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[ { "content": "You are a medical writer. Summarize the following article: evidence has emerged that inflammatory mechanisms contribute to the onset and progression of type-1 diabetes ( t1d ) . \n recent data further suggest that the initiation of autoimmunity is preceded by inflammation reflected by a proinflammatory metabolic serum profile . \n many autoimmune diseases are associated with increased histamine levels in serum and tissue fluids , such as rheumatoid arthritis , sjgren 's syndrome , multiple sclerosis , and diabetes mellitus [ 14 ] . \n similarly , it has been known for many years that histamine is elevated in tissue biopsies and secretions in the intestine of patients with crohn 's disease and ulcerative colitis [ 5 , 6 ] . during inflammatory responses , \n histamine , a hydrophilic vasoactive amine , increases capillary permeability and facilitates the immune response by enhancing leukocyte rolling , adhesion , and vascular extravasation of inflammatory cells to the site of inflammation . \n histamine is recognized not only as an inflammatory mediator but also as a regulator of immune responses , including the th1/th2 balance and hematopoiesis . \n patients with diabetes are more susceptible to vascular diseases , which led to the assumption that their increased circulating histamine levels promoted lipolysis and facilitated the transport of lipids into tissues . beside its physiological role in vascular health , excess histamine , \n a result of physical or emotional stress or a chronic disease / inflammatory state , appears to elicit atherogenic effects . \n whether histamine is a critical marker or target in the treatment of type-1 diabetes has not yet been established . \n it is still poorly understood how the expression of histidine decarboxylase ( hdc ) , the unique enzyme responsible for histamine generation , is controlled . \n hdc is induced in a variety of tissues in response to bacterial components ( lipopolysaccharides , peptidoglycan ) and to various cytokines such as il-1 , il-3 , il-12 , tnf , g - csf , and gm - csf . \n its upregulation has been associated with pathological conditions , including t1d . on the other hand , \n an impaired glucose tolerance has been reported in hdc mice , together with autoantibodies reactive to glutamic acid decarboxylase ( gad ) . \n histamine exerts important functions in allergy , inflammation , neurotransmission , and the gastrointestinal tract through g - protein - coupled specific histamine receptors , termed h1r - h4r . in the context of diabetic physiopathology , it plays a pivotal role in various physiological functions , such as feeding behaviour and energy homeostasis . \n mice with genes disrupted for hdc are prone to become obese on a high - fat diet or at advanced age . \n these metabolic changes presumably are due to the impaired regulatory loop involving hypothalamic histamine and h1r [ 12 , 13 ] . \n consequently , treatment with imetit , a h3r agonist , reduced plasma histamine , leptin , and insulin levels and has anorexigenic effects in diet - induced obesity . \n histamine has been shown to regulate t cells by enhancing th1-type responses through the h1r and downregulating both the th1 and th2 type responses through the h2r [ 15 , 16 ] . \n it has recently been described that anti - inflammatory and immunomodulatory functions of histamine are mediated through the h4r . \n thus , they can be reproduced using the h4r agonist clobenpropit , which has been shown to suppress il-12 production , modulate dendritic cell migration [ 18 , 19 ] , and regulate the growth of cancer cells through h4r . autoimmune diabetes in nod mice is characterized by a progressive mononuclear cell infiltration in the pancreatic islets of nod mice and leads to beta cell destruction and hyperglycemia . \n it has been recently demonstrated that leptin can reverse diabetes . assuming that histamine might be involved in the pathogenesis of diabetes \n , we investigated the relationship between endogenous histamine production and disease development in the nod mouse model , using histamine - deficient hdc mice backcrossed on a nod background . here , we show that the lack of endogenous histamine provides a notable protection against t1d . \n we observed an increase of ly6gcd11b immature myeloid cells in these mice that was not correlated with diabetes onset , while il-12 and ifn- levels in the serum were decreased , suggesting that endogenous histamine modulates the inflammatory pattern . \n paradoxically , we found that t1d onset was also delayed when exogenous histamine was given to nod . \n our study supports a metabolic role of histamine during the pathogenesis of autoimmune diabetes in accordance with previous findings supporting its immunomodulatory functions . \n conventional nod mice ( k , i - a , and d ) were bred in our animal facility at the hpital necker . \n hdc mice generated by ohtsu et al . received a histamine - low diet ( safe ( scientific animal food and engineering ) ) to avoid exogenous uptake . \n we generated hdc - deficient mice by speed backcrossing the hdc - deficient allele against the nod background . in order to confirm the allele maps of nod \n diabetes susceptibility genes , we analysed several representative microsatellite markers : we chose the alleles related to mhc and numerous insulin - dependent diabetes genes . \n then , mice were intercrossed to generate the hdc mice used in this study . we purchased histamine and 4-methylhistamine ( 4-mh ) from sigma - aldrich and clobenpropit dihydrobromide ( cb ) from tocris . \n l of saline once a week for 10 weeks starting at 4 weeks of age . \n h4r agonists , cb ( 10 g / kg ) , 4mh ( 10 g / kg ) , and vehicle ( control group for h4r - agonist ) were injected i.p . in 100 \n leptin ( millipore ) , ifn- ( r&d ) , il-6 ( r&d ) , and il-12 ( r&d ) were quantified in sera from mice , using elisa kits according to the manufacturer 's instructions . \n we treated nod mice with histamine , cb , or 4-mh and monitored weekly for clinical signs of diabetes using gluko - test reagent sticks , to detect glucose in urine samples ( boehringer mannheim , meylan , france ) . when needed , glycemia was also measured in a drop of blood collected from the tail vein using a reflolux s glucometer ( boehringer mannheim ) . \n incidence of diabetes was defined based on the discovery , upon serial monitoring , of glycosuria and hyperglycemia ( fasting glycemia > 2.5 g / l ) . to induce t1d by adoptive transfer , \n purified cd4 diabetogenic t cells from prediabetic nod mice were injected i.v . into eight - week - old nod rag recipient mice with a cd11b population ( 5 10 cells / mice ) \n bone marrow cells were stained with cd11b microbeads ( miltenyi biotec ) then sorted by positive magnetic separation on ls columns ( macs separator , miltenyi ) . \n a high incidence of t1d is elicited within 10 weeks after t cell transfer . bone marrow ( bm ) , spleen , and peripheral whole blood cells \n bm and spleen cells were prepared as previously reported [ 25 , 26 ] and adjusted to a final concentration of 1 10 per ml in culture medium ( pbs from gibco brl ) . \n cell suspensions were stained with the following appropriately labeled antibodies : cd11b , ly6g- ly6c , cd4 , cd25 , foxp3 , mpdca-1 , cd11c , cd115 , mhc - ii , cd80 , and cd86 ( bd biosciences , pont - de - claix , france ) . \n mononuclear cells were gated according to forward- and side - scatter properties and analysed using the facs diva software . \n diabetes incidence was plotted using the kaplan - meier method , that is , nonparametric cumulative survival plot . statistical comparison between curves \n was performed using the log rank ( mantel - cox ) test that provided the corresponding values . when needed \n to evaluate the role of histamine in autoimmune diabetes , we generated nod mice deficient for the gene encoding hdc in the nod genetic background . \n these genetically engineered mice were healthy and heavier than their wild - type controls after several months . \n as shown in figure 1 , the onset of t1d was delayed in nod hdc mice and the disease incidence remained lower than that among wild - type controls , suggesting a deleterious role of histamine during pathogenesis . it may be hypothesized that an increase in plasma and cellular histamine content contributes to disease progression by enhancing endothelial permeability , as suggested for the intimal damage in atherosclerosis . in favour of this hypothesis , \n histamine levels were effectively increased in diabetic nod mice ( data not shown ) , similarly to what has been reported for streptozotocin - diabetic rats . to assess potential changes in immunological and hormonal parameters in the absence of histamine , we analysed interleukin-12 ( il-12 ) , il-6 , and leptin . il-12 and \n ifn- levels were lower in hdc than in nod mice , but no differences were noted for il-6 or leptin ( figure 1(b ) ) . as histamine contributes to the differentiation and maturation of hematopoietic myeloid cells , we performed a comparative cytometry analysis of these populations in histamine - deficient and wild - type nod mice in various organs , such as bone marrow , spleen , and peripheral blood based on gr-1 , cd11b , ly6 g , and ly6c expression by leukocytes . \n we found an increased percentage of immature cd11bgr-1 cells in the peripheral blood and the spleen from nod hdc mice ( figure 2(a ) ) . \n a similar result was observed for the cd11bly6 g ly6c subset from peripheral blood and spleen of nod hdc mice ( figures 2(b ) and 2(c ) ) . \n these results confirmed the previously reported importance of histamine for the differentiation of cd11bgr-1 myeloid subsets in a different genetic background . \n we hypothesized that the increased proportion of immature myeloid cells ( imcs ) might be involved in the protection against type-1 diabetes in histamine - deficient mice . to test this assumption , we performed adoptive transfers of effector cd4cd62l t cells together with cd11b bone marrow cells from hdc - deficient or wt mice to nod rag recipients , which did not alter diabetes onset ( figure 2(d ) ) , ruling out the increase of imcs as a cause for diabetes progression . \n we also analysed the status of myeloid - derived suppressive cells ( mdscs ) , which can modulate diabetes onset . \n however , no difference was observed when comparing this subset in both strains ( figure 2(e ) ) . \n similarly , the proportion of cd4cd25foxp3 regulatory t cells remained unchanged ( figure 2(f ) ) . \n since the generation of endogenous histamine appears to enhance the pathogenesis of t1d , we attempted to reproduce this deleterious effect by adding exogenous histamine . \n injections of either vehicle ( 100 l pbs ) or histamine ( 4 mg / kg ) , respectively , once a week for 10 weeks . as shown in figures 3(a ) and 3(b ) \n , exogenous histamine not only failed to increase the incidence of t1d , but also delayed the onset of disease in both wild - type and hdc mice . \n as shown in figure 3(c ) , decreased expression of class ii major histocompatibility complex ( mhc - ii ) and cd86 molecules occurred only on pdcs from pancreatic lymph nodes ( lns ) in mice treated with histamine , suggesting that this mediator may modulate the activation pattern of apcs . \n recent evidence supports the notion that the inflammatory functions of histamine are mediated through the h4r [ 20 , 29 , 30 ] . for this reason , we examined whether compounds characterized as potent h4r agonists could reproduce the protective effect provided by exogenous histamine . \n we injected nod mice with clobenpropit ( cb ) , an isothiourea derivative of histamine , or 4mh for 10 weeks and found no significant protection as compared with mice having received vehicle alone ( figures 3(d ) and 3(e ) ) . \n in the present study , we provide evidence that histamine deficiency causes a delay in diabetes onset and decreases its incidence . \n this result fits with clinical observations in diabetic patients since increased histamine levels were associated with pathogenesis in these patients . \n furthermore , there is now a compelling body of evidence for the contribution of histamine to the development of autoimmune diseases from animal studies . \n it has also been reported that inhibition of histamine synthesis in experimental diabetes reduces disease complication . \n several arguments suggest that in the present study the phenotype results from vascular and/or metabolic functions of histamine . this conclusion has already been proposed in a rat diabetic model , since \n the authors have shown that the gastric histidine decarboxylase activity and plasma gastrin levels were increased in connection with the depletion of insulin . \n we can thus anticipate that decreasing histamine levels in diabetic patients can decrease the risk of vascular complications , which is one critical issue in the cure for patients . \n there is increasing evidence that diabetes is under the control of numerous cytokines and hormones . \n since leptin can influence diabetes [ 22 , 33 ] , we have measured the level of this mediator in serum of starved mice of both strains . however , we observed no change in its level . \n we observed that il-12 and ifn- levels were lower in nod hdc female mice compared with wt controls . \n these cytokines are known for promoting diabetes progression in the nod strain , possibly via induction of th1-type cytokines [ 34 , 35 ] . by contrast \n , circulating il-6 or leptin did not differ between wt and ko mice , ruling out a major involvement of these factors in the delayed disease onset in hdc - deficient mice . \n even though we detected hematopoietic anomalies in the myeloid population of hdc mice , these cells were not involved in the protective response , as demonstrated by adoptive transfer . \n although neutrophils express the same markers as imcs , the protection against diabetes in hdc - deficient mice can not be ascribed to an increase of this population among the cd11bgr-1 subset since neutrophil depletion has been shown to inhibit type-1 diabetes development in nod mice . furthermore , the expansion of imcs is not concomitant with mdscs since the two populations bear different markers and most mdscs do not express ly6 g \n . furthermore , we found that the proportion of regulatory myeloid cells was not modified comparing both strains . \n finally , the increased histamine levels associated with the pathogenesis of diabetes are expected to be a consequence rather than a cause of disease . \n therefore , adding exogenous histamine may act on the gastric mucosa to enhance the flow of the lipidic factors but will be unable to reverse the phenotype observed in hdc - deficient mice . indeed , we observed the opposite effect . \n we describe a new immunomodulatory function of histamine in our model since diabetes onset is delayed in both wild - type and hdc mice after treatment . \n for example , the role of the h3r on food intake has been well documented . \n since it has been previously established that h4r drives inflammatory responses in asthma , dermatitis , arthritis , and colitis [ 29 , 30 , 37 ] , we hypothesized that its agonists could reproduce the effects of histamine . \n this was not the case in this study , but even though we could not validate the h4r pathway using the agonists cb or 4-mh , we can not exclude that the dose , the route of administration , or the choice of the agonists tested was inadequate . \n alternatively , a mixture of h2r and h4r agonists may be required to obtain a protective effect similar to the one induced by histamine . \n histamine , which is increased in inflammatory and pathological situations , has also been shown to decrease , in vitro , the production of il-12p70 of monocyte - derived dendritic cells ( dcs ) via h2r and h4r [ 17 , 19 , 38 ] and to inhibit il-27 production in activated apcs . \n the regulation of cytokine production in maturing dcs causes an alteration of the t cell polarization that has been well described [ 15 , 40 , 41 ] . \n more recently , it has been reported that histamine decreases the migration of human pdcs and their cytokine production , while increasing regulatory t cell recruitment . in histamine - treated nod mice \n , we observed that pdcs from pancreatic lymph nodes expressed lower levels of costimulatory and mhc - ii and cd86 molecules than their counterpart from control mice , consistent with a decreased inflammatory state . \n contrary to our anticipations , injection of histamine did not promote diabetes pathogenesis but , paradoxically , decreased disease onset . \n the immunomodulatory function of histamine has been described in other inflammatory models and histamine is used therapeutically in patients suffering from acute myeloid leukemia . \n histamine release can be induced by different stimuli , such as cytokines or microbial stimulation . \n it has been previously shown that human basophils release histamine in response to a toll - like receptor 2 ( tlr2 ) agonist . \n interestingly , tlr2 agonists have also been implicated in the modulation of t1d in the nod mouse . \n similarly , parasites such as schistosoma mansoni protect nod mice and parasites stimulate basophils and induce histamine release . \n these results support our hypothesis that local histamine release upon stimulation of immune cells , such as basophils , may participate in immunoregulatory pathways in autoimmune diabetes .", "role": "user" }, { "content": "recent evidence has highlighted the role of histamine in inflammation . since \n this monoamine has also been strongly implicated in the pathogenesis of type-1 diabetes , we assessed its effect in the nonobese diabetic ( nod ) mouse model . to this end , we used mice ( inactivated ) knocked out for the gene encoding histidine decarboxylase , the unique histamine - forming enzyme , backcrossed on a nod genetic background . \n we found that the lack of endogenous histamine in nod hdc/ mice decreased the incidence of diabetes in relation to their wild - type counterpart . whereas the proportion of regulatory t and myeloid - derived suppressive cells was similar in both strains , \n histamine deficiency was associated with increased levels of immature macrophages , as compared with wild - type nod mice . \n concerning the cytokine pattern , we found a decrease in circulating il-12 and ifn- in hdc/ mice , while il-6 or leptin remained unchanged , suggesting that histamine primarily modulates the inflammatory environment . \n paradoxically , exogenous histamine given to nod hdc/ mice provided also protection against t1d . \n our study supports the notion that histamine is involved in the pathogenesis of diabetes , thus providing additional evidence for its role in the regulation of the immune response .", "role": "assistant" } ]

[ { "content": "You are a medical writer. Summarize the following article: the amyloid hypothesis states that accumulation \n of amyloid- \n ( a ) peptides in the brain is the primary driver of pathogenesis \n in alzheimer s disease ( ad ) , including synapse loss and neuronal \n cell death . the full - length a1 - 42 peptide and its a1742 fragment ( p3 ) \n are formed via cleavage of the amyloid precursor protein \n ( app ) by the action of three secretase enzymes . \n the \n ape3 - 42 fragment is post - translationally \n generated by cleavage of the first two n - terminal amino acids of a1 - 42 , leaving an exposed glutamate ( e ) residue in position \n 3 . \n the lactam ring in the pyroglutamate ( pe ) residue is subsequently \n generated by intramolecular dehydration catalyzed by the glutaminyl \n cyclase ( qc ) enzyme . \n pe - modified \n as represent the dominant fraction of a \n oligomers in brains of ad patients . \n autopsied \n brains of elderly patients with normal cognition also show accumulation \n of a1 - 40/42 , but the amount of accumulated \n ape3 - 42 is significantly lower . consequently , the ratio of ape3 - 42/a1 - 42 oligomers is higher in ad brains \n than in brains of normal elderly individuals . \n the \n larger accumulation of ape3 - 42 in ad brains \n has been attributed to its increased stability and higher aggregation \n propensity . \n these properties are attributed \n to the lactam ring in the pe - modified third residue as well as the \n loss of electrical charge in three residues during the conversion \n of a1 - 42 to ape3 - 42 , thus resulting in higher ape3 - 42 hydrophobicity \n and increased resistance to degradation by peptidases . \n significantly , it is also believed that the formation of \n ape3 - 42 plaques precedes a1 - 40/42 plaque formation . \n this is supported \n by the observation that ape3 - 42 plaques appear \n earlier than a1 - 40/42 plaques in down syndrome \n ( ds ) brains . \n the additional copy of chromosome \n 21 , characteristic of ds , is responsible for generating more app , \n and thus individuals with ds are more likely to develop ad earlier \n in life . increasing evidence suggests \n that following initial interactions \n on the cell membrane , a oligomers insert into the membrane \n and form pore structures . \n cell toxicity \n results from an abrupt change in cell ionic concentration , producing \n loss of cell homeostasis . \n pore activity has been observed for full - length \n as , a \n fragments , and point substitutions . in \n addition to pore formation , a-induced toxicity mechanisms include \n lipid extraction by peptides on the membrane surface . \n these mechanisms are not mutually exclusive , as a recent \n study suggested that pore formation precedes nonspecific fragmentation \n of the lipid membrane during amyloid fiber formation . to the best of our knowledge \n , there is currently \n no experimental \n data for amyloid pore formation in vivo . ape3 - 42 \n these ape3 - 42 mice displayed a significantly reduced survival \n rate compared with a1 - 42 transgenic mice . in vitro , \n optical patch - clamping was used to \n characterize the single - channel ca fluorescence transients \n induced by a1 - 42 pores in membranes of xenopus laevis oocytes . \n however , \n most experimental evidence of amyloid pore formation stems from model \n membrane studies . here \n we characterize the electrical properties \n of pe - modified a \n in lipid membranes . \n we discuss the activity and structure of ape3 - 42 pores in anionic model cell membranes . \n we used \n phosphoethanolamine ( pe ) and phosphoserine ( ps ) lipid headgroups to \n mimic the brains of elderly patients . \n phospholipids with ethanolamine \n ( pe ) head groups are one of the dominant components in the brains \n of the elderly , and these levels , as \n well as those of ps , have been found \n to change in ad brains . \n using planar \n lipid bilayer ( plb ) electrical recording , we show that ape3 - 42 induces pore activity in anionic membranes , producing \n increased membrane permeability with respect to a1 - 42 pores . using atomistic molecular dynamics ( md ) simulations , we model \n the architecture of ape3 - 42 pores and correlate \n them with the activity observed experimentally . \n ape3 - 42 was purchased \n from bachem ( torrance , ca ) , and a1 - 42 was \n purchased from bachem and anaspec ( fremont , ca ) . the phospholipids \n 1,2-dioleoyl - sn - glycero-3-phosphoserine ( dops ) and \n 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphoethanolamine \n ( pope ) were purchased from avanti polar lipids ( alabaster , al ) . all \n other chemicals were purchased from sigma - aldrich ( st . louis , mo ) . \n ape3 - 42 and \n a1 - 42 peptides were dissolved in milli - q \n water to a concentration of 1 mg / ml prior to being aliquoted for storage . \n these 50 l aliquots were stored at 80 c for a \n maximum of 60 days before use . \n bilayers were formed from a 1:1 ( w / w ) mixture \n of dops and pope in heptane at a total lipid concentration of 20 mg / ml . \n spontaneous membrane formation occurs following the addition of lipid \n directly over a pore with a diameter of 250 m in a \n delrin septum ( warner instruments , delrin perfusion cup , volume 1 \n ml ) . in previous studies \n , this membrane composition was shown to be \n stable for long recording times ( 4 h ) . \n as the electrolyte , we use 150 mm kcl , 10 mm hepes ( ph \n 7.4 ) , and 1 mm mgcl2 . before performing electrical \n recordings \n , we verified that the bilayer was stable for several minutes \n with low conductance ( < 10 ps ) across 100 mv applied voltage \n and that the system capacitance was > 110 pf . \n when both criteria \n were \n met , peptide was added directly to the cis ( hot wire ) side and stirred \n for 5 min . \n bilayer stability was monitored by periodic capacitance \n measurements throughout the course of the experiment . \n all traces \n were recorded in voltage clamp mode using the 2 khz \n built - in filter cutoff of our bc-535 amplifier ( warner instruments , \n hamden , ct ) . \n we used a custom - made labview program to record the current \n and clampfit 10.2 ( molecular devices , sunnyvale , ca ) to analyze traces . \n for representation in figures , \n we have filtered the recorded current \n versus time traces with a digital gaussian low - pass filter with a \n cutoff frequency of 50 hz . \n two u - shaped \n a monomer conformations with the -strand - turn--strand \n motif were extracted from a1 - 42 fibrils , \n where the structure was defined by hydrogen / deuterium - exchange nmr \n data , side - chain packing constraints from pairwise mutagenesis , ssnmr \n and em ( pdb code : 2beg ) , and small a1 - 40 protofibrils ( pdb codes : 2lmn and 2lmo ) , where the structure was based on \n a ssnmr model . in both structures , the n - terminal coordinates , residues \n 116 for the former and 18 for the latter structure , \n are missing due to disorder . \n we used the a116 coordinates , in the absence of zn ( pdb code : 1ze7 ) , for the missing portions of the peptides . for each combination \n of the n - terminal structure with the u - shaped motifs , \n conformer 1 has a turn at ser26-ile31 , and \n conformer 2 at asp23-gly29 . in the latter conformer , two c - terminal \n residues , ile41 and ala42 , \n , we removed the first two residues , asp1 and ala2 , \n from each conformer and converted glu3 into pe3 , generating ape3 - 42 . \n because the standard charmm force field does not provide a force field for pe , we first \n created the pe molecular topology using the avogadro software . \n then , we calculated partial charges , bond lengths , \n angles , and torsional angles for the atoms in the pe residue using \n the gaussian09 program on a biowulf cluster \n at the nih . \n two ape3 - 42 conformers , each derived from the wild - type ( wt ) \n a1 - 42 conformers with different turns , still \n retain the u - shaped structure with the -strand - turn--strand \n motif . \n to construct ape3 - 42 barrel structures , \n we inclined ape3 - 42 monomers 37 \n relative to the pore axis ; then , an 18-fold \n rotational symmetry operation was performed with respect to the pore \n axis creating an 18-mer ape3 - 42 barrel ( figure 1 ) . \n we modeled a barrels with the -sheet \n structure by mimicking naturally occurring -barrels observed \n in transmembrane proteins that are found frequently in the outer membranes \n of bacteria , mitochondria , and chloroplasts . \n the -barrel motif \n is a large -sheet composed of an even number of -strands . \n some known structures of -barrel membrane proteins have -strands \n ranging in size from 8 to 22 . \n we modeled \n 18-mer a barrels , with 18 -strands enclosing the solvated \n pore . \n this number is also in the range of the number of -strands \n for natural -barrels ranging from 8 to 22 , which can form a \n -barrel motif . \n our previous simulations for a channels \n indicate that different numbers of a monomers could produce \n channels with different outer and pore dimensions . \n we found that a channels obtained a preferred size range \n of 1624 -strands lining the pores . this range was also found to hold for other toxic -sheet \n channels : k3 channels with 24 -strands , 18- and 24-mer hiapp channels , pg-1 channels with 1620 -strands , and max channels with 20 -strands . in agreement with afm data , \n these channels have outer and \n pore dimensions within the range found with afm . in this work , \n the \n outer / pore diameters of the 18-mer ape3 - 42 barrels are in good agreement with the experimental afm ranges and the computational 18-mer wild - type a \n barrels . \n the afm experiments provide images of channels \n with a wide variety of sizes and shapes , but simulated a barrels \n are limited to cover all ranges of channel sizes that are imaged by \n afm . \n monomer conformations of ( a ) conformer 1 ape3 - 42 with turn at ser26-ile31 and ( b ) conformer 2 ape3 - 42 with turn at asp23-gly29 , and ( c ) highlight of pyroglutamate at \n residue 3 ( pe3 ) . \n the initial barrel structures of md \n simulations in ribbon representation for the ( d ) conformer 1 and ( e ) \n conformer 2 ape3 - 42 barrels . in the peptide \n ribbon , \n hydrophobic , polar / gly , positively charged , and negatively \n charged residues are colored white , green , blue , and red , respectively . \n the pe3 n - terminal termini are highlighted as threads . to obtain \n a lipid bilayer , we constructed a unit cell containing \n two layers of lipids . \n in the middle of the unit cell , lipid molecules \n were randomly selected from the library of the preequilibrated state \n and replaced by pseudo vdw spheres at the positions of the lipid headgroups , \n constituting the lipid bilayer topology . for a given number of lipid molecules , the optimal value of lateral \n cell dimensions can be determined . \n an anionic lipid bilayer composed \n of dops and pope with a mole ratio 1:2 , containing a total of 420 \n lipids , constitutes the unit cell with tip3p waters added at both \n sides . \n updated charmm all - atom additive \n force field for lipids ( c36 ) and the \n modified tip3p water model were used \n to construct the set of starting points and to relax the systems to \n a production - ready stage . \n the system contains mg , k , ca , and zn at the same concentration \n of 25 mm to satisfy a total cation concentration near 100 mm . \n the \n bilayer system containing an ape barrel , lipids , \n salts , and water has almost 200 000 atoms . \n we generated at \n least 10 different initial configurations for each conformer ape3 - 42 barrel for the relaxation process to obtain the \n best initial configuration for a starting point . in the pre - equilibrium \n stages , a series of minimizations was performed for the initial configurations \n to remove overlaps of the alkane chains in the lipids and to gradually \n relax the solvents around the harmonically restrained peptides . \n the \n initial configurations were gradually relaxed through dynamic cycles \n with electrostatic cutoffs ( 12 ) . \n the harmonic restraints were \n gradually diminished with the full ewald electrostatics calculation \n and constant temperature ( nos hoover ) thermostat / barostat \n at 303 k. for t < 30 ns , our simulation employed \n the npat ( constant number of atoms , pressure , surface area , and temperature ) \n ensemble with a constant normal pressure applied in the direction \n perpendicular to the membrane . \n production runs of 100 ns for \n the starting points with the namd code were performed on a biowulf cluster at the nih . \n plb electrical recording data demonstrate \n that ape3 - 42 peptides induce spontaneous \n pore activity through lipid membranes presenting the general features \n observed for a1 - 42 pores ( figure 2 ) . at a concentration of 10 m and at constant \n voltage both peptides show stepwise changes in the current flowing \n through the membrane ( figure 2a ) , characteristic \n of the opening and closing of ion channels . \n however , unlike ion channels \n that have regulated activity and possess integer values of a unitary \n conductance , amyloid pores are not regulated and present multiple \n conductance values due to the different number of monomers composing \n the pore and membrane channel interaction dynamics . \n the multilevel conductance seen here in both \n ape3 - 42 and a1 - 42 is a hallmark of amyloid pores . \n ape3 - 42 and a1 - 42 produce pore \n activity in anionic lipid membranes . \n representative \n traces for the activity of ( a ) ape3 - 42 and \n ( b ) a1 - 42 pores in stable membrane recording . \n the activity \n seen in both membranes , namely step , spike , and burst behavior is \n characteristic of amyloid ion channels . \n bilayers were formed by the \n painted technique using 1:1 ( w / w ) mixture of dops / pope . \n both sides \n of the bilayer chamber contained as electrolyte 150 mm kcl , 1 mm mgcl2 , and 10 mm hepes ( ph 7.4 ) . \n the first , as seen in figure 2 , was characterized \n by stable , long recording times ( > 30 min ) following the first onset \n of activity . \n the second type of activity was characterized by ionic \n conduction through the membrane that grows rapidly in an exponential - like \n fashion ( figure 3a ) . \n phase began within 20 min of the first observed activity and typically \n led to current saturation of the amplifier within 10 min . \n this type \n of growth was seen in 50% ( 3/6 ) of ape3 - 42 and 33% ( 2/6 ) of a1 - 42 membranes ( table 1 ) . at first , only a few pores open ( figure 3b ) , and the membrane stays in the same conductive \n state for several 10s of seconds ( region inside green rectangle ) . \n however , after an abrupt increase in the membrane conductivity due \n to the opening of a large single pore or several pores simultaneously , \n the activity is characterized by several short small steps only a \n few seconds long , suggestive of the opening of several pores in a \n cascade - like fashion . despite this rapid growth , \n discrete steps indicative \n of pore activity are still clearly observed ( figure 3c ) . \n in addition , similar to a1 - 42 , ape3 - 42 pores are voltage - independent \n and can be blocked by zn ( figure 3a ) . \n this type of membrane activity \n was observed in 50% of ape3 - 42 membranes \n and 33% of a1 - 42 membranes . \n ( a ) the current \n across the membrane increased rapidly upon disruption of the membrane , \n exceeding the 1.1 na saturation current of our amplifier . \n an abrupt increase in the membrane conductivity \n due to the opening of a large single pore or several pores simultaneously \n leads to the opening of several pores in a cascade - like fashion . \n ( c ) \n clear step behavior is still observed during exponential current growth \n ( region indicated by the short blue line an a ) . \n a mixed mechanism \n involving both channel activity and nonspecific leakage is likely \n involved . \n although the overall characteristics of the \n membrane activity induced \n by ape3 - 42 and a1 - 42 pores are similar , distinct differences are present . \n table 1 displays the characteristic parameters observed \n for the electrical activity of both ape3 - 42 and a1 - 42 . \n the onset of a1 - 42 activity in plb can be observed as soon as several minutes following \n peptide addition into the chamber and was typically seen within 60 \n min post - addition with an average of 31.6 25.7 min . \n ape3 - 42 demonstrated a significantly longer ( p < 0.05 for a one - tail t test ) and \n more variable lag time , with an average of 98.2 68.5 min . \n we next examined whether there was a difference between ape3 - 42 and a1 - 42 channel conductances . \n figure 4 shows the histogram distribution of \n single - channel conductances calculated from both step and spike activity . \n the histograms show that the conductances can be sorted into three \n groups that demonstrate a shift toward higher conductances for ape3 - 42 pores ( figure 4a ) compared \n with a1 - 42 ( figure 4b ) . \n the first group of conductances , below 100 ps , consisted of 74% \n of ape3 - 42 and 91% of a1 - 42 events . \n the second group is between 100 and 200 ps and , significantly , \n shows a second peak of activity for ape3 - 42 that is absent for a1 - 42 . \n while 21% of \n the events fall in this range for ape3 - 42 , only 5% of a1 - 42 events are in this range . \n the third grouping shows similar rates of sparse activity greater \n than 200 ps ( 5 and 4% for ape3 - 42 and a1 - 42 , respectively ) . \n while the percentage of activity \n in the high conductivity range is similar , close inspection of the \n histogram shows higher conductances for ape3 - 42 in this region . \n table 1 shows that the average conductance in this third \n region is 20% higher for ape3 - 42 . \n these results show an overall trend for higher ape3 - 42 conductance , which could have relevance to the ape3 - 42 toxicity mechanism . \n histogram analysis of ( a ) ape3 - 42 and \n ( b ) a1 - 42 conductances shows that there is \n an increased propensity for higher conductance events for ape3 - 42 . \n both sets of conductances \n can be sorted into three groups ; < 100 ps , 100200 ps and \n > 200 ps , as shown in table 1 . \n ape3 - 42 presents significantly more events than a1 - 42 in the 100200 ps interval . \n data sample \n size was 1400 for ape3 - 42 and 1192 for a1 - 42 . \n data were collected in the 50 mv range , \n with peptide at a concentration of 10 m . \n the electrolyte used \n was 150 mm kcl , 1 mm mgcl2 , and 10 mm hepes ( ph 7.4 ) . \n bilayers \n were made by the painted technique with dops / pope lipids dissolved \n in heptane . \n we performed 100 ns all - atom md simulations \n on ape3 - 42 barrels embedded in an anionic \n lipid bilayer composed of dops / pope ( mole ratio 1:2 ) . \n the ape3 - 42 barrels comprising two different u - shaped conformers \n were initially preassembled as an annular shape . \n the initial annular \n conformation is gradually lost via relaxation of the lipid bilayer , \n and no immediate peptide dissociation in the barrels was observed \n ( figure s1 of the supporting information ) . \n the u - shaped portions of the ape barrels ( residues \n 1542 and 1142 for the conformer 1 and 2 ape3 - 42 barrels , respectively ; see peptide topologies \n in figure 1 ) , which mostly include membrane \n embedded portions , reach equilibration after the initial transient \n state , while the extramembranous n - termini of the peptides ( residues \n 314 and 310 for the conformer 1 and 2 ape3 - 42 barrels , respectively ) are disordered ( figure \n s2 of the supporting information ) . small \n fluctuations in the pore and c - terminal strands in the lipid bilayer \n retain the u - shaped peptide motif in the ape3 - 42 barrels ( figure s3 of the supporting information ) . \n in our simulations , the ape3 - 42 peptide \n also presents heterogeneity in barrel conformations ( figure 5 ) , as observed in the wt a1 - 42 and mutant a barrels . \n the \n outer diameters for the membrane embedded portion are 7.6 \n and 7.1 nm for the conformer 1 and 2 ape3 - 42 barrels , respectively . \n the averaged pore diameters are 2.2 \n and 1.9 nm for the conformer 1 and 2 ape3 - 42 barrels , respectively . \n both outer / pore diameters of ape3 - 42 barrel are in the range of the wt barrel . averaged pore structures calculated with \n hole embedded in the average barrel conformations \n during the \n simulations for the ( a ) conformer 1 and ( b ) conformer 2 ape3 - 42 barrels . in the barrel structures with the surface \n ( top view ) and the ribbon representations ( angle and lateral views ) , \n hydrophobic \n , polar / gly , positively charged , and negatively charged \n residues are colored white , green , blue , and red , respectively . for \n the pore structures , \n red denotes pore diameter of d < 1.4 nm , green denotes pore diameter in the range , 1.4 nm d 2.0 nm , and blue denotes pore diameter of d > 2.0 nm . unlike the wt a1 - 42 barrels , where the \n n - terminal strands normally reside in the bulk water area ( figure \n s4 of the supporting information ) , the \n pe3 n - termini of ape3 - 42 barrels tend to \n retreat to the lipid hydrophobic core due to the hydrophobicity of \n the lactam ring . to locate the pe3 residue across the bilayer \n , we \n calculated the probability distributions for pe3 as well as few selected \n charged groups in the barrels ( figure 6 ) . \n the \n distribution curves for pe3 spanning the interior of the lipid bilayer \n indicate that several pe3 termini interact with the lipid hydrophobic \n tails . \n the interaction energy of the pe3 residue with lipids shows \n strong attraction for several pe3 n - termini ( blue bars in figure s5 \n of the supporting information ) , while no \n strong lipid interactions of the standard n - termini were observed \n in the wt a1 - 42 and mutant barrels . \n relocating \n the pe3 n - termini into the lipid hydrophobic core significantly reduces \n the fluctuations of the n - terminal portions of barrels at the lower \n bilayer leaflet , further stabilizing the barrel conformation . with \n the pe3 anchoring in the membrane , \n the ape3 - 42 barrel would provide a clear channel mouth at the lower bilayer \n leaflet and hence yield a wide - open water pore for ion leakage , suggesting \n that ape3 - 42 pores may lead to high ion conductance . \n probability \n distribution functions for pe3 and selected charged \n residues , pe3 ( purple ) , glu11 ( red ) , lys16 ( orange ) , and glu22 ( yellow ) , \n and for the phosphate group of lipid head , po4 ( green ) , \n as a function of the distance along the pore center axis for the ( a ) \n conformer 1 and ( b ) conformer 2 ape3 - 42 barrels . \n initial locations \n of pe3 are marked by arrows . to observe ion activity \n in the ape3 - 42 pores , we calculated the probability \n distribution for ions across \n the bilayer ( figure s6 of the supporting information ) . \n peaks in the distribution curves reflect the highly populated \n ion binding sites in the pore . \n the locations of ion binding sites \n in each conformer ape3 - 42 barrel are similar \n to those of each corresponding conformer of wt a1 - 42 barrels because both barrels share the same u - shaped motif in the \n lipid bilayer . to observe ion fluctuation across the pore , we calculated \n the change in total charge in the pore as a function of the simulation \n time ( figure 7 ) . \n two selected pore lengths \n along the pore axis , 1.0 < z < 1.0 \n nm and 1.8 < z < 1.8 nm , were used \n in the calculation . \n these pore lengths ensure that the charge fluctuations \n exclude a contribution of ion interactions with the lipid head groups . \n for |z| < 1.0 nm , \n the pore of the ape3 - 42 barrel conformer 1 exhibits larger charge fluctuations \n than the conformer 2 barrel , because the glu22 cationic binding site \n is located at z = 0.6 nm , attracting more cations into the \n pore . for the conformer 2 ape3 - 42 barrel , \n the glu22 cationic binding site is located at z = \n 1.8 nm near the channel mouth ( as indicated by ca peak at z = 1.8 nm in figure s6b of the supporting information ) . to correlate these charge \n fluctuations with experimental ion conductance , we calculated the \n maximum conductance , gmax , representing the ion transport , which can be \n described as1where qe is the elementary charge \n , kb denotes \n the boltzmann s constant , t is the simulation \n temperature , and l represents the pore length of \n 36 . in the bracket , d(z ) \n and gpmf(z ) denote the \n 1-d diffusion coefficient and the 1-d potential of mean force for \n ions , respectively . for mg , \n k , ca , and zn , the maximum conductances are 667 , 338 , 115 , \n 155 ps and 137 , 271 , 92 , 89 ps in the pores of conformers 1 and 2 \n ape3 - 42 barrels , respectively . averaged maximum \n conductances for ions are relatively higher than those calculated \n for the wt a1 - 42 barrels . \n change in total charge in the pore as a function of the simulation \n time for the ( a ) conformer 1 and ( b ) conformer 2 ape3 - 42 barrels . \n the pore heights with cutoff along the pore axis , 1.0 \n < z < 1.0 nm and 1.8 < z < 1.8 nm were used . \n we have shown that the toxic pe - modified a peptides produce \n pore activity in dops / pope anionic membranes . \n these pores demonstrate \n similar activity characteristics ( heterogeneous step , spike and burst \n conductance , zn blockage ) as a1 - 42 pores as well as other previously studied pores of the a \n family . however , there are also some significant differences . \n the \n onset of ape3 - 42 pore activity is generally \n delayed , but once started , ape3 - 42 pores \n show increased propensity for larger conductance events compared with \n a1 - 42 pores , particularly in the 100200 \n ps range ( figure 4 ) . \n structurally , the lactam \n ring of ape peptides induces a change in the conformation \n of the n - terminal strands of the ape3 - 42 pores . \n while the n - terminal strands of a1 - 42 pores \n normally reside in the bulk water region , the n - termini of ape pores tend to reside in the hydrophobic lipid core , providing \n the ape3 - 42 pores with higher stability . \n the longer times required for the onset of ape3 - 42 pore activity are tentatively attributed to ( i ) the higher hydrophobicity \n of ape3 - 42 , which decreases the attractive \n charge - dipole interactions with the zwitterionic lipid heads , thus \n also decreasing the number of adsorbed ape3 - 42 oligomers available for subsequent membrane insertion , and ( ii ) \n the possible longer times required for pores to assemble in the membrane . \n our plb data demonstrate that the ionic current produced by ape3 - 42 pores can rapidly rise to levels potentially \n toxic for cells . following pore formation , \n according to early \n estimations , a single pore with a gigantic \n 4 ns conductance can produce a change of 10 m / s in internal \n na concentration , producing loss of cell homeostasis in \n seconds . \n while no single catastrophic event of this nature was observed \n ( figure 4 ) , the summation of smaller ionic \n conductance events can have a similar toxic effect . \n ape3 - 42 has been shown to be more cytotoxic than a1 - 42 in vitro . \n we showed that ape3 - 42 shows a greater propensity for higher conductance values when compared \n with a1 - 42 . \n this would lead to a more prevalent \n and rapid dysregulation of cellular ionic homeostasis for ape3 - 42 , thus suggesting increased cytotoxic properties \n of ape3 - 42 pores . \n the pore structures \n found for ape3 - 42 had \n characteristics and dimensions in accordance with the pores previously \n reported . \n pore structures have been characterized by afm and modeled \n using md simulations for several a peptides . \n our previous studies \n indicate that full - length a1 - 40/42 , certain a mutants , a fragments , as well as other amyloids form heterogeneous pore structures showing 46 subunits and \n shapes varying from rectangular to hexagonal . \n our md simulations suggest \n similar pore sizes for both peptides but increased stability of the \n ape3 - 42 pores inserted in dops / pope membranes . \n stability in the membrane is likely more important for the formation \n and longevity of the larger pores with conductances in the 100200 \n ps range . as a result , \n ape3 - 42 pores with \n conductances in this range have a higher population than a1 - 42 pores ( figure 4 ) . \n pores \n with conductances > 200 ps likely have similar and fast decay rates \n for ape3 - 42 and a1 - 42 , and thus both populations are similarly low , although still somewhat \n larger for ape3 - 42 . \n such a scenario would \n also correlate with the greater in vitro cytotoxicity observed for \n ape3 - 42 peptides . \n the energy of a pore \n according to the continuum model is2where r is the pore \n radius , \n is the line tension at the pore edge , and is the surface \n tension of the membrane . \n our md results indicate that ape3 - 42 and a1 - 42 pores have \n similar diameters and general characteristics , while the main difference \n is the retreat of the n - termini of ape3 - 42 into the hydrophobic lipid core . \n therefore , this reconfiguration \n of the n - termini likely decreases the line tension of ape3 - 42 pores , minimizing their pore energy and rendering \n them more stable . \n the exponential growth of the current , seen in figure 3 , would lead to rapid cell death and was observed \n in 50% ( 3/6 ) of the ape3 - 42 membranes in \n this study . while pore activity appears to be the only mechanism present \n during the early stages ( green rectangle in figure 3b ) , the activity trace suggests that two mechanisms of ionic \n conductance act simultaneously during exponential saturation : ( i ) \n stepwise pore behavior and ( ii ) nonspecific drift leakage . \n the nonspecific \n leakage could be caused by a number of alternative mechanisms leading \n to a-induced membrane permeability , including carpeting and \n detergent effects . \n the data suggest that \n a combined mechanism ( pore and nonspecific leakage ) may be at work \n during the rapid growth of the exponential phase , \n at least some of the time . \n it is important to note that similar behavior \n was also seen for some of the a1 - 42 membranes . \n although this behavior was observed for more membranes with ape3 - 42 pores , this difference ( table 1 ) was not statistically significant to reach conclusions regarding \n specific ape3 - 42 cytotoxity . \n in summary , using plbs and md simulations , we have shown that ape3 - 42 forms ionic pores in dops / pope lipid bilayers . \n the activity of these pores follows the same general pattern as a1 - 42 pores . \n significantly , however , ape3 - 42 pores display higher conductances than a1 - 42 pores , in particular , in the 100200 ps range . \n in addition , \n the onset of ape3 - 42 pore activity is delayed \n compared with a1 - 42 pores . \n structurally , \n ape3 - 42 and a1 - 42 pores have similar dimensions , but the pe3 residue induces a change \n in the conformation of the n - terminal strands of the ape3 - 42 pores . \n while the n - terminal strands of a1 - 42 pores normally reside in the bulk water region , \n the n - termini of ape pores tend to reside in the \n hydrophobic lipid core , providing the ape3 - 42 pores with higher stability compared with the a1 - 42 pores . \n these studies are a first step in understanding the role \n of pores in the enhanced toxicity attributed to ape peptides .", "role": "user" }, { "content": "among the family of a peptides , \n pyroglutamate - modified a \n ( ape ) peptides are particularly associated with \n cytotoxicity in alzheimer s disease ( ad ) . \n they represent the \n dominant fraction of a oligomers in the brains of ad patients , \n but their accumulation in the brains of elderly individuals with normal \n cognition is significantly lower . \n accumulation of ape plaques precedes the formation of plaques of full - length a \n ( a1 - 40/42 ) . \n most of these properties appear \n to be associated with the higher hydrophobicity of ape as well as an increased resistance to enzymatic degradation . however , \n the important question of whether ape peptides induce \n pore activity in lipid membranes and their potential toxicity compared \n with other a pores is still open . \n here we examine the activity \n of ape pores in anionic membranes using planar bilayer \n electrical recording and provide their structures using molecular \n dynamics simulations . \n we find that ape pores spontaneously \n induce ionic current across the membrane and have some similar properties \n to the other previously studied pores of the a family . \n however , \n there are also some significant differences . \n the onset of ape3 - 42 pore activity is generally delayed compared with \n a1 - 42 pores . however , once formed , ape3 - 42 pores produce increased ion permeability of the \n membrane , as indicated by a greater occurrence of higher conductance \n electrical events . \n structurally , the lactam ring of ape peptides induces a change in the conformation of the n - terminal \n strands of the ape3 - 42 pores . while the n - termini \n of wild - type a142 peptides normally reside \n in the bulk water region , the n - termini of ape3 - 42 peptides tend to reside in the hydrophobic lipid core . \n these studies \n provide a first step to an understanding of the enhanced toxicity \n attributed to ape peptides .", "role": "assistant" } ]

[ { "content": "You are a medical writer. Summarize the following article: fabry disease ( fd ) is a rare x - linked disorder resulting from a deficient activity of the lysosomal enzyme -glycosidase a ( -gal a ) that leads to progressive accumulation of glycosphingolipids in several tissues and organs . in the classical form of the disease , \n renal involvement commonly ensues in the second to third decades of life with overt proteinuria ( > 300 mg / day ) rapidly leading to chronic kidney disease ( ckd ) and end - stage renal disease ( esrd ) . \n cardiac and renal atypical variants of the disease with different degrees of organ impairment and the pattern of disease have been described in males . in females , \n the disease presentation may range from a completely asymptomatic status to the classic form of fd . \n nonetheless , to the best of our knowledge , isolated proteinuria as the first sign of the disease is rare and has been described only in males . \n enzyme replacement therapy ( ert ) with agalsidase has been demonstrated to be safe and effective in the improvement of several signs and symptoms of fd in both genders , and it is accepted that the early establishment of ert might prevent organ damage . thus , ert is recommended in the presence of symptoms or organ involvement . \n however , due to the variable presentation and the slower progression of the disease in female patients , the questions of how to detect organ involvement and when to start therapy remain open . \n vv , a 20-year - old female was referred to our clinic in 2006 for a family screening programme due to one uncle , the mother and sister being diagnosed with fd [ point mutation i354k ( c1061t > a ) of the exon 7 ] . at that time , vv was completely asymptomatic for fabry disease ; leucocyte -gal a activity was 19.5 nmol / mg prot / h ( normal values 1850 nmol / mg prot / h ) , while molecular analysis showed the presence of the pathologic genotype , confirming the heterozygous status of the patient . at this time , clinical evaluation and periodic laboratory tests were unremarkable and no sign of fd could be detected . \n two years later in the first evaluation , urine analysis showed evidence of isolated persistent microalbuminuria ( 303 mg / l ; normal value < 30 mg / l ) without overt proteinuria or abnormalities in the urinary electrolytes . \n all laboratory tests were in the normal range including serum creatinine and creatinine clearance ( 0.7 mg / dl and 92 ml / min , respectively ) . \n similarly , a thorough physical examination of the patient could not elicit any sign of fd or any other disease . specifically , body mass index , blood pressure and heart rate were 20.5 kg / m , 120/70 mmhg and 68 bpm , respectively . \n audiometric evaluation did not show any evidence of hearing loss . magnetic resonance imaging ( mri ) of \n the brain was negative for infarctual encephalopathy . finally , both sweat test and electromyography were normal , with no evidence of sensitive - motor abnormalities . due to the presence of isolated and persistent microalbuminuria \n light microscopy revealed numerous vacuolated cells in both glomerular tuft and tubular cells ; myelin osmiophilic bodies were also present in glomeruli or between tubules ( figure 1a ) . \n direct immunofluorescent study showed negative staining for iga , igm , igg , c1q , c3 and c4 . \n zebra bodies in podocytes , epithelial and endothelial cells of the glomerular tuft ( figure 1b ) . \n thus , the renal biopsy confirmed that the patient presented fd with the sole involvement of the kidney . \n considering the aggressive course of the disease in the family , the optimal blood pressure control and what was suggested by the international guidelines for fd diagnosis and treatment , we started ert with agalsidase beta ( fabrazyme - genzyme corp . ) at a standard dose of 1 mg / kg every 2 weeks , in the hope to provide vv with an aetiological rather than antiproteinuric treatment . \n after 6 months of treatment , the microalbuminuria returned to the normal range ( 27 mg / l ) , while no other signs or symptoms of fd could be detected . \n renal biopsy of an asymptomatic fabry female patient presenting with isolated microalbuminuria ( 303 g / l , 24 h urine collection ) . \n light microscopy ( panel a , left ) shows numerous vacuolated cells in glomerular tufts ( upper panel ) and tubuli ( lower panel ) . \n tem ( panel b , right ) shows osmiophilic zebra bodies in podocytes and epithelial cells ( upper panel ) and endothelial cells ( lower panel ) . \n several studies have demonstrated that heterozygous patients , due to the lyon phenomenon , may present different phenotypes ranging from the complete absence of symptoms of the classical form of fabry disease . to the best of our knowledge , \n this is the first case reported of an adult heterozygous female in whom isolated microalbuminuria is the first and only presenting sign of both fd and renal involvement . \n the presentation of fd with a prevalent renal impairment ( so - called renal variant ) is rare and documented in male patients only . \n nonetheless , microalbuminuria has been recently described as a preliminary sign of renal impairment in symptomatic young female patients . \n it is the authors opinion that the present case report should question the watch and wait approach and pose several questions on what should be looked at as early signs of fd and when to start ert . indeed , the early detection of renal damage appears of paramount importance since the presence of proteinuria and/or ckd at the time of the start of ert are independent predictors of poor outcome . \n however , our case should suggest that microalbuminuria might be regarded as an early sign of fd nephropathy . \n furthermore , the disappearance of microalbuminuria after 6 months of ert without antiproteinuric agents [ such as angiotensin - converting enzyme inhibitors ( ace - i ) or angiotensin receptor blockers ( arbs ) ] should deserve attention since it might suggest that early intervention might also reverse fd nephropathy . \n this finding might be of importance if we consider data from the fabry registry that seem to corroborate the notion that females with fd are undertreated , as only 33.8% of females currently included in the fabry registry are on ert . in summary \n , this case suggests that the kidney may be the first , and sometimes only , organ damaged by fd in heterozygous female patients and that isolated and persistent microalbuminuria instead of proteinuria should be regarded as an early marker of fd nephropathy and an indication for ert . \n renal variant, as well as how to detect organ damage and when to start an early treatment in female fd patients .", "role": "user" }, { "content": "fabry disease ( fd ) is a rare x - linked disorder characterized by low or absent activity of the lysosomal enzyme -glycosidase - a that leads to progressive accumulation of glycosphingolipids in different organs and tissues . \n clinical manifestations vary from classic to atypical forms characterized by one prevalent organ involvement , and a renal variant has been described in men but not in women . \n however , little is known about renal manifestation in females affected by fd . \n we herein report a case of a 22-year - old female with isolated and persistent microalbuminuria as the only sign of fd . in light of the importance of early recognition and treatment of fd organ damage , this case should call for future studies to determine how to assess organ damage , investigate the existence of a \n renal variant in fd female patients and determine when best to start enzyme replacement therapy ( ert ) .", "role": "assistant" } ]

[ { "content": "You are a medical writer. Summarize the following article: supplementary material is available for this article at 10.1007/s13659 - 012 - 0026-z and is accessible for authorized users . \n ", "role": "user" }, { "content": "three new sesquilignans , 13 , a new sesquiterpenoid , 4 , and three known compounds were isolated from the stem barks of illicium simonsii . \n the structures of new compounds ( 14 ) were elucidated by spectroscopic methods . \n a biosynthetic pathway was proposed for simonsienols a - c ( 13 ) . \n anti - ache activity and anti - buche activity were evaluated for all compounds except for -cadinol ethyl ether ( 4 ) . as a result , \n isodunnianol ( 7 ) exhibited anti - ache activity with an ic50 value of 13.0 m . \n electronic supplementary materialsupplementary material is available for this article at 10.1007/s13659 - 012 - 0026-z and is accessible for authorized users .", "role": "assistant" } ]

[ { "content": "You are a medical writer. Summarize the following article: it is usually associated with an increased efflux of cytotoxic drugs by atp - binding cassette ( abc ) transporters including abcg2 . \n abc transporters are energy - dependent transporters that normally function in the detoxification and protection of normal cells from xenobiotics . \n overexpression of abcg2 in cancer cell lines in vitro has been shown to confer mdr to a variety of anticancer drugs including mitoxantrone , irinotecan , methotrexate , flavopiridol , and anthracyclines . moreover \n , growing evidence suggests that abcg2 underlies the mdr of clinical samples from different cancers . \n it may also contribute to the maintenance of stem or progenitor cells as a survival factor , ultimately driving tumor growth . \n identification of factors that can influence abcg2 expression and activity might lead to the development of new strategies to modulate abcg2-mediated drug transport clinically . \n they can be thought of as inhabiting within a complex milieu of normal cells , blood vessels , endogenous small molecules , and secreted factors , which collectively comprise the tumor microenvironment . \n recent advances have indicated that the tumor microenvironment is critically important for cancer initiation , progression , metastasis , and drug resistance , thus providing opportunities for therapeutic intervention . \n this study aims to evaluate the regulation of abcg2 in response to a few characteristic growth conditions within the tumor microenvironment , including hypoxia , glucose deprivation , and low ph . \n cancer cells are often confronted with a remarkable reduction in oxygen supply inside solid tumors , leading to intratumoral hypoxia . \n hypoxia - inducible factor ( hif-1 ) , the master regulator of hypoxic response , is accumulated in order to adapt to the adverse growth condition . \n the production of vascular endothelial growth factor ( vegf ) and other hypoxia - induced angiogenic cytokines to promote increase tissue vascularization , and the metabolic switch from oxidative to glycolytic metabolism represent the two major adaptive responses to enhance cell survival under tumor hypoxic condition . \n glucose depletion is often observed in malignant tumors due to insufficient blood supply in the core of solid tumors and increased rate of glycolysis . on the other hand , \n the production of lactic acid under anaerobic conditions and the hydrolysis of atp in an energy - deficient environment contribute to the acidic microenvironment that has been shown in many types of tumor ( warburg 's effect ) . \n while the effect of tumor microenvironment on oncogenes and tumor suppressor genes have been extensively studied recently , less is known about how this may modulate the xenobiotic transporters thus mediating multidrug resistance . \n a better understanding of this complicated interaction may allow us to design novel therapeutic strategies to improve treatment outcome in chemotherapy . \n mitoxantrone and 2-deoxyglucose ( dg ) were purchased from sigma chemical ( st . louis , mo ) . \n pheophorbide a ( pha ) and fumitremorgin c ( ftc ) were kind gifts obtained from dr . \n susan bates ( national cancer institute , nih , bethesda , md , usa ) . \n human colon carcinoma hct-116 , and s1 and its abcg2-overexpressing resistant s1m1 - 80 cell lines were kindly provided by dr . \n the resistant s1m1 - 80 subline used in this study was derived from the s1 clone of the ls174 human colon cancer cell line by prolonged selection in mitoxantrone , which was routinely maintained at 80 m mitoxantrone . \n there is no amplification of the abcg2 gene in s1m1 - 80 . the abcg2-mediated drug resistance in s1m1 - 80 is believed to be contributed by a balanced t(4,7 ) translocation downstream of the abcg2 gene and the escape from mir-519c repression by a shortening of its 3untranslated region . \n at the time of investigation , it has been allowed to grow in drug - free medium for at least 3 weeks . \n the cell lines were maintained in rpmi 1640 medium supplemented with 10% fetal bovine serum , 100 units / ml streptomycin sulfate , and 100 units / ml penicillin g sulfate , and incubated at 37c in 5% co2 . \n hypoxic incubation was accomplished by using the bactron anaerobe system ( sheldon manufacturing inc . , \n prior to hypoxic incubation , the culture was replenished with fresh complete medium and the cells were then subjected to 3 cycles of evacuation refilling of anaerobic gas ( 90% nitrogen and 10% co2 ) . \n the hypoxic environment was proved to be radiobiologic hypoxic ( oer = 3.0 , data not shown ) . to mimic glucose depletion , \n cells were cultured in glucose - free rpmi 1640 medium for 16 h or in regular medium but in the presence of 20 mm 2-deoxyglucose ( 2-dg ) for 16 h. under these conditions , no significant cell death was observed . to test the effect of acid , \n the glucose - containing culture medium was titrated with 0.1 m hcl to ph 5 . in order to ensure that any observed change in the transporter expression was not attributed by a change in medium osmolality , \n control cells were also grown in glucose - containing rpmi-1640 with an added volume of distilled water to achieve an osmolality equivalent to the acid - treated medium ( ~20% v / v of added water ) . \n total rna was isolated using the trizol regent ( invitrogen , carlsbad , ca ) . \n rna ( 1 g ) was reverse transcribed using the transcriptor high fidelity cdna synthesis kit ( roche applied science , indianapolis , in ) . \n quantitative real - time pcr was performed to quantify the change of abcg2 or pgk1 transcript expressions using the kapa sybr fast qpcr kit ( kapabiosystems , woburn , ma ) in a lightcycler 480 instrument i ( roche applied science , indianapolis , in ) . the human gapdh rna was amplified in parallel as the internal control . \n the specific primers used are as follows : abcg2 ( forward ) 5-tttccaagcgttcattcaaaaa-3 ( reverse ) 5-tacgactgtgacaatgatctgagc-3 ; pgk1 ( forward ) 5-tttctaacaagctgacgctg-3 , ( reverse ) 5-ttcttcctccacatgaaagc-3 ; gapdh ( forward ) 5-agccacatcgctcagacac-3 ( reverse ) 5-gttcaaacttctgctcctga-3. pcrs were performed at 95c for 5 min , followed by 50 cycles of 95c for 10 s and 60c for 10 s. fluorescence signal was acquired at the end of the elongation step of every pcr cycle ( 72c for 10 s ) to monitor the increasing amount of amplified dna . \n ct was calculated by subtracting the ct of gapdh from the ct of the transcript under investigation . \n ct was then calculated by subtracting the ct of the untreated cells ( or parental cells ) from the ct of the treated cells ( or resistant cells ) . \n fold change of gene expression was calculated by the equation 2 . a small interference hairpin - loop ( sh ) silencing vector against the human abcg2 ( pu6-abcg2 ) and a negative control shrna vector targeting firefly luciferase ( pu6-luc ) were prepared as described in to et al . . \n s1 and s1m1 - 80 cells were transfected with pu6-luc or pu6-abcg2 using lipofectamine 2000 ( invitrogen , grand island , ny ) . \n a series of human abcg2 promoter constructs with progressive deletions at the 5-end has been described previously . \n the abcg2 promoter / firefly luciferase fusion genes ( 400 ng dna ) were transfected in hct-116 or s1 cells on 24-well plates using fugene 6 ( roche , indianapolis , in ) . \n the pgl3-basic ( promoterless ) plasmid , encoding firefly luciferase ( promega , madison , wi ) , was used to determine the basal levels . in each experiment , the phrg - basic plasmid ( 100 ng ) , encoding renilla luciferase ( promega ) , was cotransfected for normalization purposes . \n luminescence was measured 48 h after transfection using the dual - luciferase reporter assay system ( promega ) with the glomax 20/20 luminometer ( promega ) . in experiments involving 2-dg or hypoxic \n treatment , the cells were treated at 24 h after transfection for 16 h. reporter activity was normalized by calculating the ratio of firefly / renilla values . \n results were expressed as mean sd of duplicate measurements from three independent transfections . \n the abcg2-mediated efflux activity in hct-116 , s1 and s1m1 - 80 cells with or without pretreatment with 2-dg or hypoxia were determined by flow cytometric assays as described previously . \n trypsinized cells was incubated in 1 m pha with or without 10 m abcg2-specific inhibitor ftc in complete medium ( phenol red - free rpmi 1640 with 10 % fbs ) at 37c in 5% co2 for 30 min . \n subsequently , the cells were washed with cold complete medium and then incubated for 1 h at 37c in pha - free medium continuing with ftc to generate the ftc / efflux histogram , or without ftc to generate the efflux histogram . \n the ftc - inhibitable pha efflux was determined as the difference in mean fluorescence intensity ( mfi ) between the ftc / efflux and efflux histograms , which indicates the abcg2-mediated transport activity . \n cells were finally washed with cold dulbecco 's pbs and placed on ice in the dark until analysis by flow cytometry . \n to measure the cell surface abcg2 expression , trypsinized cells were incubated in 2% bovine serum albumin / dulbecco 's pbs with either phycoerythrin - conjugated anti - abcg2 antibody 5d3 ( ebioscience , san diego , ca ) or phycoerythrin - conjugated mouse \n igg2b negative control antibody ( ebioscience ) according to manufacturer 's instructions for 30 min at room temperature . \n surface expression of abcg2 was calculated as the difference in mean channel numbers between the 5d3 antibody histogram and the negative control antibody histograms . \n samples were analyzed on an lsrfortessa cell analyzer ( bd biosciences , san jose , ca ) . \n phycoerythrin fluorescence was detected with a 488 nm argon laser and a 585 nm bandpass - filter , whereas pha fluorescence was detected with a 488 nm argon laser and a 670 nm bandpass filter . \n cell debris was eliminated by gating on forward versus side scatter and dead cells were excluded based on propidium iodide staining . \n growth inhibitory effect of mitoxantrone and cisplatin ( representing a typical abcg2 substrate and nonsubstrate , resp . ) , with or without the pretreatment of 2-dg or hypoxia , on hct-116 , s1 and s1m1 - 80 cells , were evaluated by the sulforhodamine b assay . \n cells were seeded into 96-well microtitre plates in 100 l at a plating density of 5,000 cells / well and allowed to incubate overnight . \n the cells were then exposed to 2-dg ( 20 mm ) or hypoxia for 16 h , before being treated with mitoxantrone or cisplatin at a range of concentrations and allowed to incubate at 37c in 5% co2 for another 72 h. each drug concentration was tested in quadruplicate and controls were tested in replicates of eight . \n each experiment was carried out independently at least three times . to determine whether differences between ic50 values were significant , \n the student 's t - test was performed with p < 0.05 being considered significant . \n soft agar plates were prepared in six - well plates with a bottom layer of 0.6% noble agar in serum - free rpmi 1640 . \n 1,000 cells , with or without a 24 h hypoxia treatment , were suspended in 0.3% noble agar in rpmi 1640 supplemented with 10% fetal bovine serum and seeded onto the bottom layer . \n the number of colonies was counted after staining with 0.05% crystal violet for 1 h. \n the abcg2 mrna expression was evaluated in three human colon carcinoma cell lines ( hct-116 , s1 and its resistant subline s1m1 - 80 ) after the pretreatment with glucose depletion , 2-dg , acidic ph and hypoxia . in hct-116 and s1 , a small but significant upregulation of abcg2 ( 2 to 4-fold ) \n since the effect was more pronounced after 2-dg and hypoxia treatment , these conditions were selected for later functional and more detailed mechanistic studies . on the other hand , \n the expression of pgk1 , a hypoxia responsive gene , was also measured to verify the hypoxia treatment . \n our data indicates that pgk1 was remarkably induced by the hypoxia treatment in all three cell lines ( figure 1 , lower panel ) . \n the increase in abcg2 mrna after 2-dg or hypoxia treatment was blocked by actinomycin d ( 5 g / ml ; data not shown ) , suggesting that these tumor microenvironment conditions regulate abcg2 mrna levels through an effect on abcg2 transcription . a series of 5-deletion reporter gene constructs harboring the abcg2 promoter region with the 3 end terminating at + 396 bp were used to elucidate the promoter region responsible for glucose depletion or hypoxia - mediated abcg2 upregulation ( figure 2(b ) ) . \n after transiently transfected into hct-116 and s1 cells , all the abcg2 promoter - luciferase constructs demonstrated good activities above the promoter - less pgl3basic background ( data for hct-116 and s1 are shown in figure 2(b ) and supplementary figure 1 ( available online at doi:10.5402/2012/746025 ) , resp . ) . \n while reporter activity of the full length abcg2 promoter construct ( 1662/+396 ) and the longer ones ( 628 and 312/+396 ) were found to be activated in hct-116 and s1 pretreated with 2-dg or hypoxia , the activation was abolished in the shortest construct ( 105/+396 ) . \n the data implies that the region between 312 and 105 , harboring two reported hif-1 response elements ( figure 2(a ) ) , is responsible for the observed induction of abcg2 transcription . \n the upregulation of abcg2 observed above suggested that all of the tumor microenvironment conditions investigated may confer a more drug - resistant phenotype . \n next we sought to demonstrate the functional evidence with special regard to drug efflux activity and in vitro drug response . by flow cytometric analysis , both 2-dg and hypoxia treatment led to a small but statistically significant elevation of abcg2-mediated drug efflux ( mfi = signal between the efflux and ftc / efflux histograms ) in all cell lines tested ( figure 3 ) . since the basal abcg2 levels in s1 and hct-116 cells are very low , we did not proceed to measure the protein expression either by western blot or surface staining by flow cytometry . \n nonetheless and more importantly , 2-dg and hypoxia were found to significantly decrease the cytotoxic effect of the abcg2 substrate anticancer drug ( mitoxantrone ) ( table 1 ) . on the other hand , \n the cytotoxicity of cisplatin was not affected , indicating the specific effect of these tumor microenvironment conditions on abcg2-mediated mdr ( table 1 ) . in the abcg2-overexpressing resistant s1m1 - 80 cells , western blot and flow cytometry - based cell surface staining analysis \n interestingly , by both assays , abcg2 protein expression was also found to be induced slightly after 2-dg and hypoxia treatment ( figures 4(a ) and 4(b ) ) . in figure 4(b ) ( flow cytometry cell surface staining ) , the channel # between the specific anti - abcg2 staining and the isotype antibody control represents the cell surface abcg2 protein expression . \n importantly , this slight induction of abcg2 protein by 2-dg and hypoxia was also found to decrease the cytotoxic effect of mitoxantrone ( an abcg2 substrate anticancer drug ) ( table 1 ) , probably through enhancing the abcg2 efflux activity ( figure 3 ) . in an attempt to further understand the induction of abcg2 protein expression by the 2-dg and hypoxia treatment , cycloheximide chase assay \n was performed to evaluate the degradation rate of abcg2 protein ( data not shown ) . \n unfortunately , abcg2 protein was found to be fairly stable and no appreciable degradation was observed with or without the 2-dg or hypoxia treatment after up to 36 h. anchorage - independent growth is one of the hallmarks of cell transformation , which is considered a reliable in vitro assay for detecting malignant transformation of cells . since abcg2 upregulation was observed after 2-dg and hypoxia treatment and that abcg2-mediated mdr may lead to a more malignant phenotype , the colony formation capability of s1 and s1m180 cells with or without 2-dg and hypoxia treatment was assessed . in both cell lines , \n hypoxia was found to significantly increase the colony formation capability ( figure 5(b ) ) . in s1m1 - 80 cells , \n 2-dg treatment was also found to enhance the tendency to form colony in a statistical significant manner . \n interestingly , the colony formation capability could be abolished when abcg2 was knocked down in the cells ( figures 5(a ) and 5(b ) ) . \n the concept of tumor microenvironment is gaining a lot of attention in recent years because it allows complex interaction between a tumor and its surrounding normal cells , molecules , and blood vessels , which all participate in tumor progression . \n a tumor can change its microenvironment and the microenvironment can affect the way the tumor grows and spreads . \n hypoxia , glucose deprivation , and acidosis are generally considered as the hallmarks of the tumor microenvironment . \n while chemotherapy is the mainstay of treatment strategy for cancer , drug resistance is seriously hindering the clinical efficacy of most anticancer drugs . \n solid tumors exhibit distinct structural abnormalities such as the leaky vasculature and thus have poor tissue perfusion , subsequently leading to physiological resistance to anticancer drugs . on the other hand , \n resistant cancer cells often express high levels of the multidrug resistance ( mdr ) transporters as their biochemical mechanism of drug resistance . \n they make use of the energy derived from atp hydrolysis to regulate intracellular drug concentration , thereby determining cell sensitivity to chemotherapeutic agents . \n abcg2 is one of these transporters , conferring mdr to a broad spectrum of antitumor agents . with high normal tissue expression in the brain endothelium , gastrointestinal tract , and placenta \n , abcg2 is believed to be important in the protection from xenobiotics , regulating oral bioavailability , forming part of the blood - brain barrier , and the maternal - fetal barrier . \n importantly , abcg2 may also contribute to the maintenance of stem or progenitor cells as a survival factor . among the various conditions within the tumor microenvironment \n this study attempted to understand the regulation of abcg2 by the various adverse growth conditions within the tumor microenvironment in colon carcinoma cell lines . \n in fact , it has been postulated that the atp - dependent drug efflux may be reduced under hypoxic condition . \n cells are known to switch from oxidative to glycolytic metabolism under hypoxia and glycolytic pathway is less efficient in generating atp . to this end \n , it has been shown that a limitation in atp supply ( by sodium azide or 2-dg ) could inhibit daunorubicin efflux in a non - pgp - overexpressing resistant cell line . \n xu et al . demonstrated that the inhibition of mitochondrial respiration leads to an increase in the cytotoxic efficacy of various chemotherapeutic drugs as a result of a reduced p - gp activity which was attributed to a depletion of atp . on the other hand \n , it has been shown that acidosis ( especially together with hypoxia ) could increase p - gp - mediated drug efflux and lead to reduced cytotoxicity of chemotherapeutic drugs . \n the discrepancy between the studies may be caused by the different cell lines used and different extent of hypoxia treatment . \n the data revealed that hypoxia , glucose deprivation , and acidosis can all upregulate abcg2 in the two parental colon cancer cell lines ( figure 1 ) and apparently leading to multidrug resistance ( table 1 ) . \n the upregulation is likely mediated at the transcription level because it can be abolished by pretreatment with actinomycin d , which inhibits rna synthesis ( data not shown ) . \n the effect on an abcg2-overexpressing resistant s1m1 - 80 cell line was much less pronounced at the rna level ( figure 1 ) . \n however and interestingly , the resistance level was further enhanced in s1m1 - 80 cells after 2-dg and hypoxia treatment ( table 1 ) . \n since hypoxic condition will commit the cells to glycolytic metabolism , a downstream glycolytic gene ( pgk1 ) was also evaluated in our study ( figure 1 ) as a surrogate for the hypoxic treatment and to demonstrate the functional linkage with glucose metabolism . \n as expected , pgk1 level was elevated remarkably after the hypoxia treatment . to evaluate the effect of glucose depletion , \n cells were grown either in glucose - free medium or in glucose - containing medium supplemented with 2-dg . \n 2-dg is a glucose analogue , which acts as a competitive inhibitor of glucose metabolism , where 2-dg competes with d - glucose to be transferred to the cells as a target for the hexokinase in the first step of the glycolytic pathway . upon transport into the cells , glucose and 2-dg are phosphorylated to glucose - po4 and 2-dg - po4 , respectively , by the hexokinase . \n however , unlike glucose - po4 , the 2-dg - po4 can not further be metabolized by the glucose phosphate isomerase ( gpi ) . \n in addition , 2-dg could inhibit gpi , and then downregulate its downstream glycolysis - related genes ( such as pgk1 ) , which agree well with our findings ( figure 1 ) . regarding the effect of acidosis , the upregulation of abcg2 was only statistically significant in hct-116 cells . \n since abcg2 upregulation is more pronounced after 2-dg and hypoxia treatment , further mechanistic and functional studies were performed under these conditions . by reporter gene assay , the abcg2 promoter region ( 312 to 105 ) close to the reported hif-1 response element was found to be required for our observed gene upregulation ( figure 2(b ) ) . \n consistent with the reduced response of cells to abcg2 substrate anticancer drug ( table 1 ) after 2-dg and hypoxia treatment , abcg2 efflux activity was found to be increased in all cell lines tested by the flow cytometry ( figure 3 ) . since mdr \n is usually mediated by overexpression of the mdr transporters , we intentionally investigated how abcg2 regulation is affected by 2-dg and hypoxia in the abcg2-overexpressing resistant s1m1 - 80 cell line . \n s1m1 - 80 has massive abcg2 overexpression probably caused by a genetic translocation and its escape from mir-519c repression reported previously . \n abcg2 protein expression was detected by western blot analysis and cell surface staining . by both assays , 2-dg and hypoxia treatment \n were found to increase abcg2 level ( figure 4 ) , which is consistent with the elevated efflux activity ( figure 3 ) and reduced response to abcg2 substrate anticancer drug ( table 1 ) . \n note that the massively overexpressed abcg2 transcript level in s1m1 - 80 was not significantly affected by these treatments ( table 1 ) ; therefore , the observed functional effect may have been caused by modulation at the posttranscriptional and/or translational levels . \n this piece of data is important because it suggests that tumor microenvironment could coordinately regulate abcg2 with preexisting mdr mechanisms to attain a higher resistance level . \n the ability of cancer cells to form colonies in vitro in an anchorage - independent manner is commonly used as a measure of their malignancy . \n after 2-dg and hypoxia treatment , both s1 and s1m1 - 80 cells were found to form more colonies in a soft agar colony formation assay ( figure 5 ) . \n the upregulation of abcg2 may play a specific role in this enhanced colony formation capability because genetic knockdown of abcg2 was found to significantly reduce the number of colonies formed ( figure 5 ) . \n taken together , the three adverse growth conditions in the tumor microenvironment tested were found to upregulate abcg2 to mediate multidrug resistance , with the possible enhancing effect on preexisting mdr mechanism , and to give rise to a more malignant phenotype . \n novel clinical interventions may be devised to help circumvent this abcg2-mediated resistance by modulating the tumor microenvironment .", "role": "user" }, { "content": "abcg2 is an efflux transporter commonly found to overexpress in multidrug resistant ( mdr ) cancer cells . \n it is also believed to be a survival factor for cancer stem cells to drive tumor growth . \n tumor microenvironment represents an attractive new drug target because it allows complex interaction between a tumor and its surrounding normal cells , molecules , and blood vessels , which all participate in tumor progression . \n hypoxia , glucose deprivation and acidosis are the hallmarks of tumor microenvironment . \n this study investigated the upregulation of abcg2 by these adverse growth conditions within the tumor microenvironment . \n reporter gene assay revealed that a region within the abcg2 promoter close to the reported hif-1 response element is responsible for abcg2 upregulation . increased abcg2 efflux activity \n was observed under the same conditions , subsequently leading to reduced response to abcg2 substrate anticancer drug . \n importantly , glucose deprivation and hypoxia were also found to enhance the resistance level of abcg2-overexpressing resistant cells with pre - existing genetic and epigenetic mdr mechanisms . \n hypoxia was further demonstrated to cause a more malignant anchorage - independent growth phenotype in the resistant cells , which can be abolished by knocking down abcg2 . \n a better understanding of abcg2 regulation by the tumor microenvironment may help design novel strategies to improve treatment outcome .", "role": "assistant" } ]

[ { "content": "You are a medical writer. Summarize the following article: the in - vivo study on ophthalmic products is done on experimental animals , usually on rabbit 's eye . \n preparation volume 50 l is instilled topically in the center of the lower cul - de - sac . \n eyes of the animal are observed for signs of irritation and injury . in vitro methods \n diclofenac sodium solid lipid nanoparticles ( slns ) , prepared with goat fat and phospholipids evaluated using bio - engineered human cornea , produced from immortalized human corneal endothelial cells ( henc ) , stromal fibroblasts , and epithelial cells . \n sustained release and high permeation of diclofenac sodium through the bio - engineered cornea were achieved . \n the bovine corneal opacity and permeability ( bcop ) assay has been accepted as a valid in - vitro alternative method to the draize eye irritation test with the development of an improved laser light - based opacitometer ( llbo ) for the analysis of the complete corneal surface instead of dual beam op - kit opacitometer . \n confocal laser scanning microscopy study on the transport mechanism through the corneal epithelium of nanostructured lipid carrier was carried out in rabbits in vivo . \n the corneas were isolated and rinsed in physiological saline to remove the adhesive fluorescence fragment . \n after 30 minute administration in to eyes , the interaction with corneal epithelia to be investigated by observing the corneal samples using confocal laser scanning microscopy ( clsm ) . \n the epiocular tissue model is a new in - vitro test method for ocular irritation , produced by mattek corporation tissue production facilities . \n the epiocular tissue model shows in - vivo like morphological and growth characteristics that are uniform and highly reproducible . \n the tissue consists of highly organized basal cells , which progressively flatten out as the apical surface of the tissue similar to the normal in - vivo corneal epithelium . \n this test could be applied as an alternate to rabbit eye test in the toxicological assessment of ophthalmic preparations . \n the in - vivo study on ophthalmic products is done on experimental animals , usually on rabbit 's eye . \n preparation volume 50 l is instilled topically in the center of the lower cul - de - sac . \n eyes of the animal are observed for signs of irritation and injury . in vitro methods \n diclofenac sodium solid lipid nanoparticles ( slns ) , prepared with goat fat and phospholipids evaluated using bio - engineered human cornea , produced from immortalized human corneal endothelial cells ( henc ) , stromal fibroblasts , and epithelial cells . \n sustained release and high permeation of diclofenac sodium through the bio - engineered cornea were achieved . \n the bovine corneal opacity and permeability ( bcop ) assay has been accepted as a valid in - vitro alternative method to the draize eye irritation test with the development of an improved laser light - based opacitometer ( llbo ) for the analysis of the complete corneal surface instead of dual beam op - kit opacitometer . \n confocal laser scanning microscopy study on the transport mechanism through the corneal epithelium of nanostructured lipid carrier was carried out in rabbits in vivo . \n the corneas were isolated and rinsed in physiological saline to remove the adhesive fluorescence fragment . \n after 30 minute administration in to eyes , the interaction with corneal epithelia to be investigated by observing the corneal samples using confocal laser scanning microscopy ( clsm ) . \n the epiocular tissue model is a new in - vitro test method for ocular irritation , produced by mattek corporation tissue production facilities . \n the epiocular tissue model shows in - vivo like morphological and growth characteristics that are uniform and highly reproducible . \n the tissue consists of highly organized basal cells , which progressively flatten out as the apical surface of the tissue similar to the normal in - vivo corneal epithelium . \n this test could be applied as an alternate to rabbit eye test in the toxicological assessment of ophthalmic preparations . \n most of the formulation efforts aim at maximizing ocular drug absorption through prolongation of the drug residence time in the eye as well as to slow drug release from the delivery system and minimize pre - corneal drug loss . \n nanostructured platforms provide the effective ways for delivery of insoluble drugs to the target site . \n as these platforms for ophthalmic delivery are very recent and the research continues , formulation factors such as loading of drug , release rate , stability , and therapeutic effectiveness for these needs to be optimized .", "role": "user" }, { "content": "the topically applied drugs as drops are washed off from the eye in very short period , resulting in low ocular bioavailability of drugs . \n number of approaches have been attempted to increase the bioavailability and the duration of action of ocular drugs . \n this review provides an insight into various novel approaches ; hydrophilic nanogels , solid lipid nanoparticles , and nanosponges applied very recently in the delivery of insoluble drugs , prolonging the ocular residence time , minimize pre - corneal drug loss and , therefore , bioavailability and therapeutic efficacy of the drugs . despite various scientific approaches , efficient ocular drug delivery remains a challenge for pharmaceutical scientists .", "role": "assistant" } ]

[ { "content": "You are a medical writer. Summarize the following article: clearance of potential cervical spine ( c - spine ) injury in the awake and cooperative patient with no distracting injury is a standardized procedure . \n the process becomes more problematic in the unconscious patient , leading to delay in c - spine collar removal and consequent complications such as tissue necrosis , raised intracranial pressure , excessive sedation and so on . \n two recent reports addressed this issue . in the first of these a postal questionnaire \n was sent to 32 neurosurgery and spinal injury departments in the uk , with the aim of determining how they assessed the c - spine in unconscious , adult trauma patients , and at what point immobilization was discontinued . \n the majority of the units questioned had no formal protocol for either screening investigations or criteria for discontinuation of c - spine immobilization . \n out of 27 units , 12 used two x - ray views alone , and only 10 out of 27 units routinely used computed tomography ( ct ) scanning . \n one unit used magnetic resonance imaging routinely and two used dynamic fluoroscopy . following negative imaging of one variety or other , 12 units discontinued immobilization immediately , 10 continued until they were able to clear spines clinically , and the remaining five were prepared to discontinue if the patient 's condition required it . \n over half of the patients had immobilization discontinued on the basis of plain x - rays alone , despite evidence that plain x - rays have poor diagnostic sensitivity for c - spine fractures [ 3 - 5 ] and are inferior to ct . \n the results suggest that there is often suboptimal and inconsistent investigation , with a subsequent lack of rationale for discontinuation of immobilization . \n it is suggested that head injured patients receiving a ct scan of the brain should routinely undergo c - spine ct scanning at the same time , and that magnetic resonance imaging and dynamic fluoroscopy are not necessary in these patients . \n the second article , also employing a postal questionnaire ( 95% response ) , looked at the major differences between clinicians of differing specialities in the management of potential c - spine injuries in unconscious adult patients . \n the report also reviews the available literature and goes on to suggest a management protocol . \n consistent with the findings of jones and coworkers , morris and mullan demonstrated great variations in practice , attitudes and perceptions with regard to management and evaluation of the potentially injured c - spine in the unconscious patient . \n no consensus existed as to the minimum standard of investigations required to clear the c - spine in these circumstances . \n recognition of the complications of prolonged c - spine immobilization in the critically ill patient was also patchy , with some clinicians suggesting that immobilization should be indefinite until clinical examination could be carried out in the awake patient . based upon a literature review and available consensus , the working group devised a protocol for the investigation and subsequent clearing , or not , of the c - spine in an unconscious patient . \n essentially , the protocol requires three - view x - rays of the c - spine , an anteroposterior and a lateral thoracolumbar x - ray , and a high - resolution ct of the craniocervical junction . \n this must be a step in the right direction , providing an evidence - based approach to an all too common dilemma in the intensive care unit ( icu ) . \n a randomized controlled trial , recently reported in jama , aimed to resolve the tricky issue of whether prone positioning for acute hypoxaemic respiratory failure improves patient survival . \n the trial involved 21 icus with a total of 791 patients being assigned to one of two groups : continually supine or intermittently prone . \n patients were eligible if they were intubated ( or tracheostomized ) with an arterial oxygen tension ( pao2)/fractional inspired oxygen ( fio2 ) ratio < 300 mmhg and had an expected duration of ventilation in excess of 48 hours . \n patients were excluded if they were at risk for harm from the prone positioning ( e.g. raised intracranial pressure ) or had recently been ventilated in the prone position . \n patients in the supine group were managed entirely in that position with a 30 head up tilt . \n patients assigned to the prone group were placed in a complete prone position for at least 8 hours per day . however , \n if a patient developed severe hypoxia in the supine position , then they could crossover to the prone group . \n if a major complication attributable to prone position occurred , then the patient was reverted to the supine position . sadly 28-day mortality rates were not significantly different between the two groups ( 31.5% for supine versus 32.4% for prone ) . \n ninety day mortality was also similar ( 42.2% for supine versus 43.3% for prone ) . \n however , ventilator associated pneumonia was found to be significantly reduced in the prone group . \n the authors acknowledged several limitations in their trial , including significant treatment group crossover and a failure rate in excess of 25% in the prone positioning protocol . \n however , the trial failed to show a reduction in mortality after early prone positioning for acute hypoxaemic failure , despite an improvement in oxygenation and a reduction in ventilator associated pneumonia ; also , it did demonstrate an increased incidence of serious side effects from ventilation in the prone position . \n this report echoes the findings of gattinoni and coworkers and begs the question , is there any evidence to justify prone ventilation ? \n the november issue of critical care medicine included reports on two retrospective studies based on the prowess ( recombinant human activated protein c worldwide evaluation in severe sepsis ) study data base , and discussed new post-28-day data . in the first of these reports , outcome beyond 28 days and health care resource utilization , employing the simplified therapeutic intervention scoring system , were examined . \n previous investigators have concluded that the cost - effectiveness of activated protein c ( apc ) in the management of severe sepsis compares favourably with that of other health care interventions , despite its high up - front cost . \n however , these findings were largely based on 28-day data , and as such they do not take the entire hospital stay into account . \n laterre and coworkers point out that , at 28 days , more than 40% of the prowess survivors were still in hospital and little is known of their health care consumption from there on . \n survival from severe sepsis is significantly better at hospital discharge for those treated with apc than for those not treated with apc , and this remained the case for the majority of subgroups . \n furthermore , this increase in survival was not associated with an increase in resource consumption , as measured using the therapeutic intervention scoring system , or in terms of icu and hospital length of stay . \n in addition , a greater number of survivors in the apc group than in the placebo group were discharged directly home . not happy with \n follow up to hospital discharge , angus and coworkers collected long - term survival data up to 3.5 years after prowess . \n they aimed to determine the effect of apc on hospital survival ( as described above ) and then to investigate the ensuing long - term survival and to analyze these results for selected subgroups based on age , premorbid functional dependency , acute physiology and chronic health evaluation ( apache ) ii score and number of organ dysfunctions , all of which are known to have prognostic significance in severe sepsis . sadly , despite survival to hospital discharge being better in those treated with apc , rates of overall median survival and survival at 3 months , 6 months , 1 year and 2.5 years were not significantly different between the apc and placebo groups . \n this finding was consistent for subgroup analyses other than for apache ii scores . in post \n hoc analysis , patients with an initial apache ii score in excess of 25 had a survival benefit when treated with apc . \n retrospective , cross - sectional observations , use of post hoc analysis and small sample sizes that are not powered for the analysis in question must temper over interpretation . \n however , it is unlikely that a new long - term prospective trial studying the effect of apc in severe sepsis is now possible , and we must therefore tailor our clinical practice with these findings in mind . \n in the past few months the journals , as has become the norm , have been peppered with studies investigating noninvasive ventilation ( niv ) in the management of acute respiratory failure ( arf ) of varying aetiology . \n the immunocompromised patient presenting with arf has been thought to carry a very poor prognosis , and as such intensive care physicians have been reluctant to institute invasive respiratory support . \n however , recent studies have demonstrated the efficacy of niv in this patient population , providing us with an alternative to endotracheal intubation . carrying this concept forward , \n rocco and colleagues reported a case control study comparing niv delivered via traditional face mask versus the much discussed helmet interface for immunocompromised patients with hypoxaemic arf and fever . accepting \n that numbers in this study were small and that patients in the helmet group were matched with historical control individuals receiving facemask niv , both groups demonstrated a similar improvement in pao2/fio2 ratio , but importantly the helmet group demonstrated a more sustained improvement . \n this was probably due to a lower incidence of complications , leading to patient intolerance of the device . \n a further study appearing in chest compared niv with invasive intubation and ventilation for this patient group . \n this was a retrospective study using a pair - wise matching system to compare the two treatment modalities and logistic regression analysis to identify factors affecting in hospital mortality . \n conclusions need to be drawn carefully because the study population was quite heterogeneous with varying types of malignancy and differing aetiologies to the respiratory failure . \n despite this the authors identified increasing severity of illness and a diagnosis of acute myeloid leukaemia as markers of poor outcome , whereas being female , intubated in the first 24 hours and the presence of recent positive blood cultures were markers of good outcome . \n make of this what you will . to end , we return to critical care medicine , december issue . \n a prospective randomized trial suggested that continuous positive airway pressure ( cpap ) and/or bilevel positive airway pressure ( bipap ) is superior to oxygen therapy alone in acute cardiogenic pulmonary oedema . \n specifically , cpap and bipap resulted in much improved pao2/fio2 ratios and a lower incidence of endotracheal intubation . \n however , there was no significant difference in mortality at hospital discharge between the groups . \n apache = acute physiology and chronic health evaluation ; apc = activated protein c ; arf = acute respiratory failure ; bipap = bilevel positive airway pressure ; cpap = continuous positive airway pressure ; ct = computed tomography ; fio2 = fractional inspired oxygen ; icu = intensive care unit ; niv = noninvasive ventilation ; pao2 = arterial oxygen tension . \n ", "role": "user" }, { "content": "' every day ' clinical conundrums are all too infrequently addressed in the mainstream literature , but in the past few months two reports attempted to tackle the thorny problem of the occult cervical spine injury on the intensive care unit . \n are we approaching the death knell for prone ventilation , and how much more can we squeeze out of the prowess study ? \n also , we must of course mention noninvasive ventilation .", "role": "assistant" } ]

[ { "content": "You are a medical writer. Summarize the following article: nuclear receptors ( nrs ) constitute a unique family of ligand - modulated transcription factors . \n nrs mediate cellular response to small lipophilic endogenous and exogenous ligands [ 1 , 2 ] and are responsible for sensing a number of hormones , including steroid and thyroid hormones , and act as positive and negative regulators of the expression of specific genes [ 35 ] . \n therefore , nrs play a central role in many aspects of mammalian development , as well as lipid homeostasis , physiology , and metabolism . \n nrs make up one of the most abundant classes of transcriptional regulators in the body and have emerged as promising pharmaceutical targets . \n classically , nrs consist of several functional domains , that is , a variable n - terminal ligand - independent transactivation domain ( which often exhibits a constitutive transcription activation function ( af-1 ) ) , a highly conserved dna - binding domain ( dbd ) that contains two zinc fingers , a hinge domain ( a variable linker region ) , and a multifunctional c - terminal domain . \n furthermore , the c - terminal domain includes the ligand binding ( lbd ) , the dimerization interface , and the ligand - dependent transactivation domain af-2 [ 1 , 6 ] . small heterodimer partner ( shp ; nr0b2 for nuclear receptor subfamily 0 , group b , member 2 ; mim number 604630 , 601665 ) is a member of the mammalian nr superfamily , due to the presence of a putative ligand - binding domain ( lbd ) . \n shp functions as a corepressor through heterodimeric interaction with a wide array of nuclear receptors and repressing their transcriptional activity . \n shp achieves its goal via several members of the nr superfamily that are able to regulate shp expression . \n however , shp is also a unique and atypical nr because it lacks the classical dna - binding domain ( dbd ) , generally present in other nrs . \n the nr0b family of nrs consists of 2 orphan receptors : shp and dax-1 ( dosage - sensitive sex reversal adrenal hypoplasia congenita ( ahc ) critical region on the x chromosome , gene 1 ) . \n dax1 is a gene whose mutation causes the x - linked adrenal hypoplasia congenita and is the only family member that lacks a conventional dbd . \n dax-1 ( nr0b1 ) is therefore seen as the closest relative of shp in the nr superfamily [ 1012 ] . \n whereas shp is different from other conventional nrs both structurally and functionally , it acts as a ligand - regulated receptor in metabolic pathways . \n shp belongs to the orphan subfamily since there is no known ligand for this receptor , except for some retinoid - related molecules . \n shp inhibits transcriptional activation by working on several other nuclear receptors , that is , directly modulating the activities of conventional nuclear receptors by acting as an inducible and tissue - specific corepressor [ 12 , 15 ] . \n the discovery of shp dates back to 1996 ; since then , this orphan nr has been identified as a key transcriptional regulator of signaling pathways [ 8 , 16 ] involving fundamental biological functions and metabolic processes . \n such processes include cholesterol , bile acid and fatty acid metabolism , glucose and energy homeostasis , and reproductive biology . \n experiments performed by fluorescence in situ hybridization ( fish ) analysis of the human metaphase chromosome have shown that shp is found at a single locus on chromosome 1 at position 1p36.1 and consists of two exons and a single intron spanning approximately 1.8 kb with 257 amino acids in humans . in mice and rats , \n shp resides on chromosomes 4 and 5 , respectively , both consisting of 260 amino acids . \n shp expression is predominantly observed in the liver [ 10 , 18 ] , but it is also detected at lower levels in other tissues , including the pancreas , spleen , small intestine , colon , gallbladder , kidney , adrenal gland , ovary , lung , brainstem , cerebellum , heart , and thymus ( table 1 ) [ 1921 ] . \n shp is indeed able to repress the transcriptional activities of its target nrs and transcriptional regulators through two functional leu - xaa - xaa - leu - leu- ( lxxll- ) like motifs [ 2224 ] . \n such motifs appear to be essential for the interaction with the ( activation function 2 ) af-2 domains of several sets of nrs [ 22 , 23 ] . \n the human shp is enriched by another 12 amino acids [ 2536 ] , and this region between helix 6 and 7 is also involved in the repression of the transactivation of nrs . \n since shp lacks dna - binding domain , it exerts the inhibitory effects through protein - protein interaction . \n shp expression seems to follow a circadian rhythm in the liver , involving the clock - bmal1 pathway and suggesting that some of the regulatory functions of shp and deriving functions must be temporal [ 19 , 20 , 38 ] . \n gene expression of shp is regulated by several factors including nrs , transcription factors , and a number of additional conditions and substances , as extensively reported in table 2 . \n also , the central role of shp is clear since this nr is able to act as a coregulator for wide range of targets , namely , nrs / transcription factors / transcriptional coregulators and few different molecules , as depicted in table 3 . in general \n , shp acts as a repressor of the transcriptional activity of the specific interacting partner ( via lbd of the partner and nr boxes of shp ) [ 12 , 3943 ] . \n however , it is also demonstrated that shp is able to upregulate gene transcription , as in the case of ppar and ppar [ 4446 ] and nf-b . \n both n - terminal nr interaction domain and c - terminal domain of shp are important for repression [ 47 , 48 ] . \n overall , the shp - mediated repression of target transcription factors occurs by at least three distinct transcriptional repression mechanisms ( figure 2 ) . \n a first mechanism involves direct interference with the af-2 coactivator domain of nrs ( competition for coactivator binding , leading to the repression of nr - mediated transcriptional activity ) . \n this is the case for the inhibition of estrogen receptor ( er ) and estrogen receptor ( er ) . a second mechanism for the shp - mediated repression involves the recruitment of corepressors including direct interactions among mammalian homolog of the saccharomyces cerevisiae transcriptional corepressor sin3p ( msin3a ) , human brahma ( brm ) , switch / sucrose nonfermentable ( swi / snf ) complexes leading to the repression of cholesterol 7-hydroxylase ( cyp7a1 ) ) . \n a third mechanism of inhibition of shp involves the direct interaction with the surface of nr or transcription factor , resulting in the blockade of dna binding and the consequent inhibition of its transcriptional activity . \n this is the case for rar - rxr heterodimers , pxr - rxr binding to dna by shp , interaction with hepatocyte nuclear factor ( hnf4 ) , or jun family of the activator protein 1 ( ap-1 ) transcription factor complex ( jund ) [ 51 , 52 ] . \n all three mechanisms might occur sequentially or alternatively according to type of cells and promoters . \n clearly , information on factors that increase or decrease shp expression and that are regulated by shp is essential for understanding the regulatory effects of this orphan nr . \n interestingly , it is suggested that targeting posttranslational modifications of shp may be an effective therapeutic strategy . \n selected groups of genes could be controlled to cure a vast range of metabolic and shp - related diseases . \n overall , the huge amount of information on shp function is currently available , making this nr essential in a number of functions involving cholesterol and bile acid metabolism , lipogenesis , glucose metabolism , steroid hormone biosynthesis , xenobiotic homeostasis / metabolism , and cell cycle . in particular , the ability of shp in interacting with different metabolic signaling pathways including bile acids and lipid homeostasis , fat mass , adipocytes , and obesity will be reviewed here \n the wide ability of shp to target multiple genes in diverse signaling pathways points to the key role of shp in various biological processes , including the metabolism of bile salts , glucose , and fatty acids . \n studies suggest that loss of shp might positively affect cholesterol and bile acid homeostasis in pathophysiologically relevant conditions . \n bile acids ( bas ) are amphipatic cholesterol metabolites which are synthesized in the liver , secreted into bile , stored in the gallbladder , and secreted postprandially into the duodenum . \n bas are synthesized from cholesterol , and this pathway provides the elimination of excess cholesterol in the body . moreover , bas should be seen as physiological detergents which , in the small intestine , are essential for the absorption , transport , and distribution of lipophilic molecules , including dietary lipids , steroids , and lipid - soluble vitamins . in the intestine , bas undergo extensive metabolism by the intestinal microflora . \n a high efficient system is the enterohepatic circulation of bas [ 55 , 56 ] , where more than 9095% of bas are returned to the liver from the terminal ileum via the portal vein . \n thus , the concentration of bas in serum , liver , and intestine is tightly regulated to prevent damage to enterohepatic tissues due to their strong detergent moiety [ 5759 ] . \n the major rate - limiting step in biosynthetic pathway of bas in humans is initiated by cholesterol 7-hydroxylase ( cyp7a1 ) , the microsomal p450 liver enzyme , to produce two primary bas , cholic acid , and chenodeoxycholic acid , essential in the overall balance of cholesterol homeostasis . \n sterol 12 hydroxylase ( cyp8b1 ) catalyzes the synthesis of cholic acid , a step which determines the cholic acid to cdca ratio in the bile . \n secondary bile acids ( deoxycholic acid and lithocholic acid ) and tertiary bile acids ( ursodeoxycholic acid ) in humans are produced following intestinal dehydroxylation of primary bile acids by intestinal bacteria [ 58 , 61 ] . \n regulation of ba biosynthesis is highly coordinated and is mediated by key nrs including the orphan receptor , liver receptor homologue-1 ( lrh1 ; nr5a2 ) , the hepatocyte nuclear factor 4 ( hnf4 ) , shp , and the bile acid receptor farnesoid x receptor ( fxr ; nr1h4 ) . \n thus , the activation of fxr initiates a feedback regulatory loop via induction of shp , which suppresses lrh-1- and hnf4-dependent expression of the two major pathway enzymes cholesterol 7hydroxylase ( cyp7a1 ) and sterol 12 hydroxylase ( cyp8b1 ) . \n the ba feedback regulation primarily occurs since bas act as transcriptional regulators for the expression of the gene encoding cyp7a1 . both cholic acid and chenodeoxycholic acid function as endogenous ligands for the nuclear bile acid receptor fxr . \n fxr expression is high in the intestine and liver , the two sites where bas reach high concentrations to activate fxr . \n the transcription by fxr includes heterodimerization with retinoid x receptors ( rxrs ) in the cytoplasm , translocation into the nucleus , and binding to dna response elements in the regulatory regions of target genes . \n when the bind of bas to fxr , shp transcription is increased [ 60 , 64 , 65 ] , this alteration leads to the inhibition of lrh-1 activity or hnf4 on the ba response elements ( bares ) of cyp7a1 and cyp8b1 promoters [ 64 , 65 ] . in this scenario , ba synthesis is downregulated by a precise feedback regulatory mechanism , which represents the major pathway under normal physiological conditions [ 6466 ] ( figure 3 ) . \n lrh1 is also a well - known activator of shp gene transcription [ 64 , 65 ] , and this step leads to an autoregulatory loop of gene expression by shp . \n this step also includes the g protein pathway suppressor 2 ( gps2 ) interacting with fxr , lrh-1 , and hnf4 to regulate cyp7a1 and cyp8b1 expression in human hepatocytes ( table 3 ) . \n a critical role in maintaining cholesterol homeostasis for cyp7a1 has been recently advocated in a model of in cyp7a1-tg mice . \n the hepatocyte nuclear factor-1 ( hnf1 ) , which haploinsufficiency causes the maturity - onset diabetes of the young type 3 ( mody3 ) , also appears to modulate shp expression via the fxr pathway . in this respect , \n hnf1 ( / ) mice displayed a defect in bile acid transport , increased bile acid and liver cholesterol synthesis , and impaired hdl metabolism . a role for shp in mediating the recruitment of msin3a - swi / \n snf to the cyp7a1 promoter , with chromatin remodeling and gene repression , has been described . in hepg2 cells , \n kemper et al . have shown that bile acid treatment resulted in shp - mediated recruitment of transcriptional coregulators msin3a and swi / snf complex to the promoter , chromatin remodeling , and gene repression ( table 3 ) . \n this is an additional mechanism involving transformation of nucleosome conformation for the repression by shp of genes activated by various nrs . in line with such results , increased synthesis and accumulation of bas occurs in shp ( / ) mice , due to the loss of shp repression and consequent derepression of the rate - limiting cyp7a1 and cholesterol 12-hydroxylase ( cyp8b1 ) ( the rate - determining enzyme of the alternative but minor ba synthesis pathway ) in the biosynthetic pathway [ 7072 ] . \n mechanisms independent of the fxr / shp / lrh pathway might also exist , since bas feeding to shp ( / ) mice reduced the levels of cyp7a1 mrna to similar levels of control mice [ 70 , 71 ] . \n such shp - independent and alternative pathways include the protein kinase c / jun n - terminal kinase ( pkc / jnk ) pathway , the fxr / fgfr4 ( fgf receptor 4 ) pathway [ 57 , 74 ] , the cytokine / jnk pathway , the pregnane x receptor ( pxr ) mediated pathway , and the jnk / c - jun signaling pathway . \n another study demonstrated , in shp ( / ) mice on a background of 129 strain , the protection against hypercholesterolemia in three different models : an atherogenic diet , hypothyroidism , and shp ( / ) mice intercrossed with ldlr ( / ) mice ( to generate shp / ldlr double ( / ) mice in a mixed 129-c57bl/6 background ) . when fed an atherogenic diet , the latter strain was almost completely resistant to diet - mediated increases in triglyceride , very low - density lipoprotein ( vldl ) cholesterol , and low - density lipoprotein ( ldl ) cholesterol but had an increase in high - density lipoprotein ( hdl ) cholesterol as compared with ldlr ( / ) mice . \n such results point to the protection against dyslipidemia following the inhibition of hepatic shp expression , although no antagonist ligands have yet been identified for shp . \n we have recently examined biliary lipid secretion and cholesterol gallstone formation in male shp ( / ) and ( + /+ ) mice before and during the feeding of a lithogenic diet for 56 days . \n deletion of the shp gene significantly increased hepatic bile salt synthesis , and doubled the increase of biliary bile salt outputs in shp ( / ) mice than in ( + /+ ) mice . \n the intestinal bile acid pool size was significantly greater in shp ( / ) mice than in ( + /+ ) mice . \n these increased bas are efficacious ligands of fxr and can stimulate the expression of intestinal fibroblast growth factor 15 ( fgf15 ) in mice through the fxr signaling pathway , which is consistent with the expanded bile acid pool size in shp ( / ) mice . at 14 days on the lithogenic diet , \n fasting gallbladder volume was significantly larger in shp ( + /+ ) mice than in ( / ) mice . \n is another fxr gene target in the intestine and appears to contribute to the fine tuning of bile acid synthesis in the liver . \n thus , a model for fxr - mediated repression of bile acid synthesis should also take into account the bile acid - mediated activation of intestinal fxr and fgf15 in the small intestine ( while the fxr - shp pathway is activated in the liver ) . according to the most plausible view , fgf15 acts as a hormone to signal between intestine and liver . \n the secreted fgf15 by the intestine circulates to the liver , likely through the portal circulation or lymph flow , and induces the activation of fgfr4 in the liver . \n as shown in figure 3 , the fgf15/fgfr4 pathway synergizes with shp in vivo to repress cyp7a1 expression . in humans , \n of note , activation of fxr transcription in the intestine protected the liver from cholestasis in mice by inducing fgf15 expression and reducing the hepatic pool of ba . \n hepatic fatty acid homeostasis is also regulated by shp since regulating these genes involves in fatty acid uptake , synthesis , and export [ 8387 ] . in a study exploring global gene expression profiling combined with chromatin immunoprecipitation assays in transgenic mice constitutively expressing shp in the liver , overexpression of shp in the liver \n was associated with the depletion of the hepatic bile acid pool and a concomitant accumulation of triglycerides in the liver . by contrast , fat accumulation induced by a high - cholesterol or high - fat diet \n the pleiotropic role of shp can also be found in the case of nonalcoholic liver steatosis since ob / shp double ( / ) mice ( a model of severe obesity and insulin resistance ) became resistant to liver steatosis and showed improved insulin sensitivity . \n another interesting role for shp emerged after it was found that bas negatively regulate the human angiotensinogen ( ang ) gene . \n ang is the precursor of vasoactive octapeptide angiotensin ii , and bas act through the shp pathway by preventing hepatocyte nuclear factor-4 ( hnf4 ) from binding to the human ang promoter . \n human obesity is considered a polygenic disorder characterized by partly known abnormal molecular mechanisms resulting in increased fat mass , with an imbalance between the energy acquired from nutrients that dissipated as heat ( i.e. , thermogenesis ) . in this respect , \n weight stability requires a balance between calories consumed and calories expended . in adipose tissue depots , two main types of adipocytes \n exist , that is , brown adipocytes and white adipocytes . in several animal species , \n some adipose tissue sites mainly include brown adipocytes ( bats ) and the other contains mainly white adipocytes ( wats ) . \n bat dissipates chemical energy to produce heat either as a defense against cold or as energy expenditure to compensate food intake [ 93 , 94 ] . \n the unusual function of bat might be better understood by considering that they share a common origin with myocytes [ 95 , 96 ] , and bat was indeed considered something in between muscle and adipose tissue . \n bat is deemed as the major site for sympathetic ( adrenergic ) mediated adaptive thermogenesis ; this pathway involves the uncoupling protein-1 ( ucp1 ) . \n wat is mainly implicated in the regulation of lipid storage and catabolism but also in the synthesis and secretion of adipokines [ 97100 ] . while the percentage of young men with bat is high , the activity of bat is reduced in men who are overweight or obese . \n thermogenesis unequivocally exists in both humans and animals , and bat is the major site of thermogenesis which can be increased by environmental factors ( i.e. , adaptive thermogenesis ) . in both human and animal species , dietary composition , \n chronic cold exposure , and exercise may increase thermogenesis . as far as adipose tissue biology is concerned \n , shp seems to play a distinct regulatory function in wat , as compared with bat . \n a number of experiments have focused on animal models of obesity and subtle molecular changes . \n shp - deficient mice are protected against high - fat - diet - induced obesity . \n peroxisome proliferator - activated receptor ( ppar ) coactivator-1 ( pgc-1 ) family members are multifunctional transcriptional coregulators . \n in particular , pgc-1 and pgc-1 regulate mitochondrial biogenesis , adaptive thermogenesis , fatty acid and glucose metabolism , fiber - type switching in skeletal muscle , peripheral circadian clock , and development of the heart . \n in particular , shp functions as a negative regulator of energy production in bat because shp is a negative regulator of pgc-1 expression in bat . in turn , pcg-1 is a coactivator of uncoupling protein 1 ( ucp1 ) which plays a major role in energy dissipation as heat in multilocular bat of different animal species and humans [ 104106 ] . \n fat - specific ( bat ) shp - overexpressed transgenic mice had increased body weight and adiposity . energy metabolism , however , was increased , and bat cold exposure function was enhanced with activation of thermogenic genes and mitochondrial biogenesis ( enhanced 1-ar gene expression and pgc1 ) . compared with wild - type mice on a high - fat diet , \n shp overexpression was associated with enhanced diet - induced obesity phenotype with weight gain , increased adiposity , and severe glucose intolerance . \n an additional feature of shp transgenic mice was a decreased diet - induced adaptive thermogenesis , increased intake of food , and decreased physical activity . \n this leads to the conclusion that , although expressed at low levels in fat , activation of shp in adipocytes has a strong effect on weight gain and diet - induced obesity . moreover , \n if mechanisms linked to energy metabolism and the development of obesity are considered , shp has distinct roles in wat and bat . \n as previously mentioned , while shp deletion in obese leptin - deficient mice ( ob / ob ) prevented the development of nonalcoholic fatty liver and improved peripheral insulin sensitivity , shp deletion did not overcome the severe obesity caused by leptin deficiency . \n a significant protective effect from obesity by shp deficiency was likely associated with the low basal level of shp expressed in fat . \n adipogenesis appears to be influenced by shp : when shp was overexpressed in 3t3-l1 preadipocytes , cell differentiation was inhibited , as well as the accumulation of neutral lipids within the cells . \n in particular , shp appears to be a potent adipogenic suppressor , and preadipocytes are kept in an undifferentiated state through the inhibition of the adipogenic transcription factors and stimulators . \n further studies will address whether the loss of shp function results in inhibition of lipid accumulation in adipocytes , similar to what is observed in hepatocytes . in a future clinical setting , treatment of obesity might also include drugs able to mimic or stimulate the effects of shp . \n mutations in the shp gene have also been reported in patients with lipodystrophy carrying four different polymorphisms . \n a number of important clinical studies have examined this issue ( table 4 ) ; however , hung et al . in uk examined the relationships between genetic variation in shp and weight at birth , adiposity , and insulin levels in three different populations ( the genetics of obesity study ) goos , the avon longitudinal study of parents and children ( alspac ) , and the ely studies ) . in the 329 cases of severe early - onset obesity ( goos study ) , two novel and rare missense mutations ( r34 g and r36 g ) \n furthermore , two common polymorphisms , namely , g171a ( 12% of subjects with higher birth weight ) and 195ctgadel ( 16% of subjects with lower birth weight ) were found . in the alspac cohort of 1,079 children , \n the g171a variant was associated with increased body mass index and waist circumference together with higher insulin secretion 30 minutes after glucose load . thus , whereas mutations in the shp gene can not be seen as a common cause of severe human obesity , genetic variation in the shp gene locus may influence birth weight and have effects on body size . \n the effect might ultimately involve insulin secretion by the negative regulation between shp and the hepatocyte nuclear factor-4 ( hfn-4 ) , a transcription factor involved in differentiation and function of pancreatic -cells . \n a possibility is that decreased shp expression or function results in increased hfn-4 activity with a cascade of events , including fetal hyperinsulinemia , and increased birth weight . at a later stage , \n mutations in the shp gene were also associated with influence on birth weight , mild obesity , and insulin levels in the study by nishigori et al . \n mutations in several genes encoding transcription factors of the hepatocyte nuclear factor ( hnf ) cascade are associated with maturity - onset diabetes of the young ( mody ) . \n mody is a monogenic form of early - onset diabetes mellitus ( defective insulin secretion with normal body weight ) , and shp is deemed as a plausible candidate mody gene ; this is because shp is able to inhibit the transcriptional activity of the hepatocyte nuclear factor-4 ( hfn-4 ) , a key member of the mody regulatory network . \n thus , further studies have looked for segregation of shp mutations with mody in a cohort of japanese patients with early - onset diabetes . in this context \n , variants in shp appeared to cosegregate with increased body mass index in families , thus contributing to obesity among japanese subjects . also , increased risk of morbidity was observed in another study from japan , examining patients with type 2 diabetes and shp mutations . \n major differences , however , might exist in the prevalence and function of shp variants in different populations . of note \n , the results from other caucasian cohorts did not confirm the association between shp mutation and obesity [ 113 , 114 ] . \n conducted an elegant study on the prevalence of shp variants by single - strand conformational polymorphism and heteroduplex analysis among 750 danish obese men with early - onset obesity . \n as control , a cohort of 795 nonobese control subjects was genotyped using pcr - rflp . \n functional analyses of the identified coding region variants were performed in both min6-m9 and hepg2 cell lines . \n five novel variants were identified ( including 3 missense variants ( c.100c > g [ p.r34 g ] , c.278g > a [ p.g93d ] , and c.415c > a [ p.p139h ] ) and 2 silent variants ( c.65c > t [ p.y22y ] and c.339g > a [ p.p113p ] ) ) . the previously reported c.512g > c [ p.g171a ] common polymorphism was identified ; however , the prevalence of functional shp variants associated with obesity was considerably lower among danish subjects ( 1 out of 750 obese , none of control subjects ) , compared to the prevalence observed in japan by nishigori et al . . \n investigated shp variants in 1927 uk subjects according to type 2 diabetes , obesity , and birth weight . \n although reporting a raised body mass index among homozygous carriers of the 171a variant ( < 1% ) , this polymorphism was unlikely to be associated with all three conditions in caucasians . \n taken together , the above - mentioned studies suggest that the 171a variant might contribute only to subsets of polygenic obesity . \n the existence of multiple interactions of shp with nrs , transcription factors and transcriptional cofactors ( tables 2 and 3 ) points to the pleiotropic and central role of shp in the body . \n shp has been hypothesized to act in glucose homeostasis via complex pathways involving the inhibition of glucocorticoid receptors ( gr ) in mammalian cells and the inhibition of pgc-1 gene , a coactivator of nrs important for gluconeogenic gene expression and the pgc-1-regulated phospho(enol)pyruvate carboxykinase ( pepck ) promoter . \n such steps underscore a physiologically relevant role for shp in modulating hepatic glucocorticoid action . following \n the bile acid - induced induction , shp inhibited a number of other pathways , including the hnf4-mediated transactivation of the pepck and fructose biphosphate ( fbp ) promoters , as well as the transactivation of the glucose-6-phosphatase ( g6pase ) promoter mediated by foxo1 . \n the interaction between shp inhibitory function and the 3 isoforms ( , , and ) of the hepatocyte nuclear factor-3 ( hnf4 ) points to the regulatory role of shp on gluconeogenesis . \n mutations in hepatocyte nuclear factor 1 ( hnf-1 ) is associated with maturity - onset diabetes of the young type 3 . \n indeed , loss of hnf-1 function in hnf-1 ( / ) mice resulted in altered expression of genes involved in glucose - stimulated insulin secretion , but also insulin synthesis , and beta - cell differentiation . \n pancreatic islets of hnf-1 ( / ) mice showed a distinctive reduction of shp expression and a downregulation of the hnf4 gene expression . since shp appears to repress its own transcriptional activation following heterodimerization with hnf4 , a feedback autoregulatory loop between shp and hnf4 has been hypothesized . \n shp ( / ) mice were characterized by hypoinsulinemia , increased glucose - dependent response of islets , increased peripheral insulin sensitivity , and increased glycogen stores . \n the role played by shp in the regulation of hepatic gluconeogenesis has also emerged in a number of additional experiments . \n for example , the liver of shp ( / ) mice showed increased glycogen stores , while hepatic shp gene expression ( induced by the antidiabetic biguanide drug metformin ) was associated with inhibition of hepatic gluconeogenesis . \n induction of shp was achieved via amp - activated protein kinase ( ampk ) and associated with downregulation of essential gluconeogenic enzyme genes , that is , phosphoenolpyruvate carboxykinase ( pepck ) , glucose-6-phosphatase ( g6pase ) , and fructose-1,6-bisphosphatase ( fbp1 ) . \n pgc-1 gene is a coactivator of nrs , and this step is relevant for gluconeogenic gene expression . \n yamagata et al . showed that bile acid ( chenodeoxycholic acid ) was able to induce the downregulation of pgc-1 gene , and this mechanism involved forkhead transcription factors ( foxo1 , foxo3a , foxo4 ) via a shp - dependent manner . \n this is also the case for excess bas : the pregnane x receptor ( pxr ) induces cyp3a and inhibits cyp7 , both involved in biochemical pathways leading to the conversion of cholesterol into primary bas , whereas cyp3a is also involved in the detoxification of toxic secondary bile acid derivatives . \n shp acts as a potent repressor of pxr transactivation , and this finding suggests that pxr can act on both bile acid synthesis and elimination detoxification . \n additional mechanisms involved in the shp - dependent control of pathways of drug metabolism have been identified . \n the expression of genes involved with the metabolism of xenobiotics might be regulated by shp in the spleen acting on ( aryl hydrocarbon receptor ( ahr)/ahr nuclear translocator ( arnt ) ) ahr / arnt heterodimers which , in turn , bind to xenobiotic response elements ( xres ) at the level of specific dna sequences . \n a number of genes involved in hormone and drug metabolism would be expressed ( i.e. , ugt16 , aldh3 , cyp1a1 , cyp1a2 , cyp1b1 , etc . ) . \n shp also appears to downregulate the constitutive - androstane - receptor- ( car- ) mediated cyp2b1 gene expression , induced by phenobarbital to form the car / rxr heterodimer which , in turn , binds to 2 dr-4 sites to form the phenobarbital responsive unit in the cyp2b gene ( table 3 ) . \n one role of shp in steroidogenesis has been identified in the testes with influence on testosterone synthesis and germ cell differentiation and in the intestine for glucocorticoid synthesis . \n a role for shp in cell proliferation and apoptosis signaling is emerging . depending on the cell type \n however , the manipulation of shp through the synthetic ligands adamantyl - substituted retinoid - related ( arr ) compounds 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid ( cd437/ahpn ) and 4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid ( 3-cl - ahpc ) induces apoptosis of a number of malignant cells ( i.e. , leukemia and breast carcinoma ) both in vitro and in vivo [ 123 , 124 ] . the complex mechanism implies binding of arr and 3-cl - ahpc to shp with formation of a corepressor complex containing sin3a and nuclear receptor corepressor ( n - cor ) which activate local control of mitochondrial function and apoptosis , with a limiting function on tumorigenesis [ 17 , 123 ] ( table 3 ) . \n shp appears to be also involved in dna methylation and acting as a tumor suppressor , at least in the human and mouse livers [ 125127 ] . \n whether manipulation of shp will be helpful in the treatment of hepatic and other gastrointestinal cancers is still a matter of research . \n the recent finding that shp negatively regulates tlr signaling to nf-b has raised the interest for the role of shp in mechanisms governing innate immunity . \n shp appears to negatively regulate the expression of genes encoding inflammatory molecules . of note , \n direct binding of nf-b seems to occur in resting cells , while binding of shp to traf6 occurs in lps - stimulated cells [ 128 , 129 ] . \n a remarkable number of metabolic functions in the body appear to be regulated by the orphan unique nr , small heterodimer partner shp , which targets a complex set of genes in multiple pathways as a transcriptional corepressor ( figure 4 ) . \n when looking at complex mechanisms leading to some important lipidopathies , that is , obesity and liver steatosis , enlightening data about the regulatory function of shp are provided by studies using shp - deleted and shp - overexpressed animal models . \n most likely , a condition of shp deficiency might counteract lipid accumulation and improve plasma lipoprotein profiles . \n further studies are urgently needed to confirm that such an important metabolic regulatory mechanism of shp is true and has high translational value . to date , however , no synthetic antagonists or agonists for shp are available , and one should keep in mind that rather divergent and somewhat elusive data have been observed regarding the loss of shp function in humans and rodents . thus , \n careful examination of subtle shp intrinsic functions is essential to dissect potential modulatory pathways of shp for a variety of metabolic abnormalities but also in tumorigenesis . \n moreover , identifying specific endogenous ligands and synthetic agonists of shp will pave the way to for therapeutic intervention . \n the effect of synthetic ligands on shp modulation in hepatocytes and adipocytes , for example , might represent therapeutic tools for the treatment of constituents of the metabolic syndrome , namely , hypercholesterolemia , overweight obesity , and liver steatosis .", "role": "user" }, { "content": "nuclear receptors ( nrs ) comprise one of the most abundant classes of transcriptional regulators of metabolic diseases and have emerged as promising pharmaceutical targets . \n small heterodimer partner ( shp ; nr0b2 ) is a unique orphan nr lacking a dna - binding domain but contains a putative ligand - binding domain . \n shp is a transcriptional regulator affecting multiple key biological functions and metabolic processes including cholesterol , bile acid , and fatty acid metabolism , as well as reproductive biology and glucose - energy homeostasis . \n about half of all mammalian nrs and several transcriptional coregulators can interact with shp . \n the shp - mediated repression of target transcription factors includes at least three mechanisms including direct interference with the c - terminal activation function 2 ( af2 ) coactivator domains of nrs , recruitment of corepressors , or direct interaction with the surface of nr / transcription factors . \n future research must focus on synthetic ligands acting on shp as a potential therapeutic target in a series of metabolic abnormalities . \n current understanding about the pleiotropic role of shp is examined in this paper , and principal metabolic aspects connected with shp function will be also discussed .", "role": "assistant" } ]

[ { "content": "You are a medical writer. Summarize the following article: among bilaterian animals , the deuterostomes include the hemichordates , the echinoderms and the chordates . \n the chordates can further be subdivided into urochordates ( = tunicates ) , cephalochordates and vertebrates ( fig . \n since human beings have often wondered about their own origins , it is not surprising that there have been many studies aimed at understanding the evolutionary history of chordates ( i.e. the origin of chordates and the diversification of vertebrates ) . \n the main characters that evolved at the base of the chordates are a notochord and a dorsal hollow nerve cord 1 , while definite neural crest cells and placodes arose at the base of the vertebrates 2 . \n however , the evolutionary origins of these structures have been controversial , mainly because of difficulties in determining homologies between chordate - specific characters and structures in non - chordate deuterostomes ( i.e. in hemichordates and echinoderms ) as well as between the vertebrate - specific characters and structures in invertebrate chordates . \n the development of many chordate- and vertebrate - specific characters is controlled , directly or indirectly , by retinoic acid ( ra ) , a vitamin a - derived morphogen . in chordate embryos , \n too much or too little ra during early embryogenesis causes malformations , which are mainly due to a mispatterning of the embryo along the anteroposterior body axis . \n ra signaling is mediated by ra binding to retinoic acid receptors ( rars ) , which form heterodimers with retinoid x receptors ( rxrs ) 3 . during chordate development , \n important functions of ra signaling are mediated by hox genes , at least some of which are direct targets of ra 4 - 6 . in many animals , including most deuterostomes , hox genes mediate anteroposterior positional patterning of the embryo 7 , 8 . moreover , in vertebrates , hox genes have been suggested to play important roles in the patterning of all embryonic tissue layers 7 , 9 . ever since it became clear that the genetic mechanisms controlling key features of embryonic development are highly conserved between organisms as diverse as fruit flies and mice , comparisons of the developmental mechanisms in different organisms have been used to address questions about animal evolution , particularly about the invertebrate - to - vertebrate transition in chordates 10 . using this so - called evo - devo \n approach , this review discusses the developmental roles of ra signaling among chordates in regard to chordate origins and vertebrate evolution . \n the focus is on the roles of ra- and hox - dependent patterning during development and their conservation during chordate evolution . \n we show that some of the control mechanisms governing ra - mediated patterning are quite conserved among vertebrates and between vertebrates and invertebrate chordates , such as the cephalochordate amphioxus . \n in contrast , evidence for roles of ra signaling outside the chordates and the evolutionary origin of ra signaling itself still remain elusive . \n retinoic acid ( ra ) is a natural morphogen synthesized from vitamin a ( retinol ) , and it has been known for over fifty years that either too much or too little ra during early development is teratogenic mainly due to anteroposterior patterning defects 3 . in general , excess ra posteriorizes , while ra deficiency anteriorizes chordate embryos 3 , 11 - 14 . \n endogenous ra is synthesized in two steps : the first is the reversible oxidation of retinol to retinal performed by alcohol dehydrogenases ( adhs or rdhs / sdrs ) and the second is the oxidation of retinal to retinoic acid , which is carried out by retinaldehyde dehydrogenases ( raldhs ) . \n ra signaling levels are also regulated by binding of retinol to cellular retinol binding proteins ( crbps ) and of ra to retinoic acid binding proteins ( crabps ) ( fig . \n the roles of these proteins in regulating ra signaling in vertebrates have been elucidated with gene knock - outs . \n for example , individual knock - outs of three of the four raldh genes ( i.e. the knock - outs of raldh1 , 2 and 3 ) have been described in the mouse , but only that of raldh2 exhibits clear developmental defects . in these mice , \n the anteroposterior axis is shortened and the embryos , which die before birth , have defects in the heart , limbs and head , which are reminiscent of vitamin a deprivation phenotypes 16 . in the ra degradation pathway , knock - out of cyp26a1 , one of the three mouse cyp26 genes , is also lethal before birth and affects anteroposterior patterning as well as hindbrain and tail development 17 , 18 . \n this cyp26a1 phenotype is comparable to the teratogenic effects of excess ra , suggesting that cyp26 genes in general may help restrict the distribution of endogenous ra to ra target tissues . \n moreover , analysis of cyp26a1 and raldh2 double knock - out mice has shown that ra and not one of its metabolic derivatives is the major ( if not the only ) active retinoid during development 19 . \n ra signaling is mediated by ra binding to retinoic acid receptors ( rars ) , which form heterodimers with retinoid x receptors ( rxrs ) . \n this complex in turn binds to retinoic acid response elements ( rares ) in the regulatory regions of target genes 13 , 15 . \n these include the rars themselves , hox genes , some other transcription factors , such as hnf-3 and cdx1 , plus genes involved in retinoic acid metabolism ( e.g. crabp1 and 2 ) 5 . in the mouse , there are 3 rars and 3 rxrs ( , and ) . \n although knock - out of any one of these has only minor , tissue - specific effects on morphogenesis due most likely to functional redundancy among them , compound mutants with two or three of the genes inactivated are much more severely affected with defects in anteroposterior patterning of pharyngeal endoderm , hindbrain and neural crest cells 3 . \n in general , rar and rxr proteins share a common organization of functional domains : an amino terminal a / b region containing a transcriptional activation domain ( af-1 ) , a centrally located c region corresponding to the dna binding domain ( dbd ) plus a weak dimerization domain and the e region , which includes the ligand binding domain ( lbd ) , a strong dimerization interface and a surface allowing binding of transcriptional co - regulators 3 , 20 , 21 . in the absence of ligand , the rar / rxr heterodimer is constitutively bound to dna on rares and associated with co - repressor complexes that induce transcriptional silencing by deacetylating histones associated with the target sequences thus increasing chromatin condensation ( fig . \n binding of ra to the rar ligand binding pocket induces a conformational change of the lbd that creates a surface allowing the association of co - activators and the release of co - repressors . \n the co - activators ( e.g. tif2 and src-1 of the p160 co - activator family ) subsequently mediate histone acetylation resulting in decondensation of the chromatin and activation of target gene expression ( fig . \n 2b ) 20 - 22 . among the known targets of the rar / rxr heterodimer \n hox genes encode transcription factors that contain a highly conserved dna binding domain of 60 amino acids , the homeodomain 7 , 8 . \n hox genes are usually linked in a cluster and their order on the chromosome correlates with both their temporal and spatial expression during embryogenesis 7 - 9 , 23 . \n this so - called collinear expression is crucial for conveying anteroposterior positional patterning information to the embryo during development and is conserved in many animals , including most deuterostomes 7 - 9 , 23 . \n however , when genomic hox clustering has been lost , as for example in tunicates , temporal collinearity is lacking and spatial collinearity is only approximate 24 , 25 . in vertebrates , hox genes are direct targets of ra signaling 5 and ra signaling is involved in regulation of collinear hox expression along the anteroposterior body axis of the developing embryo . for example , in mice , the deletion of a single rare from the hoxa1 cis - regulatory region disrupts the establishment of the anterior boundary of hoxa1 as well as the normal expression pattern of hoxa2 in the hindbrain 26 . \n moreover , the hox cluster also contains several other rares , which are largely conserved among vertebrates and at least to some extent between vertebrates and the cephalochordate amphioxus 4 , 6 . \n for example , both the amphioxus and vertebrate hox1 genes have a conserved rare in their cis - regulatory region 4 . \n in addition , although clustering of hox genes has been lost in tunicates , ascidian hox1 expression is strongly upregulated after ra treatment 27 suggesting that , as in amphioxus and vertebrates , hox genes in tunicates might also be direct targets of ra signaling . \n early during vertebrate diversification , the total number of genes has been markedly increased by two rounds of whole genome duplications 28 . \n thus , while vertebrates have 3 or more rars , there are single rars in invertebrate chordates , such as amphioxus and tunicates 14 , 29 . a comprehensive search of the sea urchin genome sequence ( www.ncbi.nlm.nih.gov/genome/guide/sea_urchin ) also reveals the presence of a single rar ( fig . \n although less is known about the ra signaling pathway in invertebrate deuterostomes than in vertebrates , it seems likely that the overall pathway is very much the same in amphioxus and vertebrates and somewhat different in tunicates ( for example , rar does not regulate its own expression in tunicates , but it does in both amphioxus and vertebrates 29 ) . \n raldh and cyp26 genes have been identified in the genomes of both amphioxus ( ftp.ncbi.nih.gov/pub/tracedb/branchiostoma_floridae ) and tunicates ( www.ensembl.org/ciona_intestinalis ) ( fig . \n too little is known about ra signaling in echinoderms , such as sea urchins , for a comparison with chordates 31 - 33 . moreover , for hemichordates , the sister group of echinoderms , it is not known if there is an rar , and roles for ra signaling during hemichordate development have yet to be established . \n even so , bioinformatic analyses of the sea urchin genome sequence ( www.ncbi.nlm.nih.gov/genome/guide/sea_urchin ) and of hemichordate est data ( ftp.ncbi.nih.gov/pub/tracedb/saccoglossus_kowalevskii ) suggest that both raldh and cyp26 genes are present in echinoderms and hemichordates ( fig . \n interestingly , these analyses also revealed a putative rare ( a so - called dr5 element ) about 3770 base pairs upstream ( 5 ' ) of the sea urchin hox1 gene . \n thus , at least some components of the ra signaling cascade were probably already present in the last common ancestor of deuterostomes . \n in addition , these results raise two very important questions about the evolution of the ra signaling network : ( 1 ) if molecular components of the ra pathway are present in non - chordate deuterostomes , what are their functional roles during development ? ( 2 ) if molecular traces of the ra pathway exist in all deuterostomes , when did ra signaling first evolve ? in the last common ancestor of deuterostomes , in the urbilaterian ancestor of deuterostomes and protostomes or even earlier during animal evolution ? \n a first tentative step towards answering the second question was recently taken by an extensive phylogenetic analysis of the nuclear hormone receptor superfamily , which includes the rar and rxr subfamilies 34 . \n the results obtained from this analysis suggest that a proto - rar gene was already present in the genome of the last common ancestor of protostomes and deuterostomes and that in protostomes this rar gene was lost at least in the lineages leading to nematode worms and insects 34 . \n in vertebrates , the roles of ra signaling in anteroposterior patterning of the central nervous system ( cns ) and pharynx have received particular attention 35 , 36 . in the cns , when vertebrate embryos are treated with ra , anterior neural structures ( like the forebrain ) are lost and posterior structures , such as the hindbrain and spinal cord , are expanded . \n in addition , expression of genes in the anterior nervous system ( like otx2 ) is lost and the hox genes , normally expressed in hindbrain and spinal cord , are upregulated and shifted anteriorly 35 . in lampreys , for example , \n conversely , decreasing ra signaling levels has the opposite effect : anterior neural structures , such as the forebrain and midbrain are expanded posteriorly , as is expression of otx2 35 . \n loss of at least two of the three rar genes in mice also leads to mispatterning of the hindbrain , such as defects in segmental ( rhombomeric ) hindbrain organization , which correlate with an induction of hox gene expression at more posterior levels of the hindbrain 38 . \n thus , in vertebrates , ra signaling influences collinear expression of hox genes , which in turn is required to set up an anteroposterior patterning code along the neural tube . not only is anteroposterior patterning of the cns affected by ra signaling , but , since hindbrain neural crest carries the hox code of the cns with it when it migrates , the hox code carried by neural crest migrating into the branchial arches is also altered when levels of ra signaling are changed . in mammals , \n excess ra causes fusion of the first two pharyngeal arches while in ra - deficient mice and rats , pharyngeal structures caudal to the first pharyngeal arch are absent 39 - 42 . \n similarly in vitamin a - deficient quail , the pharynx is extended caudally , the first pouch forms normally , the second one is abnormal and the third and fourth pharyngeal pouches never form 43 . in lampreys , \n the effects of excess ra are even more severe , with complete deletion of anterior pharyngeal structures 44 . \n thus , ra signaling in the pharynx specifies the anteroposterior position of pharyngeal structures 36 , 45 , 46 . \n it was initially believed that the effects of exogenous ra on the vertebrate pharynx were solely due to a mispatterning of neural crest cells migrating into the branchial arches 36 , 45 , 46 . however \n , this influence of neural crest cells is subordinate to regional patterning within the pharyngeal endoderm itself , since removal of neural crest does not prevent the proper patterning of pharyngeal arches and pouches 47 . \n conversely , in the zebrafish , tbx1 function in the pharyngeal endoderm is required for proper development of neural crest - derived structures in the pharyngeal arches 48 , 49 . \n interestingly , the defects obtained in the tbx1 mutant van gogh in zebrafish are similar to those obtained after loss of both rar and in mice 38 , 48 , 49 suggesting that ra signaling may be upstream of tbx1 in the pharyngeal endoderm . moreover , treatment of developing mice with an rar-specific agonistic ligand has conclusively shown that rar-dependent ra signaling in the endoderm is independent of neural crest cells 50 . \n thus , while neural crest cells migrating into the branchial arches give rise to several structures , such as the pharyngeal arch cartilage , the initial patterning of the pharynx requires ra signaling within the endoderm 36 , 45 , 46 . in the cephalochordate amphioxus \n , rar is expressed in the hindbrain and anterior spinal cord of the developing cns 14 . moreover , in the region of the amphioxus cns strongly expressing rar , hox1 , hox3 and hox4 are collinearly expressed 51 . \n ra strongly upregulates rar expression throughout the cns , whereas an ra antagonist almost completely downregulates rar 14 . \n in addition , ra pushes the anterior limits of hox1 expression anteriorly in the cns 12 , while ra antagonist shifts the hox1 domain posteriorly ( fig . \n these data suggest that in cephalochordates , as in vertebrates , ra signaling , probably acting via hox genes , controls anteroposterior patterning of the developing cns . \n similarly , in the amphioxus general ectoderm , treatments with ra and ra antagonist affect collinear expression of hox genes . \n whereas ra shifts expression of hox genes ( such as hox1 ) anteriorly in the general ectoderm , the ra antagonist completely downregulates ectodermal hox expression ( fig . \n 4b ) 53 . moreover , in amphioxus , which lacks definitive neural crest , exogenous ra shifts the posterior limit of the pharynx anteriorly , while the application of ra antagonist has the opposite effect 12 , 14 . \n the amphioxus pharyngeal endoderm strongly expresses pax1/9 and otx 54 , 55 and treatments with ra or an ra antagonist shift the posterior limit of pax1/9 and otx expression , respectively , anteriorly or posteriorly 52 . \n rar and hox1 are expressed just posterior to pax1/9 and otx in the midgut region of the endoderm suggesting that , as in vertebrates , ra signaling within the endoderm might control anteroposterior pharyngeal patterning in amphioxus 52 . \n in addition , loss of hox1 function mimics the effect of a decrease of ra signaling in the amphioxus endoderm 52 . \n taken together , these results suggest that in the amphioxus endoderm , ra signaling activates hox1 in the midgut and that hox1 in turn mediates the effects of ra signaling by limiting expression of genes that are required for specification of the pharynx ( such as pax1/9 and otx ) to the anterior endoderm 52 . \n a role for ra signaling and hox genes in neural patterning of tunicates is much less obvious than in amphioxus and vertebrates . in the ascidian ciona intestinalis , treatment with ra \n does not affect the expression of rar suggesting that , as mentioned above , rar does not regulate its own expression as it does in other chordate groups 29 . \n in addition , tunicates have lost several hox genes and both cluster organization and collinear expression have been at least partly lost . \n for example , the ascidian ciona intestinalis has lost hox7 , hox8 , hox9 and hox11 24 , while the appendicularian oikopleura dioica has lost hox3 , hox6 , hox7 and hox8 25 . \n expression of some of the remaining hox genes can not be detected at all in the cns and the domains of those that are expressed are only approximately collinear 24 , 25 . \n even so , excess ra affects morphology and even hox gene expression at least in ascidian tunicates . \n for example , treatment of embryos with exogenous ra leads to incomplete closure of the anterior neural tube of ciona intestinalis 29 and expression of the hox1 gene in the cns of the tunicate halocynthia roretzi is shifted anteriorly 11 . \n thus , a role for ra signaling via hox genes in neural patterning of tunicates remains a possibility . \n in contrast to its effects on the cns , the effects of ra on pharyngeal structures in tunicates is more similar to that in amphioxus and vertebrates . as in amphioxus , \n pax1/9 and otx genes are expressed in the tunicate pharynx during gill slit formation 56 , 57 . moreover , in the ascidian tunicate herdmania curvata , ra treatment of juveniles leads to a decrease of otx expression in pharyngeal tissues and to an eventual loss of the pharyngeal basket by respecification of anterior endoderm to a more posterior fate 57 , 58 . taken together , \n these data suggest that , in tunicates , as in amphioxus , ra signaling might play roles in patterning and development of the cns and pharyngeal endoderm . in addition \n , there is evidence that at least some of the genetic machinery mediating ra signaling in amphioxus and vertebrates ( like the hox genes ) might also be important for mediating ra signaling in tunicates . \n thus , embryonic patterning mechanisms mediated by ra signaling and hox genes are present in all chordates . in contrast to chordates , enteropneust hemichordates do not have a centralized nervous system , but instead a diffuse ectodermal nerve net ( i.e. a skin brain ) 59 , 60 . as in the cephalochordate amphioxus , \n hox genes are collinearly expressed in the hemichordate ectoderm 53 , 60 . whether ra signaling is involved in the control of hox gene expression in the ectoderm of hemichordates \n is unknown , but ra does not appear to affect anteroposterior patterning in echinoderms , the sister group of hemichordates 31 . \n these data suggest that collinear expression of hox genes was already used for anteroposterior patterning of ectodermal tissues in the last common ancestor of hemichordates and cephalochordates and that a role for ra signaling in controlling collinear expression of hox genes in the ectoderm evolved very early during chordate evolution . whether this ra - hox hierarchy evolved even earlier , in the last common ancestor of all deuterostomes , remains to be determined . \n thus , very early during chordate evolution or perhaps even very early during deuterostome diversification , ra signaling was co - opted for controlling hox - dependent anteroposterior patterning mechanisms during development . in one scenario , \n the putative deuterostome ancestor had a ventral cns that was patterned by hox genes like other tissue layers , such as the general ectoderm ( fig . \n 5 ) , but the cns was lost in hemichordates 59 . in the second scenario , \n the ancestral deuterostome lacked a cns , and the cns evolved independently from the ectoderm in protostomes and deuterostomes ( fig . \n an ancestral ectodermal patterning mechanism was carried over into the cns at the base of the chordates , leading to the creation of two independent ra - hox control hierarchies , one in general ectoderm and one in neural ectoderm ( i.e. in the cns ) ( fig . \n thus , in invertebrate chordates , ra signaling and hox genes are involved in anteroposterior patterning of the general ectoderm , the cns , the endoderm ( and possibly even of the mesoderm ) . during vertebrate evolution , with the functional elaboration of neural crest cells \n neural crest cells carry patterning information acquired at the dorsal neural tube into other tissue layers ( fig . \n 5 ) . in either case , ra signaling became linked to hox gene function at least at the base of the chordates . \n future research on hemichordates will show whether it evolved even earlier in the deuterostome lineage . \n in sum , ra signaling is a key regulator of anteroposterior patterning orchestrated by hox codes in at least general ectoderm , central nervous system and endoderm of chordates . \n in contrast to the relative conservation of developmental mechanisms within chordates , evidence for roles of ra outside the chordates and the evolutionary origin of ra signaling itself still remain elusive . \n in fact , it is unclear when ra - controlled patterning mechanisms first evolved in the animal kingdom and when the ra signaling cascade was co - opted for regulating hox gene expression . \n future research on the evolution of ra signaling should thus focus on the identification of ra signaling components , such as the ra receptor ( rar ) , in non - chordate deuterostomes ( such as hemichordates and echinoderms ) , protostomes ( such as insects and mollusks ) and maybe even cnidarians . \n furthermore , to fully understand the elaboration of this signaling pathway during evolution , the functional roles of the identified components during development will have to be assessed . \n strongylocentrotus purpuratus rar ( xm_774883 ) , ciona intestinalis rar ( ab210661 ) , branchiostoma floridae rar ( af378827 ) , takifugu rubripes rar1 ( frup00000151563 ) , takifugu rubripes rar2 ( frup00000165122 ) , takifugu rubripes rar ( frup00000079596 ) , takifugu rubripes rar1 ( frup00000158774 ) , takifugu rubripes rar2 ( frup00000143282 ) , homo sapiens rar ( nm_001033603 ) , homo sapiens rar ( nm_000965 ) , homo sapiens rar ( nm_000966 ) . \n echinoderms and hemichordates together establish the sister group of chordates . the urochordates ( = tunicates ) include ascidians and appendicularians . \n the vertebrates include agnathan groups ( hagfish and lampreys ) as well as the gnathostome chondrichthyans ( cartilaginous fish ) , actinopterygians ( ray - finned fish ) and sarcopterygians ( lobe - finned fish and tetrapods ) . within chordates , \n the phylogenetic relationships between cephalochordates and urochordates and between hagfish and lampreys are still disputed and their respective positions within the tree are thus shown as polytomies . \n two important events during deuterostome evolution are the origin of chordates and the origin of vertebrates . the two rounds of extensive gene duplications early during vertebrate diversification are highlighted with green boxes labeled ' r ' . \n deuterostome groups , for which a whole genome sequencing ( wgs ) project has already been finished are indicated with a turquoise + , those , for which a wgs project is underway , are marked with a turquoise ( + ) . \n if known , the exact number of rar , raldh and cyp26 genes in the genome of a specific deuterostome group is indicated , the certain presence of a gene is marked with + and the lack of data is highlighted by ? . \n ra is synthesized by oxidation of retinal by retinaldehyde dehydrogenases ( raldhs ) . in a reversible reaction , retinal \n is synthesized from retinol ( vitamin a ) by either aldehyde dehydrogenases ( adhs ) or short - chain dehydrogenase / reductases ( rdhs / sdrs ) . cellular retinol binding proteins ( crbps ) can bind retinol , whereas cellular retinoic acid binding proteins ( crabps ) can bind ra . \n b ) the rar / rxr heterodimer mediates the effects of ra . in the absence of ligand ( ra ) \n binding of the ligand ( ra ) induces conformational changes and the binding of co - activators leading to histone acetylation and activation of transcription . \n the tree shows the phylogenetic relationships between rars from the sea urchin strongylocentrotus purpuratus , the ascidian tunicate ciona intestinalis , the cephalochordate branchiostoma floridae , pufferfish ( takifugu rubripes ) and humans ( homo sapiens ) . \n the rar sequences were added to an alignment of nuclear hormone receptors 34 and conserved sites ( 335 positions ) were subsequently selected for phylogenetic reconstruction using phyml ( wag + 8 + i ) 61 . \n 100 bootstrap replicates were carried out to determine the robustness of the obtained phylogenetic tree . in the tree , \n the rar subfamily as a whole is strongly supported ( bootstrap support 95% ) and within the rars the sea urchin sequence is at the base ( with a moderate support of 70% ) . \n the respective branching of the cephalochordate and tunicate rars is not resolved ( bootstrap support of 49% ) . \n nonetheless , the invertebrate chordate sequences are positioned between the sea urchin rar and the duplicated rars of vertebrates , which form a single clade that is very strongly supported ( 100% ) . \n this analysis shows that a rar gene is present in the genome of echinoderms and suggests that , despite the lack of data from hemichordates , the presence of rar is an ancestral character of deuterostomes . \n ra signaling controls hox1 expression in central nervous system ( cns ) and general ectoderm of developing amphioxus . \n a ) the rostral limit of hox1 expression in the cns ( arrowheads ) is shifted , respectively , anteriorly and posteriorly by 1x10 m ra and 1x10 m ra antagonist ( bms009 ) . \n b ) in the general ectoderm , the rostral limit of hox1 ( arrowheads ) is shifted anteriorly by 1x10 m ra , whereas treatment with 1x10 m ra antagonist ( bms009 ) downregulates hox1 expression . \n evolution of ra- and hox - dependent patterning mechanisms in deuterostomes . in scenario 1 , \n the putative deuterostome ancestor had a central nervous system ( cns ) ( located ventrally ) and both cns and general ectoderm were patterned by hox genes , while a role for ra signaling remains elusive . in this scenario , chordates evolved by dorso - ventral axis inversion and the cns was secondarily lost in the hemichordate lineage . in scenario 2 , \n hox - dependent patterning codes were present in the ectoderm , but again a role for ra signaling remains elusive . \n early during chordate evolution , condensation of a central nervous system ( cns ) dorsally led to the creation of two ra - hox patterning hierarchies one in general ectoderm and one in neural ectoderm ( i.e. in the cns ) . in the vertebrate lineage , \n neural crest function was elaborated and neural crest cells contribute to patterning and development of the embryo by carrying positional information from the cns into other tissues , for example in the pharyngeal region .", "role": "user" }, { "content": "in chordates , which comprise urochordates , cephalochordates and vertebrates , the vitamin a - derived morphogen retinoic acid ( ra ) has a pivotal role during development . altering levels of endogenous \n ra signaling during early embryology leads to severe malformations , mainly due to incorrect positional codes specifying the embryonic anteroposterior body axis . in this review \n , we present our current understanding of the ra signaling pathway and its roles during chordate development . \n in particular , we focus on the conserved roles of ra and its downstream mediators , the hox genes , in conveying positional patterning information to different embryonic tissues , such as the endoderm and the central nervous system . \n we find that some of the control mechanisms governing ra - mediated patterning are well conserved between vertebrates and invertebrate chordates , such as the cephalochordate amphioxus . \n in contrast , outside the chordates , evidence for roles of ra signaling is scarce and the evolutionary origin of the ra pathway itself thus remains elusive . in sum , to fully understand the evolutionary history of the ra pathway , future research should focus on identification and study of components of the ra signaling cascade in non - chordate deuterostomes ( such as hemichordates and echinoderms ) and other invertebrates , such as insects , mollusks and cnidarians .", "role": "assistant" } ]

[ { "content": "You are a medical writer. Summarize the following article: the basic function of a tracheal tube is to provide a reliable connection between the patient 's lung and the bag or ventilator . \n ideally , this connection should be leak - proof without causing undue pressure to laryngeal or tracheal structures . \n traditional teaching was that in children under 8 - 10 years of age , this sealing should be obtained with an uncuffed tracheal tube . \n disadvantages of uncuffed tubes include air leak , difficulties in effectively ventilating patients with lung disease , environmental contamination of anaesthetic gases , risk of aspiration , and difficulty in accurate measurement of end - tidal co2 concentration . \n cuffed endotracheal tubes may be useful in special situations like laparoscopy and in surgical conditions at risk of aspiration . \n intubation depth marks and short , cylindrical cuff near tracheal tube tip allow adequate placement with a cuff - free subglottic zone , without the risk of endobronchial intubation . \n we studied the appropriateness of microcuff endotracheal tube size selection , efficacy of ventilation and monitoring and post - operative complications in children undergoing laparoscopic surgery . \n after institutional ethical committee approval and written informed consent from the parents , a prospective observational study was conducted . \n the study population included 100 patients of american society of anesthesiologists i and ii physical status , aged between 8 months and 10 years , posted for elective laparoscopic surgery . \n patients with known airway anomalies , known or suspected difficult intubation and planned post - operative ventilation in the intensive care unit were excluded from the study . all patients received injection glycopyrrolate 0.004 mg / kg , injection ketamine 0.5 mg / kg , injection midazolam 0.05 mg / kg , all intravenous ( iv ) as premedication . \n standard monitoring included electrocardiogram , pulse oximetry , non - invasive blood pressure , capnography and temperature . \n anaesthesia was induced with injection fentanyl 2 g / kg , injection propofol 2.5 mg / kg . \n air leak pressure after intubation was tested with the patient supine and head in a neutral position . in the absence of air leak at 20 cm h2o inflation pressure , \n the tube would be judged to be too large and replaced with the next smaller size ( 0.5 mm i d ) . \n tracheal tube with excessive air leak , not allowing adequate ventilation would be exchanged to the next larger size ( + 0.5 mm i d ) . \n cuff pressure was monitored continuously and limited to 20 cm h2o with a pressure release valve . \n minimal sealing pressure was assessed under steady - state ventilation conditions and maintained during the procedure . \n this was performed by reducing the cuff pressure until an audible leak appeared at the patient 's mouth and then pressure was increased until leak disappeared . \n anaesthesia was maintained with o2 : air ( 50:50% ) and isoflurane , using circle absorber with a mean flow of 1 l / min . \n patients were extubated after reversal with injection glycopyrrolate 0.008 mg / kg and injection neostigmine 0.05 mg / kg . \n parameters studied were size of microcuff tube used , tube passage characteristics , sealing pressure , capnography characteristics , effectiveness of ventilation , changes in cuff pressure during surgery , complications if any . \n coughing , laryngospasm or stridor at extubation was recorded . any change in voice or cry pitch was also noted . \n we studied 100 patients with a mean age of 5.44 years ( range 8 months 5 days9 years 11 months ) [ table 1 ] . \n leak on intermittent positive pressure ventilation ( ippv ) at airway pressure 20 cm h2o was present in all patients . \n mean sealing pressure was 11.72 ( 1.9 standard deviation [ sd ] ) cm h2o . with the creation of pneumoperitoneum , \n mean intracuff pressure increased to 12.48 ( 3.12 sd ) cm h2o [ table 3 ] . \n further , with head low positioning , mean cuff pressure recorded was 13.32 ( 2.92 sd ) . \n ventilation at low flow ( mean flow1 l / min ) , plateau - type capnography was noted in all patients . \n coughing at extubation was seen in 6 patients , which responded to gentle nasopharyngeal suctioning . \n partial laryngospasm occurred in 4 patients , which responded to continuous positive airway pressure ( cpap ) via face mask . \n age of study population , size of microcuff tube used and number of patients in each group type of laparoscopic surgery intracuff pressure variations \n cuffed tracheal tubes in smaller children are increasingly used because of the high probability of inserting a correctly sized tracheal tube at the first intubation attempt and to create an effective airway seal without the use of an oversized tracheal tube . \n the cuff of the microcuff tracheal tube differs from the conventional cuff in that it is made from ultra - thin ( 10 m ) polyurethane foil instead of the much thicker ( 5070 m ) polyvinyl chloride or polyethylene foils . \n the murphy eye has been eliminated , which has allowed the cuff to be moved more distally on the tracheal tube shaft . \n the cuff is short and when inflated , it expands below the sub - glottis , providing a seal with cuff pressure < 15 cm h2o . \n the airway is sealed at upper trachea where the posterior membranous wall can stretch and produce a complete seal , rather than at the cricoid level . \n thus , the problem that the cuff will cause airway mucosal injury , leading to sub - glottic stenosis is circumvented . \n creation of pneumoperitoneum and trendelenberg positioning during laparoscopy reduces pulmonary compliance , which may lead to ineffective ventilation or excessive peritubal air leak with an uncuffed tracheal tube . \n caudal displacement of the tracheal tube may be considerable during laparoscopy with pneumoperitoneum , especially with additional head - down tilt , increasing the risk of endobronchial migration of the tracheal tube . \n advantages of the microcuff endotracheal tube in this population , therefore , would be constant minute ventilation , precise respiratory monitoring and capnography , low fresh gas flow and lesser risk of pulmonary aspiration . \n selection of an appropriately - sized cuffed endotracheal tube is important to prevent airway mucosal injury . \n we were able to intubate the trachea without resistance in all patients , confirming the appropriateness of the size selection recommendations . \n air leak was observed with ippv at airway pressure 20 cm h2o in all patients . \n in a randomised controlled multicentre study of more than 2000 patients , weiss et al . concluded that microcuff tracheal tubes can be safely used in children if cuff pressure is controlled and size selection criteria followed . \n the difference in sealing pressure in our study could be due to the difference in age range or simply due to a difference in population characteristics . \n though the upper limit of safety for cuff pressure in adults is 2530 cm h2o , there is no data in children regarding perfusion pressures of the tracheal mucous membrane , and it is believed that a lower cuff pressure would possibly be safe . for the purpose of our study , effective ventilation meant a square wave capnogram , with etco2 values in the clinically acceptable range of 3545 mm hg and ability to ventilate with low flow ( < 2 l / min ) . \n pneumoperitoneum and head low positioning caused an increase in intracuff pressure ( mean increase 12.48 [ 3.12 sd ] cm h2o and 13.32 [ 2.92 sd ] respectively ) , but the pressure was always < 20 mm hg . in our study , \n creation of pneumoperitoneum and trendelenburg position did not affect ventilation or the effectiveness of the seal . \n the intra - abdominal pressures were limited to 10 mm hg in patients < 4 years and 12 mm hg in older patients . despite the head low positioning , we did not observe endobronchial intubation in any of the patients confirming the appropriateness of the depth marker . \n confirmation of appropriate tube position in our study was determined clinically , by auscultation in all patients as we did not have a paediatric flexible bronchoscope at our disposal . \n several studies have confirmed the safety of the cuffed endotracheal tube with respect to airway complications , as long as an appropriate size is chosen , and the cuff pressure is monitored . in a study of 204 patients , duracher et al . reported 6 cases of complications ( dysphonia , hoarse cough , and laryngeal dyspnea ) , and attributed those to the use of an incorrectly predicted larger tube size . \n incidence of stridor with microcuff tube in study by weiss et al . was 4.4% . in our study , at extubation \n partial laryngospasm was seen in 4 patients who were treated with cpap using a jackson - rees circuit , and humidified oxygen . \n history of recent upper respiratory tract infection ( within 4 weeks ) was present in 5 of these patients , which could have been a contributing factor for laryngospasm . \n our study confirms the appropriateness of size selection recommendations of microcuff tube in our patient population . \n advantages include better airway seal , economy of fresh gas flows , and effective ventilation .", "role": "user" }, { "content": "background and aims : traditionally , uncuffed endotracheal tubes have been used in children . \n cuffed tubes may be useful in special situations like laparoscopy . \n microcuff endotracheal tube is a specifically designed cuffed endotracheal tube for the paediatric airway . \n we studied the appropriateness of microcuff tube size selection , efficacy of ventilation , and complications , in children undergoing laparoscopy.methods:in a prospective , observational study , 100 children undergoing elective laparoscopy were intubated with microcuff tube as per recommended size . \n we studied appropriateness of size selection , sealing pressure , ability to ventilate with low flow , quality of capnography and post - extubation laryngospasm or stridor.results:mean age of the patients was 5.44 years ( range 8 months 5 days9 years 11 months ) . \n there was no resistance for tube passage during intubation in any patient . \n leak on intermittent positive pressure ventilation at airway pressure 20 cm h2o was present in all patients . \n mean sealing pressure was 11.72 ( 1.9 standard deviation [ sd ] ) cm h2 o. with the creation of pnemoperitoneum , mean intracuff pressure increased to 12.48 ( 3.12 sd ) cm h2 o. with head low positioning , mean cuff pressure recorded was 13.32 ( 2.92 sd ) . \n ventilation at low flow ( mean flow 1 l / min ) , plateau - type capnography was noted in all patients . \n mean duration of intubation was 83.50 min . \n coughing at extubation occurred in 6 patients . \n partial laryngospasm occurred in 4 patients , which responded to continuous positive airway pressure via face mask . \n severe laryngospasm or stridor was not seen in any patient.conclusion : microcuff tubes can be safely used in children if size selection recommendations are followed and cuff pressure is strictly monitored . \n advantages are better airway seal and effective ventilation , permitting use of low flows .", "role": "assistant" } ]

[ { "content": "You are a medical writer. Summarize the following article: the frontal bone is a singular , median , and symmetrical bone that occupies the most anterior part of the cranium . \n it has joints with the parietal , ethmoid , sphenoid , nasal , zygomatic , lacrimal , and maxillary bones , thereby contributing towards uniting the neurocranium and the viscerocranium . during the development stage , \n the frontal bone is a double bone , with right and left halves that grow together and unite along the median line at the metopic suture . \n this usually starts to undergo the fusion process at the age of two years and may have completely fused by the age of six years [ 2 , 5 ] , eight years [ 4 , 6 ] , or ten years . \n however , in approximately 8% of adults , the two halves of the frontal bone do not fuse , and the metopic suture persists . \n the suture may be incomplete or complete ( when it extends from the nasium to the bregma ) , and this condition is known as metopism [ 79 ] . \n this name originates from the greek word metopion , which means a space between the eyebrows . \n metopism has various degrees of incidence : from 7%-8% among europeans to 1% in africans and 4 - 5% in mongolians . \n overall , the range of incidence can go from 1% to 12% , and it is slightly more prevalent among males . \n the incidence of metopism and the difference in forms also vary according to geographical region . in lebanon \n it was 0.82% , in nigeria 3.14% , in india 2.66% , in southern india 6.25% , and in europe 7 to 10% [ 12 , 17 ] . prevalence of metopism is important , because , if not , this can be confused with a fracture of the frontal bone or even with a sagittal suture on radiological images [ 13 , 18 ] . \n our objective was to determine the rate of occurrence of metopism in dry crania of adult humans . \n 134 dry crania from adult humans ( 95 from males and 39 from females ) were analyzed . \n all of them belonged to anatomy laboratories in higher education institutions in macei , al , and had been obtained in accordance with law 8501 of november 30 , 1992 , which deals with use of unclaimed cadavers for teaching and research purposes . \n all the crania were macroscopically examined with regard to the presence or absence of the metopic suture . \n it was considered to be complete when it continued uninterruptedly from the nasium to the bregma and incomplete when it was not present over this entire length . \n the crania were also classified according to the cephalic index ( ci ) as follows : brachycephalic ( ci > 80 ) , dolichocephalic ( ci < 80 ) , or mesocephalic ( ci = 80 ) . \n this index was calculated by dividing the maximum width , that is , the distance between the euryons ( the widest cross - sectional distance from side to side ) , by the maximum length , that is , the distance between the glabella and the opisthocranion ( the glabella is located on the median plane , between the eyebrow arches , and the opisthocranion is the posterior point which is furthest from the longitudinal plane ) , multiplied by 100 . \n the measurements of the maximum length and width of the cranium were made using willis compasses . \n out of the 134 crania examined , the metopic suture was found in 9.7% ( 13/134 ) of the cases , of which 61.5% ( 8/13 ) were in males and 38.5% ( 5/13 ) in females . \n a complete metopic suture ( figure 1 ) was found in six cases ( 46.2% ) , while an incomplete suture ( figure 2 ) was observed in seven cases ( 53.8% ) ( table 1 ) . according to the anthropometric characteristics of the crania studied by means of the cephalic index , 47.76% ( 64 ) were of brachycephalic type , 44.03% ( 59 ) \n however , when we classified the types of cranium according to whether a metopic suture was present , 7.46% ( 10 ) were dolichocephalic , 1.49% ( 2 ) were brachycephalic , and 0.74% ( 1 ) were mesocephalic . \n there are divergences in the scientific literature in relation to the exact timing of closure of the metopic suture . \n fusion has been reported as occurring between the first year and the beginning of the second year of life , before the sixth year , in the eighth year [ 4 , 6 ] , or in the tenth year . according to sant'ana castilho et al . \n , there are several causes of failure of the metopic suture to fuse , but genetic influence is currently the factor most accepted by the scientific community . in the present study , metopism \n was found in 4.48% of the crania , which was similar to what was found in indian populations ( punjab , uttar pradesh , and southern india ) , mongolians , east asians , and nigerians ( 3.31 to 5.1% ) but was lower than what was found among scots , mongoloids , and europeans ( 7% to 10% ) ( table 2 ) . \n a study conducted in brazil by del sol et al . on 400 crania at the so paulo medical school ( escola paulista de medicina ) , federal university of so paulo , reported that the incidence of metopism was 2.75% . \n however , in an analysis on 71 crania at paranaense university , sant'ana castilho et al . found an incidence of 7% . in the present study , out of the 134 crania found in anatomy laboratories in higher education institutions in macei , al , the rate of occurrence of metopism was 4.48% and suture metopic 9.7% . \n this rate was higher than that found by either of the previous brazilian studies . among the 13 crania in which a metopic suture was seen to be present , \n the rate of occurrence was greater among males ( 61.54% ) , which corroborates the findings from studies by skrzat et al . \n , murlimanju et al . , and van der meulen , in which there was also greater occurrence among males . \n however , this was contrary to the findings of sant'ana castilho et al . , who observed that 80% of the occurrences of metopic suture were in females . according to baaten et al . \n however , when we correlated occurrences of metopic suture with the type of cranium , according to the cephalic index , we found that the occurrences in the dolichocephalic type were much greater than in the brachycephalic type . \n this differed from the findings of sant'ana castilho et al . , who reported greater occurrence in the brachycephalic type . \n on the other hand , according to bryce , what exists in reality are equal occurrences between the dolichocephalic and brachycephalic cranial types . \n the rate of occurrence of metopism in dry crania in the city of macei was 4.48% , without predominance in one sex over the other . \n incomplete metopic sutures were observed in 5.22% of the crania , with slight predominance among males . \n knowledge of the possible presence of metopism serves to alert imaging professionals to the fact that this is just a variation and does not imply any harm to the population 's health . \n moreover , this variation should not be confused with a line of fractures in the frontal bone , especially if observed close to the median line .", "role": "user" }, { "content": "the metopic suture is located between the tubercles of the frontal bone . \n there are divergences regarding the exact time at which it closes , which ranges from the first to the tenth year of life , although it may persist into adulthood . \n this study was conducted on 134 dry crania from adult brazilians , of which 95 were male and 39 were female . \n these were available in the anatomy laboratories of higher education institutions in macei , al , brazil . \n all the crania were examined macroscopically with regard to the presence ( metopism : m ) on absence of the metopic suture . \n m was considered to be complete ( mc ) when it continued uninterruptedly from the nasium to the bregma and incomplete ( mi ) when it was not present over its entire length . \n it was observed that mc was present in 4.48% ( 6/134 ) of the skull examined , of which 50% ( 3/134 ) were male and 50% ( 3/134 ) were female . \n an incomplete metopic suture was found in 5.22% ( 7/144 ) of the crania and more frequently among males ( 3.73% ; 5/134 ) . among the crania with a metopic suture , the dolichocephalic type predominated ( 7.46% ) in relation to brachycephalic crania ( 1.49% ) and mesocephalic \n crania ( 0.74% ) . \n there was no predominance of metopism between the sexes , while an incomplete metopic suture was slightly more common among males .", "role": "assistant" } ]

[ { "content": "You are a medical writer. Summarize the following article: jejunal diverticular disease is a rare clinical entity with an incidence of between 0.06 and 1.5% . \n the true incidence however may be higher as the majority of jejunal diverticula are asymptomatic , and thereby remain undiagnosed . in symptomatic cases , \n non - specific epigastric pain and bloating are the most common complaints [ 2 , 3 ] . \n however , life - threatening complications such as gastrointestinal ( gi ) bleeding , bowel perforation and obstruction have been reported in up to 18% of cases . \n it is therefore vital that the diagnosis of the condition can be made promptly to ensure optimal and timely management for the patient . \n we present a case of a 78-year - old male with massive gi bleeding secondary to jejunal diverticular disease who was surgically managed with good post - operative outcome . \n a 78-year - old male with a history of ischaemic heart disease , stage iv chronic kidney disease and chronic obstructive pulmonary disease , presented to the emergency department after a sudden collapse at home . \n whilst in the department , he experienced a further syncopal event with two episodes of large - volume melaena . \n he was pale , clammy and haemodynamically unstable with a systolic blood pressure of 75 mmhg . \n his blood results showed a haemoglobin ( hb ) of 66 g / l and a urea of 32.7 mmol / l . \n he was initially resuscitated with intravenous fluids , red blood cells ( rbcs ) , platelets , fresh frozen plasma ( ffp ) and cryoprecipitate . \n the patient proceeded to urgent upper gi endoscopy ( ogd ) but the procedure failed to identify any evidence of recent or active bleeding . \n colonoscopy showed large amounts of altered blood in the colon as well as the presence of sigmoid diverticula but without evidence of active bleeding . \n segmental ct - angiogram was contraindicated given patient 's poor renal function . despite vasopressor support and a proton - pump - inhibitor infusion , with on - going melaena , \n the patient 's hb and blood pressure remained low , requiring 19 units of rbcs in the first 48 h of admission . \n over the subsequent 36 h , the patient 's condition stabilized with an hb consistently over 90 g / l . \n however , on day 4 of admission , he produced five further episodes of melaena , and became acutely tachycardic and hypotensive . \n the decision was then made to proceed to exploratory laparotomy with a pre - operative plan of performing a subtotal colectomy . \n the presumptive diagnosis was that the bleeding was likely secondary to his known colonic diverticulosis . \n intra - operatively however , we found multiple diverticula clustered along the proximal and mid jejunum at the mesenteric edge . \n partially digested blood was seen in the portion of small bowel distal to these diverticula . \n we performed a small bowel resection of 80 cm length of diseased jejunum and an end - to - end anastomosis ( figs 1 and 2 ) . \n histology confirmed numerous true diverticula , with no evidence of malignancy . in total , since admission , the patient received 23 units of rbcs , 16 units of ffp , 4 units of platelets and 3 units of cryoprecipitate . \n note that the diverticula arise at the mesenteric border and are clustered around proximal jejunum . \n figure 2:intra - operative photography demonstrating jejunal diveritcula proximally and presence of altered blood in the small bowel distal to these diverticula . \n note that the diverticula arise at the mesenteric border and are clustered around proximal jejunum . \n intra - operative photography demonstrating jejunal diveritcula proximally and presence of altered blood in the small bowel distal to these diverticula . \n the patient was discharged on day 7 post - operation and followed - up in outpatients clinic 2 weeks later . \n there were no further reports of bleeding and he was discharged from surgical follow - up . \n jejunal diverticular disease is an uncommon condition that is usually asymptomatic but may rarely present with life - threatening complications including massive gi bleeding [ 2 , 3 ] . \n other complications such as chronic malabsorption , volvulus , diverticulitis with or without perforation or abscess occur in 1030% of patients [ 1 , 5 ] . \n the majority of patients who present with gi bleeding do not display previous gi symptoms and are reported to develop an acute onset haemorrhage per rectum . \n jejunal diverticulosis is often difficult to locate endoscopically and diagnosing jejunal diverticular bleeding remains problematic , with current imaging techniques continuing to be unreliable . whilst jejunal diverticulosis can be identified by abdominal ct and barium follow - through studies \n , enteroclysis remains the investigation of choice . although some reports demonstrate success with capsule endoscopy and double balloon endoscopy [ 9 , 10 ] such investigations are of limited use in emergency settings . in cases of jejunal diverticular bleeding , selective mesenteric angiography or ct angiogram \n can be used to localize active bleeding but may be contraindicated in patients ' with severe kidney impairment as with our patient . \n urgent laparotomy is indicated in the presence of acute complications of jejunal diverticula including diverticulitis , massive bleeding or bowel perforation both as a diagnostic and therapeutic measure [ 3 , 6 , 7 ] . \n several successful cases with complete small bowel resection with primary entero - entero anastomosis have been reported . \n due to low incidence , low clinical index of suspicion and unreliable diagnostic imaging in emergency situations , diagnosis of jejunal diverticular disease is challenging and therefore often delayed several days after initial presentation . \n this has major implications with regard to prompt and timely management , and can lead to significantly increased morbidity and mortality . \n whilst there are undoubtedly more common causes of gi bleeding , this case demonstrates that jejunal diverticular disease should remain on the differential diagnosis and investigations to confirm the diagnosis should be considered . \n however , despite investigations , the diagnosis may remain elusive and in patients with on - going bleeding , laparotomy and surgical resection is currently the treatment of choice [ 3 , 6 , 7 ] .", "role": "user" }, { "content": "jejunal diverticula are rare and the condition remains mostly asymptomatic . \n however , they can present with vague chronic abdominal symptoms and , in some cases , acute life - threatening complications , such as gastrointestinal ( gi ) bleeding , bowel obstruction and perforation . \n we present a case of an adult male who presented with life - threatening gi bleeding secondary to jejunal diverticular disease . \n whilst there are undoubtedly more common causes of gi bleeding , this case demonstrates that jejunal diverticular disease should remain on the differential diagnosis and investigations to confirm the diagnosis should be considered . however , despite investigations , the diagnosis may remain elusive and in patients with on - going bleeding , laparotomy and surgical resection is currently the treatment of choice .", "role": "assistant" } ]