo . the coil sensitivities indicate the signal intensity of the external reference phantom and the signal intensity within the imaging area . the signal intensity within the imaging area was obtained on proton - weighted images ( te / tr , 15/5000 ) with a solution phantom introduced into the measurement breast coil that was the same as the external reference solution . the spectroscopic data processing protocol was saved and linked to the measurement protocol within the syngo software ( siemens ag healthcare sector , erlangen , germany ) to ensure that data processing was identical for each measurement . the spectra were processed by zero - filling the 1028 data points to 2048 data points by applying a gaussian apodization function of 1.5 hz before fast fourier transformation . we fitted the choline peak and water peak with a gaussian function that ranged from 3.18 to 3.32 ppm for choline and was 4.7 ppm for water . the peak line widths were restricted according to the following settings ; the cho peaks were recognized as cho when the fwhm was less than 10 hz ; when the fwhm was 10 hz or more , the peaks no longer fit . the residual water signal was used for reference ( 4.7 ppm ) , and the frequency of any resonance detected in the 3.003.50 ppm spectral region was recorded . choline peaks at 3.213.23 ppm are assigned to phosphocholine ( pc ) and were evaluated by using a threshold snr of 2 [ 15 , 16 ] . when positive for choline , water subtraction and baseline correction with a sixth - order polynomial fit were applied to obtain the flat baseline of an mr spectrum . thus far , reports on clinical research on proton mr spectroscopy of the breast have centered on differentiating between benign and malignant lesions . however , the possibility of using 1.5 t to detect biomarkers of breast cancer has also been reported in recent years . below we comment on the possibilities related to the diagnostic power of proton mr spectroscopy for breast lesions . the major papers on the diagnostic performance of proton mr spectroscopy as a means of differentiating between benign and malignant tumors that were published between 1998 and 2009 are listed in table 1 [ 5 , 1523 ] . according to the data in the nine other papers after excluding our own , sensitivity ranged from 70% to 100% , and specificity ranged from 67% to 100% . sensitivity in the nine articles as a whole was 88% ( 165/187 ) , and specificity was 88% ( 126/144 ) , and the diagnostic performance reported was related to lesions of 1 cm or greater in size . the special characteristic of our own study is that we performed proton mr spectroscopy before biopsy in order to exclude any effects of bleeding or inflammation after a biopsy , and breast imaging recording and data system ( bi - rads- ) mri category 4 and 5 lesions were the subject of our study . there were 171 cases , more lesions than in any of the other studies , but a comparison with the previous studies showed that sensitivity was very low ( 44% ; 40/91 ) . we think the reasons for the low sensitivity were that the target lesions were small and that nonmass lesions were included . actually , when restricted to masses measuring 15 mm or more , sensitivity improved to 82% ( 28/34 ) . the first study of nonmass lesions by proton mr spectroscopy was reported by bartella et al . , and the results were favorable , that is , a sensitivity of 100% and a specificity of 85% . by contrast , the results for sensitivity in our own research were very low , 32% ( 9/28 ) , and specificity was 75% ( 12/16 ) ( table 2 ) . one of the reasons for this is thought to be differences between the types of cancers that were the subject of the studies . ductal carcinoma in situ ( dcis ) accounted for 17% ( 2/12 ) of the cancers in the report by bartella et al . the morphologic patterns of dcis were variable on mri and included a branching - ductal pattern and a scattered clumped pattern in cases with low tumor density at histology and confluent clustered rings in those with high tumor density at histology . therefore , the diagnostic performance for nonmass lesions of proton mr spectroscopy with the 1.5 t mr unit currently being used does not appear to be satisfactory , and we think it will be necessary to assess stronger magnetic fields in the future . it also appears that in the future , it will be necessary to improve the coils , optimize the measurement sequence , and increase the precision of the data postprocessing . however , false - negative cases have been reported even in relatively large invasive cancers , and in addition to invasive ductal carcinoma , they have been reported in medullary carcinoma [ 19 , 21 ] , mucinous carcinoma , and apocrine carcinoma . the existence of these false - negative cases corroborates the fact that biopsy must not be avoided on the basis of negative proton mr spectroscopy findings alone . fibroadenoma [ 5 , 15 , 16 , 19 , 22 , 23 ] , tubular adenoma [ 17 , 18 ] , intraductal papilloma , mastopathy ( including benign proliferative disease ) [ 5 , 18 , 22 , 23 ] , inflammatory lesions with atypia , and atypical ductal hyperplasia [ 16 , 22 ] have been reported . the elevated choline level in breast cancer may be associated with increased membrane synthesis by replicating cells , however , benign tissues , such as proliferative fibroadenoma , may also produce a positive - choline signal . when we measured the proton mr spectroscopy of the breast parenchyma in 920 cases at our hospital , however , we observed a choline peak in 12% ( 113/920 ) , and 32% of the choline peaks ( 36/113 ) were consistent with the choline that is seen in malignancy ( pc ; 3.213.23 ppm ) ( figure 2 ) . the exact reason for this phenomenon is unknown , but the increase in choline in the breast parenchyma may be greater than previously thought . this is important in terms of clinical diagnosis , and caution is required because of the possibility of intermingling by very weak choline in the surrounding area , especially when a voxel that is larger than the lesion is placed . since breast lesions are usually detected by mri after contrast medium has been injected , we tried performing proton mr spectroscopy after contrast - enhanced mri , and the gadolinium contrast medium appeared to have a very slight effect the mr spectrum of the lesion . used on a 3 t mr unit to conduct a study of the effect of six gadolinium contrast media ( magnevist , multihance , omniscan , optimark , prohance , and dotarem ) on mr spectroscopy performed on phantoms and a rat model of breast cancer . the results obtained with the phantom showed that the width of the choline peak broadened with three of the contrast media , that is , magnevist , multihance , and dotarem , and the volume of the peak decreased to an average of about 40% . the use of negatively - charged chelates may be associated with the underestimation of the choline level present in the lesion , and their conclusion was to recommend the use of neutral chelates for mr spectroscopy of the breast . we attempted to determine the effect on the choline spectrum at our hospital by using two different contrast media ( negatively - charged chelates and neutral chelates ) , magnevist ( gadopentetate dimeglumine , gd - dtpa ) and omniscan ( gadodiamide , gd - dtpa - bma ) . we used each of the two contrast media on a different day in 30 patients who had been found to have an enhanced mass , and we compared the changes in width and height of the choline peaks . the results did not show any statistically significant changes in the width or height of the choline peak due to the contrast media ( figure 3 ) , and no statistically significant differences in fwhm or t2 * during shimming were observed in the same cases . we think these findings suggested that the impact of the contrast medium in clinical settings in which 1.5 t mri is used is very mild . mr spectroscopy is more strongly impacted by the location of the tumor ( skin and vicinity of the chest wall ) and the imaging conditions , and the impact of the contrast medium appears to be trivial by comparison . the potential of proton mr spectroscopy as a means of biomarkers of breast cancer has already been investigated in many ex vivo studies . . reported being able to differentiate grade i and ii breast cancers from grade iii breast cancers based on mr spectroscopy findings in an ex vivo study of fine - needle aspiration specimens . we have reported similar findings in vivo , as described below . in our study , the choline levels measured by proton mr spectroscopy with a 1.5-t unit correlated well with the histological prognostic factors , that is , nuclear grade , estrogen receptor status , and triple - negative lesion status . it is hoped that in vivo research on proton mr spectroscopy to detect biomarkers will contribute to breast cancer therapy , including drug therapy . the role of diagnostic imaging in neoadjuvant chemotherapy lies in diagnosing the spread of residual tumors after chemotherapy and in judging the efficacy of the chemotherapy . there are two very important points in regard to the diagnosis of the spread of residual lesions after chemotherapy : the pattern of tumor spread before chemotherapy and the pattern of tumor shrinkage after chemotherapy , and when this information is taken into consideration , it appears possible to perform breast - conserving surgery safely . judging the efficacy of chemotherapy , on the other hand , means evaluating the chemosensitivity of the tumor in vivo . many reports have shown that the variety of information provided by mri ( tumor diameter , changes in volume , and changes in blood flow ) is more useful than the information provided by palpation or ultrasonography [ 3134 ] . a great deal of hope as stated above , since proton mr spectroscopy has a role in the evaluation of treatment of breast diseases by providing molecular information , it is regarded as an effective method for monitoring the activity of breast cancer and for early prediction of the efficacy of chemotherapy [ 3543 ] . accurate assessment of the response to neoadjuvant chemotherapy is one of the critical factors for optimizing the chemotherapy regimen and planning further surgery . the greatest advantages of early prediction of efficacy are being able to reduce unnecessary adverse effects , prevent treatment delays , and avoid unnecessary administration of expensive drugs that do not show promise of being effective . since methods that alter mr spectroscopy voxels according to the size of the tumor during the course of breast cancer chemotherapy are routine [ 36 , 40 ] , quantification is essential in order to compare changes in the amount of choline . however , the reproducibility of the quantitative values themselves is a problem , because when the voxels are altered , shimming accuracy and the snr change . we therefore gave priority to obtaining stabilized voxels that could be compared before and after treatment and tried a method in which we fix voxel size and position the voxel at the same site as the lesion . however , because this is not the standard method , we first investigated its accuracy by making comparisons with pet / ct in the same patients . the results of the comparisons of the amount of choline ( integral values ) obtained by mr spectroscopy and the standardized uptake value ( suv ) obtained by pet / ct showed that the changes in the two perfectly paralleled each other . when we fixed the voxel size , there appeared to be major advantages from a clinical standpoint in terms of being able to fix the scanning parameters , such as receiver gain , and easily observe increases in choline within the unit voxel . on the other hand , one of the disadvantages of this technique is that tumor metabolism can only be observed partially in tumors larger than the voxel size . the other is that the tissue surrounding the tumor is included in the voxel when the tumor becomes smaller than the voxel as a result of regression . however , if mr spectroscopy is used to identify nonresponders early , this disadvantage is avoidable . further studies are needed to examine the advantages and disadvantages of this technique in a larger number of patients . there have been more reports of using pet / ct for early prediction of the efficacy of chemotherapy for breast cancer than of using proton mr spectroscopy . the study on the largest number of cases ( n = 64 ) reported that the evaluations after 2 courses of therapy were more accurate than the evaluations after 1 course or after 3 courses . we conducted a study to determine whether proton mr spectroscopy or pet / ct is more suitable for early prediction of efficacy , and we conducted it after two courses of chemotherapy when prediction of efficacy by pet / ct also was reported to be the most accurate . the results of the study by the external reference method with a fixed voxel size ( 15 15 15 mm ) in 16 cases showed that proton mr spectroscopy and pet / ct were equivalent . however , whether mr spectroscopy is as good as pet has not been firmly established . neoadjuvant chemotherapy usually consists of an anthracycline - based regimen followed by taxane - based regimens , but because the early evaluations by mr spectroscopy are no more than evaluations of the initial anthracycline - based regimen and are not always consistent with the pathological evaluation after the completion of chemotherapy , evaluations of efficacy performed during the initial anthracycline - based regimen alone are inadequate for early prediction of the efficacy of this method . although the number of cases has been small , there have been cases in which the anthracycline - based regimen was ineffective , but the efficacy of subsequent taxane - based regimens was excellent , and cases in which the opposite was true . we therefore assessed early prediction of efficacy by mr spectroscopy in which treatment with an anthracycline - based regimen alone was performed , and the results indicated that the changes in choline after the second cycle of chemotherapy as determined by quantitative mr spectroscopy may be a more sensitive means of predicting the pathological response than changes in tumor size are . we think that it is preferable to try making early predictions of the efficacy of neoadjuvant chemotherapy for each of the individual drugs . based on the results described above , proton mr spectroscopy may not be inferior to pet / ct as a diagnostic method for early prediction of efficacy in breast cancer . in the next step , it will be necessary to determine whether it is possible to predict efficacy by proton mr spectroscopy earlier than after 2 courses . in a study on 14 cases , meisamy et al . found that the changes in the amount of choline detected at 4 t within the first 24 hours after a single course correlated with clinical efficacy after the completion of chemotherapy . we showed that the results of an evaluation by proton mr spectroscopy at 1.5 t after one course ( several days before the start of the second course ) correlated with pathological size after the completion of chemotherapy . however , the same as in the study by pet / ct , it was also possible to underestimate the efficacy of treatment after one course more than after two courses . proton mr spectroscopy is a technique that is still in the development stage in the breast disease . in the future , it will be necessary to carefully evaluate the proper timing of early prediction of the efficacy of chemotherapy because the underestimated data could be an obstacle to clinical application . the very basis of the spectroscopy method relies on the use of magnets with higher field strength to separate the diagnostic resonances . with the growing availability of 3 t systems , the use of breast mr spectroscopy for diagnosing lesions and monitoring response to neoadjuvant chemotherapy will become routine clinical practice . however , as stated above , there have not been very many reports on differential diagnosis between benign and malignant lesions or on early prediction of the efficacy of neoadjuvant chemotherapy . the reason is that many problems must be resolved , including the optimal measurement sequence for proton mr spectroscopy , differences between mr units , spectrum interpretation , postprocessing adjustments , and methods of choline quantification ( internal reference method or external reference method ) . however , if these problems are not resolved , it will be impossible to ensure the safety and reliability of being able to use it for cancer treatment ( drug therapy ) .

proton mr spectroscopy of the mammary gland area is used to be considered in the realm of basic research , but as a result of the advances in mr techniques , it is now being performed in ordinary clinical practice . it is particularly noteworthy that useful clinical data are now being accumulated with 1.5 t mr units , which are the standard units . we think that , at this point , it is very important to systematically review the techniques , clinical applications , and future prospects of proton mr spectroscopy . we have performed proton mr spectroscopy with a 1.5 t mr unit in over 3000 cases at our hospital . in this paper , we will comment on the current status of proton mr spectroscopy of the breast , primarily in regard to differentiation between benign and malignant lesions and prediction of the efficacy of chemotherapy while describing the data obtained at our hospital .

in late march and early april 2009 , an outbreak of the 2009 pandemic influenza a ( h1n1 ) virus infection was detected in mexico , with subsequent cases observed in many countries , including the united states . it was reported that the 2009 h1n1 virus was identified as the cause of outbreaks of febrile respiratory infection ranging from self - limited to severe illness , and there were severe cases that developed respiratory failure . cytokine responses to influenza virus infection have been shown to contribute to the pathogenesis and severity of influenza virus infection , particularly influenza - associated encephalopathy ( h1n1 , h3n2 ) and respiratory failure ( h5n1 ) [ 35 ] . therefore , we evaluated the systemic cytokine and chemokine response during the 2009 h1n1 virus infection and studied the expression of cytokines and chemokines in pediatric patients with pneumonia , one of the major complications of the disease . thirty - four children with the 2009 h1n1 virus infection who were admitted to our center between july and december 2009 were included in this study . there were 25 boys and 9 girls , and their ages ranged from 2 to 11 years , with a median age of 6 years . 2009 h1n1 virus infection was confirmed by the detection of the viral genomes with the real time reverse transcription polymerase chain reaction ( rt - pcr ) assay targeted at a region of swine h1 ha gene using a nasal swab specimen obtained at the time of admission . twenty - one children with the 2009 h1n1 virus infection had pneumonia with abnormal respiratory signs ( weakness of breath sound , wheezing , and hypoxia ) and definite consolidation shadows or atelectasis in their chest x - ray examinations , while another 13 patients presented without pneumonia , including vomiting , delirium , convulsion , liver dysfunction , and pancytopenia . among 21 patients with pneumonia , 10 patients had severe pneumonia with respiratory failure ; all patients were admitted to an intensive care unit ( icu ) and 5 of the 10 patients required mechanical ventilation . seven of the 10 patients with severe pneumonia had lobar or multilobar atelectasis with consolidation shadow in their chest x - ray examination . another 11 patients with pneumonia had mild respiratory disorder , and none of them had an icu admission or underwent mechanical ventilation . the concentrations of cytokines and chemokines in the serum obtained at the time of admission , which was within the first 4 days after symptoms onset , were measured . we assayed 17 cytokines in serum ( il-1 , il-2 , il-4 , il-5 , il-6 , il-7 , il-8 , il-10 , il-12 , il-13 , il-17 , g - csf , gm - csf , ifn- , mcp-1 , mip-1 , and tnf- ) by the bio - plex suspension array system and the 17-plex panel ( bio - rad laboratories , tokyo , japan ) . the mann whitney u - test was used for the statistical comparison of the two groups of patients . among the patients who presented with the 2009 h1n1 virus infection , there were no differences in the age , sex , or illness day on admission between patients with pneumonia and those without pneumonia ( table 1 ) . the patients with pneumonia were more likely to have asthma as an underlying medical condition , oxygen administration , icu admission , and mechanical ventilation than patients without pneumonia . two patients with pneumonia showing leukocytosis and elevated c - reactive protein ( crp ) were suspected to have a bacterial coinfection , but cultures of sputum or nasopharyngeal swabs were not done . table 1patient characteristicspatients with pneumonia ( n = 21)patients without pneumonia ( n = 13 ) age ( years)8 ( 211)6 ( 210 ) male sex ( no.)1510 illness day on admission ( day)2 ( 14)2 ( 13)underlying medical conditionsasthma ( no.)71seizure disorder ( no.)12neurological disorders ( no.)01renal disease ( no.)10heart disease ( no.)01obesity ( no.)01clinical signs and symptomsfever ( no.)2113cough ( no.)219vomiting ( no.)06delirium ( no.)14convulsion ( no.)01liver dysfunction ( no.)01pancytopenia ( no.)01respiratory complicationsrr ( /min)40 ( 2465)28 ( 2740)spo2 ( % ) 89 ( 8296)97 ( 9599)weakness of breath sound ( no.)100wheezing ( no.)60hypoxia ( no.)210bacterial pneumonia ( no.)20chest x - rayconsolidation : unilateral ( no.)15/210/5 bilateral ( no.)4/21atelectasis ( no.)8/210/5outcomeoxygen requirement ( no.)210icu admission ( no.)100use of mechanical ventilation ( no.)50death00duration of hospitalization ( day)6 ( 412)5 ( 26)laboratory resultswbc ( 10/l)9.7 ( 1.823.6)6.9 ( 2.610.3)ht ( % ) 37.8 ( 35.644.2)36.3 ( 33.241.8)plt ( 10/l)22.2 ( 13.070.3)20.8 ( 8.530.1)crp ( mg / dl)2.8 ( 0.38.6)0.7 ( 0.12.7)ast ( u / l)29 ( 1548)35 ( 26485)alt(u / l)13 ( 453)14(9267)ld(u / l)294 ( 220402)318 ( 231714)ck ( u / l)125(9248)87 ( 43896)data are given as medians ( range ) patient characteristics data are given as medians ( range ) among measured cytokines and chemokines in the children during the 2009 h1n1 virus infection , the serum levels of ifn- and il-5 in patients with pneumonia were significantly higher than those in patients without pneumonia . this tendency was also present for the serum levels of il-6 , il-8 , il-10 , il-13 , and mcp-1 in patients with pneumonia ( table 2 ) . comparison between patients with pneumonia and those without pneumoniapatients with pneumonia ( n = 21)patients without pneumonia ( n = 13)p - valueil-10.2 ( und.1.4)0.9 ( und.2.6)0.392il-21.0 ( und.2.3)0.4 ( und.1.8)0.150il-40.1 ( und.0.7)und . ( und.0.8)<0.001il-61.8 ( und.3.4)0.7 ( 0.42.5)0.035il-70.1 ( und.1.5)0.3 ( und.1.7)0.394il-81.1 ( 0.11.7)0.5 ( 0.61.7)0.021il-101.2 ( und.2.3)und . ( und.0.5)0.020il-17und . ( und.1.9 ) und.0.590g - csf1.3 ( und.2.4)0.7 ( und.2.1)0.068gm - csf1.3 ( und.2.7)0.3 ( und.2.4)0.282ifn-2.2 ( und.3.8)1.5 ( und.3.2)0.009mcp-12.1 ( und.3.2)1.4 ( und.2.5)0.035mip-11.7 ( 0.12.5)1.1 ( 02.7)0.074tnf-1.4 ( und.2.4)und . comparison between patients with pneumonia and those without pneumonia data are given as median log10 pg per ml ( range ) the serum levels of il-1 , il-2 , il-4 , and mcp-1 were significantly higher in the group of patients with severe pneumonia than in those with mild pneumonia ( table 3 ) . this tendency was also present for il-5 , il-13 , and tnf- , although it was not observed with the levels of il-6 , ifn- , and other chemokines , such as il-8 and mip-1. table 3level of cytokines and chemokines in the serum . comparison between patients with severepneumonia and those with mild pneumoniapneumoniap - valuesevere ( n = 10)mild ( n = 11)il-1und . ( und.0.6)0.008il-51.6 ( 1.22.5)0 ( und.2.4)0.024il-62.1 ( 0.43.4)1.5 ( und.2.4)0.078il-70.2 ( und.1.5)0.1 ( und.1.5)0.698il-81.1 ( 0.21.6)1.1 ( 0.11.7)0.725il-101.4 ( und.2.3)0.4 ( und.1.7)0.075il-120.9 ( und.2.2)und . ( und.1.9 ) und.0.056g - csf1.5 ( und.2.4)1.1 ( und.2.2)0.379gm - csf1.6 ( und.2.7)0.5 ( und.1.9)0.056ifn-2.5 ( und.3.8)1.9 ( und.3.3)0.091mcp-12.5 ( 1.03.2)1.5 ( und.2.4)0.007mip-11.8 ( 0.52.5)1.5 ( 0.12.5)0.105tnf-2.5 ( und.2.4)0.1 ( und.2.0)0.038data are given as median log10 pg per ml ( range)und . , undetectable level of cytokines and chemokines in the serum . comparison between patients with severepneumonia and those with mild pneumonia data are given as median log10 pg per ml ( range ) this study shows that the serum levels of ifn- , il-5 , il-6 , il-8 , il-10 , il-13 , and mcp-1 in patients with pneumonia were higher than that in patients without pneumonia during the 2009 h1n1 virus infection . the levels of il-1 , il-2 , il-4 , and mcp-1 were significantly different between patients with severe pneumonia and those with mild pneumonia . of note these findings suggest that mcp-1 might contribute to the pathogenesis and severity of respiratory complications in the 2009 h1n1 virus infection or the degree of mcp-1 production could reflect the severity of the infection . in this regard , it is intriguing that the mcp-1 knockout mice had a reduced antiviral clearance , together with reduced leukocytes recruitment into the lungs during infection with influenza a . in patients with severe pneumonia caused by influenza a ( h5n1 ) infection , the levels of several chemokines , such as ip-10 , mig , mcp-1 , and il-8 , in the peripheral blood experimental data in a human lung organ culture model showed that influenza viruses ( h1n1 and h3n2 ) proliferated in the bronchial epithelial cells and induced the production of mcp-1 , mip-1 , il-8 , and ip-10 . on the other hand , in pigs infected by the 2009 h1n1 virus , the levels of il-6 , ifn- , and tnf- in the broncho - alveolar lavage fluid were elevated , but the concentrations of chemokines were not measured and the roles of chemokines in respiratory complications of the 2009 h1n1 virus infection remains to be investigated . we speculate that the 2009 h1n1 virus proliferates in the bronchial epithelial cells in patients and induces the production of several chemokines , which cause the inflammation of the airway , followed by severe respiratory complications . although the pathogenesis of this complication is still unclear , the serum levels of proinflammatory cytokines , including il-1 , il-6 , and tnf- , have been shown to be elevated and related to the clinical severity of [ 3 , 4 ] . among patients with this type of encephalopathy , the serum tnf- values at onset were significantly higher in patients who died compared to those who survived [ 3 , 4 ] . the present study shows a characteristic cytokine profile in the patients with pneumonia during the 2009 h1n1 virus infection which is different from what is observed in influenza - associated encephalopathy . the secretion of cytokines and chemokines during the acute phase of the 2009 h1n1 virus infection were clearly different depending on the presenting symptoms . this suggests that an early - phase response of cytokines and chemokines during the 2009 h1n1 virus infection might have contributed to the pathogenesis of the disease . in conclusion , the present study shows that the production of several chemokines , especially mcp-1 , may have a role in the pathogenesis of respiratory complications during the 2009 h1n1 virus infection .

we report the systemic cytokine and chemokine response in children with the 2009 pandemic influenza a ( h1n1 ) virus infection . in patients with pneumonia , the serum levels of ifn- and il-5 were significantly higher than those in patients without pneumonia . this tendency was also present for il-6 , il-8 , il-10 , il-13 , and mcp-1 in patients with pneumonia . among patients with pneumonia , the levels of mcp-1 were significantly higher in the group of patients with pneumonia with severe respiratory failure than patients with mild pneumonia .

it is now recognized that the offspring of women exposed during gestation to inescapable stressors like natural disasters , adverse life events or social pressures have a higher risk of psychopathology than those not exposed to such stressors ( charil et al . , 2010 , weinstock , 2008 ) . these include , generalized anxiety states and depression ( van den bergh et al . , 2008 , van lieshout and boylan , 2010 ) , attention ( grizenko et al . , 2012 , li et al . , 2010 , park et al . , 2014 , zhu et al . , 2015 ) and learning deficits ( laplante et al . , 2008 ) , autism ( kinney et al . , 2008 ) and schizophrenia ( fineberg et al . , 2016 , khashan et al . , 2008 , levine et al . , 2016 ) . studies that are more recent have reported sex differences in the behavioral alterations induced by prenatal stress . they suggest that affective disorders are more prevalent in girls ( davis and pfaff , 2014 ) , while schizophrenia and attention deficits are more likely to occur in boys ( fineberg et al . , 2016 ) if the mother was exposed to the stressor in the second trimester ( zhu et al . , 2015 ) . autism has been associated with objective stress during the first trimester but its preponderance in boys has been disputed ( walder et al . , 2014 ) . in an attempt to provide a sounder scientific basis for these observations a large number of preclinical studies were performed , largely in rodents . the term stress has have been given different definitions in the literature ( see huizink et al . , 2004 , mcewen , 2000 , selye , 1950 ) , but for the purpose of this review the term stressor will be used as referring to the event , while stress refers to the impact on the organism and its response to it . the stressor is designed to cause distress and involves adaptive physiological responses and the release of hormones that cause emotional changes in the pregnant female and in her offspring ( graignic - philippe et al . the first study by thompson ( thompson , 1957 ) was aimed at achieving psychological stress in the pregnant rats that would not cause tissue damage to her fetuses . rat dams were trained before pregnancy in a conditioned avoidance test and were subjected to the stimulus daily throughout pregnancy . most of the subsequent studies did not use stressors that were only psychological , but may also cause pain or discomfort . yang et al . , 2006 ) or restraint in cylinders in strong light for periods of 45 min-6 h , up to three times a day ( lesage et al . , 2004 , vallee et al . , 1997 , van den hove et al . , 2005 , ward , 1972 , williams et al . , 1999 restraint can have a direct effect on the fetuses by restricting their movements ( choe et al . , 2011 ) . also , kinsley and svare ( 1986 ) reported that restraint decreased the mother 's food intake and body weight and that of her offspring . nevertheless , the majority of studies has continued to use this stressor once or thrice daily . prior to 2006 , almost all of the experiments on the effects of prenatal stress in rodents were performed only on male offspring ( weinstock , 2007 ) . recently , more reports have included females , and a few have determined the stage of the estrus cycle in association with the measurement of their behavior ( brunton and russell , 2010 , salomon et al . , 2011 ) . in order to reduce potential variability , others have performed the behavioral tests when all the females were in diestrus ( wang et al . the current review will focus on the findings in recent studies published after previous reviews ( weinstock , 2007 , weinstock , 2008 ) and explores possible reasons for any differences they report in the effects of gestational stress on various types of behavior in the offspring . these will include the influence of the strain of rat or mouse , time of stressor application during gestation , its nature , and the age of offspring at which behavior is examined . restraint , with or without bright light , is still the most popular stressor used in experimental animals in general ( buynitsky and mostofsky , 2009 ) and in pregnant rats in particular , because the duration of the stressor can easily be controlled and it is convenient for taking blood samples from the tail for hormonal measurements . the degree of rat movement can also be regulated according to the size and construction of the restraining device . almost all the studies described in this review incorporated a period of restraint in the regimen of maternal stress in rats or mice that ranged from 30 min to 6 h , either as the sole stressor , or with others . as shown in table 1 , the same or different stressors was applied up to three times daily . more recent studies have replicated the reduction of maternal body weight by restraint described earlier in sprague - dawley ( sd ) rats when it was applied thrice daily for 45 min each time ( van den hove et al . , 2014 ) , once daily for 60 min in wistar rats ( fujita et al . , 2010 ) , or for 75 min in long - evans ( le ) rats ( baker et al . , 2008 ) . interestingly , thrice daily restraint reduced body weight in sd rats ( van den hove et al . , 2014 ) , but not in the inbred fischer strain ( van den hove et al . , 2005 ) . , 2010 , palacios - garcia et al . , 2015 ) , testifying to the activation of her hypothalamic pituitary adrenal ( hpa ) axis . in only a few studies was the effect of other stressors measured on the body weight of the dam ( table 2 ) . the findings indicate that the duration of the stress rather than its nature appears to determine the weight loss . thus , when different stressors , or only restraint were applied thrice daily for 45 min , or once daily for one - six hours , maternal body weight was decreased ( fujita et al . , 2010 , palacios - garcia et al . , 2015 , no reduction in maternal weight occurred when the stressor was given once daily for no more than 45 min ( abe et al . , 2007 , goelman et al . , 2014 , zohar et al . , 2016 ) ( table 2 ) . varied stressors had no effect on birth weight in pups of either sex ( zohar and weinstock , 2011 ) , or in males ( females were not tested ) ( abe et al . , 2007 , birth weight was also unaffected in pups of le rats , restrained from day 1019 of gestation for periods of 1575 min , but a reduction was seen in growth rate in both sexes ( baker et al . , 2009 ) . a selective effect in the birth weight in female pups was induced by two forms of bystander stress from day 1214 of gestation . in one , the pregnant rat was placed in proximity of a lactating female ( brunton and russell , 2010 ) , and in the other , of a rat that was stressed by being put on an elevated platform under bright light for 30 min twice daily ( mychasiuk et al . , 2011 ) . an additional confound in these experiments is whether , or not , the rat dam adapted to the stressor , but relatively few experiments examined this in pregnant rats . adaptation to a stressor is indicated by a decline in the increase in plasma corticosterone ( cor ) after each exposure compared to that after the first time . plasma cor ceased to rise in male rodents on the fourth day after they were subjected to restraint once daily , at the same time of day ( melia et al . , 1994 ) , or to other stressors ( dhabhar et al . plasma cor increased in pregnant rats after each exposure to noise and flashing lights when these were delivered on a random basis at different times of day , but not at the same time on each successive day ( weinstock et al . , 1988 ) . furthermore , only the pups of dams subjected to random stress showed significant retardation in their development ( fride and weinstock , 1984 ) . others also showed that adaptation did not occur when different stressors were applied each day in a random order ( modir et al . , 2015 , wilson et al . , 2013 ) or when the dam was subjected to bystander stress ( brunton and russell , 2010 ) . circulating maternal cor can reach the developing fetus ( zarrow et al . , 1970 ) , impair the regulation of its hpa axis ( fujioka et al . , 1999 , henry et al . , 1994 , weinstock et al . , 1992 ) and induce behavioral alterations in the offspring ( weinstock , 2005 ) . this was demonstrated by means of maternal adrenalectomy , which prevented the impairment of hpa axis regulation by gestational stress ( barbazanges et al . , 1996 ) and the induction of anxiety - like behavior in the offspring ( salomon et al . , 2011 , zagron and weinstock , 2006 ) . administration of cor to the pregnant , adrenalectomized dams in amounts that mimicked the levels achieved by stress , reinstated anxiety and impaired hpa axis regulation . although maternal adrenalectomy also prevented learning and memory deficits in ps offspring , they were not re - instated by cor administration ( salomon et al . , 2011 ) , indicating that they resulted from the action of another adrenal hormone ( see section 7 ) . generalized anxiety and affective disorders are considered stress - related and their incidence is increased in human subjects subjected to stress during pregnancy ( davis and sandman , 2012 ) . from the 195070s , the open field ( of ) test has been used to detect potential anxiety - like behavior resulting from prenatal stress in rodents . the test is based on the assumption that fearful or anxious animals take more time than controls to enter a well - lit novel environment from their home cage and make fewer incursions into its center . in 1986 , following the description of the elevated plus maze ( epm ) by handley and mithani ( handley and mithani , 1984 ) as depicting a conflict between the animal 's desire to explore and fear of open spaces , file and her colleagues used it as a screening test for anxiolytic drugs ( pellow and file , 1986 ) . experiments were performed in bright light to deter entry by untreated rats into the open arms , thereby enabling the authors to detect an increase in those treated with the drugs . since our aim was to use the test to detect anxiety - like behavior in ps rats ( fride and weinstock , 1988 ) , we performed the experiments in dim light to encourage the controls to enter into the open arms . in subsequent experiments , we showed that a larger difference in the behavior of control and ps rats was obtained when the rats had been housed on a reversed light cycle for at least a week and experiments were carried out under dim light during the rats ' active period ( weinstock , 2015 , zohar et al . , 2015 ) . since 2008 , most of the studies have replicated our original findings and those of thompson ( thompson , 1957 ) and showed an increase in anxiety - like behavior in the epm or of tests in juvenile ( jia et al . , 2015 , xu et al . , 2013 ) and adult rat and mouse offspring of both sexes ( akatsu et al . , 2015 , glombik et al . , 2015 , palacios - garcia et al . , 2015 , salomon et al . , 2011 , wang et al . , 2015 , zohar and weinstock , 2011 ) , or when only males were tested ( barzegar et al . , 2015 , de souza et al . , 2013 , miyagawa et al . , 2011 , sun et al . , 2013 ) the stage of the estrus cycle influenced the percent of time the rats spent in the open arms of the maze , but this was reduced by prenatal stress at each stage ( salomon et al . others found an increase in anxiety - like behavior only in males ( zuena et al . , 2008 ) or females ( schulz et al . , 2011 , van den hove et al . , 2014 ) , in spite of the fact that the identical stressor was used in sd rats . this may have been due to the conditions under which offspring behavior was assessed , but such information was not supplied . for example , whether the rats were subjected to an additional stress by their transport to the experimental room only a short time before the experiment ( hogg , 1996 ) , or whether or not , the area of the maze was brightly lit ( morato and castrechini , 1989 ) . the behavior of rats in the epm was shown to be age dependent , with male rats aged 90 days or more , and females 120 days or more spending less time in the open arms than younger rats ( imhof et al . , 1993 ) . thus , it is likely that an increase in anxiety - like behavior was not seen in two of the studies because the males were aged 100 days ( van den hove et al . , 2014 ) or 170 days ( schulz et al . , 2011 this was probably also true in younger controls that , for an unknown reason , spent very little time in the open arms of the epm , thereby showing a floor effect ( wilson et al . , 2013 ) . anxiety was also not seen because the same mice ( kiryanova et al . , 2016 ) and rats ( bourke et al . were subjected to a number of different , stressful , behavioral tests , which is known to influence behavior ( voikar et al . , 2004 ) . anxiety and depression frequently occur concurrently or sequentially in childhood and adolescence ( garber and weersing , 2010 ) in association with prenatal stress ( van lieshout and boylan , 2010 ) . depression is a very complex psychological disorder comprising some , or all of these symptoms , low mood , anhedonia , feeling sad , hopeless , helpless and worthless or ashamed . clearly , it is not possible to detect and quantify such feelings in rodents . in the absence of direct methods for assessing depression , researchers have used the forced swim test ( fst ) ( porsolt et al . , 1978 ) , or a decrease in sweet preference when presented with a choice of water or a solution of sucrose as a measure of anhedonia . behavior in both tests responds to drugs that have antidepressant activity in human subjects ( moreau , 2002 ) . in the fst , a rat or mouse is exposed to inescapable forced swim for 15 min on one day and tested for learned helplessness 24 h later , characterized by floating or virtual immobility and fewer attempt to swim or climb on the walls of the cylinder . clinically effective antidepressants decrease the duration of floating and increase swimming and/or climbing . females in diestrus exhibit more , and in proestrus , less , learned helplessness than males ( jenkins et al . , it was found that different stressors , applied during the last week of gestation , increased depressive - like behavior in juvenile ( guan et al . , 2013 , . , 2015 ) and adult offspring of wistar or sd dams ( abe et al . , 2007 , , 2015 , zohar et al . , 2015 ) , but not in the offspring of dams stressed during the second week ( jia et al . , 2015 ) . those who failed to detect an effect of prenatal stress in this test either left the rats in the cylinder of water for 10 min instead of 5 , resulting in a relatively long duration of immobility in ps and controls ( van den hove et al . , others researchers applied the fst after additional tests in the same rats ( schroeder et al . , 2013 , bourke et al . , 2013 , wilson et al . , 2013 ) , unlike the majority of studies , the foregoing data from recent studies confirm earlier reports that prenatal stress can cause anxiety and depressive - like behavior in rats and mice . to date , there is no consistent evidence of a sex difference in the incidence of these behaviors in rats . the effect of prenatal stress on spatial learning and memory retention has most often been examined by means of the morris water maze ( mwm ) test in which rats are placed into a large circular pool of water from which they can escape onto a hidden platform . normal rats learn quickly to swim directly to the escape platform ( morris , 1984 ) . to assess the rate of acquisition of spatial learning , rats are given two or more trials a day and the position of entry of the rat into the maze is changed each day , while the platform remains in the same position . memory is assessed by removing the platform and measuring the time spent by the rat in the quadrant in which the escape platform was situated . like other behavioral tests , the majority of the early experiments were performed only in males and these showed impaired spatial learning in adulthood ( weinstock , 2008 ) . since then , only a few have assessed the effect of prenatal stress in both sexes . in adult female rats , no effect was found on the rate of learning ( weinstock , 2011 , zuena et al . , 2008 ) , but this was slower than in controls in pre - pubertal females ( weinstock , 2011 ) . others showed a deficit in memory consolidation in the passive avoidance test in rats of both sexes ( palacios - garcia et al . , 2015 ) . in adult males of the sd and wistar strains , prenatal stress slowed the rate of acquisition of spatial learning and memory retention , irrespective of the nature of the maternal stressor ( barzegar et al . , 2015 , lui et al . , 2011 , markham et al . , 2010 , , 2014 , ratajczak et al . , 2015 , schulz et al . , 2011 ) . in contrast to the findings in the majority of studies , thrice - daily restraint improved learning in the mwm test in adult sd male offspring ( zuena et al . , 2008 ) . impaired learning in male wister rats aged 45 weeks ( yaka et al . , 2007 , yang et al . , 2006 ) and 67 weeks ( barzegar et al . , 2015 ) was associated with a decrease in hippocampal long term potentiation ( ltp ) and an increase in long term depression ( ltd ) . others found a reduction in hippocampal ltp in ps , sd males and females aged 3 and 5 , but not 8 weeks , while ltd was only increased in rats aged 5 weeks ( yeh et al . , 2012 ) . prenatal stress also reduced spatial learning but not memory retention in pre - pubertal and adult male c57/bl mice , ( zhao et al . , 2013 ) , or in adults of both sexes ( benoit et al . , 2015 ) . those that did not detect an effect of prenatal stress either subjected the mice to multiple tests ( kiryanova et al . , 2016 ) , or housed them singly ( akatsu et al . , 2015 ) , which is very stressful and the data show clearly that prenatal stress can slow the rate of spatial learning and decrease hippocampal ltp in juvenile and adult males but in the latter , it does not always impair memory retention . the paucity of studies in females precludes a conclusion that such deficits are more prevalent in males . the protective effect of estrogens on brain regions associated with learning and memory ( liu et al . , 2008 , ping et al . , 2008 ) is consistent with the likelihood of greater resilience of female offspring to the effects of gestational stress . in rodents , the tendency to explore novel objects more than familiar ones has been exploited as a sensitive test of stimulus recognition memory ( ennaceur and delacour , 1988 ) . the test involves the substitution of a familiar object with a novel one in a memory retention trial . since rats also have an innate tendency to explore objects in a novel place , the test can be adapted for assessment of spatial memory by changing the position of one of the objects , which remain identical ( ennaceur et al . , 2005 ) . in the hooded lister strain , novel object and place recognition in young adult males and females depended on the inter - trial interval ( iti ) that differed according to the test and sex of the rat . males showed preference for new objects at itis of up to 30 min and for a new place , for up to one hour . females showed novel object recognition ( nor ) at itis of up to three hours and novel place recognition , up to an iti of one hour ( sutcliffe et al . , 2007 ) . in wistar rats , females , but not males , showed nor at itis of 40 and 60 min ( biala et al . , 2011 ) . varied forms of mild prenatal stress had no effect on the behavior of females at either time interval but increased nor in males to resemble the female phenotype ( biala et al . , 2011 , salomon et al . , 2011 ) . it may be significant that the ps males had a shorter ano - genital distance , which is associated with lower levels of testosterone ( gerardin et al . results of the assessment of prenatal stress in sd rats differed from those in wistar rats and in different studies . adult rats of both sexes showed significant nor at 15 min , one and three hours . ps females lost their discrimination at an iti of one hour , and males , at three hours . however , others found that prenatal stress abolished object discrimination in sd males at iti of 60 min . in addition to the strain of rat , it is possible that the differences in the findings cited above result from the nature of the maternal stressor and iti , but also if the nature if objects used in the test are too similar for the rats to distinguish . it has been reported that the offspring of mothers who were exposed to stress or a viral infection during gestation have an increased likelihood to develop schizophrenia ( brown et al . , 1996 , fineberg et al . , 2016 , levine et al . , 2016 , mednick et al . , 1994 ) . the period most sensitive to stressors appears to be the second trimester , when the frontal cortex ( fc ) and hippocampus develop ( bayer et al . , 1993 ) . the hippocampus is reduced in size ( harrison , 2004 ) and its cell layers are disorganized ( heckers and konradi , 2010 , stefanis et al . , 1999 ) . in the fc , excitatory neurotransmission is decreased , resulting in less inhibitory neurotransmission in the ventral tegmental area ( volk and lewis , 2010 ) . this is believed to contribute to the negative symptoms in schizophrenia including , disordered thought processes , social withdrawal , anhedonia , and blunted affect ( kirkpatrick et al . the only ones that can be identified and quantified are anhedonia and social withdrawal , indicated by a decrease in social interaction that is seen also in depression ( described in section 3.2 . ) . in mice and rats , social interaction with their novel peers is assessed by placing the two animals in a neutral cage and measuring the percentage of time during which are in direct physical contact and perform ano - genital exploration , sniffing with direct contact , crawling , grooming , and play behaviors . pre - pubertal or adult male offspring of sd dams subjected to random stressors from days 1421 ( lee et al . , 2007 , wilson et al . , 2013 ) , or unpredictable foot shocks on days 1720 of gestation ( ehrlich and rainnie , 2015 ) showed significantly less social interaction than controls . in adolescent sd rats , a decrease in social interaction was only found if both rats of the pair were stressed prenatally , but not if one was a control . prenatal stress caused a reduction in social interaction in adult male swiss albino mice ( dong et al . it is noteworthy that none of the foregoing studies examined social interactions in female offspring , which might have lent support to the suggestion that the condition is more prevalent in males . a link has been made between anxiety disorders ( martin et al . , 2009 ) and depression ( drevets et al . , 2007 ) and a decrease in the serotonergic innervation of limbic structures , prefrontal cortex ( pfc ) , amygdala and hippocampus . 5ht innervation to these areas arises in the medial and dorsal raphe nucleus ( drn ) ( azmitia and segal , 1978 ) . this has led to the examination of the effect of prenatal stress on 5ht transmission is association with alterations in behavior . in ps male mice , there was an increase in the number of 5ht positive cells in the drn , measured by staining with an antibody to tryptophan hydroxylase ( tph ) ( miyagawa et al . , 2011 ) . this , and other measurements associated with 5ht innervation , were not made in any other brain area . in ps , sd rats of both sexes , which showed increased anxiety , but no depressive - like behavior , there was an increase in tph in the dentate gyrus and ca3 region of the hippocampus and in 5ht immunoreactivity in the pfc and hippocampus in males but not in females . in the drn , there was a decrease in overall 5-ht immunoreactivity and 5-ht immunoreactive cell density in males but not in females . however , the alterations in 5ht and in tph were unrelated to the observed changes in behavior induced by prenatal stress ( van den hove et al . , 2014 ) . moreover , the authors offered no explanation as to why the effect of prenatal stress on tph in the drn of male rats was the opposite of that in mice , in spite of the similar effect on behavior . 5ht release in target areas is also dependent on the balance of activity on 5ht1a inhibitory autoreceptors on 5ht cell bodies and gabaergic inhibitory interneurons in the drn . although neither anxiety nor depressive - like behavior were assessed in the study , the number of 5ht1a receptors ( 5ht1ar ) in the whole drn region , measured by reversed transcription pcr , was significantly reduced in ps rats ( sex not defined ) ( said et al . , 2015 ) . by means of fluorescence immunohistochemistry with specific antibodies directed to different cell groups , we found that anxiety and depressive - like behavior of ps , wistar rats was accompanied by a decrease in the expression of 5ht1ar on 5ht cell bodies and gabaergic interneurons in the drn and pfc in males . in ps females , the density of 5ht1ar receptors in the pfc was unchanged , but reduced in 5ht and gabaergic interneurons , together with corticotrophin releasing factor ( crf ) type 2 receptors ( crfr2 ) in the drn . the expression of 5ht in the drn was much lower in females than in males and both were unaffected by prenatal stress ( zohar et al . , 2015 ) . we also found that prenatal stress reduced the expression of tph in the drn in both sexes ( zohar et al . , 2016 ) . chronic treatment of juvenile ps rats with the antidepressant drug , citalopram reversed the anxiety and depressive - like behavior in adulthood and also the alterations in the expression of 5ht1ar and crfr2 ( zohar et al . , 2015 ) , testifying to their relation to the behavioral measures . glutamate - gated cation channel receptors n - methyl - d - aspartic ( nmda ) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic ( ampa ) receptors are located primarily on neuronal cell membranes . their activation results in modulation of synaptic plasticity that plays an essential role in learning and memory ( debanne et al . , 2003 ) . in the hippocampus , nmdar are heteromeric assemblies of a core nr1 subunit and modulatory nr2 subunits , nr2a and nr2b ( monyer et al . , 1994 ) . an increase or decrease in the number of ionotropic nmda or ampa glutamate receptors on a postsynaptic cell may lead to changes in ltp or ltd of that cell , respectively ( asztely and gustafsson , 1996 , maren et al . , 1993 , perez - otano and ehlers , 2005 ) . metabotropic glutamate receptors ( mglur ) can also modulate synaptic plasticity by regulating postsynaptic protein synthesis through second messenger systems ( weiler and greenough , 1993 ) . they include mglu1r and mglu5r , which are coupled to polyphospho - inositide hydrolysis and mglu2 - 3r that are coupled to gi proteins ( riedel et al . , 2003 , simonyi et al . , 2005 ) . the effect of prenatal stress on the expression of the components of nmdar , ampar and mglur in rats and mice in summarized in table 3 . in ps , icr mice aged 7 weeks , which showed spatial learning deficits and a reduction in hippocampal ltp , there was a reduction in the protein levels of nr1 and nr2b measured in whole hippocampal homogenates ( son et al . , 2006 ) . gene expression of nr2b was also reduced in 3-week - old male c57bl/6j mice in conjunction with that of kif17 , a kinesin protein that helps synaptic transmission by moving nr2b along dendrites ( zhao et al . , 2013 ) . in 4 - 5 week - old male wistar rats , prenatal stress also decreased the protein levels of nr2b , together with those of ampa glur1 , but not those of nr1 and nr2a ( yaka et al . , 2007 ) . however , nr1 , nr2a or nr2b , measured only in the hippocampal ca1 region , did not differ from controls in 3 and 5-week - old ps , sd rats of both sexes , although they showed a reduction in hippocampal ltp and increase in ltd . in addition , in adult ps , sd rats in which learning and memory was not assessed , no difference was found from controls in the in the expression of hippocampal nr2b , mglur2/3 and mglur 5 ( wang et al . , 2015b ) . in the only study in adult sd males which showed a faster acquisition of the spatial learning task , there was a decrease in glur2 - 3 and mglur5 but no change in the expression of hippocampal mglur1 ( zuena et al . the forgoing data suggest that the levels of nr2b and possibly nr1 , subgroups of the nmdar and glur1 of the ampa receptor are altered in young rats and mice with impaired spatial learning and reduced ltp . failure to replicate this finding in some experiments could be a function of the methodology used to quantify them , animal species , strain , timing or nature of the maternal stressor . more experiments are needed in adults of both sexes in which ltd , learning and memory are measured , together with the expression of different forms of glutamate receptors . alteration in glutamate activity at metabotropic receptors has also been implicated in schizophrenia , depressive and anxiety - related disorders ( bauer et al . , 2002 , konradi and heckers , 2003 ) . accordingly , the effect of prenatal stress was examined on mglur in the prefrontal pfc or fc , hippocampus and amygdala , in association with anxiety or depressive - like behavior . however , the findings in these studies are inconsistent and do not show a clear relation to these behaviors . here prenatal stress that increased anxiety in 3-week old males but not in females , reduced the expression of hippocampal mglur2 - 3 and glur5 ( laloux et al . , 2012 ) , but increased expression of glur5 in the hippocampus and pfc in both sexes . however , in male and female rats aged 4 weeks , with anxiety and depressive - like behavior , prenatal stress increased mglur1 and mglur5 in the fc and hippocampus ( jia et al . , 2015 ) . in addition , in spite of the fact that adult ps males and females both showed increased anxiety - like behavior , mglur2 - 3 and mglur5 increased in the pfc of males but not in females and there was no change in the hippocampus in either sex ( wang et al . , 2015a ) . in the same study , there was no difference from controls in the expression of nr1 , nr2b or mglur1 in these brain regions in ps males or females . by contrast , others found that prenatal stress increased mglur1 expression in the fc only in females , although both sexes showed depressive - like activity ( zhang et al . , 2013 ) . these discordant findings suggest that either alterations in mglur are not a directly associated with the behaviors measured in rodents , or that their inconsistencies stem from differences in the methodology used to assess them . a possible association between behaviors associated with schizophrenia ( but also with depression ) resulting from prenatal stress has only been investigated in mice . male ps mice showing a significant reduction in social interaction had an early and long - lasting reduction in the expression of mglur2 - 3mrna and protein in the fc . this was associated with increased binding of type-1 dna methyl transferase ( dnmt1 ) ( see section below ) to cpg - rich regions of the mglu2 and mglu3 receptor promoters ( matrisciano et al . , their altered expression or methylation resulting from prenatal stress lends support to the hypothesis of glutamatergic / gabaergic dysfunction in schizophrenia . brain derived neurotrophic factor ( bdnf ) is a member of the family of neurotrophins that plays a key role in the development and survival of neurons in the central nervous system . bdnf is required for proper development and survival of dopaminergic , gabaergic , cholinergic , and serotonergic neurons and is crucial for learning and memory processes ( autry and monteggia , 2012 ) . bdnf binds to a specific tyrosine kinase receptor ( tropomyosin - related kinase b receptor ( trkb ) ) and regulates many functions related to neuron development ( bibel and barde , 2000 ) . the expression of bdnf mrna is reduced in the pfc in affective disorders ( ikegame et al . , 2013 , 2003 ) , supporting the suggestion that they have a strong developmental component ( grayson and guidotti , 2013 ) . this prompted recent studies in rodents on the effect of prenatal stress on the levels of bdnf mrna and its possible modifications by epigenetic mechanisms . ps mice or rats with a reduction in social interaction ( dong et al . , 2015 ) , memory ( ratajczak et al . , 2015 ) and ltp ( yeh et al . , 2012 ) , or an increase in measures of anxiety and depression ( jia et al . , 2015 ) , all showed a decrease in bdnf mrna in the fc and hippocampus . prenatal stress reduced the activity of the proteolytic enzyme , tissue plasminogen activator ( tpa ) , thereby increasing pro - bdnf that is converted by the enzyme to bdnf . the expression of bdnf was positively correlated with ltp and negatively correlated with ltd ( yeh et al . , 2012 ) , showing its relation to synaptic transmission . in other experiments in which no behavioral measurements were performed , no change was found in the gene expression of bdnf in the pfc of adult ps , sd male or female rats , but there was a decrease in the pool of bdnf transcripts with long 30 utr ( luoni et al . , 2014 ) . by contrast , ps , sd male rats , that showed improvement in spatial learning , but an increase in anxiety , also had higher levels of bdnf and pro - bdnf in the hippocampus . moreover , ps females that did not differ from controls in their behavior in the mwm showed no difference in hippocampal levels of bdnf ( zuena et al . , 2008 ) . these data provide strong support for a role of bdnf in the mediation of hippocampal ltp / ltd and learning and other changes induced by prenatal stress . epigenetic events in telencephalic gabaergic and glutamatergic systems are believed to play a role in the etiology of schizophrenia ( matrisciano et al . , 2016 ) . the best characterized epigenetic events that affect hippocampal learning and memory are histone acetylation and dna methylation ( levenson and sweatt , 2005 ) . acetyl groups are added by histone acetyltransferases ( hats ) and removed by histone deacetylases ( hdacs ) . lysine-14 acetylation on histone h3 leads to overall transcriptional activation ( crosio et al . , 2003 ) and increases expression of genes necessary for hippocampal synaptic plasticity ( wood et al . , 2006 ) . dna methyl transferases ( dnmt1 and 3a ) and ten - eleven translocation hydroxylase ( tet1 ) are important components of the dna - methylation / demethylation system that regulates the expression of key molecules involved in brain function ( grayson and guidotti , 2013 ) . these enzymes are overexpressed in gabaergic neurons in postmortem brains of patients with schizophrenia and are probably responsible for the downregulation of bdnf , reelin and glutamic acid decarboxylase 67 ( gad67 ) ( grayson and guidotti , 2013 ) . support for a role of gene methylation in the behavioral changes induced by prenatal stress was obtained in mice and in rats of the le strain . gene and protein expression of dnmt1 , 3a and tet1 , detected a day after birth , was increased in the fc and hippocampus of ps male mice showing a reduction in social interaction . the dnmt1 that was co - localized with gad 67 in gabaergic neurons in the fc and hippocampus was associated with a decrease in the levels of reelin and gad 67 . however , prenatal stress did not change the expression of histone tail acetylating or methylating enzymes , or other chromatin remodeling factors ( dong et al . in addition to changes in the maternal milieu induced by stress during gestation , offspring behavior can be modified by the mother - pup relationship ( caldji et al . , 1998 ) . attempts made to assess the relative contribution of pre- and postnatal factors by measuring maternal behavior in stressed rats have yielded conflicting results . some studies showed a decrease in the time spent by stressed rat ( carini and nephew , 2013 , moore and power , 1986 , power and moore , 1986 ) or mouse mothers ( akatsu et al . , 2015 , golub et al . , 2016 ) in one or more measures of maternal care , but others found no difference in maternal behavior of stressed rats ( melniczek and ward , 1994 , poltyrev and weinstock , 1999 ) or mice ( kiryanova et al . , 2016 , meek et al . , 2001 ) from that of controls . an alternative strategy used to assess the influence of postnatal rearing was to foster ps pups onto control mothers at birth and compare the behavior of the pups with those reared by their own mothers . fostering by control mothers abolished the increase in activity of the hpa axis and associated changes in cor receptors in ps rats ( maccari et al . , 1995 ) . it also prevented the increase in anxiety and decrease in social interaction in males ( barros et al . however , being reared by a control mother did not prevent the learning deficits in the mwm , reduction in ltp and ltd ( yang et al . , 2006 ; yeh et al . , 2012 ) or lack of novel object recognition ( paris and frye , 2011 ) . why does fostering prevent anxiety and alterations in the hpa axis but not memory deficits ? as mentioned in section 2 , the former , but not learning deficits in the mwm or object recognition , are mediated by excess maternal levels of cor ( barbazanges et al . , 1996 , salomon et al . , 2011 ) . while the genes for the two cor receptors are found in the rat brain on embryonic days 13 and 16 respectively , their protein expression is relatively low and continues to increase for some time after birth ( weinstock , 2008 ) . maternal stress also increases cor in the mothers ' milk and this remains elevated until the pups are weaned ( pfister and muir , 1989 ) . thus , cor could continue to contribute to the alterations in behavior in the early postnatal period . fostering ps pups onto a control dam avoids this exposure to excess postnatal cor thereby preventing only the alterations in behavior that cor mediates . maternal stress also releases catecholamines from the adrenal gland and sympathetic nervous system that reach the fetal brain ( rohde et al . , 1983 ) . noradrenaline ( na ) appears to play a role in neurodevelopment and has a strong effect on learning and memory by modifying hippocampal and neocortical functions ( berridge and waterhouse , 2003 ) through activation of -adrenergic receptors ( bramham et al . , 1997 ) . alterations in noradrenergic activity and its receptors during development cause morphological changes in the brain ( felten et al . , 1982 ) . maternal malnutrition , like gestational stress , can induce learning deficits in the offspring and impair hippocampal ltp ( austin et al . , 1986 ) , in association with down regulation of -adrenergic receptors ( flores et al . , 2011 ) . there appear to be no data showing that prenatal stress also reduces brain noradrenergic receptors . however , in hippocampal slices of ps males , ltp induced by a receptor agonist was lower than in controls in the dorsal hippocampus ( associated with spatial learning and memory ) , and higher in the ventral hippocampus , ( associated with emotional behavior ) ( grigoryan and segal , 2013 ) , that accords with the behavioral changes usually observed . this suggests that prenatal stress may selectively change the number and/or sensitivity of -receptors in a brain region - selective manner . in contrast to the effect of maternal malnutrition and possibly prenatal stress , administration of a -receptor antagonist , propranolol to pregnant rats caused up - regulation of -receptors and an increase in na activity in the brain of the offspring in later life ( erdtsieck - ernste et al . , 1993 ) . treatment of stressed rats during gestation by propranolol prevented the development of spatial memory deficits in their male offspring ( females were not tested ) ( mcgivern et al . , 1986 ) , presumably by blocking the effect of excess noradrenergic activation in the developing brain . the preclinical data described in this review support the hypothesis that alterations in early brain development induced by maternal stress are risk factors for psychopathology . this is shown in a large number of experiments in pregnant rats and mice that were subjected to different stressors at the time when the fetal limbic system develops . in their offspring , increases were found in those behaviors that are associated with anxiety , depression and schizophrenia in human subjects . prenatal stress , like schizophrenia and depression , decreased the expression of proteins like bdnf and reelin that mediate neural plasticity , and the expression of nr2b subtype of nmdar in the hippocampus . it is necessary to perform further experiments to ascertain the direction of change of mglur and their various subtypes in the hippocampus and cortex of ps offspring of both sexes showing changes consistent with those found in depression or schizophrenia . evidence from recent studies in mice supports a role of gene methylation in the down - regulation of bdnf and other components of the glutamatergic and gabaergic systems . it is less clear whether the changes in various behaviors , expression of genes and proteins differ in males and females because of the paucity of experiments in rodents , which included both sexes .

the current review focuses on studies in rodents published since 2008 and explores possible reasons for any differences they report in the effects of gestational stress on various types of behavior in the offspring . an abundance of experimental data shows that different maternal stressors in rodents can replicate some of the abnormalities in offspring behavior observed in humans . these include , anxiety , in juvenile and adult rats and mice , assessed in the elevated plus maze and open field tests and depression , detected in the forced swim and sucrose - preference tests . deficits were reported in social interaction that is suggestive of pathology associated with schizophrenia , and in spatial learning and memory in adult rats in the morris water maze test , but in most studies only males were tested . there were too few studies on the novel object recognition test at different inter - trial intervals to enable a conclusion about the effect of prenatal stress and whether any deficits are more prevalent in males . among hippocampal glutamate receptors , nr2b was the only subtype consistently reduced in association with learning deficits . however , like in humans with schizophrenia and depression , prenatal stress lowered hippocampal levels of bdnf , which were closely correlated with decreases in hippocampal long - term potentiation . in mice , down - regulation of bdnf appeared to occur through the action of gene - methylating enzymes that are already increased above controls in prenatally - stressed neonates . in conclusion , the data obtained so far from experiments in rodents lend support to a physiological basis for the neurodevelopmental hypothesis of schizophrenia and depression .

the cell nucleus contains several different structures or functional domains , among which the nucleolus , the interchromatin granule clusters ( igc ) , cajal bodies and other nuclear bodies , whose functions range from the storage of specific factors to the shuttling of proteins among the various domains . they can be found in several locations in the cell nucleus ; in many cases , the functions of these bodies are still to be completely elucidated . for some of these structures , such as promyelocytic leukaemia ( pml ) bodies or cajal bodies , , we are studying the presence of several proteins as possible markers of nuclear structures ( manuscript in preparation ) ; in particular , we are interested in possible storage structures for nucleases . in fact , as for rnas , data in the literature suggest that more than 90% of the newly synthesized rna is degraded in the nucleus and this destruction is carried out by means of several mechanisms apt to recognize damaged or nonsense rna . the reason of such nonproductive rna synthesis is not clear : one hypothesis claims that this high hnrna transcription rate would maintain chromatin domains open to prevent the formation of inactive chromatin states . there is no hint , so far , as to where rna degradation could take place in the nucleus . it is known that the perichromatin area is the site where transcription , co - transcriptional splicing and cleavage mechanism are located ; in this view , it is possible that rna degradation could be cotranscriptional or immediately subsequent to rna synthesis . it is also well known that in mammals the most actively synthesised type of rna is ribosomal rna ( rrna ) . therefore , we focused our attention on the possible localization of nucleases inside the nucleolus ; to this purpose , we have studied by means of ultrastructural immunocytochemistry the nucleolar localization of rnase a in control and aging hela cells . since nuclear degradation of rna can be accomplished - at least in part - by rnase a , we have used an anti - rnase a antibody to detect this enzyme in the cell nucleus , with the aim of finding whether any storage or shuttling system do exist . hela cells were grown in dmem medium supplemented with 10% fetal bovine serum , 2 mm glutamine and 100 units each of streptomycin and penicillin , in 25 cm plastic flasks or on 4 cm glass coverslips in multi - well dishes on a air atmosphere containing 5% of co2 ( all reagents and plastic were from euroclone , milan , italy ) . the hela - ibch cells represent a subline isolated at the institute of bioorganic chemistry , and are aged cells obtained after six month of culture under the same conditions . these cells were free from any detectable contaminations and were characterized by an increased number of apoptotic cells , which were not considered in the present investigation . the cells were harvested by trypsinization ( 0.25% trypsin in pbs containing 0.5% edta ) and immediately fixed with 4% paraformaldehyde ( 2 h at 4c ) in the culture medium . the cell pellets were embedded in 2% agar in water , thoroughly rinsed with srensen buffer ( ph 7.2 ) and dehydrated in ethanol . finally the cells were embedded in lr white resin and polymerized at 60c for 24 h. thin sections were collected on nickel grids covered with a formvar - carbon film and incubated on normal goat serum ( ngs ) for 3 min . for immunolabeling , the grids were floated for 17h at 4c onto a solution of 0.1% bovine serum albumine and 0.05% tween 20 in pbs containing the primary anti - rnase a antibody diluted 1:200 . after rinsing with pbs - tween and pbs , grids were incubated with ngs as above and then reacted with a goat - anti rabbit secondary antibody conjugated with 12 nm or 6 nm colloidal gold ( the jackson lab . , bar harbor , me , usa ) for 30 min at room temperature ( rt ) , rinsed and finally stained . to reduce chromatin contrast and selectively reveal nuclear ribonucleoprotein constituents , the sections were bleached by the edta method or selectively stained for rna with terbium citrate . the gold grains in some of the micrographs have been digitally contrasted with photoshop software . hela cells were grown on coverslips , fixed for 7 min with 1% paraformaldehyde in pbs , rinsed in pbs and left 30 min in 70% ethanol at 20c . the coverslips were rehydrated in pbs and then incubated with the anti - rnase a antibody , diluted 1:150 in pbs for 1h at rt , rinsed in pbs and incubated with an alexa 488-conjugated goat - anti rabbit antibody , diluted 1:200 for 1 h at rt , rinsed , counterstained with hoechst 33342 for 5 min , and finally mounted with mowiol . the cells were observed with an olympus bx51 microscope equipped with a 100 w mercury lamp under the following conditions : 330- to 385-nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) , for hoechst 33258 ; 450- to 480- nm excf , 500-nm dm , and 515 nm bf for alexa 488 . images were recorded with an olympus camedia c-5050 digital camera and stored on a pc by the olympus software for processing and printing . hela cells were grown in dmem medium supplemented with 10% fetal bovine serum , 2 mm glutamine and 100 units each of streptomycin and penicillin , in 25 cm plastic flasks or on 4 cm glass coverslips in multi - well dishes on a air atmosphere containing 5% of co2 ( all reagents and plastic were from euroclone , milan , italy ) . the hela - ibch cells represent a subline isolated at the institute of bioorganic chemistry , and are aged cells obtained after six month of culture under the same conditions . these cells were free from any detectable contaminations and were characterized by an increased number of apoptotic cells , which were not considered in the present investigation . the cells were harvested by trypsinization ( 0.25% trypsin in pbs containing 0.5% edta ) and immediately fixed with 4% paraformaldehyde ( 2 h at 4c ) in the culture medium . the cell pellets were embedded in 2% agar in water , thoroughly rinsed with srensen buffer ( ph 7.2 ) and dehydrated in ethanol . finally the cells were embedded in lr white resin and polymerized at 60c for 24 h. thin sections were collected on nickel grids covered with a formvar - carbon film and incubated on normal goat serum ( ngs ) for 3 min . for immunolabeling , the grids were floated for 17h at 4c onto a solution of 0.1% bovine serum albumine and 0.05% tween 20 in pbs containing the primary anti - rnase a antibody diluted 1:200 . after rinsing with pbs - tween and pbs , grids were incubated with ngs as above and then reacted with a goat - anti rabbit secondary antibody conjugated with 12 nm or 6 nm colloidal gold ( the jackson lab . , bar harbor , me , usa ) for 30 min at room temperature ( rt ) , rinsed and finally stained . to reduce chromatin contrast and selectively reveal nuclear ribonucleoprotein constituents , the sections were bleached by the edta method or selectively stained for rna with terbium citrate . the gold grains in some of the micrographs have been digitally contrasted with photoshop software . hela cells were grown on coverslips , fixed for 7 min with 1% paraformaldehyde in pbs , rinsed in pbs and left 30 min in 70% ethanol at 20c . the coverslips were rehydrated in pbs and then incubated with the anti - rnase a antibody , diluted 1:150 in pbs for 1h at rt , rinsed in pbs and incubated with an alexa 488-conjugated goat - anti rabbit antibody , diluted 1:200 for 1 h at rt , rinsed , counterstained with hoechst 33342 for 5 min , and finally mounted with mowiol . the cells were observed with an olympus bx51 microscope equipped with a 100 w mercury lamp under the following conditions : 330- to 385-nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) , for hoechst 33258 ; 450- to 480- nm excf , 500-nm dm , and 515 nm bf for alexa 488 . images were recorded with an olympus camedia c-5050 digital camera and stored on a pc by the olympus software for processing and printing . the anti - rnase a antibody used in this study recognizes the enzyme in several mammalian species , including humans . normally , a great part of the labelling is present in the cytoplasm ( not shown ) as well as in specific subnuclear locations such as the perichromatin area ( manuscript in preparation ) . intriguingly , in aging hela cells the antibody often detects the enzyme also in discrete parts of the nucleolus , and the labelling always covers roughly roundish areas about 0.20.4 m in diameter ( figure 1a ) which are more electrondense than the nucleolar body . this area is finely fibrillar and homogeneous and is morphologically different from any of the typical nucleolar components , the dense fibrillar component , the fibrilar centre or the granular component . after terbium staining to specifically reveal rna , an area similar to that labelled in figure 1a can be seen as containing rna fibrils ( figure 1b and inset ) . the terbium staining confirmed that this rna - containing structure does not seem to co - locate with any of the nucleolar components . after immunofluorescence for rnase a detection and dna counterstaining , the nuclear labelling was mainly found in the nucleolus , where rnase a distribution proved to be inhomogeneous with some more densely labelled areas ( arrow in figure 1c ) . the foci described above are clearly revealed by anti - rnase a immunolabelling and terbium staining ( figure 1d , f ) . in some cases , labelling for the nucleolar protein s3 , present in the small ribosomal subunit , can be seen in the nucleolar body at the periphery of one of the terbium - positive nucleolar foci ( figure 1e ) . these features were never found in not aging control hela cells ( not shown ) . the labelling for rnaase a is present exclusively over a dense area near the periphery of the nucleolus . c ) after immunofluorescence labeling for rnase a , the nucleolus is clearly not homogeneously labelled . d ) terbium staining and labelling for rnase demonstrate the colocalization of these components in the dense structure . e ) the dense body is labelled only at the periphery by the anti - s3 antibody . f ) terbium staining and labelling for rnase a. a dense body ( arrow ) in the close vicinity of a fibrillar centre ( fc ) . the labelling for rnaase a is present exclusively over a dense area near the periphery of the nucleolus . c ) after immunofluorescence labeling for rnase a , the nucleolus is clearly not homogeneously labelled . d ) terbium staining and labelling for rnase demonstrate the colocalization of these components in the dense structure . e ) the dense body is labelled only at the periphery by the anti - s3 antibody . f ) terbium staining and labelling for rnase a. a dense body ( arrow ) in the close vicinity of a fibrillar centre ( fc ) . the nucleolus is the site of pre - ribosome biogenesis and is also involved in other , noncanonical , functions . the main activity , however , is the synthesis of rrnas and its association with ribosomal proteins to form the ribosomal subunits . transcriptional activity is very active in hela cells , and it is known to decrease with aging in culture . interestingly , in the nucleolar body of aging hela cells we have found discrete , focal regions , which are enriched in rnase a. since the antibody used recognizes both the active and the inactive form of the enzyme , it is impossible to know whether the foci represent simple storage sites or areas where rna degradation takes place . however , since these bodies always contain detectable amounts of rna , as seen with terbium staining , rnase a is very probably inactive . the presence of these structures is not constant , and their morphology is clearly recognizable from the other nucleolar components . to our knowledge when other nuclear bodies like cajal bodies were found embedded into the nucleolus , their morphology was still recognizable . moreover , these foci are different also from the nucleolus - associated domains described by malatesta et al . in tissues from hibernating mammals . an interesting hypothesis would be that rna degradation takes place near the site of transcription and especially increases whenever the rate of error is particularly high ( as it likely occurs in ageing ) or there is a need for rapidly blocking the transcript from reaching the cytoplasm : under these circumstances , a particular structure accumulating rnase appears inside the nucleolus . it will be interesting to investigate whether this rnase - containing bodies or foci do also occur in other cell models in vitro after treatments inducing massive impairment of transcription or translation , or in vivo in aging animals .

we have studied by means of ultrastructural immunocytochemistry the localization of rnase a in nuclei of hela cells in control conditions and following cell ageing in culture . we have found that roundish , electron dense foci , which contain a significant amount of rnase a , can be detected within nucleoli of aged cells . these bodies also contain rna and lack ribosomal s3 proteins , and may represent either simple storage sites or areas where rna degradation takes place .

pigment epithelium - derived factor ( pedf ) is a 50 kda endogenous glycoprotein that was first discovered in 1991 as a factor secreted by the pigment epithelium of the human foetal eye . pedf was shown to promote differentiation of retinoblastoma cells and was implicated in a range of eye pathologies including diabetic retinopathy , macular degeneration , and retinitis pigmentosa and glaucoma . pedf is a potent antiangiogenic agent , more potent than angiostatin , endostatin and thrombospondin-1 by endothelial cell migration assay . pedf has also been shown to be an antitumorigenic agent for malignancies including osteosarcoma , melanoma , glioma , lung , breast , prostatic , ovarian and pancreatic carcinomas . the interactions between these epitopes and receptors are likely to initiate divergent signalling pathways for the different cellular effects of pedf . first characterised 34-mer ( residues 2457 ) and 44-mer ( residues 58101 ) pedf - derived peptides that conferred antiangiogenic and neurotrophic activity , respectively . additionally , another sequence , ert ( residues 7994 ) , showed both antiangiogenic and differentiation activity . the properties of these peptides were demonstrated in vitro by endothelial cell apoptosis and chemotaxis assays and y-79 retinoblastoma differentiation assay . using a subcutaneous tumour model and pc-3 prostate cancer cells , expression of the 34 mer peptide reduced tumour microvascular density and induced tumour cell apoptosis , effects not demonstrated for the 44-mer peptide . the 34 mer peptide restricted angiogenesis through a c - jun - nh2 kinase ( jnk- ) dependent pathway leading to nfatc2 deactivation and c - flip antagonism . in another study , four different pedf - derived peptides , termed stvorth-1 , stvorth-2 , stvorth-3 , and stvorth-4 , consisting of pedf residues 4064 , 78102 , 90114 , and 387411 , respectively , were tested in vitro and in vivo . in vitro , stvorth-2 was the most potent inhibitor of saos-2 osteosarcoma cell proliferation , while stvorth-3 dramatically promoted saos-2 adhesion to collagen i. stvorth-4 inhibited saos-2 cell invasion through matrigel . stvorth-2 and stvorth-3 were then evaluated in vivo using an orthotopic murine model of osteosarcoma . notably , stvorth-2 ( residues 78102 ) and stvorth-3 ( residues 90114 ) possessed sequences that overlapped with the 44-mer ( residues 58101 ) and ert ( residues 7994 ) sequences described by filleur et al . ( figure 1 ) . both stvorth-2 and stvorth-3 restricted osteosarcoma tumour growth and inhibited the development of pulmonary metastases when saos-2 cells were treated prior to intratibial injection . provide some insight into how pedf structure relates to its multidimensional ability to restrict tumour progression . however , the study design and methods of peptide delivery used in these models make it difficult to extrapolate the findings for human use . filleur et al . used a subcutaneous tumour model with transfected pc-3 cells to demonstrate the differential effects of the 34-mer and 44-mer pedf - derived peptides . gene therapies have yet to be proven safe for human application , making it unlikely that they will be used for osteosarcoma therapy in the near future . ek et al . used the saos-2 osteosarcoma cell line to achieve a spontaneously metastasizing murine model of orthotopic osteosarcoma . saos-2 cells were treated with stvorth-2 and stvorth-3 peptides prior to intraosseous injection , thus facilitating early uptake of peptides and change in phenotype . in order to evaluate the true efficacy of pedf - derived peptides against established osteosarcoma , treatment should be delayed until after the establishment of primary tumours . the saos-2 human osteosarcoma cell line ( american tissue culture collection , manassas , va , usa ) was cultured in complete medium , cm , at 37c and in humidified 5% co2 . cm consisted of mem - alpha+glutamax ( invitrogen , carlsbad , ca , usa ) supplemented with 10% foetal bovine serum ( invitrogen , carlsbad , ca , usa ) and 1% antibiotic - antimycotic ( invitrogen , carlsbad , ca , usa ) . exponentially growing cells , with passage number always less than 20 , were used for the studies . 5-week - old balb / c nude mice were purchased from the animal resource centre , perth , australia and were housed at the st vincent 's hospital bioresources centre under pc2 pathogen - free conditions . animal ethics approval was obtained from the st vincent 's hospital melbourne animal ethics committee . a 50% concentration of matrigel was used to dilute saos-2 osteosarcoma cells to a concentration of 2 10 cells / ml . following anaesthesia with intraperitoneal ketamine ( 100 mg / kg ) and xylazine ( 10 mg / kg ) , a 27-gauge needle was introduced into the left tibia of each mouse using a gentle drilling motion in order to avoid iatrogenic fracture , and a volume of 10 l of saos-2/matrigel solution was injected . tumour growth and animal weights were monitored twice weekly until the endpoint of the study . anteroposterior ( ap ) and lateral ( l ) dimensions of limbs were recorded using digital callipers . volumes were calculated from these dimensions using the formula 4/3[1/4(ap + l ) ] . the contralateral nontumour - bearing limb was used as a control to calculate actual tumour volume . primary orthotopic tumours were apparent at day 20 after saos-2 inoculation , when average tumour volume was 22.5 mm , at which point mice were randomised into treatment groups each consisting of four mice . as outlined below , these groups received either : ( 1 ) sterile water as control , ( 2 ) stvorth-2 at 50 g / kg / day , ( 3 ) stvorth-2 at 500 g / kg / day , ( 4 ) stvorth-3 at 50 g / kg / day , or ( 5 ) stvorth-3 at 500 g / kg / day . pedf - derived peptides , stvorth-2 ( residues 78102 ) and stvorth-3 ( 90114 ) , were designed and sourced previously according to the procedure outlined by ek et al . . this paper refers to the full - length human pedf sequence , and amino acid numbering is based on those sequences listed in genbank ( national institutes of health ) . high - performance liquid chromatography ( hplc ) and mass spectrometry was used to confirm the purity of these peptides . ek et al . showed that stvorth-2 inhibited saos-2 osteosarcoma cell proliferation , while stvorth-3 inhibited saos-2 osteosarcoma cell adhesion to collagen i in vitro . stvorth-2 and stvorth-3 sequences overlap with the 44-mer ( residues 58101 ) , and ert ( residues 7994 ) sequences described by filleur et al . ( figure 1 ) . sustained delivery of stvorth-2 , stvorth-3 , or sterile water ( negative control , placebo ) pumps were aseptically filled with the different treatments and surgically implanted within the peritoneal cavity of animals for systemic delivery . the mean pumping rate for the alzet microosmotic pump ( model 1002 ) is 0.25 l / hr over 14 days , as determined by the manufacturer . stvorth-2 and stvorth-3 were administered at 50 g / kg and 500 g / kg daily doses . the human physiological serum concentration of pedf ranges between 4 ng / ml ( 80 pm ) to 15 g / ml ( 300 nm ) [ 1015 ] . in one study , inhibition of vessel formation in ischemia - induced retinopathy was achieved at a 50 nm concentration . the 50 g / kg and 500 g / kg doses used in the present study are equivalent to 1 g / ml ( 20 nm ) and 10 g / ml ( 200 nm ) concentrations of pedf , respectively , which lies within the range mentioned above . this assumes an average mouse weight of 20 grams and an average blood volume of 1 ml . it is known that the pumps are capable of delivering a steady state quantity of peptides into the abdominal cavity [ 18 , 19 ] , which will eventually be taken into the microvasculature supplying the abdominal region . tumours had grown to a disabling size for control animals at day 34 after saos-2 inoculation as expected . this was the humane endpoint of the study , and all animals were euthanized under anaesthesia by cervical dislocation at this time . following this , tumour - bearing limbs , lungs , hearts , small intestines , and skin blood samples were obtained after cervical dislocation and dissection through the thoracic cage and were immediately treated with anticoagulants . affected limbs were x rayed at 35 kv for 30 s using a cabinet system ( faxitron corp . , blood collected after euthanasia was analysed for renal and hepatic biochemical parameters ( serum creatinine , alanine transaminase ( alt ) , and aspartate transferase ( ast ) ) using a sysmex xe2100 instrument . tissues were embedded in paraffin prior to histological preparation and analysis . for preparation of paraffin sections , tumours were sectioned to provide an en face surface for the quantification of per cent tumour necrosis and apoptosis . primary tumours , lungs , heart , small intestine , and skin sections were dehydrated through an ethanol series followed by xylene , then stained with haematoxylin and eosin . a terminal dutp nick end labelling ( tunel ) assay kit ( promega , madison , wi , usa ) was used to detect apoptotic cells in primary tumours . pertex mounting agent was used to seal coverslips to slide sections , and all tissues were observed using a nikon eclipse te2000-u microscope ( nikon , lidcombe , nsw , australia ) and photographed with spot advanced software ( scitech , aurora , il , usa ) . the effect of intraperitoneal stvorth-2 and stvorth-3 on the development of pulmonary metastatic disease was examined histologically . lungs were sectioned to achieve the greatest cross - sectional area and stained with haematoxylin and eosin . ten metastatic lesions were then randomly selected from each treatment group for measurement of cross - sectional areas . one - way and two - way anova analyses with bonferroni multiple comparisons test were used where appropriate ( graphpad prism 5 for mac os x ) . imagej ( version 1.45j , national institutes of health , usa ) was used for all image analysis . mice were studied for potential systemic side affects associated with delivery of stvorth-2 ( residues 78102 ) and stvorth-3 ( residues 90114 ) ( see figure 1 ) . mice were regularly monitored during the study for signs of distress and animal weights were recorded twice weekly . all mice remained well for the duration of the study , and no significant weight loss compared to control was observed ( two - way anova ) ( figure 2(a ) ) . serum , hearts , lungs , skin , and small intestines were collected postmortem for histological examination . no features of cytotoxicity were evident in these tissues , in either control or peptide - treated groups . renal and hepatic serum biochemistry parameters were within physiological limits for all treatment groups ( figure 2(b ) ) . microosmotic pumps , continuously delivering stvorth-2 and stvorth-3 at daily doses of 50 g / kg and 500 g / kg , were implanted in the peritoneal cavities of mice at day 20 of the study . tumours were well established at this time with an average tumour volume of 22.5 mm ( 1.7 sem ) . sustained delivery of stvorth-2 at both doses caused a significant reduction in primary tumour volume at the study endpoint . 50 g / kg and 500 g / kg stvorth-2 caused 49.7% and 50.9% reductions in tumour volume , respectively , at day 34 ( p < 0.01 , two - way anova with bonferroni multiple comparisons test ) ( figures 3(a ) and 3(b ) ) . a significant effect was not seen before the day 34 time point , and there was no statistical difference between the two doses of stvorth-2 . by comparison , the therapeutic effect of stvorth-2 on primary tumour volume was unable to be replicated by systemic stvorth-3 therapy . despite achieving 30.1% and 40.0% reductions in primary tumour volume at day 34 with 50 g / kg and 500 g / kg stvorth-3 , respectively , these results did not achieve statistical significance ( two - way anova ) ( figures 3(a ) and 3(b ) ) . plain radiographs obtained after disarticulation of tumour - bearing limbs showed extensive soft tissue invasion and osteolysis for all animals ( figure 4 ) . treatment with either stvorth-2 or stvorth-3 , at both 50 g / kg and 500 g / kg doses , did not affect orthotopic tumour invasion of the surrounding structures . all tumour - bearing animals showed invasion of skeletal muscles and osteolysis on both sides of the adjacent joint . treatment with stvorth-2 or stvorth-3 did not affect the degree of tumour necrosis . per cent tumour necrosis was statistically equivalent for both stvorth-2 and stvorth-3 at either dose ( one - way anova ) . similarly , treatment with stvorth-2 or stvorth-3 did not appear to affect tumour cell apoptosis . the percentage of tunel - positive staining tumour was found to be statistically equivalent across treatment groups ( one - way anova ) ( table 1 ) . this saos-2 orthotopic model of osteosarcoma gives rise to spontaneous pulmonary metastases , and the burden of metastatic disease was assessed histologically post - mortem . the numbers of micrometastases per lung section were enumerated , and treatment with stvorth-2 or stvorth-3 , at both 50 g / kg and 500 g / kg doses , had no effect on the number of lesions observed ( one - way anova ) ( figures 5(a ) and 5(b ) ) . treatment with stvorth-3 did , however , cause a significant reduction in the size of micrometastatic lesions . the mean cross - sectional area of a micrometastasis was 0.19 mm and 0.29 mm for 50 g / kg and 500 g / kg stvorth-3 . this represented a 79% and 68.2% reduction in area compared to animals that received sterile water as control ( p < 0.05 , one - way anova analysis with bonferroni multiple comparisons test ) . treatment with 50 g / kg and 500 g / kg stvorth-2 resulted in mean cross - sectional areas of 0.68 mm and 1.02 mm respectively . ek et al . evaluated four pedf - derived peptides based on the known functional epitopes of the pedf glycoprotein . stvorth-2 ( residues 78102 ) predominantly restricted saos-2 osteosarcoma cell proliferation and stvorth-3 ( residues 90114 ) increased adhesion to collagen i. both peptides restricted growth of primary osteosarcoma and the development of pulmonary metastases in vivo . in this study we aimed to assess systemically delivered stvorth-2 and stvorth-3 as treatments for established osteosarcoma ( both primary and secondary disease ) and to evaluate the therapeutic safety of these peptides . we utilised the same orthotopic murine model of osteosarcoma described by ek et al . and delivered the stvorth-2 and stvorth-3 peptides systemically via an intraperitoneal microosmotic pump . treatment was delayed until after the macroscopic appearance of primary osteosarcoma . using this optimised model , differential effects on tumour growth and metastasis we observed 49.7% and 50.9% reductions in tumour volume with 50 g / kg and 500 g / kg stvorth-2 treatments , respectively . ek et al . showed a reduction of more then 30% when these peptides were administered prior to saos-2 inoculation . ek et al . showed that stvorth-2 had a significant antiproliferative effect on saos-2 cells in vitro , while stvorth-3 did not exhibit this effect . the molecular mechanisms exploited by stvorth-2 to achieve its antitumour effect are yet to be characterised . treatment with stvorth-2 and stvorth-3 did not appear to affect local tumour invasion , tumour necrosis , or apoptosis . tumour cells were observed invading the soft tissues and destroying the local bony architecture in all animals , independent of treatment type . although groups receiving stvorth-2 and stvorth-3 were unable to be statistically differentiated based on tumour cell necrosis and apoptosis , it was evident that the percentages of tumour necrosis and apoptosis were proportional to tumour volume . this saos-2 orthotopic model of osteosarcoma gives rise to large and rapidly growing tumours . by delaying treatment until tumours were palpable , it is possible that tumour energy requirements exceeded capacity of the vascular supply , leading to spontaneous necrosis and apoptosis of the tumour core . such areas of necrosis and potential apoptosis are commonly observed in clinical tumours at our centre ( personal observation ) . ek et al . showed that both stvorth-2 and stvorth-3 dramatically restricted spontaneous pulmonary metastases , despite exhibiting differential effects on cell proliferation and collagen i invasion in vitro . is mind , the role of these peptides in the metastatic process was again investigated . pulmonary micrometastases were quantified under low - power microscopy , rather than the postmortem macroscopic method utilised by ek et al . . cross - sectional areas of metastatic lesions were quantified to estimate the burden of pulmonary disease . stvorth-3 showed a dramatic 79% reduction in pulmonary burden of disease , an effect consistent with the observation that stvorth-3 predominantly restricts adhesion to collagen i . stvorth-3 may critically inhibit the metastatic cascade via this mechanism ; however , one can not rule out a direct effect on pulmonary lesions via systemic delivery . the method used here for evaluating metastatic lesions is an improvement upon that used by ek et al . . the final aim of this study was to perform a preliminary evaluation of therapeutic safety . ek et al . showed that both stvorth-2 and stvorth-3 possess a moderate ability to inhibit vegf expression by saos-2 cells . for antiangiogenic agents to be of clinical use , it is critical that these effects are not achieved at the expense of homeostatic processes such as wound healing and inflammation . serum analysis and histology of harvested viscera did not show any evidence of treatment - related toxicity . all animals appeared to be healthy and exhibited normal grazing and behaviour for the duration of the study . we also did not note any gross differences in surgical wound healing between the treatment groups . we also clarify that both stvorth-2 and stvorth-3 were nonimmunogenic , despite their length ( 25-mer ) . there are two direct items that need to be highlighted : ( i ) both peptides were effective in vivo when administered systemically ( despite any potential immunogenicity ) , and ( ii ) we did not notice any inflammation- or immunostimulation - mediated exudates around the region of the pump outlet . if the peptides were immunogenic , this would be clearly visible in these mice , as we have noticed this before with other anticancer agents ( dass , unpublished data ) . stvorth-2 has been shown to be most effective at inhibiting primary tumour growth while stvorth-3 predominantly restricts progression of pulmonary metastases . the molecular mechanisms utilised by these peptides are yet to be fully studied and elucidated . mechanistic studies have focused on the parent pedf glycoprotein that interacts with regulatory pathways that induce apoptosis of both endothelial and tumour cells . in vitro and in vivo studies have implicated the fas - fasl death receptor pathway [ 2527 ] , bcl-2 family proteins , caspases , and c - flip . the current study did not look at molecular markers in vivo , apart from the blood analyses for creatinine , alanine transaminase and aspartate transferase . in vitro though , we have previously examined several biological and molecular features of pedf - based peptide treatment of saos-2 cells . in that earlier study , stvorth-2 was the most potent inhibitor of saos-2 osteosarcoma cell proliferation , while stvorth-3 dramatically promoted saos-2 adhesion to collagen i. stvorth-2 and -3 induced osteoblastic differentiation , while stvorth-3 and stvorth-4 reduced vegf expression in saos-2 cells . the current set of in vivo results , from a focussed pharmacodynamic study , reflect quite closely what we expected from the earlier in vitro study . peptides have a number of advantages as targeted therapies , being neither genotoxic nor genotype specific . peptides have excellent tissue penetration and bind with high affinity and specificity to therapeutic targets [ 3133 ] . in this study we demonstrated pronounced differences in action between the two peptides and this begs the question as to precisely which are the aminoacid sequences essential for these effects . stvorth-2 consisted of 78lspls vatal salsl gaeqr tesii102 , and stvorth-3 consisted of 90lslga eqrte siihr alyyd lissp114 . the overlapping 90lsl gaeqr tesii102 sequence may in fact be nonessential for the described effects . further studies utilising the shortened , nonoverlapping sequences of stvorth-2 and stvorth-3 are the next logical step . beyond this , development of a hybrid version of stvorth-2 and stvorth-3 peptides may then be possible , leading potentially to a compound that possesses antitumorigenic and antimetastatic properties superior to that of the parent pedf glycoprotein and hopefully of the individual peptides used in isolation . multiple functional epitopes for pedf have been identified , as well as several distinct pedf receptors [ 34 , 35 ] . first characterised 34-mer ( residues 2457 ) and 44-mer ( residues 58101 ) pedf - derived peptides that conferred antiangiogenic and neurotrophic activity , respectively . additionally , another sequence , ert ( residues 7994 ) , showed both antiangiogenic and differentiation activity . derived three different shorter peptides from the 34-mer pedf - based peptide and demonstrated that a 18-mer peptide was in fact able to inhibit angiogenesis and the growth of prostate cancer in mice . amaral and becerra showed that the 34 mer peptide suppresses choroidal neovascularization following subconjunctival administration . the interactions between these various identified epitopes and receptors are likely to initiate divergent signalling pathways for the different cellular effects of pedf . we did not identify which pedf receptor the peptides were interacting with in this study . there is also the possibility that the peptides may be interacting with other cell surface ligands given that the quaternary structure of the peptides is expected to be very different from the full length pedf protein . this study provides a number of insights into the mechanisms of the antiosteosarcoma activity of pedf and supports the use of pedf - derived peptides as therapeutic agents . stvorth-2 ( residues 78102 ) primarily restricts growth of primary osteosarcoma , while stvorth-3 ( residues 90114 ) restricts pulmonary metastatic disease . these findings are particularly significant given that the orthotopic model of osteosarcoma gives rise to large and rapidly growing tumours , and that these peptides were administered at a stage of advanced disease .

the potent antiangiogenic pigment epithelium - derived factor ( pedf ) has shown promise against osteosarcoma , a tumour that originates in the bone and metastasises to the lungs . neurotrophic , antiangiogenic , antiproliferative , and antimetastatic properties of pedf have been attributed to a number of functional epitopes on the pedf glycoprotein . stvorth-2 ( residues 78102 ) and stvorth-3 ( residues 90114 ) are two pedf - derived peptides based on these functional epitopes . stvorth-2 has previously been shown to inhibit osteosarcoma cell proliferation , while stvorth-3 increased osteosarcoma cell adhesion to collagen i in vitro . in this paper , we have evaluated systemically and continuously delivered stvorth-2 and stvorth-3 using a clinically relevant murine model of osteosarcoma with spontaneous metastasis . treatment with stvorth-2 or stvorth-3 with microosmotic pumps was initiated after primary osteosarcoma was established in the tibia . while treatment with stvorth-2 and stvorth-3 did not appear to affect local tumour invasion , tumour necrosis or apoptosis , stvorth-2 predominantly restricted the growth of primary tumours , while stvorth-3 restricted the burden of pulmonary metastatic disease . no peptide caused gross toxicity in mouse tissues as assessed by measuring weight of animals , serum biochemistry , and gross tissue observation . the differential effects exhibited by stvorth-2 and stvorth-3 in this orthotopic model of osteosarcoma may be related to the functional epitopes on the pedf glycoprotein that they represent .

a 51-year - old woman was hospitalized due to posterior neck pain and left arm weakness after a traffic accident while riding a bicycle . on admission , there was a four - fifths decrease in motor function of the left arm . plain radiographs of the cervicothoracic spine revealed compression fractures of c7-t3 with anterior subluxation of facets at c6 - 7 . ct and magnetic resonance imaging ( mri ) showed anterior wedging of the c7 vertebral body , ligamentous disruption at the c6 - 7 level and acute compression fractures at t1 - 3 ( fig . vertebral compression fractures have been treated mainly with bed rest , the use of analgesics , braces and various physical therapies . however , unlike lumbar compression fracture patients , the patient was uncomfortable in the supine position and even lying down in bed . the patient had to keep her posture in the sitting or a semi - fowler position all day , and the patient had slight difficulty in deep breathing . the patient underwent ct - guided vertebroplasty at t1 - 3 and an anterior discectomy and instrumented interbody fusion at c6 - 7 . the procedure was performed in strictly sterile manner in the ct ( somatom sensation open scanner , siemens medical solutions , erlangen , germany ) room . after the patient was placed on the ct table , blood pressure and pulse oximetry were monitored continuously . the area to be treated was prepared in a strictly sterile manner and then ct topography for the upper thoracic spine , including the symptomatic lesion , was checked . on this topographic image , axial ct scanning was performed for the t3 vertebral body and a sectional image for needle insertion was selected . the image mode was converted to ct fluoroscopy , and the image size was magnified for precise monitoring . with the administration of local anesthesia with 1% lidocaine , a 10-gauge vertebroplasty cannula was inserted to the affected vertebral body via the unilateral transpedicular route . the tip of the cannula was placed in the anterior half of the vertebral body ( fig . a total of 3 cc pmma was applied to the t3 vertebral body . during the procedure , needle advancement , venous leakage , the injection process of pmma and extravasation out of the vertebral body compression fractures of the t1 and t2 vertebral bodies were managed under the same conditions . six days after the procedure , the patient underwent an anterior discectomy and instrumented interbody fusion and interspinous wiring with a songer cable at c6 - 7 . postoperative radiographs and ct images demonstrated complete reduction of the c7 vertebral body and a good position of instruments ( fig . the patient was discharged six days after surgery with significant improvement in the left arm weakness and neck pain . however , paresthesia on the left arm still remained . at a 5-month follow - up examination , a radiological study of the cervical spine performed five months after surgery revealed evidence of solid bone fusion and no cervical instability ( fig . a 51-year - old woman was hospitalized due to posterior neck pain and left arm weakness after a traffic accident while riding a bicycle . on admission , there was a four - fifths decrease in motor function of the left arm . plain radiographs of the cervicothoracic spine revealed compression fractures of c7-t3 with anterior subluxation of facets at c6 - 7 . ct and magnetic resonance imaging ( mri ) showed anterior wedging of the c7 vertebral body , ligamentous disruption at the c6 - 7 level and acute compression fractures at t1 - 3 ( fig . vertebral compression fractures have been treated mainly with bed rest , the use of analgesics , braces and various physical therapies . however , unlike lumbar compression fracture patients , the patient was uncomfortable in the supine position and even lying down in bed . the patient had to keep her posture in the sitting or a semi - fowler position all day , and the patient had slight difficulty in deep breathing . the patient underwent ct - guided vertebroplasty at t1 - 3 and an anterior discectomy and instrumented interbody fusion at c6 - 7 . the procedure was performed in strictly sterile manner in the ct ( somatom sensation open scanner , siemens medical solutions , erlangen , germany ) room . after the patient was placed on the ct table , blood pressure and pulse oximetry were monitored continuously . the area to be treated was prepared in a strictly sterile manner and then ct topography for the upper thoracic spine , including the symptomatic lesion , was checked . on this topographic image , axial ct scanning was performed for the t3 vertebral body and a sectional image for needle insertion was selected . the image mode was converted to ct fluoroscopy , and the image size was magnified for precise monitoring . with the administration of local anesthesia with 1% lidocaine , a 10-gauge vertebroplasty cannula was inserted to the affected vertebral body via the unilateral transpedicular route . the tip of the cannula was placed in the anterior half of the vertebral body ( fig . a total of 3 cc pmma was applied to the t3 vertebral body . during the procedure , needle advancement , venous leakage , the injection process of pmma and extravasation out of the vertebral body compression fractures of the t1 and t2 vertebral bodies were managed under the same conditions . six days after the procedure , the patient underwent an anterior discectomy and instrumented interbody fusion and interspinous wiring with a songer cable at c6 - 7 . postoperative radiographs and ct images demonstrated complete reduction of the c7 vertebral body and a good position of instruments ( fig . the patient was discharged six days after surgery with significant improvement in the left arm weakness and neck pain . however , paresthesia on the left arm still remained . at a 5-month follow - up examination , the patient reported no neck pain or limb weakness . a radiological study of the cervical spine performed five months after surgery revealed evidence of solid bone fusion and no cervical instability ( fig . percutaneous vertebroplasty was developed by deramond and colleagues ( 4 ) in 1987 and has been widely used in the therapy of vertebral compression fractures . the procedure provides benefits in terms of pain relief , increased activity and decreased analgesic drug consumption in patients with an osteoporotic compression fracture . the use of pmma leads to stabilization and immobilization of compressed vertebral bodies and the heat effect of cement on nerve endings plays a role in pain reduction ( 4 , 5 ) . the average transverse diameter of the pedicle at t1 vertebral body is 6.4 mm in women and 7.3 mm in men . at t3 , however , the mean diameter is 3.4 mm in women and 3.9 mm in men ( 3 ) . the thoracic vertebral arch encloses a small , round spinal canal that will not admit the tip of an index finger . for this reason , the percutaneous transpedicular approach to the upper thoracic vertebrae is difficult and even a small amount of cement leakage can cause serious neurological complications . cho et al . ( 1 ) have used the posterolateral percutaneous vertebral body access technique ( pvba ) to avoid the pedicle and provide direct access to the vertebral body in a middle thoracic compression fracture . this technique at the thoracic level is very safe in that direct injury to the nervous system is prevented as the needle passes through the costotransverse facet , rib head and lateral margin of the pedicle . this is further from the spinal canal than is the case with the transpedicular approach . however , cho and colleagues were concerned about the potential risk of pneumothorax due to the posterolateral trajectory of the use of the pvba technique . it is also very difficult to visualize the upper thoracic spine in a lateral view under fluoroscopy because of shoulder overlap . with the use of our ct guided technique , however , we could obtain good access to the high thoracic vertebra with monitoring of the full passage of needle advance from the skin to the vertebral body . inadvertent leakage of pmma into spinal canal is the most serious complication , and can cause a major postprocedural neurological deficit ( 4 - 8 ) . ( 9 ) have advocated dose - dependent epidural leakage of pmma after pvp in patients with osteoporotic vertebral compression fractures . with the help of the use of ct fluoroscopy , we could monitor the volume of pmma injected until the optimal filling of the vertebral body , thus avoiding the risk of epidural leakage . the use of ct fluoroscopy can help detect unintended pmma leakage at an early stage of the procedure ( 2 , 6 , 8 , 10 ) . this is another advantage of the use of the ct guidance procedure as compared with the use of a conventional fluoroscopic guide . early detection of pmma leakage can help prevent a serious neurological complication and a pulmonary embolism . the pmma leakage rate is variable as reported in the literature ( range , 8 - 87.5% ) ( 2 , 4 - 10 ) . no pmma leaking to the epidural space or paravertebral area occurred as seen on ct monitoring . in spite of the feasibility and benefits , the use of ct fluoroscopic guidance one of the limitations is that the radiation dose associated with ct fluoroscopy is much higher than that of c - arm fluoroscopy ( 2 ) . therefore , the operator should avoid radiation exposure during the procedure by use of lead gloves or other protective devices . another limitation is the inability to monitor intraoperative venography as the image of ct fluoroscopy is confined to an axial two - dimensional view . however , the necessity of venography during pvp in osteoporotic compression fractures has recently been questioned . in addition , the risk of contamination is relatively high due to the small working space , so operators should pay close attention to the possibility of postoperative infection . in conclusion , we have presented a case of upper thoracic compression fracture treated with ct - guided pvp . this procedure might be a good treatment option for compression fractures , especially at the upper thoracic level . the procedure can overcome the problem of shoulder interference in the lateral x - ray image by providing a clear and precise trajectory toward the target point of the upper thoracic spine and can help to prevent a serious neurological complication by early detection of unintended pmma leakage .

percutaneous vertebroplasty ( pvp ) has been used to relieve pain and to prevent further collapse of the vertebral body in patients with an osteoporotic compression fracture . the most commonly affected site for the use of pvp is the thoracolumbar junction . there are few reports that have described on the usefulness of pvp in the treatment of a high thoracic compression fracture . we report a case of an upper thoracic compression fracture that was treated with computed tomography ( ct)-guided pvp . it was possible to obtain easy access to the narrow thoracic pedicle and it was also possible to monitor continuously the proper volume of polymethylmethacrylate employed , under ct guidance .

systemic lupus erythematosus ( sle ) is a chronic autoimmune disease with acute periodic flare - ups of symptoms impacting any organ system and resulting in potentially life - threatening complications.1,2 some of the significant complications of treatment include hirsutism , weight gain , osteoporosis , osteonecrosis , accelerated atherosclerosis , and retinal damage.3,4 these side effects and complications can lead to significant functional and emotional challenges . patients often experience a high degree of psychological symptoms , including anxiety , depression , mood disorders , and decreased health - related quality of life.58 this article reports on the findings from a preliminary exploratory study on how patients living with sle perceive their sle - related challenges . this was a hypothesis - generating study to tease out some of the nuances of the psychosocial challenges for this population . while there are various empirical studies across the health care disciplines on the psychosocial impact of sle illness , these studies identified general psychosocial experiences without identifying some of the more complex emotional needs of those living with sle in the us.811 this study included several instruments , including one that had only been used once previously , ie , the systemic lupus erythematosus needs questionnaire ( slenq ) and another known as the multidimensional health locus of control , that had not ever been used with the sle population previously . there are many biopsychosocial implications of sle that have been shown to precipitate depression and anxiety . the disease itself , unexpected exacerbations , medical regimen side effects , and medical care issues are often identified as the sources of depressed feelings.1418 one complex nuance , that has not been addressed fully , is how much does disease activity influence emotional states such as depression and anxiety ? and which disease manifestations create the most emotional distress ? a patient being treated for disease activity will have medical and nursing needs , and will likely have needs for occupational and physical therapy , as well as social work counseling.811 the disease disproportionally impacts women ( 9:1 ) , and women will experience physical changes , such as rashes or a cushingoid appearance , which can trigger feelings of low self - esteem , depression , and anxiety at significantly higher rates than those of healthy women.1220 danoff - burg and friedberg studied the unmet needs of 112 sle patients . key findings regarding the impact of sle included tiredness ( 94% ) , need for assistance about feeling anxious or depressed ( 78% and 71% , respectively ) , and nearly half ( 48% ) desired assistance related to maintaining relationships with friends.5 these findings are consistent with similar international research on the psychosocial impact of sle.11 moses et al developed and used an slenq specifically for 386 sle patients from a support association in australia to ascertain their unmet psychosocial needs.12 five of the highest levels of unmet needs were in the psychological domain . they found that need for help with psychosocial and lifestyle problems outranked the needs for information.12 a key implication from this study was that sle patients should be assessed early on for the likelihood of depressive sequelae . the current range of multidisciplinary literature indicates that sle patients have a high vulnerability for self - reported feelings of depression and anxiety.1519 it is unclear which sle manifestations contribute to the forms of psychosocial distress occurring most often , which sociodemographic cohorts may be at higher risk for such psychosocial distresses , and the nature of the physical and emotional sequelae of sle medication regimens . we therefore performed a cross - sectional study of 378 sle patients to identify these psychosocial experiences and which ethnicities may be at risk for which psychosocial stressors . all 899 individuals in the new york sle lupus foundation contact database were sent the survey instrument . each respondent received a six - page survey to investigate their psychosocial experiences of living with sle . the survey instrument was written at an eighth - grade reading level and was also available in spanish . an informed consent letter was sent along with each survey that explained the purpose of the study , its voluntary nature , that participants could discontinue without any penalty , and that the information would be used in the aggregate with no identifying information . to maintain the anonymity of patients responses , packets were distributed to home mailing addresses with stamped envelopes , so that completed surveys could be bulk - mailed to the researchers at yeshiva university with complete anonymity . of the 899 questionnaires originally distributed , 19 were returned unopened due to a change of address . of 880 received , a total of 378 individuals responded to the survey in english or spanish ( 336 in english and 42 in spanish ) , with an overall return rate of 42.9% . the survey instrument comprised four components . part one included sociodemographic variables including gender , race , and age , as well as time of diagnosis , length of diagnosis , education , employment , and relationship status . part two consisted of two scales , ie , the slenq , developed and validated by moses et al,12 which uses a 5-point likert scale ( 1 = no need , 3 = moderate need , 5 = high need ) for 12 different psychosocial factors . for example , the slenq asks : how much assistance do you need with your change in appearance due to sle ? and how much assistance do you need because you have anxiety about sle.12 for the purpose of analysis , three subscales were created from the psychosocial need items , ie , depression , anxiety , and socioeconomic coping . depression was assessed by the following items : feeling depressed due to limitations caused by sle ; feeling depressed because of changes in the body ; and feeling depressed because of side effects . anxiety regarding sle was assessed by the following items : feeling confused about why this disease happened to you ; anxiety about changes in your appearance ; feeling angry about having sle ; feeling uncertain about the future ; dealing with anxiety about sle ; and anxiety about side effects . socioeconomic coping consisted of the following items : concerns about gaining employment ; satisfactory performance in job ; and coping with extra costs . the slenq subscales have been validated , with higher scores reflecting higher need.11 reliability of the subscales was high , with coefficent alphas of 0.91 for depression , 0.90 for anxiety , and 0.76 for economic coping . one - way analysis of variance was utilized to test how various factors like age , educational level , employment , and race impact psychosocial need . in part three , the second scale was used , ie , the multidimensional health locus of control scale measuring the respondents subjective perceptions of how much control they had over their sle.21 two subscales , ie , chance and internal , were utilized in this research . chance refers to the mindset that the course of one s illness is out of one s control . if i manage my illness with diet , exercise , compliance with medication regimens , i can control its course . each is a six - item self - report questionnaire that uses a 6-point likert scale , with items ranging from 1 ( disagree very much ) to 6 ( agree very much ) . examples of items included in the chance subscale are : no matter what i do , i am going to get sick , and examples of items included in the internal subscale are : if i get sick , it is my own behavior which determines how soon i get well again and i am in control of my health . the internal reliability for these subscales was good , with a coefficent alpha of 0.76 for chance and 0.77 for internal . each subscale can range between 1 ( lowest need ) and 6 ( highest need ) . part four concluded with open - ended questions about the range of medication regimens , side effects , and psychosocial impact of those medication regimens . the data were analyzed using spss ( v 17.0 ; spss , inc , chicago , il ) and stata ( v 11.0 ; stata corp , college station , tx ) . statistical tests used in this analysis included the chi - square , t - test , and analysis of variance . ordinary least squares regression was used to analyze the likert scale questions on the slenq and multidimensional health locus of control scales . ordinary least squares regression was used to perform a multivariate analysis in order to evaluate how the different variables affected the outcome measures of depression , anxiety , and socioeconomic coping derived from the slenq . african - american , hispanic , and asian were contrasted with white ; education was coded as some college , college , and advanced degree , and contrasted with high school or less education . how the respondents rated their experience with sle was also coded as chronic symptoms and frequent flares , and contrasted with infrequent flares . finally , insurance was coded as medicaid , medicare , and no insurance , and contrasted with private insurance . coef in table 2 indicates the slope which shows how much the degree of an outcome variable ( depression , anxiety , or socioeconomic coping ) changes for every point increase in a covariate ( chance , internal ) . for example , when chance increases by 1 , a respondent s level of depression increases by 0.17 points . a respondent who had a score of 5 on this scale would have a 0.85-point increase ( 5 0.17 ) in their degree of depression . postestimation wald tests were utilized to test the significance of indicator variables , such as race and insurance . with regards to missing data , some respondents did not respond to every question , so some items were tabulated with less than the total number of respondents . list - wise removal of missing data was utilized because missing cases were not missing at random . each of the subscales on the slenq , ie , depression , anxiety , and socioeconomic coping , ranges from 1 ( no need ) to 5 ( high need ) . it has been reported that formal statistical tests for normality are especially undesirable as they will have low power in the small samples where the distribution matters and high power only in large samples where the distribution is unimportant.22 given the relatively large sample size of nearly 400 subjects reported here , the means and the medians were compared for overall skew . when the mean and median are equal , it indicates that the distribution is symmetrical . parametric tests perform well with large samples ( more than 100 ) even when the data are non - normal.23 as a result , the t - test and one - way analysis of variance were utilized to compare groups of respondents . regarding ordinary least squares regression analysis , each of the regression models was tested for normality of residuals , homoscedasticity , multicollinearity , model specification , and linearity . linear regression does not require any assumption of normal distribution in sufficiently large samples . the variance inflation factor was calculated to test for the presence of collinearity in each model . all models had mean variance inflation factors close to 1 , and no independent variable had a variance inflation factor above 2 , indicating that the independence assumption was met . this type of error occurs when a relevant variable is omitted or an irrelevant one is included . the results indicated that the coefficents in each of the models were not influenced by a model specification error . finally , by utilizing scattergrams between the outcome variables and key independent variables , the models met the linearity assumption . all 899 individuals in the new york sle lupus foundation contact database were sent the survey instrument . each respondent received a six - page survey to investigate their psychosocial experiences of living with sle . the survey instrument was written at an eighth - grade reading level and was also available in spanish . an informed consent letter was sent along with each survey that explained the purpose of the study , its voluntary nature , that participants could discontinue without any penalty , and that the information would be used in the aggregate with no identifying information . to maintain the anonymity of patients responses , packets were distributed to home mailing addresses with stamped envelopes , so that completed surveys could be bulk - mailed to the researchers at yeshiva university with complete anonymity . of the 899 questionnaires originally distributed , 19 were returned unopened due to a change of address . of 880 received , a total of 378 individuals responded to the survey in english or spanish ( 336 in english and 42 in spanish ) , with an overall return rate of 42.9% . part one included sociodemographic variables including gender , race , and age , as well as time of diagnosis , length of diagnosis , education , employment , and relationship status . part two consisted of two scales , ie , the slenq , developed and validated by moses et al,12 which uses a 5-point likert scale ( 1 = no need , 3 = moderate need , 5 = high need ) for 12 different psychosocial factors . for example , the slenq asks : how much assistance do you need with your change in appearance due to sle ? and how much assistance do you need because you have anxiety about sle.12 for the purpose of analysis , three subscales were created from the psychosocial need items , ie , depression , anxiety , and socioeconomic coping . depression was assessed by the following items : feeling depressed due to limitations caused by sle ; feeling depressed because of changes in the body ; and feeling depressed because of side effects . anxiety regarding sle was assessed by the following items : feeling confused about why this disease happened to you ; anxiety about changes in your appearance ; feeling angry about having sle ; feeling uncertain about the future ; dealing with anxiety about sle ; and anxiety about side effects . socioeconomic coping consisted of the following items : concerns about gaining employment ; satisfactory performance in job ; and coping with extra costs . the slenq subscales have been validated , with higher scores reflecting higher need.11 reliability of the subscales was high , with coefficent alphas of 0.91 for depression , 0.90 for anxiety , and 0.76 for economic coping . one - way analysis of variance was utilized to test how various factors like age , educational level , employment , and race impact psychosocial need . in part three , the second scale was used , ie , the multidimensional health locus of control scale measuring the respondents subjective perceptions of how much control they had over their sle.21 two subscales , ie , chance and internal , were utilized in this research . chance refers to the mindset that the course of one s illness is out of one s control . if i manage my illness with diet , exercise , compliance with medication regimens , i can control its course . each is a six - item self - report questionnaire that uses a 6-point likert scale , with items ranging from 1 ( disagree very much ) to 6 ( agree very much ) . examples of items included in the chance subscale are : no matter what i do , i am going to get sick , and examples of items included in the internal subscale are : if i get sick , it is my own behavior which determines how soon i get well again and i am in control of my health . the internal reliability for these subscales was good , with a coefficent alpha of 0.76 for chance and 0.77 for internal . each subscale can range between 1 ( lowest need ) and 6 ( highest need ) . part four concluded with open - ended questions about the range of medication regimens , side effects , and psychosocial impact of those medication regimens . the data were analyzed using spss ( v 17.0 ; spss , inc , chicago , il ) and stata ( v 11.0 ; stata corp , college station , tx ) . statistical tests used in this analysis included the chi - square , t - test , and analysis of variance . ordinary least squares regression was used to analyze the likert scale questions on the slenq and multidimensional health locus of control scales . ordinary least squares regression was used to perform a multivariate analysis in order to evaluate how the different variables affected the outcome measures of depression , anxiety , and socioeconomic coping derived from the slenq . african - american , hispanic , and asian were contrasted with white ; education was coded as some college , college , and advanced degree , and contrasted with high school or less education . how the respondents rated their experience with sle was also coded as chronic symptoms and frequent flares , and contrasted with infrequent flares . finally , insurance was coded as medicaid , medicare , and no insurance , and contrasted with private insurance . coef in table 2 indicates the slope which shows how much the degree of an outcome variable ( depression , anxiety , or socioeconomic coping ) changes for every point increase in a covariate ( chance , internal ) . for example , when chance increases by 1 , a respondent s level of depression increases by 0.17 points . a respondent who had a score of 5 on this scale would have a 0.85-point increase ( 5 0.17 ) in their degree of depression . postestimation wald tests were utilized to test the significance of indicator variables , such as race and insurance . with regards to missing data , some respondents did not respond to every question , so some items were tabulated with less than the total number of respondents . list - wise removal of missing data was utilized because missing cases were not missing at random . each of the subscales on the slenq , ie , depression , anxiety , and socioeconomic coping , ranges from 1 ( no need ) to 5 ( high need ) . formal statistical tests for normality are especially undesirable as they will have low power in the small samples where the distribution matters and high power only in large samples where the distribution is unimportant.22 given the relatively large sample size of nearly 400 subjects reported here , the means and the medians were compared for overall skew . when the mean and median are equal , it indicates that the distribution is symmetrical . parametric tests perform well with large samples ( more than 100 ) even when the data are non - normal.23 as a result , the t - test and one - way analysis of variance were utilized to compare groups of respondents . regarding ordinary least squares regression analysis , each of the regression models was tested for normality of residuals , homoscedasticity , multicollinearity , model specification , and linearity . linear regression does not require any assumption of normal distribution in sufficiently large samples . the variance inflation factor was calculated to test for the presence of collinearity in each model . all models had mean variance inflation factors close to 1 , and no independent variable had a variance inflation factor above 2 , indicating that the independence assumption was met . this type of error occurs when a relevant variable is omitted or an irrelevant one is included . the results indicated that the coefficents in each of the models were not influenced by a model specification error . finally , by utilizing scattergrams between the outcome variables and key independent variables , the models met the linearity assumption . as expected , the vast majority of respondents ( n = 357 , 96.5% ) were women . age ranged from 20 to over 67 years , with approximately one - third under 35 years ( n = 97 , 26% ) , one - third aged 3645 years ( n = 100 , 27% ) , and one - third aged 46 years ( n = 123 , 33% ) . the majority of respondents were non - white women , with 40% ( n = 144 ) identifying themselves as african - american and 38% ( n = 135 ) as hispanic . a large majority of the group was either unemployed ( 19.4% ) or receiving disability due to sle ( 44% ) . most of the respondents ( 70.4% ) had been diagnosed with sle more than 5 years earlier and , in the last 12 months , more than one - third ( 37.3% , n = 139 ) had been hospitalized because of complications from sle . the most frequent type of medical coverage for the respondents was medicaid ( 44.7% , n = 168 ) , followed by private coverage ( 29.1% ) . the primary source of medical care for the majority of respondents was provided by a private physician ( 53.95% , n = 191 ) followed by clinics ( 37.9% , n = 134 ) . the sociodemographic variables of this sample were representative of the national profile of this population regarding age , race , and ethnicity.19 the only significant divergence was for education . this sample had a higher level of education than what is reported in most lupus studies.611 two hundred and twenty - eight ( 60.58% ) respondents indicated that their sle was marked by a chronic set of symptoms . another 16.23% ( n = 61 ) had frequent flares , while 19.13% ( n = 72 ) reported infrequent flares . joint aches , fatigue , and muscle pain were present for at least two - thirds of the respondents . respondents reporting chronic symptoms or frequent flares had higher psychosocial needs as determined by their mean scores , ie , depression ( 3.8 1.1 , p = 0.000 ) , anxiety ( 3.7 1.1 , p = 0.000 ) , and socioeconomic coping ( 3.3 1.2 , p = 0.043 ) , as compared with those having infrequent flares . specific disease manifestations , such as retinal damage and renal damage , did not emerge . participants responded concerning their level of psychosocial needs using the slenq , as previously detailed . figure 1 displays the median scores for each factor from lowest to highest need for psychosocial support or assistance . needing some form of psychosocial assistance for coping with their feelings of depression because of changes in body and changes in appearance each of the subscales , ie , depression , anxiety , and socioeconomic coping , ranged from 1 ( no need ) to 5 ( high need ) . the scales had the following overall means and medians : depression mean = 3.5 1.3 , median = 3.7 , interquartile range = 2 ; anxiety mean = 3.3 1.2 , median = 3.3 , interquartile range = 2 ; and socioeconomic coping mean = 2.9 1.3 , median = 2.7 , interquartile range = 2.3 . the means and medians indicate that respondents had the most difficulty coping with depression , followed by anxiety and socioeconomic coping . lumley et al point out that respondents reporting chronic symptoms or frequent flares are more likely to have higher psychosocial needs with their depression , anxiety , and socioeconomic coping , as compared with those having infrequent flares.22 those who reported frequent flares had a mean score of 3.8 1.1 for depression ( p = 0.000 ) , a mean of 3.7 1.1 for anxiety ( p = 0.000 ) , and a mean of 3.3 1.2 ( p = 0.043 ) . respondents reporting chronic symptoms also reported significantly higher psychosocial needs on depression and anxiety compared with those reporting infrequent symptoms . education impacts the level of perceived psychosocial need , mediating levels of self - reported depression and anxiety associated with sle . for depression , respondents with a high school education or lower rated their psychosocial need as 4.0 1.1 compared with those who obtained college or advanced degrees ( 3.2 1.3 and 3.0 1.5 , respectively , p < 0.001 ) . a similar pattern existed for anxiety , where respondents with high school education or less had an average need of 3.8 1.2 compared with 3.0 1.2 for respondents who had obtained at least a college degree ( p = 0.001 ) . respondents who were unemployed or receiving disability insurance had higher psychosocial needs on all three subscales . those who were more fully employed reported less distress with depression , anxiety , and socioeconomic coping . for depression , respondents on disability rated their need as 3.8 1.2 compared with 3.1 1.4 and 3.1 1.3 for those working part time or full time ( p < 0.001 ) . a similar pattern was true for anxiety , where respondents receiving disability had a mean of 3.6 1.2 compared with 3.0 1.2 and 3.1 1.2 for respondents working full time and part time , respectively ( p = 0.001 ) . hispanic respondents demonstrated the highest need for psychosocial assistance on all three subscales . for feelings of depression , hispanic respondents rated their need for assistance as 3.8 1.2 compared with 3.5 3.5 for african - americans , 2.9 1.3 for asians , and 3.2 1.4 for white respondents ( p = 0.009 ) . on anxiety , hispanic respondents rated their need for psychosocial assistance as 3.5 1.2 compared with 2.8 1.1 for asians , 3.3 1.2 for african - americans and 3.0 1.2 for whites ( p = 0.013 ) . for socioeconomic coping , white respondents displayed little need for assistance , with a mean of 2.3 2.3 compared with 3.1 1.3 for hispanics , 2.9 1.3 for african - americans , and 3.0 1.0 for asians . those who indicated a lack of insurance rated the highest need for assistance with socioeconomic coping . the mean on the depression subscale for medicaid recipients was 3.9 1.2 compared with 3.2 1.2 for those who utilized private insurance ( p < 0.001 ) . medicaid recipients rated the anxiety subscale highest , with a mean of 3.6 1.1 compared with a mean of 3.1 1.3 for medicare beneficiaries ( p = 0.006 ) . respondents without insurance had the highest need for assistance with socioeconomic coping , with a mean of 3.6 1.2 as compared with a mean of 2.5 1.3 for medicare recipients , 3.2 1.3 for medicaid recipients , and 2.6 1.2 for those receiving private insurance ( p = 0.0001 ) . the scales had the overall means of 3.5 1.3 for depression , 3.3 1.2 for anxiety , and 2.9 1.3 for socioeconomic coping . these means indicate that respondents had the most difficulty coping with depression , followed by anxiety and socioeconomic coping . the analyses of sle manifestations revealed that those with muscle pain and hair loss were the most likely to report feelings of sle - related depression and anxiety . respondents reporting muscle pain had a mean score of 3.8 1.2 for depression compared with 2.9 1.3 for those who did not ( p < 0.001 ) . the respondents experiencing muscle pain also had higher levels of anxiety with a mean of 3.7 1.1 compared with 2.8 1.1 for those who were not experiencing pain ( p < 0.001 ) . those who experienced hair loss had a higher level of depression , with a mean of 3.9 1.2 compared with 3.1 1.3 for those who did not experience this side effect ( p = 0.000 ) . respondents reporting chronic symptoms or frequent flares were more likely to have higher psychosocial needs with their depression , anxiety , and socioeconomic coping , as compared with those having infrequent flares . those who reported frequent flares tested higher for depression , with a mean of 3.8 1.1 ( p = 0.0000 ) and higher for anxiety , with a mean of 3.7 1.1 ( p = 0.000 ) . those who experienced hair loss also had higher levels of anxiety , with a mean of 3.6 1.2 compared with 3.0 1.2 for those who did not experience this side effect ( p = 0.000 ) . similarly , those with muscle pain had higher levels of socioeconomic need , with a mean of 3.0 1.3 compared with 2.6 1.3 for those who did not ( p = 0.006 ) . the more respondents perceived they had some control over the illness , the less likely they were to report high levels of depression or anxiety . the mean and median scores for the chance and internal subscales on the multidimensional health locus of control scale were : mean 2.84 1.2 , median 2.7 , interquartile range 1.5 , and mean 2.98 1.2 , median 3.0 , interquartile range = 1.6 , respectively , across all patients . respondents who reported their sle as having mostly infrequent flares ( mean 2.5 1.2 ) perceived that they had more control over their health compared with those with chronic symptoms ( mean 2.9 1.2 ) or infrequent flares ( 2.8 0.98 , p = 0.002 ) . in table 2 , this indicates how much the degree of an outcome variable ( depression , anxiety , and socioeconomic coping ) changes for every point increase in a covariate ( eg , chance , internal ) . the critical locus of control finding was that the more control a patient felt they had over their disease , the less likely they were to report feelings of depression and anxiety , with the specific variances detailed in table 2 . the most significant locus of control finding was that for those who reported the sensation of having no control over their disease ; chance ( coef = 0.169 , p = 0.007 ) were positively associated with more chronic symptoms ( coef = 0.648 , p = 0.001 ) , frequent flares ( coef = 0.796 , p = 0.001 ) and depression ( coef = 0.648 , p = 0.001 ) . a college degree ( coef = 0.561 , p = 0.010 ) or advanced degree ( coef = 0.644 , p = 0.026 ) compared with a high school degree or less was associated with requiring less assistance with depression , as was the case with having medicare as compared with having private insurance , medicaid patients required more assistance than those with insurance . the results of the postestimation test indicated that , compared with medicaid recipients , respondents receiving medicare had a decreased need for psychosocial assistance with depression ( 0.589 , p = 0.008 ) . the following covariates significantly increased the need for psychosocial assistance for feelings of anxiety : chance ( coef = 0.138 , p = 0.019 ) and frequent flares ( coef = 0.686 , p = 0.003 ) as compared with infrequent flares , and the following covariates decreased the degree of need for psychosocial assistance for feelings of anxiety : college degree ( coef = 0.538 , p = 0.011 ) compared with high school . the results of the postestimation test indicated that compared with respondents with no health insurance , respondents receiving medicare had a decrease in need for psychosocial assistance for feelings of anxiety ( coef = 0.483 , p = 0.039 ) . being african - american ( coef = 0.488 , p = 0.041 ) , having muscle pain ( coef = 0.380 , p = 0.038 ) and having no insurance ( coef = 1.09 , p = 0.001 ) all significantly increased the need for psychosocial assistance for socioeconomic needs . the results of postestimation indicated that , compared with respondents with no health insurance , respondents receiving medicare had a statistically significant decrease in the need for assistance with economic coping ( p = 0.034 ) . in analyzing the medication regimens and side effects , it is important to note that respondents could and often did report use of various combinations of drugs . because the side effects can be a result from any one medication , a combination of sle medications , or indeed a disease manifestation , the significance of their responses is biased . because most patients were taking more than one medication at a time , it is difficult to ascertain which specific medications gave rise to which side effects nevertheless , their perceptions of medication side effects is significant because they may have misinterpreted side effects incorrectly , and may have titrated their own medication regimens based on erroneous perceptions and beliefs . it is also important to glean which side effects are experienced as being the most distressing to these patients , so that treating physicians can assess these issues as they develop or adjust treatment plans . at least one - third of the respondents used hydroxychloroquine , azathioprine , vitamins , methotrexate , steroids , or anti - inflammatory medications . there are many expected and some unexpected side effects of sle medications that present a myriad of physical and emotional challenges . most respondents experienced either hair loss ( 51.1% ) or weight gain ( 32.7% ) as side effects from the use of their respective medications . table 3 displays the relationship between medications for sle and the types of side effects respondents experienced from them . those utilizing hydroxychloroquine and steroids experienced the most side effects . over two - thirds of those who experienced hair loss ( 66.4% ) or weight gain ( 67.4% ) were taking hydroxychloroquine ( p = 0.02 ) . another set of interesting findings involved respondents perceptions of the advantages of sle medications , as well as their respective emotional preferences for medication treatment plans . the chief reported benefit of these medications was the reduction in frequency and intensity of flares . when respondents were queried about what they would desire from a new medication for sle , a majority ( 55% , n = 207 ) desired fewer flares and almost one - third ( 32% , n = 120 ) desired fewer side effects . although most respondents experienced side effects of hair loss or weight gain from their medications , they still expressed that their primary desire from a new medication was fewer flares . almost two - thirds ( 62.4% ) of those who experienced hair loss desired fewer flares ( p = 0.01 ) in a new medication , and 44% who experienced weight gain also desired fewer flares ( p = 0.01 ) . as expected , the vast majority of respondents ( n = 357 , 96.5% ) were women . age ranged from 20 to over 67 years , with approximately one - third under 35 years ( n = 97 , 26% ) , one - third aged 3645 years ( n = 100 , 27% ) , and one - third aged 46 years ( n = 123 , 33% ) . the majority of respondents were non - white women , with 40% ( n = 144 ) identifying themselves as african - american and 38% ( n = 135 ) as hispanic . a large majority of the group was either unemployed ( 19.4% ) or receiving disability due to sle ( 44% ) . most of the respondents ( 70.4% ) had been diagnosed with sle more than 5 years earlier and , in the last 12 months , more than one - third ( 37.3% , n = 139 ) had been hospitalized because of complications from sle . the most frequent type of medical coverage for the respondents was medicaid ( 44.7% , n = 168 ) , followed by private coverage ( 29.1% ) . the primary source of medical care for the majority of respondents was provided by a private physician ( 53.95% , n = 191 ) followed by clinics ( 37.9% , n = 134 ) . the sociodemographic variables of this sample were representative of the national profile of this population regarding age , race , and ethnicity.19 the only significant divergence was for education . this sample had a higher level of education than what is reported in most lupus studies.611 two hundred and twenty - eight ( 60.58% ) respondents indicated that their sle was marked by a chronic set of symptoms . another 16.23% ( n = 61 ) had frequent flares , while 19.13% ( n = 72 ) reported infrequent flares . joint aches , fatigue , and muscle pain were present for at least two - thirds of the respondents . respondents reporting chronic symptoms or frequent flares had higher psychosocial needs as determined by their mean scores , ie , depression ( 3.8 1.1 , p = 0.000 ) , anxiety ( 3.7 1.1 , p = 0.000 ) , and socioeconomic coping ( 3.3 1.2 , p = 0.043 ) , as compared with those having infrequent flares . specific disease manifestations , such as retinal damage and renal damage , did not emerge . participants responded concerning their level of psychosocial needs using the slenq , as previously detailed . figure 1 displays the median scores for each factor from lowest to highest need for psychosocial support or assistance . needing some form of psychosocial assistance for coping with their feelings of depression because of changes in body and changes in appearance each of the subscales , ie , depression , anxiety , and socioeconomic coping , ranged from 1 ( no need ) to 5 ( high need ) . the scales had the following overall means and medians : depression mean = 3.5 1.3 , median = 3.7 , interquartile range = 2 ; anxiety mean = 3.3 1.2 , median = 3.3 , interquartile range = 2 ; and socioeconomic coping mean = 2.9 1.3 , median = 2.7 , interquartile range = 2.3 . the means and medians indicate that respondents had the most difficulty coping with depression , followed by anxiety and socioeconomic coping . lumley et al point out that respondents reporting chronic symptoms or frequent flares are more likely to have higher psychosocial needs with their depression , anxiety , and socioeconomic coping , as compared with those having infrequent flares.22 those who reported frequent flares had a mean score of 3.8 1.1 for depression ( p = 0.000 ) , a mean of 3.7 1.1 for anxiety ( p = 0.000 ) , and a mean of 3.3 1.2 ( p = 0.043 ) . respondents reporting chronic symptoms also reported significantly higher psychosocial needs on depression and anxiety compared with those reporting infrequent symptoms . education impacts the level of perceived psychosocial need , mediating levels of self - reported depression and anxiety associated with sle . for depression , respondents with a high school education or lower rated their psychosocial need as 4.0 1.1 compared with those who obtained college or advanced degrees ( 3.2 1.3 and 3.0 1.5 , respectively , p < 0.001 ) . a similar pattern existed for anxiety , where respondents with high school education or less had an average need of 3.8 1.2 compared with 3.0 1.2 for respondents who had obtained at least a college degree ( p = 0.001 ) . respondents who were unemployed or receiving disability insurance had higher psychosocial needs on all three subscales . those who were more fully employed reported less distress with depression , anxiety , and socioeconomic coping . for depression , respondents on disability rated their need as 3.8 1.2 compared with 3.1 1.4 and 3.1 1.3 for those working part time or full time ( p < 0.001 ) . a similar pattern was true for anxiety , where respondents receiving disability had a mean of 3.6 1.2 compared with 3.0 1.2 and 3.1 1.2 for respondents working full time and part time , respectively ( p = 0.001 ) . hispanic respondents demonstrated the highest need for psychosocial assistance on all three subscales . for feelings of depression , hispanic respondents rated their need for assistance as 3.8 1.2 compared with 3.5 3.5 for african - americans , 2.9 1.3 for asians , and 3.2 1.4 for white respondents ( p = 0.009 ) . on anxiety , hispanic respondents rated their need for psychosocial assistance as 3.5 1.2 compared with 2.8 1.1 for asians , 3.3 1.2 for african - americans and 3.0 1.2 for whites ( p = 0.013 ) . for socioeconomic coping , white respondents displayed little need for assistance , with a mean of 2.3 2.3 compared with 3.1 1.3 for hispanics , 2.9 1.3 for african - americans , and 3.0 1.0 for asians . those who indicated a lack of insurance rated the highest need for assistance with socioeconomic coping . the mean on the depression subscale for medicaid recipients was 3.9 1.2 compared with 3.2 1.2 for those who utilized private insurance ( p < 0.001 ) . medicaid recipients rated the anxiety subscale highest , with a mean of 3.6 1.1 compared with a mean of 3.1 1.3 for medicare beneficiaries ( p = 0.006 ) . respondents without insurance had the highest need for assistance with socioeconomic coping , with a mean of 3.6 1.2 as compared with a mean of 2.5 1.3 for medicare recipients , 3.2 1.3 for medicaid recipients , and 2.6 1.2 for those receiving private insurance ( p = 0.0001 ) . the scales had the overall means of 3.5 1.3 for depression , 3.3 1.2 for anxiety , and 2.9 1.3 for socioeconomic coping . these means indicate that respondents had the most difficulty coping with depression , followed by anxiety and socioeconomic coping . the analyses of sle manifestations revealed that those with muscle pain and hair loss were the most likely to report feelings of sle - related depression and anxiety . respondents reporting muscle pain had a mean score of 3.8 1.2 for depression compared with 2.9 1.3 for those who did not ( p < 0.001 ) . the respondents experiencing muscle pain also had higher levels of anxiety with a mean of 3.7 1.1 compared with 2.8 1.1 for those who were not experiencing pain ( p < 0.001 ) . those who experienced hair loss had a higher level of depression , with a mean of 3.9 1.2 compared with 3.1 1.3 for those who did not experience this side effect ( p = 0.000 ) . respondents reporting chronic symptoms or frequent flares were more likely to have higher psychosocial needs with their depression , anxiety , and socioeconomic coping , as compared with those having infrequent flares . those who reported frequent flares tested higher for depression , with a mean of 3.8 1.1 ( p = 0.0000 ) and higher for anxiety , with a mean of 3.7 1.1 ( p = 0.000 ) . those who experienced hair loss also had higher levels of anxiety , with a mean of 3.6 1.2 compared with 3.0 1.2 for those who did not experience this side effect ( p = 0.000 ) . similarly , those with muscle pain had higher levels of socioeconomic need , with a mean of 3.0 1.3 compared with 2.6 1.3 for those who did not ( p = 0.006 ) . the more respondents perceived they had some control over the illness , the less likely they were to report high levels of depression or anxiety . the mean and median scores for the chance and internal subscales on the multidimensional health locus of control scale were : mean 2.84 1.2 , median 2.7 , interquartile range 1.5 , and mean 2.98 1.2 , median 3.0 , interquartile range = 1.6 , respectively , across all patients . respondents who reported their sle as having mostly infrequent flares ( mean 2.5 1.2 ) perceived that they had more control over their health compared with those with chronic symptoms ( mean 2.9 1.2 ) or infrequent flares ( 2.8 0.98 , p = 0.002 ) . in table 2 , the column labeled coef is the slope . this indicates how much the degree of an outcome variable ( depression , anxiety , and socioeconomic coping ) changes for every point increase in a covariate ( eg , chance , internal ) . the critical locus of control finding was that the more control a patient felt they had over their disease , the less likely they were to report feelings of depression and anxiety , with the specific variances detailed in table 2 . the most significant locus of control finding was that for those who reported the sensation of having no control over their disease ; chance ( coef = 0.169 , p = 0.007 ) were positively associated with more chronic symptoms ( coef = 0.648 , p = 0.001 ) , frequent flares ( coef = 0.796 , p = 0.001 ) and depression ( coef = 0.648 , p = 0.001 ) . a college degree ( coef = 0.561 , p = 0.010 ) or advanced degree ( coef = 0.644 , p = 0.026 ) compared with a high school degree or less was associated with requiring less assistance with depression , as was the case with having medicare as compared with having private insurance , medicaid patients required more assistance than those with insurance . the results of the postestimation test indicated that , compared with medicaid recipients , respondents receiving medicare had a decreased need for psychosocial assistance with depression ( 0.589 , p = 0.008 ) . the following covariates significantly increased the need for psychosocial assistance for feelings of anxiety : chance ( coef = 0.138 , p = 0.019 ) and frequent flares ( coef = 0.686 , p = 0.003 ) as compared with infrequent flares , and the following covariates decreased the degree of need for psychosocial assistance for feelings of anxiety : college degree ( coef = 0.538 , p = 0.011 ) compared with high school . the results of the postestimation test indicated that compared with respondents with no health insurance , respondents receiving medicare had a decrease in need for psychosocial assistance for feelings of anxiety ( coef = 0.483 , p = 0.039 ) . being african - american ( coef = 0.488 , p = 0.041 ) , having muscle pain ( coef = 0.380 , p = 0.038 ) and having no insurance ( coef = 1.09 , p = 0.001 ) all significantly increased the need for psychosocial assistance for socioeconomic needs . the results of postestimation indicated that , compared with respondents with no health insurance , respondents receiving medicare had a statistically significant decrease in the need for assistance with economic coping ( p = 0.034 ) . in analyzing the medication regimens and side effects , it is important to note that respondents could and often did report use of various combinations of drugs . because the side effects can be a result from any one medication , a combination of sle medications , or indeed a disease manifestation , the significance of their responses is biased . because most patients were taking more than one medication at a time , it is difficult to ascertain which specific medications gave rise to which side effects nevertheless , their perceptions of medication side effects is significant because they may have misinterpreted side effects incorrectly , and may have titrated their own medication regimens based on erroneous perceptions and beliefs . it is also important to glean which side effects are experienced as being the most distressing to these patients , so that treating physicians can assess these issues as they develop or adjust treatment plans . at least one - third of the respondents used hydroxychloroquine , azathioprine , vitamins , methotrexate , steroids , or anti - inflammatory medications . there are many expected and some unexpected side effects of sle medications that present a myriad of physical and emotional challenges . most respondents experienced either hair loss ( 51.1% ) or weight gain ( 32.7% ) as side effects from the use of their respective medications . table 3 displays the relationship between medications for sle and the types of side effects respondents experienced from them . those utilizing hydroxychloroquine and steroids experienced the most side effects . over two - thirds of those who experienced hair loss ( 66.4% ) or weight gain ( 67.4% ) were taking hydroxychloroquine ( p = 0.02 ) . another set of interesting findings involved respondents perceptions of the advantages of sle medications , as well as their respective emotional preferences for medication treatment plans . the chief reported benefit of these medications was the reduction in frequency and intensity of flares . when respondents were queried about what they would desire from a new medication for sle , a majority ( 55% , n = 207 ) desired fewer flares and almost one - third ( 32% , n = 120 ) desired fewer side effects . although most respondents experienced side effects of hair loss or weight gain from their medications , they still expressed that their primary desire from a new medication was fewer flares . almost two - thirds ( 62.4% ) of those who experienced hair loss desired fewer flares ( p = 0.01 ) in a new medication , and 44% who experienced weight gain also desired fewer flares ( p = 0.01 ) . because this was an exploratory study with a broad focus , it has several inherent limitations and biases . nevertheless , these were patients who reported themselves as being sle patients , and were being treated for sle by rheumatologists . the second critical limitation is that the majority of this sample was on various combinations of sle medications , and therefore it is difficult to discern which side effects were the results of which treatments . this study reveals an association between chronic symptoms and the likelihood of higher reports of depression and anxiety , but not specifically the cause and dynamic effect . this may be a bidirectional association , in which chronic disease activity influences emotional states or , in fact , that feelings of depression and anxiety triggers more disease activity . the sequelae of sle that may predict higher vulnerability for a depressive reaction include more reports of depression attributed to changes in appearance ( particularly hair loss and weight gain ) and limitations due to sle ( particularly due to muscle pain and joint pain ) as was found by moses12 in australia , shorthall et al24 in england , and dobkin et al.25 this finding was also consistent with that of ng,26 who found that self - esteem for women living with sle was negatively impacted by changes in body appearance , such as weight gain and hair loss . when responding to the slenq , the sample overwhelmingly reported that the highest general cause of depressive and anxious feelings was changes in appearance and limitations in physical abilities due to sle , primarily muscle and joint pain . the more chronic the symptoms , the more likely that feelings of depression would arise . . these correlations between symptomatology and emotional distress are consistent with the findings of other psychosocial researchers in this area , and our sample represents a larger and more ethnically diverse sample.27 learning that african - americans and hispanics report a higher vulnerability for sle - related depression and anxiety , as well as poorer socioeconomic coping than their white and asian counterparts , is an important finding . this should alert the health care team to integrate social supports into treatment for this population and inform those who develop program service delivery for this population . this finding is consistent with those of other studies reporting health disparities among race and class , ie , the larger burden of disease that hispanics and african - americans carry , as well as higher mortality and complication rates.28,29 from our data , it is unclear what role language or cultural barriers play in the higher rates of depression reported by hispanics . while all sle patients should be assessed comprehensively , hispanic and african - american women may require more psychosocial resources and support , and every effort has to be made to provide culturally competent assessment and intervention.30,31 it is critical to appreciate the psychological impact of this illness and the negative side effects of various medical regimens and how these may impact mood changes , and as moses and ng found , feelings of low - self - esteem , capacity for self - care , and medication compliance.12,2426 the hispanic cohort reported the most emotional distress and reported facing the most socioeconomic distress . the data also found that the more one perceives control over the illness and the more education one has , the less likely one is to report feeling depressed or anxious about sle challenges . this is an important finding because there have been no other psychosocial studies which were able to confirm this important relationship . a related study demonstrated that sle patients with lower levels of education tend to be more anxious , as well as less compliant with visiting their physician.27,28 some possible suppositions about this correlation are that the more educated one is , the better one may be able to understand sle , and the more resources one may have to cope more adaptively.3,4 identifying that those affected by muscle pain and hair loss tend to be more vulnerable to feelings of depression and anxiety is significant for treating physicians , as well as for individual and group counselors . one has to consider the dynamic interplay of the disease manifestations and the emotional state , because muscle pain and hair loss could influence depression and anxiety . the converse is also possible , ie , that depression and anxiety could result in both muscle pain and hair loss . individual and group counseling with sle patients should allow for exploration and expression of feelings about these specific sle manifestations and the range of potentially troubling side effects from sle medications.29 clinical initiatives and program development should emphasize patient education and advocacy because this has been found to be a mitigating factor in both depression and anxiety for this population.31 the great majority of sle patients on medication experience a wide range of side effects , the most prominent of which is hair loss and weight gain , and yet when asked what they wanted from a new medication , the answer was fewer flares . it is difficult to be definitive regarding certain side effects because some patients may interpret an sle symptom as a side effect , eg , they may report that plaquenil is causing hair loss , but it is also possible that increased sle activity is the culprit . for many , if they are on steroids , they have active disease , which has its own sequelae . the prevalence of emotional distress for this population would indicate that intake , assessment , and treatment include inquiry into the psychosocial impact of the illness , with a focus on depression , anxiety , and socioeconomic coping . assessment should pay particular attention to how a patient has been coping with any changes in appearances and limitations due to sle , as well as the emotional impact of any side effects they may be experiencing . any adjustments that can be made to medical regimens with negative side effects should be considered wherever possible . the issue of control for an sle patient may include information , patient education , and advocacy , as well as emphasizing self - actualization for patients in the use of their resources and psychosocial support.12 sle patients manifesting signs of depressive thinking , anxiety , and difficulty in socioeconomic coping , should be referred to social workers who should employ active therapeutic approaches , such as crisis intervention and task - centered counseling , to combat the unpredictable nature of sle.32 identifying and confirming that feelings of depression and anxiety occur at significant rates for those living with sle is critical . those working in health care should be alerted to which sle populations are at higher risk , ie , hispanic and african - american women , and which manifestations trigger the greatest likelihood of feeling depressed and anxious , ie , changes in appearance due to sle , limitations in physical abilities , joint pain , and weight gain . depression and anxiety are broad terms that can reflect very different levels of intensity and frequency for each patient . the slenq should be accompanied by the iverson depression scale in future research in order to gain more precision for health care treatment.33 the overriding study question for future research is how does the depressed state influence disease activity , disease management , and psychosocial experience of the disease ? in addition , further studies should examine sle patients longitudinally to determine how patients cope adaptively and what strengths ( internal and external ) aid in the resilience of patients coping with this chronic disease over time . this study explored self - reported states of depression and anxiety in sle patients using the slenq instrument , as well as the relationship between perceived sense of control and states of depression and anxiety for the sle patient . key findings demonstrated that individuals with sle were more likely to experience depression and anxiety when there were changes in body appearance due to sle , and to experience limitations in physical abilities due to sle , primarily due to muscle and joint pain . the higher the perceived sense of control over sle , the less likely respondents were to report feeling depressed and anxious . african - american and hispanic sle patients reported a higher level of unmet psychological needs than did their other ethnic counterparts . weight gain and hair loss were the most likely medication side effects , and the most likely to trigger sle - related depression and anxiety . whether the health care provider is the treating physician , nurse , social worker , or any other member of a health care team , it is essential to assess this population for how emotional states may impact disease activity , self - care , and medication compliance . the slenq proved a reliable , valid , and comprehensive assessment tool for identifying psychosocial needs related to sle . the health care team treating this population should be alert to the potential psychosocial impact of sle , such as depression and anxiety . health care providers should include comprehensive biopsychosocial screening and assessment wherever possible , and make referrals where needed in order to address the emotional sequelae of living with sle .

background : the purpose of this exploratory study was threefold , ie , to clarify the unique psychosocial challenges facing those living with systemic lupus erythematosus ( sle ) , to distinguish which sociodemographic variables impact the lives of sle patients , and generate knowledge regarding the way patients perceive sle medication regimens.methods:this was a cross - sectional exploratory study in 378 patients diagnosed with sle and receiving services from the sle lupus foundation in new york city . in addition to sociodemographic variables , the instrument used consisted of two scales , ie , the systemic lupus erythematosus needs questionnaire ( slenq ) and the multidimensional health locus of control scale , as well as questions regarding subjective perceptions of side effects from sle medication.results:the highest general cause of self - reported depressive and anxious feelings was changes in appearance due to sle , and limitations in physical abilities due to sle ( primarily from muscle and joint pain ) . the higher the sense of control over sle , the less likely respondents were to report feeling depressed and anxious . african - american and hispanic sle patients reported a higher level of unmet psychological needs due to sle than did their other ethnic counterparts . weight gain and hair loss were the most likely medication side effects and also the most likely causes of sle - related depression and anxiety.conclusion:those living with sle are at risk for feelings of depression and anxiety . african - american and hispanic women are at higher risk for these emotional states . comprehensive assessment across the disciplines should screen this group of patients for depression and anxiety , and be prepared to refer them to patient education and social work counseling as indicated .

anxiety disorders are among the most abundant affective disorders , with a prevalence of 1% in the western countries . the neurochemistry behind this set of conditions are relatively well described : the imbalance of the serotoninergic system has been implicated in regulating permanently elevated anxious mood , and a prominent class of anxiolytic drugs used in the clinic exert their action via the serotonergic system including serotonin reuptake inhibitors and agonists of the serotonin 1a receptor ( 5-ht1ar ) [ 1 , 2 ] . this receptor is most abundant in the hippocampal ca1 [ 3 , 4 ] , suggesting a primary role of this brain part in regulating anxiety . brain oscillations are thought to govern cognitive processes , providing temporal functional linkage between different brain parts and allowing the temporal separation of functionally distinct neuronal assemblies . among the rich repertoire of the prominent hippocampal oscillations , theta activity is implicated in various cognitive processes , including locomotion , learning and memory , and alert ( fear and anxiety ) [ 5 , 6 ] . this rhythm is subject to serotonergic influences originating in the raphe nuclei [ 711 ] . of particular interest here , almost all classes of clinically effective anxiolytics and compounds in the preclinical stage reduce the frequency of hippocampal theta activity elicited by stimulation of the reticular formation in freely behaving or anesthetized animals . notable exception includes direct bilateral histamine infusion into the lateral septum , which decreased anxiety - like responses in two models of anxiety , the elevated plus maze and novelty - induced suppression of feeding test in rats , but , instead of decreasing , the same infusion significantly increased hippocampal theta frequency elicited by reticular stimulation in urethane - anesthetized rats . similar results were reported for direct septal infusion of muscimol , which also increased theta frequency evoked by brainstem stimulation and reduced anxiety - like behaviors . moreover , if the above hypothesis holds true , then compounds that generate anxiety ( anxiogenics ) should increase hippocampal theta frequency . in contrast , two benzodiazepine receptor inverse agonists and an 2 noradrenergic receptor antagonist , all having strong anxiogenic effect , do not have any effect on hippocampal theta . in this study , we took advantage of the two inbred mouse lines we breed in our laboratory to test the effect of an anxiolytic on hippocampal theta . we also tested the hypothesis that if theta frequency is in correlation with anxious phenotype , then these mice will show different theta frequency . these strains show extremes in anxiety - related behavior in the open field , elevated plus maze , and light / dark test . inbred mouse strains having either high or low anxiety level ( ax and nax ) were bred in our animal facility . these strains were originally developed at egis pharmaceuticals co. ( budapest , hungary ) by bidirectional inbreeding based on anticipatory anxiety . male mice of 2.53 months were housed individually under a light / dark 12 h cycle ( lights on at 08:00 ) at 24 1c and given ad libitum food and water . mice were handled for 7 days prior to testing , the procedure is described by hurst and west . for the experiments , the study conformed to eu directive 2010/63/eu and was approved by the regional station for animal health and food control under project license xxxi/2012 . the elevated plus maze apparatus ( epm ) was made of stainless steel ( painted matt black ) consisting of two opposite open arms ( 35 cm 7 cm ) and two opposite closed arms surrounded by 15 cm high walls of the same dimensions . the middle section that allows the animal to transit from arm to arm consisted of a square with dimension of 7 cm 7 cm . the apparatus was elevated 50 cm from the floor and the open arms were equipped with 1.5 cm 1.5 cm ledges to ensure that no animals would fall off the maze . each mouse was placed in the central square facing an open arm and the behavior was recorded for 5 min . before each test , the apparatus was cleared with 20% ( v / v ) alcohol and wiped thoroughly to eliminate the residual odor . the behavior of the mice was recorded and analyzed with an ethovision software package ( ethovision xt 8.5 noldus technology , the netherlands ) . the open field ( of ) arena consisted of circular shape ( 92 cm ) plastic arena with 40 cm high black wall . two 55 w light bulbs were positioned 50 - 50 cm above the center of the arena , which were the only source of illuminations in the testing room . the of arena was divided into centrum ( diameter 77 cm ) and periphery zones . briefly , mice were anesthetized with 1 g / kg urethane i.p . and placed in a stereotaxic frame . under anesthesia , a multichannel electrode having 16 pt / ir contact points separated by 100 m ( the impedance of a 15 m contact point is between 300700 kohm ) and insulated with formvar ( neuronelektrd kft . , budapest , hungary ) was lowered into the dorsal hippocampus ( ap , 2.0 mm ; l , + 2.0 mm ; h , 2.2 mm ) and a fhc concentric bipolar stimulating electrode ( bowdoin , me , usa ) was lowered in the contralateral hippocampus ( ap , 2.0 mm ; l , 2.0 mm ; h , 5.8 mm ) . the location of the recording electrode was set by continuously monitoring the local field potential ( lfp ) activity . similarly , the coordinates of the stimulation electrode were set to evoke the largest response . field epsps were evoked by a bipolar constant current paired - pulse stimuli separated by 50 ms delivered every 20 sec with an am systems isolated pulse stimulator ( model 2100 , carlsborg , wa , usa ) . to establish baseline amplitude levels , the current used for paired - pulse stimulation was adjusted to obtain ~30% of the maximal amplitude under control conditions . one hour of recording was used to establish baseline frequency and amplitude measures followed by the injection of saline or buspirone . and we waited for at least 60 sec . before applying the following tail pinch . paired - pulse stimulation was applied right before and after 45 min of i.p . injection . hippocampal lfp activity was preamplified by a plexon headstage ( dallas , tx , usa ) and amplified 100x and filtered ( 15000 hz ) with an am systems model 2100 differential amplifier and digitized with an a - d converter ( 1401 micro 3 , 15-khz sampling rate , cambridge electronic design , cambridge , uk ) and commercially available software ( spike2 , cambridge electronic design ) . power spectral analyses of hippocampal lfp were performed on 20-s sweeps following the onset of each tail pinch . changes in power and peak frequency in response to drug treatment were assessed by paired t - test unless otherwise indicated . electrophysiological features between strains were evaluated by using independent t - test . for behavioral data , independent t - test or paired sampled t - test ax and nax mice were subjected to elevated plus maze in order to determine their level of anxiety . there was a robust difference between ax and nax both in the total time spent in the open ( 110 12 sec . versus 193 5 ; p 0.001 , independent t - test , n = 8 , 10 ) and closed ( 129 11 sec . versus 57 3 sec . ; p 0.001 , independent t - test ) arms , indicating difference in anxious phenotype . this dose of the anxiolytic was only effective in the ax strain , as treated mice spent less time in the closed arm compared to untreated counterparts ( 81 10 sec ; p 0.01 , independent t - test , n = 6 ) . there was no difference between untreated and bus treated nax mice ( 200 5 sec . for open arm , we observed a significant difference in the time spent in the center of the arena ( 55 2 sec . for ax versus 71 4 sec . for nax ; p 0.05 , independent t - test ; n = 33 and 28 , resp . ) and in the periphery ( 245 2 sec . for ax versus 229 4 sec . for nax ; p 0.05 , independent t - test ) between control mice . bus treatment significantly elevated the time exploring the center ( 80 7 sec . ; p 0.01 versus control , independent t - test ) , while it decreased the time spent in the periphery of the arena ( 220 7 sec . ; p 0.01 versus control , independent t - test ) in the ax mice ( n = 6 ) , a clear sign of anxiolysis . on the other hand , the same bus treatment did not modify the behavior of nax mice ( time in center : 54 6 sec ; time in periphery 246 6 sec ; n = 10 , figure 2 ) . urethane anaesthetized mice were subjected to a brief tail flick which evoked prominent oscillation in the ca1 with frequency between 2.6 and 3.5 hz . this theta activity lasted for several seconds , as is shown at figures 3 , 4 , and 5 . we did not observe any difference in the peak frequency nor in the peak power of theta between ax and nax mice ( 2.7 0.2 hz for ax versus 2.9 0.3 hz for nax ; 0.015 0.004 mv for ax versus 0.015 0.005 mv for nax ; n = 11 and 11 , resp . ; figure 3 ) . after several tail flick epochs , saline ( control ) or buspirone was injected intraperitoneally , and theta was evoked every 2 - 3 minutes . no change was seen in the oscillation following saline application ( 2.8 0.2 hz before and 2.7 0.2 hz after for ax ; 2.9 0.2 hz before and 2.8 0.3 hz after for nax ; n = 5 , 5 , resp . ; data not shown ) . in contrast , peak frequency continuously increased in the ax mice having received buspirone . the change peaked at 1520 min after bus application and remained for the rest of the recording ( 2.9 0.3 hz before versus 3.4 0.2 hz after ; p 0.05 , paired t - test ; n = 6 ; figure 4 ) . no effect of bus on the peak theta frequency was detected in the nax strain ( 2.8 0.2 hz before versus 2.9 0.2 hz after ; n = 6 ; figure 5 ) . buspirone did not alter the peak power of the evoked theta neither in ax ( 0.018 0.005 mv before versus 0.016 0.006 mv after ) nor in nax ( 0.013 0.004 mv before versus 0.011 0.006 mv after ) . buspirone slightly decreased the amplitude of evoked fepsps in both ax and nax mice , as was reported previously by o'connor et al . . we did not observe such a great reduction , as is reported in that study ( around 47% for 2.5 mg / kg buspirone ) . under our experimental conditions , there was no difference between the two strains in this regard ( 94% for ax and 96% for nax ; data not shown ) . similarly , no difference was found between the ratio of 2nd/1st evoked fepsps of ax and nax ( 1.37 0.04 for ax versus 1.4 0.06 for nax ; n = 11 and 11 , resp . ; figure 6 ) . paired - pulse facilitation ( ppf ) was not changed by saline injection ( 1.36 0.03 before versus 1.34 0.06 after ; n = 5 for ax and 1.39 0.05 before versus 1.38 0.04 after ; n = 5 ) . interestingly , we saw a decrease of ppf after bus application in the ax strain , which was not significant ( 1.38 0.06 before versus 1.31 0.12 after ; n = 6 ) . on the other hand , a significant increase of ppf was detected in nax strain following bus administration ( 1.41 0.07 before versus 1.56 0.06 after ; n = 6 ; p 0.05 paired t - test ) . hippocampal theta activity was implicated to play a role in regulating anxious mood . enhanced theta activity can be recorded from rodents subjected to a potentially anxiety provoking situation , like the open arm of the elevated plus maze and lesions of the septohippocampal pathway , which drives theta oscillation , disrupts anxiety - like behavior in tests measuring anxiety ( elevated plus maze , open field , and light / dark box ) , social interaction , and hyponeophagia [ 1921 ] . serotonin can modulate theta through a number of different receptors and pathways [ 2225 ] . buspirone , a clinically effective anxiolytic is an agonist of 5ht1ar , and other anxiolytics , like benzodiazepines , exert their calming effects through the 5ht1ar . recently , several papers have questioned the direct correlation of hippocampal theta and anxious phenotype , suggesting that the previous correlation between anxiety level and theta frequency might be an epiphenomenon . by using our inbred mouse strains with different anxiety levels , we tested whether there is any difference in the theta oscillation parameters of these mice under urethane anesthesia . unlike in undrugged animals , theta oscillation emerges at a lower frequency ( 2.56 hz ) under urethane anesthesia and is sensitive to atropine or scopolamine ( [ 31 , 32 ] reviewed in ) , independent of the method of evocation . this type of oscillation is referred to as type 2 theta , which , unlike the movement related type 1 theta , is connected to sensory processing [ 33 , 34 ] . based on several reports and observation , it was proposed that nucleus pontis oralis ( npo ) stimulation evoked hippocampal theta can be used as a screen for potential anxiolytic compounds , because all classes of clinically effective anxiolytics decrease theta frequency . moreover , a hyperpolarization - activated cation ( ih ) channel blocker and an antiepileptic , which decrease evoked theta but were not shown to reduce anxiety before , proved to be anxiolytic in subsequent behavioral tests [ 35 , 36 ] , lending additional support for the hypothesis . we did not observe any difference in the peak frequency nor in the peak power of theta between ax and nax mice . since there is a robust difference in the anxiety - related behavior between these strains , this observation does not support the straightforward connection between hippocampal theta and anxious phenotype . moreover , the anxiolytic we have used , buspirone , affected only ax mice in the current dose and acute application ( reduced anxiety ) but , oddly , increased hippocampal theta frequency . we did not observe any effect on nax mice of this low concentration of buspirone . importantly , we have used a different kind of approach for triggering hippocampal theta , namely , tail pinch . the mechanisms behind theta evoked by either tail pinch ( sensory evoked ) or npo stimulation seem to be the same . moreover , blockade of the caudal npo abolishes sensory evoked theta in the hippocampus , suggesting that the two approaches to evoke theta share common final pathways . other groups have also raised concerns about the direct relationship between evoked theta and anxiety level . in addition , an otherwise anxiolytic treatment , histamine infusion into the brain failed to reduce npo stimulation evoked theta , showing the lack of direct connection between the two phenomena . a recent paper describes a similar phenomenon that we have found . direct infusion of muscimol into the lateral septum increased theta frequency evoked by brainstem stimulation , but , importantly , it reduced anxiety - like behaviors . it has to be noted that hippocampal theta is usually recorded from anaesthetized animals from more easily accessible dorsal hippocampus . interestingly , electrophysiological recordings from freely moving , behaving 5ht1a ko mice , which have increased anxious phenotype , have shown that theta is not changed in the dorsal hippocampus but is elevated in the ventral part of hippocampus in a novel environment . basal synaptic transmission was slightly reduced in both strains by bus , similarly as was reported by o'connor et al . for rats . we did not see such a great fepsp amplitude reduction as the authors did observe . a possible explanation for this contradiction might be that the mentioned study has used wistar rats , not mice . paired - pulse facilitation ( ppf ) ratio was the same in the two strains , but the effect of bus on ppf was divergent : the compound increased ppf in the nax , while it did not modify ppf in the ax strain . ppf is usually regarded as an indicator of presynaptic function ; however local inhibitory networks were also shown to regulate hippocampal ppf . have examined a decreased ca1 ppf ratio in slices taken from the 5ht1a ko animals , but this was only significant at 10 ms interstimulus interval ( we have used 50 ms interstimulus interval in this study ) . however , the same group reported on decreased ppf in the same ko strain in the gyrus dentatus using longer interstimulus intervals ( isi , 4090 ms ) , but not at 10 ms isi . activation of the same receptor therefore might lead to increase of ppf , as was observed in the nax animals . however , 5ht1a receptors are localized at both pre- and postsynaptic sites in the ca1 ; therefore , slight changes in the distribution might lead to divergent functional effects and different ppf ratio , as was seen in our mice . in summary , we have found that two mouse strains having robust difference in anxiety - related behavior do not display difference in sensory evoked hippocampal theta and basal synaptic transmission . however , buspirone increased theta frequency only in ax strains , parallel to the anxiolytic effect . elicited hippocampal oscillation under urethane anesthesia is proposed to correlate with the level of anxiety.anxious ( ax ) and nonanxious ( nax ) mice display robust difference in anxiety level , but no difference was found in ca1 oscillation under urethane.buspirone ( 2.5 mg / kg ) is anxiolytic in the ax , but not in nax mice.buspirone increased frequency of sensory evoked oscillation in the ax , but not in the nax.paired-pulse facilitation was enhanced by buspirone in the nax , but not in the ax strain . elicited hippocampal oscillation under urethane anesthesia is proposed to correlate with the level of anxiety . anxious ( ax ) and nonanxious ( nax ) mice display robust difference in anxiety level , but no difference was found in ca1 oscillation under urethane . buspirone ( 2.5 mg / kg ) is anxiolytic in the ax , but not in nax mice . buspirone increased frequency of sensory evoked oscillation in the ax , but not in the nax . paired - pulse facilitation was enhanced by buspirone in the nax , but not in the ax strain .

hippocampal oscillations recorded under urethane anesthesia are proposed to be modulated by anxiolytics . all classes of clinically effective anxiolytics were reported to decrease the frequency of urethane theta ; however , recent findings raise concerns about the direct correlation of anxiolysis and the frequency of hippocampal theta . here , we took advantage of our two inbred mouse strains displaying extremes of anxiety ( anxious ( ax ) and nonanxious ( nax ) ) to compare the properties of hippocampal activity and to test the effect of an anxiolytic drugs . no difference was observed in the peak frequency or in the peak power between ax and nax strains . buspirone ( bus ) applied in 2.5 mg / kg decreased anxiety of ax but did not have any effect on nax as was tested by elevated plus maze and open field . interestingly , bus treatment increased hippocampal oscillatory frequency in the ax but left it unaltered in nax mice . saline injection did not have any effect on the oscillation . paired - pulse facilitation was enhanced by bus in the nax , but not in the ax strain . collectively , these results do not support the hypothesis that hippocampal activity under urethane may serve as a marker for potential anxiolytic drugs . moreover , we could not confirm the decrease of frequency after anxiolytic treatment .

inflammatory response induced by leukocyte activation during extracorporeal circulation ( ecc ) is one of the major causes of organ dysfunction [ 13 ] . blocking leukocyte activation may attenuate acute lung and renal injuries [ 4 , 5 ] . therefore , some studies have suggested that leukocyte filters could reduce expression of cd11b / cd18 in leukocytes , alleviate systemic inflammatory responses [ 7 , 8 ] , and improve outcomes [ 7 , 9 , 10 ] . however , other studies have shown that leukocyte filters can not reduce , and may even increase , the release of inflammatory cytokines and proteases , and thus could have a negative effect [ 1214 ] . leukocyte filtration activates leukocytes [ 1417 ] , which may enhance inflammatory response during ecc . our previous in vivo study showed that short - term filtration effectively reduced leukocyte count , and preserved lung function better than long - term filtration . for example , neutrophil elastase ( ne ) and il-8 increased during long - term filtration . the results suggest that long - term application of leukocyte filtration may be contraindicated . in the present study the study was approved by the animal hospital ethics committee and all animals in the study received standard care in compliance with the guide for the care and use of laboratory animals . eighteen healthy adult hybrid dogs were provided by the animal experiment center of sichuan university for the study . dogs ( either sex , 2335 kg bw ) were anesthetized with sodium pentobarbital ( 25 mg / kg ) . after heparinization ( 3 mg / kg ) , 300 ml of blood was harvested from the left femoral artery to a blood bag . meanwhile , 6% hydroxyethyl starch 130/0.4 ( fresenius kabi , germany ) was infused from the femoral vein to maintain blood pressure . a closed ecc loop was established with a blood storage device ( xi - jing medical ltd , xi'an , china ) and a regular arterial filter ( xi - jing medical ltd , xi'an , china ) or a leukocyte filter ( heart - care system , separator haemo - technology beijing co ltd . china ) , a roller pump ( stckert ii , munich , germany ) , and tubing . the ecc loop was primed with 300 ml of preserved blood harvested above and 300 ml hydroxyethyl starch 130/0.4 . the circulating flow rate was set at 1 l / min , and ecc lasted for 60 min in all groups . our pilot study showed that the leukocyte count decreased sharply with low levels of ne after 5 min . therefore , 5 min was used for short - term test in this study . in clinical trials , however , a leukocyte filter is used throughout ecc , often longer than 60 min . . established eccs were divided randomly into 3 groups ( n = 6 in each group ) . blood from the control group ( group c ) was filtrated throughout ecc with a standard arterial filter . blood from groups l ( long - term ) and s ( short - term ) was filtrated with a leukocyte filter for 60 and 5 min ( the filter was bypassed in the remaining 55 min ) . blood samples were obtained at 0 , 5 , 30 , and 60 min of ecc for indexes analyses . cell count and differentiation were performed by an automatic blood cell analyzer ( mindray , bc-3000 , china ) . for measurements of ne and free hemoglobin ( fhb ) levels ne levels were determined by enzyme linked immunoadsorbent assay ( ne elisa kits , uscn life science inc . the levels of fhb were determined by the orthotolidine method . for determination of expression of cd11/cd18 on leukocyte , red blood cells were lysed with ammonium chloride solution , and acquired leukocytes were resuspended in 100 l of phosphate - buffered saline . nonspecific antibody binding was blocked with fcr - blocking reagent ( miltenyi biotec ) for 20 min before staining with conjugated antibodies . immunofluorescent cell staining was performed with rat anti dog cd11/cd18:fitc ( clone : ykix490.6.4 , abd serotec co. , ltd . data acquisition was performed on a facsaria cytometer equipped with facs diva 5.0 software ( bd ) and analyzed by flowjo software ( tree star ) [ 4 , 21 ] . briefly , red blood cells were lysed ; the sample was centrifuged at 302 g for 5 min and washed with pbs 3 times . one hundred nucleated cells per slide were counted under a microscope . only naked nuclei that stained blue were calculated . at the end of experiment , the membrane of the leukocyte filter was taken for transmission electron microscopy as reported previously [ 3 , 19 ] . samples were fixed in 3% glutaraldehyde at 4c overnight and then fixed with 1% osmium tetroxide , dehydrated with acetone , and embedded in epon812 . the ultrathin sections were double stained with uranyl acetate and lead citrate and examined under a transmission electron microscope ( h-600iv , hitachi , osaka , japan ) . data were analyzed by spss 16.0 ( spss , inc , chicago , ill ) . quantitative data were expressed by the mean sd , and one - way anova with student - newman - keuls test was used to compare differences among the 3 groups . segmental data were expressed as a percentage , and the differences between the 3 groups were compared using the chi - square test . leukocyte counts were similar among the 3 groups before ecc and decreased by 90% after 5 min of filtration in groups l and s ( figure 1(a ) ) . then , it continued to decrease throughout 60 min of ecc in group l. mean fluorescent intensities ( mfis ) of cd11/cd18 on leukocyte increased significantly in both groups l and s compared with that in group c at 5 min ecc ( figure 1(a ) , p < 0.01 ) . thereafter , mfis of cd11/cd18 in the leukocyte increased sharply in group l , while it increased gradually in groups c and s. neutrophils and lymphocytes , and expression of cd11/cd18 had a similar pattern ( figures 1(b ) , 1(c ) , 1(b ) , and 1(c ) ) . ne is released most from the activated leukocyte , and induces an inflammatory response in blood and tissues . to confirm leukocyte activation , ne level in group l increased significantly at 30 min of filtration , and continued to increase to 60 min ( figure 2(c ) ) . l than groups s and c. to determine whether erythrocyte destruction is associated with activated neutrophils during ecc , fhb levels in plasma were measured . hemoglobin ( a ) and fhb ( b ) levels were similar among the 3 groups before ecc ( figure 2 ) . l than in the other 2 groups at 30 and 60 min ( p < 0.01 ) . furthermore , the fhb level was lower in group s than in group c. wright 's stain and transmission electron microscopy analyses showed significant leukocyte rupture in group l ( figure 3 ) , indicated by cellular membrane rupture and cytoplasmic leakage ( ( a ) , yellow arrow ) . our study is the first in vitro approach that focused on filtration induced inflammation . using the extracorporeal circulation ( ecc ) model in vitro , we clearly showed that effectiveness of leukocyte filtration depends on exposure time during ecc . short - term use of a leukocyte filter reduced circulating leukocytes ; with continued usage , leukocytes that adhered to the filter membrane disrupted , leading to leakage of protease , inflammatory reaction , and tissue destruction . our results may explain why leukocyte depletion could not improve the outcomes in some clinical studies [ 1214 ] . studying the effect of filtration on inflammation is difficult in vivo , because inflammatory products are processed by the immune system . therefore , the in vitro model may be a necessary approach . in this in vitro model , circulating leukocytes were significantly reduced with a 5 min short - term filtration ( about 57 circles ) . the leukocyte filter was then bypassed , and leukocytes adhered to the membrane were no longer exposed to the circulation . our results support a short - term usage of the filter because the concentration of fhb was lower in group s than in group c. this is in agreement with several clinic trials . . found leukocyte depletion before ecc significantly attenuated both myocardial and lung injuries induced by cardiopulmonary bypass . in patients with systemic inflammatory response syndrome , a short - term leukocyte depletion significantly improved organ function and outcomes . in patients with chronic obstructive pulmonary diseases , leukocyte filtration at early reperfusion ( 30 min ) improved oxygenation and shortened intubation and icu and hospital times . the filtration would reach its maximal capacity by prolonging filtering time . then , the number of circulating leukocytes would not change with continued use of the filter ; however , damaging effects on leukocytes develop , which compromises the beneficial effect of the filter . previous studies have observed that leukocyte filtration did not inhibit , but activated leukocytes [ 1417 ] . this is consistent with our results that cd11/cd18 expression on leukocytes was significantly upregulated with long - term filtration , which induces leukocyte sequestration in the tissues . furthermore , leukocytes adhered to the membrane were elongated and ruptured under the transmission electron microscope after long - term filtration . this result was confirmed by a high level of circulating naked nucleus count and neutrophil elastase . taken together , our results indicate that long - term filtration during ecc induces leukocyte rupture . activated leukocytes induce tissue injury via obstruction of microcirculation and release of proteases , such as neutrophil elastase . in the present study , neutrophil elastase and free hemoglobin increased in group l significantly , suggesting that long - term usage of leukocyte filtration exerts deleterious side effects . this statement can be verified by the fact that aprotinin , a serine proteases inhibitor , significantly improves lung function and reduces the incidence of atrial fibrillation in patients treated with a leukocyte filter . although our results showed that short - term filtration may effectively inhibit inflammatory reactions and reduce erythrocyte destruction , whether it would improve patient outcomes requires further in vivo studies . in conclusion , our results clearly show that long - term filtration can induce leukocyte destruction and evoke inflammatory reactions and should be avoided .

filtration during extracorporeal circulation ( ecc ) not only removes but also activates leukocytes ; therefore , long - term leukocyte filtration may cause adverse effects . in the present study , we tested this hypothesis by priming ecc with 300 ml of canine blood and examining filtration effects in 3 groups ( n = 6 each ) during 60 min ecc . in the control group ( group c ) blood was filtrated with an arterial filter for 60 min ; in long - term ( group l ) and short - term ( group s ) groups , blood was filtrated with a leukocyte filter for 60 and 5 min . we found that about 90% of leukocytes were removed after 5 min of filtration in both groups l and s. although leukocyte count continued to reduce , mean fluorescent intensities of cd11/cd18 , free hemoglobin , and neutrophil elastase increased in group l and were higher than those in groups c and s at 60 min . leukocyte rupture , cytoplasmic leakage , and circulating naked nuclei were also found in group l. the data support our hypothesis that long - term filtration can induce inflammation and lead to leukocyte destruction .

systemic lupus erythematosus ( sle ) is an autoimmune disease that causes damage of multiple organs . disease activity and stages can be generally divided into three patterns the remitting relapsing pattern , chronically active disease , and long quiescence based on various clinical manifestations that include , but are not limited to , skin rash , arthritis , nephritis , hematological disorders , and neurological disorders . during sle pathogenesis , autoreactive t cells are activated which in turn activate autoreactive b cells to produce high affinity autoantibodies against self - antigens . immune complexes ( ics ) formed by aggregation of autoantibodies and self - antigens circulate in the blood and eventually deposit in peripheral tissues where the complement system is activated , ultimately inducing the release of signals that further recruit and activate autoreactive cells to feed forward a vicious cycle of chronic inflammation . different innate and adaptive immune cell populations , including monocytes / macrophages , neutrophils , dendritic cells ( dc ) , and lymphocytes , are recruited into peripheral tissues following the inflammatory signals to amplify inflammation and cause tissue damage [ 1 , 46 ] . dc were discovered as the professional antigen - presenting cells ( apc ) with a primary function of priming nave t cell activation . since their discovery , our understanding of how dc contribute to immune responses has much expanded , and dc have been divided into many subpopulations with distinct phenotypes and functions . two main subpopulations are classical dc ( cdc ) and plasmacytoid dc ( pdc ) . common dendritic cell progenitors ( cdp ) originated from macrophage dendritic cell progenitors ( mdp ) are the first dedicated dc progenitors that can differentiate into pre - cdc and pdc in bone marrow . murine cdc is characterized by high expression of cd11c and mhc - ii on surface , while human cdc also express nonoverlapping makers cd1c ( blood dendritic cell antigen 1 or bdca1 ) or cd141 ( bdca3 ) on different subsets besides cd11c and mhc - ii . different from cdc , murine pdcs express low level cd11c and mhc - ii but are positive for b220 and siglec - h on surface , and human pdcs are defined by the expression of mhc - ii , bdca2 , and bdca4 . functionally , cdc are professional apc that prime nave t cells upon antigen uptake and maturation induced by appropriate maturation signals ( e.g. , upon tlr ligation ) . mature cdc start to prime nave t cells with the interaction between mhc - ii - peptide complex on cdc and t cell receptor on t cells . the ligation of costimulatory molecules , with cd80 and cd86 on cdc and cd28 on t cells , further mutually activates cdc and t cells . finally the cytokines secreted by cdc induce the differentiation of nave t cells into different effector helper t cell subsets . pdcs , on the other hand , are professional interferon ( ifn ) producing cells that , through producing a high level of ifn , activate multiple immune cell populations that express type i ifn receptor ( ifnar ) . interestingly , pdc can also upregulate mhc - ii upon activation and act like cdc to activate t cells . immature cdc when presenting self - antigens in the absence of maturation stimuli express low level mhc - ii on the surface and induce immune tolerance to self . upon activation by maturation stimuli , however , cdc mature with upregulation of mhc - ii and activation markers ( cd40 , cd80 , cd86 , pd - l1 , pd - l2 , etc . ) to facilitate inflammation . for pdcs , while their primary function is to control infections , pdcs in thymus are involved in the negative selection to maintain the central tolerance . not surprisingly , studies have shown that both cdc and pdc play important roles in the development of autoimmune diseases , such as sle . peripheral blood mononuclear cells ( pbmc ) from sle patients can be used to study in vitro dc responses . lupus - prone mouse models that develop lupus - like symptoms spontaneously or artificially can be used to better understand dc - mediated mechanisms of lupus progression under both in vivo and in vitro conditions . in this review , we summarize recent results obtained from studies of sle patients and lupus - prone mice on the roles of cdc and pdc in lupus development . a direct strategy to study whether a cell population is critical for the development of a disease is to deplete the population in vivo . depletion of dc in wild - type mice and lupus - prone mice shows differential contributions of dc to immune homeostasis , with a tolerogenic role of dc in wild - type mice versus an immunogenic role of dc in lupus - prone mice . in wild - type mice , constitutive depletion of cd11c cdc showed normal development of regulatory t ( treg ) cells and normal negative selection of cd4 t cells in the thymus without an autoimmune response . constitutive depletion of both cdc and pdc in wild - type mice , however , led to increased autoimmune inflammation with elevated autoantibodies , increased ifn/il-17-secreting t cells in peripheral tissues , and abnormal negative selection of cd4 t cells in the thymus . this suggests that pdc , or the combination of pdc and cdc , may contribute to immune tolerance to self . interestingly , regardless of the presence of pdc , the absence of cdc consistently resulted in dramatic expansion of myeloid cells , particularly neutrophils and macrophages [ 12 , 13 ] . in mrl / lpr lupus - prone mice , constitutive depletion of cdc and pdc however , it led to fewer splenic treg cells and less cd25 expression on the surface of these cells , suggesting compromised immune tolerance in mrl / lpr mice in the absence of dc . importantly , even though myeloid cells expanded dramatically as in wild - type mice [ 12 , 13 ] , glomerulonephritis and dermatitis were significantly reduced with dc depletion in mrl / lpr mice , which was accompanied by a significant decrease of the proliferation of total t cells and ifn-producing effector t cells . the lack of dc also led to significantly fewer plasmablasts and impaired autoantibody production and class switching to igg , the primary autoantibody isotype in lupus . these results demonstrate a critical role of dc in promoting lupus - like disease in mrl / lpr mice . interestingly , the initiation of t cell activation in lupus may be dc - independent , as the ratio of nave to activated t cells in the spleen did not change with dc depletion . it appears that autoreactive b cells , instead of dc , initiate the activation of autoreactive t cells through antigen presentation in mrl / lpr mice . these data suggest that although dc can maintain immune tolerance to self in wild - type mice , overall their functions have switched to promoting autoimmune responses in lupus - prone mice . for pdc , early transient depletion of these cells from bxsb ( yaa ) lupus - prone mouse model inhibited type i ifn signature , reduced t and b cell activation , decreased autoantibody production , and improved lupus nephritis . while pdc reappeared later on , the effect of early depletion was sustained , suggesting that pdcs contribute to lupus disease at the initiation stage . these depletion studies indicate the importance of cdc and pdc in the development of lupus . therefore , we will next summarize in detail how cdc and pdc , respectively , break down immune tolerance to self and facilitate lupus progression . changes of cell number and phenotype may reflect changes of the cells ' activation status and/or their dynamic trafficking into different tissues . studies on the changes of cdc number and phenotype in lupus will help us understand whether cdc are activated and where they function to break down self immune tolerance . in sle patients , a general sense is that cdc number and frequency in the blood are lower with higher disease activity [ 1823 ] . the decrease of blood cdc may be due to increased migration of cdc into peripheral tissues . for example , more cdc were found to infiltrate the tubulointerstitial region in the kidney biopsy of sle patients with proliferative or active nephritis than the healthy control ( hc ) or patients with nonproliferative nephritis , and the increase in renal infiltration was accompanied by a decrease of cdc number in the peripheral blood [ 20 , 24 ] . murine cdc , particularly those expressing cd11b , also accumulated in the kidney of various types of lupus - prone mice as lupus nephritis progressed [ 2527 ] . in addition , we and others showed increased cdc accumulation in the spleen and lymph nodes of lupus - prone mice [ 2832 ] . how cdc infiltrated inflamed tissues is unclear , but studies have shown that chemokine receptors chemr23 and ccr7 may be important for cdc migration into the kidney and secondary immune tissues , respectively [ 3235 ] . renal expression of chemerin the chemokine ligand of chemr23and increased chemr23 dc in the kidney of sle patients suggest chemerin - dependent migration of cdc into inflamed kidney in lupus . ccr7 , on the other hand , mediates migration of cdc to lymph nodes . upon ifn priming and lipopolysaccharide ( lps ) stimulation , monocyte - derived cdc ( modc ) from sle patients expressed a significantly higher level of ccr7 . besides ifn and lps , ics can also induce the migration of modc towards ccr7 ligands both in vitro and in vivo . the phenotype of cdc is different between tolerogenic cdc , which suppress inflammation , and immunogenic cdc that stimulate inflammation . cdc in the blood of sle patients or secondary immune tissues of lupus - prone mice have been shown to exhibit elevated expression of cd40 , cd80 , cd86 , pd - l1 , and pd - l2 , suggesting that cdc in lupus may be activated and immunogenic [ 18 , 3638 ] . however , in vitro studies using modc from sle patients or lupus - prone mice have shown inconsistent results regarding the activation phenotype of cdc [ 18 , 36 , 3941 ] . some showed higher activation state of modc and enhanced t cell activation with lupus , while others showed either comparable activities or reduced modc and t cell activation . the inconsistency may be due to different methods used for modc differentiation , maturation , and activation , as different amounts of granulocyte macrophage colony - stimulating factor ( gm - csf ) and il-4 were used to generate immature modc , and different stimuli ( e.g. , lps , tnf , cpg , or ifn ) were used to mature or activate modc in different studies . however , whether monocytes are a precursor of cdc in lupus is still an open question . monocytes incubated with sera from sle patients could differentiate into cdc , but the differentiation depended on the presence of ifn in the serum . later studies also showed that igg - containing ics in the serum , tumor necrosis factor ( tnf ) receptor i on monocytes , and the interaction between monocytes and t cells are all important for the differentiation of monocytes into cdc in lupus [ 35 , 43 , 44 ] . regarding the function of differentiated modc , only those generated in the presence of sle sera , rather than modc generated by ifn/gm - csf alone , could promote differentiation of igg- and iga - producing plasmablasts from b cells . this suggests that factors other than ifn in the sle patient sera affect the function of modc in lupus . as discussed earlier , it is important to understand how they are activated in the context of lupus . in vitro studies suggest that self - dna and/or self - rna containing antigens could activate cdc [ 4648 ] . in vitro generated modc from both healthy human pbmc and wild - type mouse bone marrow can be activated by necrotic or apoptotic cell particles containing self - dna and self - rna to produce inflammatory cytokines ( il-6 , tnf ) , upregulate mhc - ii and costimulatory molecules ( cd40 , cd80 , and cd86 ) , and activate allogeneic t cells that in turn produce il-2 , ifn , and il-17 . it has been demonstrated that cdc generated in vitro or isolated directly from human or mouse could be activated by dna- and rna - containing self - antigens through the signaling of toll - like receptor ( tlr)9 and tlr7/8 , respectively [ 4954 ] . however , it is still unclear whether cdc can be activated by nucleic acid - containing self - antigens in vivo , because natural igm antibodies and complement c1q - opsonized apoptotic particles , both present in vivo but not necessarily in in vitro experiments , have the ability to suppress cdc activation [ 5557 ] . the suppression of p38 mapk phosphorylation by mapk phosphatase-1 appears to be important for cdc tolerance induced by natural igm . studies using gene knockouts in mice have shown that tlr7 , myd88 , and interferon regulatory factor ( irf)5 are important for cdc activation in lupus , and tlr8 , a20 , lyn , b lymphocyte - induced maturation protein-1 ( blimp1 ) , and bim can downregulate cdc activation [ 5865 ] . while tlr7 promotes cdc activation in lupus , tlr8 downregulates tlr7 expression and tlr7-dependent cdc activation . irf5-deficient cdc exhibited a reduced ability to produce tnf , il-6 , and il-10 in lupus - prone mice . dc - specific deficiency of a20 , lyn , or blimp1 led to lupus - like disease in mice [ 60 , 6264 ] . cdc isolated from bim mice compared to wild - type mice induced higher t cell proliferation in vitro , and autoantibodies can be generated in non - lupus - prone mice upon transfer of bim - deficient cdc . one study using myd88-deficient mrl / lpr mice showed no obvious change of lupus nephritis , while another study using dc - specific myd88 and lyn double - deficient mice showed attenuated lupus disease compared to dc - specific lyn - deficient mice . interestingly , polymorphisms within tlr7 , irf5 , tlr8 , a20 , lyn , and blimp1 gene loci have all been shown to be associated with sle [ 6670 ] . activation of cdc can be regulated by several additional factors according to studies of sle patient samples . expression of immunoglobulin - like transcript ( ilt)3 , an inhibitory receptor , was found to be decreased on circulating cdc of sle patients , and the decrease was correlated with higher levels of proinflammatory cytokines ( type i ifn , tnf ) in the plasma of these patients . not surprisingly , sle - susceptible single nucleotide polymorphisms were identified in the ilt3 gene locus . sex hormones may also affect the activation of cdc . in a minichromosome maintenance protein ( mcm)6 dependent manner , 17beta - estradiol , a female hormone , could induce upregulation of cd40 on in vitro - generated modc that in turn increased t cell activation . cdc purified from sle patients compared to hc expressed a higher level of mcm6 , and mcm6 expression was positively correlated with the level of 17beta - estradiol in the sera of sle patients . moreover , cdc activation is affected by complement c1q , although the effect of complement c1q on cdc is still unclear . one study showed that immobilized c1q coated on plates induced maturation of immature modc differentiated in vitro from healthy pbmc by gm - csf / il-4 . mature modc , compared to immature modc , had increased production of il-12 , tnf , and il10 and enhanced t cell proliferation and secretion of ifn. however , another study showed that , when immobilized c1q was added concurrently with gm - csf / il-4 during modc differentiation from pbmc , modc stayed at immature state . upon lps or lps / ifn stimulation , these modc did mature , but they produced less il-12 , tnf , and il-6 but more il-10 . mature cdc generated by lps or lps / ifn also had reduced ability to activate t cells . the timing of c1q addition appears to be important , and further studies are required to uncover the roles of c1q in regulating cdc maturation and activation . apoptosis of activated cdc is important for immune tolerance to self . under normal condition , activated cdc undergo apoptosis through either fas - dependent or mitochondria - dependent pathways , the latter by interacting with activated treg cells that express lymphocyte - activation gene ( lag)3 [ 75 , 76 ] . dc - specific deficiency in either fas - dependent or fas - independent apoptosis in mice could induce lupus - like symptoms , suggesting that abnormal accumulation of activated cdc may contribute to breakdown of self - tolerance and lupus development [ 7577 ] . upon activation by self - antigens , cdc can promote lupus development by interacting with t cells and b cells . while in vivo studies of how cdc affect autoreactive t cells are still lacking , in vitro evidence suggests that modc derived from the bone marrow of lupus mice or from pbmc of sle patients , upon activation , can promote t cell activation and hamper treg response [ 39 , 52 , 7880 ] . it is demonstrated in both mouse and human cell studies that modc activated by apoptotic cells or cytosolic dsdna could induce the activation of t cells , including that of autoreactive t cells [ 52 , 79 ] . in addition , compared to bone marrow - derived macrophages , bone marrow - derived cdc ( bmdc ) from lupus - prone mice possessed higher ability to activate autoreactive t cells , suggesting that cdc rather than macrophages are the apc for autoreactive t cell activation . moreover , in vitro generated tolerogenic bmdc from sle patients were less capable of generating treg cells in vitro than hc bmdc . furthermore , lps - activated bmdc from lupus - prone mice suppressed treg function by producing more il-6 , which indirectly promoted proliferation of cd4 t cells . several studies using in vitro systems have indicated possible roles of cdc in promoting autoreactive b cell activation [ 45 , 52 , 8183 ] . a couple of them have shown that gm - csf / il-4-induced bmdc from b6.sle1.sle2.sle3 lupus - prone mice , compared to bmdc from b6 mice , promoted better b cells proliferation and igm / igg production in in vitro coculture system upon anti - cd40 ligation [ 81 , 82 ] . injection of ics , splenic cd11c dc from b6.sle1.sle2.sle3 mice produced more il-6 and ifn than those from b6 mice . in human cell studies , modc derived from healthy pbmc in vitro activated by either the sera from sle patients or cytosolic dsdna promoted b cell antibody class switch to igg and iga [ 45 , 52 ] . contradictory to these observations , however , one study showed that bmdc from several lupus - prone mouse models , when activated by lps , possessed reduced il-6-producing ability compared to bmdc from b6 mice . due to the decrease of il-6 production , lps - activated bmdc from mrl / lpr mice failed to suppress autoreactive igm production by b cells . the discrepancy may have been due to different lupus - prone mouse models used or different activation methods ( anti - cd40 versus lps ) , although another study has shown that lps could increase il-6 production from bmdc of b6.sle1.sle2.sle3 mice . besides activating t cells and b cells , cdc may also promote lupus development by producing high - mobility group box 1 ( hmgb1 ) protein that not only binds nucleosomes to facilitate activation of cdc as a positive feedback but also enhances ifn production by pdc , the latter of which will be discussed below [ 46 , 49 , 84 ] . since cdc can promote lupus development , they are a potential target for the development of new drugs against lupus . to target innate immune cells such as cdc , nanogel - based immunosuppressive drugs have been tested in lupus - prone mice that led to prolonged survival and reduced lupus nephritis [ 85 , 86 ] . the lipid coating of nanogel enables better uptake of the drug by cdc , thus increasing the amount of immunosuppressive drug inside the cells . in addition , in vitro studies have shown that bmdc incubated with immunosuppressive drug - containing nanogel had lower production of inflammatory cytokines compared to cells incubated with free drug . the ability of pdc to produce ifn was also suppressed , with less ifn produced in the presence of nanogel . it appears that cdc - targeted therapies may benefit from nanogel - based delivery with minimal side effects . several studies have shown that tolerogenic cdc generated by transgenic method or induced in vitro can rebuild immune tolerance to self after adoptive transfer to lupus - prone mice [ 8789 ] . tolerogenic cdc can also be induced from pbmc of sle patients in vitro to suppress t cell activation [ 18 , 90 ] . human studies of pdc frequency and number in the blood of sle patients have shown inconsistent results [ 1921 , 9195 ] . the inconsistency may reflect the dynamic change of cell number and migration of pdc corresponding to different disease stages and/or treatments . the decrease of pdc in the circulation of some sle patients may indicate increased migration of the cells into peripheral tissues . notably , increased infiltration of pdc to the kidney of sle patients has been confirmed by several studies [ 20 , 24 , 95 ] , although the location of the infiltrate is still a matter of debate . it has been suggested that pdc may use il-18 receptor and chemr23 to migrate into the inflamed kidney that expresses il-18 and chemerin , respectively [ 33 , 34 , 95 ] . in mice , however , one study showed no change of pdc in the kidney as lupus progressed . pdc can also accumulate in the skin of sle patients and lupus - prone mice [ 96 , 97 ] . in mrl / lpr lupus - prone mice , uvb irradiation induces skin infiltration of pdc , while ifn response in the skin has been shown to be positively correlated with the level of chemerin that can attract pdc through chemr23 . conversely , the increase of pdc in the circulation of some sle patients may be due to increased generation and emigration of pdc from the bone marrow . our study using mrl / lpr mice demonstrated that the number of pdc was increased in the bone marrow compared to mrl control mice . a higher percentage of pdc was also found in the bone marrow of sle patients compared to hc . it is worth noting that phenotypic identification of pdc varies from one study to another and that the surface markers used to define pdc in healthy individuals may not be appropriate under the disease environment . however , we and others have consistently observed the expansion of pdc in secondary immune tissues during lupus progression . we have found that pdc are increased in the mln of young mrl / lpr mice compared to age - matched mrl controls . others using nzb / w f1 mice and nzm2328 mice have found similar results in mln and renal lymph nodes [ 38 , 100 ] . pdcs also accumulate in the spleen of lupus - prone mice , particularly in the marginal zone ( mz ) of the spleen [ 30 , 38 , 82 , 101 , 102 ] . the increase of pdc in secondary lymph tissues on one hand may be caused by inflammation - induced migration and/or self - expansion in situ , as will be discussed later . on the other hand , pdcs appear to be able to survive better in lupus [ 102104 ] , as their expression of antiapoptotic bcl-2 was found to be increased . survival signal in pdc from both humans and lupus - prone mice is activated by tlr7/9-induced nfb pathway [ 103 , 105 ] . pdcs in lupus are constantly stimulated by tlr7/9 ligands , which are known to suppress mir-29b and mir-29c , allowing for upregulation of the target of these micrornas , including bcl-2 . many functional markers expressed on pdc are altered in sle patients and lupus - prone mice . the expression of three inhibitory receptors , bdca2 , leukocyte - associated immunoglobulin - like receptor 1 ( lair-1 ) , and ilt3 , on human pdc is reduced in sle patients compared to hc [ 94 , 106 , 107 ] . on the contrary , mhc - ii and costimulatory molecules are increased on pdc of both sle patients and lupus - prone mice , suggesting an increased ability to present self - antigens and activate autoreactive t cells [ 28 , 37 , 38 , 101 , 108 , 109 ] . one major function of pdc in immune responses against foreign pathogens is to produce a large amount of type i ifn . many studies have shown that type i ifn , particularly ifn , is critical for lupus development . it is well known that sle patients have elevated serum ifn level that is positively correlated with disease severity . administration of ifn into humans for antivirus or antitumor treatment , or into preautoimmune lupus - prone mice , can induce or accelerate lupus - like symptoms [ 110112 ] . deficiency of the receptor of type i ifn and ifnar in several lupus - prone mouse models resulted in ameliorated lupus symptoms [ 100 , 113 , 114 ] . interestingly , anti - ifnar treatment transiently ameliorated lupus disease in mrl / lpr mice , but constitutive depletion of ifnar in the same model deteriorated lupus symptoms [ 115 , 116 ] . ifn deficiency in bxsb mice failed to modify lupus progression , indicating that the ifn subtype is the principal type i ifn important for lupus development . recent studies have shown that by either depleting pdc or abrogating ifn production of pdc , lupus disease is reduced [ 16 , 17 , 117 ] . however , only the depletion of pdc or blockade of ifn signaling at early stage of disease could prevent lupus development [ 17 , 116 ] . together , these studies suggest that through secreting ifn , pdc may play a critical role in the development of lupus disease at the early initiation stage . many types of leukocytes can express ifnar on the surface and respond to ifn , including monocytes , cdc , pdc , t cells , and b cells . sera from sle patients can induce normal monocytes to differentiate into cdc in an ifn-dependent manner . ifn can also expand splenic cdc , particularly cd11bcx3cr1 cdc , that may have been derived from monocytes . in addition , ifn is able to precondition the immunogenic status of monocytes . without ifn priming , monocytes incubated with rna - containing ics from sle patients failed to upregulate activation markers . the same phenomenon was observed for modc differentiated by apoptotic blebs or apoptotic cells , where ifn priming enabled these modc , which were tolerogenic without ifn , to activate t cells [ 119 , 120 ] . the molecular mechanism of how ifn activates monocytes is still unclear , but studies have shown increased expression of two ifn inducible genes , ifi202 in bone marrow - derived dc from lupus - prone mice and ifit4 in monocytes from sle patients [ 121 , 122 ] . overexpression of these genes can activate normal modc with enhanced il-12 production , which promotes th1 differentiation . besides activation , ifn also affects the migration of modc . ifn/gm - csf - induced rather than il-4/gm - csf - induced modc from healthy human pbmc can upregulate mmp-9 and migrate towards ccl5 and ccl3 that are expressed in inflamed tissues . ifn also influences pdc themselves as well as non - monocyte - derived cdc . in lupus - prone mice , both cell number and surface activation markers of splenic pdc were reduced . in the case for non - monocyte - derived cdc , studies of ifnar - i - deficient nzm2328 mice have shown reduced splenic cd8 and cd8 cdc with decreased activation markers . il-12- and tnf-producing ability of cd8 cdc was also reduced in the absence of ifnar - i . regarding t cells , an in vitro study showed that normal cdc primed by ifn could promote nave t cells to differentiate into th1/th17 t cells . however , if ifn was constantly present in the cdc - t cell coculture system , it had a suppressive effect for th1/th17 differentiation . ifn can also promote inflammatory t cell function by inducing the migration of effector t cells into inflamed tissues in a cxcr3-dependent manner . studies on lupus - prone mouse models have shown that ifn-producing pdc can directly influence autoreactive b cell response . in bxd2 lupus - prone mice , it was demonstrated that the accumulation of activated pdc in the mz of spleen resulted in the upregulation of cd86 on mz b cells , which was important for germinal center ( gc ) formation and autoantibody production . in addition , mz b cells increased their migration into the follicular region in response to ifn produced by the accumulated pdc . such migration of b cells reduced the interaction with mz macrophages , causing the macrophages to decrease in number in the mz . this would compromise clearance of apoptotic cells in the spleen of lupus - prone mice and promote exposure of autoantigens to dc , autoreactive t cells , and b cells . due to the critical role of ifn in lupus development , pdcs produce a large amount of ifn upon tlr7 and tlr9 stimulation by bacterial or viral nucleic acids . one study showed that epstein - barr virus ( ebv ) infection was associated with lupus . in addition , nucleic acid self - antigens and/or nucleic acid - containing ics are another potential inducer of tlr7/9-dependent ifn production by pdc in lupus . nucleic acid self - antigens derived from apoptotic or necrotic cells are increased significantly in sle patients and lupus - prone mice compared to respective controls . when the sera of sle patients were mixed with healthy pbmc , more ifn production was induced from pdc . the patient sera contained ics formed between igg and apoptotic cells , which were found to activate pdc to produce ifn through tlr7/9 [ 53 , 130133 ] . interestingly , igg alone or ics with nucleic acid digestion failed to induce ifn production by normal pdc , suggesting a critical role of tlr7/9 stimulation by nucleic acids within the ics . however , dna / rna alone or nucleic acid - containing ics in the presence of fcraii blockade also could not trigger pdc to produce ifn , indicating that the interaction between igg in ics and fcraii on pdc is important for ic - induced ifn production by pdc [ 130 , 133 ] . moreover , it has been shown that cpg motif in dsdna of dna - containing ics is required for ifn production by normal pdc . nucleic acid self - antigens can also induce ifn production by pdc in an fc receptor- ( fcr- ) independent pathway free from the formation of ics . ll37 , an antimicrobial peptide , has been shown to complex with self - dna and self - rna to form nanoscale aggregates that trigger ifn production by normal pdc in a tlr7/9-dependent manner [ 54 , 134 ] . neutrophils from sle patients possess an increased ability to release neutrophil extracellular traps ( nets ) , which contain ll37 [ 108 , 135 ] . when ll37 was digested , nets were no longer able to induce ifn production by pdc , suggesting a critical role for this peptide . ifn in turn can upregulate ll37 and hnp ( another antimicrobial peptide ) on the surface neutrophils as seen in the blood of sle pateints . the levels of anti - ll37 and anti - hnp antibodies in the patient sera are also increased , which , when ligated with transmembrane expressed ll37 and hnp , respectively , can trigger the release of nets by neutrophils . these results suggest that a positive feedback loop between nets release by neutrophils and ifn production by pdc may initiate and/or promote lupus development in sle patients . interestingly , ll37 has been found to be also important for fcriia - dependent ifn production from pdc , likely through facilitating the internalization of ics . signaling molecules in the tlr7/9 pathway are important for autoantigen - induced ifn production from pdc . slc15a4- , myd88- , irf8- , or irf5-deficient lupus - prone mice have shown ameliorated lupus symptoms with reduced ifn protein level in the serum , decreased ifn transcript level in pdc , downregulation of type i ifn inducible genes , and suppressed activation of both t cells and b cells [ 59 , 61 , 136138 ] . in addition , pdc from irf-5- or irf7-deficient mice failed to produce ifn upon stimulation with rna - containing ics from the sera of sle patients [ 50 , 139 ] . moreover , interleukin-1 receptor - associated kinase ( irak)1 and irak4 are required for ifn induction from pdc , as their inhibition abrogates the production of ifn from healthy pdc stimulated with the sera of sle patients . the ability of pdc to produce ifn is also regulated by many other factors that may influence the outcome of lupus development . high - mobility group box ( hmgb ) proteins , for example , function as universal sentinels for nucleic acid - mediated immune response through both cytosolic receptors and those in endosomes including tlr9 and tlr7 . it has been shown that , compared to cpg - a alone , hmgb1-bound cpg - a could induce higher ifn and tnf production by normal pdc . this is due to increased recruitment of myd88 to tlr9 in the presence of hmgb1 . in addition , hmgb1 can facilitate the formation of cpg - tlr9 complexes and retain the complexes in early endosome rather than lysosome , resulting in sustained ifn production by pdc . studies on sle patient samples have shown that the level of hmgb1 in the circulation was positively correlated with the concentration of ifn [ 46 , 107 ] . moreover , the interaction between hmgb1 and receptor for advanced glycation endproducts ( rage ) is required , as pbmc from hc incubated with the sera of sle patients produce much less ifn when the interaction is blocked [ 46 , 142 ] . amyloid fibrils can also regulate ifn production from pdc by modulating the trafficking of nucleic acid - tlr complexes . these are stable insoluble aggregates of misfolded protein products with extensive -sheet structure that can facilitate the maintenance of nucleic acid antigens in early endosomes of pdc . albeit rare , amyloid fibrils have been found to be associated with sle cases and complicate lupus nephritis . immunization of healthy mice with dna - containing amyloid fibrils induces lupus - like disease , promoting autoantibody production and lupus nephritis . c - reactive protein ( crp ) , an acute - phase reactant produced by liver in response to inflammation , can suppress ifn production from normal pdc by increasing the trafficking to ics into late endosomes in pdc . in sle patients , the elevation of crp in response to inflammation is modest and much less than expected , suggesting compromised regulation of ifn production . when c1q is added simultaneously , rna - containing ics or cpg stimulated less production of ifn , il-6 , il-8 , and tnf from pbmc or purified healthy pdc . the suppressive effect of c1q on ifn production from pdc has been shown to be dependent on the ligation of c1q to lair-1 expressed on pdc . one study has shown that that tlr7 agonist induced higher ifn production by pbmc from healthy women than those from healthy men . in addition , 17beta - estradiol , a female hormone , can increase ifn production from pdc upon cpg stimulation . pdc may interact with other cell types in vivo that affect their ability to produce ifn. studies have shown that b cells , platelets , nk cells , and monocytes can differentially influence ifn production by pdc [ 152156 ] . b cells facilitate ifn production by normal pdc stimulated with rna - containing ics or cpg - a . interestingly , the mechanisms of b cell involvement are different depending on the stimulation . for rna - containing ics , the contact between b cells and pdc through adhesion molecule cd31 is required , while the elevation of cpg - induced ifn production is b cell contact - independent . the latter may be dependent on an unknown secreted molecule , as the supernatant from cpg - a - stimulated b cell culture could promote ifn production from pdc . in addition , activated platelets , found to be more abundant in the blood of sle patients , can promote ifn production from normal pdc stimulated with nucleic acid - containing ics through interaction between cd154 on platelets and cd40 on pdc . in lupus - prone mice , depletion of platelets improved , while administration of activated platelets worsened , lupus disease , suggesting the involvement of platelets in lupus development . moreover , cd56cd16 nk cells can promote ifn production from pdc upon stimulation with rna - containing ics in the coculture of pdc and nk cells through both secreted mip-1 and cd11a - dependent direct contact between the two cell types [ 154 , 155 ] . nk cells isolated from sle patients , however , produced less ifn than nk cells from hc , since most of them were cd56cd16 rather than cd56cd16 nk cells . furthermore , cd14 monocytes , contrary to b cell , platelets , and nk cells , can suppress ifn production from pdc through various mechanisms . it has been shown that upon rna - containing ics stimulation , cd14 monocytes produced tnf , prostaglandin e2 , and reactive oxygen species , all of which suppressed ifn production from normal pdc in coculture . additionally , monocytes can suppress ifn production from normal pdc through competitive binding of c1q - coated ics to reduce internalization of ics in pdc . monocytes isolated from sle patients have less suppressive effect on ifn production from pdc compared to those isolated from hc . while the essential role of ifn-producing pdc in lupus is inarguable , questions remain on whether pdcs are the major ifn-producing cells during the entire course of lupus progression . it has been demonstrated in several studies that pbmc or pdc purified from pbmc of sle patients produced much less ifn upon tlr9-ligand stimulation compared to hc [ 93 , 157159 ] . we have shown in our recent study that pdc isolated from older mrl / lpr mice in the late stage of lupus development produced significantly less ifn upon cpg stimulation in vitro compared to pdc purified from younger mice in the early stage . the reduced ifn-producing ability may be due to continuous exposure to nucleic acid self - antigens , as pdc from hc produced much less ifn after repeated stimulation with cpg or dna - containing ics . notably , one study showed comparable ifn production between pdc from sle patients versus healthy individuals . in their study , however , il-3 was added in cell culture medium , which may have enhanced ifn production by pdc from sle patients . resting or the addition of ifn , ifn , and gm - csf could also recover ifn-producing ability of pdc from sle patients to some extent [ 157 , 159 ] . moreover , ifn production by pdc from sle versus hc was comparable upon stimulation with influenza viruses or tlr7 agonist [ 43 , 158 ] . it is possible that pdc in sle patients and lupus - prone mice can still produce a normal level of ifn through the tlr7 pathway . collectively , the results of these studies have raised two important questions : ( 1 ) do pdcs gradually lose the ability to produce ifn in vivo during lupus progression ? ( 2 ) if pdc fail to produce ifn in late stage lupus , what is the source of ifn that stays at a high level in sle patients and lupus - prone mice ? . an early study showed that pbmc from sle patients could still produce detectable ifn when pdcs were depleted , suggesting that other cell types besides pdc may have the ability to produce ifn in sle . neutrophils isolated from hc , sle patients , and b6 mice were able to do so upon nucleosomes or cpg - b stimulation . interestingly , neutrophils from tlr9-deficient mice retained their ability to produce ifn upon nucleosomes stimulation , suggesting that the production ifn in neutrophils is tlr9-independent . moreover , neutrophils from both sle patients and lupus - prone mice possessed increased ifn transcript level compared to hc , although the protein level of ifn was not measured in these studies [ 162164 ] . besides neutrophils , monocytes and cdc can also produce ifn. with ifn priming , monocytes purified from healthy human pbmc , as well as cdc derived in vitro from bone marrow of normal mice , were shown to produce ifn through lps - activated tlr4 pathway . monocytes from healthy human pbmc also produced ifn upon stimulation with liposome - coated rna . in addition , ly6c monocytes are the primary source of ifn in pristine - induced lupus - prone mice , as depletion of these monocytes abrogated ifn production . cdc , on the other hand , have been shown to produce ifn through a cytosolic pattern recognition pathway via stimulator of interferon genes ( sting ) . due to the critical role of pdc and ifn in the development of lupus , one example is intravenous immunoglobulin ( ivig ) therapy , where igg , the major antibody in ivig , inhibits ic- or cpg - a - mediated production of ifn from pdc . it has been suggested that fc fragment of igg through blocking fcriia on pdcs directly suppresses the uptake of nucleic acid - containing ics by pdc . through the function of igg glycan hydrolysis , endoglycosidase s ( endo s ) can also inhibit the uptake of ics . sialylated subfraction positive ( sna ) fab fragment of igg , targeting unknown receptor on monocytes , induces production of pge2 by monocytes , which in turn suppresses tlr7/9 agonist - mediated ifn production by pdc . another potential treatment targeting pdc and ifn is dna - like class r inhibitory oligonucleotides ( inh - odns ) , which block trl7/9-mediated activation of pdc upon stimulation with nucleic acid - containing ics . administration of inh - odn in mrl / lpr lupus - prone mice dramatically ameliorated lupus disease with reduced pathology and autoantibodies . moreover , proteasome inhibitors have been shown to suppress ifn production from normal pdc by inhibiting tlr9 translocation from endoplasmic reticulum to endosomes and lysosomes [ 172 , 173 ] . furthermore , hmg - coa reductase inhibitors ( statins ) and histone deacetylases inhibitors can suppress ifn production by healthy human pdc through inhibiting irf7 translocation into the nucleus [ 174 , 175 ] . lastly , by neutralizing ifn directly , sifalimumab , a monoclonal antibody against human ifn , was able to reduce ifn - signature in phase i clinical trial . subcutaneous injection of h471 - 94 peptide from histone proteins into nsf1 lupus - prone mice at a low dose induced tolerogenic pdc that promoted treg cells . adoptive transfer of tolerogenic pdc into lupus - prone mice was able to reduce autoantibodies against dna - containing antigens , decrease il-17 production in spleen , and delay the development of lupus nephritis . many questions remain regarding the mechanisms by which dc modulate lupus pathogenesis that needs to be revealed by additional studies . many different lupus - prone mouse models have been generated , making it feasible to investigate whether dc are important for lupus development in vivo . depletion studies of whole dc populations , including both cdc and pdc , in mrl / lpr lupus - prone mice suggest the involvement of dc in promoting lupus development , but not activation of nave t cells . two additional studies that selectively deplete pdc or abrogate ifn-producing ability of pdc in lupus - prone mouse models other than mrl / lpr further demonstrate the importance of pdc in lupus pathogenesis however , selective depletion of cdc populations in lupus - prone mice has not been reported . the second question is which tlr , tlr9 , or tlr7 is critical for the role of pdc in lupus pathogenesis . studies have shown that the pathogenic role of tlr7 in lupus - prone mice is partially dependent on ifn induction , and tlr9 on the contrary can regulate lupus progression by suppressing tlr7 signaling [ 178181 ] . however , pdc - specific tlr7 or tlr9 deficiency in lupus - prone mice has not been reported , as b cells and some other innate immune cell types also express tlr7 and tlr9 . a third question is how to develop new treatment strategies targeting dc populations for lupus . current treatments for lupus are nonspecific immunosuppressive drugs that suppress general immune responses from both innate and adaptive immune system . future direction for lupus treatment should be focused on specific targeting with minimal side effects , where dc are a valuable target . new drugs targeting dc should avoid blocking the mechanism by which they defend against pathogens or cancer cells . therefore , a better understanding of how dc are activated in lupus versus cancer / infection will be particularly useful . how to translate results obtained from in vitro studies is another question . through either purifying dc directly from pbmc of sle patients or in vitro generating modc , researchers have investigated activation of dc by self - antigens , activation / maturation markers on dc , cytokine production by dc , and the ability of dc to activate t cells . similar studies have also been done with bone marrow cells or sorted splenic dc from lupus - prone mice . however , the results from different studies are not always consistent or even contradictory to each other , likely due to differences in stimulation protocols . it is also unclear whether in vitro stimulation methods would create the actual environment for dc in sle patients or lupus - prone mice . in many cases , in vitro studies have revealed that the stimuli for dc activation have to be of certain concentrations or given at specific time points , making it difficult to translate the results . based on the reviewed studies above , we summarize how cdc and pdc may be involved in lupus pathogenesis . at the initiation stage of lupus , dysregulated cdc and pdc are activated by accumulated self - antigens ( e.g. , self - nucleic acids bound with associated molecules ) and cytokines in genetically predisposed individuals and accumulate in peripheral immune and nonimmune tissues . activated pdcs through secreting ifn then provide immunogenic signals to other immune cells including cdc , monocytes , neutrophils , t cells , and b cells . , monocytes differentiate into activated cdc , which , together with cdp - derived activated cdc , sustain and amplify primed adaptive immune responses in both immune and nonimmune tissues , thus exacerbating the disease .

dendritic cells ( dc ) play an important role in the pathogenesis of systemic lupus erythematosus ( sle ) , an autoimmune disease with multiple tissue manifestations . in this review , we summarize recent studies on the roles of conventional dc and plasmacytoid dc in the development of both murine lupus and human sle . in the past decade , studies using selective dc depletions have demonstrated critical roles of dc in lupus progression . comprehensive in vitro and in vivo studies suggest activation of dc by self - antigens in lupus pathogenesis , followed by breakdown of immune tolerance to self . potential treatment strategies targeting dc have been developed . however , many questions remain regarding the mechanisms by which dc modulate lupus pathogenesis that require further investigations .

orthopaedic surgeons play a critical role in evaluating potential cases of child abuse because fractures represent the second most common presentation of abuse behind only soft tissue injuries . there are numerous studies that have focused on the characteristics of fractures considered typical of abuse , including fractures of the femur [ 1 , 4 , 5 , 9 , 14 , 19 , 28 , 29 ] , humerus [ 3 , 12 , 19 , 31 ] , tibia [ 4 , 12 , 18 ] , and ribs [ 6 , 33 ] . yet , orthopaedic surgeons are less likely than general pediatric and emergency room colleagues to identify abuse as the potential etiology of these fractures , thus causing delay for appropriate multidisciplinary intervention [ 15 , 26 ] . this is concerning because of the million children annually in the united states who are the victims of substantiated abuse , orthopaedic surgeons are often the first clinicians to evaluate these patients , and if the cause of injury is not recognized , these children will return to an abusive environment with a 50% risk for reinjury and a 10% risk of death [ 7 , 13 ] . it is therefore essential orthopaedic surgeons not only treat the fractures these children present with , but also recognize the associated etiologic characteristics of the injury that may suggest child abuse . when examining a fracture in the emergency room , the following questions should arise : what patient characteristics may be indicative of abuse versus accidental trauma ? is the history and mechanism of injury inconsistent with the presenting injury ? does this fracture type , pattern , and location represent potential abuse ? fractures of the femur in young children provide an ideal model to develop a systematic examination process to differentiate an abusive from accidental etiology . fracture of the femur in children is the most common musculoskeletal injury requiring hospitalization [ 5 , 20 ] . numerous studies describe fractures of the femur in young children , and many of these have attempted to identify fracture characteristics that may indicate abuse [ 2 , 5 , 8 , 11 , 20 , 21 , 24 , 25 , 28 , 29 ] . the most common presentation cited is the presence of a femur fracture in a patient occurring either before walking age or before the second birthday [ 5 , 9 , 13 , 20 , 28 , 30 ] . in addition , several authors have classified the radiographic appearance and location of femur fractures as predictors of abuse [ 2 , 15 , 25 , 28 , 29 ] as well as elements of the history ( inconsistent history , inappropriate delay , multiple presentations ) , physical examination ( examination inconsistent with history , head injury or fracture in a child not of walking age ) , and socioeconomic background [ 16 , 22 , 32 ] . a number of other reports previously examined fracture patterns in children younger than 48 months of age [ 6 , 14 , 16 , 17 , 26 ] . these studies do not , however , allow a clinician to decide with confidence whether a given femur fracture is likely caused by abuse . we therefore asked whether femur fractures caused by abusive trauma could be distinguished from those caused by accidental trauma using presumed risk factors in the history , physical examination , and radiographic characteristics . our institution is a large pediatric tertiary care center with level i trauma status with a large referral base . institutional review board approval was obtained before data collection . since 1998 , we have maintained an extensive database ( suspected child abuse and neglect [ scan ] ) that examines all potential cases of suspected child abuse and/or neglect . this database contains information identifying child abuse victims , demographic factors , and icd-9 and cpt codes relating to their concomitant diagnoses and treatment . from that database , we identified all children from birth to 48 months of age who had the diagnosis of child abuse ( icd-9 code 995.5x ) and the diagnosis of femur fracture ( icd-9 code 820.x and 821.x ) . in addition , we reviewed the general trauma database from our institution for all children presenting to the emergency room and/or hospitalized with traumatic injuries for children of the same age from 2000 to 2003 with the diagnosis of femur fracture ( icd-9 code 820.x and 821.x ) . the general trauma database contains data identifying trauma patients that fit these characteristics , demographic information , information regarding injury diagnoses , and subsequent treatment . information other than demographic information , injury code data and identifying information were abstracted from the electronic medical record . we calculated a power analysis for multiple logistic regression as described by hsieh et al . . the power analysis assumes a background abuse rate of 30% in patients with femur fractures in this age group with a difference in the rate in any given independent variable of 20% . the power analysis was conducted with a desired two - sided alpha of 0.05 and a desired power of 0.80 . the analysis assumes a variance inflation rate ( adjustment for increased variability caused by multiple regressors ) of 25% for multiple regression . with these characteristics , because our analysis assumes a 30% event ( abuse ) rate in femur fractures , we would need 30% of our total cases to be from abuse and 70% from accidental trauma ( 59 abuse cases and 137 accidental trauma cases ) . patients were identified in the study as a potential control ( accidental ) trauma case if they were found in the general trauma database and did not have a diagnosis of child abuse in their record and were not included in the scan database . we excluded patients from the study if they were younger than 48 months of age ( except for one patient , the scan database contained only patients 48 months of age and younger ) , if we could not adequately confirm whether the case represented child abuse or accidental injury as a result of incomplete medical records ( three children ) , and if cases were erroneously placed in either of the two databases ( ie , did not represent either child abuse or accidental trauma ) . this was the case in one child in whom a diagnosis of femur fracture by icd-9 code was found but no record of this injury could be identified . seventy femur fractures were from the child abuse cohort and 139 femur fractures were from the accidental trauma cohort . these 209 total femur fractures represented the cases and controls that would be analyzed in our study . of these 70 patients , 63 were younger than 18 months of age and seven were older than 18 months of age . of these 139 patients , 44 were younger than 18 months of age and 95 were older than 18 months of age . patients were included in the scan database if hospital personnel determined the patient was a victim of abuse or neglect . the scan team consisted of an independent group of nonorthopaedic physicians , nurses , and social workers with advanced training in child abuse who examined these children independently in the emergency room and/or as inpatients and made the determination whether the patients should be diagnosed with child abuse and when child protective services should be activated . patients were included in the general trauma database if they presented to the emergency room and/or were hospitalized with organ system trauma that necessitated an evaluation from the hospital s trauma service , died in the emergency department as a result of their traumatic injuries , or were initially seen in the emergency department and transferred to another trauma center for management of traumatic injuries . both databases were collected and maintained outside of the orthopaedic department at our institution and were initiated many years before the retrospective review of the data that was performed by the authors . data parameters collected from the patients medical records included age at the time of injury , gender , insurance status , presence of current polytrauma ( another concurrent long bone , clavicle or axial skeletal fracture , or other body system injury that would require hospitalization ) , and physical and/or radiographic evidence of prior trauma . furthermore , one of the authors ( hw ) who was blinded to the scan status of patients examined the paper and electronic medical records of the children and briefly described the history of each patient . subsequently , this author categorized in a separate list the plausibility of each patient s history as either suspicious for abuse ( unwitnessed accident , witnessed abuse , delayed presentation greater than 48 hours , a mechanism that would not normally cause a fracture , or different stories provided by different witnesses ) , or consistent with accidental trauma in cases in which these criteria were not fulfilled , and then transferred the list to the database . two other authors ( np and kb ) reviewed the patient histories to determine if they were consistent with accidental trauma or suspicious for abuse . an intraclass correlation coefficient was generated based on average measures ( icc 0.902 , p value < 0.001 ) indicating agreement . since many of the original radiographs for patients were no longer available , we examined radiology reports to determine the location of the femur fracture in all patients . proximal , diaphyseal , or distal ( each representing roughly one - third of the length of the femur : subtrochanteric region , shaft region , and distal metaphyseal region ) . raw data from the databases were pooled , and means , sds , and/or percentages were calculated for age at time of injury , gender , insurance status , presence of current polytrauma , physical and/or radiographic evidence of prior trauma , history plausibility ( consistent with accidental trauma or suspicious for abuse ) , and radiographic femur fracture location ( proximal , diaphyseal , distal ) . because our age data were skewed we used the mann whitney u test for independent samples to determine if there was a difference in age at the time of injury between the child abuse and accidental trauma patients . the chi square test with yates correction for independence or the fisher s exact test in cases where the assumptions of a chi square test were violated were used to determine differences in binary variables . we used binary logistic regression to calculate adjusted odds ratios for a femur fracture representing abuse as opposed to accidental trauma using age at time of injury , gender , insurance status , presence of current polytrauma , physical and/or radiographic evidence of prior trauma , history plausibility , and femur fracture location as risk factors . ninety - five percent confidence intervals were calculated for proportions and interquartile ranges for median values . child abuse victims were younger than accidental trauma victims ( median age 4.0 months compared with 26.2 months ) and were more often female ( 49% versus 32% ) ( table 1 ) . there was no difference ( p = 0.77 ) between the abuse and accidental trauma groups in terms of insurance status . patients with femur fractures who were victims of abuse had a greater frequency of current polytrauma ( 53% versus 8% ) , physical and/or radiographic evidence of prior trauma ( 62% versus 4% ) , and histories that were deemed suspicious for abuse ( 33% versus 4% ) ( table 2 ) . patients with accidental trauma more often had diaphyseal femur fractures ( 46% versus 66% ) , abuse victims more often had distal femur fractures ( 37% versus 20% ) , and there was no difference in proximal femur fractures between groups ( table 3 ) . the odds of a femur fracture being the result of abuse rather than accidental trauma was greater for children younger than 18 months ( 19.4 times ) and patients of female gender ( 2.0 times ) ( table 1 ) . furthermore , abuse was associated more frequently with current polytrauma ( 13.0 times ) , physical and/or radiographic evidence of prior trauma ( 37.5 times ) , and patients with a suspicious history ( 10.8 times ) ( table 2).table 1demographic information for child abuse and accidental trauma patients for age , gender , and insurance statusvariableall abuse ( 95% ci)all accidentalodds ratio ( 95% ci)p valuenumber of patients70139n / an / aage younger than 18 months90.0% ( 83.0% , 97.0%)31.7% ( 23.9% , 39.4%)19.4 ( 8.3 , 45.0 ) < 0.001*age ( months)4.0 ( 2.0 , 8.3**)26.2 ( 11.9 , 34.8**)n / a < 0.001gender ( percent female)48.6 % ( 36.9% , 60.2%)32.4% ( 24.6% , 40.2%)2.0 ( 1.1 , 3.6)0.033*percentage without insurance7.1% ( 1.1% , 13.2%)9.4% ( 4.5% , 14.2%)0.7 ( 0.3 , 2.1)0.795 * chi square test with yates correction ; mann whitney u test ; fisher s exact test ; * * interquartile range n / a = not applicable.table 2comparison of current polytrauma , prior trauma , and history plausibility in child abuse and accidental trauma patients with femur fracturesvariablenumber of abusespercent abuse ( 95% ci)number accidental traumapercent accident ( 95% ci)odds ratio ( 95% ci)p valuecurrent polytrauma37/7052.8% ( 41.2% , 52.9%)11/1397.9% ( 3.4% , 12.4%)13.0 ( 6.1 , 28.1 ) < 0.001*physical and/or radiologic evidence of prior trauma44/7062.3% ( 51.5% , 74.2%)6/1394.3% ( 0.9% , 7.7%)37.5 ( 14.8 , 94.7 ) < 0.001*history suspicious for abuse23/7032.9% ( 21.9% , 43.9%)6/1394.3% ( 0.9% , 7.7%)10.8 ( 4.3 , 27.5 ) < 0.001*raw number in parentheses ; p values generated by yates chi square test for independence ; * statistically significant.table 3comparison of femur fracture location in child abuse and accidental trauma patientsvariablenumber of abusespercent abuse ( 95% ci)accidental traumapercent accident ( 95% ci)odds ratio ( 95% ci)p valueproximal femur14/7020.0% ( 10.6% , 29.4%)19/13913.7% ( 8.0% , 19.4%)1.5 ( 0.7 , 3.3)0.33femoral diaphyseal32/7045.7% ( 34.0% , 53.4%)92/13966.1% ( 58.3% , 74.1%)0.4 ( 0.2 , 0.8)0.007*distal femur26/7037.1% ( 25.8% , 48.5%)28/13920.1% ( 13.5% , 26.8%)2.3 ( 1.2 , 4.4)0.01*raw number in parentheses ; p values generated by yates ; chi square test for independence ; odds ratios compare patients who are positive for each fracture type as the outcome and abuse as the risk factor ; * statistically significant . demographic information for child abuse and accidental trauma patients for age , gender , and insurance status * chi square test with yates correction ; mann whitney u test ; fisher s exact test ; * * comparison of current polytrauma , prior trauma , and history plausibility in child abuse and accidental trauma patients with femur fractures raw number in parentheses ; p values generated by yates chi square test for independence ; * statistically significant . comparison of femur fracture location in child abuse and accidental trauma patients raw number in parentheses ; p values generated by yates ; chi square test for independence ; odds ratios compare patients who are positive for each fracture type as the outcome and abuse as the risk factor ; * statistically significant . finally , we used a multiple logistic regression model to calculate a prediction rule for the chance of abuse given the aforementioned risk factors . all of the variables that were significant in any portion of the study were used . in addition , variables previously judged important were included in the initial multiple logistic regression model . all variables were entered and criteria of 0.10 by the 2 log likelihood method was used for removal of variables . in the backward regression analysis , only age younger than 18 months , physical and/or radiographic evidence of prior injury , and history of plausibility were predictive of an abusive etiology for child abuse . in this model , patients with a femur fracture whose age was younger than 18 months had a 10 times greater chance of being the victim of abuse than older children . patients who had the presence of prior trauma on physical and/or radiographic examination had a 16.8 times greater chance of having been a victim of abuse than patients who had no such findings . finally , patients with a suspicious history had a 7.4 times greater chance of having been abused than patients with a plausible history after considering the other factors in the model . given these findings , a new simpler logistic regression model was built based on the outcome of the first model ( table 4 ) . the variables entered were whether the patient had one , two , or three risk factors ( risk factors from the first model : age younger than 18 months , physical and/or radiographic evidence of old trauma , and history suspicious for abuse ) . this final model did not differ ( p = 0.69 ) from the old model using the 2 log likelihood method of comparison.table 4multiple logistic regression model with number of risk factors ( risk factors : age younger than 18 months , physical and/or radiologic evidence of prior trauma , and suspicious history)variablebeta statisticodds ratio ( 95% confidence interval)p valueone risk factor2.07.2 ( 2.223.5 ) < 0.001*two risk factors5.0155.5 ( 41.6581.0 ) < 0.001*three risk factors5.6273.0 ( 28.12649.0 ) < 0.001 * * statistically significant . multiple logistic regression model with number of risk factors ( risk factors : age younger than 18 months , physical and/or radiologic evidence of prior trauma , and suspicious history ) * statistically significant . a simple model using number of risk factors ( age younger than 18 months , physical or radiographic evidence of prior injury , and suspicious history ) predicted abuse in this age group . odds ratios were calculated based on the logistic regression model versus a patient with no risk factors ( table 4 ) . the logistic regression equation was then solved for each number of risk factors to develop a prediction tool . in our population , patients with no risk factors had a 4% chance of having abuse as the etiology for their femur fracture , patients with one risk factor had a 24% chance abuse was the etiology of their femur fracture , patients with two risk factors had an 87% chance abuse was the etiology of their femur fracture , and patients with three risk factors had a 92% chance abuse was the etiology of their femur fracture ( fig . 1the figure shows our algorithm for determining whether a femur fracture stems from abuse or accidental trauma based on our regression model . the figure shows our algorithm for determining whether a femur fracture stems from abuse or accidental trauma based on our regression model . pediatric femur fractures are the most common musculoskeletal injury requiring hospitalization [ 5 , 20 ] , and their presence in children raises suspicion for abuse [ 2 , 5 , 8 , 11 , 20 , 21 , 24 , 28 , 29 ] . in fact , it has been proposed that one - third of femur fractures in children younger than age 4 years and 80% of femur fractures in children who are not yet walking have an abusive etiology . multiple studies have attempted to classify the demographic and fracture characteristics of children whose fracture may have an abusive etiology , including age ( particularly before walking age or the second birthday ) [ 5 , 9 , 13 , 20 , 28 , 30 ] , radiographic appearance and location of the fracture [ 2 , 15 , 28 , 29 ] as well as elements of the history , physical examination , and socioeconomic background [ 16 , 22 , 32 ] . because femur fractures are a common entity that the orthopaedic clinician will evaluate in the emergency room and are linked to child abuse [ 2 , 5 , 8 , 11 , 20 , 21 , 24 , 28 , 29 ] , it is essential for the orthopaedic clinician to be able to differentiate an abusive from an accidental etiology so that proper multidisciplinary action can be initiated to protect children from an abusive environment . in light of this fact , and the fact that one recent investigation suggests fracture pattern may not be as useful in determining fracture etiology , there exists no prediction algorithm with which to help a clinician assign etiology to an injury . we presumed femur fractures resulting from accidental trauma could be differentiated from femur fractures resulting from abuse using history , physical examination , demographic factors , and radiographic findings , and a prediction model based on these parameters can be generated to allow clinicians to more accurately identify victims of abuse and distinguish abuse from accidental trauma . first , our prediction rule was generated from an urban level i pediatric trauma center . it is unclear if the prediction rule would be different for pediatric patients presenting to lower - level pediatric trauma centers in nonurban environments and/or in different countries . however , our tertiary referral status allows us to say we attract patients from a broad catchment area , hence enhancing our external validity . second , it can be argued our assessment of history status ( consistent versus suspicious ) is subjective . specifically , arguments for mitigating individual circumstances such as unwitnessed injury could be due to a momentary lapse in supervision ; delay in seeking care could be due to lack of health insurance or a child who is not expressing substantial discomfort . however , our criteria for suspicion are consistent with previous reports [ 6 , 27 , 30 ] . rater was instructed to look at only the history and physical records to determine the patient history without viewing other aspects of the medical record , she was not strictly blind to the scan status of the patient because she had access to the medical record . the use of corroborating investigators with a high intraclass correlation coefficient somewhat mitigates this risk . third , as a result of the fact that child abuse is largely a social diagnosis , there exists no test that can confirm the presence or absence of child abuse with a high level of specificity or sensitivity . yet , we believe evaluation by independent specialists constitutes the best method available in the current literature . inclusion in the scan database is determined by an independent group of social workers , nurses , and physicians who all have advanced training in child abuse ( and are independent from the authors ) at the time the child presents to the emergency room , and is also used for a clinical purpose ( ie , activating child protective interventions ) . as with any study of child abuse , the study risks circuitous logic , that is to say , child abuse is often diagnosed by suspicious history , physical examination findings , and other social factors . however , this could be said about any condition that is a clinical diagnosis ; as such , we believe guidelines for diagnosis of this condition are helpful . the use of age , history , physical findings , and radiographic findings to assess for child abuse is not new ( table 5 ) . although these multiple characteristics are essential in providing a general picture of a pediatric patient presenting with child abuse , the question arises as to how these characteristics can be applied in a quantifiable , objective manner to help to reliably predict etiology for a clinician . whereas other studies have limited their analysis to descriptions of abusive and accidental femur fracture , the prediction model developed from our study allows for research to be translated into clinical action . of the multiple demographic characteristics in our study , the multiple regression model identified the following three predictors after accounting for other confounders when differentiating abusive from accidental femur fractures in this age group : ( 1 ) age younger than 18 months ; ( 2 ) physical and/or radiographic evidence of prior trauma ; and ( 3 ) history suspicious for abuse clinicians can use this rule in the following fashion : patients with no risk factors have a 4% chance of having a femur fracture stemming from child abuse ; patients with one risk factor have a 24% chance ; those with two risk factors have an 87% chance ; and those with three risk factors have a 92% chance of having a femur fracture stemming from abuse.table 5risk factors found to be important or used to help diagnose child abusefirst authorage as a risk factor for abusehistorical featuresphysical examination featuresradiographic or other featuresanatomic locationcoffey et al . 67% ( extremity injuries were in patients younger than 18 months)n / an / an / anot available within specific fracture groupsrex and kay 92% ( younger than 1 year)n / an / an / ano difference in site of fracture ( proximal / middle / distal)schwend et al . 42% of children younger than walking age with femur fracturessuspicious , inconsistent history or delayed presentation used to characterizebruises or polytraumamultiple fracturesall were shaft fracturesleventhal et al . 60% ( younger than 1 year femur fractures)suspicious history , change in behavior or medicaid payornone listed for femursnone listed for femursnone listed for femur fracturespierce et al . 50% younger than 1 year with probable abuse.suspicious , inconsistent history or delayed presentationbruises or multiple injuriesmultiple fracturesno specific pattern , study speaks in terms of fracture plausibilityloder and bookout 15% ( younger than 2 years)*n / an / an / an / ascherl et al . 13% of total cohort , but average age of confirmed abuse 0.83 yearssuspicious historybilateral injuries worked up more often , but only 1/7 were positiveassociated injuriesno particular distributionfong et al . younger than 3 years old ( all fractures)suspicious , inconsistent history or delayed presentationnone specifiedfractures in various stages of healingno anatomic distribution noted within femur fracturesworlock et al . 80% abusive fractures in those younger than 18 monthsno historical information availablebruising of the head and neckrib fractures in the absence of chest trauma , multiple fractures50% metaphyseal chip in the femur in abuse patients ; in other long bones , spiral or oblique fractures more commonbaldwin et al . [ current study]90% abused children younger than 18 monthsinconsistent or suspicious history , or delayed presentationconsistent with other injuries or prior injuryconsistent with prior injurydistal more often abuse shaft more often accident * based on the 2000 kids inpatient database ; n / a = no available data in the study . risk factors found to be important or used to help diagnose child abuse * based on the 2000 kids inpatient database ; n / a = no available data in the study . it is essential for clinicians to be able to differentiate the etiology of pediatric femur fractures as stemming from abuse or accidental trauma . not only are femur fractures a common injury seen by orthopaedic clinicians , but identification of the etiology of the injury is vital to ensure proper multidisciplinary intervention can be initiated for the safety of the child . prediction based on the multiple logistic regression model that was developed in our study can help determine the etiology of pediatric patients presenting with a fractured femur . although further studies will be necessary to validate the rule prospectively , we believe the rule is a good common sense approach to a young patient with a femur fracture . we believe this method will help all clinicians determine whether child abuse occurred and thus enhance their approach to management .

backgroundchild abuse represents a serious threat to the health and well - being of the pediatric population . orthopaedic specialists will often become involved when child abuse is suspected as a result of the presence of bony injury . distinguishing abuse from accidental trauma can be difficult and is often based on clinical suspicion.questions/purposeswe sought to determine whether accidental femur fractures in pediatric patients younger than age 4 could be distinguished from child abuse using a combination of presumed risk factors from the history , physical examination findings , radiographic findings , and age.methodswe searched our institution s scan ( suspected child abuse and neglect ) and trauma databases . we identified 70 patients in whom the etiology of their femur fracture was abuse and compared that group with 139 patients who had a femur fracture in whom accidental trauma was the etiology.resultsa history suspicious for abuse , physical or radiographic evidence of prior injury , and age younger than 18 months were risk factors for abuse . patients with no risk factors had a 4% chance , patients with one risk factor had a 29% chance , patients with two risk factors had an 87% chance , and patients with all three risk factors had a 92% chance of their femur fracture being a result of abuse.conclusionsclinicians can use this predictive model to guide judgment and referral to social services when seeing femur fractures in very young children in the emergency room.level of evidencelevel iii , diagnostic study . see guidelines for authors for a complete description of levels of evidence .

texture is one of the important features characterizing many natural and man - made images . texture characterization and analysis are usually performed according to the spatial as well as frequency variations of brightness , pixel intensities , color , and texture orientation in the different regions of the image corresponding to different types of textures . for example , the roughness or bumpiness of an image usually refers to variations in the intensity values , or gray levels . texture segmentation , recognition , and interpretation are critical for human visual perception and processing . as a result , a large number of approaches has been proposed for texture classification and segmentation [ 116 ] . in general , texture analysis methods fall into two categories : statistical methods which analyze the fourier power spectrum , gray level values , and various variance matrices of the input image , and structural methods which are knowledge - based algorithms with an emphasis on the structural primitives and their placement rules . some examples of such methods include markov random field models [ 17 , 18 ] , simultaneous autoregressive model , and fractal models . among many existing approaches , local variation minimization has been a popular and powerful technique in image analysis with applications to the texture modeling . more recently , multiresolution approaches have become more important in texture analysis [ 19 , 2426 ] , where fixed - size neighborhood and window size are used to derive features at varying scales corresponding to the input image at different resolutions . in general , the total texture extraction has become a mature technique in real applications . however , despite the progress in the past few decades , selective extraction of entangled textures encounters a number of difficulties . another difficulty is due to gray - scale entanglement , especially the near - continuous merging of various textures . the other difficulty is due to frequency entanglement when two similar but different textures share overlapping frequency band in the frequency domain . this difficulty would especially plague texture analysis when many high - frequency textures coexist . in this work , we propose an adaptive partial differential equation ( pde ) transform approach for selective extraction of entangled textures . by using arbitrarily high - order pdes , the pde transform is able to decompose signals , images , and data into functional modes , which exhibit appropriate time - frequency localizations [ 2731 ] . additionally , the pde transform is able to provide a perfect reconstruction . unlike wavelet transform or fourier transform , the pde transform offers results in the physical domain , which enables straightforward mode analysis and secondary processing . based on the image mode functions generated by the pde transform method , the adaptive pde transform algorithm calculates the variance of the local variation of the image mode functions followed by the corresponding thresholding analysis . in the past two decades , pde - based image processing approaches have raised a strong interest in the image processing and applied mathematical communities and have opened new approaches for image denoising , enhancement , edge detection , restoration , segmentation , and so forth . the use of pdes for image analysis started as early as 1980s when witkin first introduced diffusion equation for image denoising . the time evolution of an image under a diffusion operator is formally equivalent to the lowpass filter . after perona and malik introduced anisotropic diffusion equation in 1990 , nonlinear pdes have found great applications for a variety of image processing tasks such as edge detection and denoising . two important advances in the history of image processing , namely , the perona - malik equation and the total variation methods , employ second - order nonlinear pdes for image analysis . the willmore flow , proposed in 1920s , is a fourth - order geometric pde and has also been used for surface analysis . in the past decade , fourth - order nonlinear pdes arbitrarily high - order nonlinear pdes were introduced by wei in 1999 to more efficiently remove image noise in edge - preserving image restoration : ( 1)ut(r , t)=q[dq(u,|u|)2qu ] + e(u,|u| ) , ( q=0,1 , ) , where u u(r , t ) is the image function , dq(u(r ) , |u(r)| , t ) and e(u(r ) , |u(r)| , t ) are edge - sensitive diffusion coefficients and enhancement operator , respectively . the perona - malik equation is recovered at q = 0 and e(u(r ) , |u(r)| , t ) = 0 . as in the original perona - malik equation , the hyperdiffusion coefficients dq(u(r ) , |u(r)| , t ) in ( 1 ) can be chosen in many different ways . for instance , one can set ( 2)dq(u(r),|u(r)|,t)=dq0exp[|u|22q2 ] , where the values of constants dq0 depend on the noise level , and 0 and 1 are chosen as the local statistical variance of u and u : ( 3)q2(r)=|ququ|2 ( q=0,1 ) . the notation y(r) above denotes the local average of y(r ) centered at position r. in this algorithm , the statistical measure based on the variance is important for discriminating image edges from noise . as such , one can bypass the image preprocessing , that is , the convolution of the noise image with a test function or smooth mask . in general , the nonlinear pde operators described above serve as lowpass filters . pde - based nonlinear highpass filters were introduced by wei and jia in 2002 . , this approach has been combined with wei 's earlier arbitrarily high - order nonlinear pde operator to give ( 4)t(umvn)=(j=0m1duj2jum,vnum,j=0n1dvj2jvn)(umvn ) , where um um(|um| ) and un un(|vn| ) are made edge sensitive . as lowpass filters , both duj duj(|um| ) 0 and dvj dvj(|vn| ) 0 when j is even . similarly , both duj(|um| ) 0 and dvj(|um| ) 0 when j is odd . we can define a pde transform as ( 5)wm , n(r , t)=um(r , t)vn(r , t)=hmn(r , t)x(r ) , where hmn(r , t ) can be regarded as a coupled nonlinear pde operator . in order for ( 5 ) to work properly , we choose |dvj(|vn|)||duj(|um|)| . as shown in our earlier work , by increasing the order of the highest derivative , one can increase frequency localization and accuracy of the pde transform for mode decomposition . the frequency selection of wm , n(r , t ) also depends on the evolution time . high - order pdes are integrated by using the fourier pseudospectral method . in the pde transform , intrinsic mode functions w are systematically extracted from residues x , that is , ( 6)wmnk = hmnxmnk , k=1,2 , , where wmn is the kth mode function . here , the residue function is given by ( 7)xmnk = xmn1j=1k1wmnj , k=2,3 , , where xmn = x(r ) . therefore , x = j=1wmn + xmn is a perfect reconstruction of x in terms of all the mode functions and the last residue . the mode decomposition algorithm given in ( 6 ) is inherently nonlinear , even if a linear pde operator might be used . the pde transform is applied to figure 1(a ) to extract the three textures in figures 1(b ) , 1(c ) , and 1(d ) . note that only one texture is isolated at each time , which means the proposed pde transform is able to perform a controlled or selective segmentation of textures . the pdes of up to order 200 have been used for the selective texture segmentation . numerically , such high - order linear pde needs to be solved in the frequency domain . due to the ideal frequency localization the separation of textures that are highly entangled in spatial locations , frequency ranges , and gray scales become a challenge , and conventional segmentation techniques are in general not applicable for such cases . for example , highly oscillatory textures can be separated from slowly varying background but can not be separated from another texture with overlapping frequency distribution purely based on frequency fingerprints . to selectively distinguish such entangled textures of high frequency , one needs a mode decomposition algorithm that is able to be highly localized in frequency . however , the pde transform by itself does not perform well for the separation of entangled textures . to this end the essence of the adaptive pde algorithm lies in the realization that features of various textures are closely correlated with both the magnitude and smoothness of the gray - scale values , or , equivalently , the local variation of the image mode functions . nonlinear pdes have been widely applied to detect images with noises . however , despite better image edge protection , the nonlinear anisotropic diffusion operator may still break down when the gradient generated by noise is comparable to image edges and features . application of a preconvolution with a smoothing function to the image can practically alleviate the instability and reduce gray - scale oscillation , but the image quality is often degraded . one alternative solution introduced by wei is to statistically discriminate noise from image edges by a measure based on the local statistical variance of the image or its gradient . such a local statistical variance based edge - stopping algorithm was found to work very well for image restoration . similar statistical analysis can be employed to perform selective texture extraction for images containing highly entangled and overlapping textures . in the present approach , we first compute the local variation of each pixel of the image mode functions obtained by the high - order pde transform . unlike the total variation , the local variation is still a function , of which the variance can be calculated : ( 8)e(x(r))=||xk(r)||xk(r)||2 , where x(r ) is the kth mode function obtained by the pde transform ( 7 ) , and |x(r)| is evaluated locally over the neighbor pixels . equation ( 8) yields a statistical analysis which is used for various texture separation and segmentation with appropriate threshold values . various threshold values need to be chosen to select the range of the variance corresponding to the particular texture of interest . a flowchart of the adaptive algorithm of pde transform is shown in figure 2 . figure 1(e ) shows the edge mode obtained by applying the pde transform to figure 1(a ) . figure 1(f ) shows the variance of the local variation of gray scale calculated using the adaptive pde transform . figures 1(g ) and 1(h ) show the projection , or average , of the variance in figure 1(f ) along x- and y - direction , respectively . by slicing out different domain of the variance in figure 1(f ) , three different textures in figures 1(b) 1(d ) are then perfectly separated from each other . in this section , the adaptive pde transform is applied to three different cases to illustrate its superior capability of selective texture separation . figures 5(a ) and 6(a ) contain spatially segmented textures overlapping in the frequency domain . figure 7 contains textures with overlapping textures highly entangled in both the frequency and spatial domains . the adaptive pde transform method employing the variance of the local variation of the image mode functions is applied to several benchmark test cases . in particular , separation of text and texture the separation of english title from both background image and chinese characters is a challenging task in terms of texture analysis because of the high degree of entanglement of very similar textures . due to the font size difference in this application , high - order pde transform plays an extremely important role in differentiating modes with slightly different frequency characteristics . in figure 3(b ) , the pde transform successfully suppresses the low - frequency parts and extracts the mode with frequency band mainly corresponding to texts . a suitable threshold value is used to cut off the region with low variance and yields only the texts as shown in figure 3(c ) . the present algorithm of selective texture extraction is also tested on one of the most widely used images , the barbara , in figure 5 . it contains fine details of different textures such as the table cloth , curtain behind barbara , scarf , and clothes on her . distinctions between all these textures and the background are much larger than those among these textures , which leads to the difficulty of selective texture separation and segmentation . due to the tiny difference between the frequency or spectrum features of different textures mentioned above , a highly frequency - selective separation method is required . however , the conventional fourier method is not applicable for this case since the textures are entangled in the frequency domain . moreover , conventional statistical segmentation approaches do not perform well for this case due to the gray - scale entanglement . the present adaptive pde transform method performs well for the selective texture extraction in the barbara image . the total texture , or image edge , is extracted from the high - frequency mode of the pde transform as shown in figure 5(b ) . the variance of the local variation is shown in figure 4 , which is calculated and employed for selective texture extraction and separation with appropriate thresholding values . the resulting textures are shown in figures 5(c)5(f ) which correspond to those of clothes , curtain , and table cloth , respectively . the four textures in figure 5 are superimposed on the original image for the purpose of a clearer visualization . in figure 6 , the present adaptive pde transform is applied to detect a sniper hidden in the forest ( figure 6(a ) ) . the whole image is composed of highly entangled textures . the boundaries between these textures are very challenging to be identified appropriately . in our approach , variance of the local variation is calculated and used for texture separation as in the previous examples . by appropriate thresholding , the variance can be decomposed into three regions corresponding to those of the forest , the tree trunk , and the sniper . the resulting texture modes are shown in figures 6(b)6(d ) . in the previous introduction to the adaptive pde transform algorithm and applications , local variation is defined and calculated for the intensity of image mode functions to selectively extract textures beyond the total texture extraction . the selective texture extraction can be generalized to indicate any spatial parts of the image characterized with specific ( and usually functionally important ) spatial orientation and/or frequency oscillation , such as different parts in the neuron synapses , brain cells , and retina vasculatures . in figure 7(a ) , the image of a typical neuron is shown . with advanced imaging techniques made available , research scientists have been able to obtain more and clearer 2d images and 3d data of various neuron cells and networks , whose study will be important for identifying the relation between phenotype and genotype patterns in physiology and molecular biology . closely related to the advancement in the experimental imaging techniques , various improved computational image processing techniques have been proposed to better analyze neuron images . neuron morphology study has become more and more important since the shape and branching of dendrites in neurons are closely related to the structure and functioning of the neuron network . advancements in both experimental imaging techniques and computational image enhancements have led to better visualization and exploration of neuron morphology [ 3945 ] . in the study of neuron morphology , image processing and segmentation of cultured neuron skeletons provide details of how neuron grow and branches . in this work , we apply the adaptive pde transform to the study of natural neuron skeleton to segment and classify neuron skeletons into desirable classes according to the spatial extension and frequency oscillation of neuron dendrites , very much like the way of dividing a total image texture into several selective fine textures . such separation and classification enable secondary processing and analysis of neuron morphology , such as the computation of surface areas ( for 2d images ) or volumes ( for 3d data ) for different classes of neuron skeletons . specifically , we aim to separate different parts , or textures , such as soma , dendrites , axon , terminal or lobe , and numerous ramifications , from the neuron imaging as shown in figures 7(b)7(d ) , where three classes of neuron parts are separated according to the spatial extension and frequency oscillation . ratios of these surface areas and many other geometric ratios of neuron morphology are related , on both molecular and cellular levels , to the many physiological diseases as well as the classification of neuron synapses . selective extraction and separation of image textures involving spatial entanglement , gray - scale mixing , and high - frequency overlapping are challenging tasks in image analysis . in this work , we introduce an appropriate adaptation to our earlier partial differential equation ( pde ) transform to construct an adaptive pde transform algorithm . the adaptation is realized via a proper thresholding with the statistical variance of the local variation of image functional mode functions . the present pde transform enables one to decompose and separate modes with entanglement in both spatial and frequency domains . textures of very similar features in the same image are successfully decomposed and separated using the present adaptive pde transform method .

texture and feature extraction is an important research area with a wide range of applications in science and technology . selective extraction of entangled textures is a challenging task due to spatial entanglement , orientation mixing , and high - frequency overlapping . the partial differential equation ( pde ) transform is an efficient method for functional mode decomposition . the present work introduces adaptive pde transform algorithm to appropriately threshold the statistical variance of the local variation of functional modes . the proposed adaptive pde transform is applied to the selective extraction of entangled textures . successful separations of human face , clothes , background , natural landscape , text , forest , camouflaged sniper and neuron skeletons have validated the proposed method .

parkinson 's disease ( pd ) is the most frequent neurodegenerative disorder in europe , with a prevalence of 1/1,000 in the general population and 1.5% in subjects more than 65 years of age . it originates in destruction of the dopaminergic nigrostriatal circuit ; and it is manifested in parkinson 's syndrome , which entails cognitive and psychic complications . in fact , depression is frequent in this disease , with an average prevalence of 40% . many other neuropsychiatric subcortical manifestations have been described in the literature [ 35 ] , but apathy is among the most frequent : current estimates of its prevalence in parkinson 's disease vary between 16.5% and 42% [ 6 , 7 ] . apathy refers to a wide - ranging behavioural , emotional , and motivational constellation including reduced interest and participation in normal purposeful behaviour , lack of initiative with problems initiating or sustaining an activity to completion , lack of concern or indifference , and affective flattening [ 8 , 9 ] . with that said , apathy syndrome may be partially secondary , with regard to dysfunction of a fronto - subcortico - striato - thalamo - cortical loop [ 8 , 1012 ] . furthermore , these circuits are in all likelihood the same as those involved in the motor and cognitive dysfunction typical of parkinson 's disease [ 10 , 13 , 14 ] . in fact , apathy may be considered as a multicomposite entity consisting in dysfunction of associative and limbic loops and accompanied by emotional and motivational aspects . in most cases of apathy , the emotional and motivational dimension arises from intricate links between the generally unconscious mobilization of attentional resources and their purposeful utilization , in which the automatic attentional process assumes a major role . have underscored the basic role of the prefrontal cortex ( particularly the dorsolateral prefrontal cortex ) in attentional process . their studies were primarily based on analyses of patients with frontal lobe injury ( with chronic infarction in the dorsolateral prefrontal cortex ) in which subjects were made to view repetitive frequent ( for voluntary attention ) and infrequent ( for automatic attention ) background stimuli : they demonstrated a correlation between decrease of attentional level , prefrontal lesions , and increase of apathy level ( evaluated with marin apathy scale ) . another study showed a strong correlation between decrease of the electrophysiological attentional marker p300a ( novelty p3 ) and increase of apathy . the same correlation between p300a and apathy has been found in alzheimer 's disease and in cerebral trauma with selective lesion of prefrontal cortex , as illustrated in functional cerebral imaging studies we nonetheless wish to hypothesise that , in parkinson 's disease , there exists a correlation between increase of apathy level and decrease of p300a amplitude , which constitutes an electrophysiological marker of automatic attention . twenty - five patients ( 13 women and 12 men ) , hospitalized in the department of neurology ( chu poitiers ) for evaluation of parkinson 's disease , were included with their agreement ; written informed consent for research purposes was obtained for each patient . the general characteristics of this population were age = 64.1 6.4 years ; duration of pd = 11.5 4.8 years ; daily dose of dopa therapy = 1567.8 725.4 mg ; total updrs score during off period = 25.8 11.8 ; total updrs score during on period = 9.0 7.1 ; hoehn and yahr score = 2.5 0.4 . patients presented with the usual parkinson 's disease criteria . the severity of the disease was evaluated with the hoehn and yahr score , which maps out stages from i through v . the daily dose of dopa therapy was calculated by addition of a daily dose of levodopa and the dopaminergic agonists transcribed as dose - equivalent dopa [ 21 , 22 ] . patients with dementia ( mmse < 24 ) or with serious depression , melancholy , or depression with delirium ( according to the diagnostic and statistical manualcriteria ) were excluded from the study . each subject included in the study was evaluated with cognitive , motor , psychiatric , and subsequent electrophysiological tests during off ( without dopaminergic treatment ) and on ( with dopaminergic treatment ) periods . the study took into account the fluctuating characteristics of pd over the daytime . each period ( on or off ) was selected alternatively in order to have as many patients during the on period as during the off period ( 1 subject ) . only patients able to withstand the off period , or without important dyskinesia in the on period , were included in the study . patients were analyzed during the off period , before taking a levodopa test , patients stopped their treatments the previous evening , and the study began at 8:30 the following morning ( prior to the levodopa test ) . for each patient , the study took place over 3 days ( d0 , d1 , and d2 ) ; the order of tests was systematically the same for all patients . the cognitive assessment included 4 tests systematically taken in 30 minutes in the following order ( in both on and off periods ) : verbal fluency test , stroop test , wisconsin sorting card test , and fab ( frontal assessment battery ) test . in the verbal fluency test , patients were asked to name in one minute as many items as they could from one semantic category ( animals ) and then as many words as they could beginning with the letter m ( one minute ) ; category switching ( boys ' names and fruits ) was then assessed ( one minute ) . in an alternative form , the last one of these verbal fluency tests was used for evaluation of mental flexibility . the stroop test was used for evaluation of inhibitory control . with the wisconsin card sorting test ( wcst ) , we collected data ( number of series , number of mistakes , and perseveration percentage ) in order to evaluate mental flexibility . finally , the frontal assessment battery ( fab ) was used for the purposes of general evaluation of executive functions . after day 0 ( d0 ) assessment , we applied the updrs iii scale and then the hoehn and yahr score , for motor evaluation of pd . in order to assess apathy , we used the starkstein apathy scale . as a variant of the marin apathy scale each item was rated from 0 to 3 so as to calculate an apathy score ranging from 0 to 42 . cut - off score was 14 with 66% sensitivity and 100% specificity . in our study , only patients with score > 14 were considered apathetic . for purposes of psychiatric evaluation , we used the dsm ( diagnostic and statistical manual ) criteria ( d0 ) and the had scale ( d1 and d2 ) . the had scale ( hospital anxiety and depression scale ) was also used during the on and off periods in order to quantify the depression and anxiety levels . we calculated a depression and anxiety score with cut - off > 7 for each of two categories . each patient was subjected to the recording of long - latency auditory evoked potentials according to a previously described protocol [ 3133 ] , and according to the guidelines for event - related potentials ( erp ) , 140 tone bursts ( intensity : 80 db ; duration : 20 ms ; 0.9 ms rise - fall time ) were presented binaurally through earphones at a rate of 1 tone every 0.8 sec , including 100 low frequency sounds ( 1000 hz ) , 20 high target sounds ( 2000 hz ) , and 20 randomized unexpected novelty stimulations ( the word airplane in french , i.e. , avion ) . a disc electrode was affixed to the midline site cz and referred to linked mastoids ; responses to rare and frequent stimuli were averaged separately . the curves were numerically filtrated ( high - pass filter : 0.5 hz ; low - pass filter : 15 hz ) . the horizontal and vertical bipolar electrooculogram ( eog ) was recorded during the task to monitor artefacts . trials with artefacts were automatically excluded from the averages : if any data point ( beyond the first 2.5 ms of the sweep ) was greater than 96% full scale , the entire sweep was rejected and was not added to the memory block . after this first run , all subjects were able to distinguish high - pitched from low - pitched tones and were then instructed to keep a mental record of the rare tones and to report their number at the end of the run ( active condition ) . the p300 wave ( target p3 ) was clearly identifiable only in the active condition in response to rare 2000 hz tones ; this condition was used in measurement of amplitude and latency of n200 and p300 potentials . the p300a ( novelty p3 ) was clearly identifiable in response to the randomized unexpected novelty stimulation ( word avion ) . latency values were calculated from the intersection of extrapolated lines from the ascending and descending slopes of each peak . latencies of n200 and p300 were determined for each subject , as well as the peak - to - peak amplitude of n200-p300 . latency and maximum amplitude of p300a were identified in an interval ranging from 250 ms to 360 ms after stimulation ( on fz , cz , and pz electrodes ) . in addition , average p300a amplitude was calculated in an interval varying from 250 to 360 ms after stimulation and was evaluated in comparison with the basal line ( 60 ms before the stimulation ) . correlations between apathy score ( starkstein apathy scale ) and each feature ( electrophysiological , cognitive , psychiatric , and motor ) were carried out independently ( for data during on and off periods ) with correlation index ( nonparametric spearman 's test ) . the roles of dopaminergic innervation ( reflected by the percentage of improvement after updrs iii score ) or of nondopaminergic lesions ( reflected by the residual updrs iii score in on period ) on the different features ( electrophysiological , cognitive , psychiatric , and motor ) were determined with a matched nonparametric signed - rank wilcoxon test ( z ) with regard to averages during the on and off periods for each feature . differences for each feature ( electrophysiological , cognitive , psychiatric , and motor ) between apathetic and nonapathetic subjects ( a cut - off score was established at 14 ) were determined by using a nonparametric mann - whitney u test for quantitative data and a chi - square test ( corrected chi - square test and exact fisher 's test , according to the conditions of application ) for qualitative data . if a significant correlation between the apathy score and an electrophysiological data appeared , this electrophysiological variable is further dichotomized ( median as cut - off value ) for subsequent logistic regression to predict high or low value of electrophysiological data , apathy score , and other potential confounding factors . after univariate data screening , an ascending stepwise procedure for multivariate analysis was used with significant p values at 10% level to include it into logistic regression . correlations between electrophysiological data and the other variables were calculated using the spearman nonparametric correlation coefficient . the risk was 5% and the statistical tests were carried out in bilateral situations . the software we used for data capture and analysis was statview 5.0 ( from sas institute ) . the apathy score was correlated , during off periods , with a decrease of p300 amplitude in cz and pz locations and with average p300 amplitude in cz and fz locations ( see table 1 and figure 1 ) . during on periods , there was a significant decrease of the amplitude of p300a wave ( in fz ) in apathetic subjects ( see table 1 and figure 2 ) . during off periods , there was a tendency towards decrease of p300a amplitude in fz ( statistically insignificant : p = 0.0707 ) . the apathy score was positively correlated , on the one hand with alteration of mental flexibility ( wcst and verbal fluency test ) and on the other hand with severity of executive dysfunctions ( fab ) during on and off periods , whereas there was no correlation with disturbed inhibitory control ( explored by the stroop test ) ( table 1 ) . in the entire studied population ( 25 patients ) , 6 patients ( 24% ) were depressive according to the dsm criteria , independently of on or off period . the apathy score was positively correlated with the depression score ( had depression ) , during on and off periods , whereas anxiety was not correlated with apathy ( table 1 ) . lastly , the apathy score was correlated with the treated motor score ( total updrs iii during on period ) and the axial updrs iii score . during the off period , the apathy score tended to be correlated with the axial updrs iii score ( statistically insignificant : p = 0.0594 ) ( table 1 ) . the dopaminergic treatment had no consequence on the latency of p300 and p300a amplitude ( in all localizations ) , but it brought about an electrophysiological modification by increasing n200 amplitude during the on period ( p = 0.045 ) . after administration of dopaminergic treatment , the apathy score was significantly lower ( p = 0.0394 ) , whereas there was no significant modification for the other cognitive data . there existed a dopaminergic effect on anxiety ( p = 0.0348 ) but not with regard to depression ( p = 0.3325 ) . lastly and logically , the motor score improved with dopa therapy ( p < 0.0001 ) . during off periods ( table 2 ) , no electrophysiological feature showed a detectable difference between apathetic and nonapathetic patients , but average p300a amplitude ( in fz ) tended to decrease in apathetic subjects ( statistically insignificant : p = 0.0592 ) . on the other hand , apathetic subjects during off period presented with alteration of mental flexibility ( verbal fluency and wcst ) and impairment of executive functions ( fab ) and a higher , more significant depression score ( had depression ) than in nonapathetic subjects . finally , the motor axial score ( axial updrs iii ) and patient age were higher in apathetic subjects ( during off period ) . during on period ( table 3 ) , apathetic patients in on showed a significant decrease of average p300a amplitude ( in fz ) ( figure 3 ) and an increase of n200 latency ( in cz ) . mental flexibility ( wcst ) was lower in apathetic patients during on . apathetic patients during on recorded higher significant depression ( had depression ) and anxiety scores ( had anxiety ) . no modification was observed for motor score or age during on period . following the correlation highlighted by spearman 's test between p300a amplitude ( in fz and on period ) and other variables , the logistic regression was applied only for four variables ( p 0.01 ) : age , verbal fluency ( letter m ) , fab , and apathy score ( starkstein ) . results indicated that apathy is the only variable correlated with the decrease of the amplitude of p300a ( in fz area and on period ) ( table 4 ) . to the best of our knowledge , this study is the first to explore characteristics of apathy in pd by analysing the role of a dysfunction in automatic attention and through use of a neurophysiological approach designed to test patients ' capacity of automatic reactivity to an unexpected sound . the first wave collected ( p300a ) is considered to be a reflection of the automatic attentional process . we have shown a correlation between the apathy score and the decrease of p300a amplitude ( only in fz localization ) and between apathy and treated motor score , severity of frontosubcortical cognitive alteration ( mental flexibility and executive functions ) , and depression . lastly , the dopaminergic treatment increased n200 amplitude ( in pz ) and lessened apathy . the decrease of p300a amplitude in pd is in agreement with other studies , one of which showed , in treated pd , a decrease of p300a amplitude with regard to frontal disturbances . our result nonetheless differs on account of its selectivity : it is observed only during on period ( but with a pronounced tendency during off period ) and it is correlated with the apathy score ( not explored in previous studies ) . the decrease of p300a amplitude has in fact been reported in other studies using a similar electrophysiological protocol , but with a solely visual modality . in patients with alzheimer 's disease , decreased p300a amplitude has been shown and was more pronounced in those with a higher apathy level . p300a wave abnormality was found to be maximal in the central area and interpreted as a consequence of a specific dysfunction of the automatic attentional process ( and not as a consequence of the cognitive disturbance ) . our results consequently suggest , in pd during on period ( and off period ) , the existence of a disturbance of automatic attention as one possible precursor of apathy . this result is in agreement with the hypothesis of marin : apathy could be the partial consequence of a deficit in mobilization of the appropriate attentional resources in a new or unusual situation . in this study , data obtained by linear regression analysis showed that , independently of other data , decreased p300a amplitude predicts severity of the apathy score , thereby confirming this hypothesis . if this hypothesis is admitted , how can we explain the lack of correlation between the apathy score and a modification of the p300a wave during the on period , whereas it is present during off period ? moreover , varied and at times contradictory results with regard to the effect of dopaminergic treatment on p300 and p300a waves have been reported : shortened or unmodified p300 latency with dopa therapy , lengthened p300 latency with dopaminergic agonists , or decrease of p300 amplitude with dopa therapy after acute levodopa and dopaminergic agonist treatment . the improved apathy score with dopaminergic treatment corroborates the results of starkstein et al . and of researchers exploring the influence of dopaminergic treatments on motivation . in a population of 30 nondepressive patients with pd ( without dementia ) , these authors have shown , in comparison with control subjects , that apathy or lessened motivation was more serious during the off period and less serious after the dopaminergic treatment . the clinical result is also coherent with experimental data suggesting a role for dopaminergic mesolimbic innervation in the regulation of the cerebral circuits involved in reward . in these circuits , secretion of dopamine strengthens the signalling of new stimuli for reward , and permits mobilization of attentional resources towards the latter . alternatively , in our work , the hypothesis of a different effect of dopaminergic treatments on attentional process can not be excluded : sensitivity of voluntary attention ( reflected by the p300 wave ) and insensitivity of automatic attention ( likewise reflected by the p300a wave ) for dopaminergic treatment . the results of our study indicate a correlation between the apathy score and severity of frontosubcortical dysfunction . indeed , apathetic patients present executive function disorders ( deterioration of fab ) such as disruption of mental flexibility ( verbal fluency and wcst ) and working memory ( verbal fluency ) . our findings confirm those of pluck and brown , who have shown in their study of apathy in 45 patients with pd a strong link between severity of apathy and disruption of executive functions ( wcst ) and verbal fluency . interestingly , another study using a visual modality of our neurophysiological protocol in patients with frontal lobe injury underscored a correlation between decrease of p300a amplitude and intensity of executive dysfunctions . the authors suggested that prefrontal dysfunction ( particularly dorsolateral prefrontal cortex ) contributed to the disruption of mobilization of attentional resources toward new stimuli . the prefrontal cortex assumes an important role in the appropriate direction of attentional resources toward new stimuli [ 15 , 41 ] . in our patients , executive dysfunctions appear to contribute ( through a similar mechanism suggested by daffner et al . ) to the electrophysiological anomaly we have reported . the frontosubcortical dysfunction probably reflects the progression of nondopaminergic lesions , since it is insensitive to dopaminergic treatment and is correlated with the treated motor score ( reflected by updrs iii in on period ) and the axial motor score during the on period . illustrate one possible mechanism , by showing that apathetic parkinsonian subjects , in comparison with reference subjects , present an attentional disorder ( in situations where attention precedes action ) due to insufficient activation of connections between the prefrontal cortex and premotor cortical areas . and our data strongly suggest that while dopaminergic probably mesolimbic denervation contributes to apathy , nondopaminergic lesions ( bringing about a dysfunction of the frontosubcortical loops probably involving the dorsolateral prefrontal cortex ) likewise play a crucial role . if this is true , how do we explain the results with regard to noncorrelation with the stroop test ? in fact , similar results have been reported in studies concerning healthy [ 43 , 44 ] or parkinsonian subjects . as is the case with ours , these results are compatible with the hypothesis according to which the stroop effect does not apply to perceptive mechanisms but rather to stimulation assessment and response elaboration . and , as we have found in this study , the stroop test requires an adequate attentional level but does not directly necessitate the presence of the loops involved in attentional process . lastly , imaging studies have reported a possible anatomical functional substrate for this hypothesis by showing a lack of activation during the stroop effect this result is similar to the one reported by czernecki et al . who found , in a nondepressive parkinsonian population , using bdi depression scale , a correlation with depression . but it contrasts with the results given by pluck and brown who found no correlation between apathy and depression . furthermore , prevalence of 24% of depression in our study may have reflected a bias . with that said , three arguments counteract this hypothesis : ( a ) prevalence of depression in our population is lower than the average of 40% reported in the literature [ 2 , 48 ] ; ( b ) in this work , the decrease of p300a amplitude ( in fz , during on period ) effectively predicts the severity of apathy score , independently from depression ( table 4 ) ; ( c ) finally , if modifications of p300a wave are reported in depression , they concern mainly a lengthening of latencies , and only melancholy is associated with a decrease of p300a amplitude . in fact , none of our patients present severe depression or melancholy . therefore , these different arguments confirm the postulate that it is possible to distinguish apathy from depression . the absence of a control group and the lack of event - related potential ( erp ) spatial resolution also represent methodological limits . on this subject , some studies have clearly shown modifications of p300a wave with age , but without any observed modification of amplitude . in our case , a control group would have facilitated confirmation that neurophysiological modifications are age independent . it may nonetheless be difficult to explain this result because , in pd , intensity of cognitive troubles is correlated with severity of treated motor score , duration of evolution of the disease , and age of the patient [ 53 , 54 ] , but due to subject distribution , we are prudent when interpreting the data assembled in table 3 . finally , in our study , use of erp does not allow for topographical indication of the neuronal loops involved in apathy . this additional limit is inherent to erp , which presents the advantage of excellent temporal resolution and the drawback of spatial resolution so poor as to preclude precise topographical indication of the dysfunctions . in our exploration of apathy with an electrophysiological approach , it appears that apathy in pd is correlated with a decrease of p300a amplitude : it represents a reliable neurophysiological marker , independently from depression . our results confirm on the one hand the involvement of a dorsolateral prefrontal cortex dysfunction in the antecedents of apathy and on the other hand the beneficial effect on apathy of dopaminergic treatment . enhanced and more purposeful allocation and mobilization of attentional resources are likely to result . from a physiopathological aspect , our results allow us to put forward the following hypothesis : in pd , apathy is the consequence of dopaminergic denervation ( probably mesolimbic ) and nondopaminergic lesions ( linked to evolution of the disease and particularly affecting prefrontal subcorticodorsolateral circuits ) . unfortunately , the low spatial resolution inherent to erp does not allow for a sufficiently accurate approach to the topography of the neuronal pathways involved ; the success of such an approach will require an adroit combination of erp and functional imaging .

in parkinson 's disease ( pd ) , apathy ( or loss of motivation ) is frequent . nevertheless , the contribution of attentional disorders to its genesis is still not clearly known . we want to determine the relation existing between apathy and attentional disorders by using p300a ( or novelty p3 ) as a marker of the attentional process . the study included 25 patients ( 13 women and 12 men ) with pd for whom we have determined the relationship between automatic attention ( represented by p300a ) and motor status , apathy , executive dysfunction , mental flexibility , inhibitory control , and depression / anxiety . we have found a correlation between the apathy score and amplitude of novelty p300 during the on period and also a correlation of the apathy score with a decrease in amplitude of p300 during the off period . in a linear regression model , changes in the p300a predicted the severity of apathy independently of any other variable . we concluded firstly that the reduction in amplitude of the p300a wave was a neurophysiological marker of apathy in pd and secondly that apathy led to both dopaminergic denervation ( mesolimbic ) and nondopaminergic ( dorsolateral prefrontal - subcortical ) dysfunction .

the online version of this article ( doi:10.1007/s40801 - 015 - 0009 - 6 ) contains supplementary material , which is available to authorized users . the amounts of intravenous immunoglobulin ( ivig ) used for short- and long - term treatment of immune thrombocytopenia ( itp ) are forecasted to be approximately 5,000 and 11,000 g per year , respectively , up to 2018 at these two tertiary care centers.the estimated provincial cost of ivig use in itp may be as high as $ 5 million annually.physicians and policy makers should reflect on the impact of treating itp with ivig and should consider alternatives , where appropriate , to improve patient quality of life and decrease economic costs . immune thrombocytopenia ( itp ) is a heterogeneous autoimmune disorder characterized by the presence of platelet autoantibodies , low platelet counts , and an increased risk of bleeding [ 1 , 2 ] . some patients will present with asymptomatic thrombocytopenia , while others will experience bleeding complications , which range in severity from skin bruises to fatal intracranial hemorrhage . itp can be classified by duration as newly diagnosed ( acute ) , persistent ( lasting 312 months ) , or chronic ( lasting 12 months ) . treatments for patients with itp are aimed at preventing serious bleeding , improving quality of life , and achieving a safe platelet count . for newly diagnosed patients , treatment with a brief course of corticosteroids , intravenous anti - d , or intravenous immunoglobulin ( ivig ) results in rapid yet transient responses . durable remissions of more than 6 months occur in fewer than 20 % of patients following first - line therapy with prednisone [ 5 , 6 ] . higher durable response rates from 59 to 75 % may occur with high - dose dexamethasone , depending on the age of the patients studied and the duration of follow - up [ 79 ] . chronic maintenance therapy with corticosteroids is limited by toxicities , such as neuropsychiatric symptoms , glucose intolerance , and osteoporosis ; hence , other therapies are often used in relapsed or refractory itp patients to achieve a hemostatic platelet count , thereby preventing or minimizing bleeding complications . treatment choices include thrombopoietin receptor agonists , immunosuppressive agents , and ivig [ 1 , 2 ] . ivig , a blood product manufactured from pooled human plasma , is a therapeutic option in the acute and chronic management of patients with itp . patients with itp are among the highest users of ivig in canada , representing 1017 % of utilization for all indications [ 10 , 11 ] . ivig administered at a dose of 12 g / kg causes rapid transient increases in platelet counts in over 80 % of patients ; however , platelet counts generally return to pretreatment levels within 4 weeks . while repeated infusions of ivig at regular intervals may be useful as maintenance therapy for patients with itp who require ongoing treatment because of bleeding , where a reasonable alternative exists , persistent use should be limited because of finite supplies and high associated costs [ 1315 ] . further , ivig is associated with bothersome side effects , including headache , nausea , flushing , fevers , chills , fatigue , and diarrhea . less commonly , it may result in more serious complications , including anaphylaxis , hemolysis , thrombosis , renal failure , and aseptic meningitis [ 1 , 4 , 16 , 17 ] . over the past 5 years , in the province of ontario , ivig utilization for all indications has risen to 1.56 million units per year at a cost of $ 97.9 million in 2010/2011 , representing an increase of 44 % in the number of units and a 53 % increase in costs . given the growth in the utilization of ivig in various clinical conditions , it is forecasted that the supply may not be able to meet the demand . in 2006 , the ontario ministry of health and long term care ( mohltc ) identified the unsustainable increases in ivig utilization as a key priority ; however , there is currently no provincial mechanism for routinely tracking and accurately quantifying and characterizing ivig use . this study aimed to assess the utilization of ivig in patients with itp at two large tertiary care centers ; determine the extent of short- and long - term utilization ; assess the proportion of ivig usage for itp compared with all indications ; compare utilization between these two unique centers ; and forecast future demand . a retrospective analysis of all adult patients who received ivig for the treatment of itp at these two participating centers during the study period was performed . patients were eligible for inclusion if they met the following criteria : age 18 years or older ; a diagnosis of itp ; and receipt of at least one dose of ivig for the treatment of itp between january 1 , 2003 , and september 30 , 2012 , at either of two large tertiary care centers , hamilton health sciences ( hhs ) and london health sciences centre ( lhsc ) . data collected at both centers included the date of birth , sex , weight ( if available ) , date of ivig infusion , and amount of ivig administered . at hhs , potential itp patients were identified and cases were confirmed by chart review . the transfusion registry for utilization , surveillance and tracking ( trust ) database , developed by the mcmaster transfusion research program ( mtrp ) , was used as the primary source of data extraction in hamilton . trust comprises data primarily from two sources of electronic data capture : meditech ( meditech circle , westwood , ma , usa ) , and the discharge abstract database ( dad ) . meditech is a laboratory information system used at hhs , which houses laboratory values and transfusion / infusion product information . the dad is the electronic database at both institutions that collects clinical data for the canadian institute for health information ( cihi ) . it was used to identify patients with a diagnosis of itp , extract patient information , and determine the indication for ivig , etc . to confirm the diagnosis , information was obtained from patients clinic charts and electronic medical records . patient diagnoses are coded using the cihi international classification of diseases and related health problems , 10th revision , canada ( icd-10-ca ) . during the study period , two of the three hamilton hospitals issued ivig from the transfusion medicine laboratory ; hence , the information on ivig disposition was in the laboratory information system ( meditech ) and had been captured in trust . the third hospital ( at the mcmaster site ) issued ivig from the transfusion medicine laboratory between 2009 and 2012 ; however , from 2002 to 2009 , the ivig product was issued from the pharmacy and recorded manually . the information on these paper logs was entered into a spreadsheet and cross - linked with the dad data to identify all potential itp patients . a chart review was then performed on all potential itp patients to confirm their diagnosis and eligibility for final inclusion in the analysis . at lhsc , the transfusion medicine laboratory information system contained all infusion episodes and data on ivig utilized within the study period , including all inpatient and outpatient ivig utilization at all hospital sites ( i.e. , university hospital and victoria hospital of lhsc , and st . this database did not identify icd-10 codes , but all ivig requested through the transfusion medicine laboratory required an ivig request form , which documented the indications . further , one of the investigators ( ch ) retrospectively reviewed the clinical data from the recipients patient electronic records to differentiate and confirm the diagnosis of itp versus an error in coding due to another cause of thrombocytopenia . where electronic records were not available , paper charts were reviewed . the de - identified data were submitted to the mtrp , where the data from the two sites were combined and analyzed . the primary outcome of this study was a description of the proportion of patients using ivig in the short - term and long - term chronic itp settings , and the corresponding durations of therapy . secondary outcomes included the number of ivig infusions administered ; number of ivig infusions administered per patient ; average total amount administered per infusion , per course , and per treatment period ; average number of infusions and courses given per treatment period ; days between courses ; percentage of total ivig use ( for all indications ) that was used for itp ; estimated ivig product usage cost per year ; and future forecasts of ivig utilization from 2012 to 2018 . to determine the primary outcome of quantifying short- and long - term utilization , definitions of acute , short - term and long - term treatments were developed on the basis of clinical judgment by experts in the treatment of itp , both individually and by consensus as part of an advisory board meeting sponsored by glaxosmithkline inc . infusions of ivig were grouped to form courses , and courses were grouped to form treatment periods , which were then classified as acute , short - term , or long - term ( fig . 1 ) . course was defined as the number of ivig infusions administered within a 5-day period . this definition allowed for variable practice patterns , as physicians often order ivig 12 g / kg divided over 1 , 2 , or 5 days . a treatment period was defined as a collection of courses where consecutive courses were given within a 6-month ( 182-day ) time frame of each other . thus , a single treatment period could last for years if consecutive courses during that treatment period were given within 6 months of each other . consecutive courses that were given more than 6 months apart were considered to be in separate treatment periods ( i.e. , a patient may have been treated in multiple treatment periods ) . an acute treatment was defined as involving only one course in the treatment period . clinically , for example , this could correspond to using ivig once for emergency treatment of bleeding associated with low platelet counts . a short - term treatment contained 25 courses , possibly representing a longer bridging treatment until another therapy started to work , and a long - term treatment was defined as containing six or more courses in that treatment period , perhaps denoting maintenance therapy.fig . 1summary of steps used to classify intravenous immunoglobulin ( ivig ) infusions into courses , treatment periods , and acute , short - term , and long - term treatment periods summary of steps used to classify intravenous immunoglobulin ( ivig ) infusions into courses , treatment periods , and acute , short - term , and long - term treatment periods lhsc data were sent to hhs for centralized analysis . a data dictionary was developed for the data from each site , and variable names were mapped to standardize data coding . sas 9.3 software ( sas institute , cary , nc , usa ) was used to perform this cross - mapping and for data analysis . descriptive statistics were used to determine the proportion of ivig being used for patients with itp compared with all other indications . time series forecasting utilization for 20122018 was performed using a stepwise autoregressive method ( sas 9.3 , proc forecast ) with the history data from 2003 to 2011 . the stepwise autoregressive method ( stepar ) that was used combines a time trend regression with an autoregressive model for departures from the trend . a retrospective analysis of all adult patients who received ivig for the treatment of itp at these two participating centers during the study period was performed . patients were eligible for inclusion if they met the following criteria : age 18 years or older ; a diagnosis of itp ; and receipt of at least one dose of ivig for the treatment of itp between january 1 , 2003 , and september 30 , 2012 , at either of two large tertiary care centers , hamilton health sciences ( hhs ) and london health sciences centre ( lhsc ) . data collected at both centers included the date of birth , sex , weight ( if available ) , date of ivig infusion , and amount of ivig administered . at hhs , potential itp patients were identified and cases were confirmed by chart review . the transfusion registry for utilization , surveillance and tracking ( trust ) database , developed by the mcmaster transfusion research program ( mtrp ) , was used as the primary source of data extraction in hamilton . trust comprises data primarily from two sources of electronic data capture : meditech ( meditech circle , westwood , ma , usa ) , and the discharge abstract database ( dad ) . meditech is a laboratory information system used at hhs , which houses laboratory values and transfusion / infusion product information . the dad is the electronic database at both institutions that collects clinical data for the canadian institute for health information ( cihi ) . it was used to identify patients with a diagnosis of itp , extract patient information , and determine the indication for ivig , etc . to confirm the diagnosis , patient diagnoses are coded using the cihi international classification of diseases and related health problems , 10th revision , canada ( icd-10-ca ) . during the study period , two of the three hamilton hospitals issued ivig from the transfusion medicine laboratory ; hence , the information on ivig disposition was in the laboratory information system ( meditech ) and had been captured in trust . the third hospital ( at the mcmaster site ) issued ivig from the transfusion medicine laboratory between 2009 and 2012 ; however , from 2002 to 2009 , the ivig product was issued from the pharmacy and recorded manually . the information on these paper logs was entered into a spreadsheet and cross - linked with the dad data to identify all potential itp patients . a chart review was then performed on all potential itp patients to confirm their diagnosis and eligibility for final inclusion in the analysis . at lhsc , the transfusion medicine laboratory information system contained all infusion episodes and data on ivig utilized within the study period , including all inpatient and outpatient ivig utilization at all hospital sites ( i.e. , university hospital and victoria hospital of lhsc , and st . this database did not identify icd-10 codes , but all ivig requested through the transfusion medicine laboratory required an ivig request form , which documented the indications . further , one of the investigators ( ch ) retrospectively reviewed the clinical data from the recipients patient electronic records to differentiate and confirm the diagnosis of itp versus an error in coding due to another cause of thrombocytopenia . where electronic records were not available , paper charts were reviewed . the de - identified data were submitted to the mtrp , where the data from the two sites were combined and analyzed . the primary outcome of this study was a description of the proportion of patients using ivig in the short - term and long - term chronic itp settings , and the corresponding durations of therapy . secondary outcomes included the number of ivig infusions administered ; number of ivig infusions administered per patient ; average total amount administered per infusion , per course , and per treatment period ; average number of infusions and courses given per treatment period ; days between courses ; percentage of total ivig use ( for all indications ) that was used for itp ; estimated ivig product usage cost per year ; and future forecasts of ivig utilization from 2012 to 2018 . to determine the primary outcome of quantifying short- and long - term utilization , definitions of acute , short - term and long - term treatments were developed on the basis of clinical judgment by experts in the treatment of itp , both individually and by consensus as part of an advisory board meeting sponsored by glaxosmithkline inc . infusions of ivig were grouped to form courses , and courses were grouped to form treatment periods , which were then classified as acute , short - term , or long - term ( fig . 1 ) . course was defined as the number of ivig infusions administered within a 5-day period . this definition allowed for variable practice patterns , as physicians often order ivig 12 g / kg divided over 1 , 2 , or 5 days . a treatment period was defined as a collection of courses where consecutive courses were given within a 6-month ( 182-day ) time frame of each other . thus , a single treatment period could last for years if consecutive courses during that treatment period were given within 6 months of each other . consecutive courses that were given more than 6 months apart were considered to be in separate treatment periods ( i.e. , a patient may have been treated in multiple treatment periods ) . an acute treatment was defined as involving only one course in the treatment period . clinically , for example , this could correspond to using ivig once for emergency treatment of bleeding associated with low platelet counts . a short - term treatment contained 25 courses , possibly representing a longer bridging treatment until another therapy started to work , and a long - term treatment was defined as containing six or more courses in that treatment period , perhaps denoting maintenance therapy.fig . 1summary of steps used to classify intravenous immunoglobulin ( ivig ) infusions into courses , treatment periods , and acute , short - term , and long - term treatment periods summary of steps used to classify intravenous immunoglobulin ( ivig ) infusions into courses , treatment periods , and acute , short - term , and long - term treatment periods a data dictionary was developed for the data from each site , and variable names were mapped to standardize data coding . sas 9.3 software ( sas institute , cary , nc , usa ) was used to perform this cross - mapping and for data analysis . descriptive statistics were used to determine the proportion of ivig being used for patients with itp compared with all other indications . time series forecasting utilization for 20122018 was performed using a stepwise autoregressive method ( sas 9.3 , proc forecast ) with the history data from 2003 to 2011 . the stepwise autoregressive method ( stepar ) that was used combines a time trend regression with an autoregressive model for departures from the trend . there were 383 adult itp patients who received a total of 2,098 ivig infusions at the two major ontario tertiary care centers participating in this retrospective study between january 1 , 2003 , and september 30 , 2012 ( tables 1 , 2 ) . comparing the two centers , in london , 547 infusions were given to 150 patients , and in hamilton , 1,551 infusions were given to 233 patients ( table 2 ) . despite this difference , the proportions of male and female recipients were similar at the two sites . the mean age of the patients at the first ivig infusion was 51.3 years , with a range of ages from 18 to 96 years of age ( table 1 ) . the mean weight ( 80.4 kg ) was available and calculated from 195 recipients ( london 81/150 , 54 % ; hamilton 114/233 , 49 % ) and was also similar at the two centers ( table 1 ) . patients received , on average , 5.5 ivig infusions ( london 3.6 , hamilton 6.7 ; range 1196 ) at 0.96 g / kg per infusion ( table 2).table 1demographic characteristics by site and overallcharacteristiclondon , n = 150hamilton , n = 233total , n = 383male sex [ n ( % ) ] 56 ( 37.3)88 ( 37.8)144 ( 37.6)age at the first ivig infusion [ years ; mean / sd ] 54.0/20.0449.5/19.1651.3/19.60patients with available weight data [ n]81114195average weight at time of first infusion [ kg ; mean / sd]80.1/18.2380.6/20.1080.4/19.30 ivig intravenous immunoglobulin , sd standard deviation age was calculated using the birth year of each patient , so for some patients the age may have been overcalculated by 1 yeartable 2intravenous immunoglobulin ( ivig ) usage : infusions and courses by site and overalllondon , n = 150hamilton , n = 233total , n = 383total number of ivig infusions 5471,5512,098number of ivig infusions per patient [ mean / sd]3.6/6.796.7/15.655.5/13.00amount per infusion [ g / kg ; mean / sd]0.91/0.19020.98/0.21810.96/0.2121total number of ivig courses4281,1751,603number of ivig courses per patient [ mean / sd]2.9/6.395.0/9.624.2/8.56number of infusions per course [ mean / sd ] 1.3/0.501.3/0.491.3/0.49total grams of ivig38,980121,096160,076 sd standard deviation infusions occurring on the same day were counted as one infusion demographic characteristics by site and overall ivig intravenous immunoglobulin , sd standard deviation age was calculated using the birth year of each patient , so for some patients the age may have been overcalculated by 1 year intravenous immunoglobulin ( ivig ) usage : infusions and courses by site and overall sd standard deviation infusions occurring on the same day were counted as one infusion when ivig infusions were combined into courses , there were a total of 1,603 courses of therapy given ( london 428 courses ; hamilton 1,175 courses ) , with a mean of 4.2 courses given per patient and 1.3 infusions given per course ( table 2 ) . the majority of patients received one or two infusions per course , which was similar at the two centers . further , the majority of patients receiving infusions at both centers received approximately 1 or 2 g / kg per course , with 1 g / kg being the most commonly prescribed amount , followed by 2 g / kg . when grouped into treatment periods ( as defined earlier ) , there were a total of 492 treatment periods ( london 172 , hamilton 320 ) with 264 ( 53.7 % ) defined as acute , 172 ( 35.0 % ) short - term , and 56 ( 11.4 % ) long - term treatments ( tables 3 , 4 ) . the majority of patients , 306 ( 79.9 % ) , received all of their ivig infusions in one treatment period , but some patients had up to six treatment periods . the average number of courses given in a treatment period was 3.3 ( london 2.5 , hamilton 3.7 ; range 199 ) . this correlated with an average 4.3 ivig infusions given per treatment period ( london 3.2 , hamilton 4.8 ; range 1196 ) [ table 3 ] . the number of days between consecutive courses within these treatment periods was 29.5 days ( london 32.2 , hamilton 28.7 ) , and the number of days between consecutive treatment periods , for those patients who received ivig in more than one treatment period , was 636 days and was similar at the two centers ( london 632 , hamilton 637).table 3intravenous immunoglobulin ( ivig ) usage categorized by treatment type by site and overalllondon , n = 150hamilton , n = 233total , n = 383total number of ivig treatment periods ( % ) 172320492 acute treatment ( % ) 101 ( 58.7)163 ( 50.9)264 ( 53.7 ) short - term treatment ( % ) 60 ( 34.9)112 ( 35.0)172 ( 35.0 ) long - term treatment ( % ) 11 ( 6.4)45 ( 14.1)56 ( 11.4)number of ivig treatment periods received per patient [ mean / sd]1.1/0.441.4/0.781.3/0.68courses per treatment period [ mean / sd]2.5/5.863.7/7.793.3/7.19infusions per treatment period [ mean / sd]3.2/6.134.8/12.854.3/11.00total grams per treatment period [ mean / sd]226.6/475.08378.4/1,121.75325.4/949.41 sd standard deviationtable 4intravenous immunoglobulin ( ivig ) utilization analysis by treatment type ( overall population)key findingslong - term treatment : 6 or more coursesshort - term treatment : 25 coursesacute treatment : 1 coursenumber of classified treatment periods ( % ) 56 ( 11.4)172 ( 35.0)264 ( 53.7)patients [ n]52152228total ivig amount per treatment type [ g ; mean / sd]1,559.5/2,482.91281.6/136.2092.1/36.11average number of infusions per treatment period [ mean / sd]19.9/27.903.8/1.661.3/0.50days of therapy per treatment period [ mean / sd]427/385.1884.2/79.44 sd standard deviation intravenous immunoglobulin ( ivig ) usage categorized by treatment type by site and overall sd standard deviation intravenous immunoglobulin ( ivig ) utilization analysis by treatment type ( overall population ) sd standard deviation the number of grams of ivig used in london and hamilton for all indications at both sites was seen to be trending upward from 144,605 g in 2003 to 245,763 g in 2012 ( see table a1 in the electronic supplementary material ) . utilization for itp also increased , but , relative to all other indications , the proportion of ivig used for adult itp patients remained relatively stable ( see table a1 ) . the actual and forecasted ivig usage in grams is provided in table 5 . the proportion of ivig given to adult itp patients ranged from 5.6 to 9.1 % and was generally lower in london than in hamilton ( see table a2 in the electronic supplementary material ) . in total , 160,076 g of ivig was administered in 2,098 infusions for patients with itp , representing 7.9 % of the overall amount of ivig usage for all indications , which differed between the centers ( london 4.6 % , hamilton 10.3 % ) [ see table a1].table 5annual actual and forecasted intravenous immunoglobulin ( ivig ) short- and long - term usage in patients with immune thrombocytopeniayearamount of ivig used [ g]londonhamiltontotalshort - term treatmentlong - term treatmentshort - term treatmentlong - term treatmentshort - term treatmentlong - term treatment20031,4606002,9506,0904,4106,69020041401,2603,46711,3533,60712,61320051,18004,4227,8385,6027,83820062,3971,2603,2485,6955,6456,95520071,3001,0203,50311,1374,80312,15720081,2959602,1656,5863,4607,54620091,9771,6001,8106,5003,7878,10020102,3952,7854,5802,3906,9755,17520111,8052,9802,19012,5153,99515,4952012 2,2702,8012,6487,5424,91810,3432013 2,4143,0852,5487,4924,96210,5772014 2,5583,3682,4477,4425,00510,8102015 2,7023,6512,3477,3935,04911,0442016 2,8463,9342,2477,3435,09311,2772017 2,9904,2182,1477,2945,13711,5122018 3,1344,5012,0477,2445,18111,745 the 20122018 data were forecasted using the complete annual data from 2003 to 2011 annual actual and forecasted intravenous immunoglobulin ( ivig ) short- and long - term usage in patients with immune thrombocytopenia the 20122018 data were forecasted using the complete annual data from 2003 to 2011 this 10-year , retrospective study at two tertiary care centers analyzed data from both sites to provide comprehensive information on ivig utilization in adult itp patients . these two large centers represent 19.9 % of the total ivig utilization ( for all indications ) in the province of ontario . during the study period , there were 383 adult itp patients who received a total of 160,076 g of ivig in 2,098 infusions . this represented 7.9 % of the total ivig utilization for all indications somewhat lower than the 1017 % previously reported in atlantic canada [ 10 , 11 ] . on average , there were roughly 1.5 times more women than men who received ivig , which is in keeping with the higher prevalence of itp in women than in men , with estimated female to male ratios of 1.2:1 and 1.7:1 being previously reported [ 20 , 21 ] . the range of patient ages ( 1896 years ) was broad , reflecting the diverse patient population who develop itp and require treatment . ivig utilization patterns were generally similar at the two study centers , including the age of the patients treated , the proportions of male and female recipients , and the amount of ivig infused . however , there were more ivig infusions given to more itp patients in hamilton than in london . these two sites are independent of one another and have dissimilar catchment areas and referral practices . hamilton has a very specialized dedicated itp practice , with referrals from other tertiary care centers to its itp clinics , reflecting a potentially sicker , usually more heavily pretreated population , who may require longer and perhaps more intensive treatments . london has a more community - based referral pattern , where patients are typically seen by a specialist for the first time after diagnosis by a family physician or emergency room physician . despite the clinical heterogeneity in the two patient populations , the majority of patients received approximately 1 or 2 g / kg per course , with 1 g / kg being the most commonly prescribed dose at both centers , followed by 2 g / kg . this is in keeping with infusions used in clinical practice and guidelines [ 1 , 2 ] . the recently updated american society of hematology evidence - based guidelines for itp recommend 1 g / kg as an initial dose . however , it is noted that patients who fail to respond to 1 g / kg may respond to a higher dose of 2 g / kg . at both centers , the majority of ivig was given for acute treatment of itp ( as per our definition ) . less ivig was used for short - term treatment , and it was infrequently used for long - term treatment . it is possible that acute treatments were given for itp patients who required therapy for active bleeding , a bridge to a procedure or surgery with an increased bleeding risk ( i.e. , splenectomy ) , or a trial to assess platelet response . compared with the london site , hamilton treated a larger cohort of itp patients with slightly more ivig infusions per patient , gave more courses per patient , had a higher proportion of itp patients relative to those with other indications , and administered more grams of ivig for itp relative to other indications , as would be expected with its highly specialized itp referral pattern . unlike acute treatments , short - term and long - term therapy with ivig may be provided to patients with the intent of using it as the sole maintenance therapy or as a longer bridging solution until another therapy works to control itp . from the present data , we can predict the impact of ivig utilization in adult itp patients requiring short- and long - term treatments ( table 5 ) . the total amount of ivig used for long - term treatment of itp at these two centers from 2012 to 2018 is forecasted to remain at approximately 11,000 g per year , at an estimated cost of $ 693,000 per year , based on a unit price of approximately $ 63 per gram in 2011 , published in bloody easy 3 by callum et al . . however , short - term ivig treatments make up an additional 172 ( 35 % ) of the total number of ivig treatment periods . with 281.6 g administered per short - term treatment period , this could contribute to an estimated additional 5,000 g of ivig being used , costing $ 315,000 per year , for a total of roughly $ 1 million per year for both short- and long - term treated patients . the estimated costs from these two centers represent 19.9 % of the overall provincial utilization . assuming that the patient populations and practice patterns are similar across ontario , the overall cost in the province for short- and long - term treatments of itp may be upward of fivefold greater , or $ 5 million per year . it was not feasible to include any kind of in - depth resource utilization discussion , since various components , such as nursing costs , chair time , and monitoring for reactions , were not included as part of this study . thus , a complete and accurate cost analysis of all components was not performed , and the true cost impact of ivig to the health care system , based only on the product cost of ivig , is underestimated . as forecasted by our data , ivig usage for itp will remain a substantial burden on the public health care budget and will continue to increase by approximately 2 % per year from 2013 to 2018 . ivig usage for itp should be reduced for several reasons other than its high cost . while ivig safety and toxicity were not studied in this review , ivig is associated with numerous potential side effects . bothersome side effects include headache , nausea , flushing , fevers , chills , fatigue , and diarrhea ( provan et al . ) . less commonly , it may result in serious complications , including anaphylaxis , hemolysis , thrombosis , renal failure , and aseptic meningitis [ 1 , 4 , 16 , 17 ] . further , ivig is a limited resource , which is widely utilized and can impact the quality of life of patients , including the need for travel to an infusion clinic , the need to sit for several hours during the infusion , and anxiety over possible reactions . limiting the use of ivig for itp is one of the items highlighted by choosing wisely canada and the canadian hematology society . in the last 5 years , newer therapies , such as thrombopoietic mimetics , have become available to patients with itp , and the role of these agents as an alternative to ivig , particularly for short- and long - term treatments , requires consideration . however , most of the data came from the laboratory information system at each site , which is considered the gold - standard data repository for blood product utilization . while these two large centers represent 19.9 % ( london 6.5 % , hamilton 13.4 % ) of total ivig utilization ( for all indications ) in the province of ontario , it is not clear if the findings are representative of the province as a whole ( i.e. , community / rural settings ) . the thrombopoietin mimetics were approved for itp treatments in the past few years and may have impacted the utilization of ivig as reflected in this retrospective analysis . further , clinical trials with these new medications at the hamilton site , and a provincial ivig audit , may have reduced the enthusiasm for using ivig during the past few years and may have affected our estimates . this retrospective review of comprehensive data at the london and hamilton sites has helped to characterize ivig utilization in adult itp patients and may improve the understanding of its impact on provincial utilization and inform future clinical practice and policy decisions . with the current practice pattern , short- and long - term ivig utilization for physicians treating itp and policy makers should consider the impact of treating itp with ivig and should consider alternatives , where appropriate , to improve both patient quality of life and economic impact . below is the link to the electronic supplementary material . supplementary material 1 ( docx 20 kb ) supplementary material 1 ( docx 20 kb ) cyrus c. hsia , neerav monga , julia elia - pacitti , and nancy heddle conceived the study . cyrus c. hsia , yang liu , kathleen eckert , and nancy heddle were involved in the collection of data . cyrus c. hsia drafted the manuscript , and all authors provided comments on the drafts and have read and approved the final version submitted for publication .

introductionintravenous immunoglobulin ( ivig ) is an immune thrombocytopenia ( itp ) therapy , which is associated with toxicities , limited availability , increasing utilization , and high cost . this study aimed to assess short- and long - term ivig utilization in patients with itp at two tertiary care centers in ontario , canada , to determine the proportion of ivig used in itp compared with all usage , and to forecast ivig demand in itp.methodsrecords from all adult itp patients who received ivig between january 1 , 2003 , and september 30 , 2012 , at hamilton health sciences and london health sciences centre were reviewed retrospectively.resultsduring the study period , 383 adult itp patients ( mean age 51.3 years ) received a total of 2,098 ivig infusions ( london 547 infusions in 150 patients ; hamilton 1,551 infusions in 233 patients ) . itp accounted for 5.6 and 9.1 % of all ivig usage in london and hamilton , respectively . the treatments included 264 ( 53.7 % ) acute , 172 ( 35.0 % ) short - term , and 56 ( 11.4 % ) long - term treatments . the amounts of ivig used for short- and long - term treatment of itp are forecasted to be approximately 5,000 and 11,000 g per year , respectively , up to 2018 . together , these two centers represent 19.9 % of the provincial ivig utilization . assuming similar patient populations and practice patterns in ontario , the overall provincial cost of ivig use in itp may be as high as $ 5 million annually.conclusionshort- and long - term ivig utilization for itp will remain an expensive resource within the ontario provincial health care system . physicians and policy makers should reflect on the impact of treating itp with ivig and should consider alternatives , where appropriate , to improve patient quality of life and decrease economic costs.electronic supplementary materialthe online version of this article ( doi:10.1007/s40801 - 015 - 0009 - 6 ) contains supplementary material , which is available to authorized users .

due to a 3-fold increase in the consumption of herbal remedies in the united states along with a staggering popularity of the ginseng herb as a method of sustaining good health , significant focus have been placed on two widely used types of ginseng , american ( panax quinquefolius l. ) ( 1 ) and asian ginseng ( panax ginseng ca meyer ) ( 24 ) . the usage of ginseng root for medicinal purposes have been recorded for millennia , known as a tonic capable of sustaining longevity as well as maintaining viability ( 5,6 ) . the two species display different effects , possibly due to the different active chemical makeup of each other . asian ginseng is said to facilitate blood flow , alleviate fatigue as well as relieve oxidative stress in diabetic conditions through various mechanisms such as the inhibition of lipid peroxidation ( 7 ) ( table 1 ) . american ginseng has been reported to have stress - relieving qualities , anti - aging effects and aids in digestion ( 3 ) . clinical studies have reported that ginseng improves psychological function , immune function and conditions associated with diabetes ( 8) . ginseng root has been reported to treat diabetic symptoms in the compendium of materia medica ( ben cao gang mu ) by dr li ( li , shizhen , ming dynasty from 1368 to 1644 , in china ) . the symptom was called xiao ke described as over eating , drinking and losing energy , body weight which are typical diabetic symptoms in modern medicine . common methods of treatment for hyperglycemia ( high glucose ) include both mono - therapy and combination therapy with insulin , metformin , rosiglitazone and acarbose . these treatments have been shown to help control blood glucose levels but do not solve the problem no pancreatic -cell function , which results in numerous diabetic complications ( 9 ) . table 1.differing effects of american and asian ginsengamerican ginseng ( panax quinquefolius l.)asian ginseng ( panax ginseng ca meyer)differing effectsanti - aging effects ( human , rat ) ( 37,38)increases blood flow ( human ) ( 13,39)anti - hyperglycemia ( human , mice ) ( 3,4,40,41)decreases fatigue ( human ) ( 13,42)aids in digestion ( rat ) ( 43)stimulates nervous system ( rat , cell ) ( 2,44)reduces stress ( rat ) ( 45)anti - inflammatory agent ( cell ) ( 4648)enhances memory ( rat ) ( 49)liver - protection ( rat)(50)reduces cholesterol ( cell ) ( 5)endurance ( rat ) ( 51)improves sexual function ( rat ) ( 52)anti - depression ( mice ) ( 53 ) differing effects of american and asian ginseng a range of chemical components of ginseng such as ginsenosides , alkaloids , polypeptides and polysaccharides has been identified with their biological activity . the elements phosphorus ( p ) , potassium ( k ) , calcium ( ca ) , thallium ( ti ) , manganese ( mn ) , iron ( fe ) , copper ( cu ) , zinc ( zn ) and strontium ( sr ) were detected by screening the components of ginseng ( 10 ) . other minor components include volatile oils ( member of vitamin b complex ) , manganese , vanadium , copper , cobalt , fatty acids , amino acids , simple sugars and other carbohydrates ( 11 ) . aside from the basic elemental components of ginseng , glycosides have been reported to be the active component in ginseng responsible for ginseng 's medicinal value . glycoside is a naturally occurring substance consisting of a sugar and non - sugar moiety . a group of saponins ( glycoside which produces froth by reducing water surface tension ) in ginseng were named ginsenosides , and were classified into subclasses ro , ra , rb , rc , rd , re , rf , rg and rh . these ginsenosides were differentiated based on their retention factor values in thin layer chromatography ( tlc ) , which is the distance that the ginsenosides travel up the tlc column ( 12 ) . more than 60 ginsenosides have been found in different parts of the ginseng plant ( 13 ) . asian ginseng can be distinguished from american using ginsenoside profiles since asian and american ginseng contains various ginsenosides ( 14 ) . a recent study analyzed the ginsenoside composition of asian and american ginseng through observing 12 commercially available ginsenosides . asian ginseng contains ginsenoside rf ( 15 ) and rg2 , but american ginseng does not . also , rg1 since the usage of ginseng root extracts lacks standardization due to the different levels of ginsenosides in different batches , purified ginsenosides have been used to test whether the effects of ginseng root lies on one ginsenoside or a combination of various ginsenosides . the anti - hyperglycemic effects of the total ginsenosides extracted from asian ginseng were evaluated in diabetic c57bl/6j ob / ob mice . total ginsenosides had significant anti - hyperglycemic and anti - obesity properties in a diabetic ob / ob mouse model ( 17 ) . in the same model , ginsenoside re showed to improve muscle metabolism and reduce inflammation through increase c - reactive protein levels , leading to an anti - hyperglycemic effect ( 18 ) . ginsenoside rh2 was able to lower plasma glucose in stz - diabetic rats in a dosage dependent manner ( 19 ) . rh2 has also been noted to have the ability to regulate glucose by increasing insulin secretion through the control of nerve terminals , signifying that the effects of ginsenosides encompass the entire body ( 20 ) . it is possible that ginsenoside components create an anti - hyperglycemic effect , which aids pancreatic function as well . there have been numerous reports of ginseng root ( american and asia ginseng ) improving diabetic conditions in both humans as well as animal studies . in animal studies , orally administered ginseng root was able to counteract the effects of high - fructose induced insulin resistance in rats after 4 weeks , decreasing glucose concentrations , as well as inhibit insulin resistance ( 21 ) . ethanol extract of ginseng root prevented weight gain , fasting blood glucose , triglyceride and high free fatty acid levels in a high - fat induced hyperglycemia mouse model ( 22 ) . ginsenoside re reduced blood glucose levels , cholesterol and triglyceride levels as well as reduced oxidative stress in the eye and kidney of diabetic rats ( 2 ) . clinical studies have reported that american ginseng has the ability to lower blood glucose in diabetic patients . ( 3 ) in some cases , both type ii diabetic patients and non - diabetic subjects showed to benefit from an intake of american ginseng in terms of stabilizing post - prandial glycemia after meals , suggesting that the intake of ginseng may also benefit healthy individuals . american ginseng did not have adverse effects on healthy individuals but rather beneficial effects in post - prandial glycemia compared with placebo when administered together with the glucose challenge . in subjects with type ii diabetes mellitus , significant reductions were observed when ginseng was taken 40 min before the glucose challenge ( 3 ) . in both clinical and animal studies , a limited amount of mechanism studies have been done on the effects of american ginseng on either the pancreas or any other organ . according to the mechanistic studies , which have been performed , ginseng root and components seem to exert its effect through several different mechanisms , suggesting that we have merely scraped the surface in discovering the effects of ginseng and its components . studies have shown that ginseng and its components attenuate hyperglycemia in two ways , the first through enhancing pancreatic -cell function and the other through reducing insulin resistance . this leads us to believe that ginseng may have benefits for both type i and type ii diabetes . american ginseng root extracts have been shown to affect pancreatic -cells through altering cell metabolism , increasing insulin production and reducing apoptosis in a dosage dependent manner ( 23 ) . ginseng extracts were able to enhance atp production and in turn increase insulin production , as insulin deficiency is often linked to a lack of atp produced ( 24,25 ) . along with an increase in atp production , ginseng reduced mitochondrial protein ucp-2 , which negatively regulates insulin secretion ( 13,2528 ) . aside from affecting insulin production , ginseng may have the ability to target various glucose receptors , creating an antilipolytic effect , thus attenuating hyperglycemia ( 12,29 ) . overall , ginseng is able to enhance insulin production through regulating cell metabolism . though ucp-2 regulates insulin secretion , resulting in the destruction of genetic material , apoptosis is regulated by factors such as bcl-2 , which protects against apoptosis and caspase-3/9 , which promotes death through the caspase cascade ( 8,31,32 ) . american ginseng reduces apoptosis by promoting caspase-3/9 and enhanced cell protective bcl-2 protein levels , resulting in protecting cells against apoptosis ( 23 ) . preventing apoptosis allows for further cell function and insulin production in pancreatic -cells . aside from affecting cell metabolism and longevity , ginseng has the ability to change neuropeptide through rh2 , a ginsenoside derived from panax ginseng , in stz - diabetic rats . rh2 lowered plasma glucose due to an increase in beta - endorphin secretion that activates opioid mu - receptors thereby resulting in an increased expression of glut 4 , a glucose transporter in fat and muscle tissue ( 19 ) . protopanaxatriol , a ginsenoside metabolite , also increased glut4 and improved insulin resistance ( 33 ) . the up - regulation of glut-4 signalizes that ginseng has an effect on fat / muscle tissue , possibly decreasing insulin resistance . along with mechanisms pertaining to specific organs and tissues , ginsenoside re prevented oxidative stress in ( 2 ) and reduce inflammation in stz - diabetic rats ( 34 ) . in cellular models , ginseng reduces h2o2 induced oxidative damage and enhances superoxide dismutase and catalase to create a protective effect in a dosage dependent manner ( 1 ) . ginseng leaf and berry extracts also show high antioxidant activities which detoxify free radicals excessively produced in diabetic environments ( 35 ) . currently , studies show ginseng as having general anti - oxidative properties in specific cells and tissue such as pancreatic -cells and fat / muscle tissue . the usage of herbs and spices has long been documented than modern pharmaceuticals . unlike modern pharmaceuticals , which utilize small molecular weight compounds consumed in a purified form , herbs are eaten in combinations of unmeasured quantities . it is almost certain the herbs contain beneficial qualities and that the goal should not be to ask if they have effects but rather what effects do they have ( 36 ) . since herbs have been consumed in its natural form for the major duration of history , very little attention has been paid to the standardization in this industry . it could be possible that the positive effects of ginseng on hyperglycemia may not hold true for all ginseng products due to differences in batches . sievenpiper , arnason et al.discovered that asian ginseng showed both null and opposing effects on indices of acute post - prandial plasma glucose and insulin , which contrasted the findings with ginseng , thus showing that different species attribute to different functions ( 3 ) . as with all chemical components , the metabolized form could generate three possibilities : enhance activity , become toxins , or have no effect . three main concerns limiting the usage of ginseng include the ubiquitous lack of standardization , in depth mechanism studies , as well as widespread clinical studies ( 3 ) . standardization of ginseng components would be the first step to ensuring that there are no unnecessary variations in the data . although single ginsenosides have shown to have positive effects , whether a single component or a mixture of components maximizes the therapeutic effect of ginseng on diabetes is still unclear . many steps have been taken to standardize the usage of ginseng root through isolating specific ginsenosides , which is an effective way to maintain dosages and specificity . it is more than likely that ginseng affects not only the pancreas to increase insulin production but also other tissue to utilize insulin as well as decrease insulin resistance through its various components ( 15 ) . ginseng root has been shown to be effective in cell cultures , animal studies , as well as clinical practice . root extracts and components exhibited anti - hyperglycemic activities and reduced insulin resistance and increased insulin production . since the mechanisms behind ginseng 's effects have not yet been fully discovered , we can not understand the full extent of its potential . ginseng root is able to increase insulin production and decrease cell apoptosis in pancreatic -cells , which signifies that ginseng affects the pancreas directly . also , ginseng has been shown to mediate various mechanisms related to muscle and fat tissue such as the glut4 pathway ( see proposed mechanism of ginseng 's activation in fig . 1 ) . despite the lack of sufficient widespread clinical , mechanistic studies and standardization for immediate therapeutic uses greatly hinders the possibility of practical applications , current reports of ginseng and ginsenosides point to the possibility of ginseng as a candidate for complementary diabetes therapy . figure 1.the mechanisms of ginseng activation proposed here in scheme summarizes that the active components of ginseng regulate uncoupling protein-2 ( ucp-2 ) , which results in a decrease in apoptosis and an increase of atp production and thus insulin production , which will stimulates cellular glut4 activation to promote metabolism . ginseng also activates cytochrome - c related bcl-2 expressing , leading to an inhibition of caspase-9 triggered apoptosis cascade ( 23 ) . + : positive effects - : negative effects solid - line arrow : known actions dotted - line arrow : recently discovered actions . the mechanisms of ginseng activation proposed here in scheme summarizes that the active components of ginseng regulate uncoupling protein-2 ( ucp-2 ) , which results in a decrease in apoptosis and an increase of atp production and thus insulin production , which will stimulates cellular glut4 activation to promote metabolism . ginseng also activates cytochrome - c related bcl-2 expressing , leading to an inhibition of caspase-9 triggered apoptosis cascade ( 23 ) . + : positive effects - : negative effects solid - line arrow : known actions in order to effectively apply ginseng on diabetes therapy , three fronts must be expanded . first , standardization of ginseng root has to be taken into consideration as different batches contain different concentrations of ginsenosides . second , more clinical studies have to be performed to confirm effects of ginseng on both type i and type ii diabetic patients . third , the mechanisms behind ginseng 's therapeutic effects have to be examined closely to see the mechanisms which specific components of ginseng act through . taking these three routes into consideration , the evident yet mysterious effects of ginseng

it has been reported that american ginseng attenuates hyperglycemia and may present itself as a supplement to diabetes therapy . however , the lack of standardization in the usage of ginseng root leads to inconclusive results when applied to diabetes treatment . the mechanisms of american ginseng root in the treatment of diabetes remains a mystery . this greatly limits the effective utilization of american ginseng in facilitating diabetic therapy . initiating studies have shown that american ginseng increases insulin production and reduces cell death in pancreatic -cells . also , studies have revealed american ginseng 's ability to decrease blood glucose in type ii diabetes patients as well as in streptozotocin - induced diabetic animals ( stz - diabetic mice ) . these data suggest that effects of ginseng in improving hyperglycemia may alter mitochondrial function as well as apoptosis cascades to ensure cell viability in pancreatic islet cells . this review briefly summarizes current knowledge of ginseng components and clinical studies related to diabetes . further research will be needed to explore and identify the component(s ) of ginseng , which may be responsible for the beneficial effects observed in animal studies which could then be extrapolated to human islets .

kmmell 's disease ( kd ) is rare form of post - traumatic delayed avascular necrosis of the vertebral body . to best of authors knowledge , there is no reported case of kd from india . it 's close radiological and histological resemblance to endemic tuberculosis of the vertebrae posses a diagnostic dilemma . we are reporting a case , whose clinical , radiological , and histological profile were compatible with kd and discuss the dilemma of diagnosing kd in indian context . a 60-year - old gentleman , a laborer by profession , presented with the complaints of severe low back ache of 3 days duration and low grade fever for the past 2 weeks . one year back , he had low backache of moderate intensity , precipitated by lifting and carrying heavy loads on his back , which resolved spontaneously . on clinical examination , there was focal tenderness over l4 - 5 vertebral levels with gross restriction of spinal movements and marked paraspinal spasm . routine hematological and biochemical parameters were normal except for high esr of 94 mm/1 h ( westergren ) . magnetic resonance imaging ( mri ) of lumbosacral spine [ figure 1 ] revealed collapse of l4 body with relatively preserved disc spaces . the collapsed l4 vertebral body was hypointense on t1w image and hyperintense on t2w image [ figure 1 ] . computed tomography ( ct ) of spine [ figures 2 and 3 ] revealed l4 vertebral body collapse . he underwent l4 laminectomy , l3 - 5 transpedicular fixation , and the biopsy of the l4 body . histopathology revealed thin irregular osteoporotic bony lamellae and fragments of pale ischemic bony bits [ figure 5a ] . the enclosed marrow spaces revealed fresh hemorrhage bordered by clusters of foamy histiocytes as response to fresh hemorrhage reflecting trauma [ figure 5b ] . there was abundant fibrin and early necrosis of the marrow without any osteoblastic or osteoclastic activity . there was no evidence of tuberculosis ( absence of granulomatous pathology or acid fast bacilli ) . the possibility of post - traumatic avascular necrosis ( kd ) was suggested . as tuberculosis mri t2w and t1w images show collapse of l4 vertebra and signal changes within the collapsed body ( hyperintense on t2w image and hypointense on t1w image ) , without pre or postvertebral collection / soft tissue and relatively preserved disc ct axial scan through l4 vertebra show fracture of left lamina sagittal reformatted ct shows collapse of vertebra with increased attenuation without evidence of bone destruction or erosion coronal reformatted ct shows collapse of vertebra and presence of air within the collapsed body ( kmmell 's sign ) histopatholgy reveal osteoporotic bony lamellae with preserved calcium lines contiguous with fragmented , ischemic bone with loss of calcium ( asterix , a ) closely apposing ischemic fragments , densely eosinophillic necrotic bone with remnants of osteocytes are seen . marrow spaces showed fresh hemorrhage walled in by foamy histiocytes ( b ) reflecting reparative process . [ a : he16 , b : he240 ] on subsequent reviewing of mri , radiological features were not favoring tuberculosis , as the disc space was well preserved . there was no pre or paravertebral collection of inflammatory granulation tissue and the collapsed vertebral body did not have abnormal enhancement or signal changes . review of the ct scan revealed l4 vertebral body collapse with cortical breach , linear fracture of lamina on the left side , and presence of intravertebral vacuum cleft on radiograph [ kmmell 's sign a retrospective observation ( figure 4 ) ] . clinical history was suggestive of mild trauma and delayed acute presentation , and hence , diagnosis of kd was made and att was stopped . serum ada performed at 3 months of follow up , which was normal . at 12 months kd is a rare form of delayed post - traumatic osteonecrosis , typically precipitated by mild trauma to the lower thoracic and lumbar spine . the initial pain following trauma resolves after a few days and reappear some months later . though first described in 1895 by hermann kmmell , this disease is uncommon and occasionally reported in english literature , possibly because of nonspecific symptomatology , difficulty in recalling trivial trauma , spontaneous resolution of initial pain , and absence of specific diagnostic tests . there is neither a consensus on the degree of initial trauma precipitating the event nor on the duration and/or degree of symptoms during the asymptomatic period . the term kmmell 's disease has been inappropriately applied to vertebral collapses with vacuum phenomenon , resulting in great confusion about the exact nature of cases reported as reviewed the available english literature on kd since the 1950s and authors felt that only five cases meet the criteria set out by kmmell . most of reported cases are isolated case reports affecting the elderly and more frequent in males . osteoporosis , chronic corticosteroid use , and local radiotherapy are the associated risk factor for this disorder . repetitive spinal loading in hyperflexion has been described as triggering factor for young onset kd . diagnosis usually requires exclusion of other causes of vertebral body collapse and osteonecrosis , as various disorders can cause vertebral body collapse beside trauma , which includes neoplasm , infection ( especially tuberculosis ) , metabolic disorders , radiation , and osteoporosis . there is no diagnostic imaging for the disease and the diagnosis remains a diagnosis of exclusion ; however , careful review of imaging and exclusion of other common cause of vertebral body collapse on radiology is vital to avoid missing the diagnosis . an intravertebral vacuum cleft on radiograph , referred to as kmmell sign , is highly suggestive of ischemic necrosis and corresponds to the one of the most characteristic features of kd is normal radiological study immediately following trauma and subsequent development of rarefaction and loss of bone tissue leading to collapse of the vertebral body after a delay of weeks or months . probably , the ideal investigation is serial imaging . it is suggested that the initial trigger of trivial trauma results in subtle disruption in the bony spongiosa with microhemorrhages . the etiological contribution of vascular insufficiency is supported by finding of delayed vertebral body collapse with normal appearance on routine ct imaging but with an abnormal bone scan . in our case , though there was no histological evidence of tuberculosis , however , the diagnostic possibility of tuberculosis could not be excluded , especially in an endemic area like india and in the presence of systemic symptoms of fever and raised esr . histopathological review revealed osteoporotic bony lamellae , a described risk factor for kmmell 's disease in addition to the presence of necrosis , and histiocytes . our patient was not investigated after initial trauma and we did not have the opportunity to establish the delayed radiological vertebral body collapse . however , review of the initial radiological imaging of l4 body revealed cortical breach and fracture line extending to involve whole body with presence of kmmell 's sign with conspicuous absence of features of tuberculosis . reconsideration and review of the clinicoradiological and histopathology is crucial to avoid under diagnosing the kd . in one of the published cases of kd , authors could diagnose only after clinical and histopathological review , who had two biopsies at different center and even received radiation for suspected malignancy . it is really difficult not to favor the diagnosis of tuberculosis , in endemic countries like india . it is therefore possible that kd is probably overlooked / under - reported rather than nonexistent . a high degree of clinical suspicion with careful review of imaging and histopathology and clinical correlation may aid in diagnosis and avoid unnecessary work up and prolonged treatment .

kmmell 's disease is a rare form of vertebral body osteonecrosis , which develops as a delayed post - traumatic event . it is infrequently reported in literature and to the best of our knowledge , has not been reported from india . we describe the clinical , radiological , and pathological features of a case occurring in a 60-year - old man and relevant brief review of the literature of this rare disease . its close resemblance to more commonly occurring bony tuberculosis poses a diagnostic dilemma particularly in developing country like india , where tuberculosis is endemic . awareness of this entity , though rare , is essential to avoid unnecessary diagnostic work up and treatment .

pulmonary hypertension ( ph ) is characterized by high pulmonary arterial pressure , increased pulmonary vascular resistance , right ventricular ( rv ) hypertrophy , and rv failure [ 13 ] . hypoxic exposure induces a selective pulmonary arterial vasoconstriction and an increase in pulmonary arterial pressure . when hypoxic pulmonary vasoconstriction ( hpv ) is persistent , pro - proliferative signals are activated in vascular cells , which leads to vascular remodeling and chronic hypoxic ph . pulmonary vascular remodeling is characterized by intimal thickening , medial smooth muscle cell ( smc ) hypertrophy and hyperplasia , adventitial fibroblast proliferation , and matrix protein synthesis . the magnitude of the fixed structural changes depends on many factors , including developmental stage , genetic factors and type of insult . protein kinase c ( pkc ) is an important signaling , intermediate in many of the vascular responses such as contraction and growth , and may play a key role in chronic hypoxic ph . the pkc family of serine / threonine protein kinases is involved in multiple biochemical processes , including cell growth , differentiation and transformation . so far , at least 10 pkc isoforms have been identified in mammals and classified into 3 subgroups based on their molecular structures and sensitivity to activators : conventional pkc ( cpkc , i , ii and ) , requiring both ca and diacylglycerol for activation ; novel pkc ( npkc , , and ) ; and atypical pkc ( apkc/ and ) . numerous pkc isozymes are expressed in pulmonary vascular media smooth muscle and adventitial fibroblasts , and their activations are generally marked by its translocation from the cytosolic fraction to the particulate or membrane - related fraction . ph is a major cause of disability in patients with chronic lung , heart and sleep disorders , and supplemental oxygen and supportive care are the only treatments currently available . pkc - based therapy might be a complementary therapeutic tool , but the role of individual pkc isoforms in the regulation of chronic hypoxic ph remains unclear . previous studies have suggested that pkc mediates hypoxia - induced proliferation and growth of pulmonary artery adventitial fibroblasts and smooth muscle cells [ 1619 ] . ca - dependent cpkc and expressed in pulmonary artery smooth muscle cells ( pasmcs ) are particularly important in their hypoxic growth and , ultimately , the development of chronic hypoxic ph . found that bovine pulmonary artery adventitial fibroblasts expressed cpkc , i and ii , npkc and , and apkc , but only cpkci and apkc activations were associated with the exaggerated growth responses of pulmonary artery adventitial fibroblasts under chronic hypoxia condition . however , short et al . found that apkc attenuates hypoxia - induced proliferation of fibroblasts by regulating map kinase phosphatase-1 ( mkp-1 ) expression . unfortunately , most of the experiments were carried out in vitro and some results are contradictory . the systematic investigation of pkc isoform - specific protein expression and activation in pulmonary vascular regions were rare in whole animal models with chronic hypoxia . therefore , the primary aim of this study was to determine the pkc isoform - specific involvement in the development of hypoxic ph by observing their protein expression and membrane translocation in pulmonary arteries of hypoxia - induced ph rats . the following materials were obtained from the indicated sources : proteinase inhibitors ( leupeptin , aprotinin , pepstatin a and chymostatin ) ; phosphatase inhibitors ( okadaic acid , sodium pyrophosphate and potassium fluoride ) ; and monoclonal anti--actin antibody , as well as other reagents , such as dithiothreitol ( dtt ) , nonidet p-40 , ethylene diamine tetraacetic acid ( edta ) , ethylene glycol tetraacetic acid ( egta ) , and sodium dodecyl sulfate ( sds ) , were purchased from sigma - aldrich company ( st . protein assay reagent , horseradish peroxidase - conjugated goat anti - rabbit igg or goat anti - mouse igg were purchased from bio - rad company ( hercules , ca , usa ) . all procedures conducted in this study were approved by the animal care and use committee of capital medical university and were consistent with the nih guide for the care and use of laboratory animals ( nih publications no . 80 - 23 ) . specific pathogen - free adult male sprague - dawley ( sd ) rats , weighing 200250 g , were exposed for the specified time periods ( 1 , 3 , 7 , 14 and 21 d [ days ] ) to normobaric hypoxia ( 10% oxygen ) in a ventilated plexiglas chamber while age- and weight - matched control rats were maintained in a 21% oxygen environment ( n=12 per group ) . to establish the hypoxic conditions as previously reported , the chamber was flushed with a mixture of oxygen and nitrogen from high - pressure cylinders , and an oxygen analyzer was used to monitor the chamber environment . co2 was removed with soda lime , excess humidity was prevented by drierite granules , and boric acid was used to keep ammonia levels within the chamber to a minimum . the chamber was opened every other day for 30 min ( minutes ) to clean the cages and replenish food and water . at the end of the hypoxic exposure , the animals were anesthetized with pentobarbital sodium ( 50 mg / kg , i.p . ) , and a 1.4f microtip pressure transducer ( spr-671 ; millar instruments ; houston , tx , usa ) was inserted into the right ventricle ( rv ) through the jugular vein for hemodynamic measurements . rv systolic pressure ( rvsp , an indirect index of pulmonary artery pressure ) was measured with a polygraph system ( powerlab ; ad instruments , australia ) . the rv was dissected from the left ventricle ( lv ) and the septum ( s ) , and weighed separately to determine the ratio of rv to lv plus s , rv/(lv+ s ) as rv hypertrophy . the animals were anesthetized with pentobarbital sodium ( 50 mg / kg , i.p . ) , exsanguinated in the hypoxic chamber , and the lungs were removed from the thoracic cavity . under a microscope , pulmonary arteries , including the main trunk plus the right and left branches , were isolated in ice - cold phosphate - buffered saline ( pbs ) containing 120 mm nacl , 2.7 mm kcl , and 10 mm phosphate buffer , and were quickly frozen in liquid nitrogen for later analysis . as in our previous reports , the frozen samples were placed into 100 l freshly prepared homogenization buffer a ( 50 mm tris - cl , ph 7.5 , 2 mm dtt , 2 mm edta , 2 mm egta , 5 mg / ml each of leupeptin , aprotinin , pepstatin a and chymostatin , 50 mm potassium fluoride , 50 m okadaic acid , 5 mm sodium pyrophosphate ) and homogenized . the homogenate was centrifuged at 30 000 g for 30 min , and the supernatant was collected as cytosolic fraction . the pellet was further solubilized in 100 l homogenization buffer b ( buffer a containing 0.5% nonidet p-40 ) before being sonicated and centrifuged at 30 000 g for 30 min again , and the resulting supernatants were taken as particulate fractions . both fractions were prepared for pkc isoform - specific membrane translocation analysis . to determine pkc isoform - specific protein expression levels , frozen pulmonary arteries were homogenized in 250 l homogenization buffer c ( buffer a containing 2% sds ) and sonicated to dissolve completely as the whole tissue homogenate . the protein concentration was determined by the bca protein assay ( pierce company , rockford , il , usa ) . as described previously , equal amounts of vascular protein ( 50 g from either cytosolic or particulate fractions , and 20 g from whole tissue homogenate ) per lane were loaded on 10% sds - page gels . after separation , the proteins were transferred onto polyvinylidene difluoride ( pvdf ) membrane ( ge healthcare , buckinghamshire , uk ) at 4c . after several rinses with ttbs ( 20 mm tris - cl , ph7.5 , 0.15 m nacl and 0.05% tween-20 ) , the transferred pvdf membrane was blocked with 10% non - fat milk in ttbs for 1 h ( hour ) and incubated with the primary rabbit polyclonal antibodies against cpkc , i , ii and , npkc , , and , and apkc/ and ( santa cruz biotechnology inc . , ca , usa ) at 1:5000 dilution for 3 h. to verify equal loading of protein , the blots were stripped by incubating the membranes for 45 min in stripping buffer containing 62.5 mm tris - cl ( ph 6.7 ) , 2% sds and 100 mm 2-mercaptoethanol at 55c and reprobed with primary mouse monoclonal antibody against -actin ( sigma - aldrich company , st . the horseradish peroxidase - conjugated goat anti - rabbit or anti - mouse igg ( stressgen biotechnologies corporation , victoria , bc , canada ) were used as second antibodies at a 1:3000 dilution for 1 h incubation . following incubation with the primary and secondary antibodies , the enhanced chemiluminescence ( ecl ) kit ( ge healthcare , buckinghamshire , uk ) the ratio of pkc isoform ( band density in particulate / band densities in both particulate and cytosolic fractions ) of the control group was normalized to 100% , and hypoxia groups were expressed as percentages of the control group . for the quantitative analysis of pkc isoform - specific protein expressions , the optical density of each band corresponding to pkc isoforms ( from whole tissue homogenate ) was normalized to that of -actin . the protein expression ratio in the control group was regarded as 100% , and data from the hypoxia groups were also expressed as percentages of the control group . the animals were anesthetized with pentobarbital sodium ( 50 mg / kg , i.p . ) and lungs were removed from the thoracic cavity . right lower lungs were fixed in 10% formaldehyde for at least 24 h. paraffin slices ( 6 m thickness ) were stained with hematoxylin and eosin ( he ) solution . he - stained images of the slices were examined under a microscope to look for the structural changes of pulmonary artery walls in response to hypoxic exposure . to determine the changes of pkc isoform - specific expressions in pulmonary arteries of rats after chronic hypoxia - induced pulmonary hypertension , immunostaining with cpkc , i , ii , and npkc ( santa cruz biotechnology inc . , ca , usa ) were performed as reported . lungs were infused by the fixative ( 3% formaldehyde in pbs ) via the trachea , and then immersed in the fixative at 4c for 24 h. the tissues were dehydrated , embedded in paraffin , and sectioned at a thickness of 5 m . the slices were incubated with 0.3% h2o2 for 10 min to exhaust the endogenous peroxidase and washed in 0.1 m phosphate - buffered saline ( pbs , ph7.4 ) . next , they were blocked with 10% bovine serum for 1 h. the blocked slices were incubated with primary rabbit polyclonal antibodies against cpkc , i , ii and ( santa cruz biotechnology inc . , ca , usa ) at a dilution of 1:200 for 12 h at 4c . the specimens were incubated in secondary antibodies of horseradish peroxidase - conjugated goat anti - rabbit igg for 2 h. finally , the slices were thoroughly washed in pbs , and a solution containing h2o2 ( 0.03% ) and 3,3-diaminobenzidine ( dab , 60% ) was added to visualize the slices . the images were captured by a leica microscope imaging system ( leica dm4000b , wetzlar , germany ) . quantitative analysis for immunoblotting was done after scanning of the x - ray film with quantitative - one software ( geldoc 2000 imaging system , bio - rad , hercules , ca , usa ) . statistical analysis was conducted by one - way analysis of variance followed by all pair - wise multiple comparison procedures using the bonferroni test , and the significance was regarded as p<0.05 . all procedures conducted in this study were approved by the animal care and use committee of capital medical university and were consistent with the nih guide for the care and use of laboratory animals ( nih publications no . 80 - 23 ) . specific pathogen - free adult male sprague - dawley ( sd ) rats , weighing 200250 g , were exposed for the specified time periods ( 1 , 3 , 7 , 14 and 21 d [ days ] ) to normobaric hypoxia ( 10% oxygen ) in a ventilated plexiglas chamber while age- and weight - matched control rats were maintained in a 21% oxygen environment ( n=12 per group ) . to establish the hypoxic conditions as previously reported , the chamber was flushed with a mixture of oxygen and nitrogen from high - pressure cylinders , and an oxygen analyzer was used to monitor the chamber environment . co2 was removed with soda lime , excess humidity was prevented by drierite granules , and boric acid was used to keep ammonia levels within the chamber to a minimum . the chamber was opened every other day for 30 min ( minutes ) to clean the cages and replenish food and water . at the end of the hypoxic exposure , the animals were anesthetized with pentobarbital sodium ( 50 mg / kg , i.p . ) , and a 1.4f microtip pressure transducer ( spr-671 ; millar instruments ; houston , tx , usa ) was inserted into the right ventricle ( rv ) through the jugular vein for hemodynamic measurements . rv systolic pressure ( rvsp , an indirect index of pulmonary artery pressure ) was measured with a polygraph system ( powerlab ; ad instruments , australia ) . the rv was dissected from the left ventricle ( lv ) and the septum ( s ) , and weighed separately to determine the ratio of rv to lv plus s , rv/(lv+ s ) as rv hypertrophy . the animals were anesthetized with pentobarbital sodium ( 50 mg / kg , i.p . ) , exsanguinated in the hypoxic chamber , and the lungs were removed from the thoracic cavity . under a microscope , pulmonary arteries , including the main trunk plus the right and left branches , were isolated in ice - cold phosphate - buffered saline ( pbs ) containing 120 mm nacl , 2.7 mm kcl , and 10 mm phosphate buffer , and were quickly frozen in liquid nitrogen for later analysis . as in our previous reports , the frozen samples were placed into 100 l freshly prepared homogenization buffer a ( 50 mm tris - cl , ph 7.5 , 2 mm dtt , 2 mm edta , 2 mm egta , 5 mg / ml each of leupeptin , aprotinin , pepstatin a and chymostatin , 50 mm potassium fluoride , 50 m okadaic acid , 5 mm sodium pyrophosphate ) and homogenized . the homogenate was centrifuged at 30 000 g for 30 min , and the supernatant was collected as cytosolic fraction . the pellet was further solubilized in 100 l homogenization buffer b ( buffer a containing 0.5% nonidet p-40 ) before being sonicated and centrifuged at 30 000 g for 30 min again , and the resulting supernatants were taken as particulate fractions . both fractions were prepared for pkc isoform - specific membrane translocation analysis . to determine pkc isoform - specific protein expression levels , frozen pulmonary arteries were homogenized in 250 l homogenization buffer c ( buffer a containing 2% sds ) and sonicated to dissolve completely as the whole tissue homogenate . the protein concentration was determined by the bca protein assay ( pierce company , rockford , il , usa ) . as described previously , equal amounts of vascular protein ( 50 g from either cytosolic or particulate fractions , and 20 g from whole tissue homogenate ) per lane were loaded on 10% sds - page gels . after separation , the proteins were transferred onto polyvinylidene difluoride ( pvdf ) membrane ( ge healthcare , buckinghamshire , uk ) at 4c . after several rinses with ttbs ( 20 mm tris - cl , ph7.5 , 0.15 m nacl and 0.05% tween-20 ) , the transferred pvdf membrane was blocked with 10% non - fat milk in ttbs for 1 h ( hour ) and incubated with the primary rabbit polyclonal antibodies against cpkc , i , ii and , npkc , , and , and apkc/ and ( santa cruz biotechnology inc . , ca , usa ) at 1:5000 dilution for 3 h. to verify equal loading of protein , the blots were stripped by incubating the membranes for 45 min in stripping buffer containing 62.5 mm tris - cl ( ph 6.7 ) , 2% sds and 100 mm 2-mercaptoethanol at 55c and reprobed with primary mouse monoclonal antibody against -actin ( sigma - aldrich company , st . the horseradish peroxidase - conjugated goat anti - rabbit or anti - mouse igg ( stressgen biotechnologies corporation , victoria , bc , canada ) were used as second antibodies at a 1:3000 dilution for 1 h incubation . following incubation with the primary and secondary antibodies , the enhanced chemiluminescence ( ecl ) kit ( ge healthcare , buckinghamshire , uk ) the ratio of pkc isoform ( band density in particulate / band densities in both particulate and cytosolic fractions ) of the control group was normalized to 100% , and hypoxia groups were expressed as percentages of the control group . for the quantitative analysis of pkc isoform - specific protein expressions , the optical density of each band corresponding to pkc isoforms ( from whole tissue homogenate ) was normalized to that of -actin . the protein expression ratio in the control group was regarded as 100% , and data from the hypoxia groups were also expressed as percentages of the control group . the animals were anesthetized with pentobarbital sodium ( 50 mg / kg , i.p . ) and lungs were removed from the thoracic cavity . right lower lungs were fixed in 10% formaldehyde for at least 24 h. paraffin slices ( 6 m thickness ) were stained with hematoxylin and eosin ( he ) solution . he - stained images of the slices were examined under a microscope to look for the structural changes of pulmonary artery walls in response to hypoxic exposure . to determine the changes of pkc isoform - specific expressions in pulmonary arteries of rats after chronic hypoxia - induced pulmonary hypertension , immunostaining with cpkc , i , ii , and npkc ( santa cruz biotechnology inc . , ca , usa ) were performed as reported . lungs were infused by the fixative ( 3% formaldehyde in pbs ) via the trachea , and then immersed in the fixative at 4c for 24 h. the tissues were dehydrated , embedded in paraffin , and sectioned at a thickness of 5 m . the slices were incubated with 0.3% h2o2 for 10 min to exhaust the endogenous peroxidase and washed in 0.1 m phosphate - buffered saline ( pbs , ph7.4 ) . next , they were blocked with 10% bovine serum for 1 h. the blocked slices were incubated with primary rabbit polyclonal antibodies against cpkc , i , ii and ( santa cruz biotechnology inc . , the specimens were incubated in secondary antibodies of horseradish peroxidase - conjugated goat anti - rabbit igg for 2 h. finally , the slices were thoroughly washed in pbs , and a solution containing h2o2 ( 0.03% ) and 3,3-diaminobenzidine ( dab , 60% ) was added to visualize the slices . the images were captured by a leica microscope imaging system ( leica dm4000b , wetzlar , germany ) . quantitative analysis for immunoblotting was done after scanning of the x - ray film with quantitative - one software ( geldoc 2000 imaging system , bio - rad , hercules , ca , usa ) . statistical analysis was conducted by one - way analysis of variance followed by all pair - wise multiple comparison procedures using the bonferroni test , and the significance was regarded as p<0.05 . to confirm the establishment of hypoxia - induced ph in the rats , rvsp ( a marker of systolic pulmonary arterial pressure ) and the rv/(lv+s ) ratio ( a marker of rv hypertrophy ) were measured . rvsp ( mmhg ) increased significantly ( p<0.05 vs. 0 day 23.80.8 , n=12 ) with the time of hypoxic exposure ( 1 d : 34.61.4 ; 3 d : 40.41.7 ; 7 d : 48.31.1 ; 14 d : 46.22.4 ; 21 d : 50.11.3 , n=12 per group ) . there was also a significant increase in the rv/(lv+s ) ratio ( p<0.05 vs. 0 day 100% , n=12 ) of rats following hypoxic exposure ( 1 d : 111.78.4 ; 3 d : 127.89.3 ; 7 d : 133.910.1 ; 14 d : 148.79.8 ; 21 d : 156.58.6 , n=12 per group ) . in addition , we also observed the pathologic changes at days 3 , 7 and 21 in pulmonary arteries of rats after hypoxic exposure . there was marked vascular remodeling , including medial hypertrophy and adventitial proliferation , in both the distal and the proximal pulmonary arteries of the ph rats when compared with that of control rats . these results suggest that prolonged exposure to hypoxia can induce ph with rv hypertrophy of rats . the typical western blot results in figure 1a showed that cpkc ( 80 kda ) , i ( 79 kda ) and ii ( 79 kda ) , not ( 78 kda ) , presented in pulmonary arteries of rats . in addition , we found a decrease in membrane translocation of cpkcii , but not and i , in pulmonary arteries of ph rats ( figures 1b and 2a ) . the translocation of cpkcii from cytosolic to particulate fraction significantly decreased at 3 d ( 68.95.1% ) and 7 d ( 90.35.5% ) after hypoxia when compared with that of the normoxic control group ( 0 day 100% ) . as shown in figures 1c and 2b , cpkc isoform - specific protein levels decreased significantly during 114d ( 83.96.2% , 71.64.7% , 55.69.7% and 73.65.7% , p<0.05 ) for cpkc , 121d ( 68.62.9% , 47.47.4% , 25.02.7% , 38.52.7% and 40.65.5% , p<0.05 ) for cpkci and 121d ( 72.13.1% , 41.12.8% , 62.43.2% and 72.55.1% , p<0.05 ) for cpkcii after hypoxia when compared with that of the normoxic control group ( 0 day 100% ) . however , we could only detect the protein expressions of npkc ( 77.5kda ) , ( 82kda ) and ( 82kda ) , but not npkc and apkc/ and , in pulmonary arteries of rats by western blot ( figure 3a ) . unlike cpkcii , significant increases of membrane translocations ( figures 2a and 3b ) and protein expressions ( figures 2b and 3c ) of npkc , not and , were observed in pulmonary arteries of rats following hypoxia exposures . to further characterize the cellular localization of cpkc , i , ii and npkc in the pulmonary arteries of hypoxia - induced ph rats , we performed immunostaining and found that the immunolabeled cpkc , i , ii and npkc proteins were diffusely located throughout the cytoplasmic areas of pulmonary artery smooth muscle cells and fibroblasts ( figure 4a , c , e , g , n=6 per group ) . furthermore , the decreased protein expressions of cpkc , i and ii , and the increased expression of npkc could be observed at 14 d after hypoxia ( figure 4b , d , f , h , n=6 per group ) . to confirm the establishment of hypoxia - induced ph in the rats , rvsp ( a marker of systolic pulmonary arterial pressure ) and the rv/(lv+s ) ratio ( a marker of rv hypertrophy ) were measured . rvsp ( mmhg ) increased significantly ( p<0.05 vs. 0 day 23.80.8 , n=12 ) with the time of hypoxic exposure ( 1 d : 34.61.4 ; 3 d : 40.41.7 ; 7 d : 48.31.1 ; 14 d : 46.22.4 ; 21 d : 50.11.3 , n=12 per group ) . there was also a significant increase in the rv/(lv+s ) ratio ( p<0.05 vs. 0 day 100% , n=12 ) of rats following hypoxic exposure ( 1 d : 111.78.4 ; 3 d : 127.89.3 ; 7 d : 133.910.1 ; 14 d : 148.79.8 ; 21 d : 156.58.6 , n=12 per group ) . in addition , we also observed the pathologic changes at days 3 , 7 and 21 in pulmonary arteries of rats after hypoxic exposure . there was marked vascular remodeling , including medial hypertrophy and adventitial proliferation , in both the distal and the proximal pulmonary arteries of the ph rats when compared with that of control rats . these results suggest that prolonged exposure to hypoxia can induce ph with rv hypertrophy of rats . the typical western blot results in figure 1a showed that cpkc ( 80 kda ) , i ( 79 kda ) and ii ( 79 kda ) , not ( 78 kda ) , presented in pulmonary arteries of rats . in addition , we found a decrease in membrane translocation of cpkcii , but not and i , in pulmonary arteries of ph rats ( figures 1b and 2a ) . the translocation of cpkcii from cytosolic to particulate fraction significantly decreased at 3 d ( 68.95.1% ) and 7 d ( 90.35.5% ) after hypoxia when compared with that of the normoxic control group ( 0 day 100% ) . as shown in figures 1c and 2b , cpkc isoform - specific protein levels decreased significantly during 114d ( 83.96.2% , 71.64.7% , 55.69.7% and 73.65.7% , p<0.05 ) for cpkc , 121d ( 68.62.9% , 47.47.4% , 25.02.7% , 38.52.7% and 40.65.5% , p<0.05 ) for cpkci and 121d ( 72.13.1% , 41.12.8% , 62.43.2% and 72.55.1% , p<0.05 ) for cpkcii after hypoxia when compared with that of the normoxic control group ( 0 day 100% ) . however , we could only detect the protein expressions of npkc ( 77.5kda ) , ( 82kda ) and ( 82kda ) , but not npkc and apkc/ and , in pulmonary arteries of rats by western blot ( figure 3a ) . unlike cpkcii , significant increases of membrane translocations ( figures 2a and 3b ) and protein expressions ( figures 2b and 3c ) of npkc , not and , were observed in pulmonary arteries of rats following hypoxia exposures . to further characterize the cellular localization of cpkc , i , ii and npkc in the pulmonary arteries of hypoxia - induced ph rats , we performed immunostaining and found that the immunolabeled cpkc , i , ii and npkc proteins were diffusely located throughout the cytoplasmic areas of pulmonary artery smooth muscle cells and fibroblasts ( figure 4a , c , e , g , n=6 per group ) . furthermore , the decreased protein expressions of cpkc , i and ii , and the increased expression of npkc could be observed at 14 d after hypoxia ( figure 4b , d , f , h , n=6 per group ) . this study reports the first description of the pattern of pkc isoform - specific membrane translocation and protein expression in pulmonary arteries of rats with hypoxia - induced ph . analyzed the expression of pkc isoforms in smooth muscle cells ( smc ) by using western blots and thermocycle amplification of mrna , and demonstrated the presence of cpkc , npkc , and apkc in smc in various states of differentiation . showed the presence of cpkc , i , npkc , , apkc in rat aortic vascular smc using western blot analysis . cpkc , i and ii , npkc and , and apkc were reported in neonatal bovine pulmonary artery adventitial fibroblasts and pulmonary artery smcs from chronically hypoxic neonatal calves . in addition , the expressions of cpkc , ii and , npkc and , and apkc were observed in ventricles of chronically hypoxic rats . similarly , our in vivo experiment demonstrated that 6 isoforms of cpkc , i and ii , npkc , and expressed in smc and fibroblasts of rat pulmonary arteries by using western blot and immunohistochemistry methods . pulmonary artery smc and fibroblast proliferation occurs with hypoxic ph in vivo . pkc is a key protein kinase cascade leading to cell proliferation in response to growth factors , hormones and antigens . however , it is not known whether pkc is involved in the signaling of hypoxia - induced pulmonary remodeling in an in vivo model . reported that the ca - dependent pkc , especially cpkc , is particularly important in hypoxia - induced pulmonary artery smc growth and ultimately the development of chronic ph by observing the cultured pulmonary artery smc in vitro[17,1921 ] . found that cpkc and ii contribute to the augmented proliferative response of immature bovine pulmonary artery adventitial fibroblasts . zhu et al . reported that specific pkc isozymes mediate forskolin - induced activation of large - conductance , calcium- and voltage - activated potassium ( bkca ) channels in pulmonary vascular smooth muscle ( pvsm ) . although investigators have reported cpkc activation in hypoxic condition , we found in this study that the decreases of cpkcii membrane translocation and cpkc , i and ii protein expressions , as well as the increases of npkc membrane translocation and protein expression , were involved in the development of hypoxia - induced ph of rats . these results are consistent with previous observations from in vivo cerebral ischemia and primary neuronal cultures damaged by glutamate , which suggest that decreased cpkcii activation probably provides a negative feedback mechanism in cell hypoxic growth , and the down - regulation of cpkc isoform - specific protein expression might be linked to the augmented growth capacity of pulmonary artery smc . the decrease of cpkc , i and ii protein expressions may be the result of enhanced proteolytic degradation by ca - activated proteinases during chronic hypoxia . the discrepancy of studies shows the role of pkc isoform in proliferation might be cell type- and species - specific . in this study , we also found that the development of hypoxia - induced rat ph was not accompanied by exaggerated pulmonary vascular remodeling at the early stage , suggesting that vasoconstriction is a major contributor to the increased pulmonary artery pressure . pkc activity was reported to be involved in the transduction pathway forwarding pulmonary vasoconstriction in response to alveolar hypoxia , but which pkc isoform is responsible for vasoconstriction is unclear . demonstrated that npkc - null mice have decreased hypoxic pulmonary vasoconstriction ( hpv ) ; the ca - independent vascular contraction appears to be associated with plasmalemmal translocation of npkc . our finding of increased npkc membrane translocation and protein expression in pulmonary arteries of hypoxia - induced ph rats is consistent with the observation that npkc was involved in modulating contraction via actions on the myofilaments . these data show that the different pkc isoforms appear to have distinct functions regarding phosphorylation of specific substrate proteins such as cell proliferation and vasoconstriction . in conclusion , our study is the first to demonstrate the pkc isoform - specific membrane translocation and protein expression systematically in a preparation of a whole animal model of hypoxia - induced rat ph . the present results indicate that the changes in membrane translocation and protein expression of cpkc , i , ii and npkc are involved in the development of hypoxia - induced rat ph . future experiments will focus on evaluating the pkc isoform - specific role in pulmonary artery constriction and proliferation .

summarybackgroundevidence indicates that protein kinase c ( pkc ) plays a pivotal role in hypoxia - induced pulmonary hypertension ( ph ) , but pkc isoform - specific protein expression in pulmonary arteries and their involvement in hypoxia - induced ph are unclear.material/methodsmale sd rats ( 200250 g ) were exposed to normobaric hypoxia ( 10% oxygen ) for 1 , 3 , 7 , 14 and 21 d ( days ) to induce ph . pkc isoform - specific membrane translocation and protein expression in pulmonary arteries were determined by using western blot and immunostaining.resultswe found that only 6 isoforms of conventional pkc ( cpkc ) , i and ii , and novel pkc ( npkc ) , and were detected in pulmonary arteries of rats by western blot . hypoxic exposure ( 121 d ) could induce rat ph with right ventricle ( rv ) hypertrophy and vascular remodeling . the cpkcii membrane translocation at 37 d and protein levels of cpkc at 314 d , i and ii at 121 d decreased , while the npkc membrane translocation at 321 d and protein levels at 314 d after hypoxic exposure in pulmonary arteries increased significantly when compared with that of the normoxia control group ( p<0.05 vs. 0 d , n=6 per group ) . in addition , the down - regulation of cpkc , i and ii , and up - regulation of npkc protein expressions at 14 d after hypoxia were further confirmed by immunostaining.conclusionsthis study is the first systematic analysis of pkc isoform - specific membrane translocation and protein expression in pulmonary arteries , suggesting that the changes in membrane translocation and protein expression of cpkc , i , ii and npkc are involved in the development of hypoxia - induced rat ph .

despite the widespread use of bone - targeted therapies such as bisphosphonates ( e.g. zoledronate , pamidronate , clodronate ) or receptor activator of nuclear factor kappa - b ligand ( rankl ) antibodies ( i.e. denosumab ) in patients with metastatic bone disease , many questions remain about their optimal use , . bone - targeted agents are usually given every 34 weeks , a dosing interval that is based predominantly on their clinical development as an add - on treatment to standard anti - cancer therapies such as chemotherapy , along with data derived from the treatment of hypercalcemia from malignancy , , . one size fits all approach to the dosing intervals is sub - optimal however , as it ignores the long half - life of these agents in bone and the substantial variability in individual patient risk of skeletal related events ( sres ) . given the modest magnitude of absolute benefit of bone - targeted agents on skeletal related event reductions , it is important to investigate whether to not less frequent administration could affect the efficacy of these agents . this would not only result in reduced financial costs to both the patient and to the health care system , but would also likely reduce drug - associated toxicity . the latter is particularly important as toxicity of these agents has been shown to be related to both the potency and the cumulative dose of the bone - targeted agent . to date , two trials have been presented assessing reduced frequencies of administration of these agents in metastatic breast cancer patients , and others are still on going . despite this , the results of the published trials would suggest that there is still a need for larger definitive studies . in addition , we are not aware of any similar studies planned for prostate cancer where again the benefits of bone - targeted agents in reducing sres are likely even more modest than that seen in breast cancer patients . we are however aware of considerable variability in clinical practice , not only between cancer centers , but also within centers with respect to a number of questions around optimal use of bone - targeted agents , despite various clinical practice guidelines . before contemplating a trial to formally assess the feasibility of de - escalated bone - targeted treatment in both prostate and breast cancer patients , we wished to conduct a survey of potential collaborating physicians at canadian hospitals regarding their current clinical practice in these populations and their views on this matter to assist with design of future trials . in particular , we hoped a survey would help establish current standards of care , the extent of clinical equipoise with respect to de - escalation , physician comfort with entering patients on such a trial , and finally , what the most important outcomes and related effect sizes might be for clinicians in order to establish the comparability of these approaches to treatment . the survey was designed by the authors through three rounds of question development and refinement , and consists of three components . the first component was devised to collect pertinent demographic information of the population of respondents , as well as to determine what proportion amongst them use bone - targeted agents to treat their patients . the second component was designed to collect information from those respondents who prescribe bone - targeted agents with regard to intended benefits from bone - targeted agent use , scenarios in which they prescribe bone - targeted agents , and choice of agent for their patients in order to gain an understanding of current canadian clinical practice . in the third component , respondents were presented with a series of questions related to the design of a future clinical trial geared toward studying the clinical benefits of de - escalated therapy , with topics of interest including outcome selection and patient inclusion criteria . a member of the authorship team ( mc ) has held a national annual meeting related to the study of bone in oncology patients since 2005 , and participants from past years ' meetings were considered an accessible , representative , and appropriate group to approach as a population for this survey . a list of all participants ' email addresses was compiled , and these individuals were then sent a link via electronic mail inviting them to participate in the survey . the survey was designed and implemented using the online tool www.fluidsurveys.com . the survey was initiated at the start of july 2012 and remained open until september 1 , 2012 . electronic invites were sent to a total of 193 clinicians , and a total of 90 invitees responded ; 11 of the email invites were associated repeatedly with automatic out of office responses , and were thus excluded from the denominator ( survey response rate=49.5% ) . all measures of respondent characteristics including profession , type of center for clinical practice , province , populations treated ( breast cancer , prostate cancer , or both ) , number of new patients and follow up patients seen annually , and use of bone - targeted agents were compiled and reported as proportions of the total number of respondents . for summary statistics calculated in relation to components two and three of the survey as described above , a denominator of 66 respondents was used as physicians not using bone - targeted agents in their practice were not asked to respond to the questions associated with these components . where relevant , we stratified findings according to the type of patients treated by the respondents ( i.e. breast or prostate cancer ; where respondents treated both populations , they were included within both groups ) . data were analyzed using microsoft excel 2010 ( microsoft corporation , seattle , washington ) . the survey was designed by the authors through three rounds of question development and refinement , and consists of three components . the first component was devised to collect pertinent demographic information of the population of respondents , as well as to determine what proportion amongst them use bone - targeted agents to treat their patients . the second component was designed to collect information from those respondents who prescribe bone - targeted agents with regard to intended benefits from bone - targeted agent use , scenarios in which they prescribe bone - targeted agents , and choice of agent for their patients in order to gain an understanding of current canadian clinical practice . in the third component , respondents were presented with a series of questions related to the design of a future clinical trial geared toward studying the clinical benefits of de - escalated therapy , with topics of interest including outcome selection and patient inclusion criteria . a member of the authorship team ( mc ) has held a national annual meeting related to the study of bone in oncology patients since 2005 , and participants from past years ' meetings were considered an accessible , representative , and appropriate group to approach as a population for this survey . a list of all participants ' email addresses was compiled , and these individuals were then sent a link via electronic mail inviting them to participate in the survey . the survey was designed and implemented using the online tool www.fluidsurveys.com . the survey was initiated at the start of july 2012 and remained open until september 1 , 2012 . electronic invites were sent to a total of 193 clinicians , and a total of 90 invitees responded ; 11 of the email invites were associated repeatedly with automatic out of office responses , and were thus excluded from the denominator ( survey response rate=49.5% ) . all measures of respondent characteristics including profession , type of center for clinical practice , province , populations treated ( breast cancer , prostate cancer , or both ) , number of new patients and follow up patients seen annually , and use of bone - targeted agents were compiled and reported as proportions of the total number of respondents . for summary statistics calculated in relation to components two and three of the survey as described above , a denominator of 66 respondents was used as physicians not using bone - targeted agents in their practice were not asked to respond to the questions associated with these components . where relevant , we stratified findings according to the type of patients treated by the respondents ( i.e. breast or prostate cancer ; where respondents treated both populations , they were included within both groups ) . data were analyzed using microsoft excel 2010 ( microsoft corporation , seattle , washington ) . the distribution of characteristics shows that the majority work in teaching hospitals , consistently manage moderate to large numbers of patients , and that 55.6% ( 50/90 ) see at least one new patient per month , suggesting the population is an experienced group of oncologists . approximately two thirds of the respondents were medical oncologists , with the remainder consisting of radiation oncologists and urologists . physicians were located predominantly in ontario ( 60% ) , quebec ( 13.3% ) and alberta ( 11.1% ) . totals of 43 , 26 and 18 respondents treated breast cancer , prostate cancer , or both , respectively ; 3 respondents failed to indicate their specialty . amongst the 90 respondents , 73.3% ( 43/43 treating breast cancer patients , 12/26 treating prostate cancer patients , and 11/18 treating both ) indicated that use of bone - targeted agents is a part of their clinical practice , and thus continued on to complete component two of the survey . respondents were first asked to describe the primary reasons they provide bone - targeted agents to their patients , with the ability to select multiple reasons as deemed relevant ( see online questionnaire in supplementary data ) . most respondents primarily provide bone - targeted agents to reduce fracture risk ( 95.45% ) , to reduce risk of surgery to bone or radiotherapy ( 87.88% ) , to reduce metastasis pain ( 89.39% ) , to improve quality of life ( 72.13% ) , to reduce hypercalcaemia risk ( 71.21% ) , and to reduce risk of spinal compression ( 68.18% ) . few do so based on beliefs that these agents will improve progression - free survival ( 18.18% ) or overall survival ( 4.55% ) . response profiles were generally consistent across sub - populations of those treating breast cancer , prostate cancer , or both diseases ( fig . 1 ) . to assess the situations in which bone - targeted treatment might be prescribed , respondents were presented a series of five scenarios and asked whether they would prescribe a bone - targeted agent to the patient in question . the physician was asked , for a typical patient seen in your clinic with newly diagnosed bone metastases , i would usually recommend a bone - targeted agent even if this patient has little or no pain . for the responding clinicians 93.75% indicated they would recommend treatment in patients with newly diagnosed bone metastases , even if there is little to no pain present . for scenario 2 clinicians were asked , for a typical patient seen in your clinic with newly diagnosed bone metastases , i would usually recommend a bone - targeted agent even if the metastases were blastic . results from scenario 3 showed that approximately 60% of respondents would still recommend bone - targeted therapy in patients who have a prognosis of 6 months or less due to visceral metastases . data from scenario 4 showed that only 28.13% of responding clinicians would recommend treatment in patients who display poor oral hygiene or who require considerable dental work . finally , in scenario 5 , amongst the 23 respondents managing prostate cancer patients who use bone - targeted agents , 21.74% indicated that they would only start therapy in conjunction with chemotherapy . three of the 90 respondents indicated regular monitoring of patients with new bone metastases using biomarkers such as urinary n - telopeptide to assess response to treatment . there were some differences in the bone - targeted agents used for breast cancer when compared to prostate cancer ( fig . pamidronate was the agent most consistently used ( 52/61=85.25% ) , while denosumab ( 42/61=68.85% ) , zoledronic acid ( 40/61=65.57% ) and oral clodronate ( 31/61=50.82% ) had also been used to various degree by large proportions of clinicians . amongst respondents who treat prostate cancer , zoledronic acid ( 20/23=86.96% ) and denosumab ( 19/23=82.61% ) were both used . for breast cancer patients , there was a variation in the frequency of administration of bone targeted agents ( fig . a total of 16/52 ( 30.77% ) treat patients every 34 weeks from the start , 8/52 ( 15.38% ) responded every 34 weeks unless there is a fall in performance status , 1/52 ( 1.92% ) indicated every 34 weeks for 3 months before modifying to once every 3 months , 11/52 ( 21.15% ) indicated every 34 weeks for a year before modifying to once every 3 months , 11/52 ( 21.15% ) indicated every 34 weeks for 2 years before modifying to once every 3 months . finally , 5/52 ( 9.61% ) respondents noted other scenarios , which relied upon patients ' sre history , extent of disease , and presence of concurrent chemotherapy . for prostate cancer patients , 8/23 ( 34.78% ) treat patients every 34 weeks from the start , 5/23 ( 21.74% ) every 34 weeks unless there is a fall in performance status , 3/23 ( 13.04% ) indicated every 34 weeks for 3 months before modifying to once every 3 months , 1/23 ( 4.35% ) indicated every 34 weeks for a year before modifying to once every 3 months . finally , 3/23 ( 13.04% ) indicated every 34 weeks for 2 years before modifying to once every 3 months , and 3/23 ( 13.04% ) noted other frequencies . the answers showed that cessation was not common ; 28/64 ( 43.75% ) indicated never or < 5% of the time , 21/64 ( 32.80% ) between 5% and 25% of the time , 8/64 ( 12.5% ) between 26% and 50% of the time , and 6/64 ( 9.4% ) > 50% of the time . this trend was generally consistent amongst the sub - groups of those treating breast cancer , prostate cancer , or both . clinicians did not feel that worsening of bone pain , the presence of radiographic evidence of progression or the occurrence of an sre while on a bone - targeted agent was an indication to stop this therapy . indeed , the most common reasons for treatment cessation were ; a marked deterioration of performance status ( 57.81% ) , the occurrence of intolerable side effects ( 18.75% ) and progression of disease outside the bone ( 4.7% ) . other assorted reasons ( 18.75% ) included ; presence of renal failure , duration of treatment > 12 years , rising creatinine levels , and proximity to death . in the third survey component respondents were asked to share their views regarding potential patient enrollment criteria , clinical outcomes of interest and minimum clinically important differences for these measures , to explore opinions regarding key parameters for the planning of future trials . when presented with several alternatives regarding potential patient populations that could be studied , physicians were asked to assess whom they would be comfortable enrolling into a randomized controlled trial . the question around duration of prior bone - targeted agent use prior to study randomization ( i.e. 3 months vs. 1 year ) did not produce a majority decision . however , in a situation of clinical equipoise the finding that 45.5% of respondents were comfortable randomising those who have received 3 months of prior therapy and 59.1% were comfortable with those who have received 1 year treatment tells us that such trials are still very feasible . physicians were asked to rank order the potential primary study outcomes for a clinical trial of de - escalated treatment from the most to the least clinically meaningful . sre was the measure that was most commonly ranked to be most meaningful ( 57/66=86.4% of respondents ) , while pain ( 10.60% ) and patient preference ( 3.03% ) were chosen much less frequently . when asked what between - group difference in the brief pain inventory ( bpi ) score would be an appropriate amount to suggest equivalent effectiveness between groups , 22 ( 34.4% ) suggested 1 point , 30 ( 46.9% ) suggested 2 points , 6 ( 9.4% ) suggested 3 points , and 6 ( 9.4% ) were not comfortable giving a response . when asked what between - group difference in the number of sres would be an appropriate amount to suggest equivalent effectiveness between groups , 17 ( 26.6% ) suggested a null difference , 24 ( 37.5% ) suggested a difference within 1 sre , 16 ( 25% ) suggested a difference within 2 sres , and 3 ( 4.7% ) suggested within 3 sres ( 4 ( 6.3% ) were not comfortable providing a response ) . while designing a clinical trial to compare different frequencies of drug administration should use expected differences in each outcome based on prior trial results , it is important to also obtain feedback on subjective evaluations and feedbacks from physicians who use these agents . this type of feedback also helps ensure that the results of such a trial will likely have a clinical impact on patient care . the distribution of characteristics shows that the majority work in teaching hospitals , consistently manage moderate to large numbers of patients , and that 55.6% ( 50/90 ) see at least one new patient per month , suggesting the population is an experienced group of oncologists . approximately two thirds of the respondents were medical oncologists , with the remainder consisting of radiation oncologists and urologists . physicians were located predominantly in ontario ( 60% ) , quebec ( 13.3% ) and alberta ( 11.1% ) . totals of 43 , 26 and 18 respondents treated breast cancer , prostate cancer , or both , respectively ; 3 respondents failed to indicate their specialty . amongst the 90 respondents , 73.3% ( 43/43 treating breast cancer patients , 12/26 treating prostate cancer patients , and 11/18 treating both ) indicated that use of bone - targeted agents is a part of their clinical practice , and thus continued on to complete component two of the survey . respondents were first asked to describe the primary reasons they provide bone - targeted agents to their patients , with the ability to select multiple reasons as deemed relevant ( see online questionnaire in supplementary data ) . most respondents primarily provide bone - targeted agents to reduce fracture risk ( 95.45% ) , to reduce risk of surgery to bone or radiotherapy ( 87.88% ) , to reduce metastasis pain ( 89.39% ) , to improve quality of life ( 72.13% ) , to reduce hypercalcaemia risk ( 71.21% ) , and to reduce risk of spinal compression ( 68.18% ) . few do so based on beliefs that these agents will improve progression - free survival ( 18.18% ) or overall survival ( 4.55% ) . response profiles were generally consistent across sub - populations of those treating breast cancer , prostate cancer , or both diseases ( fig . 1 ) . to assess the situations in which bone - targeted treatment might be prescribed , respondents were presented a series of five scenarios and asked whether they would prescribe a bone - targeted agent to the patient in question . the physician was asked , for a typical patient seen in your clinic with newly diagnosed bone metastases , i would usually recommend a bone - targeted agent even if this patient has little or no pain . for the responding clinicians 93.75% indicated they would recommend treatment in patients with newly diagnosed bone metastases , even if there is little to no pain present . for scenario 2 clinicians were asked , for a typical patient seen in your clinic with newly diagnosed bone metastases , i would usually recommend a bone - targeted agent even if the metastases were blastic . results from scenario 3 showed that approximately 60% of respondents would still recommend bone - targeted therapy in patients who have a prognosis of 6 months or less due to visceral metastases . data from scenario 4 showed that only 28.13% of responding clinicians would recommend treatment in patients who display poor oral hygiene or who require considerable dental work . finally , in scenario 5 , amongst the 23 respondents managing prostate cancer patients who use bone - targeted agents , 21.74% indicated that they would only start therapy in conjunction with chemotherapy . three of the 90 respondents indicated regular monitoring of patients with new bone metastases using biomarkers such as urinary n - telopeptide to assess response to treatment . there were some differences in the bone - targeted agents used for breast cancer when compared to prostate cancer ( fig . pamidronate was the agent most consistently used ( 52/61=85.25% ) , while denosumab ( 42/61=68.85% ) , zoledronic acid ( 40/61=65.57% ) and oral clodronate ( 31/61=50.82% ) had also been used to various degree by large proportions of clinicians . amongst respondents who treat prostate cancer , zoledronic acid ( 20/23=86.96% ) and denosumab ( 19/23=82.61% ) , there was a variation in the frequency of administration of bone targeted agents ( fig . a total of 16/52 ( 30.77% ) treat patients every 34 weeks from the start , 8/52 ( 15.38% ) responded every 34 weeks unless there is a fall in performance status , 1/52 ( 1.92% ) indicated every 34 weeks for 3 months before modifying to once every 3 months , 11/52 ( 21.15% ) indicated every 34 weeks for a year before modifying to once every 3 months , 11/52 ( 21.15% ) indicated every 34 weeks for 2 years before modifying to once every 3 months . finally , 5/52 ( 9.61% ) respondents noted other scenarios , which relied upon patients ' sre history , extent of disease , and presence of concurrent chemotherapy . for prostate cancer patients , 8/23 ( 34.78% ) treat patients every 34 weeks from the start , 5/23 ( 21.74% ) every 34 weeks unless there is a fall in performance status , 3/23 ( 13.04% ) indicated every 34 weeks for 3 months before modifying to once every 3 months , 1/23 ( 4.35% ) indicated every 34 weeks for a year before modifying to once every 3 months . finally , 3/23 ( 13.04% ) indicated every 34 weeks for 2 years before modifying to once every 3 months , and 3/23 ( 13.04% ) noted other frequencies . the answers showed that cessation was not common ; 28/64 ( 43.75% ) indicated never or < 5% of the time , 21/64 ( 32.80% ) between 5% and 25% of the time , 8/64 ( 12.5% ) between 26% and 50% of the time , and 6/64 ( 9.4% ) > 50% of the time . this trend was generally consistent amongst the sub - groups of those treating breast cancer , prostate cancer , or both . clinicians did not feel that worsening of bone pain , the presence of radiographic evidence of progression or the occurrence of an sre while on a bone - targeted agent was an indication to stop this therapy . indeed , the most common reasons for treatment cessation were ; a marked deterioration of performance status ( 57.81% ) , the occurrence of intolerable side effects ( 18.75% ) and progression of disease outside the bone ( 4.7% ) . other assorted reasons ( 18.75% ) included ; presence of renal failure , duration of treatment > 12 years , rising creatinine levels , and proximity to death . respondents were first asked to describe the primary reasons they provide bone - targeted agents to their patients , with the ability to select multiple reasons as deemed relevant ( see online questionnaire in supplementary data ) . most respondents primarily provide bone - targeted agents to reduce fracture risk ( 95.45% ) , to reduce risk of surgery to bone or radiotherapy ( 87.88% ) , to reduce metastasis pain ( 89.39% ) , to improve quality of life ( 72.13% ) , to reduce hypercalcaemia risk ( 71.21% ) , and to reduce risk of spinal compression ( 68.18% ) . few do so based on beliefs that these agents will improve progression - free survival ( 18.18% ) or overall survival ( 4.55% ) . response profiles were generally consistent across sub - populations of those treating breast cancer , prostate cancer , or both diseases ( fig . 1 ) . to assess the situations in which bone - targeted treatment might be prescribed , respondents were presented a series of five scenarios and asked whether they would prescribe a bone - targeted agent to the patient in question . the physician was asked , for a typical patient seen in your clinic with newly diagnosed bone metastases , i would usually recommend a bone - targeted agent even if this patient has little or no pain . for the responding clinicians 93.75% indicated they would recommend treatment in patients with newly diagnosed bone metastases , even if there is little to no pain present . for scenario 2 clinicians were asked , for a typical patient seen in your clinic with newly diagnosed bone metastases , i would usually recommend a bone - targeted agent even if the metastases were blastic . results from scenario 3 showed that approximately 60% of respondents would still recommend bone - targeted therapy in patients who have a prognosis of 6 months or less due to visceral metastases . data from scenario 4 showed that only 28.13% of responding clinicians would recommend treatment in patients who display poor oral hygiene or who require considerable dental work . finally , in scenario 5 , amongst the 23 respondents managing prostate cancer patients who use bone - targeted agents , 21.74% indicated that they would only start therapy in conjunction with chemotherapy . three of the 90 respondents indicated regular monitoring of patients with new bone metastases using biomarkers such as urinary n - telopeptide to assess response to treatment . respondents were asked to comment on their choice of bone - targeted agent . there were some differences in the bone - targeted agents used for breast cancer when compared to prostate cancer ( fig . pamidronate was the agent most consistently used ( 52/61=85.25% ) , while denosumab ( 42/61=68.85% ) , zoledronic acid ( 40/61=65.57% ) and oral clodronate ( 31/61=50.82% ) had also been used to various degree by large proportions of clinicians . amongst respondents who treat prostate cancer , zoledronic acid ( 20/23=86.96% ) and denosumab ( 19/23=82.61% ) , there was a variation in the frequency of administration of bone targeted agents ( fig . a total of 16/52 ( 30.77% ) treat patients every 34 weeks from the start , 8/52 ( 15.38% ) responded every 34 weeks unless there is a fall in performance status , 1/52 ( 1.92% ) indicated every 34 weeks for 3 months before modifying to once every 3 months , 11/52 ( 21.15% ) indicated every 34 weeks for a year before modifying to once every 3 months , 11/52 ( 21.15% ) indicated every 34 weeks for 2 years before modifying to once every 3 months . finally , 5/52 ( 9.61% ) respondents noted other scenarios , which relied upon patients ' sre history , extent of disease , and presence of concurrent chemotherapy . for prostate cancer patients , 8/23 ( 34.78% ) treat patients every 34 weeks from the start , 5/23 ( 21.74% ) every 34 weeks unless there is a fall in performance status , 3/23 ( 13.04% ) indicated every 34 weeks for 3 months before modifying to once every 3 months , 1/23 ( 4.35% ) indicated every 34 weeks for a year before modifying to once every 3 months . finally , 3/23 ( 13.04% ) indicated every 34 weeks for 2 years before modifying to once every 3 months , and 3/23 ( 13.04% ) noted other frequencies . the answers showed that cessation was not common ; 28/64 ( 43.75% ) indicated never or < 5% of the time , 21/64 ( 32.80% ) between 5% and 25% of the time , 8/64 ( 12.5% ) between 26% and 50% of the time , and 6/64 ( 9.4% ) > 50% of the time . this trend was generally consistent amongst the sub - groups of those treating breast cancer , prostate cancer , or both . clinicians did not feel that worsening of bone pain , the presence of radiographic evidence of progression or the occurrence of an sre while on a bone - targeted agent was an indication to stop this therapy . indeed , the most common reasons for treatment cessation were ; a marked deterioration of performance status ( 57.81% ) , the occurrence of intolerable side effects ( 18.75% ) and progression of disease outside the bone ( 4.7% ) . other assorted reasons ( 18.75% ) included ; presence of renal failure , duration of treatment > 12 years , rising creatinine levels , and proximity to death . in the third survey component respondents were asked to share their views regarding potential patient enrollment criteria , clinical outcomes of interest and minimum clinically important differences for these measures , to explore opinions regarding key parameters for the planning of future trials . when presented with several alternatives regarding potential patient populations that could be studied , physicians were asked to assess whom they would be comfortable enrolling into a randomized controlled trial . the question around duration of prior bone - targeted agent use prior to study randomization ( i.e. 3 months vs. 1 year ) did not produce a majority decision . however , in a situation of clinical equipoise the finding that 45.5% of respondents were comfortable randomising those who have received 3 months of prior therapy and 59.1% were comfortable with those who have received 1 year treatment tells us that such trials are still very feasible . physicians were asked to rank order the potential primary study outcomes for a clinical trial of de - escalated treatment from the most to the least clinically meaningful . sre was the measure that was most commonly ranked to be most meaningful ( 57/66=86.4% of respondents ) , while pain ( 10.60% ) and patient preference ( 3.03% ) were chosen much less frequently . when asked what between - group difference in the brief pain inventory ( bpi ) score would be an appropriate amount to suggest equivalent effectiveness between groups , 22 ( 34.4% ) suggested 1 point , 30 ( 46.9% ) suggested 2 points , 6 ( 9.4% ) suggested 3 points , and 6 ( 9.4% ) were not comfortable giving a response . when asked what between - group difference in the number of sres would be an appropriate amount to suggest equivalent effectiveness between groups , 17 ( 26.6% ) suggested a null difference , 24 ( 37.5% ) suggested a difference within 1 sre , 16 ( 25% ) suggested a difference within 2 sres , and 3 ( 4.7% ) suggested within 3 sres ( 4 ( 6.3% ) were not comfortable providing a response ) . while designing a clinical trial to compare different frequencies of drug administration should use expected differences in each outcome based on prior trial results , it is important to also obtain feedback on subjective evaluations and feedbacks from physicians who use these agents . this type of feedback also helps ensure that the results of such a trial will likely have a clinical impact on patient care . there is considerable interest in optimizing the frequency of administration of bone - targeted agents as the current one size fits all approach is likely inefficient , expensive , and there is reason to believe that some patients may achieve equivalent benefit and improved safety with less frequent treatment administration . the results of the two published trials , assessing reduced frequencies of administration of these agents , would suggest there is still a need for larger definitive studies , . in addition , we are not aware of any similar studies planned for prostate cancer where again the benefits of bone - targeted agents in reducing sres are likely even more modest than that seen in breast cancer patients . we are aware that physician surveys have been previously used to assess ; bisphosphonate use for bone pain , bisphosphonate use in metastatic breast cancer patients and radiotherapy use for painful bone metastases but are not aware of physician surveys being used to help design trials of bone - targeted agents . given the need for larger definitive studies this survey was designed to develop a greater understanding of the current clinical practice at canadian centers in terms of frequency of use of bone - targeted agents and preferred choice of agent and frequency of administration . it was also designed to explore the degree of clinical equipoise which would underlie potential future trials of de - escalated bone - targeted therapy in breast and prostate cancer patients . the results from this survey tell several important aspects of current clinical practice that can be explored in trials . the first component was devised to collect pertinent demographic information of the population of respondents , as well as to determine what proportion amongst them that use bone - targeted agents to treat their patients . firstly , it is evident that bone - targeted agents are commonly used for both breast and prostate patients with metastatic bone disease . it is also clear that the rationale for starting these agents are indeed based on practice guidelines , . the second component was designed to gain an understanding of current canadian clinical practice by collecting information from those respondents who prescribe bone - targeted agents . specifically we sought to collect information with regard to intended benefits from bone - targeted agent use , scenarios in which they prescribe bone - targeted agents and their choice of agent for their patients . from this part of the study it was clear that the rationale for starting these agents was based on practice guidelines , . respondents primarily provide bone - targeted agents to reduce the risk of skeletal related events and improve pain control . few felt that these agents had any effect on either progression - free or overall survival . the choice of bone - targeted agent reflected funding and therefore more agents were used in breast cancer patients ( pamidronate , clodronate , zoledronic acid or denosumab ) than in prostate cancer patients ( zoledronic acid or denosumab ) . also of interest was the fact that few clinicians use biomarkers of bone turnover to assess response to therapy . with respect to discontinuation of bone - targeted therapy clinicians did not feel that worsening of bone pain , the presence of radiographic evidence of progression or the occurrence of an sre while on a bone - targeted agent was an indication to stop this therapy . indeed , the most common reasons for treatment cessation was a marked deterioration in patient performance status . in the third component , respondents were presented with a series of questions related to the design of a future clinical trial geared toward studying the clinical benefits of de - escalated therapy , with topics of interest including outcome selection and patient inclusion criteria . with respect to potential future trials , given that once started these agents are rarely stopped , it would appear that initiating a study of effect of drug cessation would be extremely difficult to do from a practical standpoint . it is also evident that there is more variability in the choice of bone - targeted agent in the treatment of breast cancer patients than in prostate cancer , which is likely a reflection of differential funding for these agents . this means that for a de - escalation trial to be performed in breast cancer patients the design would need to incorporate the use of different bone - targeted agents . the results do however confirm that physicians are very interested in de - escalation trials and that they would consider de - escalating therapy from every 34 weeks to 3 monthly therapy and entering patients on such a study at anytime from 3 months to 2 years after starting treatment . however , physicians do want the primary study endpoint to be sre and not biomarker studies using biomarkers of bone turnover as a surrogate for skeletal related event risk . ultimately the results from our survey confirms that trials exploring de - escalated use of bone targeted agents are wanted for both breast and prostate cancer and that ultimately there will remain a need for further very large randomised trials with skeletal related events as the primary endpoint . , there is an inherent bias in those that are contacted and in those that respond . obtaining 90 responses on this topic is clearly important , however , the study would be strengthened by a greater number of responses . in addition , the majority of respondent were from the two most highly populated provinces in canada , however 70% were from ontario and quebec , representing around 66% of the total canadian population . as the respondents tend to have an interest in cancer and bone health and the majority is based in academic centers , there is a bias that the results of our study provide greater insight into practice at an academic center , but potentially less so in a broad scope of practice . however , it is important to appreciate that , in canada , most cancer care is delivered in large centers and most anti - cancer drugs are funded through central regulatory bodies with specific funding policies , thus reducing variability . another limitation of this type of survey is that it might not necessarily reflect what physicians actually prescribe . bisphosphonate prescribing has suggested that different treatment schedules may result in differences in skeletal morbidity . this study was retrospective and funded by the manufacturer of zoledronate and therefore does not replace the need for prospective randomised studies . we believe that our objectives of developing a sense of current canadian practice involving bone - targeted agents in patients with metastatic bone disease and exploring methodological considerations for future trials have been achieved . these data will provide valuable information to guide future efforts regarding this area of research .

objectivequestions remain regarding the optimal use of bone - targeted agents in patients with metastatic bone disease . the purpose of this study was to assess current clinical practice regarding the use and administration of bone - targeted agents by canadian oncologists in patients with metastatic breast and prostate cancer.methodsa survey was designed to explore ; bone - targeted agent use in metastatic bone disease , variability in the choice and the frequency of administration of these agents . opinions were sought on potential outcomes for future trials.resultsa total of 193 clinicians were contacted and 90 completed our survey ( response rate 49% after adjustment for inactivity ) . survey respondents were medical oncologists ( 71.1% ) , radiation oncologists ( 21.1% ) and urologists ( 7.8% ) . the findings suggest that once bone - targeted agents are started they are rarely discontinued . more agents are used in breast cancer than in prostate cancer . there was considerable interest in performing studies of de - escalated therapy in both breast and prostate cancer . physicians requested ( 86% ) that the primary study endpoint be the occurrence of skeletal related events and not biomarker driven.conclusionsdespite clinical practice guidelines and widespread use , significant areas of clinical equipoise with respect to use of bone - targeted agents exist . findings from this survey suggest that physicians are interested in de - escalated therapy for both breast and prostate patients . however , the use of multiple agents in breast cancer and the desire for skeletal related events to be the primary endpoint means that very large randomized studies will be required .

patient - oriented therapy represents a modern approach in the treatment of patients with diabetes , an approach which is supported in the most recent guidelines by the ada and the european association for the study of diabetes ( easd ) ( 1 ) . the progressive nature of diabetes demands the introduction of insulin therapy much earlier in order to prevent the development of late complications of the disease . though often this is not enough , it is necessary to combine other anti - diabetic agents in order to achieve target blood glucose values . it is necessary to consider multiple parameters for each individual during the introduction of anti - diabetic agents which will allow minimizing hypoglycemia and weight gain . results of major world studies clearly indicate that efforts for better glucose control often lead to the risk of hypoglycemia which is a significant risk factor for development of cardiovascular complications . it is necessary to consider the duration of the disease for each and every individual , as well as the presence of late complications and comorbidities with consideration of potential adverse effects of the treatment ( 2 ) . the current therapeutic algorithms are more and more the subject of a debate , which is why there is a constant need for seeking to improve the therapeutic approach by the introduction of new anti - diabetic agents . the prevalence of diabetes in the world is caused by the increasing number of obese people , and at the same time , the treatment of patients with type 2 diabetes leads to weight gain . evidence provided from the world studies show that patients who were treated with insulin during the twelve - year period , on average gained about 8 kg in weight . similar effects are shown by the treatment with sulfonylurea and thiazolidinedione derivatives which leads to hypoglycemia and weight gain . all these facts clearly indicate that we must pay special attention to the treatment of obese patients with type 2 diabetes . bmi before and after the test hba1c before and after the test fasting glycaemia level blood glucose level after meals ideal treatment of type 2 diabetes involves a treatment of glycemia , blood pressure , dyslipidemia , and body weight . increntin therapy that includes agonists of the glp 1 receptors , acts on lowering the level of hba1c and body weight , together with the reduction of cardiovascular risk factors such as blood pressure and dyslipidemia ( 3 , 4 ) . to compare the effects of therapy with basal insulin analogues combined with a glp 1 analogue and metformin with therapy of basal insulin analogues in combination with metformin . the study included 30 patients who had been treated with long - acting insulin analogue and metformin in doses of 3 x 850 mg at least 6 months prior to study entry and in which a good glycaemic control had not been achieved , or with hba1c > 7% . patients who had a bmi > 28 kg /m2 were included in the study . the parameters monitored prior and after the study were : the patients were switched with new therapy using basal insulin analogs combined with glp1 analogues and metformin for 12 weeks . the study included 30 patients who were treated in the counseling clinic of the endocrinology clinic in sarajevo . . the average age of the involved population group amounted to 54.15.06 years ; duration of diabetes was 4.061.73 years . average baseline parameter values covered in the study amounted to : hba1c 8.810.77% , the average value of fasting blood glucose level was 8.40.61 mmol / l , postprandial glycaemic level was 9.610.84 mmol / l , systolic blood pressure level with included stable dose of antihypertensive 149.08.15 and diastolic blood pressure level 90.33.2 mmhg . the patients were switched to combined therapy with long - acting basal analog , metformin and liraglutide in a dosage of 0.6 mg of 1x1 . after 12 weeks of the new therapeutic regimen we recorded a significant reduction in the parameter levels that we monitored in the study . bmi value after the test was 28.21.39 kg / m2 , p=0.025 , hba1c 7.240.47% , p=0.030 , fasting blood glucose level 7.040.32 mmol / l , p=0.023 , postprandial glucose level 7.60.46 mmol / l , p=0.012 , systolic blood pressure level 1235.75 mmhg , p=0.015 , diastolic blood pressure level 79.12.91 mmhg , p=0.03 . diabetes mellitus type 2 has a progressive course which requires patientsconstant monitoring of glycemia together with supervision of attending physicians in order to act in a timely manner and include additional antidiabetic drug . it is clear that hyperglycemia leads to further destruction of pancreatic beta cells because of their gluco - toxicity and further progression of diabetes disease and complications afterwards . in recent decades , attempts are made to find the optimal therapeutic solution that will enable action on complete pathophysiology mechanism of type 2 diabetes and also at the same time , reduce the risk of weight gain and the occurrence of hypoglycemia . long - acting basal insulin analogues provide 24- hour supply of insulin , which compensates for the lack of endogenous insulin that has arisen because of the destruction of beta cells . exogenous insulin also suppresses hyperinsulinemia that develops due to insulin resistance which is one of the characteristics of type 2 diabetes . this explains the significance of including long - acting insulin analogues in the treatment of type 2 diabetes . origin trial clearly represents the significance of the basal insulin analogues in the treatment of diabetes and impaired glucose tolerance . during the six years of treatment with basal long - acting insulin analogues , number of newly diagnosed patients with type 2 diabetes was reduced in the treated population with impaired glucose tolerance . exogenous insulin prevents and delays at the same time further destruction of the beta cells of the pancreas , thus reducing glucotoxicity ( 5 ) . incretin hormone glucagon - like - peptide-1 ( glp-1 ) and glucose - dependent insulinotropic polypeptide ( gip ) are released into the small intestine during the food intake and increase in blood glucose in the body . incretin hormones stimulate glucose - dependent insulin secretion , whereas glp 1 also suppresses glucagon secretion thus delaying the gastrointestinal emptying and lowering postprandial glycemia . the pharmacological effect of glp1 involves stimulation of endogenous insulin secretion , suppressing the secretion of glucagon , which provides good glycaemic control . at the same time , this is the basic pathophysiological disorder in type 2 diabetes . it is important to be pointed out that the effect is observed only at elevated blood glucose , thereby reducing the risk of hypoglycemia . delayed emptying of the stomach and glp1 action in the central nervous system on appetite decrease enables the reduction of body weight . it is demonstrated through study that after 8 years of insulin therapy application leads to increase in body weight by 5 kg . this justifies the importance of the use of glp1 analogues in combination with long - acting insulin analogue ( 6,7,8 ) . metformin , which is a mild insulin senziter but has a significant effect on suppression of gluconeogenesis , prevents destruction of the beta cells and has an anti - atherogenic effect . with all this being said , metformin is a significant addition to the regulation of postprandial glucose . this therapeutic approach enables action on different pathophysiological processes in type 2 diabetes reducing the risk of weight gain and hypoglycemia which were leading therapeutic challenges over the last decade in the treatment of type 2 diabetes . during research that we have conducted over 12 weeks , a reduction of body weight was achieved while improving the value of parameters significant for the study . there was a significant lowering of hba1c , fasting blood glucose levels , postprandial glucose levels and better blood pressure control by which we have proved that glp1 analogues in combination with basal insulin and metformin provide a good glycaemic control with a cardio protective effect , and reduce the risk of late complications ( 9,10,11 ) . intensification of treatment of type 2 diabetes by adding liraglutide in addition to existing basal insulin analogue and metformin therapy provides improvements in hba1c levels together with better glycaemic control , fasting / postprandial glucose levels and body weight . significant reductions in blood pressure poster an additional vasodilatory effect and reduce the risk of cardiovascular complications .

abstractintroduction : patient - oriented therapy represents a modern approach in the treatment of patients with diabetes , an approach which is supported in the most recent guidelines by the ada and the european association for the study of diabetes ( easd ) . the progressive nature of diabetes demands the introduction of insulin therapy much earlier in order to prevent the development of late complications of the disease.material and methods : the study included 30 patients who had been treated with long - acting insulin analogue and metformin in doses of 3 x 850 mg at least 6 months prior to study entry and in which a good glycaemic control had not been achieved , or with hba1c > 7% . patients who had a bmi > 28 kg /m2 were included in the study.results and discussion : at the beginning of the study the patients were switched to combined therapy with long - acting basal analog , metformin and liraglutide in a dosage of 0.6 mg of 1x1 . after 12 weeks of the new therapeutic regimen we recorded a significant reduction in the parameter levels that we monitored in the study . bmi value after the test was 28.21.39 kg / m2 , p=0.025 , hba1c 7.240.47% , p=0.030 , fasting blood glucose level 7.040.32 mmol / l , p=0.023 , postprandial glucose level 7.60.46 mmol / l , p=0.012 , systolic blood pressure level 1235.75 mmhg , p=0.015 , diastolic blood pressure level 79.12.91 mmhg , p=0.03 . during research that we have conducted over 12 weeks , a reduction of body weight was achieved while improving the value of parameters significant for the study.conclusion:there was a significant lowering of hba1c , fasting blood glucose levels , postprandial glucose levels and better blood pressure control by which we have proved that glp1 analogues in combination with basal insulin and metformin provide a good glycaemic control with a cardio protective effect , and reduce the risk of late complications .