Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios) and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish chronic infections.The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed ‘cassettes’ that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium,  Borrelia burgdorferi, evades immune detection through a system consisting of numerous unexpressed ‘cassette’ sequences that alter the expressed protein via recombination. Importantly, the potential for evolutionary change in the expressed protein, and thus its ability to repeatedly escape a directed immune response, is correlated with the diversity among the cassettes. We analyzed genetic data from diverse B. burgdorferi strains to test the hypothesis that natural selection favors diversity among the unexpressed cassettes in order to promote evolvability. The data provide significant evidence of natural selection acting to increase among-cassette diversity, but only in regions that are targeted by immune antibodies when expressed. Further, these antigenically-important regions contain mutation-prone DNA sequence structures that are conserved despite high levels of sequence divergence among strains. The empirical evidence of selection favoring mutation-prone sequences and favoring among-cassette diversity supports the hypothesis that natural selection can shape the evolvability of populations.The hypothesis that natural selection shapes the ability of a population to evolve is highly controversial due primarily to a paucity of empirical evidence. However, the ability to constantly and rapidly evolve may be selectively adaptive in pathogens due to the constantly changing environment created by the vertebrate immune response. The Lyme disease bacterium,  The ability of a biological trait to evolve by natural selection, or evolvability, varies substantially among species, among populations within species, and even among traits within populations. The hypothesis that differences in evolvability result from past natural selection acting on the ability to evolve, however, remains highly controversial for two primary reasons The major objections to the evolvability-as-adaptation hypothesis lose force when applied to many microbial pathogens as a result of two biological features of these organisms. First, microbial pathogen cells within a host are often nearly clonal such that the fitness interests of individuals and groups are closely aligned Borrelia burgdorferi, rapid evolution of the surface antigen, VlsE, is required for immune evasion and long-term infection in vertebrate hosts In vivo studies have shown that the vlsE antigen expression locus is indeed highly evolvable; a near-complete replacement of vlsE alleles occurs every 14–28 days in experimentally infected mice vls cassettes into the vlsE expression site; six regions in the unexpressed vls cassettes are known to vary among cassettes and correspond to the antigenically important extracellular loop structures of the VlsE protein vls cassettes and vlsE are not recognized by antibodies that target previously detected VlsE antigens vlsE locus during infection is tightly correlated with the amount of diversity among the unexpressed vls cassettes, as mutations in the vlsE locus are rare except by recombination  are substantially more frequent than synonymous differences (per synonymous site) among the six regions of the unexpressed vls cassettes that correspond to the antigenically important loop regions of VlsE , nor the ospC or erp loci Sequence alterations in the eletions , Fig. S5B. burgdorferi, the rate of genetic change, or antigenic evolvability, at VlsE is tightly correlated with the amount of genetic diversity contained in the unexpressed vls cassettes  and thus be more likely to persist within hosts vls cassettes and promotes antigenic evolvability was supported by molecular evolutionary analyses of the cassettes of 12 B. burgdorferi strains  mutations at unstable tandem-repeat motifs, which are present in all  in VlsE , Fig. S1 in VlsE . Tandem B. burgdorferi occur as triplets in line with the reading frame and thus have the potential to alter antigenic epitopes, when recombined into VlsE, without introducing stop codons or frameshifts that would have deleterious effects on the protein structure. The tri-nucleotide repeats show little variation at the first and third codon positions but are significantly more variable at second codon positions resulting in differences in the amino acids they encode  from the beneficial alleles they create (VlsE escape antigens), thereby inhibiting hitchhiking Sexual eukaryotic pathogens conceivably experience fluctuating selective pressures similar to those experienced by B. burgdorferi strains that differ only in the diversity among vls cassettes in a repeated mouse-tick-mouse transmission cycle would provide an experimentally-controlled evaluation of the strength of selection on evolvability in this system. Analyses of the mutations introduced into the cassettes of each strain at each mouse-tick-mouse transmission could also elucidate the role of tandem repeats and other mechanisms that result in greater diversity among cassettes. In particular, comparing experimental infections in immunologically active and immunocompromised mice can determine the role of cassette diversity in establishing and maintaining persistent infection during an adaptive host immune response.The data reported here exhibit the genetic signatures expected given selection on antigenic evolvability. Future experimental evolution assays can be used to experimentally validate these conclusions and to further dissect the molecular mechanisms involved. For example, assays competing isogenic vls unexpressed cassettes in which strong diversifying selection leads to elevated amino acid diversity in regions that correspond to antigenically important domains in order to promote the evolvability at VlsE that allows for continual immune evasion. Such selection for cassette diversity could be a common strategy for maintaining antigenic evolvability in a diverse range of pathogens that generate antigenic variation by intragenomic recombination pilE locus of Neisseria gonorrhoeae are maintained despite common gene conversion events Our results support a model of evolution in the vls unexpressed cassettes both within and among B. burgdorferi strains for which genome sequence data was available We analyzed the genetic diversity of the The hypothesis that diversity among unexpressed cassettes is favored by natural selection to promote antigenic evolvability was tested using three molecular evolutionary analyses. First, the proportion of non-synonymous polymorphic nucleotides (dN) and synonymous polymorphic nucleotides (dS) were estimated Tandem-repeat motifs were identified using the mreps server B. burgdorferi strains JD1 and N40 were predicted using the SWISS-MODEL server B. burgdorferi strain JD1 were determined using the reported VlsE sequence (Genbank [CP002306]), whereas the protein structure in strain N40 was predicted by replacing the cassette region of vlsE in B31 with the unexpressed cassettes of N40.Structural models of VlsE in A nucleotide Neighbour-Joining (NJ) phylogeny  was constructed in Geneious v. 5.3 B. burgdorferi strain N40 7 cells/ml and the genomic DNA was purified using the DNeasy Blood and Tissue Kit protocol for gram negative bacteria . The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit  by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector . Long-range PCR amplification was conducted using the primers N40-vlsLR-R (5′ Phos - GCT GGA CTT GAA TTT GGT AGG GAT TC 3′) and N40-vlsLR-F (5′ Phos - GGT GAT GGT GCC GAT TCA AAA TCT GG 3′) which anneal to the unique conserved regions flanking the unexpressed cassettes. The PCR reactions contained 2–6 ng/µl of genomic DNA, 0.2 mM of each dNTP, 0.4 µM of each primer, 7% DMSO, 1× GC buffer and 0.02 U/µl of Phusion Hot Start II DNA Polymerase (NEB) and were amplified with 25 cycles of 30 s at 98°C and 90 s at 72°C.A clonal isolate of E.coli and isolated using a Qiagen Mini-prep kit. The TSA cell line used in transformation of the BigEasy vector (Lucigen) contains RecA and EndA mutations that make them recombination-deficient and minimize the chance that gene-conversion was introduced during cloning. Purified plasmid DNA was sheared using a Nebulizer  to 500–3000 bps, purified by ethanol precipitation, and subcloned using the Zero Blunt PCR cloning kit for sequencing (Invitrogen). Additionally, the ospA and rrs-rrlA IGS loci were PCR amplified from each culture as previously described BigEasy vectors containing an intact cassette region were amplified in Figure S1Indels are more abundant in antigenic loop regions containing 3 bp tandem repeat motifs. The average number of indel events in regions of the cassettes corresponding to antigenic loop regions (ALR) of VlsE was significantly larger in those that contained tandem repeats compared to those in which no repeats were detected . This suggests that tandem repeats contribute to diversification among the cassettes by increasing the rate of indel mutations and contributing to length variation among cassettes in the antigenic loop regions.(TIF)Click here for additional data file.Figure S2Greater diversity at second codon position of tandem repeats. Diversity at the second positions of identified 3 bp tandem repeat motifs, which are inline with the reading frame, were significantly greater than diversity at the first and third positions . All differences at the second positions resulted in amino acid changes in the expressed proteins. Diversity was measured by parsing each of the 3 bp elements within a repetitive region and calculating the entropy at the first, second, and third position.(TIF)Click here for additional data file.Figure S3Concerted evolution of the vls unexpressed cassettes.A) The unrooted Neighbor-Joining phylogeny of individual unexpressed cassettes demonstrates high degrees of similarity within B. burgdorferi strains and high degrees of divergence among cassettes of different strains. This pattern of concerted evolution is typical of multi-copy sequences and results from sequence homogenization within strains due to gene conversion. The nucleotide Neighbour-Joining (NJ) phylogeny  was constructed in Geneious v. 5.3 B) Diversity among the vls unexpressed cassettes within each strain is localized to six antigenic loop regions. In both antigenic loop and alpha helical regions, cassettes are far more divergent between strains compared to diversity contained among cassettes within individual strains. Although recombination from the cassettes into the vlsE expression locus is unidirectional and does not affect the sequence of unexpressed cassettes C) Moderate sequence divergence was observed between cassettes belonging to strains that co-occurred on the same branch of the phylogeny. Three evolutionary lineages in the phylogenetic analyses consisted of unexpressed cassettes from more than one strain. The average pair-wise genetic distance among strains at alpha helical regions  and antigenic loop regions  shows a slight increase in diversity between strains compared to divergence within strains  indicating incipient divergence. Divergence among strains was not sufficient to produce a signal in the phylogenetic analysis, possibly due to recent ancestry, which is further supported by the observation that strains with similar unexpressed cassettes contain the vls antigenic variation system on the same linear plasmid ( plasmid .(TIF)Click here for additional data file.Figure S4Experimental evolution of B. burgdorferi reveals mutations caused by gene conversions among unexpressed cassettes. Evolutionary changes observed in the cassette regions during experimental infections are characterized primarily by gene-conversions and large deletions. The parental N40 isolate differs from a previously sequenced N40 laboratory isolate in a 70 bp segment of cassette 19 which contains 12 substitutions and a 6 bp deletion. These sequence changes are identical to the homologous region of cassette 20 and are shared in all derived strains. Mutations that occurred during infection consisted of gene conversion events in derived isolates 1 and 3 and large deletions in derived isolates 2 and 3. The mutations in cassette 13 of derived isolate 3 are depicted with different colors to represent gene conversion from different donor cassettes. At least 6 overlapping gene conversion events from other cassettes are necessary to account for the mutations that occurred in this cassette. Of the two single-nucleotide substitutions in cassettes occurred during experimental evolution, the substitution that occurred in an antigenically important region (cassette 2 of derived isolate 1) was non-synonymous, whereas the substitution that occurred in a alpha helical region (cassette 15 of derived isolate 3) was synonymous.(TIF)Click here for additional data file.Figure S5Southern hybridization confirms large deletions in the vls cassette region. The large deletions observed in the unexpressed cassette region of derived strains 2 and 3  which cuts at the recognition site, TTTAAA. Recognition sites for this enzyme occur throughout the AT rich genome of B. burgdorferi but are present at only one site within the vls unexpressed cassette region of each strain. The digested DNA was separated for four hours on a 0.4% agarose gel, transferred to a nylon membrane, and allowed to hybridize with two fluorescein labeled probes specific to the vls cassette sequences (5′ Fluor- TTC GGT TRG TGG KGA GCA GA 3′ and 5′ Fluor- ACA ATC CCC TTC ATC CCC TTA 3′). Southern blot analysis revealed two fragments (∼3 kb and ∼4.5 kb) in both the parental strain (cN40) and derived isolate 1 (36B) consistent with the single DraI site expected 2949 bp from the beginning of cassette 1 of a 7.467 kb fragment given the sequence data obtained in this study. The single fragment (∼4.3 kb) apparent in derived isolate 3 (39B) is consistent with the single DraI site expected 4204 bp from the beginning of cassette 1 of a 4.381 kb fragment. The 177 bp fragment was likely not detected because it ran off the gel. Similarly, derived isolate 2 (44B) revealed only a single fragment (∼5.9 kb) which is consistent with the single expected DraI site 5893 bp from the beginning of cassette 1 in a 6.073 kb fragment. 2 and 3  were con(PDF)Click here for additional data file.Table S1Summaries of the silent cassette loci in B. burgdorferi strains analyzed in this study.(DOC)Click here for additional data file.Table S2Statistical analysis of positive selection within silent cassettes of B. burgdorferi strains.(DOC)Click here for additional data file.Table S3Genbank accession numbers of the vls cassette region, ospA and IGS loci sequenced in isolates after one year of experimental infection.(DOC)Click here for additional data file.


Lung cancer is the leading cause of cancer related death in the world. Current therapies are only partially successful and relapse is frequent in a large number of patients. Hence, new therapeutic strategies are needed to improve the quality of life and survival of NSCLC patients.The Notch pathway is a key pathway involved in cell fate determination during development in many tissues and also in tissue homeostasis during adulthood . DeregulKras that can be experimentally induced on adult mice resulting in NSCLC [Kras-driven lung carcinogenesis is strictly dependent on the presence of a functional Notch pathway.In our recent work , we havein NSCLC . Importain NSCLC . This inKras-driven NSCLC [Previous studies have demonstrated that phosphorylated ERK is a critical mediator of oncogenic en NSCLC -8. Interen NSCLC . This isen NSCLC .Kras-driven NSCLC, detected by positron emission tomography (PET), arrested their growth after 15 days of GSI treatment (with Eli Lilly´s GSI LSN-411575). This therapeutic response was associated to biomarkers of Notch pathway inhibition and ERK dephosphorylation [Gamma-secretase inhibitors (GSIs) have been used during the last years to inhibit the Notch pathway . ImportaKras-driven NSCLC [Our results open a new therapeutic opportunity to treat NSCLC using GSIs. Interestingly, GSIs have been used in long-term treatments in Alzheimer´s patients without major side effects . The accumulated knowledge on the pharmacology of GSIs should pave the way to test these compounds in NSCLC patients. MEK inhibitors (MEKi) have shown some therapeutic activity in murine en NSCLC . In this


Current clinical assessment of borderline aortic coarctation may involve cardiovascular magnetic resonance(CMR) but if inconclusive, invasive catheterization pressure measurements are required to evaluate the pressure gradient at rest and during pharmacological stress.To predict the aortic pressure distribution in patients with aortic coarctation at rest and pharmacological stress using a transient rigid-walled computation fluid dynamics model(RW-CFD).The study cohort comprises 5 patients with native or recurrent aortic coarctation and 2 control patients with healthy aortic archesTable , who und1)Ascending aortic root: The inlet flow is extracted from the phase-contrast CMR flow. Supra-aortic vessels2) :The flow rate is calculated as a proportion of the inlet flow based on the assumption of a constant wall shear stress .Diaphragmatic aorta3) : The pressure waveform is extracted from the invasive catheter investigation.The model workflowFigure  requiresThe clinically invasive aortic pressure gradients were compared with the predicted pressure distribution along the centreline in the RW-CFD model at the time of peak flowTable .mean±standard deviation) and invasive pressure gradient drop across the coarctation .For patients with aortic coarctation,the RW-CFD simulations accurately predict the pressure gradient at rest and give indication of the gradient severity during stress.Furthermore, no gradient was predicted in control patients with normal aortae.These preliminary results, whilst using a simple CFD approach and a small cohort of patients, are quite promising. This study represents the first step towards an image-based fluid-solid-interaction CFD analysis. This more sophisticated approach is likely to overcome the current limitations and might grant additional information.In the future, it is envisaged that CFD models could be based on a patient-specific, non-invasive and non-ionising radiation assessment such as CMR in order to predict the hemodynamic conditions in the aorta and avoid invasive cardiac catheterization.The pressures assessed by clinically invasive catheterization at the level of the ascending aorta, diaphragmatic aorta and the pressure gradient across the coarctation  are compared with the RW-CFD model pressure gradient prediction (ΔP CFD). The absolute pressure differences values are shown in the final column. Pressures expressed as mean ± standard deviation. bpm, beats per minute.


Right ventricular apical pacing-related heart failure is reported in some patients after long-term pacing. The exact mechanism is not yet clear but may be related to left ventricular dyssynchrony induced by right ventricular apical pacing. Right ventricular septal pacing is thought to deteriorate left ventricular function less frequently because of a more normal left ventricular activation pattern.We report the case of a 55-year-old Tunisian woman with preserved ventricular function, implanted with a dual-chamber pacemaker for complete atrioventricular block. Right ventricular septal pacing induced a major ventricular dyssynchrony, severe left ventricular ejection fraction deterioration and symptoms of congestive heart failure. Upgrading to a biventricular device was associated with a decrease in the symptoms and the ventricular dyssynchrony, and an increase of left ventricular ejection fraction.Right ventricular septal pacing can induce reversible left ventricular dysfunction and heart failure secondary to left ventricular dyssynchrony. This complication remains an unpredictable complication of right ventricular septal pacing. Right ventricular apical (RVA) pacing results in abnormal left ventricular (LV) electrical and mechanical activation and is associated with an increased risk of developing heart failure -3. RightWe report the case of a 55-year-old Tunisian woman with preserved ventricular function, implanted with a dual-chamber pacemaker for complete atrioventricular block. RVS pacing induced a major ventricular dyssynchrony, severe left ventricular ejection fraction deterioration and symptoms of congestive heart failure. Upgrading to a biventricular device was associated with a decrease in the symptoms and ventricular dyssynchrony, and increased left ventricular ejection fraction (LVEF).A 55-year-old Tunisian woman presented with syncope. An electrocardiogram (ECG) upon admission showed complete heart block with a narrow QRS complex <120 ms) and an escape ventricular rate of 45 bpm. Our patient's medical history included arterial hypertension. She did not have diabetes mellitus, and had no family history of coronary artery disease. A two-dimensional echocardiography showed normal LV function with a 60% EF, the absence of significant valvulopathy and no regional wall motion abnormalities or pulmonary artery hypertension. A conventional dual chamber pacemaker  was implanted with the right ventricular (RV) lead positioned to her RV septum. The septal position was confirmed by fluoroscopic images; defined as a leftward orientation of the lead confirmed by 40° left anterior oblique projection . The pac20 ms andDespite instauration of optimal medical therapy, our patient remained at NYHA functional class III. She was upgraded to a cardiac resynchronization therapy (CRT)-device with implantation of a lateral left ventricular lead Figure . After oAfter one month, an echocardiography showed a decrease in the aortic PEP (130 ms), LV reverse remodeling, with a reduction of the LVEDV from 153 mL to 135 mL, and significant improvement in LVEF to 40%. She had no symptoms of heart failure or syncope afterwards and device interrogation showed that her cumulative biventricular pacing was 100%.Pacing from RVS sites has been suggested as an alternative to RVA pacing in an attempt to avoid long-term adverse consequences on LV function . This caThere is an increasing body of literature in which the authors investigate the acute and chronic effects of RVS pacing on electrical and mechanical synchrony, systolic and diastolic ventricular function and cardiovascular morbidity and mortality. Alternative RV pacing sites appear advantageous when compared to RVA pacing but their superiority has not been uniformly proven.et al. [et al. [Ten Cate et al.  have dem [et al.  demonstrIn patients with standard indications for pacing, the prediction of heart failure is difficult and the exact mechanism of RV pacing-related heart failure is not clear but may be related to LV dyssynchrony induced by RV pacing . The besThe Pacing to Avoid Cardiac Enlargement study  showed tRVS pacing can induce reversible LV dysfunction and heart failure secondary to LV dyssynchrony. This remains an unpredictable complication of RV pacing. It should be highlighted that not all patients develop LV dyssynchrony and new onset heart failure after RV pacing. Therefore, early predictive factors -15, suchWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.SO was the major contributor in writing the manuscript. All authors read and approved the final manuscript.


Arterial Spin Labeling (ASL) is a well-known technique that allows the non-invasive acquisition of angiograms without the need of a contrast agent 2]. ASL . ASL 2].The proposed method is a modified Quadruple Inversion Recovery  sequenceRenal arteries including small branches were successfully visualized in all subjects with excellent suppression of background Figure ,4. The pWe demonstrate a new ASL approach for non-contrast enhanced MR angiography with excellent background tissue suppression and without the need of subtraction. In comparison to DIR-ASL, mQIR-ASL yielded better background suppression and improved vessel delineation.


Acute ascending aortic dissections is the most common catastrophe of the aorta that requires emergency surgery. There is still a debate regarding the optimal surgical approaches for construction of the distal anastomosis in replacing the ascending aorta. The purpose of this study was to evaluate early clinical outcomes of two different surgical techniques: open distal anastomosis in hypothermic circulatory arrest (OHCA) compared to anastomosis with clamped aorta while continuing on extracorporeal circulation (CECC).A prospective randomized study in 84 consecutive patients who were operated, between September 2008 and September 2010, for acute type A aortic dissection with isolated replacement of the ascending aorta. The influence of two techniques  on clinical outcome was investigated.No significant difference was found between the two groups in terms of age and sex distribution. The groups were comparable with respect to all preoperative and intraoperative predictive risk factors for early clinical outcome and complications. The mean period of circulatory arrest lasted 30,46 min (15-61 min) at an average nasopharyngeal temperature of 23.57°C (16-28°C). Treatment method (OHCA vs CECC) was not associated with differences in intrahospital mortality  nore in early major postoperative complications: de novo focal neurolocigal deficits , acute renal failure , blood loss  or reexploration for hemorrhage .While there is no difference in clinical outcome in surgical treatment of acute type A aortic dissection with or without circulatory arrest, there are some practical technical advantages if the distal anastomosis is performed in an open manner. Probably the long-term outcome would also be better with this anastomosis technique.


Acute renal failure after exposure to contrast media in patients at risk for CIN increases substantially 90-day mortality and morbidity. Biomarkers for outcome after CIN are missing so far.We analyzed in a prospective study 233 patients with either diabetes or preexisting impaired kidney function getting intra-arterial contrast media (CM) for coronary angiograms. Urinary cGMP concentrations were analyzed 24 hours after exposure to intra-arterial CM in spot urine. The patients were followed for 90 day for the following events: death, need for transient or permanent dialysis, doubling of serum creatinine and unplanned re-hospitalization. 51 out of the 233 patients had such an event during the follow up period of 90 days after contrast media exposure. The urinary cGMP to creatinine ratio was significantly increased in patients having an event during the follow up period (p=0.011). We furthermore performed a multivariate regression analysis considering age, baseline kidney function before contrast media exposure and preexisting diabetes - all important risk factors for CIN. These analysis revealed that the urinary cGMP to creatinine ratio is an in dependent predictor of the occurrence of the composite endpoint: death, need for transient or permanent dialysis, doubling of serum creatinine and unplanned re-hospitalization .The urinary cGMP to creatinineratio isan independent predictor of major advers events after contrast media exposure during a follow-up of 90 days.


HIV-1 evolves rapidly within the host, resulting in the development of diverse variants called a viral “quasispecies” population. A major goal of vaccine efforts is the design of Envelope (Env)-based immunogens effective at eliciting broadly neutralizing antibodies. We hypothesize that B cells become programmed to develop broad NAbs by exposure to Envs presented by the viral quasispecies variants. We propose that similar programming could be achieved by a vaccine concept exposing the host to such Env quasispecies variants isolated from an individual who developed broad NAbs over time.Full-length functional env genes were cloned longitudinally from elite neutralizer CI10014 by single genome amplification, and a combination of in silico sequence analysis and in vitro neutralization was used to select vaccine candidates. Four immunization strategies were tested in rabbits: (1) sequential env evolution as it occurred in CI10014, with multiple clones per timepoint ; (2) sequential vaccine approach using only one clone per timepoint ; (3) an approach uniquely focused on env clones derived from timepoints where env evolution drove the development of breadth (Jump into Breadth); and (4) single env variant . The gp160-DNA and gp140-trimer immunogens were co-administered.NAbs were detected at six weeks, after only two immunizations and increased after additional immunizations. The Jump into Breadth strategy elicited significantly higher NAbs than the Clonal and Sequential strategies. Modest heterologous neutralization was obtained against Tier 1 clade A and B viruses.Exposure to env immunogens derived from timepoints preceding and contemporaneous with the appearance of neutralization breadth elicited higher NAbs than exposure to a single variant or a longitudinal collection of Envs. This study explores the use of multiple native, related HIV-1 Envs as immunogens and emphasizes the critical importance of understanding the development of breadth in an elite neutralizer subject.


Left ventricular (LV) myocardial wall thickness is influenced by non-ischemic conditions like cardiomyopathies (CMP) as well as ischemic disorders, i. e. coronary artery disease (CAD). For CMP, regional wall thickness under resting conditions has prognostic value and serves as risk predictor for future cardiovascular events. In CAD, aside from perfusion also inadequate dobutamine wall thickening indicates myocardial ischemia and leads to coronary intervention. Therefore, quantitative resting and stress regional wall thickness provides relevant diagnostic and prognostic information. To date there is only scarce data regarding reference values of regional wall thickness in healthy volunteers.We sought to investigate differences in age and gender of LV rest and stress regional wall thickness with a state-of-the-art SSFP sequence in a modified AHA 17-segment model and present reference values for myocardial rest and stress wall thickness.We studied 119 healthy volunteers in two gender groups  which both consisted to equal parts of three age groups to minimize its possible influence. 30 of the male and 29 of the female participants were pharmacologically stressed with dobutamine up to their age-depended maximal heart rate according to present guidelines. Long axis were obtained on a 1.5T whole body MRI scanner (Philips Achieva) using the SSFP sequence. Images of the left ventricle were analyzed applying a modified 17-segment model. P<0.05 was considered significant.The acquired normal values are shown in figure Applying a state-of-the-art SSFP MRI sequence in three different age groups of male and female healthy volunteers, we provide reference values for myocardial LV wall thickness at rest and under dobutamine stress which could lead to a clinical reference frame for the detection of abnormal myocardial thickness in CMP or thickening in CAD. The values for each segment of the left ventricle are significantly different between men and women as well as rest and stress. Further investigations with larger patient cohorts may address the issue of age dependency.None.


Arithmatic computational modeling permits improved prognostic detection via a combinatorial or independent method.Quantitative analysis can improve myocardial perfusion imaging (MPI) for prediction of prognosis. We have designed a novel approach, Decisions Informed by Computed Entities (DICE), to enhance the prognostic ability of MPI data to predict major adverse cardiovascular events (MACE) in women with suspected ischemic heart disease. We applied DICE to cardiovascular magnetic resonance imaging (CMRI) and Single Positron Computed Tomography (SPECT) MPI data sets in the Women’s Ischemia Syndrome Evaluation (WISE) Study.Women (n=228), mean age 59±11yrs, with symptoms suggestive of myocardial ischemia underwent MPI and cardiac function evaluation separately by CMRI and SPECT and were followed (40±17mo) for time to MACE . Abnormal perfusion defects were noted for each MPI modality . Cardiac regions were separately evaluated and at least one abnormal region was considered a positive study. The CMRI data were evaluated using qualitative (QLMR) and semi-quantitative (SQMR) approaches. Multiple linear regression models (DICE models) were generated, each predicting MPI status from one modality (e.g. CMRI) using data from a second modality (e.g. SPECT). Two DICE models were constructed from pairs of data sets and included variables such as end-systolic volume and myocardial wall thickness in addition to the clinical MPI reading.MACE occurred in 29 women (13%). The percentage of MACE occurring in pts with abnormal perfusion was 43% using the average of the clinical MPI readings vs. 75% for the average of the DICE models (p<0.01). The average percentage of pts having abnormal perfusion was 27% for the clinical readings vs. 35% for the average of the DICE models (p=0.09). Increasing the number of variables in each DICE model increased the percentage of MACE captured.DICE modeling, incorporating the clinical MPI reading and cardiac variables derived from one imaging modality predicted the clinical MPI reading of a second modality while increasing the prognostic value as compared to just the clinical reading. The predictive power of the DICE model increases with the number of variables measured while simultaneously reducing noise and bias.NHLBI/Internal.


Type 2 Diabetes Mellitus (T2DM) and prediabetes are associated with an increased cardiovascular risk and heart failure independent of the presence of obstructive coronary artery disease, caused in part by myocardial microvascular dysfunction. Recent studies have reported contractile dysfunction and increased rotation with the onset of diabetic cardiomyopathy in uncomplicated T2DM patients. Independently, higher resting myocardial blood flow (MBF) has been reported in T2DM. The objective of this study was to determine whether there is a correlation between LV Epicardial Rotation (LV ER) and resting MBF in T2DM patients with angiographically normal coronaries, measured by tissue tagging and perfusion cardiovascular Magnetic Resonance (CMR).Sixty five patients with no coronary stenosis >30% on invasive angiography were recruited and categorized into T2DM, prediabetes and normal controls groups according to American Diabetes Association guidelines. All patients underwent rest and adenosine stress myocardial perfusion CMR at a single mid-LV location, cine imaging covering the entire heart and myocardial tissue tagging in three LV short axis locations . LV volumes and mass were calculated from cine images. LV rotation in three myocardial layers  was calculated from CSPAMM tissue images using Tag Track . For this study, only rest MBF was calculated using Fermi-constrained deconvolution for the entire myocardium.Patient characteristics and results are shown in Table T2DM patients when compared to age-matched patients with prediabetes and normal controls have higher peak LV ER. Resting MBF correlates with LV ER. These results suggest that LV ER analysis in uncomplicated T2DM patients may be useful for non-invasive screening of early subtle contractile dysfunction associated with microvascular dysfunction in diabetes patients.This work has been funded by British heart foundation in part with the Libyan ministry for higher education and scientific research.


PfHslV, a homolog of β subunit of 20S proteasome forms the proteolytic core of the PfHslUV machinery in P. falciparum [PfHslV has no homolog in the human host and it is a promising drug target essential to the plasmodial metabolism. The use of single proteasome inhibitor targeting these threonine proteases has a potential to be antimalarial drug candidate. One of our recent studies identified several promising inhibitors against 20S β5 subunit of P. falciparum [. The present study adopts a similar knowledge based virtual screening strategy using Support Vector Machines (SVM) and molecular docking to build a focused library of potential PfHslV inhibitors. SVM model has been trained using 170 molecular descriptors of 64 inhibitors and 208 putative non-inhibitors. The non-linear classifier based on Radial Basis Function (RBF) kernel yielded classification accuracy of 97%. The SVM model rapidly predicted inhibitors from NCI library and were subsequently docked in to the active site of an optimised three-dimensional model of PfHslV. The novel drug-like PfHslV inhibitors with very good binding affinity and novel scaffold can be a good starting point to develop new antimalarial drugs.lciparum ,2. PfHsllciparum . The pre


Abnormal diffusion tensor imaging (DTI) measurements have been found in human and experimental subjects with severe hydrocephalus (HCP) [1-3]. Because ventriculomegaly can often be variable, the present study aimed to investigate the degree of DTI abnormality in neonatal rats with less severe HCP.Seventeen Sprague-Dawley rats were divided into three groups: (1) HCPS - 5 rats with intracisternal kaolin injections leading to severe hydrocephalus; (2) HCPM - 5 rats with the same kaolin injection procedure that developed mild HCP; (3) Control - 7 rats with intracisternal saline injections. Either kaolin or saline injection was performed at P2. DTI were acquired at P9-10 on a 7T Bruker MRI scanner. Evan’s ratios of ventricular size and DTI metrics  and mean diffusivity (MD)) were calculated and compared using ANOVA and other statistical comparisons.The three groups were significantly different (p<0.0001) in Evan’s ratios with HCPM rats  slightly higher than controls  but lower than the HCPS group . Both FA and MD values were significantly different among the three groups in cortex, corpus callosum and internal capsule . Post-hoc comparisons showed that the HCPM group was not significantly different from the control group, while the HCPS group was significantly different  from the control group  and the HCPM group. In the HCPM group, it was found that the MD values in the external capsule increased proportionally with ventricle size , while FA values in the internal capsule decreased with increasing ventricle size .Although the DTI measurements in the HCPM rats with mild ventriculomegaly were not significantly different from normal controls, the correlation between ventricle size and DTI values in this group suggested that different levels of abnormality in brain tissues can be detected with DTI during various stages of hydrocephalus. Our results demonstrate that DTI is potentially a sensitive biomarker for monitoring the progression of ventricle enlargement and may help to determine the optimal window for surgical intervention.


Of patients with left-sided endocarditis 20–47% have septic embolization to the spleen with varying degrees of clinical presentation. The natural history varies based on the size and location of the infarct. Larger and more peripheral infarctions are more prone to form abscesses and cause splenic rupture, whereas smaller infarcts may go largely unnoticed and only be detected on incidental imaging. Splenic abscesses are felt due to embolized septic material or secondary infection in a patient with bacteremia.The patient in this case is a 45-year-old female with a history of substance abuse, end-stage renal disease on dialysis, and aortic endocarditis status post bioprosthetic valve replacement. The patient presented to the emergency department with a 1-day history of 10/10 left upper quadrant abdominal pain. A computed tomography (CT) scan of the abdomen revealed a large splenic infarct . A subseThe CT scan appearance of splenic infarction includes a typically triangular region of low radiodensity on portal phase images, following intravenous contrast administration which does not show enhancement on the delayed images. Splenic abscess is strongly suspected if there is gas within the hypodense region. Additional signs of bulging of the splenic capsule, a focal fluid collection, or perisplenic inflammatory stranding are non-specific and each can be seen with bland infarction. Cardiac technique CT scan has shown excellent sensitivity and specificity for detecting aortic valve vegetations as well as for complications such as adjacent abscess or pseudoaneurysm. Pre-operative scanning has been recommended for those who need valve surgery.


Ecological communities are increasingly exposed to multiple chemical and physical stressors, but distinguishing anthropogenic impacts from other environmental drivers remains challenging. Rarely are multiple stressors investigated in replicated studies over large spatial scales (>1000 kms) or supported with manipulations that are necessary to interpret ecological patterns. We measured the composition of sediment infaunal communities in relation to anthropogenic and natural stressors at multiple sites within seven estuaries. We observed increases in the richness and abundance of polychaete worms in heavily modified estuaries with severe metal contamination, but no changes in the diversity or abundance of other taxa. Estuaries in which toxic contaminants were elevated also showed evidence of organic enrichment. We hypothesised that the observed response of polychaetes was not a ‘positive’ response to toxic contamination or a reduction in biotic competition, but due to high levels of nutrients in heavily modified estuaries driving productivity in the water column and enriching the sediment over large spatial scales. We deployed defaunated field-collected sediments from the surveyed estuaries in a small scale experiment, but observed no effects of sediment characteristics (toxic or enriching). Furthermore, invertebrate recruitment instead reflected the low diversity and abundance observed during field surveys of this relatively ‘pristine’ estuary. This suggests that differences observed in the survey are not a direct consequence of sediment characteristics  but are related to parameters that covary with estuary modification such as enhanced productivity from nutrient inputs and the diversity of the local species pool. This has implications for the interpretation of diversity measures in large-scale monitoring studies in which the observed patterns may be strongly influenced by many factors that covary with anthropogenic modification. The intense and extensive development by humans across the planet has subjected much of the world's biological diversity to multiple severe anthropogenic stressors Environmental monitoring studies often rely on ecological measures of community composition and diversity as indicators of anthropogenic change Chemical stressors that are released into estuaries accumulate in benthic sediments We tested whether impacts of large-scale anthropogenic stress in estuaries could be detected over and above natural spatial variation across regional scales (100 s of kms) using an observational approach to assessing the outcome of contamination on benthic diversity. We surveyed benthic sediments in three heavily modified and four relatively ‘pristine’ estuaries This study surveyed benthic invertebrate distributions across estuaries and experiments were conducted on invertebrate communities. Sampling in each location was approved and carried out in strict accordance with the New South Wales Department of Primary Industries and New South Wales Marine Parks (Permit No. P09/0072-1.0).We investigated variation in soft-sediment assemblages in relation to anthropogenic contaminants and other environmental variables at seven sites within seven estuaries. Estuaries were assigned a ‘modification category’ according to the level of anthropogenic modification and the study was conducted in three heavily modified and four relatively unmodified estuaries stretching across 400 km of the coast of New South Wales, Australia . In this3 for a minimum of 24 h and then rinsed in deionised water (Milli-Q™). All sediments for chemical analyses were kept in the dark on ice for transport to the laboratory and then frozen at −20°C until analysis.Benthic sediments were collected between February and March 2010. Two replicate sediment grabs and water quality measures were collected at each site from 5 m depth using a Van Veen grab to target 5 kg surficial sediments. Each sediment grab was homogenised in a clean tray and sub-sampled for infauna (250 mL) and measures of toxic contaminants  (150 g), organic enrichment indicators (sediment Chl-a levels and organic carbon) (150 g) and sediment grain size (50 g). An additional two sediment grabs were taken from sites in 6 of the surveyed estuaries  for the experimental deployment (details below). Plasticware used in sediment collection was previously soaked in 5% HNOTemperature, salinity, turbidity, pH, oxygen and Chl-a levels in the water column directly above sediment collection sites were measured using a multiprobe  calibrated according to the manufacturer's instructions.We used benthic recruitment containers (10 cm diameter ×20 cm height Perspex cylinders) to experimentally investigate the contribution of sediment characteristics to observed patterns by deploying sediments collected during our field survey. Bulk surficial sediment was collected from 5 m depth at 7 sites with unvegetated sediments within 6 estuaries . The sediments collected from multiple sites within each estuary were combined and homogenised then sieved (2 mm) to remove shells and larger fauna and flora. This created one sediment homogenate to represent each estuary. The six homogenates represented different sediment characteristics. Homogenate sediments were then divided into 7 replicate sub- samples (0.5 kg), which were applied as a top sediment layer (∼2 cm) to replicate recruitment containers (N = 42). Approximately 1 kg of sand was used to line the bottom of each of the recruitment containers with the homogenate sediments layered on top. Sand was collected from a site in Port Jackson where the chemical composition had been previously characterised as uncontaminated Infaunal sub-samples (125-ml) were stained with Rose Bengal and preserved in a 7% formalin solution then passed through a 2 mm mesh (to remove large debris) and onto a 500-µm sieve. The remaining organisms were sorted with a dissecting microscope and identified to the lowest feasible taxonomic level (mostly order). Polychaetes comprised >70% of all assemblages and were therefore identified to family and we used this higher taxonomic resolution to focus on patterns in polychaete family diversity and polychaete abundance in response to anthropogenic modification. A reference collection was deposited at the Australian Museum.Sediment characteristics were investigated from benthic sediments collected at each site in the survey and analyses included concentrations of major metal and PAH contaminants, total organic carbon and Chl-a and sediment grain size according to the techniques described in Sediment metal analyses were undertaken using a microwave-assisted acid digestion according to Method 3051A 10, phenanthrene-d10, chrysene-d12 and perylene-d12) were spiked into all samples and recoveries were 111±19%. Individual PAHs were summed for each sample to give a value of total PAHs Sixteen PAHs were analysed including naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluofranthene, pyrene, benz(a)anthracene, chrysene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, indenopyrene, dibenzoanthracene and benzoperylene. Analyses of PAHs in sediments followed Method 8260 Sediment Chl-a analysis were undertaken following extractions of pigments in acetone with a spectrophotometer (Lambda 35 UV/Vis) according to Greenberg et al. Porewaters were extracted from the sediments by centrifugation at 800 g for 5 min and then filtered  immediately to minimise exposure to air. Samples were taken for ammonia . Dissolved ammonia was analysed colorimetrically using a Merck Spectroquant Kit (14752).In all analyses ‘Modification Category’ (heavily modified or relatively unmodified) was treated as a fixed orthogonal factor and ‘Estuary (within Modification Category)’ was a random nested factor. Replicates in each analysis were either sites (survey) or recruitment containers (experiment). Infaunal community composition was compared between modification categories with permutational multivariate analysis of variance (MANOVA) and Principal Components Analysis (PCO). We also investigated changes in taxa richness and abundance between modification categories because these measures are closely linked to contaminants (both inorganic and organic) Principal components analysis (PCA) was used to visualize the entire surveyed infaunal assemblages at each site or in each recruitment container. All analyses were done in PRIMER v6 with PERMANOVA+ Benthic sediments had higher metal  and PAH concentrations in heavily modified estuaries than relatively unmodified estuaries . Many exThe infaunal community composition differed significantly between heavily modified and relatively unmodified estuaries and also among individual estuaries . A combiPolychaete richness (number of families) and abundance  differed significantly between modification categories ; Table 2In summary, sediments from heavily modified estuaries were more contaminated with metals and PAHs and organically enriched than the relatively unmodified estuaries. These measures of toxic and enriching contamination also varied between estuaries Grain sizes were finer in heavily modified estuaries; conversely most variation in water quality was between estuaries rather than between heavily modified and relatively unmodified estuaries. Therefore differences in the infaunal community and measures of polychaete richness and abundance with modification category are likely to be related to contamination or sediment grain size. Infaunal differences between estuaries could be related to metal concentrations, sediment grain size or environmental variables.Characteristics  of the field-collected sediments differed consistently between modification categories at the start and the end of the field experiment . AdditioMost national biomonitoring guidelines recommend the use of multiple lines of evidence when assessing environmental impacts including the use of experiments to establish causation Habitats that are severely stressed by anthropogenic activities are reported to have substantially reduced species richness  that would enrich the nutrients within the estuary and may increase the diversity of the local species pool However, large-scale anthropogenic impacts may not be reflected in smaller-scale manipulations The outcomes of this study have relevance for environmental managers as the patterns we observed across regional scales provided important information about the ecological condition of estuaries and useful directions for future research. The scale of field manipulations may also be an important consideration, as the patterns of increased polychaete richness we observed in heavily modified estuaries across regional scales were not found in our small-scale manipulations within one estuary. This suggests that increases in diversity are related to parameters that co-vary with estuary modification over larger scales such as the productivity of the entire system and the diversity of the regional species pool, and patches of contaminated sediments will not result in similar effects. If environmental managers rely solely on small-scale surveys or manipulations together with meta-analyses from the literature this may lead to a poor understanding of actual impacts over regional scales. Ecological monitoring of human impacts should, as a matter of course, use multiple lines of evidence including field surveys and field experiments in order to reliably identify the drivers of biological patterns and initial observational studies can inform the design of manipulative experiments to further investigate causation.Figure S1Mean (+S.E.) proportional abundances of different taxa analysed from benthic sediment grabs collected in seven estuaries.(DOCX)Click here for additional data file.Figure S2Mean (+S.E.) metal and total PAH concentrations (dw), sediment Chl-a, TOC, porewater ammonia and percent fines (<63 µm) analysed from benthic sediment grabs collected in heavily modified (filled bars) and relatively unmodified (open bars) estuaries.(DOCX)Click here for additional data file.Figure S3Mean (+S.E) values for water quality variables  collected in heavily modified (filled bars) and relatively unmodified (open bars) estuaries.(DOCX)Click here for additional data file.Figure S4Mean (+S.E.) end metal concentrations (dw) and percent fines (<63 µm) analysed from deployed sediments sourced from heavily modified (filled bars) and relatively unmodified (open bars) estuaries. Starting concentrations are indicated as a single figure above the bars.(DOCX)Click here for additional data file.Table S1Details of limits of detection for each of the analytes sampled from sediments.(DOCX)Click here for additional data file.Table S2Soft sediment infauna sampled in benthic sediment surveyed from seven sites in seven NSW estuaries that were either heavily modified or relatively unmodified.(DOCX)Click here for additional data file.Table S3Soft sediment infauna sampled in field manipulated sediment.(DOCX)Click here for additional data file.Table S4Permutational ANOVA results for sediment quality measures in benthic sediment surveyed from seven NSW estuaries.(DOCX)Click here for additional data file.Table S5Permutational ANOVA results for water quality variables surveyed from seven NSW estuaries.(DOCX)Click here for additional data file.Table S6Permutational ANOVA results for metal contaminants and sediment quality variables measured at the start and end in sediment field experiments deployed in the Clyde estuary.(DOCX)Click here for additional data file.


Cervus elaphus) to playbacks of red deer vs. sika deer (Cervus nippon) male mating calls, with the prediction that females will prefer conspecific calls. While on average female red deer preferred male red deer roars, two out of twenty females spent more time in close proximity to the speaker broadcasting male sika deer moans. We suggest that this absence of strict vocal preference for species-specific mating calls may contribute to the permeability of pre-zygotic reproductive barriers observed between these species. Our results also highlight the importance of examining inter-individual variation when studying the role of female preferences in species discrimination and intraspecific mate selection.Males of all polygynous deer species (Cervinae) give conspicuous calls during the reproductive season. The extreme interspecific diversity that characterizes these vocalizations suggests that they play a strong role in species discrimination. However, interbreeding between several species of Cervinae indicates permeable interspecific reproductive barriers. This study examines the contribution of vocal behavior to female species discrimination and mating preferences in two closely related polygynous deer species known to hybridize in the wild after introductions. Specifically, we investigate the reaction of estrous female red deer ( According to the biological species concept, reproductive isolating mechanisms are integral to the process of speciation Acoustic signals can play an important role in the process of mate choice, functioning in both intraspecific mating decisions  and interspecific mating decisions (species discrimination) Cervus elaphus) and Asian sika deer (Cervus nippon). Although closely related This study examines the possible role of acoustic communication in hybridization between two species of Cervinae: European red deer  vs. heterospecific (sika deer) male mating calls on female red deer during estrus and comparing the females' behavioral reactions to calls from these two species exemplars. Given the strong acoustic differences that characterize the species-specific male mating calls of these two species, we predicted that female red deer should show more attention and preference towards the conspecific red deer roars over the heterospecific sika deer moans.Playback trials were conducted during the 2008 breeding season (August 28–29) at the Institut National de la Recherche Agronomique (INRA) Redon Experimental Farm, Clermont-Ferrand, France. Twenty female red deer of reproductive age (age range = 2–15 years old) were used in the experimental trials. This work follows the Association for the Study of Animal Behavior/Animal Behavior Society guidelines for the ethical use of animals in research, and was carried out under the procedural and ethical authorization of the French Ministry of Agriculture .Female reactions to male mating calls may be influenced by the hormonal state of the female The 20 females used in this study were randomly separated into two groups of 10 females each (Group A and Group B). Group A underwent the protocol for estrus synchronization (and the subsequent playback experiments) one day before Group B.Playbacks were created using mating calls recorded from four different male red deer and four different male sika deer. All male exemplars used in this study are unfamiliar to the current experimental subjects. Roars from four adult male red deer were recorded at Redon in 1996 using a Telinga pro-III-S/(DAT) microphone used without the parabolic reflector (flat frequency response = 80−16 kHz±3 dB) and a DAT Sony TCD-D7 recorder  at distances of 2–10 m. Moans from four adult male sika deer were recorded in 2007 at a farm in Waterford, Ireland using a Sanken CS-1 directional condenser microphone (flat frequency response = 50−20 kHz±3 dB) and a Fostex FR-2 digital field recorder . Male red deer roars were re-sampled to 44.1 kHz and the intensity all red and sika deer calls were normalized to 98% of maximum intensity using Cool Edit Pro 2.0 (Syntrillium).Calls were arranged into bouts (or groups) of calls for each male exemplar. To create playbacks that mimicked natural calling patterns for each species exemplar, red deer bouts consisted of one to four roars (separated by 0.5 sec) while sika deer bouts contained only one moan.Playbacks consisted of paired sequences of consecutive calling bouts from both species exemplars. Each playback contained six call bouts from both species exemplars, broadcast in a paired dyadic fashion from two different speakers . The ‘leader’ of each bout pair was alternated for each consecutive pair. The individual bouts within a matched pair of consecutive bouts were separated by 2 sec while bout pairs were separated by 20 sec . A LatinThe total duration of all calls from each species played during the playbacks was equalized so that females were exposed to each species for a similar total amount of time. Each individual red deer playback contained 16 roars (constructed from a pool of 11 different roars with no roars repeated consecutively or within the same bout and no roars used more than twice) arranged in six calling bouts with an average bout duration of 4.93 sec and an average total call duration of 29.59 sec per playback. Each individual sika deer playback contained six different single moan bouts with an average bout duration of 4.96 sec and an average total call duration of 29.81 sec per playback. Overall, the total duration of each dyadic playback was approximately three minutes.2), two Anchor Liberty 6000HIC amplified speakers were hidden behind mesh screens (2 m high×4 m wide) on two ends of the enclosure, facing the entrance (enclosure similar to 2) were demarcated with white chalk lines starting at 8 m outward from the speakers.In a roughly triangular shaped experimental enclosure  SPL at 1 m as measured by Radio Shack Sound Level Meter set for C-weighted fast response. Female behavior was video recorded with a Sony Mini DV DCR-TRV19E camcorder from a hidden, elevated observational room.s = +0.999, p<0.01) on all behavioral measures . Because we could not normalize the data distribution for all the response variables, two-tailed Wilcoxon matched-pair signed-rank tests were used to determine if significant differences were present between the behavioral responses of the females to playbacks of the two species exemplars. All statistical tests were performed using SPSS  and 0.05 levels of significance are quoted.We predicted that females would show more attention and preference towards conspecific calls than heterospecific calls. ‘Attention’ was measured as looking towards the speakers and ‘preference’ was measured as entering proximity zones in front of the speakers. The number of instances and the cumulative duration of these two behaviors were quantitatively coded using the digital video analysis software Gamebreaker 7.0.121  at a 25 fps. Behaviors were coded from the initiation of the playback sequence until 2 min after the last call was broadcast. Looking towards a speaker was defined as occurring when the animal was in a standing position or if moving, when the animal stopped within two steps of initiating the look. A look began when the head first started to orientate to a fixed position facing the speaker and ended when the head started moving away from this fixed position. Entering a proximity zone was defined as starting when the first front leg breaks the outer plane of the zone boundary  and ending when the last front leg passes out of the zone boundary. Video analyses were carried out by MTW with 20% of trials (n = 4) double-coded by DR, resulting in an overall agreement of 99.9%  or in the total duration of looks given by females towards red deer roars or sika deer moans,     . However= 0.015) . Interes= 0.091) , with twOur experiment simulated a situation where allopatric species are brought together and females in estrus are confronted with an unfamiliar species with whom they can hybridize. While these females had been exposed to male red deer and their roars , they had never been exposed to male sika deer. Thus, the playbacks presented during each trial contrasted a familiar conspecific stimulus  with an unfamiliar heterospecific stimulus .p = 0.091) for females to spend more total time in the conspecific proximity zone. Mating preferences for conspecifics over heterospecifics is well documented While female red deer showed no significant difference in attention behaviors between the novel heterospecific and the conspecific stimulus, they entered the conspecific proximity zones significantly more times than the heterospecific proximity zones, indicating an overall preference for conspecific calls. There was also a non-significant trend  combined with geographic isolation and phylogenetic contingencies. Indeed, several experimental studies have confirmed the function of key acoustic properties of male red deer roars in female choice decisions While most female red deer in our experiment preferred conspecific calls, some individuals displayed preferences towards heterospecific calls, possibly as a result of the mechanisms discussed above. Regardless of why some female red deer approach male sika deer moans, in the absence of a harem-holding male red deer, the act of moving towards a reproductively compatible heterospecific caller in the wild could lead to hybridization if the female does not oppose or successfully avoid mating attempts by a male sika deer. Inter-individual differences in mate choice or species discrimination have been correlated with a variety of factors such as the environment This study is an important step in investigating the role that vocalizations may play in intra- and interspecific mating decisions in the context of hybridization. We demonstrated that in a simulated situation where female red deer in estrus encounter both conspecific male red deer roars and novel heterospecific male sika deer moans, most, but crucially, not all females approached the conspecific call. The minority of females that spend more total time near the sika deer speaker may represent cases of incorrect species discrimination where hybridization could occur in the wild. Additionally, we highlight the potential importance of inter-individual differences in mate preferences. Additional studies are being conducted to quantify interspecific and intersexual differences in male and female behavioral and physiological responses to conspecific, heterospecific, and hybrid male mating calls.


Laparoscopic ventral mesh rectopexy using a composite mesh is a technique gaining more recognition for management of pelvic floor disorders such as full thickness rectal prolapse, obstructive defecation symptoms and vaginal vault prolapse. A recent Cochrane review concluded that laparoscopic rectopexy results in fewer post-operative complications and an earlier dischargeTwo longitudinal pieces of the standard 3cm x 20cm strip of polypropylene or polyester mesh are used; the second piece is placed at right angles to prepare a T shape. The second piece is stitched with four sutures, forming a T shape .This prepared mesh is cheaper than pre-packed/shaped meshes. The T shape allows better sitting in the pelvis. It improves area for fixation to the anterior rectum and offers better support.


Rapid diagnosis of influenza not only facilitates timely clinical management, time series test results can also serve for disease surveillance purpose. We evaluated the performance of a community based system using QuickVue Influenza A+B test  for influenza surveillance in Hong Kong.As part of a large community study, subjects older than 2 years reporting at least two symptoms of influenza-like-illness were recruited from 30 outpatient clinics in Hong Kong prospectively from February 2007 to July 2010 around the influenza seasons [A total of 5,824 subjects were recruited throughout the study. We define each study week as either high influenza activity or low influenza activity using fixed thresholds after removing extreme values two standard deviations above the mean. The sensitivity of QuickVue data for picking up high influenza activity was 78% when compare with the laboratory data and was 100% when comparing with the community data.Although the sensitivity of QuickVue Influenza A+B test decreases with specimen viral load, in which may miss out mild influenza infection cases , its exc


Understanding how the intervention works is a primary objective in most pragmatic trials. Although good evidence exists for the effectiveness of cognitive behaviour therapy (CBT), there is little evidence presently that cognitive factors, such as dysfunctional attitudes (DAS) or meta-cognitive awareness (MAQ), mediate the effect of CBT on depression outcomes. Standard regression methods adjusting for such mediators produces biased estimates. Using data from the CoBalT trial, we use a ‘causal inference' approach to examine the mechanism through which CBT affects depression outcomes.The proportion of the causal effect of CBT on depressive symptoms mediated by DAS and MAQ was estimated  using the ‘potential outcomes' approach. The outcome was the Beck Depression Inventory (BDI-II) score at 12 months. Mediators were measured at 6 months post-randomisation.Approximately 25% of the treatment effect observed at 12 months was mediated through changes in DAS  or MAQ at 6 months . This equated, on average, to a one point reduction in the difference in mean BDI-II scores between treatment groups. Sensitivity analyses suggested that a modest degree of ‘hidden' confounding (ρ≅0.3) would violate the key assumption of no confounding between any of the three sets of variables.Changes in cognitive variables contributed a modest proportion of the observed treatment effect. Causality is intuitively counterfactual and use of potential outcomes provides valid causal inference. However, sensitivity analyses suggested that our findings need to be interpreted with caution as hidden confounding may explain the mediated effect.


Participant retention is challenging in long term trials and attrition can threaten trial validity, especially for patient-reported outcomes which cannot be ascertained otherwise. The evidence base to maximise retention is limited so we investigated factors influencing questionnaire response rates with a mixed methods research approach.ProtecT is an ongoing randomised trial of localised prostate cancer treatments in men currently aged 53-76 years (ISCRTN 20141297) with six years follow-up. Symptoms and QoL were collected annually by postal questionnaire with a reminder letter for non-responders at six weeks (phase I). Three additional interventions were sequentially investigated to increase retention. Firstly, clinical centre staff commenced telephoning non-responders at four weeks (phase II). A study pen was included with reminder letters and a shortened questionnaire was sent to non-responders at nine weeks (phase III). Questionnaire response rates over six months were compared by phase. 1279 participants were asked in the questionnaires about online completion. 18 participants gave in-depth interviews about trial follow-up which were transcribed verbatim and analysed thematically.The questionnaire response rate was 76.4% (phase I: 1045/1367) with a median return of 13 days (IQ range 8-22 d) and the reminder letter increased this to 86.8% (1187). Telephoning increased rates to 90.5% (phase II: 1105/1221). The additional incentive of a study pen was ineffective  whilst the short questionnaire had some impact . One quarter of men wished to complete on-line questionnaires .In interviews, most men found questionnaires acceptable and understood their purpose although they were regarded as a less enjoyable aspect. Some men saw questionnaires becoming less relevant over time either because they felt they were cured or they had become repetitive, although they continued to complete them. Participant newsletters were interesting and gave a sense of belonging to a group although some men wanted preliminary trial findings. The study website was infrequently accessed (four men did not use the web), partly because men assumed it contained no extra information.A reminder letter and telephone call increased questionnaire response rates in a trial with older men. A study pen provided no incentive but response rates were already 90%. Men understood the purpose of questionnaires but some attitudes could increase attrition and need to be addressed by trialists. Newsletters maintained participant interest, unlike the website.The ProtecT study is funded by the Health Technology Assessment programme.


Dual left anterior descending artery (LAD) is a rare coronary anomaly that is important to recognize at coronary imaging as it may influence reperfusion strategies. Four types of dual LAD are described by the traditional literature. We present a novel case of dual LAD with coronary computed tomographic angiographic correlation that does not fit into this classification system. Rather, our case supports the recently proposed notion of adding a fifth variant to the traditional dual LAD classification system. Dual left anterior descending artery (LAD) is an extremely rare coronary anomaly traditionally classified into one of four types. Recognition at coronary imaging is critical as both surgical and percutaneous reperfusion strategies may be altered by its presence .A 37-year-old Caucasian male presented with a 1-year history of atypical left-sided chest discomfort. Past medical history was negative for major cardiac risk factors but positive for borderline diabetes, increased body mass index, and gastroesophageal reflux. Physical exam was unremarkable. EKG demonstrated a left anterior fascicular block. Biochemical markers of cardiac ischemia were negative.Single photon emission computed tomography stress myocardial perfusion imaging (SPECT-MPI) revealed a heterogeneous inferolateral and apical myocardial perfusion defect with partial reversibility. Conventional coronary angiography identified an aberrant left anterior descending artery (LAD) originating from the right coronary cusp without evidence of atherosclerosis. To further characterize the anatomy of this aberrant artery, coronary computed tomography angiography (CTA) was performed.CTA revealed a novel case of dual LAD. The long LAD segment arises from a common coronary ostium on the right coronary cusp. It takes an aberrant course posterior to the right ventricular outflow tract. The long LAD has a hammock-like downward slope that closely resembles, on coronal imaging, the hammock sign described on conventional angiography . The lonDual LAD is a rare type of coronary artery anomaly that is important to recognize and characterize as it may critically influence surgical or angiographic reperfusion strategies , 4. In dThere are four types of dual LAD described in the traditional literature that are distinguished by the origins and courses of the long and short segments of the dual LAD system, as well as the supply of the septal perforators and diagonal branches , 4 Tabl. RecentlIn summary, we present an unusual case of dual LAD that does not fit within the traditional classification system. A similar case of this anatomic variant has recently been described in the literature, which the authors have presented as a novel Type V dual LAD  Table 1Table 1.


It is often recommended to test patients presenting for HIV care for tuberculin skin test reactivity at the initial visit if they have a prior stay in tuberculosis-endemic areas.To assess whether incident cases of tuberculosis (tb) in our cohort could have been prevented by such testing and subsequent preventive treatment of patients with a positive skin test.HIV clinic in northern Europe caring for about 1000 patients. During the observation time preventive treatment was only rarely prescribed.File review of all tb cases occurring more than two months after presenting for HIV care during the ten years 2000-2009. Cases were classified as possibly preventable, probably non-preventable or non-preventable based on residence and travel history, strain typing by restriction fragment length polymorphism technique and/or skin test reactivity.24 cases in 23 patients were identified. 9 (38%) were classified as possibly preventable, 3 (13%) as probably non-preventable and 12 (50%) as non-preventable.A minority of tb cases occurring during HIV care is preventable by a policy of testing new patients from endemic areas and treating patients with positive skin tests.


A consistent line of investigation suggests that autonomic nervous system dysfunction may explain the multi-system features of fibromyalgia (FM); and that FM is a sympathetically maintained neuropathic pain syndrome. Dorsal root ganglia (DRG) are key sympathetic-nociceptive short-circuit sites. Sodium channels located in DRG (particularly Nav1.7) act as molecular gatekeepers for pain detection. Nav1.7 is encoded in gene SCN9A of chromosome 2q24.3 and is predominantly expressed in the DRG pain-sensing neurons and sympathetic ganglia neurons. Several SCN9A sodium channelopathies have been recognized as the cause of rare painful dysautonomic syndromes such as paroxysmal extreme pain disorder and primary erythromelalgia. The aim of this study was to search for an association between fibromyalgia and several SCN9A sodium channels gene polymorphisms.We studied 73 Mexican women suffering from FM and 48 age-matched women who considered themselves healthy. All participants filled out the Fibromyalgia Impact Questionnaire (FIQ). Genomic DNA from whole blood containing EDTA was extracted by standard techniques. The following SCN9A single-nucleotide polymorphisms (SNP) were determined by 5' exonuclease TaqMan assays: rs4371369; rs4387806; rs4453709; rs4597545; rs6746030; rs6754031; rs7607967; rs12620053; rs12994338; and rs13017637.P = 0.036) mostly due to an absence of the GG genotype in controls. Interestingly; patients with this rs6754031 GG genotype had higher FIQ scores  than patients with the GT genotype  and the TT genotype .The frequency of the rs6754031 polymorphism was significantly different in both groups (In this ethnic group; a disabling form of FM is associated to a particular SCN9A sodium channel gene variant. These preliminary results raise the possibility that some patients with severe FM may have a dorsal root ganglia sodium channelopathy. The background information leading to the present investigation has been already published as a hypothesis  and is sDRG are nodules that lie along the spinal column. They play a key role in pain perception. DRG house the cell bodies of sensory neurons; the dendrites of which are located in the skin; muscles; tendons; joints; and internal organs. DRG act also as viral sanctuaries. Under normal circumstances; DRG have scant sympathetic innervation. Nevertheless; trauma and/or infection trigger sympathetic sprouting within DRG via nerve growth factor over-expression. Such aberrant neuroplasticity enables catecholamines and sympathetic traffic to induce sensory neuron firing. Sodium channels play a pivotal role in this hyperexcitability. These mechanisms are the basis of the sympathetically-maintained pain concept .Sodium channels located in DRG act as molecular gatekeepers of pain detection at peripheral nociceptors. Nine sodium channel subunits have been identified (Nav1.1-Nav1.9); each with a unique central and peripheral nervous system distribution. An isoform Nav1.7) encoded in gene SCN9A of chromosome 2q24.3 is predominantly expressed in the DRG pain-sensing neurons and sympathetic ganglia neurons . Several encoded This information lead us to the present investigation whose objective was to search for an association between several SCN9A sodium channels gene polymorphisms and fibromyalgia.We studied 73 Mexican women suffering from FM according to the 1990 American College of Rheumatology criteria. These patients were referred from different private rheumatology practices in Mexico. As controls; we studied 48 Mexican women who considered themselves to be healthy and who denied having chronic pain. Patients and controls were matched by age. Mexicans have diverse ethnicity. The great majority have European and Native American background. "Mestizo" is the term used to name this mixed heritage. All individuals included in the study were born in Mexico and were catalogued as Mestizo. No further genealogical characterization was done. Both patients and controls filled out a validated Spanish translation of the Fibromyalgia Impact Questionnaire (FIQ) [This research complies with the Helsinki Declaration. Informed consent was obtained from all participants; and the Human Research Committee of the National Institute of Cardiology of Mexico approved the study. This group of subjects is the same reported in our previous genetic studies in FM looking for polymorphisms of the adrenergic receptor  and cateGenomic DNA from whole blood containing EDTA was extracted by standard techniques . Ten difP > 0.05 by Shapiro-Wilk statistics); therefore; data were measured as median and interquartile range. The Kruskal-Wallis test was used to compare visual analog scale (VAS) scores on the FIQ with SNPs and genotypes. Proportions of patients and controls per SNP were estimated and compared using chi square or Fisher's exact tests; as required; and presented as absolute frequencies and proportions. Statistical significance was set at an alpha level of less than or equal to 0.05. Pair wise linkage disequilibrium  estimations between polymorphisms and haplotype reconstruction were performed with Haploview version 3:32 .Statistical analysis was carried out with Stata 8.0 software for Windows . A chi-square test was used to evaluate Hardy-Weinberg equilibrium for each polymorphism. In the exploratory analysis; numeric data showed a non-normal (non-Gaussian) distribution (P = 0.036) mostly due to an absence of the GG genotype in controls. Correlation analysis between SNPs and total FIQ score showed that patients with the rs6754031 GG genotype had higher total FIQ scores  than patients with the GT genotype  or the TT genotype   had strong LD. One SNP (rs6746030) was not studied because too few individuals were determined for this variant. In light of the strong LD; we analyzed the most frequent haplotypes in patients and healthy controls to determine whether some of these haplotypes could be associated with the risk of developing FM. The distribution of the different haplotypes was similar in patients and healthy controls (data not shown).This group of Mexican patients suffering from FM displayed different distribution of SCN9A rs6754031 genotypes when compared to ethnic; age; and gender matched controls. The difference was due mostly to the absence of the rs6754031 GG genotype in controls. Additionally; in this group of FM patients; the rs6754031 GG genotype is associated with severe disease; as assessed by the FIQ. To our knowledge there is no prior research linking rs6754031 with any particular illness. Although no causality could be derived from the present investigation; these preliminary results raise the possibility that some patients with severe FM may have a DRG sodium channelopathy.We are not aware of previous studies looking at SCN9A sodium channels gene variation in FM. However; Reimann et al. studied several SCN9A polymorphisms in patients with different painful syndromes; including osteoarthritis; sciatica; amputee phantom pain; spine surgery and pancreatitis. They found a link of rs6746030 with pain intensity. They did not study the SNP rs6754031 that we found associated to FM severity . Nav1.7 After the submission of this manuscript Faber et al. published a gain of function SCN9A mutation associated to idiopathic small fiber neuropathy. This novel finding points to a broader role of Na(V) 1.7 mutations in painful neurological disease than previously considered from studies on rare genetic syndromes .Our previous genetic studies in this group of patients found an association of two catechol-o-methyl transferase (COMT) enzyme gene polymorphisms with FIQ subscales. COMT rs6269 was associated with pain and fatigue; and COMT rs165599 with morning stiffness and disability . The resWe recognize limitations in this pilot study. The sample size is not large enough to withstand multiple comparisons of common gene polymorphisms; therefore a type II statistical error is possible. Nevertheless; the following two findings favor a real linkage between rs6754031 GG genotype and Mexican FM patients: 1) rs6754031 polymorphism was significantly different in both groups mostly due to an absence of the GG genotype in controls. 2) In the patients group the same rs6754031 GG genotype was associated to a severe disease. Studies in other ethnic groups with a larger sample size are needed to verify or amend these preliminary observations.Another limitation of our study is the lack of rs6754031 functional profiling. Techniques such as patch methods or functional expression assays are beyond our capabilities. So; at the present time; the link between SCN9A polymorphism and severe FM is just associative. Nevertheless; our findings raise the possibility that DRG could be an important element in the pathogenesis of FM; a site in which physical; emotional; or infective stimuli could be converted into chronic pain . GeneticThe results of the present investigation may have potential therapeutic implications. If future investigations confirm the Nav1.7 sodium channels dysfunction in FM; then sodium channel blockers may become a therapeutic option. Preliminary evidence supports this contention. Lignocaine; a non-specific sodium channel blocker; appears to reduce FM pain . DuloxetOur results show that; in this group of Mexican women; there is an association of the rs6754031 polymorphism with the risk of developing FM; as well as with the FIQ score. This association raises the possibility that some patients with severe FM may have a DRG sodium channelopathy.FM: Fibromyalgia; DRG: Dorsal root ganglia; FIQ: Fibromyalgia Impact Questionnaire; SNP: Single nucleotide polymorphism; COMT: Catechol o methyl transferase; VAS: Visual analogue scale; AR: Adrenergic receptorThe authors declare that they have no competing interests.GVA; EAL and VMF carried out the genetic studies. AM and AG recruited patients and were involved in the clinical part of the investigation. MV performed the statistical analysis. All authors read and approved the final manuscript. MML conceived the study; participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.We gratefully recognize the financial support of the American Fibromyalgia Syndrome Association.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/13/23/prepub


Navicular stress fracture (NSF) is a relatively uncommon injury according to Australian Rules Football (AFL) injury surveillance data. Like all stress fractures, a disproportionate amount of compression / strain (dose) versus remodeling (response) accounts for bone breakdown. The exact mechanism of injury or anthropometric factors involved in navicular stress fracture remains largely unknown however; several authors speculate a short first metatarsal and metatarsus adductus as contributing factors. It is thought that a long second metatarsal transmits forces unevenly through the medial cuneiform creating shearing stress at the avascular central zone of the navicular. To date no prospective data is available to support these theories. Navicular stress fracture is deemed high risk due to the potential of delayed or non union and often excessive time away from activity.2), Force and Pressure Time Integrals were used for further anaylsis.Retrospective plantar pressure mapping of two elite AFL footballers who’d sustained NSF injury were recorded. Testing involved unshod across a floor mounted pressure sensing mat  using a two-step method following familiarisation. Five (5) trials were performed for each foot with the averages of the middle three (3) trials used for further analyses. Subsequent testing using an in-shoe pressure mapping system,  was performed with custom moulded foot orthoses in situ. Data from twenty (20) steps were recorded, allowing for acceleration and deceleration with the middle ten (10) steps used for further analyses. Computer software generated data for Peak Pressure across 10 masked areas (kPa), Instant to Peak Pressure (ms), Contact Area  beneath the 2Presented is the use of plantar pressure mapping in NSF injury in two Elite AFL footballers. The results suggest increased FF pressure and anatomical variations such as a short 1st metatarsal and metatarsus adductus could contribute to NSF injury. Technology such as plantar pressure mapping may be used prospectively to assess or screen at risk groups for NSF injury.


Neuropeptides play a critical role in regulating these processes. Neuropeptide Y (NPY) is highly expressed in limbic brain areas, including the amygdala. Depending on the receptor subtypes involved  mice display a dramatically accelerated acquisition of conditioned fear while fear extinction was impaired. Interestingly this phenotpye was only reproduced in mice lacking both the Y1 and Y2 receptors are involved in these processes. Y1 receptors in the basolateral amygdala are modulating the acquisition and extinction of fear while Y2 receptors in the central amygdala are preferentially inhibiting the expression but facilitating the extinction of learned fear. Furthermore, Y2 receptors are crucially involved in the discrimination of fear-related stimuli.Taken together, our data demonstrate that NPY delays acquisition and reduces expression of conditioned fear whereas it promotes fear extinction. Both Y


Acute respiratory failure (ARF), a common syndrome, is still poorly clinically appreciated. Literature review reports only a few attempts in neonatology (Silverman score) and in adults (Patrick score ) constru2/FiO2 <250 and/or ventilatory support).A prospective study among 70 patients with ARF on previously healthy lungs. ARF was rated in a randomized blinded manner respectively by residents and seniors. An inter-rater reliability analysis using the kappa statistic was performed to determine consistency among raters. Clinimetric properties were assessed by examining the prognostic prediction by the ROC curve using a composite gold standard (PaOP < 0.001), indicating an almost perfect agreement [The inter-rater reliability for the raters was found to be κ = 0.82 (This new and validated score could drive some advantages in daily practice, allowing accurate assessment of ARF severity, more objective monitoring of patients and easier communication between care providers. It may accurately guide oxygen supplementation and ventilatory support and afford accurate monitoring of patho-physiological and etiological treatment of ARF. It could be a valuable tool in randomized clinical trials or physiological studies evaluating treatments in ARF. Finally it could be used as an educational tool.


The rewarming benefit of anterior torso heat pad application in mildly hypothermic conscious adult trauma patients remains inconclusive in this randomized comparative clinical trial. There was no between-group rewarming gain in ear canal temperature when an anterior torso chemical heat pad was compared with blankets. Patient awareness, and favorable perception of, being administered the active intervention (heat pad) could explain the significant improvement in patient-rated cold discomfort discerned with the heat pad. In the context of marginal demonstrated benefit, it would have been informative to ascertain adverse effects related to the heat pad, including burn injury to the chest wall. In their randomized clinical trial of managing hypothermia in trauma, Lundgren at al  attempt Improvements in heart rate, systolic blood pressure and respiratory rate seem to not be clinically consequential; the heat pad was associated with - 4 beats/min for heart rate compared with -1 beat/min in patients that were allocated blankets. The favourably biased view that a patient holds of any active treatment in a non-blinded intervention trial  could suA heat pad weighing 1400 grams  could thAlthough spontaneous hypothermia in conjunction with acidosis and coagulopathy is harmful in severe trauma , there i°C: degrees centigradeThe author declares that they have no competing interests.


Cyanobacteria are a rich source of natural products with interesting biological activities. Many of these are peptides and the end products of a non-ribosomal pathway. However, several cyanobacterial peptide classes were recently shown to be produced through the proteolytic cleavage and post-translational modification of short precursor peptides. A new class of bacteriocins produced through the proteolytic cleavage and heterocyclization of precursor proteins was recently identified from marine cyanobacteria. Here we show the widespread occurrence of bacteriocin gene clusters in cyanobacteria through comparative analysis of 58 cyanobacterial genomes. A total of 145 bacteriocin gene clusters were discovered through genome mining. These clusters encoded 290 putative bacteriocin precursors. They ranged in length from 28 to 164 amino acids with very little sequence conservation of the core peptide. The gene clusters could be classified into seven groups according to their gene organization and domain composition. This classification is supported by phylogenetic analysis, which further indicated independent evolutionary trajectories of gene clusters in different groups. Our data suggests that cyanobacteria are a prolific source of low-molecular weight post-translationally modified peptides. Bacteriocins are secondary metabolites and have been found in all major lineages of bacteria in silico screening for bacteriocins in Gram-negative bacteria had revealed the presence of sixteen double-glycine-type precursors and ten cognate transporters from strains of the cyanobacteria Nostoc, Prochlorococcus, Synechococcus and Synechocystis3E(I/L)X2(I/V)XG(G/A) Prochlorococcus marinus MIT9313 Cyanobacteria are a prolific source of natural products and secondary metabolites In order to explore the genetic potential for bacteriocin production in cyanobacteria, we mined 58 cyanobacterial genomes to identify the organization of bacteriocin-processing gene clusters. Surprisingly, we found more than a hundred new putative bacteriocin gene clusters from genomes of nearly all examined cyanobacterial species. Nearly 300 putative precursor genes were encoded in close proximity to the bacteriocin gene clusters. Our results demonstrate the widespread presence of bacteriocin gene clusters in cyanobacteria. The genetic diversity of the core peptides of these bacteriocin precursors is enormous with little sequence conservation.A total of 145 putative bacteriocin gene clusters were identified in 43 cyanobacteria , Table 1Cyanothece PCC 7425 and Nostoc punctiforme PCC 73102 each have four LanM encoding gene clusters, the other strains each have only one. Twelve gene clusters belong to group V were found in nine genomes . Protein sequences of these genomes were extracted and formatted for local BLAST searches. Three tailored query files containing FASTA format protein sequences of bacteriocin synthesis genes were constructed. The first one consisted of 30 representative proteins of the C39 peptidase domain-containing ABC transporter (cd02259), the second contained 14 HlyD family proteins (TIGR01843). Sequences in these two files were collected from the NCBI Conserved Domains database Genomic data of 58 cyanobacterial strains  were dowE < 0.00001) The three query files were utilized for searching against the database containing all proteins of the collected cyanobacterial genomes. Protein hits of blastP (E < 0. 1) Precursor genes were searched in a 20 Kb range upstream and downstream for each gene cluster. Small ORFs and intergenic regions were manually checked by searching for the double glycines. These predicted precursors were compared to known precursor families Gammaproteobacterium HdN1, Bradyrhizobium japonicum USDA 110, and Escherichia coli UMN026. The second midpoint tree  int tree  is generFigure S1Sequence alignments of putative novel cyanobacterial bacteriocin precursors. (A) Ten selected HetP substrates are shown in a ClustalW alignment (PDF)Click here for additional data file.Figure S2Sequence logo of motif with double-glycine found in the putative HetP precursors in cyanobacteria. A conserved region was found near the peptide cleavage site with Gly-Gly motif from the putative HetP precursor proteins in cyanobacteria. Here the sequence logo with relative frequency of acidic residues of the conserved sequences is demonstrated. This figure was generated by web-based software (PDF)Click here for additional data file.Table S1Cyanobacterial genomes analyzed in this study .(PDF)Click here for additional data file.Table S2Conserved domains identified in cyanobacterial bacteriocin gene clusters.(PDF)Click here for additional data file.Table S3Cyanobacterial precursors identified in this study.(XLS)Click here for additional data file.