ALBERT

Description: The predicted effect of effective population size on the distribution of fitness effects and substitution rate is critically dependent on the relationship between sequence and fitness. This highlights the importance of using models that are informed by the molecular biology, biochemistry, and biophysics of the evolving systems. We describe a computational model based on fundamental aspects of biophysics, the requirement for (most) proteins to be thermodynamically stable. Using this model, we find that differences in population size have minimal impact on the distribution of population-scaled fitness effects, as well as on the rate of molecular evolution. This is because larger populations result in selection for more stable proteins that are less affected by mutations. This reduction in the magnitude of the fitness effects almost exactly cancels the greater selective pressure resulting from the larger population size. Conversely, changes in the population size in either direction cause transient increases in the substitution rate. As differences in population size often correspond to changes in population size, this makes comparisons of substitution rates in different lineages difficult to interpret.

Description: O -GlcNAcylation is an inducible, highly dynamic and reversible post-translational modification, mediated by a unique enzyme named O -linked N -acetyl- d -glucosamine ( O -GlcNAc) transferase (OGT). In response to nutrients, O -GlcNAc levels are differentially regulated on many cellular proteins involved in gene expression, translation, immune reactions, protein degradation, protein–protein interaction, apoptosis and signal transduction. In contrast to eukaryotic cells, little is known about the role of O -GlcNAcylation in the viral life cycle. Here, we show that the overexpression of the OGT reduces the replication efficiency of Kaposi's sarcoma-associated herpesvirus (KSHV) in a dose-dependent manner. In order to investigate the global impact of O -GlcNAcylation in the KSHV life cycle, we systematically analyzed the 85 annotated KSHV-encoded open reading frames for O -GlcNAc modification. For this purpose, an immunoprecipitation (IP) strategy with three different approaches was carried out and the O -GlcNAc signal of the identified proteins was properly controlled for specificity. Out of the 85 KSHV-encoded proteins, 18 proteins were found to be direct targets for O -GlcNAcylation. Selected proteins were further confirmed by mass spectrometry for O -GlcNAc modification. Correlation of the functional annotation and the O -GlcNAc status of KSHV proteins showed that the predominant targets were proteins involved in viral DNA synthesis and replication. These results indicate that O -GlcNAcylation plays a major role in the regulation of KSHV propagation.

Description: Galectins are potent adhesion/growth-regulatory effectors with characteristic expression profiles. Understanding the molecular basis of gene regulation in each case requires detailed information on copy number of genes and sequence(s) of their promoter(s). Our report reveals plasticity in this respect between galectins and species. We here describe occurrence of a two-gene constellation for human galectin (Gal)-7 and define current extent of promoter-sequence divergence. Interestingly, cross-species genome analyses also detected single-copy display. Because the regulatory potential will then be different, extrapolations of expression profiles are precluded between respective species pairs. Gal-4 coding in chromosomal vicinity was found to be confined to one gene, whereas copy-number variation also applied to Gal-9. The example of rat Gal-9 teaches the lesson that the presence of multiple bands in Southern blotting despite a single-copy gene constellation is attributable to two pseudogenes. The documented copy-number variability should thus be taken into consideration when studying regulation of galectin genes, in a species and in comparison between species.

Description: In studying the molecular basis for the potent immune activity of previously described gamma and delta inulin particles and to assist in production of inulin adjuvants under Good Manufacturing Practice, we identified five new inulin isoforms, bringing the total to seven plus the amorphous form. These isoforms comprise the step-wise inulin developmental series amorphous -〉 alpha-1 (AI-1) -〉 alpha-2 (AI-2) -〉 gamma (GI) -〉 delta (DI) -〉 zeta (ZI) -〉 epsilon (EI) -〉 omega (OI) in which each higher isoform can be made either by precipitating dissolved inulin or by direct conversion from its precursor, both cases using regularly increasing temperatures. At higher temperatures, the shorter inulin polymer chains are released from the particle and so the key difference between isoforms is that each higher isoform comprises longer polymer chains than its precursor. An increasing trend of degree of polymerization is confirmed by end-group analysis using 1 H nuclear magnetic resonance spectroscopy. Inulin isoforms were characterized by the critical temperatures of abrupt phase-shifts (solubilizations or precipitations) in water suspensions. Such (aqueous) "melting" or "freezing" points are diagnostic and occur in strikingly periodic steps reflecting quantal increases in noncovalent bonding strength and increments in average polymer lengths. The (dry) melting points as measured by modulated differential scanning calorimetry similarly increase in regular steps. We conclude that the isoforms differ in repeated increments of a precisely repeating structural element. Each isoform has a different spectrum of biological activities and we show the higher inulin isoforms to be more potent alternative complement pathway activators.

Description: The methylotrophic yeast, Pichia pastoris , is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N -linked glycans but up to now no one has addressed engineering the O -linked glycosylation pathway. Typically, O -linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with β- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O -linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O -linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein- O -linked-mannose β-1,2- N -acetylglucosaminyltransferase 1, resulted in the capping of the single O -linked mannose residues with N -acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O -linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N -linked glycosylated biotherapeutics to include molecules possessing O -linked glycans.

Description: Neurons and other cells require intracellular transport of essential components for viability and function. Previous work has shown that while net amyloid precursor protein (APP) transport is generally anterograde, individual vesicles containing APP move bi-directionally. This discrepancy highlights our poor understanding of the in vivo regulation of APP-vesicle transport. Here, we show that reduction of presenilin (PS) or suppression of gamma-secretase activity substantially increases anterograde and retrograde velocities for APP vesicles. Strikingly, PS deficiency has no effect on an unrelated cargo vesicle class containing synaptotagmin, which is powered by a different kinesin motor. Increased velocities caused by PS or gamma-secretase reduction require functional kinesin-1 and dynein motors. Together, our findings suggest that a normal function of PS is to repress kinesin-1 and dynein motor activity during axonal transport of APP vesicles. Furthermore, our data suggest that axonal transport defects induced by loss of PS-mediated regulatory effects on APP-vesicle motility could be a major cause of neuronal and synaptic defects observed in Alzheimer's Disease (AD) pathogenesis. Thus, perturbations of APP/PS transport could contribute to early neuropathology observed in AD, and highlight a potential novel therapeutic pathway for early intervention, prior to neuronal loss and clinical manifestation of disease.

Description: With age, muscle mass and integrity are progressively lost leaving the elderly frail, weak and unable to independently care for themselves. Defined as sarcopenia, this age-related muscle atrophy appears to be multifactorial but its definite cause is still unknown. Mitochondrial dysfunction has been implicated in this process. Using a novel transgenic mouse model of mitochondrial DNA (mtDNA) double-strand breaks (DSBs) that presents a premature aging-like phenotype, we studied the role of mtDNA damage in muscle wasting. We caused DSBs in mtDNA of adult mice using a ubiquitously expressed mitochondrial-targeted endonuclease, mito- Pst I. We found that a short, transient systemic mtDNA damage led to muscle wasting and a decline in locomotor activity later in life. We found a significant decline in muscle satellite cells, which decreases the muscle's capacity to regenerate and repair during aging. This phenotype was associated with impairment in acetylcholinesterase (AChE) activity and assembly at the neuromuscular junction (NMJ), also associated with muscle aging. Our data suggests that systemic mitochondrial dysfunction plays important roles in age-related muscle wasting by preferentially affecting the myosatellite cell pool.

Description: Background: Despite their widespread use, the biological mechanisms underlying the efficacy of psychotropic drugs are still incompletely known; improved understanding of these is essential for development of novel more effective drugs and rational design of therapy. Given the large number of psychotropic drugs available and their differential pharmacological effects, it would be important to establish specific predictors of response to various classes of drugs. Results: To identify the molecular mechanisms that may initiate therapeutic effects, whole-genome expression profiling (using 324 Illumina Mouse WG-6 microarrays) of drug-induced alterations in the mouse brain was undertaken, with a focus on the time-course (1, 2, 4 and 8 h) of gene expression changes produced by eighteen major psychotropic drugs: antidepressants, antipsychotics, anxiolytics, psychostimulants and opioids. The resulting database is freely accessible at www.genes2mind.org. Bioinformatics approaches led to the identification of three main drug-responsive genomic networks and indicated neurobiological pathways that mediate the alterations in transcription. Each tested psychotropic drug was characterized by a unique gene network expression profile related to its neuropharmacological properties. Functional links that connect expression of the networks to the development of neuronal adaptations (MAPK signaling pathway), control of brain metabolism (adipocytokine pathway), and organization of cell projections (mTOR pathway) were found. Conclusions: The comparison of gene expression alterations between various drugs opened a new means to classify the different psychoactive compounds and to predict their cellular targets; this is well exemplified in the case of tianeptine, an antidepressant with unknown mechanisms of action. This work represents the first proof-of-concept study of a molecular classification of psychoactive drugs.

Description: Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5[prime]and 3[prime]junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the position effect of identical transgene located in diverse chromosomal contexts. These findings also form the basis for targeted pig genome engineering and may be used to produce genetically modified pigs for agricultural and biomedical uses.

Description: Background: The molecular epidemiology of C. jejuni and C. coli clinical strains isolated from children with gastroenteritis, was investigated using the multilocus sequence typing method (MLST). This analysis establishes for the first time in Greece and constitutes an important tool for the epidemiological surveillance and control of Campylobacter infection in our country. Methods: The MLST genotypes were compared with those gained by other typing methods (HS-typing, PFGE and FlaA typing) and were also phylogenetically analyzed, in order to uncover genetic relationships. Results: Among 68 C. jejuni strains, 41 different MLST-Sequence Types (MLST-STs) were found. Fifty six strains or 34 MLST-STs could be sorted into 15 different MLST-Sequence Type Complexes (MLST-STCs), while twelve strains or seven MLST-STs did not match any of the MLST-STCs of the database. Twenty C. coli strains belonged to 14 different MLST-STs. Eleven MLST-STs were classified in the same MLST-STC (828), and three were unclassifiable. There was no significant association between the MLST-STs and the results of the other typing methods.Phylogenetic analysis revealed that some strains, classified to the species of C. jejuni, formed a separate, phylogenetically distinct group. In eight strains some alleles belonging to the taxonomic cluster of C. jejuni, were also detected in C. coli and vice versa, a phenomenon caused by the genetic mosaic encountered inside the genus Campylobacter. Conclusions: The MLST-ST determination proved to be a very useful tool for the typing as well as the identification of Campylobacter on the species level.

Description: Background: Opsins have been found in the majority of animals and their most apparent functions are related to vision and light-guided behaviour. As an increasing number of sequences have become available it has become clear that many opsin-like transcripts are expressed in tissues other than the eyes. Opsins can be divided into three main groups: rhabdomeric opsins (r-opsins), ciliary opsins (c-opsins) and group 4 opsins. In arthropods, the main focus has been on the r-opsins involved in vision. However, with increased sequencing it is becoming clear that arthropods also possess opsins of the c-type, group 4 opsins and the newly discovered arthropsins but the functions of these opsins are unknown in arthropods and data on their localisation is limited or absent. Results: We identified opsins from the spider Cupiennius salei and the onychophoran Euperipatoides kanangrensis and characterised the phylogeny and localisation of these transcripts. We recovered all known visual opsins in C. salei, and in addition found a peropsin, a c-opsin and an opsin resembling Daphnia pulex arthropsin. The peropsin was expressed in all eye types except the anterior median eyes. The arthropsin and the c-opsin were expressed in the central nervous system but not the eyes. In E. kanangrensis we found: a c-opsin; an opsin resembling D. pulex arthropsins; and an r-opsin with high sequence similarity to previously published onychophoran onychopsins. The E. kanangrensis c-opsin and onychopsin were expressed in both the eyes and the brain but the arthropsin only in the brain. Conclusion: Our novel finding that opsins of both the ciliary and rhabdomeric type are present in the onychophoran and a spider suggests that these two types of opsins were present in the last common ancestor of the Onychophora and Euarthropoda. The expression of the c-opsin in the eye of an onychophoran indicates that c-opsins may originally have been involved in vision in the arthropod clade. The lack of c-opsin expression in the spider retina suggests that the role for c-opsin in vision was lost in the euarthropods. Our discovery of arthropsin in onychophorans and spiders dates the emergence of arthropsin to the common ancestor of Onychophora and Euarthropoda and their expression in the brain suggests a non-visual function.

Description: Populations of widely distributed species encounter and must adapt to local environmental conditions. However, comprehensive characterization of the genetic basis of adaptation is demanding, requiring genome-wide genotype data, multiple sampled populations, and an understanding of population structure and potential selection pressures. Here, we used single-nucleotide polymorphism genotyping and data on numerous environmental variables to describe the genetic basis of local adaptation in 21 populations of teosinte, the wild ancestor of maize. We found complex hierarchical genetic structure created by altitude, dispersal events, and admixture among subspecies, which complicated identification of locally beneficial alleles. Patterns of linkage disequilibrium revealed four large putative inversion polymorphisms showing clinal patterns of frequency. Population differentiation and environmental correlations suggest that both inversions and intergenic polymorphisms are involved in local adaptation.

Description: Background: Statin-fibrate combination therapy has been used to treat patients with acute coronary syndrome (ACS) complicated by elevated triglycerides (TG) and decreased high density lipoprotein cholesterol (HDL-C). The purpose of this study was to evaluate the influence of the combination therapy on lipids profile and apolipoprotein A5 (apoA5) level in patients with ACS. Methods: One hundred and four patients with ACS were recruited and randomly assigned into two groups: one was statin group (n = 52), given atorvastatin (20 mg QN) or other statins with equivalent dosages; the other was combination group (n = 52), given the same dose of statin plus bezafibrate (200 mg BID). Follow-up visits were scheduled at the end of 6 and 12 weeks post treatment. Serum apoA5 levels were determined using a commercial available ELISA kit. Results: (1) Compared with that of statin monotherapy, statin-bezafibrate combination treatment not only resulted in a significant reduction of TG, TC and LDL-C levels , (all p 〈 0.05), but also led to increases in HDL-C and apoA5 levels (p 〈 0.05).(2) The percentage changes of TC, TG, LDL-C and apoA5 levels in both groups were even bigger at 12 weeks after treatment than that at 6 weeks.(all p 〈 0.05). Similarly, the rates of achieving lipid-control target were higher in statin-bezafibrate combination treatment group than those in statin monotherapy group (all p 〈 0.05).(3) Spearman rank correlation analysis showed that the pre-treatment apoA5 level was positively correlated with TG (r = 0.359, p = 0.009). However, a negative correlation was observed between apoA5 and TG (r = -0.329, p = 0.017) after 12 weeks treatment. Conclusions: Statin and fibrate combination therapy is more effective than statin alone in achieving a comprehensive lipid control for ACS patients. Serum apoA5 elevation after statin and fibrate combination treatment could be due to the synergistic effect of both drugs on hypertriglyceridemia control.

Description: Not all calcite ballast is created equal: differing effects of foraminiferan and coccolith calcite on the formation and sinking of aggregates Biogeosciences Discussions, 10, 14861-14885, 2013 Author(s): K. Schmidt, C. L. De La Rocha, M. Gallinari, and G. Cortese Correlation between particulate organic carbon (POC) and calcium carbonate sinking through the deep ocean has led to the idea that ballast provided by calcium carbonate is important for the export of POC from the surface ocean. While this idea is certainly to some extent true, it is worth considering in more nuance, for example, examining the different effects on the aggregation and sinking of POC of small, non-sinking calcite particles like coccoliths and large, rapidly sinking calcite like planktonic foraminiferan tests. We have done that here in a simple experiment carried out in roller tanks that allow particles to sink continuously without being impeded by container walls. Coccoliths were efficiently incorporated into aggregates that formed during the experiment, increasing their sinking speed compared to similarly sized aggregates lacking added calcite ballast. The foraminiferan tests, which sank as fast as 700 m d −1 , became associated with only very minor amounts of POC. In addition, when they collided with other, larger, foraminferan-less aggregates, they fragmented them into two smaller, more slowly sinking aggregates. While these effects were certainly exaggerated within the confines of the roller tanks, they clearly demonstrate that calcium carbonate ballast is not just calcium carbonate ballast- different forms of calcium carbonate ballast have notably different effects on POC aggregation, sinking, and export.

Description: Background: The geographic distribution of evolutionary lineages and the patterns of gene flow upon secondary contact provide insight into the process of divergence and speciation. We explore the evolutionary history of the common lizard Zootoca vivipara (= Lacerta vivipara) in the Iberian Peninsula and test the role of the Pyrenees and the Cantabrian Mountains in restricting gene flow and driving lineage isolation and divergence. We also assess patterns of introgression among lineages upon secondary contact, and test for the role of high-elevation trans-mountain colonisations in explaining spatial patterns of genetic diversity. We use mtDNA sequence data and genome-wide AFLP loci to reconstruct phylogenetic relationships among lineages, and measure genetic structure Results: The main genetic split in mtDNA corresponds generally to the French and Spanish sides of the Pyrenees as previously reported, in contrast to genome-wide AFLP data, which show a major division between NW Spain and the rest. Both types of markers support the existence of four distinct and geographically congruent genetic groups, which are consistent with major topographic barriers. Both datasets reveal the presence of three independent contact zones between lineages in the Pyrenean region, one in the Basque lowlands, one in the low-elevation mountains of the western Pyrenees, and one in the French side of the central Pyrenees. The latter shows genetic evidence of a recent, high-altitude trans-Pyrenean incursion from Spain into France. Conclusions: The distribution and age of major lineages is consistent with a Pleistocene origin and a role for both the Pyrenees and the Cantabrian Mountains in driving isolation and differentiation of Z. vivipara lineages at large geographic scales. However, mountain ranges are not always effective barriers to dispersal, and have not prevented a recent high-elevation trans-Pyrenean incursion that has led to asymmetrical introgression among divergent lineages. Cytonuclear discordance in patterns of genetic structure and introgression at contact zones suggests selection may be involved at various scales. Suture zones are important areas for the study of lineage formation and speciation, and our results show that biogeographic barriers can yield markedly different phylogeographic patterns in different vertebrate and invertebrate taxa.

Description: Background: Loss of CpG dinucleotides in genomic DNA through methylation-induced mutation is characteristic of vertebrates and plants. However, these and other eukaryotic phyla show a range of other dinucleotide frequency biases with currently uncharacterized underlying mutational or selection mechanisms. We developed a parameterized Markov process to identify what neighbour context-dependent mutations best accounted for patterns of dinucleotide frequency biases in genomic and cytoplasmically expressed mRNA sequences of different vertebrates, other eukaryotic groups and RNA viruses that infect them. Results: Consistently, 11- to 14-fold greater frequencies of the methylation-association mutation of C to T upstream of G (depicted C[rightwards arrow]T,G) than other transitions best modelled dinucleotide frequencies in mammalian genomic DNA. However, further mutations such as G[rightwards arrow]T,T (5-fold greater than the default transversion rate) were required to account for the full spectrum of dinucleotide frequencies in mammalian sequence datasets. Consistent with modeling predictions for these two mutations, instability of both CpG and CpT dinucleotides was identified through SNP frequency analysis of human DNA sequences. Different sets of context-dependent mutations were modelled in other eukaryotes with non-methylated genomic DNA. In contrast to genomic DNA, best-fit models of dinucleotide frequencies in transcribed RNA sequences expressed in the cytoplasm from all organisms were dominated by mutations that eliminated UpA dinucleotides, observations consistent with cytoplasmically driven selection for mRNA stability. Surprisingly, mRNA sequences from organisms with methylated genomes showed evidence for additional selection against CpG through further context-dependent mutations (eg. C[rightwards arrow]A,G). Similar mutation or selection processes were identified among single-stranded mammalian RNA viruses; these potentially account for their previously described but unexplained under-representations of CpG and UpA dinucleotides. Conclusions: Methods we have developed identify mutational processes and selection pressures in organisms provide new insights into nucleotide compositional constraints and a wealth of biochemical and evolutionarily testable predictions for the future.

Description: Background: Ramie fiber extracted from stem bark is one of the most important natural fibers. Drought is a main environment stress which severely inhibits the stem growth of ramie and leads to a decrease of the fiber yield. The drought stress-regulatory mechanism of ramie is poorly understood.Result: Using Illumina sequencing, approximately 4.8 and 4.7 million (M) 21-nt cDNA tags were respectively sequenced in the cDNA libraries derived from the drought-stressed ramie (DS) and the control ramie under well water condition (CO). The tags generated from the two libraries were aligned with ramie transcriptome to annotate their function and a total of 23,912 and 22,826 ramie genes were matched by these tags of DS and CO library, respectively. Comparison of gene expression level between CO and DS ramie based on the differences of tag frequencies appearing in the two libraries revealed that there were 1516 potential drought stress-responsive genes, in which 24 genes function as transcription factor (TF). Among these 24 TFs, the unigene19721 encoding the DELLA protein which is a key negative regulator in gibberellins (GAs) signal pathway was probably markedly up-regulated under water stress for a increase of tag abundance in DS library, which is possibly responsible for the inhibition of the growth of drought-stressed ramie. In order to validate the change of expression of these potential stress-responsive TFs under water deficit condition, the unigene19721 and another eleven potential stress-responsive TFs were chosen for further expression analysis in well-watered and drought-stressed ramie by real-time quantitative PCR (qRT-PCR) and the result showed that all 12 TFs were authentically involved in the response of drought stress. Conclusion: In this study, twelve TFs involving in the response of drought stress were first found by Illumina tag-sequencing and qRT-PCR in ramie. The discovery of these drought stress-responsive TFs will be helpful for further understanding the drought stress-regulatory mechanism of ramie and improving the drought tolerance ability of ramie.

Description: Background: The great majority of afferent neurons of insect legs project into their segmental ganglion. Intersegmental projections are rare and are only formed by sense organs associated with the basal joints of the legs. Such intersegmental projections never ascend as far as the brain and they form extensive ramifications within thoracic ganglia. A few afferents of chordotonal organs of the subcoxal joints ascend as far as the suboesophageal ganglion. Results: We describe novel afferent neurons in distal segments of locust legs that project directly into the brain without forming ramifications in other ganglia. In the brain, the fibres terminate with characteristic terminals in a small neuropile previously named the superficial ventral inferior protocerebrum. The somata of these neurons are located in the tibiae and tarsi of all legs and they are located within branches of peripheral nerves, or closely associated with such branches. They are not associated with any accessory structures such as tendons or connective tissue strands as typical for insect internal mechanoreceptors such as chordotonal organs or stretch receptors. Morphologically they show great similarity to certain insect infrared receptors.We could not observe projections into the superficial ventral inferior protocerebrum after staining mandibular or labial nerves, but we confirm previous studies that showed projections into the same brain neuropile after staining maxillary and antennal nerves, indicating that most likely similar neurons are present in these appendages also. Conclusion: Because of their location deep within the lumen of appendages the function of these neurons as infrared receptors is unlikely. Their projection pattern and other morphological features indicate that the neurons convey information about an internal physiological parameter directly into a special brain neuropile. We discuss their possible function as thermoreceptors.

Description: Background: Multidrug-resistant (MDR) Salmonella isolates are associated with increased morbidity compared to antibiotic-sensitive strains and are an important health and safety concern in both humans and animals. Salmonella enterica serovar Typhimurium is a prevalent cause of foodborne disease, and a considerable number of S. Typhimurium isolates from humans and livestock are resistant to three or more antibiotics. The majority of these MDR S. Typhimurium isolates are resistant to tetracycline, a commonly used and clinically and agriculturally relevant antibiotic. Because exposure of drug-resistant bacteria to antibiotics can affect cellular processes associated with virulence, such as invasion, we investigated the effect tetracycline had on the invasiveness of tetracycline-resistant MDR S. Typhimurium isolates. Results: The isolates selected and tested were from two common definitive phage types of S. Typhimurium, DT104 and DT193, and were resistant to tetracycline and at least three other antibiotics. Although Salmonella invasiveness is temporally regulated and normally occurs during late-log growth phase, tetracycline exposure induced the full invasive phenotype in a cell culture assay during early-log growth in several DT193 isolates. No changes in invasiveness due to tetracycline exposure occurred in the DT104 isolates during early-log growth or in any of the isolates during late-log growth. Real-time PCR was used to test expression of the virulence genes hilA, prgH, and invF, and these genes were significantly up-regulated during early-log growth in most isolates due to tetracycline exposure; however, increased virulence gene expression did not always correspond with increased invasion, and therefore was not an accurate indicator of elevated invasiveness. This is the first report to assess DT193 isolates, as well as the early-log growth phase, in response to tetracycline exposure, and it was the combination of both parameters that was necessary to observe the induced invasion phenotype. Conclusions: In this report, we demonstrate that the invasiveness of MDR S. Typhimurium can be modulated in the presence of tetracycline, and this effect is dependent on growth phase, antibiotic concentration, and strain background. Identifying the conditions necessary to establish an invasive phenotype is important to elucidate the underlying factors associated with increased virulence of MDR Salmonella.

Description: Background: Mutations in the PTRF gene, coding for cavin-1, cause congenital generalized lipodystrophy type 4 (CGL4) associated with myopathy. In CGL4, symptoms are variable comprising, in addition to myopathy, smooth and skeletal muscle hypertrophy, cardiac arrhythmias, and skeletal abnormalities. Secondary features are atlantoaxial instability, acanthosis nigricans, hepatomegaly, umbilical prominence and metabolic abnormalities related to insulin resistance, such as diabetes mellitus, hyperlipidemia and hepatic steatosis.Case presentationWe describe a 3 year-old child of Moroccan origin with mild muscle phenotype, mainly characterized by mounding, muscle pain, hyperCKemia and mild caveolin 3 reduction on muscle biopsy. No CAV3 gene mutation was detected; instead we found a novel mutation, a homozygous single base pair deletion, in the PTRF gene. Only after detection of this mutation a mild generalized loss of subcutaneous fat, at first underestimated, was noticed and the diagnosis of lipodystrophy inferred. Conclusions: The PTRF gene should be investigated in patients with hyperCKemia, mild myopathy associated with spontaneous or percussion-induced muscle contractions like rippling or mounding, and no CAV3 mutation. The analysis should be performed even if cardiac or metabolic alterations are absent, particularly in young patients in whom lipodystrophy may be difficult to ascertain.

Description: Background: Magnetotactic bacteria produce membrane-enveloped magnetite crystals (magnetosomes) whose formation is controlled primarily by a gene island termed the magnetosome island (MAI). Characterization of single gene and operon function in MAI has elucidated in part the genetic basis of magnetosome formation. The mamX gene, located in the mamXY operon, is highly conserved in the MAI of all Magnetospirillum strains studied to date. Little is known regarding the function of mamX in the process of biomineralization. Results: A mamX deletion mutant ([increment]mamX) and its complemented strain (CmamX) by conjugation in M. gryphiswaldense strain MSR-1 were constructed. There were no striking differences in cell growth among [increment]mamX, CmamX, and wild-type strain (WT). [increment]mamX displayed a much weaker magnetic response than WT. Transmission electron microscopy revealed the presence of irregular, superparamagnetic magnetite particles in [increment]mamX, in contrast to regular, single-domain particles in WT and CmamX. The phenotype of [increment]mamX was similar to that of an ftsZ-like deleted mutant and mamXY operon deleted mutant reported previously. Quantitative real-time RT-PCR (qPCR) results indicated that the deletion of mamX had differential effects on the transcription levels of the other three genes in the operon. Conclusions: The MamX protein plays an important role in controlling magnetosome size, maturation, and crystal form. The four MamXY proteins appear to have redundant functions involved in magnetosome formation. Our findings provide new insights into the coordinated function of MAI genes and operons in magnetosome formation.

Description: Background: Host plant roots, mycorrhizal mycelium and microbes are important and potentially interacting factors shaping the performance of mycorrhization helper bacteria (MHB). We investigated the impact of a soil microbial community on the interaction between the extraradical mycelium of the ectomycorrhizal fungus Piloderma croceum and the MHB Streptomyces sp. AcH 505 in both the presence and the absence of pedunculate oak microcuttings. Results: Specific primers were designed to target the internal transcribed spacer of the rDNA and an intergenic region between two protein encoding genes of P. croceum and the intergenic region between the gyrA and gyrB genes of AcH 505. These primers were used to perform real-time PCR with DNA extracted from soil samples. With a sensitivity of 10 genome copies and a linear range of 6 orders of magnitude, these real-time PCR assays enabled the quantification of purified DNA from P. croceum and AcH 505, respectively. In soil microcosms, the fungal PCR signal was not affected by AcH 505 in the absence of the host plant. However, the fungal signal became weaker in the presence of the plant. This decrease was only observed in microbial filtrate amended microcosms. In contrast, the PCR signal of AcH 505 increased in the presence of P. croceum. The increase was not significant in sterile microcosms that contained plant roots. Conclusions: Real-time quantitative PCR assays provide a method for directly detecting and quantifying MHB and mycorrhizal fungi in plant microcosms. Our study indicates that the presence of microorganisms and plant roots can both affect the nature of MHB-fungus interactions, and that mycorrhizal fungi may enhance MHB growth.

Description: Background: Little is known about the Phasmatodea gut microbial community, including whether phasmids have symbiotic bacteria aiding in their digestion. While symbionts are near ubiquitous in herbivorous insects, the Phasmatodea's distinctively thin body shape precludes the gut enlargements needed for microbial fermentation. High-throughput sequencing was used to characterize the entire microbiota of the fat bodies, salivary glands, and anterior and posterior midguts of two species of walking stick. Results: Most bacterial sequences belonged to a strain of Spiroplasma (Tenericutes) found primarily in the posterior midgut of the parthenogenetic species Ramulus artemis (Phasmatidae). Beyond this, no significant differences were found between the R. artemis midgut sections or between that species and Peruphasma schultei (Pseudophasmatidae). Histological analysis further indicated a lack of bacteriocytes. Conclusions: Phasmids are unlikely to depend on bacteria for digestion, suggesting they produce enzymes endogenously that most other herbivorous insects obtain from symbionts. This conclusion matches predictions based on phasmid anatomy. The role of Spiroplasma in insects warrants further study.

Description: Background: High-throughput sequencing technologies are improving in quality, capacity and costs, providing versatile applications in DNA and RNA research. For small genomes or fraction of larger genomes, DNA samples can be mixed and loaded together on the same sequencing track. This so-called multiplexing approach relies on a specific DNA tag or barcode that is attached to the sequencing or amplification primer and hence appears at the beginning of the sequence in every read. After sequencing, each sample read is identified on the basis of the respective barcode sequence.Alterations of DNA barcodes during synthesis, primer ligation, DNA amplification, or sequencing may lead to incorrect sample identification unless the error is revealed and corrected. This can be accomplished by implementing error correcting algorithms and codes. This barcoding strategy increases the total number of correctly identified samples, thus improving overall sequencing efficiency. Two popular sets of error-correcting codes are Hamming codes and Levenshtein codes.ResultLevenshtein codes operate only on words of known length. Since a DNA sequence with an embedded barcode is essentially one continuous long word, application of the classical Levenshtein algorithm is problematic. In this paper we demonstrate the decreased error correction capability of Levenshtein codes in a DNA context and suggest an adaptation of Levenshtein codes that is proven of efficiently correcting nucleotide errors in DNA sequences. In our adaption we take the DNA context into account and redefine the word length whenever an insertion or deletion is revealed. In simulations we show the superior error correction capability of the new method compared to traditional Levenshtein and Hamming based codes in the presence of multiple errors. Conclusion: We present an adaptation of Levenshtein codes to DNA contexts capable of correction of a pre-defined number of insertion, deletion, and substitution mutations. Our improved method is additionally capable of recovering the new length of the corrupted codeword and of correcting on average more random mutations than traditional Levenshtein or Hamming codes.As part of this work we prepared software for the flexible generation of DNA codes based on our new approach. To adapt codes to specific experimental conditions, the user can customize sequence filtering, the number of correctable mutations and barcode length for highest performance.

Description: Background: Angiogenesis is the main therapeutic mechanism of cell therapy for cardiovascular diseases, but diabetes is reported to reduce the function and number of progenitor cells. Therefore, we studied the effect of streptozotocin-induced diabetes on the bone marrow-mesenchymal stem cell (MSC) function, and examined whether diabetes-impaired MSC could be rescued by pretreatment with oxytocin. Results: MSCs were isolated and cultured from diabetic (DM) or non-diabetic (non-DM) rat, and proliferation rate was compared. DM-MSC was pretreated with oxytocin and compared with non-DM-MSC. Angiogenic capacity was estimated by tube formation and Matrigel plug assay, and therapeutic efficacy was studied in rat myocardial infarction (MI) model.The proliferation and angiogenic activity of DM-MSC were severely impaired but significantly improved by pretreatment with oxytocin. Kruppel-like factor 2 (KLF2), a critical angiogenic factor, was dramatically reduced in DM-MSC and significantly restored by oxytocin. In the Matrigel plug assay, vessel formation of DM-BMSCs was attenuated but was recovered by oxytocin. In rat MI model, DM-MSC injection did not ameliorate cardiac injury, whereas oxytocin-pretreated DM-MSC improved cardiac function and reduced fibrosis. Conclusions: Our results show that diabetes influenced MSC by reducing angiogenic capacity and therapeutic potential. We demonstrate the striking effect of oxytocin on stem cell dysfunction and suggest the use of oxytocin as a priming reagent in autologous stem cell therapy.

Description: Background: Temperature is one of key environmental parameters that affect the whole life of fishes and an increasing number of studies have been directed towards understanding the mechanisms of cold acclimation in fish. However, the adaptation of larvae to cold stress and the cold-specific transcriptional alterations in fish larvae remain largely unknown. In this study, we characterized the development of cold-tolerance in zebrafish larvae and investigated the transcriptional profiles under cold stress using RNA-seq. Results: Pre-exposure of 96hpf zebrafish larvae to cold stress (16[degree sign]C) for 24 h significantly increased their survival rates under severe cold stress (12[degree sign]C). RNA-seq generated 272 million raw reads from six sequencing libraries and about 92% of the processed reads were mapped to the reference genome of zebrafish. Differential expression analysis identified 1,431 up- and 399 down-regulated genes. Gene ontology enrichment analysis of cold-induced genes revealed that RNA splicing, ribosome biogenesis and protein catabolic process were the most highly overrepresented biological processes. Spliceosome, proteasome, eukaryotic ribosome biogenesis and RNA transport were the most highly enriched pathways for genes up-regulated by cold stress. Moreover, alternative splicing of 197 genes and promoter switching of 64 genes were found to be regulated by cold stress. A shorter isoform of stk16 that lacks 67 amino acids at the N-terminus was specifically generated by skipping the second exon in cold-treated larvae. Alternative promoter usage was detected for per3 gene under cold stress, which leading to a highly up-regulated transcript encoding a truncated protein lacking the C-terminal domains. Conclusions: These findings indicate that zebrafish larvae possess the ability to build cold-tolerance under mild low temperature and transcriptional and post-transcriptional regulations are extensively involved in this acclimation process.

Description: Background: Apoptosis is a highly controlled process of cell death that can be induced by periodontopathogens. The present study aimed to investigate the expression of Fas and Bcl-2 proteins by CD3+ T cells in vitro under stimulation by total Porphyromonas gingivalis antigens and purified recombinant P. gingivalis HmuY protein. Results: CD3+ T cells derived from CP patients and stimulated with HmuY expressed higher levels of Bcl-2 compared to identical cells stimulated with P. gingivalis crude extract or cells derived from NP control subjects (p = 0.043). Conclusion: The authors hypothesize that P. gingivalis HmuY plays a role in the pathogenesis of chronic periodontitis, possibly by reducing or delaying apoptosis in T cells through a pathway involving the Bcl-2 protein.

Description: Background: It is increasingly recognized that the bacteria that live in and on the human body (the microbiome) can play an important role in health and disease. The composition of the microbiome is potentially influenced by both internal factors (such as phylogeny and host physiology) and external factors (such as diet and local environment), and interspecific comparisons can aid in understanding the importance of these factors. Results: To gain insights into the relative importance of these factors on saliva microbiome diversity, we here analyze the saliva microbiomes of chimpanzees (Pan troglodytes) and bonobos (Pan paniscus) from two sanctuaries in Africa, and from human workers at each sanctuary. The saliva microbiomes of the two Pan species are more similar to one another, and the saliva microbiomes of the two human groups are more similar to one another, than are the saliva microbiomes of human workers and apes from the same sanctuary. We also looked for the existence of a core microbiome and find no evidence for a taxon-based core saliva microbiome for Homo or Pan. In addition, we studied the saliva microbiome from apes from the Leipzig Zoo, and found an extraordinary diversity in the zoo ape saliva microbiomes that is not found in the saliva microbiomes of the sanctuary animals. Conclusions: The greater similarity of the saliva microbiomes of the two Pan species to one another, and of the two human groups to one another, are in accordance with both the phylogenetic relationships of the hosts as well as with host physiology. Moreover, the results from the zoo animals suggest that novel environments can have a large impact on the microbiome, and that microbiome analyses based on captive animals should be viewed with caution as they may not reflect the microbiome of animals in the wild.

Description: Background: The choice of variable selection methods to identify important variables for binary classification modeling is critical to produce stable models that are interpretable, that generate accurate predictions and have minimum bias. This work is motivated by data on clinical and laboratory features of severe dengue infections (dengue hemorrhagic fever, DHF) obtained from 51 individuals enrolled in a prospective observational study of acute human dengue infections. Results: We carry out a comprehensive performance comparison using several classification models for DHF over the dengue data set. We compared variable selection results by Multivariate Adaptive Regression Splines, Learning Ensemble, Random Forest, Bayesian Moving Averaging, Stochastic Search Variable Selection, and Generalized Regularized Logistics Regression. Model averaging methods (bagging, boosting and ensemble learners) have higher accuracy, but the generalized regularized regression model has the highest predictive power because the linearity assumptions of candidate predictors are strongly satisfied via deviance chi-square testing procedures. Bootstrapping applications for evaluating predictive regression coefficients in regularized regression model are performed. Conclusions: Feature reduction methods introduce inherent biases and therefore are data-type dependent. We propose that these limitations can be overcome using an exhaustive approach for searching feature space. Using this approach, we results suggest that IL-10, platelet and lymphocyte counts are the major features for predicting dengue DHF on the basis of blood chemistries and cytokine measurements.

Description: Background: Sjogren's syndrome is characterized by lymphocytic infiltration of the exocrine glands, together with polyclonal B-cell activation, and lung diseases are well-known complications of the disease. Therefore, in most cases associated with Sjogren's syndrome, infiltrating lymphocytes in the lung specimen exhibit the features of B-cells. We herein report an atypical case of lymphoproliferative pulmonary involvement in a patient with Sjogren's syndrome.Case presentationA 46-year-old female was admitted to our hospital because of an abnormal chest roentgenogram finding on a medical checkup. Chest computed tomography showed randomly-distributed micronodules and patchy ground-glass opacities. A surgical biopsied specimen showed an atypical pattern of interstitial pneumonia with numerous lymphoid follicles. Among the infiltrating lymphocytes in the lung, only the monoclonality of the T-cells was proven by a gene rearrangement analysis, but there was no cytological atypicality or genetic disorder revealed by testing the bone marrow aspirate. A diagnosis of Sjogren's syndrome was made based on the patient's other symptoms and these negative findings. The patient's pulmonary lesions have been successfully treated and remission has been maintained for over three years with corticosteroid treatment alone. Conclusion: The present patient was an atypical case of lymphoproliferative pulmonary involvement in a patient with Sjogren's syndrome. Although monoclonality of the infiltrating T-cells was proven, the clinical course and the findings of the imaging and laboratory examinations were inconsistent with the previously-reported cases of primary pulmonary T-cell lymphoma. This suggests that the monoclonality of lymphocytes does not always define malignancy. The diagnosis of malignant lymphoma or lymphoproliferative diseases should be made clinically, pathologically and cytogenetically to rule out other similar diseases.

Description: There is much interest in using high-throughput DNA sequencing methodology to monitor microorganisms, complex plant and animal communities. However, there are experimental and analytical issues to consider before applying a sequencing technology, which was originally developed for genome projects, to ecological projects. Many of these issues have been highlighted by recent microbial studies. Understanding how high-throughput sequencing is best implemented is important for the interpretation of recent results and the success of future applications. Addressing complex biological questions with metagenomics requires the interaction of researchers who bring different skill sets to problem solving. Educators can help by nurturing a collaborative interdisciplinary approach to genome science, which is essential for effective problem solving. Educators are in a position to help students, teachers, the public and policy makers interpret the new knowledge that metagenomics brings. To do this, they need to understand, not only the excitement of the science but also the pitfalls and shortcomings of methodology and research designs. We review these issues and some of the research directions that are helping to move the field forward.

Description: We believe that undergraduate biology students must acquire a foundational background in computing including how to formulate a computational problem; develop an algorithmic solution; implement their solution in software and then test, document and use their code to explore biological phenomena. Moreover, by learning these skills in the first year, students acquire a powerful tool set that they can use and build on throughout their studies. To address this need, we have developed a first-year undergraduate course that teaches students the foundations of computational thinking and programming in the context of problems in biology. This article describes the structure and content of the course and summarizes assessment data on both affective and learning outcomes.

Description: Bioinformatics is an integral part of modern life sciences. It has revolutionized and redefined how research is carried out and has had an enormous impact on biotechnology, medicine, agriculture and related areas. Yet, it is only rarely integrated into high school teaching and learning programs, playing almost no role in preparing the next generation of information-oriented citizens. Here, we describe the design principles of bioinformatics learning environments, including our own, that are aimed at introducing bioinformatics into senior high school curricula through engaging learners in scientifically authentic inquiry activities. We discuss the bioinformatics-related benefits and challenges that high school teachers and students face in the course of the implementation process, in light of previous studies and our own experience. Based on these lessons, we present a new approach for characterizing the questions embedded in bioinformatics teaching and learning units, based on three criteria: the type of domain-specific knowledge required to answer each question (declarative knowledge, procedural knowledge, strategic knowledge, situational knowledge), the scientific approach from which each question stems (biological, bioinformatics, a combination of the two) and the associated cognitive process dimension (remember, understand, apply, analyze, evaluate, create). We demonstrate the feasibility of this approach using a learning environment, which we developed for the high school level, and suggest some of its implications. This review sheds light on unique and critical characteristics related to broader integration of bioinformatics in secondary education, which are also relevant to the undergraduate level, and especially on curriculum design, development of suitable learning environments and teaching and learning processes.

Description: Next-generation sequencing (NGS) is increasingly being adopted as the backbone of biomedical research. With the commercialization of various affordable desktop sequencers, NGS will be reached by increasing numbers of cellular and molecular biologists, necessitating community consensus on bioinformatics protocols to tackle the exponential increase in quantity of sequence data. The current resources for NGS informatics are extremely fragmented. Finding a centralized synthesis is difficult. A multitude of tools exist for NGS data analysis; however, none of these satisfies all possible uses and needs. This gap in functionality could be filled by integrating different methods in customized pipelines, an approach helped by the open-source nature of many NGS programmes. Drawing from community spirit and with the use of the Wikipedia framework, we have initiated a collaborative NGS resource: The NGS WikiBook. We have collected a sufficient amount of text to incentivize a broader community to contribute to it. Users can search, browse, edit and create new content, so as to facilitate self-learning and feedback to the community. The overall structure and style for this dynamic material is designed for the bench biologists and non-bioinformaticians. The flexibility of online material allows the readers to ignore details in a first read, yet have immediate access to the information they need. Each chapter comes with practical exercises so readers may familiarize themselves with each step. The NGS WikiBook aims to create a collective laboratory book and protocol that explains the key concepts and describes best practices in this fast-evolving field.

Description: Background: In cross breeding, it is important to choose a good parental combination that has high probability of generating offspring with desired characteristics. This study examines a method for predicting the segregation of target traits in a progeny population based on genome-wide markers and phenotype data of parental cultivars. Results: The proposed method combines segregation simulation and Bayesian modeling for genomic selection. Marker segregation in a progeny population was simulated based on parental genotypes. Posterior marker effects sampled via Markov Chain Monte Carlo were used to predict the segregation pattern of target traits. The posterior distribution of the proportion of progenies that fulfill selection criteria was calculated and used for determining a promising cross and the necessary size of the progeny population. We applied the proposed method to Japanese pear (Pyrus pyrifolia Nakai) data to demonstrate the method and to show how it works in the selection of a promising cross. Verification using an actual breeding population suggests that the segregation of target traits can be predicted with reasonable accuracy, especially in a highly heritable trait. The uncertainty in predictions was reflected on the posterior distribution of the proportion of progenies that fulfill selection criteria. A simulation study based on the real marker data of Japanese pear cultivars also suggests the potential of the method. Conclusions: The proposed method is useful to provide objective and quantitative criteria for choosing a parental combination and the breeding population size.

Description: Background: Gene reshuffling, point mutations and horizontal gene transfer contribute to bacterial genome variation, but require the genome to rewire its transcriptional circuitry to ensure that inserted, mutated or reshuffled genes are transcribed at appropriate levels. The genomes of Epsilonproteobacteria display very low synteny, due to high levels of reshuffling and reorganisation of gene order, but still share a significant number of gene orthologs allowing comparison. Here we present the primary transcriptome of the pathogenic Epsilonproteobacterium Campylobacter jejuni, and have used this for comparative and predictive transcriptomics in the Epsilonproteobacteria. Results: Differential RNA-sequencing using 454 sequencing technology was used to determine the primary transcriptome of C. jejuni NCTC 11168, which consists of 992 transcription start sites (TSS), which included 29 putative non-coding and stable RNAs, 266 intragenic (internal) TSS, and 206 antisense TSS. Several previously unknown features were identified in the C. jejuni transcriptional landscape, like leaderless mRNAs and potential leader peptides upstream of amino acid biosynthesis genes. A cross-species comparison of the primary transcriptomes of C. jejuni and the related Epsilonproteobacterium Helicobacter pylori highlighted a lack of conservation of operon organisation, position of intragenic and antisense promoters or leaderless mRNAs. Predictive comparisons using 40 other Epsilonproteobacterial genomes suggests that this lack of conservation of transcriptional features is common to all Epsilonproteobacterial genomes, and is associated with the absence of genome synteny in this subdivision of the Proteobacteria. Conclusions: Both the genomes and transcriptomes of Epsilonproteobacteria are highly variable, both at the genome level by combining and division of multicistronic operons, but also on the gene level by generation or deletion of promoter sequences and 5[prime] untranslated regions. Regulatory features may have evolved after these species split from a common ancestor, with transcriptome rewiring compensating for changes introduced by genomic reshuffling and horizontal gene transfer.

Description: Phenology as a strategy for carbon optimality: a global model Biogeosciences Discussions, 10, 15107-15152, 2013 Author(s): S. Caldararu, D. W. Purves, and P. I. Palmer Phenology is essential to our understanding of biogeochemical cycles and the climate system. We develop a global mechanistic model of leaf phenology based on the hypothesis that phenology is a strategy for optimal carbon gain at the canopy level so that trees adjust leaf gains and losses in response to environmental factors such as light, temperature and soil moisture, to achieve maximum carbon assimilation. We fit this model to five years of satellite observations of leaf area index (LAI) using a Bayesian fitting algorithm. We show that our model is able to reproduce phenological patterns for all vegetation types and use it to explore variations in growing season length and the climate factors that limit leaf growth for different biomes. Phenology in wet tropical areas is limited by leaf age physiological constraints while at higher latitude leaf seasonality is limited by low temperature and light availability. Leaf growth in grassland regions is limited by water availability but often in combination with other factors. This model will advance the current understanding of phenology for ecosystem carbon models and our ability to predict future phenological behaviour.

Description: Background: Hypersensitivity pneumonitis is defined as an allergic lung disease that occurs in response to inhalation of fungal antigens, bacterial antigens, chemicals, dusts, or animal proteins. The incidence of summer-type hypersensitivity pneumonitis is higher in the summer season, especially in Japan, due to the influence of the hot and humid environment and the common style of wood house or old concrete condominiums.Case presentationThe present report describes a case of a middle-aged married couple who lived in the same house and who simultaneously suffered from summer-type hypersensitivity pneumonitis. This report analyzes these two cases in terms of environmental research and its microbiological, radiological, and pathological aspects. This case report is followed by a review of family occurrences of summer-type hypersensitivity pneumonitis from 22 studies with a total of 49 patients (including the two present cases) in Japan. Conclusion: Summer-type hypersensitivity pneumonitis may be unrecognized and misdiagnosed as pneumonia or other respiratory diseases. A greater understanding of the clinical, pathologic, and environmental features of summer-type hypersensitivity pneumonitis might help improve diagnosis and delivery of appropriate management for this condition.

Description: Background: Cell migration is a fundamental biological process and has an important role in the developing brain by regulating a highly specific pattern of connections between nerve cells. Cell migration is required for axonal guidance and neurite outgrowth and involves a series of highly co-ordinated and overlapping signalling pathways. The non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) has an essential role in development and is the most highly expressed kinase in the developing CNS. FAK activity is essential for neuronal cell adhesion and migration. Results: The objective of this study was to optimise a protocol for the differentiation of the neuroblastoma cell line, SH-SY5Y. We determined the optimal extracellular matrix proteins and growth factor combinations required for the optimal differentiation of SH-SY5Y cells into neuronal-like cells and determined those conditions that induce the expression of FAK. It was confirmed that the cells were morphologically and biochemically differentiated when compared to undifferentiated cells. This is in direct contrast to commonly used differentiation methods that induce morphological differentiation but not biochemical differentiation. Conclusions: We conclude that we have optimised a protocol for the differentiation of SH-SY5Y cells that results in a cell population that is both morphologically and biochemically distinct from undifferentiated SH-SY5Y cells and has a distinct adhesion and spreading pattern and display extensive neurite outgrowth. This protocol will provide a neuronal model system for studying FAK activity during cell adhesion and migration events.

Description: Background: Life history tradeoffs may result from temporal and physiological constraints intrinsic to an organism. When faced with limited time and energy, compromises occur and these resources are allocated among essential activities, such as body growth, maintenance, foraging, mating, and offspring care. We investigated potential tradeoffs that may occur between reproductive activities and feeding performance in female Arizona Bark Scorpions (Centruroides sculpturatus) by comparing the time taken to capture prey between non-reproductive and reproductive females (gravid females and females exhibiting maternal care, i.e. carrying offspring on their backs). Results: Gravid females were as efficient at catching prey as non-gravid females. To control for variation in the duration of the maternal care period, we removed all offspring from all post-parturient females after 5 days. Brooding females and females 24 hours following offspring removal (FOR) did not successfully capture prey within the 900-second trial period. Twenty-eight days FOR, females caught prey faster than females displaying maternal care and females 24 hours FOR, but were not as efficient at catching prey as non-gravid and gravid females. When pursuing prey, C. sculpturatus exhibiting maternal care used an active foraging strategy more frequently than non-gravid, gravid, and females 28 days FOR. In contrast, non-gravid, gravid, and females 28 days FOR used active and ambush foraging with similar frequency. Conclusions: Our data suggest that reproduction does not significantly reduce the predatory efficiency of gravid C. sculpturatus, and that these females can cope with increasing body mass and the physiological costs of gestation. However, the observation that brooding females and females 24 hours FOR did not catch prey within the trial period indicates that maternal care significantly reduces predatory efficiency in these scorpions. Females 28 days FOR were still not as efficient at catching prey as non-gravid and gravid females, suggesting that reproductive costs extend for at least 4 weeks after the end of the maternal care period. Preferential use of an active foraging strategy by brooding females may increase prey encounter rates, allowing the scorpions to more rapidly replenish energy reserves depleted during reproduction. However, active foraging may be energetically costly and increase predation risk for brooding females. Our findings regarding antagonistic interactions between reproduction and feeding in female C. sculpturatus demonstrate the pervasive nature of reproductive costs for viviparous females, and may provide insight on factors that influence the diversity of reproductive strategies observed in nature.

Description: Background: External development and optical transparency of embryos make zebrafish exceptionally suitable for in vivo insertional mutagenesis using fluorescent proteins to visualize expression patterns of mutated genes. Recently developed Gene Breaking Transposon (GBT) vectors greatly improve the fidelity and mutagenicity of transposon-based gene trap vectors. Results: We constructed and tested a bipartite GBT vector with Gal4-VP16 as the primary gene trap reporter. Our vector also contains a UAS:eGFP cassette for direct detection of gene trap events by fluorescence. To confirm gene trap events, we generated a UAS:mRFP tester line. We screened 270 potential founders and established 41 gene trap lines. Three of our gene trap alleles display homozygous lethal phenotypes ranging from embryonic to late larval: nsftpl6, atp1a3atpl10 and flrtpl19. Our gene trap cassette is flanked by direct loxP sites, which enabled us to successfully revert nsftpl6, atp1a3atpl10 and flrtpl19 gene trap alleles by injection of Cre mRNA. The UAS:eGFP cassette is flanked by direct FRT sites. It can be readily removed by injection of Flp mRNA for use of our gene trap alleles with other tissue-specific GFP-marked lines. The Gal4-VP16 component of our vector provides two important advantages over other GBT vectors. The first is increased sensitivity, which enabled us to detect previously unnoticed expression of nsf in the pancreas. The second advantage is that all our gene trap lines, including integrations into non-essential genes, can be used as highly specific Gal4 drivers for expression of other transgenes under the control of Gal4 UAS. Conclusions: The Gal4-containing bipartite Gene Breaking Transposon vector presented here retains high specificity for integrations into genes, high mutagenicity and revertibility by Cre. These features, together with utility as highly specific Gal4 drivers, make gene trap mutants presented here especially useful to the research community.

Description: Background: Little is known about the Phasmatodea gut microbial community, including whether phasmids have symbiotic bacteria aiding in their digestion. While symbionts are near ubiquitous in herbivorous insects, the Phasmatodea’s distinctively thin body shape precludes the gut enlargements needed for microbial fermentation. High-throughput sequencing was used to characterize the entire microbiota of the fat bodies, salivary glands, and anterior and posterior midguts of two species of walking stick. Results: Most bacterial sequences belonged to a strain of Spiroplasma (Tenericutes) found primarily in the posterior midgut of the parthenogenetic species Ramulus artemis (Phasmatidae). Beyond this, no significant differences were found between the R. artemis midgut sections or between that species and Peruphasma schultei (Pseudophasmatidae). Histological analysis further indicated a lack of bacteriocytes. Conclusions: Phasmids are unlikely to depend on bacteria for digestion, suggesting they produce enzymes endogenously that most other herbivorous insects obtain from symbionts. This conclusion matches predictions based on phasmid anatomy. The role of Spiroplasma in insects warrants further study.

Description: Background: Very little is known about manganese (Mn)-toxicity-responsive genes in citrus plants. Seedlings of 'Xuegan' (Citrus sinensis) and 'Sour pummelo' (Citrus grandis) were irrigated for 17 weeks with nutrient solution containing 2 muM (control) or 600 muM (Mn-toxicity) MnSO4. The objectives of this study were to understand the mechanisms of citrus Mn-tolerance and to identify differentially expressed genes, which might be involved in Mn-tolerance. Results: Under Mn-toxicity, the majority of Mn in seedlings was retained in the roots; C. sinensis seedlings accumulated more Mn in roots and less Mn in shoots (leaves) than C. grandis ones and Mn concentration was lower in Mn-toxicity C. sinensis leaves compared to Mn-toxicity C. grandis ones. Mn-toxicity affected C. grandis seedling growth, leaf CO2 assimilation, total soluble concentration, phosphorus (P) and magenisum (Mg) more than C. sinensis. Using cDNA-AFLP, we isolated 42 up-regulated and 80 down-regulated genes in Mn-toxicity C. grandis leaves. They were grouped into the following functional categories: biological regulation and signal transduction, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, lipid metabolism, cell wall metabolism, stress responses and cell transport. However, only 7 up-regulated and 8 down-regulated genes were identified in Mn-toxicity C. sinensis ones. The responses of C. grandis leaves to Mn-toxicity might include following several aspects: (1) accelerating leaf senescence; (2) activating the metabolic pathway related to ATPase synthesis and reducing power production; (3) decreasing cell transport; (4) inhibiting protein and nucleic acid metabolisms; (5) impairing the formation of cell wall; and (6) triggering multiple signal transduction pathways. We also identified many new Mn-toxicity-responsive genes involved in biological and signal transduction, carbohydrate and protein metabolisms, stress responses and cell transport. Conclusions: Our results demonstrated that C. sinensis was more tolerant to Mn-toxicity than C. grandis, and that Mn-toxicity affected gene expression far less in C. sinensis leaves. This might be associated with more Mn accumulation in roots and less Mn accumulation in leaves of Mn-toxicity C. sinensis seedlings than those of C. grandis seedlings. Our findings increase our understanding of the molecular mechanisms involved in the responses of plants to Mn-toxicity.

Description: Background: Two-partner secretion systems in Gram-negative bacteria consist of an outer membrane protein TpsB that mediates the secretion of a cognate TpsA protein into the extracellular milieu. TpsA proteins have diverse, often virulence-related functions, and some of them inhibit the growth of related bacteria. In Neisseria meningitidis, several functions have been attributed to the TpsA proteins. Downstream of the tpsB and tpsA genes, several shorter tpsA-related gene cassettes, called tpsC, are located interspersed with intervening open-reading frames (IORFs). It has been suggested that the tpsC cassettes may recombine with the tpsA gene as a mechanism of antigenic variation. Here, we investigated (i) whether TpsA of N. meningitidis also has growth-inhibitory properties, (ii) whether tpsC cassettes recombine with the tpsA gene, and (iii) what the consequences of such recombination events might be. Results: We demonstrate that meningococcal TpsA has growth-inhibitory properties and that the IORF located immediately downstream of tpsA confers immunity to the producing strain. Although bioinformatics analysis suggests that recombination between tpsC cassettes and tpsA occurs, detailed analysis of the tpsA gene in a large collection of disease isolates of three clonal complexes revealed that the frequency is very low and cannot be a mechanism of antigenic variation. However, recombination affected growth inhibition. In vitro experiments revealed that recombination can be mediated through acquirement of tpsC cassettes from the environment and it identified the regions involved in the recombination. Conclusions: Meningococcal TpsA has growth-inhibitory properties. Recombination between tpsA and tpsC cassettes occurs in vivo but is rare and has consequences for growth inhibition. A recombination model is proposed and we propose that the main goal of recombination is the collection of new IORFs for protection against a variety of TpsA proteins.

Description: Background: Sequencing of the genome of Propionibacterium acnes produced a catalogue of genes many of which enable this organism to colonise skin and survive exposure to the elements. Despite this platform, there was little understanding of the gene regulation that gives rise to an organism that has a major impact on human health and wellbeing and causes infections beyond the skin. To address this situation, we have undertaken a genome--wide study of gene regulation using a combination of improved differential and global RNA-sequencing and an analytical approach that takes into account the inherent noise within the data. Results: We have produced nucleotide-resolution transcriptome maps that identify and differentiate sites of transcription initiation from sites of stable RNA processing and mRNA cleavage. Moreover, analysis of these maps provides strong evidence for 'pervasive' transcription and shows that contrary to initial indications it is not biased towards the production of antisense RNAs. In addition, the maps reveal an extensive array of riboswitches, leaderless mRNAs and small non-protein-coding RNAs alongside vegetative promoters and post-transcriptional events, which includes unusual tRNA processing. The identification of such features will inform models of complex gene regulation, as illustrated here for ribonucleotide reductases and a potential quorum-sensing, two-component system. Conclusions: The approach described here, which is transferable to any bacterial species, has produced a step increase in whole-cell knowledge of gene regulation in P. acnes. Continued expansion of our maps to include transcription associated with different growth conditions and genetic backgrounds will provide a new platform from which to computationally model the gene expression that determines the physiology of P. acnes and its role in human disease.

Description: Background: Nutritional guidance and diet control play important roles in the treatment of obesity and non-alcoholic fatty liver. However, in Japan, nutritional guidance is difficult to provide in practice. Therefore, we evaluated the effects of providing the 'once-a-day' intervention of a healthy lunch on various metabolic parameters. Methods: For a 1-month preparatory period, 10 subjects generally consumed the lunches that were provided by the worksite cafeteria. This was followed by a 1-week washout period, after which, the subjects consumed healthy, low-calorie, well-balanced lunches for a 1-month test period. After the preparatory and test periods, blood samples were obtained from all subjects. The serum levels of indices relevant to metabolic syndrome and fatty liver were measured. Results: Serum alanine aminotransferase activity significantly decreased by 20.3% after the healthy intervention. However, the indices of metabolic syndrome did not significantly change. Analysis of the relationship between serum alanine aminotransferase activity and nutrient content indicated that the improvement of serum alanine aminotransferase status was due to the higher vegetable content and lower animal-source protein of the meals provided. Conclusions: In summary, the 'once-a-day' intervention of providing a healthy lunch improved serum alanine aminotransferase status. A diet high in vegetables and low in animal-based protein is important in maintaining a healthy condition.

Description: The genomes of related species contain valuable information on the history of the considered taxa. Great apes in particular exhibit variation of evolutionary patterns along their genomes. However, the great ape data also bring new challenges, such as the presence of incomplete lineage sorting and ancestral shared polymorphisms. Previous methods for genome-scale analysis are restricted to very few individuals or cannot disentangle the contribution of mutation rates and fixation biases. This represents a limitation both for the understanding of these forces as well as for the detection of regions affected by selection. Here, we present a new model designed to estimate mutation rates and fixation biases from genetic variation within and between species. We relax the assumption of instantaneous substitutions, modeling substitutions as mutational events followed by a gradual fixation. Hence, we straightforwardly account for shared ancestral polymorphisms and incomplete lineage sorting. We analyze genome-wide synonymous site alignments of human, chimpanzee, and two orangutan species. From each taxon, we include data from several individuals. We estimate mutation rates and GC-biased gene conversion intensity. We find that both mutation rates and biased gene conversion vary with GC content. We also find lineage-specific differences, with weaker fixation biases in orangutan species, suggesting a reduced historical effective population size. Finally, our results are consistent with directional selection acting on coding sequences in relation to exonic splicing enhancers.

Description: Genetic control of male or female gonad development displays between different groups of organisms a remarkable diversity of "master sex-determining genes" at the top of the genetic hierarchies, whereas downstream components surprisingly appear to be evolutionarily more conserved. Without much further studies, conservation of sequence has been equalized to conservation of function. We have used the medaka fish to investigate the generality of this paradigm. In medaka, the master male sex-determining gene is dmrt1bY , a highly conserved downstream regulator of sex determination in vertebrates. To understand its function in orchestrating the complex gene regulatory network, we have identified targets genes and regulated pathways of Dmrt1bY. Monitoring gene expression and interactions by transgenic fluorescent reporter fish lines, in vivo tissue-chromatin immunoprecipitation and in vitro gene regulation assays revealed concordance but also major discrepancies between mammals and medaka, notably amongst spatial, temporal expression patterns and regulations of the canonical Hedgehog and R-spondin/Wnt/Follistatin signaling pathways. Examination of Foxl2 protein distribution in the medaka ovary defined a new subpopulation of theca cells, where ovarian-type aromatase transcriptional regulation appears to be independent of Foxl2. In summary, these data show that the regulation of the downstream regulatory network of sex determination is less conserved than previously thought.

Description: Background: Pancreatic cancer is a deadly disease with a five-year survival of less than 5%. A better understanding of the underlying biology may suggest novel therapeutic targets. Recent surveys of the pancreatic cancer genome have uncovered numerous new alterations; yet systematic functional characterization of candidate cancer genes has lagged behind. To address this challenge, here we have devised a highly-parallel RNA interference-based functional screen to evaluate many genomically-nominated candidate pancreatic cancer genes simultaneously. Results: For 185 candidate pancreatic cancer genes, selected from recurrently altered genomic loci, we performed a pooled shRNA library screen of cell growth/viability across 10 different cell lines. Knockdown-associated effects on cell growth were assessed by enrichment or depletion of shRNA hairpins, by hybridization to barcode microarrays. A novel analytical approach (COrrelated Phenotypes for On-Target Effects; COPOTE) was used to discern probable on-target knockdown, based on identifying different shRNAs targeting the same gene and displaying concordant phenotypes across cell lines. Knockdown data were integrated with genomic architecture and gene-expression profiles, and selected findings validated using individual shRNAs and/or independent siRNAs. The pooled shRNA library design delivered reproducible data. In all, COPOTE analysis identified 52 probable on-target gene-knockdowns. Knockdown of known oncogenes (KRAS, MYC, SMURF1 and CCNE1) and a tumor suppressor (CDKN2A) showed the expected contrasting effects on cell growth. In addition, the screen corroborated purported roles of PLEKHG2 and MED29 as 19q13 amplicon drivers. Most notably, the analysis also revealed novel possible oncogenic functions of nucleoporin NUP153 (ostensibly by modulating TGFbeta signaling) and Kruppel-like transcription factor KLF5 in pancreatic cancer. Conclusions: By integrating physical and functional genomic data, we were able to simultaneously evaluate many candidate pancreatic cancer genes. Our findings uncover new facets of pancreatic cancer biology, with possible therapeutic implications. More broadly, our study provides a general strategy for the efficient characterization of candidate genes emerging from cancer genome studies.

Description: Molecular interactions are often represented as network models which have become the common language of many areas of biology. Graphs serve as convenient mathematical representations of network models and have themselves become objects of study. Their topology has been intensively researched over the last decade after evidence was found that they share underlying design principles with many other types of networks.Initial studies suggested that molecular interaction network topology is related to biological function and evolution. However, further whole-network analyses did not lead to a unified view on what this relation may look like, with conclusions highly dependent on the type of molecular interactions considered and the metrics used to study them. It is unclear whether global network topology drives function, as suggested by some researchers, or whether it is simply a byproduct of evolution or even an artefact of representing complex molecular interaction networks as graphs.Nevertheless, network biology has progressed significantly over the last years. We review the literature, focusing on two major developments. First, realizing that molecular interaction networks can be naturally decomposed into subsystems (such as modules and pathways), topology is increasingly studied locally rather than globally. Second, there is a move from a descriptive approach to a predictive one: rather than correlating biological network topology to generic properties such as robustness, it is used to predict specific functions or phenotypes.Taken together, this change in focus from globally descriptive to locally predictive points to new avenues of research. In particular, multi-scale approaches are developments promising to drive the study of molecular interaction networks further.

Description: Background: Acute adrenal insufficiency is a potentially lethal condition rarely caused by bilateral adrenal haemorrhage due to heparin use. Most of the times, it is difficult to establish the diagnosis, as symptoms are not specific. Few cases have been reported in the literature.Case presentationA 52-year-old Caucasian woman presented with abdominal pain, vomiting and weakness nine days after arthroplasty and heparin use. Hyperkalemia, low cortisol and high adrenocorticotropic hormone levels were found, indicating adrenal insufficiency. Magnetic resonance imaging of the upper abdomen was compatible with preceding adrenal haemorrhage. Hydrocortisone and fludrocortisone were administered. Review of the literature revealed 36 cases of postoperative adrenal haemorrhage which are presented briefly. Conclusion: Postoperative acute adrenal insufficiency due to haemorrhage is a rare condition. If patients are treated based on clinical suspicion, they have good chances to survive. Hydrocortisone is given permanently in the majority of the patients.

Description: Timing of sea ice retreat can alter phytoplankton community structure in the western Arctic Ocean Biogeosciences Discussions, 10, 15153-15180, 2013 Author(s): given_name prefix surname suffix, A. Fujiwara, T. Hirawake, K. Suzuki, I. Imai, and S.-I. Saitoh This study assesses the response of phytoplankton assemblages to recent climate change, especially with regard to the shrinking of sea ice in the northern Chukchi Sea of the western Arctic Ocean. Distribution patterns of phytoplankton groups in the late summers of 2008–2010 were analyzed based on HPLC pigment signatures and, the following four major algal groups were inferred via multiple regression and cluster analyses: prasinophytes, diatoms, haptophytes and dinoflagellates. A remarkable interannual difference in the distribution pattern of the groups was found in the northern basin area. Haptophytes dominated and dispersed widely in warm surface waters in 2008, whereas prasinophytes dominated in cold water in 2009 and 2010. A difference in the onset date of sea ice retreat was evident among years – the sea ice retreat in 2008 was 1–2 months earlier than in 2009 and 2010. The spatial distribution of early sea ice retreat matched the areas in which a shift in algal community composition was observed. Steel-Dwass's multiple comparison tests were used to assess the physical, chemical and biological parameters of the four clusters. We found a statistically significant difference in temperature between the haptophyte-dominated cluster and the other clusters, suggesting that the change in the phytoplankton communities was related to the earlier sea ice retreat in 2008 and the corollary increase in sea surface temperatures. Longer periods of open water during the summer, which are expected in the future, may affect food webs and biogeochemical cycles in the western Arctic due to shifts in phytoplankton community structure.

Description: Background: The apolipoprotein M (APOM) T-778C gene polymorphism has been associated with serum lipid levels and the risk of coronary artery disease (CAD), but the results are inconclusive. The purpose of this meta-analysis was to detect the association between the APOM T-778C polymorphism and serum lipid levels and the risk of CAD in the Chinese population. Methods: Databases of MEDLINE, EMBASE, the Cochrane Library and CNKI were systematically searched. Data were extracted using standardized methods. The association was assessed by mean difference (MD) with 95% confidence intervals (CI) or odds ratio (OR) with 95% CI. Results: Ten studies with 4,413 patients were included in this meta-analysis. Pooled effects indicated that CT+CC group had higher levels of total cholesterol (TC) (MD:-0.36, 95% CI: -0.53 -- -0.19, P 〈 0.0001) and low-density lipoprotein cholesterol (LDL-C) (MD: -0.08, 95% CI: -0.16 -- -0.01, P = 0.03) than TT group. There was no difference in the levels of triglyceride (MD: 0.06, 95% CI: -0.04 -- 0.15, P = 0.22) and high-density lipoprotein cholesterol (MD: 0.00, 95% CI: -0.03--0.03, P = 0.93) between TT and CT+CC groups. Pooled effects showed that CAD group had higher CT+CC genotype frequency than control group (OR: 1.97, 95% CI: 1.62--2.39, P 〈 0.00001; heterogeneity test x2 = 2.96, P = 0.71, I2 = 0%). Conclusions: The results of the current meta-analysis show that the CT+CC group has higher levels of TC and LDL-C than the TT group. Moreover, there is also a prominent association between APOM T-778C polymorphism and the risk of CAD in the Chinese population, the CT+CC genotype is associated with increased risk of CAD.

Description: Background: The adhesion of lactobacilli to the vaginal surface is of paramount importance to develop their probiotic functions. For this reason, the role of HeLa cell surface proteoglycans in the attachment of Lactobacillus salivarius Lv72, a mutualistic strain of vaginal origin, was investigated. Results: Incubation of cultures with a variety of glycosaminoglycans (chondroitin sulfate A and C, heparin and heparan sulfate) resulted in marked binding interference. However, no single glycosaminoglycan was able to completely abolish cell binding, the sum of all having an additive effect that suggests cooperation between them and recognition of specific adhesins on the bacterial surface. In contrast, chondroitin sulfate B enhanced cell to cell attachment, showing the relevance of the stereochemistry of the uronic acid and the sulfation pattern on binding. Elimination of the HeLa surface glycosaminoglycans with lyases also resulted in severe adherence impairment. Advantage was taken of the Lactobacillus-glycosaminoglycans interaction to identify an adhesin from the bacterial surface. This protein, identify as a soluble binding protein of an ABC transporter system (OppA) by MALDI-TOF/(MS), was overproduced in Escherichia coli, purified and shown to interfere with L. salivarius Lv72 adhesion to HeLa cells. Conclusions: These data suggest that glycosaminoglycans play a fundamental role in attachment of mutualistic bacteria to the epithelium that lines the cavities where the normal microbiota thrives, OppA being a bacterial adhesin involved in the process.

Description: Background: Many scientific disciplines rely on correct taxon delineations and identifications. So does a great part of the general public as well as decision makers. Researchers, students and enthusiastic amateurs often feel frustrated because information about species remains scattered, difficult to access, or difficult to decipher. Together, this affects almost anyone who wishes to identify species or verify identifications. Many remedies have been proposed, but we argue that the role of natural history collections remains insufficiently appreciated. We suggest using state-of-the-art mass imaging technology and to join forces to create a global natural history metacollection on the internet, providing access to the morphology of tens of millions of specimens and making them available for automated digital image analysis.DiscussionRobotic high-resolution imaging technology and fast (high performance) computer-based image stitching make it now feasible to digitize entire collection drawers typically used for arthropod collections, or trays or containers used for other objects. Resolutions of 500 megapixels and much higher are already utilized to capture the contents of 40x50 cm collection drawers, providing amazing detail of specimens. Flanked by metadata entry, this helps to create access to tens of thousands of specimens in days. By setting priorities and combining the holdings of the most comprehensive collections for certain taxa, drawer digitizing offers the unique opportunity to create a global, virtual metacollection.The taxonomic and geographic coverage of such a collection could never be achieved by a single institution or individual. We argue that by joining forces, many new impulses will emerge for systematic biology, related fields and understanding of biodiversity in general.Digitizing drawers containing unidentified, little-curated specimens is a contribution towards the beginning of a new era of online curation. It also will help taxonomists and curators to discover and process the millions of "gems" of undescribed species hidden in museum accessions.SummaryOur proposal suggests creating virtual, high-resolution image resources that will, for the first time in history, provide access for expert scientists as well as students and the general public to the enormous wealth of the world's natural history collections. We foresee that this will contribute to a better understanding, appreciation and increased use of biodiversity resources and the natural history collections serving this cause.

Description: Motivation: Pyrosequencing technology provides an important new approach to more extensively characterize diverse sequence populations and detect low frequency variants. However, the promise of this technology has been difficult to realize, as careful correction of sequencing errors is crucial to distinguish rare variants (~1%) in an infected host with high sensitivity and specificity. Results: We developed a new approach, referred to as Indel and Carryforward Correction (ICC), to cluster sequences without substitutions and locally correct only indel and carryforward sequencing errors within clusters to ensure that no rare variants are lost. ICC performs sequence clustering in the order of (i) homopolymer indel patterns only, (ii) indel patterns only and (iii) carryforward errors only, without the requirement of a distance cutoff value. Overall, ICC removed 93–95% of sequencing errors found in control datasets. On pyrosequencing data from a PCR fragment derived from 15 HIV-1 plasmid clones mixed at various frequencies as low as 0.1%, ICC achieved the highest sensitivity and similar specificity compared with other commonly used error correction and variant calling algorithms. Availability and implementation: Source code is freely available for download at http://indra.mullins.microbiol.washington.edu/ICC . It is implemented in Perl and supported on Linux, Mac OS X and MS Windows. Contact: jmullins@uw.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : Tiki proteins appear to antagonize Wnt signalling pathway by acting as Wnt proteases, thereby affecting Wnt solubility by its amino-terminal cleavage. Tiki1 protease activity was shown to be metal ion-dependent and was inhibited by chelating agents and thus was tentatively proposed to be a metalloprotease. Nevertheless, Tiki proteins exhibit no detectable sequence similarity to previously described metalloproteases, but instead have been reported as being homologues of TraB proteins (Pfam ID: PF01963), a widely distributed family of unknown function and structure. Here, we show that Tiki proteins are members of a new superfamily of domains contained not just in TraB proteins, but also in erythromycin esterase (Pfam ID: PF05139), DUF399 (domain of unknown function 399; Pfam ID: PF04187) and MARTX toxins that contribute to host invasion and pathogenesis by bacteria. We establish the core fold of this enzymatic domain and its catalytic residues. Contact: luis.sanchezpulido@dpag.ox.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: The research area metabolomics achieved tremendous popularity and development in the last couple of years. Owing to its unique interdisciplinarity, it requires to combine knowledge from various scientific disciplines. Advances in the high-throughput technology and the consequently growing quality and quantity of data put new demands on applied analytical and computational methods. Exploration of finally generated and analyzed datasets furthermore relies on powerful tools for data mining and visualization. Results: To cover and keep up with these requirements, we have created MeltDB 2.0, a next-generation web application addressing storage, sharing, standardization, integration and analysis of metabolomics experiments. New features improve both efficiency and effectivity of the entire processing pipeline of chromatographic raw data from pre-processing to the derivation of new biological knowledge. First, the generation of high-quality metabolic datasets has been vastly simplified. Second, the new statistics tool box allows to investigate these datasets according to a wide spectrum of scientific and explorative questions. Availability: The system is publicly available at https://meltdb.cebitec.uni-bielefeld.de . A login is required but freely available. Contact: nkessler@cebitec.uni-bielefeld.de

Description: Motivation: High - throughput next - generation sequencing technologies enable increasingly fast and affordable sequencing of genomes and transcriptomes, with a broad range of applications. The quality of the sequencing data is crucial for all applications. A significant portion of the data produced contains errors, and ever more efficient error correction programs are needed. Results: We propose RACER (Rapid and Accurate Correction of Errors in Reads), a new software program for correcting errors in sequencing data. RACER has better error-correcting performance than existing programs, is faster and requires less memory. To support our claims, we performed extensive comparison with the existing leading programs on a variety of real datasets. Availability: RACER is freely available for non-commercial use at www.csd.uwo.ca/~ilie/RACER/ . Contact: ilie@csd.uwo.ca Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : Interactions between various types of molecules that regulate crucial cellular processes are extensively investigated by high-throughput experiments and require dedicated computational methods for the analysis of the resulting data. In many cases, these data can be represented as a bipartite graph because it describes interactions between elements of two different types such as the influence of different experimental conditions on cellular variables or the direct interaction between receptors and their activators/inhibitors. One of the major challenges in the analysis of such noisy datasets is the statistical evaluation of the relationship between any two elements of the same type. Here, we present SICOP (significant co-interaction patterns), an implementation of a method that provides such an evaluation based on the number of their common interaction partners, their so-called co-interaction. This general network analytic method, proved successful in diverse fields, provides a framework for assessing the significance of this relationship by comparison with the expected co-interaction in a suitable null model of the same bipartite graph. SICOP takes into consideration up to two distinct types of interactions such as up- or downregulation. The tool is written in Java and accepts several common input formats and supports different output formats, facilitating further analysis and visualization. Its key features include a user-friendly interface, easy installation and platform independence. Availability: The software is open source and available at cna.cs.uni-kl.de/SICOP under the terms of the GNU General Public Licence (version 3 or later). Contact: agnes.horvat@iwr.uni-heidelberg.de or zweig@cs.uni-kl.de

Description: : Molecular recognition features (MoRFs) are small, intrinsically disordered regions in proteins that undergo a disorder-to-order transition on binding to their partners. MoRFs are involved in protein–protein interactions and may function as the initial step in molecular recognition. The aim of this work was to collect, organize and store all membrane proteins that contain MoRFs. Membrane proteins constitute ~30% of fully sequenced proteomes and are responsible for a wide variety of cellular functions. MoRFs were classified according to their secondary structure, after interacting with their partners. We identified MoRFs in transmembrane and peripheral membrane proteins. The position of transmembrane protein MoRFs was determined in relation to a protein’s topology. All information was stored in a publicly available mySQL database with a user-friendly web interface. A Jmol applet is integrated for visualization of the structures. mpMoRFsDB provides valuable information related to disorder-based protein–protein interactions in membrane proteins. Availability: http://bioinformatics.biol.uoa.gr/mpMoRFsDB Contact: shamodr@biol.uoa.gr

Description: : The signaling Petri net (SPN) simulator, designed to provide insights into the trends of molecules’ activity levels in response to an external stimulus, contributes to the systems biology necessity of analyzing the dynamics of large-scale cellular networks. Implemented into the freely available software, BioLayout Express 3D , the simulator is publicly available and easy to use, provided the input files are prepared in the GraphML format, typically using the network editing software, yEd, and standards specific to the software. However, analysis of complex networks represented using other systems biology formatting languages (on which popular software, such as CellDesigner and Cytoscape, are based) requires manual manipulation, a step that is prone to error and limits the use of the SPN simulator in BioLayout Express 3D . To overcome this, we present a Cytoscape plug-in that enables users to automatically convert networks for analysis with the SPN simulator from the standard systems biology markup language. The automation of this step opens the SPN simulator to a far larger user group than has previously been possible. Availability and implementation: Distributed under the GNU General Public License Version 3 at http://apps.cytoscape.org/apps/spnconverter . Contact: christine@picb.ac.cn

Description: Motivation: Conformational diversity is a key concept in the understanding of different issues related with protein function such as the study of catalytic processes in enzymes, protein-protein recognition, protein evolution and the origins of new biological functions. Here, we present a database of proteins with different degrees of conformational diversity. Conformational Diversity of Native State (CoDNaS) is a redundant collection of three-dimensional structures for the same protein derived from protein data bank. Structures for the same protein obtained under different crystallographic conditions have been associated with snapshots of protein dynamism and consequently could characterize protein conformers. CoDNaS allows the user to explore global and local structural differences among conformers as a function of different parameters such as presence of ligand, post-translational modifications, changes in oligomeric states and differences in pH and temperature. Additionally, CoDNaS contains information about protein taxonomy and function, disorder level and structural classification offering useful information to explore the underlying mechanism of conformational diversity and its close relationship with protein function. Currently, CoDNaS has 122 122 structures integrating 12 684 entries, with an average of 9.63 conformers per protein. Availability: The database is freely available at http://www.codnas.com.ar/ . Contact: gusparisi@gmail.com

Description: Background: A number of studies have implicated the direct involvement of the liver in dengue virus (DENV) infection, and it has been widely shown that liver cells subsequently undergo apoptosis. The mechanism by which liver cells undergo apoptosis in response to DENV infection remains unclear. To provide further information on the mechanism of apoptosis in DENV infected liver cells, HepG2 cells were infected with DENV 2 and analyzed for the induction of ER stress, apoptosis and autophagy. Results: In response to DENV infection, HepG2 cells showed the induction of both the ER resident unfolded protein response as well as the Noxa/PUMA stress response pathways. Proteolytic activation of caspases 4, 7, 8 and 9 was observed as well as changes in mitochondrial transmembrane potential. Increased monodansylcadaverine staining was observed in DENV infected cells, consistent with the previously reported induction of autophagy. Conclusions: These results are consistent with a model in which the induction of multiple ER stress pathways is coupled with the induction of multiple cell death pathways as a mechanism to ensure the removal of infected liver cells from the system.

Description: Motivation: Recent experimental advancements allow determining positions of nucleosomes for complete genomes. However, the resulting nucleosome occupancy maps are averages of heterogeneous cell populations. Accordingly, they represent a snapshot of a dynamic ensemble at a single time point with an overlay of many configurations from different cells. To study the organization of nucleosomes along the genome and to understand the mechanisms of nucleosome translocation, it is necessary to retrieve features of specific conformations from the population average. Results: Here, we present a method for identifying non-overlapping nucleosome configurations that combines binary-variable analysis and a Monte Carlo approach with a simulated annealing scheme. In this manner, we obtain specific nucleosome configurations and optimized solutions for the complex positioning patterns from experimental data. We apply the method to compare nucleosome positioning at transcription factor binding sites in different mouse cell types. Our method can model nucleosome translocations at regulatory genomic elements and generate configurations for simulations of the spatial folding of the nucleosome chain. Availability: Source code, precompiled binaries, test data and a web-based test installation are freely available at http://bioinformatics.fh-stralsund.de/nucpos/ Contact: gero.wedemann@fh-stralsund.de Supplementary Information: Supplementary data are available at Bioinformatics online.

Description: Wolbachia , endosymbiotic bacteria of the order Rickettsiales, are widespread in arthropods but also present in nematodes. In arthropods, A and B supergroup Wolbachia are generally associated with distortion of host reproduction. In filarial nematodes, including some human parasites, multiple lines of experimental evidence indicate that C and D supergroup Wolbachia are essential for the survival of the host, and here the symbiotic relationship is considered mutualistic. The origin of this mutualistic endosymbiosis is of interest for both basic and applied reasons: How does a parasite become a mutualist? Could intervention in the mutualism aid in treatment of human disease? Correct rooting and high-quality resolution of Wolbachia relationships are required to resolve this question. However, because of the large genetic distance between Wolbachia and the nearest outgroups, and the limited number of genomes so far available for large-scale analyses, current phylogenies do not provide robust answers. We therefore sequenced the genome of the D supergroup Wolbachia endosymbiont of Litomosoides sigmodontis , revisited the selection of loci for phylogenomic analyses, and performed a phylogenomic analysis including available complete genomes (from isolates in supergroups A, B, C, and D). Using 90 orthologous genes with reliable phylogenetic signals, we obtained a robust phylogenetic reconstruction, including a highly supported root to the Wolbachia phylogeny between a (A + B) clade and a (C + D) clade. Although we currently lack data from several Wolbachia supergroups, notably F, our analysis supports a model wherein the putatively mutualist endosymbiotic relationship between Wolbachia and nematodes originated from a single transition event.

Description: Background: Mediating DNA damage-induced apoptosis is an important genome-maintenance function of the mismatch repair (MMR) system. Defects in MMR not only cause carcinogenesis, but also render cancer cells highly resistant to chemotherapeutics, including alkylating agents. To understand the mechanisms of MMR-mediated apoptosis and MMR-deficiency-caused drug resistance, we analyze a model alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine, MNNG)-induced changes in protein phosphorylation and abundance in two cell lines, the MMR-proficient TK6 and its derivative MMR-deficient MT1. Results: Under an experimental condition that MNNG-induced apoptosis was only observed in MutSalpha-proficient (TK6), but not in MutSalpha-deficient (MT1) cells, quantitative analysis of the proteomic data revealed differential expression and phosphorylation of numerous individual proteins and clusters of protein kinase substrates, as well differential activation of response pathways/networks in MNNG-treated TK6 and MT1 cells. Many alterations in TK6 cells are in favor of turning on the apoptotic machinery, while many of those in MT1 cells are to promote cell proliferation and anti-apoptosis. Conclusions: Our work provides novel molecular insights into the mechanism of MMR-mediated DNA damage-induced apoptosis.

Description: Background: The specialisation of mammalian cells in time and space requires genes associated with specific pathways and functions to be co-ordinately expressed. Here we have combined a large number of publically available microarray datasets derived from human primary cells and analysed large correlation graphs of these data. Results: Using the network analysis tool BioLayout Express3D we identify robust co-associations of genes expressed in a wide variety of cell lineages. We discuss the biological significance of a number of these associations, in particular the coexpression of key transcription factors with the genes that they are likely to control. Conclusions: We consider the regulation of genes in human primary cells and specifically in the human mononuclear phagocyte system. Of particular note is the fact that these data do not support the identity of putative markers of antigen-presenting dendritic cells, nor classification of M1 and M2 activation states, a current subject of debate within immunological field. We have provided this data resource on the BioGPS web site (www.biogps.org) and on macrophages.com (www.macrophages.com).

Description: Many insects rely on bacterial symbionts with tiny genomes specialized for provisioning nutrients lacking in host diets. Xylem sap and phloem sap are both deficient as insect diets, but differ dramatically in nutrient content, potentially affecting symbiont genome evolution. For sap-feeding insects, sequenced symbiont genomes are available only for phloem-feeding examples from the suborder Sternorrhyncha and xylem-feeding examples from the suborder Auchenorrhyncha, confounding comparisons. We sequenced genomes of the obligate symbionts, Sulcia muelleri and Nasuia deltocephalinicola , of the phloem-feeding pest insect, Macrosteles quadrilineatus (Auchenorrhyncha: Cicadellidae). Our results reveal that Nasuia- ALF has the smallest bacterial genome yet sequenced (112 kb), and that the Sulcia- ALF genome (190 kb) is smaller than that of Sulcia in other insect lineages. Together, these symbionts retain the capability to synthesize the 10 essential amino acids, as observed for several symbiont pairs from xylem-feeding Auchenorrhyncha. Nasuia retains genes enabling synthesis of two amino acids, DNA replication, transcription, and translation. Both symbionts have lost genes underlying ATP synthesis through oxidative phosphorylation, possibly as a consequence of the enriched sugar content of phloem. Shared genomic features, including reassignment of the UGA codon from Stop to tryptophan, and phylogenetic results suggest that Nasuia -ALF is most closely related to Zinderia , the betaproteobacterial symbiont of spittlebugs. Thus, Nasuia / Zinderia and Sulcia likely represent ancient associates that have co-resided in hosts since the divergence of leafhoppers and spittlebugs 〉200 Ma, and possibly since the origin of the Auchenorrhyncha, 〉260 Ma.

Description: Background: Performing multiple tests in primary research is a frequent subject of discussion. This discussion originates from the fact that when multiple tests are performed, it becomes more likely to reject one of the null hypotheses, conditional on that these hypotheses are true and thus commit a type one error. Several correction methods for multiple testing are available. The primary aim of this study was to assess the quantity of articles published in two highly esteemed orthopedic journals in which multiple testing was performed. The secondary aims were to determine in which percentage of these studies a correction was performed and to assess the risk of committing a type one error if no correction was applied. Methods: The 2010 annals of two orthopedic journals (A and B) were systematically hand searched by two independent investigators. All articles on original research in which statistics were applied were considered. Eligible publications were reviewed for the use of multiple testing with respect to predetermined criteria. Results: A total of 763 titles were screened and 127 articles were identified and included in the analysis. A median of 15 statistical inference results were reported per publication in both journal A and B. Correction for multiple testing was performed in 15% of the articles published in journal A and in 6% from journal B. The estimated median risk of obtaining at least one significant result for uncorrected studies was calculated to be 54% for both journals. Conclusion: This study shows that the risk of false significant findings is considerable and that correcting for multiple testing is only performed in a small percentage of all articles published in the orthopedic literature reviewed.

Description: It has been argued that scale is the central problem in ecology ( Levin, 1992 ). Studies on carbon cycles and global climate change, the current major themes in modern ecology, require the interfacing of phenomena that occur on different scales of space, time, and ecological organization. For several decades, tremendous efforts have been made to reveal the general patterns of, and the mechanisms for the global carbon cycles. However, many uncertainties remain, particularly on local to regional scales. To reduce these uncertainties, regional collaborations across the board of nations are required.

Description: Aims Boreal forest is the largest and contains the most soil carbon among global terrestrial biomes. Soil respiration during the prolonged winter period may play an important role in the carbon cycles in boreal forests. This study aims to explore the characteristics of winter soil respiration in the boreal forest and to show how it is regulated by environmental factors, such as soil temperature, soil moisture and snowpack. Methods Soil respiration in an old-growth larch forest ( Larix gmelinii Ruppr.) in Northeast China was intensively measured during the winter soil-freezing process in 2011 using an automated soil CO 2 flux system. The effects of soil temperature, soil moisture and thin snowpack on soil respiration and its temperature sensitivity were investigated. Important Findings Total soil respiration and heterotrophic respiration both showed a declining trend during the observation period, and no significant difference was found between soil respiration and heterotrophic respiration until the snowpack exceeded 20cm. Soil respiration was exponentially correlated with soil temperature and its temperature sensitivity (Q 10 value) for the entire measurement duration was 10.5. Snow depth and soil moisture both showed positive effects on the temperature sensitivity of soil respiration. Based on the change in the Q 10 value, we proposed a ‘freeze–thaw critical point’ hypothesis, which states that the Q 10 value above freeze–thaw critical point is much higher than that below it (16.0 vs . 3.5), and this was probably regulated by the abrupt change in soil water availability during the soil-freezing process. Our findings suggest interactive effects of multiple environmental factors on winter soil respiration and recommend adopting the freeze–thaw critical point to model soil respiration in a changing winter climate.