This review presents a comprehensive overview on selected synthetic routes towards commercial drug compounds as published in both journal and patent literature. Owing to the vast number of potential structures, we have concentrated only on those drugs containing five-membered heterocycles and focused principally on the assembly of the heterocyclic core. In order to target the most representative chemical entities the examples discussed have been selected from the top 200 best selling drugs of recent years. Over the past few decades organic chemistry has seen tremendous progress and this has enabled the synthetic chemist to assemble virtually any molecular structure imaginable given reasonable time and sufficient resources. A steady increase in architectural complexity and the incorporation of more diverse molecular functionality has been a notable feature of pharmaceutical research and development. This general trend has emerged as a consequence of the better understanding of the genome and has resulted in many highly specific therapeutic targets being elucidated.However, even today it might be argued that because of the perceived simpler structures of drug molecules when compared to complex natural products, only a limited repertoire of synthetic transformations are utilised for their construction. Furthermore, many of the modern pioneering developments in organic synthesis including new highly selective and mild bond forming reactions such as metathesis and C–H activation, asymmetric transformations as well as polymer- and technology-assisted syntheses are underused.In order to evaluate the validity of this hypothesis we decided to investigate the syntheses of the best-selling pharmaceutical substances focusing not only on the type of transformations involved but more importantly on the way the heterocyclic components were assembled. Aromatic and non-aromatic heterocyclic rings are a predominant architectural constant of pharmaceuticals and allow for variable interactions with the biological target which are not possible using simpler carbocyclic motifs. Based on our observations, we will moreover be able to evaluate the degree these privileged structures utilise innovative and challenging synthetic strategies. In addition, it will be possible to establish which reactions are most frequently employed and which ones are surprisingly rare or notably absent. Furthermore, from this study it will be possible to judge whether novel methods and transformations developed within the academic community are commonly applied in the later stages of drug research.In order to illustrate the diversity of synthetic methods used by the pharmaceutical industry to generate heterocycle containing molecules we decided in the first part of this review to focus mainly on five-membered aromatic heterocycles represented within the top 200 best selling drugs –2. This 1, Lipitor; Pyrrole, a five-membered nitrogen containing heterocycle is present in some of the most common biologically important molecules, i.e., chlorophyll and haem. However, while the pyrrole ring is not widely represented in pharmaceutical compounds, the core structure of atorvastatin , this method proved to be less successful for more complex pyrroles of which atorvastatin was an example  and an in situ generated mesoionic species 21 furnished the desired product 23 regiospecifically [By modification of the coupling partners a more defined dipolar [3 + 2] cycloaddition between ifically   and an advanced Michael acceptor obtained from an initial Knoevenagel condensation  is Pfizer’s novel receptor tyrosine kinase inhibitor (RTK) approved in 2006 by the FDA for the treatment of both renal cell carcinoma and gastrointestinal stromal tumors. This drug has been suggested as a second-line therapy for patients developing mutation-related resistance to other cancer medications such as imatinib (Gleevec). Sunitinib binds to the inactivated, auto-inhibited conformation of the KIT kinase by partially occupying the space normally filled by the ADP’s adenine in the phosphorylated protein. The indolinone section is located in a deeper pocket with the heteroatoms being involved in H-bonding glutamate and tyrosine residues whilst the pyrrole ring and the diethylaminoethyl appendage are exposed to the solvent environment .–43.42–43153, Lodine) is a racemic non-steroidal anti-inflammatory tetrahydrocarbazole derivative used to treat inflammation and prescribed for general pain relief. Two general routes have been described for the preparation of this drug. In the first route the hydrazine 149 is prepared via the reduction of diazonium salt 148 with tin(II) chloride and subjected to a Fischer indole reaction with aldehyde 150. The resulting indole is then condensed with ethyl 3-oxopropanoate followed by saponification of the ester to yield etodolac -(+)-enantiomer. The resolution of the racemate can be readily accomplished using cinchonine or the glucose derivative N-methyl-D-glucamine. The latter provides the pure (S)-(+)-enantiomer in 25.3% yield (>96% de) after two crystallisations. In addition, the racemisation of the undesired enantiomer via methyl ester formation and treatment with sodium hydroxide was found to be feasible allowing efficient recycling of this material . Th. Th119),5% yield . Also, a0% yield .H-carbazol-4-one (140) by the reaction of phenylhydrazine (138) with cyclohexan-1,3-dione , omeprazole , lansoprazole , pantoprazole  and rabeprazole  are proton pump inhibitors. The common feature in their synthesis is the double condensation of a 1,2-diaminobenzene with potassium ethylxanthate (196) ..257) (26269 with triazine (270)   80]..269 with271 which then immediately reacts with a second molecule of triazine to yield the 1,2,4-triazole . In. In76) hnt (274) .275, Januvia) is a recently developed oral anti-diabetic drug which belongs to the dipeptyl peptidase DPP-IV inhibitor class. The inhibition of DPP-IV leads to increased incretin levels and the inhibition of glucagon release. Consequently, insulin secretion increases leading to decreased blood glucose levels in diabetes type II patients. Structurally, this novel antihyperglycaemic consists of a fluorinated β-amino acid which is coupled to a trifluoromethylated triazolopiperazine  and excess hydrazine. Subsequent treatment of the substituted intermediate with trifluoroacetic anhydride furnished the corresponding bis-hydrazide 278 which underwent cyclisation at elevated temperatures in the presence of polyphosphoric acid. Finally, the partial hydrogenation of triazolopyrazine derivative 279 with palladium on carbon gave the core triazolopiperazine 280. This was then coupled with carboxylic acid unit 281 under standard peptide bond forming conditions and subjected to palladium catalysed debenzylation to liberate the free amine functionality of sitagliptin (275) (In the discovery chemistry route  the hetein (275) .283 was then subjected to cyclodehydration using phosphoryl chloride to give a chloromethyl oxadiazole derivative 284. In a cleverly staged transformation, this compound was treated with diaminoethane to yield the piperazine ring 285, which, on heating under reflux in methanol, undergoes a further condensation with the attached hydrazide to furnish the desired triazolopiperazine ring 280 directly.An inherent problem with this synthesis was the necessity for excess hydrazine in the first step of the sequence in addition to a requirement for an expensive and only moderately efficient palladium reduction in the penultimate step. Furthermore, the chloropyrazine starting material also proved to be unstable under a series of reaction conditions and gave rise to numerous by-products. An improved route was developed for the compounds large scale manufacture. The refined process route  starts w286, Celsentri). HIV uses a member of the G-protein coupled receptor family called CCR-5 as an anchor to attach itself to white blood cells such as T-cells and macrophages followed by viral fusion and entry into white blood cells. Maraviroc blocks this pathway by acting as an antagonist for the CCR-5 receptor hence disrupting HIV life cycle. The structural features of this molecule are a geminal difluorocyclohexyl carboxamide which is linked to a β-aminoacid, and a tropinone-type unit bound to a 1,2,4-triazole ring. Relatively simple and straightforward chemical transformations are used to assemble the main fragments of maraviroc such as amide bond formation and reductive amination (N-acylation of the tropinone fragment 287 with 2-methylpropanoyl chloride (288). The resulting amide 289 is then converted to the corresponding imidoyl chloride 290 using phosphorous pentachloride in dichloromethane (which proved to be superior to phosphoryl chloride) followed by condensation with acetic hydrazide (291). It was found that the dryness of the acetic hydrazide was crucial in order to minimise the hydrolysis of the starting amide 289.A novel and very promising HIV treatment is Pfizer’s maraviroc  which contains a 1,2,4-triazole fused to the benzodiazepine core. The synthesis of this molecule .,94.157) 325, Pletal) which is a selective PDE3 phosphodiesterase inhibitor used as a platelet aggregation inhibitor. The tetrazole ring 327 is prepared via the reaction of an in situ generated imidoyl chloride and hydrazoic acid delivered as a 10% solution in benzene isothiazol-3(2H)-one (369), a saccharin derivative ..382, Mac385, Cordarone) is an antiarrhythmic drug containing a benzofuran ring system. It is one of the most effective antiarrhytmic drugs. Although it is considered a class III antiarrhytmic with its mode of action being principally the blocking of potassium channels, it is anticipated that it is also capable of targeting additional sodium and calcium channels. This might explain its general effectiveness, but could also account for its potentially dangerous side effects. The basic benzofuran framework is commonly prepared by alkylation of salicylic aldehyde (386) with chloroacetic acid (387) to give the dihydrobenzofuran carboxylic acid which after intramolecular condensation readily decarboxylates  and olanzapine (399 Zyprexa). The former, which is a benzo[b]thiophene, is widely used as an oral selective estrogen receptor modulator displaying estrogenic actions on bone (prevention of osteoporosis) and anti-estrogenic actions on breast and uterus, especially in the postmenopausal women [b]thiophene core 396 was accomplished by condensing 3-mercaptoanisole (393) with 4-methoxyphenacyl bromide (394) firstly under basic conditions to affect the SN2 displacement followed by dehydration with polyphosphoric acid at elevated temperatures  which was first introduced to the market by Eli Lilly in 1996. The thiophene unit is synthesised by a multi-component reaction between malononitrile, elemental sulfur and propionaldehyde (400) in the presence of triethylamine. This Gewald-type thiophene synthesis is thought to proceed via an initial Knoevenagel reaction (400→402) followed by the addition of the sulfur into the nitrile to form thiocyanate 403 which then ring closes to the aminothiophene product 405. This species is then employed in a nucleophilic aromatic substitution with 2-fluoronitrobenzene (406) to give the coupled product 407. Reduction of the nitro functionality produces the corresponding aniline that readily undergoes ring closure to furnish ultimately the thienobenzodiazepine 408. Substitution of the pendant amine group with excess N-methylpiperazine (409) then affords olanzapine in modest overall yield  whi410) to give the alternative compound 411 , a milder palladium catalysed hydrogenation was used. Finally, a one pot intramolecular amide formation followed by a TiCl4-mediated reaction to introduce the amidine function yields olanzapine  to 414, Cymbalta), tiotropium  and Cosopt [dorzolamide (416)  for tiotropium [419) for dorzolamide [Three additional thiophene-containing drugs are duloxetine (de (416)  and its de (416) ]. Howeveloxetine , or a Grotropium  and thiozolamide .420, Plavix) which is a chiral tetrahydrothienopyridine derivative (421) with a secondary amine 422. Subsequent hydrolysis of the secondary nitrile under phase transfer conditions delivers the free acid which is converted to the methyl ester 424. In order to obtain the desired S-enantiomer, a classical resolution with ca. 0.5 equiv of L-camphorsulfonic acid (L-CSA) in toluene is used. The desired enantiomer 420 is collected as a crystalline salt in greater than 98% ee and 88% of the expected yield  which was subjected to a Henry reaction with nitromethane. Reduction of the nitro olefin function to the corresponding alkylamine followed by reaction with formaldehyde gave the corresponding imine 426 [Although the tetrahydrothieno[3,2-mine 426 . TreatmeN-protected 4-piperidone 427 is subjected to Vilsmeier conditions to produce the reactive chloroaldehyde species 428 which, upon treatment with ethyl mercaptoacetate (429), cyclises to the heterocyclic structure although in only low isolated yield. Simple base hydrolysis of the ester followed by decarboxylation generates the desired product , an analogue of the previously mentioned carvedilol (136) β-blocker, shows the binding of these molecules , which can be prepared from cyanogen (434) and sulfur dichloride [436 is alkylated with dichloroacetone to yield a carbonyl compound 437 susceptible to enzymatic reduction with baker’s yeast. The levorotatory enantiomer thus obtained can be subjected to Mitsunobu conditions with benzoic acid as the nucleophile and leads to clean inversion of the stereocentre which thus gives access to the other enantiomer of timolol. Under basic conditions the chlorohydrin 438 can be converted to the corresponding epoxide which can be ring opened by tert-butyl amine to furnish timolol (Whilst the synthesis of the thiophene-containing component (dorzolamide) of Cosopt starts from 2-mercaptothiophene and elaborates the thienothiopyran motif in a linear multi-step fashion, the thiadiazole ring of timolol is prepared from an acyclic precursor . The actchloride , the two timolol .440, Zanaflex). This substituted 2,1,3-benzothiadiazole 442 can be prepared by the reaction of an aromatic diamine 441 on heating with thionyl chloride in the presence of DMF. Selective nitration followed by an iron-mediated reduction affords the corresponding aniline 443 which partakes in a nucleophilic substitution of 2-chloro-3,4-dihydroimidazole (generated in situ from the reaction of the urea 444 and phosphoryl chloride). Removal of the acetate group under basic conditions furnishes tizanidine  and sulfamethoxazole .The isoxazole ring is a common heterocyclic motif represented by several of the top-selling small molecule pharmaceuticals. This ring structure is often encountered as a surrogate of other nitrogen containing heterocycles such as pyrazoles, pyridines or pyrimidines . Two spe39) and triethyl orthoformate in the presence of acetic anhydride. The resulting ethyl ethoxymethylene acetoacetate (448) is next condensed with hydroxylamine hydrate in methanol to yield ethyl 5-methylisoxazole-4-carboxylate (449). The ethyl ester is hydrolysed under acidic conditions and the carboxylic acid activated with thionyl chloride in DMF for amide formation with 4-trifluoromethylaniline (450) (Leflunomide is a pyrimidine synthase inhibitor of the DMARD-type (disease-modifying anti-rheumatic drug) marketed by Sanofi-Aventis. Unlike NSAIDs, which only deal with symptoms of rheumatoid arthritis, DMARDs target the cause of it. DMARDs are not necessarily structurally or mechanistically related. The effect of leflunomide is possibly due to its regulation of the immune system via affecting lymphocytes. Its synthesis  is relatne (450) .E)-4-(dimethylamino)but-3-en-2-one (451) and hydroxylamine is used in the synthesis of bactriostatic antibiotic sulfamethoxazole (447) to yield the equivalent 5-methylisoxazole (452)  in a aluminium-amalgam mediated reduction [454 yields the antibiotic agent 447 (A related condensation between (le (452) . This baeduction –136. Sulgent 447 .455, Risperdal). For this molecule the benzisoxazole ring is formed via an intramolecular nucleophilic aromatic substitution between an in situ generated oxime 459 and the adjacent aromatic ring [458 arises from a Friedel–Crafts acylation of the difluoroaromatic 457 (the acetate N-protecting group is presumably lost in the work up). Finally, alkylation of the piperidine ring under Finkelstein conditions is used to complete the synthesis of risperidone (The most prescribed therapeutic for schizophrenia is the benzisoxazole containing antipsychotic risperidone (tic ring . The preperidone .Having collected data with respect to many different reported syntheses of drugs containing heteroaromatic five-membered rings, a number of observations can be drawn. For instance, it was established that the most common transformations used are indeed condensations and nucleophilic substitution reactions  138–139139. ThisAn additional comment should be made about the time frame of new reaction uptake within the process environment. For certain reaction types such as metathesis and C–H activation, which obviously offer significant synthetic potential, it could be argued that the length of time required for these reactions to evolve from research tools to production processes are outwith the pipeline development times of the drugs under discussion. A significant body of the literature representing the synthesis of these drugs date back many years to origins in the late seventies and early eighties. Consequently, it is probably not unexpected to see the absence of many of the ‘newer’ chemical methodologies.Interestingly, the above-mentioned classes of chemical reactions are most widely applied to a limited number of heterocyclic cores with indoles, triazoles and benzimidazoles being the most prominent examples to date . It can One constant in all the drug syntheses is the shortness of the reported routes, which typically do not exceed 6–8 steps. Furthermore, chiral centres are still less well represented in drugs unless they can be sourced cheaply from the chiral pool or accessed from commercial suppliers specialised in asymmetric synthesis, for instance, hydrogenations or enzymatic transformations/resolutions. It is predicted  that theBased on the material presented in this review it can be concluded that most of the reported routes towards well established drugs are based on conventional transformations to furnish the desired compound quickly. However, with increasing competition between the pharmaceutical companies and additional pressures from regulatory/government authorities, chemical producers will need to adopt improved processing techniques that encompass a reduced environmental footprint and deliver the APIs at significantly reduced cost. In order to achieve the necessary productivity gains future chemistry programmes will have to employ a wider range of synthetic transformations targeted at delivering molecules covering an expanded area of chemical space. This will not be solely achieved by minor improvements in the efficiency of individual chemical reactions but will need the industry to integrate actively all the new tools and enabling technologies within their research and manufacturing efforts .


No studies to date have formally investigated the role of epigenetics in food allergy. Many believe that disruption in epigenetic marks may lead to loss of gene control in relevant immune pathways, and this may predispose to allergic disease. There is a growing body of evidence to suggest DNA methylation marks in key locus control regions are the primary regulators of naïve T-helper cell differentiation. To address these contemporary research questions, we undertook genome-wide methylation profiling of CD4+ T-cells harvested from children with and without food allergy, before and during the onset of disease. This data was analysed in the context of genome-wide expression data from the same cohort.Genome wide DNA methylation profiling of CD4+ cells from children with diagnosed food allergy (n=30) and non-allergic children (n=30) was undertaken at birth  and 12 months (during onset). A comparative analysis of DNA methylation profiles was performed and this data was correlated with gene expression data and functional T-cell assays.We report the first lines of evidence for epigenetic disruption in association with food allergy. 85 loci were differentially methylated between allergics and non-allergics after adjusting for age , and genetic effects. This represented a change in the promoter methylation status in 25 unique genes involved in cellular response to stress, fatty acid beta-oxidation pathways, calcium-activated potassium channel activity, small molecule and vitamin metabolism. Approximately 40% of methylation changes occurred outside known gene-associated regions with unknown significance. An examination of the effects of SNPs on methylation profiles revealed HLA-DQB1 as differentially methylated between allergics and non-allergics, resulting in a quantitative change in gene expression.DNA methylation profiling of CD4+ cells reveals disruption of several epigenetic pathways that appear to be programmed into the T-cell compartment. Although a proxy marker, the methylation array has genotyping utility and suggests a novel role for SNPs in HLA-DQB1 in association with changes in methylation and gene expression.


The pathophysiology of episodic memory dysfunction after infarction is not completely understood. It has been suggested that infarctions located anywhere in the brain can induce widespread effects causing disruption of functional networks of the cortical regions. The default mode network, which includes the medial temporal lobe, is a functional network that is associated with episodic memory processing. We investigated whether the default mode network activity is reduced in stroke patients compared to healthy control subjects in the resting state condition. We assessed the whole brain network properties during resting state functional MRI in 21 control subjects and 20 ‘first-ever’ stroke patients. Patients were scanned 9–12 weeks after stroke onset. Stroke lesions were located in various parts of the brain. Independent component analyses were conducted to identify the default mode network and to compare the group differences of the default mode network. Furthermore, region-of-interest based analysis was performed to explore the functional connectivity between the regions of the default mode network. Stroke patients performed significantly worse than control subjects on the delayed recall score on California verbal learning test. We found decreased functional connectivity in the left medial temporal lobe, posterior cingulate and medial prefrontal cortical areas within the default mode network and reduced functional connectivity between these regions in stroke patients compared with controls. There were no significant volumetric differences between the groups. These results demonstrate that connectivity within the default mode network is reduced in ‘first-ever’ stroke patients compared to control subjects. This phenomenon might explain the occurrence of post-stroke cognitive dysfunction in stroke patients. The majority of stroke survivors suffer from post-stroke cognitive impairment. These cognitive deficits after infarction are usually well explained by size and location of the infarction The MTL is a part of the default mode network (DMN) that also comprises of posterior cingulate cortex, medial prefrontal cortex and bilateral parietal cortices A novel approach in understanding the intrinsic state of brain activity is the study of the spatiotemporal patterns of the blood-oxygen level-dependent (BOLD) fluctuations in the task-independent condition In this study, we performed independent component analysis (ICA) to identify the resting-state DMN and region-of-interest (ROI) analysis to examine the inter-regional functional connectivity between the regions of DMN as the independent component may not capture all the variance of the data. We hypothesized that patients following an infarction would have an altered DMN compared to the control subjects. More specifically, the functional connectivity between the key regions involved in DMN (including MTL) would be reduced in the patient group compared with controls.Acute ischemic stroke patients, admitted at the Department of Neurology of the Radboud University Nijmegen Medical Centre between September 2005 and May 2007, were considered for enrollment in this study. The selection of patients and controls has been described in detail before All subjects underwent an extensive neuropsychological assessment between six and eight weeks after stroke onset that included an MMSE, IQCODE, Hospital Anxiety Depression Scale 3, 300 volumes, FoV = 224 mm). All participants were scanned on the same scanner.Magnetic resonance imaging was performed on a 1.5 Tesla Siemens Magneton Sonata scanner  which included T1 3D magnetization-prepared rapid gradient-echo (MPRAGE) imaging  and resting state data using a gradient-echo echo planar imaging (EPI) sequence . The first five resting state images were discarded because of the possible spin saturation effects. Remaining images were motion corrected and the mean image was coregistered to each subject’s T1-weighted image. Subsequently, the images were normalized to the Montreal Neurological Institute (MNI) T1 template and resampled at an isotropic voxel size of 2 mm. To reduce the effects of low and high non-neuronal noise, the images were then temporally band-pass filtered between 0.01 Hz and 0.1 Hz using fifth order Butterworth filter. Finally, the images were spatially smoothed using a Gaussian kernel of 8 mm full width at half maximum (FWHM).Image processing and statistical analysis was performed using the statistical parametric mapping software SPM5 (http://icatb.sourceforge.net/version 2.0c) t-tests using a threshold of P<0.05 family wise error (FWE)-corrected for multiple comparisons. For the group comparisons, two sample t-tests were applied with a height and extent threshold of P<0.05 The independent component analysis (ICA) was performed using Group ICA of fMRI Toolbox  to estimate the hippocampal volumes more precisely. This was done in a standardized way according to the previously published protocol Automated segmentation was conducted on the high-resolution T1-weighted anatomical image using SPM5. Independent two-tailed t-tests were applied on the normal distributed baseline characteristics and neuropsychological scores, Chi-square tests for nominal data and Mann-Whitney U-analyses for the non-Gaussian distributed data.Independent The study protocol was approved by the medical ethics committee region Arnhem-Nijmegen and all the participants gave written informed consent according to the Declaration of Helsinki. None of the participants had a compromised capacity or ability to consent, which was established by neurological examination.Data were available of 49 subjects, of whom eight subjects were excluded because of the use of different resting state protocol (n = 7) and absence of resting state data (n = 1). Twenty-one healthy control subjects  and twenty stroke patients  were included. The group analyses of the independent components revealed seven distinct RSN’s . These RFor every pair-wise region, the correlation coefficients were calculated after removing the non-neural BOLD fluctuations. This resulted in correlation matrices and corresponding graphs for visualization of the matrices for both control and stroke subjects . The regThere were no significant differences of the total intracranial, gray matter, white matter and CSF volume between control and stroke subjects . VBM anaWe found a decreased functional connectivity in posterior cingulate, medial prefrontal and left MTL within the DMN in ‘first-ever’ stroke patients compared to the control subjects. In addition, reduced inter-regional functional connectivity between these regions was observed when comparing stroke patients with healthy control subjects.Several methodological issues in this study need to be considered. A potential limitation of functional connectivity analyses is the contribution of physiological noise, such as cardiac and respiratory pulsations, on the functional connectivity In terms of the interpretation of our findings, the altered functional connectivity is commonly considered to reflect the occurrence of the cognitive disturbances in various brain disorders Theoretical explanations for widespread effects of local structural damage have been postulated for long time. This is referred to as ‘disconnection’ syndrome This study complements the emerging evidence that infarctions not only result in focal neurological symptoms dependent on their location, but can also induce widespread effects in remote regions. Altered DMN connectivity in stroke patients might underlie reduced episodic memory after stroke. To further support these findings, future studies should incorporate stroke patients without impaired episodic memory function as extra validation. Future studies should also evaluate other resting state networks, which could be disrupted due to the effects of stroke lesions and should incorporate follow-up of patients in order to determine long-term consequences of such lesions on the functional and structural connectivity network and cognitive function, which can give us valuable information about the neural plasticity and functional recovery. This is important for the practitioners to identify stroke patients at risk for post-stroke dementia and to determine adequate medical treatment and/or cognitive or physical rehabilitation


Scientific research has supported the effectiveness of brief motivational intervention (BMI) to reduce young-adult alcohol use and related consequences. Often, BMI includes a decisional balance component examining the “pros and cons” of alcohol use, which attempts to explore client ambivalence about drinking though a discussion of its good (and not-so-good) aspects. The aim of this qualitative study was to investigate the link between the need for social inclusion and engagement in alcohol-related behavior. Participants included 28 young adult (18-24 years old) alcohol users enrolled in a large hospital-based clinical trial. All BMIs were recorded and transcribed, and results were analyzed using NVIVO 9.0 software. Responses to the pros-and-cons dialogue were coded and thematically organized using grounded theory methods. The results identify the role of social group membership in alcohol use, including the need to avoid social exclusion and the desire to facilitate social interaction. Alcohol consumption allowed for such facilitation by providing an “escape” from social anxiety and promoting social behavior. Patterns of alcohol use as influenced by social factors may depend on rejection sensitivity, suggesting that an emphasis on social skills and abstinence-supportive relationships can be used to assist young people trying to reduce alcohol consumption. Social-skills training may prove useful as a preventative measure as well. If young adults feel more confident socially, they may feel less of an impulse to drink to increase the likelihood of social group acceptance.


Background. Expectations held by health professionals and their patients are likely to affect treatment choices in subacute inpatient rehabilitation settings for older adults. There is a scarcity of empirical evidence evaluating whether health professionals expectations of the quality of their patients' future health states are accurate. Methods. A prospective longitudinal cohort investigation was implemented to examine agreement  between physiotherapists' (n = 23) prediction of patients' discharge health-related quality of life (reported on the EQ-5D-3L) and the actual health-related quality of life self-reported by patients (n = 272) at their discharge assessment (using the EQ-5D-3L). The mini-mental state examination was used as an indicator of patients' cognitive ability. Results. Overall, 232 (85%) patients had all assessment data completed and were included in analysis. Kappa coefficients (exact agreement) ranged between 0.37–0.57 (58%–83%) across EQ-5D-3L domains in the lower cognition group and 0.53–0.68 (81%–85%) in the better cognition group. Conclusions. Physiotherapists in this subacute rehabilitation setting predicted their patients' discharge health-related quality of life with substantial accuracy. Physiotherapists are likely able to provide their patients with sound information regarding potential recovery and health-related quality of life on discharge. The prediction accuracy was higher among patients with better cognition than patients with poorer cognition. Many older adults do not return to their premorbid health states following hospital admissions requiring a period of subacute inpatient rehabilitation –4. PatieThere is currently a scarcity of research investigating whether health-professionals' and patients' expectations of future health-states are accurate among patient groups receiving health interventions in settings that utilise patient centred models of care . A recenPhysiotherapists are one group of health professionals of particular interest in subacute hospital settings where older adults receive rehabilitative therapies to address functional concerns in order to maximize health-related quality of life on discharge from hospital , 9–12. IThis study investigated whether physiotherapists' expectations of their patients' discharge health-related quality of life (formulated while conducting their admission assessment) were in agreement with their patients' actual self-reported health-related quality of life on discharge from a subacute rehabilitation unit. Due to the potential influence of patient cognition on self-reported health-related quality of life at discharge, the levels of agreement between physiotherapist predicted health-related quality of life and patients' self-reported discharge health-related quality of life were examined for an entire cohort, as well as two cognition subgroups based on mini-mental state examination scores –17. A prospective longitudinal cohort design was implemented to examine agreement between physiotherapists' expectations of their patients discharge health-related quality of life versus the patients' actual discharge health-related quality of life as self-reported on the EQ-5D-3L . n = 23) from a subacute geriatric assessment and rehabilitation unit at tertiary hospital participated in this investigation. Participants were consecutive admissions to this unit following a period of acute management on a specialised hospital ward appropriate for their condition . The participating unit admits subacute older adults with a range of primary diagnoses for rehabilitative interventions to improve their functioning and prepare them for discharge from hospital. Patients admitted to the participating rehabilitation unit received medical and allied health input designed to maximize functioning, independence, and health-related quality of life on discharge. The typical length of stay in this rehabilitation unit at the time of the study was approximately six weeks. However, length of stay was determined on an individual basis and may have been less or more than six weeks dependent on a range of clinical and external factors.A total of 272 patients and their treating physiotherapists  was used as a general indicator of patient cognitive ability . The MMSOn admission and discharge from the unit, patients completed a standard battery of clinical assessments. This assessment included physical performance tests  as wellImmediately following the completion of the admission clinical assessment battery (but before patients completed their admission EQ-5D-3L self-report), physiotherapy staff completed their discharge prediction rating on the EQ-5D-3L instrument. Physiotherapist participants received a 1-hour training session regarding their role in this study, including how to follow the assessment protocols at the admission and discharge assessments. The training included completing a practice admission and discharge assessment under the supervision of a member of the research team. Additionally, instructions for providing the predicted discharge EQ-5D-3L report were included in each patient's chart. As part of the training session, therapists were instructed to rate the EQ-5D-3L according to how they believed the patient would answer EQ-5D-3L items on discharge from rehabilitation, regardless of whether clinicians agreed with the patient's perspective. The MMSE was completed by hospital occupational therapists or medical staff for each patient admitted to the unit. MMSE results along with other patient demographic variables were collated from the medical history, and patients were grouped into a better cognition group (MMSE >23/30) and a poorer cognition group (MMSE ≤23/30) for use in data analysis. This research investigation was approved by the local human research ethical review board and complied with the declaration of Helsinki. Patients completed the EQ-5D-3L at discharge as a component of their routine clinical assessments, and gatekeeper consent was provided for physiotherapists' participation in this research.t-test (age) and a Mann-Whitney U test (length of stay in the subacute rehabilitation unit). For the individual EQ-5D-3L domain scores, levels of agreement between physiotherapist predicted discharge rating and actual self-reported discharge EQ-5D-3L responses were calculated using weighted Cohen's Kappa with disagreements of only one level assigned a 0.5 weighting [Demographic and clinical characteristics of the sample were tabulated . Conventeighting  , limits of agreement (LOA) , and intt cohort .P < 0.001). The median (IQR) length of stay for the entire sample was 42 (25–66), with no difference between cognition groups (P = 0.60). The median (interquartile range) years of experience of physiotherapists (n = 23) providing a prediction rating was 6 (2–11). Overall, 232 (85%) patients had complete data and were included in analyses; 81 (35%) were in the lower cognition group and 151 (65%) were in the higher cognition group. The demographic and clinical characteristics of the sample are presented in The levels of agreement (Kappa statistics and exact matches) between physiotherapist predicted discharge health-related quality of life (EQ-5D-3L domain scores) and patients' self-reported health-related quality of life (EQ-5D-3L domain scores) are reported in P = 0.038). No differences were observed in mean utility ratings  for the overall sample or for the better cognition group. Agreement (intraclass-correlation coefficients and limits of agreement) between physiotherapist predicted discharge health-related quality of life and patients' self-reported health related quality of life at discharge is displayed in This investigation revealed substantial agreement between physiotherapists' predictions of their patients' health-related quality of life at discharge and the patients' actual self-reported health-related quality of life at discharge. Interestingly, patient cognitive ability seemed to be a mediating factor in the accuracy of the prediction. The agreement between physiotherapist predicted and self-reported quality of life was not only lower for patients in the poorer cognition group, but physiotherapists tended to underestimate the level of health-related utility (EQ-5D-3L utility) that patients' in the lower cognition group would report at discharge. The size of this underestimation was of a sufficient magnitude to be considered clinically meaningful –34. On tThe purpose of this investigation was not to probe the cause of any underestimation for patients with poorer cognition. However, the authors speculate that there are at least two potential reasons why physiotherapists may have underestimated the summary utility score generated by the EQ-5D-3L instrument for patients with poorer cognitive abilities. First, the physiotherapists would have had some knowledge of patients' cognitive ability from reviewing their medical record and receiving a clinical handover of pertinent information from their colleagues prior to conducting their assessment in this subacute hospital setting. It is plausible that physiotherapists perceived a level of impact from the cognitive impairment on the patients' potential for recovery that was not accurate, and patients subsequently experienced more recovery and better health-related quality of life (EQ-5D-3L utility score) at discharge than the physiotherapists had anticipated. Second, it is also plausible that patients from the poorer cognition group may have lacked some level of insight into their level of functioning when responding to the EQ-5D-3L questions at their discharge assessment. For example, a patient with poor cognition who had some problems with their ability to wash and dress themselves may have reported having no problems with personal care. Potential lack of insight of this nature may have led to difficulty predicting how patients might respond at their discharge assessment rather than difficulty predicting the level of functional recovery a patient may experience relevant to their health-related quality of life . There was no clear pattern in the level of prediction accuracy across the five EQ-5D-3L domains for patients in the better cognition group, with the level of exact agreement consistently between 81% and 85% for all five domains. Similarly, there was no clear pattern in the level of prediction accuracy within the poorer cognition group across the five health-related quality of life domains, despite the agreement statistics being lower than those observed for the better cognition group. This was an interesting finding, given that some prior research has indicated that proxies may be better able to predict how patients will respond to health survey questions for more observable domains like mobility than for less easily observable domains like depression –37. One There are several important implications for patient centred models of care in subacute rehabilitation settings from this research. Patients that experience debilitating injuries or illnesses are likely to derive information about their potential for recovery from their treating health professionals when planning goals and making joint treatment decisions intended to maximize their health-related quality of life on discharge and thereafter , 6. PhysThere may be other explanatory factors beyond the scope of this investigation that influenced patient self-reports among some patients and contributed to some discrepancies between predicted and actual health-related quality of life reports in both cognition groups. Some previous reports have demonstrated paradoxical improvements in self-reported health-related quality of life as people have aged despite declining physical functioning and increased depression –40. ChanThere are several strengths and limitations for this research. First, all participating physiotherapists and patients were from a single geographical location. Physiotherapists from other geographical locations may not have responded in the same way. On the other hand, the inclusion of 23 different physiotherapists and 224 patients provides some ability to generalize findings to other similar populations. Second, only the EQ-5D-3L instrument was used. While this instrument is among the most widely used generic health-related quality of life measures, is suitable for use among older adults , 23, andIt may be useful for future research in this field to examine the prediction of health state outcomes across transition periods. This may include transitions from inpatient rehabilitation to returning to live in the community, particularly among older patients at risk of requiring institutional care. Intervention decisions in the subacute setting may influence the likelihood of subsequent hospital or nursing care admissions. The ability for clinical teams to be able to inform their patients about the likelihood of functional abilities and health-related quality of life in the post-discharge period may assist patients (in conjunction with their treating teams) to make sound decisions regarding postdischarge care arrangements. Additionally, specific physiotherapist-related factors that may have influenced their ability to predict patients' discharge health-related quality of life were beyond the scope of this investigation. However, future investigations of health-professional attributes that may influence their ability to anticipate their patients' future health states may also be worthy of consideration to advance this field of research. Physiotherapists in this subacute rehabilitation setting predicted their patients' discharge health-related quality of life with substantial accuracy. They are likely to be able to provide their patients with sound information regarding the potential recovery and health-related quality of life on discharge. The prediction accuracy was higher among patients with better cognition than patients with poorer cognition. Physiotherapists tended to underestimate the self-reported discharge health-related quality of life of patients in the poorer cognition group by a small, but potentially clinically meaningful, margin.


These membrane-integral pMHC molecules are present on surface of all nucleated cells and allow T-cells to detect aberrant intracellular activity, be this infection with microorganisms or abnormal host biochemistry such as neoplastic division. Scanning of pMHC molecules occurs via the αβ T-cell receptor (TCR), a clonotypic, heterodimeric, and membrane-integral molecule on the T-cell surface  cradled in multiple major histocompatibility complex (MHC) molecules, termed An interesting consequence of the low antigen affinity and high antigen cross-reactivity characteristics of TCRs is that many, and perhaps all, cognate antigens could potentially be improved upon. Through rational structural modifications of native blueprint antigens we now know it is possible to engineer “optimal fit antigens” which exhibit logarithmic increases in affinity and immunogenicity. Compared with the native antigens, if these optimal antigens prove more effective at stimulating antigen-specific T-cell populations during experimental priming then the compounds may fundamentally redefine how we think about vaccine design.T-cell epitopes can be optimized by: (i) improving antigen affinity for MHC; (ii) improving antigen affinity for TCR; and (iii) improving antigen pharmacology through synthetic biology. Enhancing the stability of the epitope within the MHC cleft is the simplest engineering strategy as MHC anchoring preferences have been determined from MS elution data   where the side chains are appended to the nitrogen atom of the peptide backbone . The motive behind this strategy is that proteogenic amino acid-based polypeptides are susceptible to acid degradation and rapid proteolytic cleavage and have half-lives of less than 5 min in the presence of proteases  antigens which generally exhibit very low immunogenicity  blueprint from human cytomegalovirus (HCMV) which were 10,000-fold more active. This achievement is striking given that the native A2-NLV epitope is one of the most immunogenic across the HCMV proteome (Khan et al., L-amino acids as demonstrated by recognition of peptidomimetics. To date, such compounds have been made by rational design and have failed to exceed the potency of optimal peptide ligands. It is possible that the screening of combinatorial chemistries so that T-cells can select their recognition preference may overcome this limitation. While these synthetic compounds are unable, at this time, to exceed the sensitivity of the model epitope during completive titrations, they are considerably more resistant to digestion from proteases and serum and demonstrate remarkable biostability.Table in vitro priming from healthy and patient samples. We also need to determine what effects priming with optimal T-cell epitopes has on the responding T-cell repertoire, both in terms of polyfunctionality and TCR usage. Studies that directly test whether optimal T-cell epitopes can outperform native ligands during in vivo priming of naïve animals are required. Importantly, these responses must be tested in relevant in vivo models of infection. It is hoped that optimal epitopes can be found that amplify greater numbers of T-cells than the native ligand alone and that T-cells maintain the desired functional profiles required for effective immunity. The development of effective synthetic T-cell epitopes will be particularly exciting as these epitopes have many attractive characteristics. First, they can be engineered to show absolute resistance to temperature fluctuations and environmental degradation, effectively nullifying the need for cold chain storage and transport. Indeed, the cold chain transport of vaccines costs WHO–UNICEF around USD 11.5 billion every year (Wolfson et al., It is clear that optimal T-cell epitopes can be generated with relative ease but the big question of whether they really work as therapeutic and prophylactic vaccines remains unanswered. Future studies should determine whether optimal T-cell epitopes can outperform native ligands during in vivo and could have the potential to change the way we build vaccines and immunotherapies in the future.In summary, optimization of T-cell epitopes can be achieved using a number of different techniques. Whether the resultant compounds can be used as effective prophylactic or therapeutic vaccines in humans remains to be determined. However, such approaches may allow the precise targeting of the most effective T-cell clonotypes (Ekeruche-Makinde et al.,


These apoptotic cells expressed the early neuronal marker PSA-NCAM, suggestive of newly generated immature neurons. In contrast, aged rats had a significantly higher number of TUNEL+ cells following TBI than injured juveniles, which were NeuN-positive mature neurons located predominantly in the granule cell layer. Fluorescent triple labeling revealed that microglial cells were closely associated to the apoptotic cells. In concert with these cellular changes, proteomic studies revealed both age-associated and injury-induced changes in the expression levels of three apoptotic-related proteins: hippocalcin, leucine-rich acidic nuclear protein and heat shock protein 27. Taken together, this study revealed distinct apoptotic responses following TBI in the juvenile and aged brain which may contribute to the differential cognitive recovery observed.The elderly are among the most vulnerable to traumatic brain injury (TBI) with poor functional outcomes and impaired cognitive recovery. Of the pathological changes that occur following TBI, apoptosis is an important contributor to the secondary insults and subsequent morbidity associated with TBI. The current study investigated age-related differences in the apoptotic response to injury, which may represent a mechanistic underpinning of the heightened vulnerability of the aged brain to TBI. This study compared the degree of TBI-induced apoptotic response and changes of several apoptosis-related proteins in the hippocampal dentate gyrus (DG) of juvenile and aged animals following injury. Juvenile (p28) and aged rats (24 months) were subjected to a moderate fluid percussive injury or sham injury and sacrificed at 2 days post-injury. One group of rats in both ages was sacrificed and brain sections were processed for TUNEL and immunofluorescent labeling to assess the level of apoptosis and to identify cell types which undergo apoptosis. Another group of animals was subjected to proteomic analysis, whereby proteins from the ipsilateral DG were extracted and subjected to 2D-gel electrophoresis and mass spectrometry analysis. Histological studies revealed age- and injury-related differences in the number of TUNEL-labeled cells in the DG. In sham animals, juveniles displayed a higher number of TUNEL Traumatic brain injury (TBI) is a major cause of death and disability world-wide. With the increase in numbers of the aging population in the United States, the epidemiology of TBI has shifted toward this demographic, with falls representing the leading cause of brain injuries involving the elderly. Although no overt neuronal cell loss is observed in the brain with aging, several subtle structural, chemical, and metabolic changes occur that render the aged brain more vulnerable to TBI as compared to the young brain. These changes include a reduction in the complexity of dendritic arborization, dendritic length, and spine numbers , increasThe hippocampus, a region responsible for learning and memory functions, is particularly vulnerable to TBI. The learning and memory deficits observed following TBI are likely a reflection of differential susceptibility of neurons in different hippocampal subregions to injury . Under nAs neuronal generation and degeneration concomitantly exist following TBI, the observed age-related differences in recovery may be due to not only the level of neurogenesis but also differences in the degree of cell death occurring after brain injury. Neural cell loss in the hippocampus has been linked to multiple neurochemical pathways and cell death cascades leading to necrosis and apoptosis . Apart fIn order to ascertain the potential link between apoptotic cell death and the observed age-related differences in functional recovery following TBI, the current study was undertaken to investigate the levels of aging and injury associated apoptotic cell death and the proteomic profiles of apoptosis-related proteins in the DG of the juvenile and aged rats.ad libitum. All procedures were approved by our Institutional Animal Care and Use Committee.Juvenile  and aged  male Sprague–Dawley rats  were used. Animals were housed in the animal facility, with a 12-h light/dark cycle, water and food provided n = 7 for each age group) or sham injury (n = 7 for each age group) using the lateral fluid percussion injury (FPI) model as previously described  was administered through the craniotomy onto the intact dura, using a pre-calibrated FPI device. After injury, the Luer lock fitting was removed, the wound sutured and after a 3-h observation, the rats were returned to the vivarium. Sham animals underwent the same surgical procedure, but did not receive the injury pulse. Animals were allowed to survive for 48 h following injury, at which point they were anesthetized and brain tissue processed for either histochemical or proteomic analysis.Animals were subjected to moderate TBI  were anesthetized with isofluorane, euthanized with euthasol , and transcardially perfused with phosphate buffered saline (PBS) immediately followed by 1% paraformaldehyde in PBS. The brains were rapidly dissected and immediately frozen in dry ice-chilled isopentane at -30°C. Ten μm-thick coronal sections spanning the rostro-caudal extent of the hippocampal DG (corresponding to the Paxinos and Watson stereotaxic rat atlas coordinates of -2.5 to -5.2 relative to bregma (®/Plus Slides (Fisher Scientific) and stored at -80°C until histochemical procedures were conducted.Forty-eight hours following injury, animals . Briefly, sections were post-fixed in pre-cooled ethanol:acetic acid (2:1) for 5 mins at -20°C, followed by two 5 mins washes in PBS. An equilibration buffer was applied for 10 s followed by 1 h incubation in a labeling solution of working strength TdT enzyme (composed of reaction buffer and TdT enzyme) in a humidified 37°C chamber. Following a 15 s agitation, sections were incubated for 10 mins in the stop/wash buffer to terminate the reaction and subsequently washed three times (one minute each) in PBS. In order to visualize the DNA fragments, sections were incubated in working strength anti-digoxigenin conjugate (composed of blocking solution and anti-digoxigenin antibody conjugated to fluorescein) for 30 min in a dark, humidified chamber at room temperature. Following four PBS washes, a mounting medium containing 0.5 μg/mL of a DAPI nuclear counter stain  was applied to sections and slides were coverslipped.In order to assess apoptosis levels in the DG, TUNEL histochemistry was performed according to the manufacturer’s protocol using the ApopTagFigure 1A). A cell was considered apoptotic only if its nucleus was labeled with both fluorescein and a DAPI nuclear counter stain. The apoptotic state of these TUNEL-positive cells was further confirmed by the presence of condensed or fragmented nuclei when visualized by DAPI . The estimated number of TUNEL-positive cells reported in the results represents the sum of apoptotic cell counts from all quantified sections throughout the DG of an individual rat multiplied by 10 to account for the intervals between quantified sections. The number of TUNEL-positive cells reported was the average of four animals in each group. In addition to evaluating TUNEL-positive cells in the DG of injured juvenile and aged animals, the extent of apoptotic cell death was also examined in age-matched shams.TUNEL-stained sections were examined at 40× with an Olympus BX51 microscope with a BX-RFA fluorescence illuminator . To quantify the degree of apoptotic cell death in the DG, every tenth coronal section throughout the rostro-caudal extent of the hippocampus was examined  and TUNEL-positive cells were systematically counted in a defined sampling region. The sampling region of interest extended from the hippocampal fissure down to the border of the lateral ventricle, including the suprapyramidal and infrapyramidal blades, granule cell layer (GCL), subgranular zone, and hilus of the DG  with 1% Triton for 1 h at room temperature. Sections were then incubated overnight at 4°C with mouse monoclonal NeuN , PSA-NCAM , or rabbit polyclonal GFAP  combined with goat polyclonal Iba1  diluted in the serum blocking solution. Following over night primary antibody incubation at 4°C, sections were washed in PBS and then incubated for 1 h at room temperature with Alex Fluor 568 anti-mouse IgG (for NeuN), IgA (for PSA-NCAM), or anti-rabbit IgG (for GFAP) combined with Alex Fluor 488 anti-goat IgG  in the serum blocking solution. Following incubation, sections were then washed three times in PBS and incubated with DAPI (1:1000) for 10 min. After a PBS wash, sections were coverslipped with Vectorshied. Sections were examined by confocal microscopy (Leica TCS SP2).n = 3 for TBI and sham animals in each age group) were anesthetized with isofluorane, euthanized with euthasol  and transcardially perfused with ice-cold PBS. Brains were then rapidly dissected on ice and placed in a rat brain mold with coronal divisions, so that three 1 mm coronal sections encompassing the rostro-caudal extent of the DG were cut using a single edge razor blade. The hippocampus ipsilateral to the site of injury in each slice was visualized using an Olympus SZX9 dissecting microscope and the dentate gyri  from the three slices were dissected, pooled, and snap frozen on dry ice. This was repeated on three samples for each of the four groups . Tissues samples were then thawed on ice and homogenized in an osmotic lysis buffer containing protease inhibitors and nucleases. Samples were quantified with a bicinchoninic acid (BCA) protein assay to determine the protein concentration of each sample and to ensure that equal quantities of proteins (50 μg) were loaded onto each gel for two dimensional polyacrylamide gel electrophoresis (2D-PAGE).Forty-eight hours following injury, animals (n = 3/group) resulting in a total of six gels per experimental group.In order to assess group differences in protein expression profiles, a comparative proteomic analysis was performed on tissue extracted from the DG of juvenile and aged rats subjected to either a sham or FPI. To identify candidate proteins in the DG that exhibited differential expression patterns with aging and injury, tissue was processed for 2D-PAGE  according to the method of The objective of this proteomic analysis was twofold: (1) to identify apoptosis-related proteins that are differentially expressed in the DG during the normal aging process and (2) to identify apoptosis-associated proteins that exhibit age-dependent alterations in expression following TBI. To this end, a manual and computer-automated subtractive comparative analysis was performed using Discovery Series PDQuest 2D-Gel Analysis software . Briefly, gels were digitized using a Bio-Rad GS-800 scanner (BioRad) and digital gels were cropped, prepped for spot detection, filtered, and smoothed to clarify spots using PDQuest software. Spot volume and density  parameters were used to quantitatively compare corresponding protein spots between experimental groups. To account for any inconsistencies in silver staining, the total staining intensity in a gel image was used to normalize spot density. Optical density measurements were further normalized via background subtraction and according to the internal tropomyosin standard. A conservative twofold selection threshold was applied to control the number of 2D gel spots processed for tandem mass spectrometric identification. Thus, only the more prominent protein changes were selected in this initial study and were subsequently excised from the 2D gels, tryptic digested, and processed for mass spectrometric analysis.Liquid chromatography-electrospray ionization-tandem MS  was then used to determine the amino acid sequence of each protein, which was then compared to theoretical MS spectra of known proteins using a MASCOT database search in order to determine the identity of the protein.post hoc Fisher LSD test or the Student t-test with an applied Bonferroni correction for multiple groups was utilized, with p value less than 0.05 considered statistically significant. Densitometric data for identified gel spots for log(2) transformed to a normal distribution and tested using a two-way ANOVA method for factors of injury and age and the interaction between the two using a Holm–Sidak method for multiple comparisons based on an initial alpha of 0.05. All values are reported as mean ± SEM in all figures.The TUNEL data was analyzed using SPSS software with analysis of variance (ANOVA) with Figures 1 and 2). Quantitative analysis to compare the number of TUNEL labeled cells between juvenile and aged animals revealed an age-related difference, both in the uninjured (sham) condition as well as following TBI . Specifically, sham juvenile animals exhibited significantly higher levels of apoptosis as compared to aged sham counterparts . Furthermore, while significantly increased TUNEL labeling was observed in the DG of both age groups following TBI, the magnitude of the injury-induced apoptotic response was significantly more pronounced in aged animals as compared to juveniles . By comparing the number of apoptotic cells in the injured brain to the sham base level between age-matched counterparts, the injured-aged brain exhibited a 38.9-fold increase while the injured-juvenile showed a more modest 4.8-fold increase.Age-associated differences in the apoptotic response of the brain to traumatic insult may represent a mechanistic underpinning of the heightened vulnerability of the aged brain to TBI, as well as contributing to the poor cognitive recovery observed in elderly patients following TBI. In this study, TUNEL labeling was employed to assess the level of apoptosis in the DG of juvenile and aged rats following a moderate lateral fluid percussion or sham injury. Varying levels of TUNEL-positive cells were observed in the DG in all groups assessed , localized predominately in the GCL and subgranular zone . The majority of apoptotic cells in the DG of the juvenile rats as characterized by dense DAPI labeling were NeuN-negative, PSA-NCAM-positive immature neurons located in the subgranule layer of the DG . In contrast, there were very few PSA-NCAM-positive cells in the DG in the aged brain. Rather, most of the apoptotic cells with dense DAPI labeling observed in the aged DG were NeuN-positive, PSA-NCAM-negative localized to the GCL . In both age groups, the apoptotic cells were enveloped by Iba+ microglial cells suggesting that the apoptotic cells were taken up by microglial cells . No GFAP-labeled astrocytes displayed apoptotic nuclei morphology (data not shown).To determine the cellular constituents of apoptotic cells in the DG, we used neuronal markers NeuN (for mature neurons) and PSA-NCAM (for immature neurons) and the astrocytic marker GFAP combined with microglial cell marker Iba1. Apoptotic cells were identified by the presence of condensed, fragmented nuclei with DAPI staining . Sixty distinct protein spots were detected recurrently across 2D gels with 10 exhibiting a twofold or greater change in abundance with injury or aging as a factor. Using tandem MS, 3 of the 10 gel spots were identified as proteins implicated in apoptotic processes: hippocalcin (P23k), acidic nuclear phosphoprotein pp32 (LANP), and heat shock protein (Hsp27).To gain insight into the underlying mechanisms of the observed aging/injury-associated differences in the DG apoptotic response, we examined protein expression profiles in the DG in sham and injured juvenile and aged rats. Specifically, each individual protein sample from the DG was subjected to a 2D electrophoresis. Proteins spots from each 2D gel were then semi-quantitatively analyzed to identify apoptosis-associated proteins that are differentially expressed in the DG during aging process as well as following TBI. A comparison of 2D gel protein expression profiles between the experimental groups revealed several age- and injury-induced alterations in the DG proteome  in the DG of sham aged animals as compared to sham juveniles . The elevated expression of this anti-apoptotic protein in the aging brain appears related to the declining levels of apoptosis that are observed during the aging process within the DG (as assessed by TUNEL staining). In addition to the differential expression of hippocalcin between sham animals of different ages, alterations in the expression levels of this anti-apoptotic protein were also observed in response to injury. A comparison between sham and injured aged animals revealed a significant decrease  in hippocalcin expression following TBI  which corresponded to the marked injury-induced increase in TUNEL-positive cells observed in this aged population . In contrast to that seen in aged animals, no significant change in hippocalcin expression level was observed in juvenile rats as a consequence of injury. This hippocalcin expression pattern observed by proteomic analysis was further confirmed by immunohistochemistry. As shown in Figure 7, strong hippocalcin immunoreactivity was observed in the stratum oriens, stratum lucidum, and stratum radiatum of the CA1 region in all groups but particularly in the sham aged brain . In the DG, hippocalcin was expressed in the molecular layers (ML) and hilus region, with robust expression observed in the aged sham animals  as compared to that of juvenile sham animal . Furthermore, while hippocalcin immunoreactivity remained relatively unchanged in the DG of juvenile animals following injury , there was a substantial decline in hippocalcin immunoreactivity in the DG of aged rats in response to TBI .Hippocalcin, a calcium binding protein that has been shown to protect neurons against apoptosis, exhibited a significantly higher level  in the DG of juvenile sham animals as compared to aged shams . The significantly decreased expression of this apoptosis-promoting protein in the DG during the normal aging process parallels the declining levels of apoptosis observed with aging (as assessed by TUNEL staining). Furthermore, while no change in LANP abundance was observed in the DG of injured juveniles, a significant increase  in LANP expression over sham levels was observed in aged animals following TBI . This injury-induced increase in LANP expression in the aged DG following TBI corresponds to the markedly enhanced levels of apoptosis in this population.LANP, a protein that has been shown to exhibit pro-apoptotic properties, revealed a significantly greater level  in Hsp27 expression were observed in the DG as a factor of normal aging . However, the expression of this anti-apoptotic/anti-necrotic protein was significantly enhanced  as a factor of injury, irrespective of age.Unlike the aforementioned proteins, no significant alterations , which suggested the presence of differential regulating pathways of apoptosis in the normal aging process and following TBI.+ cells are predominantly localized to the SGZ and that the number of TUNEL+ cells decline with aging, are in agreement with these published studies.Apoptosis is an important mechanism during brain development for regulating neuronal cell numbers and to ensure the appropriate formation of neuronal circuitries. Apoptosis also occurs in the neurogenic regions of the adult brain, with a significant portion of newly generated cells in the adult brain eliminated within the first months following generation . Cells u+ cells in the injured juvenile brain as compared to sham counterparts, with apoptotic cells localizing predominantly to the SGZ and displaying an immature neuronal phenotype (PSA-NCAM+). This observation is in agreement with previously published findings that adult-generated immature neurons in the SGZ are vulnerable to TBI, with many cells undergoing cell death due to the injury impact  was present on 2D-electrophoresis gels run for this study at a MW of 23 kDa and with an isoelectric point (pI) of 6.12. In our study, Hsp27 expression level was low in sham animals regardless of age. Its expression level was drastically increased after injury in both age groups indicating that Hsp27 is an injury-induced protein, which is in agreement with previous findings that report enhanced synthesis of Hsp27 after stress. To date, no published literature has reported HsP27 expression following TBI. Hsp27 is a member of the small heat shock or stress protein (shsp) families that are known to display enhanced synthesis after heat or oxidative stress . The incpoptosis .Hsp27 has also been studied in relation to necrosis, a cell death process that has been shown to dominate after moderate to severe experimental TBI . NecrosiHippocalcin, also known as p23k, was present on 2D-electrophoresis gels at a MW of 23 kDa and with a pI of 4.87. This protein displayed an increase in expression with aging in sham animals, and a decrease in expression after injury in the aged brain only. The differential expression correlating with aging is in conflict with a previously published study reporting decreased hippocalcin immunostaining in the hippocampus of aged rats during normal aging . This di++ extrusion and maintaining ideal intracellular Ca++ levels  . In the + levels . It is ativities . In additivities . It alsompal LTD  suggesti+ cells observed. As hippocalcin plays roles in neuronal viability and plasticity, the decreased expression of hippocalcin in the DG following injury may contribute to the vulnerability of the aged brain to TBI leading to increased neuronal cell death and cognitive dysfunction. Taken together, our findings suggest that hippocalcin may play a role in endogenous repair or homeostasis after TBI.In the current study, in the injured brain, hippocalcin level was sharply decreased in the aged DG while remaining constant in the juvenile animals. This decrease of hippocalcin expression in the injured aged DG corresponds to the sharp increase in the number of TUNELLANP, also known as ANP21-A, PHAPI (putative HLA-associated protein 1), and pp32 (phosphoprotein with a molecular mass of 32 kDa), was present on 2D-electrophoresis at a MW of 29 kDa and with a pI of 4.00. In sham animals, this protein was expressed at high level in the DG of juvenile brain and was low in the aged brain. Following TBI, an increase in expression was observed only in the aged animal while remaining constant in the juvenile animal. This expression pattern suggests that the aged brain was stimulated to produce high level of LANP to recapitulate the level in the juvenile brain.+ cells observed in these groups, which support the role of LANP as an apoptosis promoter. Nevertheless, other roles of LANP such as its function in regulating neurite extension , we may miss out on some other proteins that may have significant association to age and injury related changes. Nevertheless, the differential expression of the three apoptosis proteins that we have identified in the current study may suggest any one of these proteins may play an important role in contributing to the decreased capacity of recovery of the aged population following injury. Our results suggest that therapeutic strategies for treating TBI sufferers need to consider age-related differences in pathological changes and cellular pathways in order to be effective for the particular age group.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In resource-rich areas, risky sexual behavior (RSB) largely diminishes after initiation of anti-retroviral therapy, with notable exceptions among some populations who perceive a protected benefit from anti-retroviral therapy (ART). Yet, there is limited data about long-term trends in risky sexual behavior among HIV-infected people in sub-Saharan Africa after initiation of anti-retroviral therapy.We administered questionnaires every three months to collect sexual behavior data among patients taking ART in southwestern Uganda over four years of follow-up time. We defined RSB as having unprotected sex with an HIV-negative or unknown status partner, or unprotected sex with a casual partner. We fit logistic regression models to estimate changes in RSB by time on ART, with and without adjustment for calendar year and CD4 count.506 participants were enrolled between 2005 and 2011 and contributed a median of 13 visits and 3.5 years of observation time. The majority were female (70%) and median age was 34 years (interquartile range 29–39). There was a decrease in the proportion of men reporting RSB from the pre-ART visit to the first post-ART visit  but not women . With each year of ART, women reported decreasing RSB . In contrast, men had increasing odds of reporting RSB with each year of ART to near pre-treatment rates , which was partially confounded by changes in calendar time and CD4 count .Men in southwestern Uganda reported increasing RSB over four years on ART, to levels approaching pre-treatment rates. Strategies to promote long-term safe sex practices targeted to HIV-infected men on ART might have a significant impact on preventing HIV transmission in this setting. Approximately 2.5 million people continue to be infected with HIV each year in sub-Saharan Africa, and the majority of new infections in the region are caused by sexual transmission between sero-discordant, heterosexual partners Recent reviews summarizing data in Sub-Saharan Africa reported similar reductions in risky sexual behavior (RSB) after ART initiation We enrolled participants in the Uganda AIDS Rural Treatment Outcomes (UARTO) study from the adult HIV clinic at Mbarara regional Referral Hospital beginning in 2005. Adult PLWHA living within 20 kilometers of the clinic (to facilitate study procedures) and initiating ART were eligible. We administered surveys at baseline (pre-ART visit) and quarterly after ART initiation to collect data on sociodemographic and sexual behavior characteristics, and collected blood samples for CD4 count testing. Trained research assistants fluent in the local language performed interviews in private study rooms. They asked about number of partners, age at first sexual encounter, number of sexual partners in the past three months , condom use, and HIV serostatus of partners. All participants gave written informed consent to participate.The study procedures were approved by ethical review committees at Partners Healthcare, University of California, San Francisco, the Mbarara University of Science and Technology and the Uganda National Council for Science and Technology.∧3), divided into a two-piece segmented linear model (<300 cells/mm∧3 and ≥300 cells/mm∧3) to allow for the changing slope of RSB that were apparent in graphical models. We implemented the Huber-White estimator to account for within cluster dependence from repeated measures in participants over time Our outcome of interest was risky sexual behavior (RSB). We defined RSB as having unprotected sex with a sero-discordant or unknown status partner, or unprotected sex with a casual partner in the prior three months. We fit logistic regression models to estimate changes in RSB by year of ART use, with and without adjustment for calendar year (per year) and CD4 count (per 100 cells/mm∧3 (IQR 75-202). The median age at first sexual encounter was 17 years (IQR 15–19) in women and 18 years (IQR 16–20) in men. Women reported a median of 3 lifetime sexual partners (IQR 2–4) versus men who reported a median of 6 ). At baseline, 49% of the entire cohort reported sexual contact in the past three months, and 16% of men and 14% of women met criteria for RSB in the past three months.A total of 506 participants  were enrolled from the time of ART initiation from November 2005– June 2011 . Median ∧3 . In the multivariable model adjusted for calendar year and CD4 count, the association between years on ART and RSB remained positive, but not statistically significant . However, men reported increasing RSB for each year of ART use . In a univariable regression model of change in RSB by year of ART, women reported decreasing RSB for each additional year of ART use  and attributed to a “disinhibition” phenomenon Further discernment of long-term trends and causes of increasing RSB will serve multiple purposes. Measurement of the association between RSB and ART adherence is a priority. Though HIV transmission can be reduced among discordant heterosexual couples when one of the partners is using ART Our study was limited to four years of follow-up. As ART availability continues to expand in sub-Saharan Africa, and because our estimates suggest fluid trends in reporting of RSB up to four years, continued observance of the long-term trends in RSB is recommended. Moreover, we primarily focused on describing global trends of RSB and did not evaluate time varying correlates of RSB aside from CD4 count and calendar time. Future studies should measure predictors of RSB to identify at-risk sub-populations and whether adherence changes correlate with RSB. Finally, our data are limited to self-report of RSB. The validity of self-reporting sensitive information can be poor due to motivational biases, cognitive factors, and privacy concerns, with a predicted bias towards under-reporting In summary, while women in rural Uganda report decreasing RSB over four years after initiation of ART, men reported an initial decrease followed by increased RSB during time on ART to pre-baseline levels. Future research should focus on associations between RSB and ART adherence as well as correlates of the heterogeneous patterns of risk. Intervention strategies that are aimed specifically at high-risk groups will be needed to effectively impact RSB and HIV transmission in the region.


The model should be able to show the influence of cellular components as i.e. plasmacytoid dendritic cells (pDC) or drug candidates. This might allow the avoidance of animal experiments and relates more closely to the human situation. For example, plasmacytoid dendritic cells (pDC) representing a DC subpopulation with major immunologic regulatory functions, were investigated to show a clear analogy to the in vivo situation in the model.The description of complex regulatory immunologic pathways represents a major difficulty during preclinical testing. Animal models are widely used for this purpose. In the study described herein, a human Antigen-presenting cells were generated from CD14-positive monocytes obtained from whole blood of healthy or allergic donors. On day 5, APC were stimulated with house dust mite extract (HDM). On day 7, cells were harvested and DC were co-cultured with autologous CD4+ lymphocytes (ratio 1:10) in the presence of allergen with or without pDC or a immunomodulatory compound. Alternatively, PBMC were isolated from whole blood of healthy donors and stimulated with HDM ± pDC. The effects of pDC on the allergen-induced T cell proliferation were analyzed by ³H-thymidine incorporation. Cytokine secretion was measured in cell culture supernatants.The generated and HDM-pulsed APC induced strong proliferation of the cocultured CD4+ lymphocytes compared to the unpulsed APC. The presence of pDC in the cocultures inhibited this proliferative response by about 50%. Likewise, the proliferation of HDM-stimulated PBMC was dose-dependently reduced by pDC, with the highest pDC dose nearly completely abolishing the allergen induced proliferation showing a significant deregulating functionality.in vitro allergy model. The presence of pDC in this model clearly reduced the allergen-induced immune response, demonstrating the regulatory properties of this DC subpopulation depending on the stimulatory APC to pDC cell ratio. Hence, this model enables to mimic allergen-specific immune responses and furthermore regulatory mechanisms. In the future, this model thus might be applicable for efficacy testing of potential immunoregulatory cellular therapeutics or biomolecules with the advantages of avoiding animal experiments and enabling greater predictivity for the human situation.An allergen-specific T cell immune response can reproducibly be performed using the human


Genome-wide association studies (GWAS) have become an effective tool to map genes and regions contributing to multifactorial human diseases and traits. A comparably small number of variants identified by GWAS are known to have a direct effect on protein structure whereas the majority of variants is thought to exert their moderate influences on the phenotype through regulatory changes in mRNA expression. MicroRNAs (miRNAs) have been identified as powerful posttranscriptional regulators of mRNAs. Binding to their target sites, which are mostly located within the 3′-untranslated region (3′-UTR) of mRNA transcripts, they modulate mRNA expression and stability. Until today almost all human mRNA transcripts are known to harbor at least one miRNA target site with an average of over 20 miRNA target sites per transcript. Among 5,101 GWAS-identified sentinel single nucleotide polymorphisms (SNPs) that correspond to 18,884 SNPs in linkage disequilibrium (LD) with the sentinels ( The state of knowledge regarding development, progression and inheritability of human diseases has long since outrun the classical understanding. There are only few disorders where monogenic causes could be determined. Also, the genetic links to disease incident are missing to a substantial part and the complexity of pathways encompassing pathogenic effects can still not be limited upwards. In recent years, more and more evidence is emerging that microRNAs (miRNAs), a class of small non-coding RNAs, play an important role in human traits. Databases collecting information on miRNAs mediating human disease such as miR2Disease MiRNAs are key posttranscriptional regulators of most known cellular processes and have been associated with cell fate decision, development, and stress response. Additionally, miRNAs have been identified to be usable as biomarkers for human diseases As soon as the importance of miRNA functioning for human health was realized, approaches were undertaken with the objective to identify potential interrelations of miRNA dysregulation and genetic variation. However, until now neither the data on trait-associated polymorphisms nor experimentally verified miRNA targeting information provided a sufficient basis for such analyses. For instance, mutations in the 3′-UTR, the major target of miRNA-mediated regulation, have long been (and are still) neglected for the most part in association studies. Therefore, only few particular cases of polymorphisms affecting miRNA regulation pathways have been identified, yet The success of genome-wide association studies (GWAS) in determining the genetic causes of some common diseases led to an immense increase of the efforts put on screening common alleles for disease involvement. Since then, more and more loci associated with trait susceptibility are detected. GWAS can, however, neither identify causal genes in associated loci nor provide functional mechanisms behind observed association signals. Consequently, many identified GWAS signals are awaiting mechanistic characterization and the rate at which GWAS signals are currently discovered necessitates systematic and scalable functional approaches cis-acting regulatory effects.When it became clear that only few diseases are due to common risk alleles, sequencing endeavors have been performed to identify putatively rare variants hiding behind the signals detected in GWAS. In some cases these approaches successfully identified rare causative variants predisposing to disease. However, the expected breakthrough failed to appear as in several cases sequencing of risk loci did not provide further knowledge rs10923 in the 3′-UTR of SMC4 we show how our findings can be utilized in the biological interpretation of GWAS results in a systems biological manner.We assess the potential impact of trait-associated SNPs on miRNA regulation pathways using publicly available GWAS data r2 thresholds.We compared the amount of trait-associated variants within the predefined five function classes of SNPs , coding sequence (CDS), and 3′-UTR) to examine a potential location bias of these markers. Of 18,884 SNPs contained in the extended GWAS-SNP set . To investigate if 3′-UTR SNP enrichment values hold only for specific MAFs, we recalculated the ORs against the HapMap-SNP set in dependency of the MAF. The ORs resulting for the five MAF intervals follow roughly the pattern of over−/underrepresentation observed in the comparison with the other extended GWAS-SNPs but never lose significance or fall below an OR of 2.0 . By analyzing the MAF, we found the extended GWAS-SNPs to hold a commonly higher MAF than the HapMap-SNPs, regardless of their chromosomal location. However, the comparison of the MAF distribution of the 3′-UTR SNPs to the MAF distribution of other extended GWAS-SNPs revealed a slight trend of 3′-UTR SNPs towards moderate MAF frequencies between 0.1 and 0.4. This trend becomes more explicit when comparing the 3′-UTR SNPs to the combined extended GWAS-SNPs in the other two exonic regions (i.e. 5′-UTR and CDS). In comparison, 3′-UTR SNPs show underrepresentation of the intervals 0.0–0.1 (OR = 0.88), 0.2–0.3 (OR = 0.70) and 0.4–0.5 (OR = 0.78) whereas the other two intervals are significantly  revealed only four significantly enriched  reported in the study is in high LD  is associated with the damaging of the small bile ducts and is mediated by auto-antibodies rs10923 is localized within an experimentally validated RISC binding site hsa-mir-299-5p, a miRNA that has been shown to be up-regulated in PBC patients rs10923 disrupts the 6mer seed complementary region  and thus the ability of the miRNA to bind the transcript , expression data from the MuTHER study SMC4 expression in lymphoblastoid cell lines. We suggest that, by this process, rs10923 contributes to the phenomenon of DNA repair perturbation in cirrhosis SMC4 in the generation of the Condensin I-PARP1-XRCC1 complex. Furthermore, PBC patients present elevated risk to develop different types of cancer The SNP y region  and thusy region  and thusy region  and thusAlthough thousands of SNP-trait associations have been published and the applicability of GWAS is broadly appreciated, the associations identified by GWAS hold predictive power for only a small fraction of disease incidence so far Here, we suggest that associated common or unknown rare variants in the 3′-UTR depict posttranscriptionally regulatory variants which, to differing extent, affect disease development and progression by interfering with miRNA regulatory networks. The differing impact of 3′-UTR SNPs can be explained with two facts: first, human mRNAs are mostly targeted by multiple miRNAs at different target sites which may allow for compensating the loss of a single binding site; and second, the extent to which mRNAs are silenced by miRNAs depends on multiple factors such as tissue-specific miRNA expression, the binding affinity of the RISC and the degradation rate of the mRNA rs2168518) was found to be located in the hairpin of hsa-mir-4513. But we observed a significant overrepresentation of trait-associated SNPs in the 3′-UTR that strongly suggests a functional coherence between genetic variants and miRNA regulation pathways on the cis-regulatory level.Based on recently available data, our results clearly show that several lines of evidence become obvious that miRNAs play an important role in genetically determined posttranscriptional disease development and progression. There is no evidence for considerable direct interference of miRNA processing: only one SNP  signals, our extensive analysis of the potential impact of GWAS-identified SNPs on functional elements in the 3′-UTR revealed several mechanisms whereby variants may affect miRNA-mediated regulation. The smallest fraction of potentially functional 3′-UTR SNPs affects 3′-UTR splicing. These SNPs are predicted to mediate miRNA target site loss, mostly through the gain of acceptor splice sites (donor splice site gain only occurred once) resulting in shortened 3′-UTRs. That altered splicing is the rarest found mechanism can be explained by the huge impact it mediates on miRNA targeting which manifests in the high fraction of target site loss (46% on average) for the affected transcripts. The second mechanism is the SNP-mediated alteration of RNA secondary structure of a RISC binding region. The impact of RNA folding on the binding affinity of RBPs has already been described In general, our findings indicate that miRNAs play an important role in genetic variants causing trait development. Therefore, novel aspects not only for the interrelations in pathogenic disturbance of cellular processes, but also for the coherence of different traits can be addressed. For instance, differential tissue-specific expression patterns of miRNAs in combination with genetic variants may shed light on the still unknown functions driving the same cellular pathways to feature different effects in diseased and healthy individuals. Thus, closing the gap between trait-associated mutations and impaired miRNA-mediated gene regulation may lead the way to a new understanding of the interconnection of those functional entities.SMC4 we show that the incorporation of miRNA related information can be used to construct models of potential disease-driving mechanisms in a systems biological fashion. In this case, we present a very simple one-gene/one-miRNA scenario. However, up to now SMC4 and PBC have never been connected, as well as an association of PBC and cancer only exists in clinical ratios. But our suggestion of a highly active, SMC4-dependent DNA repair machinery is not only a likely cellular response mechanism to apoptosis signals in cirrhotic tissue. It also provides the link to cell transformation and cancerogenesis. Therefore, in the context of miRNA biology plausible coherences can be established for common variants with unknown effect. This also points out that, beyond statistical evaluations, case-specific detailed analyses may greatly outrange global approaches in the biological interpretation of GWAS results.We found 326 candidate genes, and on the example of Our findings highlight numerous starting points for biomedical research. To advance the potential of conclusion drawing in GWAS results interpretation, it may be invaluable to overcome the GWAS bias towards the coding sequence and extend approaches based on 3′-UTR mutations. Conversely, more comprehensive data on miRNA binding sites might enhance our understanding of miRNA regulation functioning. It may also be of interest to investigate the most suspicious cases described in this work further. Moreover, determination of miRNA regulation via RISC binding to other mRNA locations than the 3′-UTR might shed light on the effect of coding-synonymous trait-associated alleles. The combination, i.e. extensive measurements of miRNA-mediated gene regulation in patients with traits for which a plausible model of miRNA involvement can be created in the context of associated mutations in the 3′-UTR, may further provide new perspectives of disease progression. But the implications of our findings go beyond GWAS and miRNAs. Considering the advances made in the exploration of the still poorly understood elements of the genome, the impact of the presented results are pointed out. Just recently, the approaches of the modENCODE project We downloaded the Catalog of Published Genome-Wide Association Studies As background distribution for localization enrichment we used the 2.7 million SNPs from the CEU panel of the joint HapMap Phases I, II and III  for which genotype information was available r2 values in the localization enrichment analysis was performed accumulatively in 5% steps. For binning of HapMap-SNPs into LD blocks, we searched for all SNP sets with an all-vs.-all We mapped all SNPs on genomic locations of protein-coding genes and miRNA genes. The genome annotations were obtained from the UCSC Table Browser We used the algorithm PhastCons from the PHAST package Argonaute and TNRC6 in human HEK293 cells To analyze the single nucleotide mutation effect on miRNA targeting, we used the high-throughput transcriptome-wide CLIP-Seq interaction maps describing sites of the RBPs We obtained chromosomal positions of poly(A) signals from the PolyA DB for mRNA polyadenylation sites We applied the NNSplice algorithm from the Berkeley Drosophila Genome project . All SNPs inducing a minimal correlation coefficient of less than 0.55 between wild-type and mutated structure were filtered.To account for structural changes caused by SNPs we used the RNAfold algorithm from the Vienna RNA Package version 1.8.5  To find all possible MREs we searched for sites complementary to a canonical miRNA seed sequence We evaluated the enrichment of functional annotations of the 326 genes containing 3′-UTR SNPs using DAVID For retrieval of phenotype-specific microRNA expression alterations we used PhenomiR Table S1SNPs predicted to affect 3′-UTR splicing. The first column lists the SNP rs-numbers, in the second column the respective transcripts are given and the third column contains the genomic locus of the SNP including strand information of the transcripts. The fourth column shows the conservation of the SNP in binary code, i.e. 1 means conserved, 0 means not conserved. The fifth column gives the type of the gained splice site, the score column contains the likelihood of NNSplice and the last column provides the percentage of lost RBP binding sites for the accordant transcripts.(XLS)Click here for additional data file.Table S2SNPs predicted to affect 3′-UTR secondary structure. The first column lists the SNP rs-numbers, in the second column the respective transcripts are given and the third column contains the genomic locus of the SNP including strand information of the transcripts. The fourth column shows the conservation of the SNP in binary code, i.e. 1 means conserved, 0 means not conserved. The fifth column lists the correlation coefficient of the wild-type structure to the mutated structure of the respective transcripts.(XLS)Click here for additional data file.Table S3SNPs disrupting existing MREs. The first column lists the SNP rs-numbers, in the second column the respective transcripts are given and the third column contains the genomic locus of the SNP including strand information of the transcripts. The fourth column shows the conservation of the SNP in binary code, i.e. 1 means conserved, 0 means not conserved. The fifth column lists the miRNAs the MREs of which are affected.(XLS)Click here for additional data file.Table S4SNPs creating new MREs. The first column lists the SNP rs-numbers, in the second column the respective transcripts are given and the third column contains the genomic locus of the SNP including strand information of the transcripts. The fourth column shows the conservation of the SNP in binary code, i.e. 1 means conserved, 0 means not conserved. The fifth column lists the miRNAs for which MREs are created.(XLS)Click here for additional data file.