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bioRxiv2018

Protein-Protein Interaction Network Analysis and Identification of Key Players in N-hydroxy-nor-L-Arg (nor-NOHA) and N(omega)-hydroxy-L-arginine (NOHA) Mediated Pathways for Treatment of Cancer Through Arginase Inhibiton: Insights from Systems Biology

L-arginine is involved in a number of biological processes in our bodies. Metabolism of L-arginine by the enzyme arginase has been found to be associated with cancer cell proliferation. Arginase inhibition has been proposed as a potential therapeutic means to inhibit this process. N-hydroxy-nor-L-Arg (nor-NOHA) and N (omega)-hydroxy-L-arginine (NOHA) has shown promise in inhibiting cancer progression through arginase inhibition. In this study, nor-NOHA and NOHA-associated genes and proteins were analyzed with several Bioinformatics and Systems Biology tools to identify the associated pathways and the key players involved so that a more comprehensive view of the molecular mechanisms including the regulatory mechanisms can be achieved and more potential targets for treatment of cancer can be discovered. Based on the analyses carried out, 3 significant modules have been identified from the PPI network. Five pathways/processes have been found to be significantly associated with nor-NOHA and NOHA associated genes. Out of the 1996 proteins in the PPI network, 4 have been identified as hub proteins-SOD, SOD1, AMD1, and NOS2. These 4 proteins have been implicated in cancer by other studies. Thus, this study provided further validation into the claim of these 4 proteins being potential targets for cancer treatment.

systems biology↗

bioRxiv2018

Long-read DNA metabarcoding of ribosomal rRNA in the analysis of fungi from aquatic environments

DNA metabarcoding is now widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints have limited most studies to marker lengths of ca. 300-600 bp. Longer sequencing reads of several 5 thousand bp are now possible with third-generation sequencing. The increased marker lengths provide greater taxonomic resolution and enable the use of phylogenetic methods of classifcation, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most well-established bioinformatics tools for DNA metabarcoding were originally 10 designed for short reads and are therefore not suitable. Here we used Pacifc Biosciences circular consensus sequencing (CCS) to DNA-metabarcode environmental samples using a ca. 4,500 bp marker that included most of the eukaryote ribosomal SSU and LSU rRNA genes and the ITS spacer region. We developed a long-read analysis pipeline that reduced error rates to levels 15 comparable to short-read platforms. Validation using fungal isolates and a mock community indicated that our pipeline detected 98% of chimeras de novo i.e., even in the absence of reference sequences. We recovered 947 OTUs from water and sediment samples in a natural lake, 848 of which could be classifed to phylum, 486 to family, 397 to genus and 330 to species. By 20 allowing for the simultaneous use of three global databases (Unite, SILVA, RDP LSU), long-read DNA metabarcoding provided better taxonomic resolution than any single marker. We foresee the use of long reads enabling the cross-validation of reference sequences and the synthesis of ribosomal rRNA gene databases. The universal nature of the rRNA operon and our recovery of >100 25 non-fungal OTUs indicate that long-read DNA metabarcoding holds promise for the study of eukaryotic diversity more broadly.

ecology↗

bioRxiv2018

Rapid Paediatric Sequencing (RaPS): Comprehensive real-life workflow for rapid diagnosis of critically ill children

BackgroundRare genetic conditions are frequent risk factors for, or direct causes of, organ failure requiring paediatric intensive care unit (PICU) support. Such conditions are frequently suspected but unidentified at PICU admission. Compassionate and effective care is greatly assisted by definitive diagnostic information. There is therefore a need to provide a rapid genetic diagnosis to inform clinical management.\n\nTo date, Whole Genome Sequencing (WGS) approaches have proved successful in diagnosing a proportion of children with rare diseases, but results may take months to report or require the use of equipment and practices not compatible with a clinical diagnostic setting. We describe an end-to-end workflow for the use of rapid WGS for diagnosis in critically ill children in a UK National Health Service (NHS) diagnostic setting.\n\nMethodsWe sought to establish a multidisciplinary Rapid Paediatric Sequencing (RaPS) team for case selection, trio WGS, a rapid bioinformatics pipeline for sequence analysis and a phased analysis and reporting system to prioritise genes with a high likelihood of being causal. Our workflow was iteratively developed prospectively during the analysis of the first 10 children and applied to the following 14 to assess its utility.\n\nFindingsTrio WGS in 24 critically ill children led to a molecular diagnosis in ten (42%) through the identification of causative genetic variants. In three of these ten individuals (30%) the diagnostic result had an immediate impact on the individuals clinical management. For the last 14 trios, the shortest time taken to reach a provisional diagnosis was four days (median 7 days).\n\nInterpretationRapid WGS can be used to diagnose and inform management of critically ill children using widely available off the shelf products within the constraints of an NHS clinical diagnostic setting. We provide a robust workflow that will inform and facilitate the rollout of rapid genome sequencing in the NHS and other healthcare systems globally.\n\nFundingThe study was funded by NIHR GOSH/UCL BRC: ormbrc-2012-1

genomics↗

bioRxiv2018

Genome-specific histories of divergence and introgression between an allopolyploid unisexual salamander lineage and two sexual species

Quantifying genetic introgression between sexual species and polyploid lineages traditionally thought to be asexual is an important step in understanding what factors drive the longevity of putatively asexual groups. However, the presence of multiple distinct subgenomes within a single lineage provides a significant logistical challenge to evaluating the origin of genetic variation in most polyploids. Here, we capitalize on three recent innovations--variation generated from ultraconserved elements (UCEs), bioinformatic techniques for assessing variation in polyploids, and model-based methods for evaluating historical gene flow--to measure the extent and tempo of introgression over the evolutionary history of an allopolyploid lineage of all-female salamanders and two ancestral sexual species. We first analyzed variation from more than a thousand UCEs using a reference mapping method developed for polyploids to infer subgenome specific patterns of variation in the all-female lineage. We then used PHRAPL to choose between sets of historical models that reflected different patterns of introgression and divergence between the genomes of the parental species and the same genomes found within the polyploids. Our analyses support a scenario in which the genomes sampled in unisexuals salamanders were present in the lineage [~]3.4 million years ago, followed by an extended period of divergence from their parental species. Recent secondary introgression has occurred at different times between each sexual species and their representative genomes within the unisexuals during the last 500,000 years. Sustained introgression of sexual genomes into the unisexual lineage has been the defining characteristic of their reproductive mode, but this study provides the first evidence that unisexual genomes have also undergone long periods of divergence without introgression. Unlike other unisexual, sperm-dependent taxa in which introgression is rare, the alternating periods of divergence and introgression between unisexual salamanders and their sexual relatives could reveal the scenarios in which the influx of novel genomic material is favored and potentially explain why these salamanders are among the oldest described unisexual animals.

evolutionary biology↗

bioRxiv2018

Single-molecule optical mapping enables accurate molecular diagnosis of facioscapulohumeral muscular dystrophy (FSHD)

Facioscapulohumeral Muscular Dystrophy (FSHD) is a common adult muscular dystrophy in which the muscles of the face, shoulder blades and upper arms are among the most affected. FSHD is the only disease in which \"junk\" DNA is reactivated to cause disease, and the only known repeat array-related disease where fewer repeats cause disease. More than 95% of FSHD cases are associated with copy number loss of a 3.3kb tandem repeat (D4Z4 repeat) at the subtelomeric chromosomal region 4q35, of which the pathogenic allele contains less than 10 repeats and has a specific genomic configuration called 4qA. Currently, genetic diagnosis of FSHD requires pulsed-field gel electrophoresis followed by Southern blot, which is labor-intensive, semi-quantitative and requires long turnaround time. Here, we developed a novel approach for genetic diagnosis of FSHD, by leveraging Bionano Saphyr single-molecule optical mapping platform. Using a bioinformatics pipeline developed for this assay, we found that the method gives direct quantitative measurement of repeat numbers, can differentiate 4q35 and the highly paralogous 10q26 regions, can determine the 4qA/4qB allelic configuration, and can quantitate levels of post-zygotic mosaicism. We evaluated this approach on 5 patients (including two with post-zygotic mosaicism) and 2 patients (including one with post-zygotic mosaicism) from two separate cohorts, and had complete concordance with Southern blots, but with improved quantification of repeat numbers resolved between haplotypes. We concluded that single-molecule optical mapping is a viable approach for molecular diagnosis of FSHD and may be applied in clinical diagnostic settings once more validations are performed.

genomics↗

bioRxiv2018

Controlling false discoveries in Bayesian gene networks with lasso regression p-values

MotivationBayesian networks can represent directed gene regulations and therefore are favored over co-expression networks. However, hardly any Bayesian network study concerns the false discovery control (FDC) of network edges, leading to low accuracies due to systematic biases from inconsistent false discovery levels in the same study.\n\nResultsWe design four empirical tests to examine the FDC of Bayesian networks from three p-value based lasso regression variable selections -- two existing and one we originate. Our method, lassopv, computes p-values for the critical regularization strength at which a predictor starts to contribute to lasso regression. Using null and Geuvadis datasets, we find that lassopv obtains optimal FDC in Bayesian gene networks, whilst existing methods have defective p-values. The FDC concept and tests extend to most network inference scenarios and will guide the design and improvement of new and existing methods. Our novel variable selection method with lasso regression also allows FDC on other datasets and questions, even beyond network inference and computational biology.\n\nAvailabilityLassopv is implemented in R and freely available at https://github.com/lingfeiwang/lassopv and https://cran.r-project.org/package=lassopv.\n\nContactLingfei.Wang@roslin.ed.ac.uk\n\nSupplementary informationSupplementary data are available at Bioinformatics online.

systems biology↗

bioRxiv2018

THE MITOCHONDRIAL DNA CONTROL REGION MIGHT HAVE USEFUL DIAGNOSTIC AND PROGNOSTIC BIOMARKERS FOR THYROID TUMORS

BackgroundIt is currently present in the literature that mitochondrial DNA (mtDNA) defects are associated with a great number of diseases including cancers. The role of mitochondrial DNA (mtDNA) variations in the development of thyroid cancer is a highly controversial topic. In this study, we investigated the role of mt-DNA control region (CR) variations in thyroid tumor progression and the influence of mtDNA haplogroups on susceptibility to thyroid tumors.\n\nMaterial & methodFor this purpose, totally 108 hot thyroid nodules (HTNs), 95 cold thyroid nodules (CTNs), 48 papillary thyroid carcinoma (PTC) samples with their surrounding tissues and 104 healthy control subjects blood samples were screened for entire mtDNA CR variations by using Sanger sequencing. The obtained DNA sequences were anaysed with the mistomaster, a web-based bioinformatics tool.\n\nResultsMtDNA haplogroup U was significantly associated with susceptibility to benign and malign thyroid entities on the other hand J haplogroup was associated with a protective role for benign thyroid nodules. Besides, 8 SNPs (T146C, G185A, C194T, C295T, G16129A, T16304C, A16343G and T16362C) in mtDNA CR region were associated with the occurrence of benign and malign thyroid nodules in Turkish population. By contrast with the healthy Turkish population and HTNs, frequency of C7 repeats in D310 polycytosine sequence was found higher in cold thyroid nodules and PTC samples. Beside this, the frequency of somatic mutations in mtMSI regions including T16189C and D514 CA dinucleotide repeats were found higher in PTC samples than the benign thyroid nodules. Conversely, the frequency of somatic mutations in D310 was detected higher in HTNs than CTNs and PTCs.\n\nConclusionmtDNA D310 instability do not play a role in tumorogenesis of the PTC but the results indicates that it might be used as a diagnostic clonal expansion biomarker for premalignant thyroid tumor cells. Beside this, D514 CA instability might be used as prognostic biomarker in PTCs. Also, we showed that somatic mutation rate is less frequent in more aggressive tumors when we examined micro- and macro carcinomas as well as BRAFV600E mutation.

cancer biology↗

bioRxiv2018

Natural selection on gene-specific codon usage bias is common across eukaryotes

Although the actual molecular evolutionary forces that shape differences in codon usage across species remain poorly understood, majority of synonymous mutations are assumed to be functionally neutral because they do not affect protein sequences. However, empirical studies suggest that some synonymous mutations can have phenotypic consequences. Here we show that in contrast to the current dogma, natural selection on gene-specific codon usage bias is common across Eukaryota. Furthermore, by using bioinformatic and experimental approaches, we demonstrate that specific combinations of rare codons contribute to the spatial and sex-related regulation of some protein-coding genes in Drosophila melanogaster. Together, these data indicate that natural selection can shape gene-specific codon usage bias, which therefore, represents an overlooked genomic feature that is likely to play an important role in the spatial and temporal regulation of gene functions. Hence, the broadly accepted dogma that synonymous mutations are in general functionally neutral should be reconsidered.

evolutionary biology↗

bioRxiv2018

LIkelihood-based Fits of Folding Transitions (LIFFT) for Biomolecule Mapping Data

SummaryBiomolecules shift their structures as a function of temperature and concentrations of protons, ions, small molecules, proteins, and nucleic acids. These transitions impact or underlie biological function and are being monitored at increasingly high throughput. For example, folding transitions for large collections of RNAs can now be monitored at single residue resolution by chemical mapping techniques. LIkelihood-based Fits of Folding Transitions (LIFFT) quantifies these data through well-defined thermodynamic models. LIFFT implements a Bayesian framework that takes into account data at all measured residues and enables visual assessment of modeling uncertainties that can be overlooked in least-squares fits. The framework is appropriate for multimodal techniques ranging from chemical mapping including multi-wavelength spectroscopy.\n\nAvailabilityFreely available MATLAB package at https://ribokit.stanford.edu/LIFFT/.\n\nContactrhiju@stanford.edu\n\nSupplementary informationSupplementary data are available at Bioinformatics online.

biochemistry↗

bioRxiv2018

Earthworms and Cadmium - heavy metal resistant gut bacteria as indicators for heavy metal pollution in soils?

Preservation of the soil resources stability is of paramount importance for the ecosystem, particularly in the current era of environmental change, which presents a severe pollution burden (e.g. by heavy metals) to soil ecosystems and its fauna. Gut microbiomes are becoming recognized as important players in organism health, with comprehension of their perturbations in the polluted environment offering new insights into the nature and extent of heavy metal effects on the health of soil biota. Our aim was therefore to evaluate the effect of environmentally relevant heavy metal concentrations of cadmium (Cd) on the earthworm gut microbiota. Cd exposure led to perturbations of several heavy metal resistant taxa as well as taxa able to bind heavy metals, revealing the potential of the earthworm-microbiota system in overcoming humancaused heavy metal pollution. An indicator species analysis linked bacterial genera Paenibacillus and Flavobacterium, and members of the order Actinomycetales with Cd treatment, suggesting the possible use of these bacterial taxa as biomarkers of exposure for Cd stressed soils. The results of this study will be essential to understanding of the soil fauna health, under anthropogenic disturbance, and will have implications for environmental monitoring and protection of soil resources.\n\nImportanceSoil heavy metal pollution presents a severe burden for soil invertebrates and can have impact on their health, which in turn reflects on the health of the entire ecosystem. Gut microbiome is recognized as a central driver of the host health and its shifts can have severe consequences for the host. In this study we investigated the impact of cadmium (Cd) on earthworm gut microbiota, in a controlled experiment using cutting edge next generation sequencing and state of the art bioinformatics tools. The significance of this study is in identifying the gut bacterial taxa which are indicators for Cd treatment and are potential biomarkers of exposure to Cd. Therefore, this study contributes to develop efficient measures to qualify environmental pollution and to protect fragile soil resources and ultimately human health.

microbiology↗

bioRxiv2018

Complementary information on single nucleotide variants, INDELs and functional translocations can be obtained with RNAseq using different library preparations

BackgroundRNA-seq represents an attractive methodology for the detection of functional genomic variants because it allows the integration of variant frequency and their expression. However, although specific statistic frameworks have been designed to detect SNVs/INDELS/gene fusions in RNA-seq data, very little has been done to understand the effect of library preparation protocols on transcript variant detection in RNA-seq data.\n\nResultsHere, we compared RNA-seq results obtained on short reads sequencing platform with two protocols: one based on polyA+ RNA selection protocol (POLYA) and the other based on exonic regions capturing protocol (ACCESS). Our data indicate that ACCESS detects 10% more coding SNV/INDELs with respect to POLYA, making this protocol more suitable for this goal. Furthermore, ACCESS requires less reads for coding SNV detection with respect to POLYA. On the other hand, if the analysis aims at identifying SNV/INDELs also in the 5and 3 UTRs, POLYA is definitively the preferred method. No particular advantage comes from the usage of ACCESS or POLYA in the detection of fusion transcripts.\n\nConclusionData show that a careful selection of the \"wet\" protocol adds specific features that cannot be obtained with bioinformatics alone.

genomics↗

bioRxiv2018

MicroRNA-205 affects mouse granulosa cell apoptosis and estradiol synthesis by targeting CREB1

MicroRNAs-205 (miR-205), were reportedly to be involved in various physiological and pathological processes, but its biological function in follicular atresia remain unknown. In this study, we investigated the expression of miR-205 in mouse granulosa cells (mGCs), and explored its functions in primary mGCs using a serial of in vitro experiments. The result of qRT-PCR demonstrated that miR-205 expression was significantly increased in early atretic follicles (EAF), and progressively atretic follicles (PAF) compared to healthy follicles (HF). Our results also revealed that overexpression of miR-205 in mGCs significantly promoted apoptosis, caspas-3/9 activities, and inhibited estrogen E2 release, and cytochrome P450 family 19 subfamily A polypeptide 1 (CYP19A1, a key gene in E2 production) expression. Bioinformatics and luciferase reporter assays revealed that the gene of cyclic AMP response element (CRE)-binding protein 1 (CREB1) was a potential target of miR-205. qRT-PCR and western blot assays revealed that overexpression of miR-205 inhibited the expression of CREB1 in mGCs. Importantly, CREB1 upregulation partially rescued the effects of miR-205 on apoptosis, caspase-3/9 activities, E2 production and CYP19A1 expression in mGCs. Our results indicate that miR-205 may play an important role in ovarian follicular development and provide new insights into follicular atresia.

evolutionary biology↗

bioRxiv2018

Glycoprotein 3 of porcine reproductive and respiratory syndrome virus exhibits an unusual hairpin-like membrane topology

The glycoprotein GP3 of the Arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) consists of a cleaved signal peptide, a highly glycosylated domain, a short hydrophobic region and an unglycosylated C-terminal domain. GP3 is supposed to form a complex with GP2 and GP4 in virus particles, but secretion of GP3 from cells has also been reported.\n\nWe analyzed the membrane topology of GP3 from various PRRSV strains. A fraction of the protein is secreted from transfected cells; GP3 from PRRSV-1 strains to a greater extent than GP3 from PRRSV-2 strains. This secretion behavior is reversed after exchange of the variable C-terminal domain. A fluorescence protease protection assay shows that the C-terminus of GP3, fused to GFP, is resistant against proteolytic digestion in permeabilized cells. Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. Both experiments indicate that the C-terminus of GP3 is translocated into the lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic region greatly enhances secretion of GP3 and fusion of this domain to GFP promotes membrane anchorage. Bioinformatics suggests that the hydrophobic region might form an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents and in its hydrophobic face enhances secretion of GP3. Exchanging the latter amino acids in the context of the viral genome did not affect release of virions, but released particles were not infectious. In sum, GP3 exhibits an unusual hairpin-like membrane topology that might explain why a fraction of the protein is secreted.\n\nIMPORTANCEThe porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the pork industry. It causes persistent infections that lead to reduced weight gain of piglets; highly pathogenic strains even kill 90% of an infected pig population. PRRSV cannot be eliminated from pig farms by vaccination due to the large amino acid variability between the existing strains, especially in the glycoproteins. Here we analyzed basic structural features of glycoprotein 3 (GP3) from various PRRSV strains. We show that the protein exhibits an unusual hairpin-like membrane topology; membrane anchoring might occur via an amphipathic helix. This rather weak membrane anchor explains why a fraction of the protein is secreted from cells. Interestingly, PRRSV-1 strains secrete more GP3 than PRRSV-2. We speculate that secreted GP3 might play a role during PRRSV infection of pigs; it might serve as a decoy to distract antibodies away from virus particles.

biochemistry↗

bioRxiv2018

Intratumoral MAPK and PI3K signaling pathway heterogeneity in glioblastoma tissuecorrelates with selective CREB signaling and specific functional gene signatures

Limitations in discovering useful tumor biomarkers and drug targets is not only due to patient-to-patient differences but also due to intratumoral heterogeneity. Heterogeneity arises due to the genetic and epigenetic variation of tumor cells in response to microenvironmental interactions and cytotoxic therapy. We explored specific signaling pathway activation in glioblastoma (GBM) by investigating the intratumoral activation of the MAPK and PI3K pathways. We present data demonstrating a striking preponderance for mutual exclusivity of MAPK and PI3K activation in GBM tissue, where MAPK activation correlates with proliferation and transcription factor CREB activation and PI3K activation correlates with CD44 expression. Bioinformatic analysis of signaling and CREB-regulated target genes supports the immunohistochemical data, showing that the MAPK-CREB activation correlates with proliferative regions. In-silico analysis suggests that MAPK-CREB signaling activates a pro-inflammatory molecular signature and correlates with a mesenchymal GBM subtype profile, while PI3K-CREB activation correlates with the proneural GBM subtype and a tumor cell invasive gene signature. Overall, the data suggests the existence of intratumoral subtype heterogeneity in GBM and that using combinations of both MAPK and PI3K drug inhibitors is necessary for effective targeted therapy.

pathology↗

bioRxiv2018

Characterization of the apicomplexan amino acid transporter (ApiAT) family in Toxoplasma gondii

Apicomplexan parasites are auxotrophic for a range of amino acids which must be salvaged from their host cells, either through direct uptake or degradation of host proteins. Here, we describe a family of plasma membrane-localized amino acid transporters, termed the Apicomplexan Amino acid Transporters (ApiATs), that are ubiquitous in apicomplexan parasites. Functional characterization of the ApiATs of Toxoplasma gondii indicate that several of these transporters are important for intracellular growth of the tachyzoite stage of the parasite, which is responsible for acute infections. We demonstrate that the ApiAT protein TgApiAT5-3 is an exchanger for aromatic and large neutral amino acids, with particular importance for L-tyrosine scavenging and amino acid homeostasis, and that TgApiAT5-3 is critical for parasite virulence. Our data indicate that T. gondii expresses additional proteins involved in the uptake of aromatic amino acids, and we present a model for the uptake and homeostasis of these amino acids. Our findings identify a family of amino acid transporters in apicomplexans, and highlight the importance of amino acid scavenging for the biology of this important phylum of intracellular parasites.\n\nAuthor SummaryThe Apicomplexa comprise a large number of parasitic protozoa that have obligate intracellular lifestyles and cause significant human and animal diseases, including malaria, cryptosporidiosis, toxoplasmosis, coccidiosis in poultry, and various cattle fevers. Apicomplexans must scavenge essential nutrients from their hosts in order to proliferate and cause disease, including a range of amino acids. The direct uptake of these nutrients is presumed to be mediated by transporter proteins located in the plasma membrane of intracellular stages, although the identities of these proteins are poorly defined. Using a combination of bioinformatic, genetic, cell biological, and physiological approaches, we have characterized a family of plasma membrane-localized transporter proteins that we have called the Apicomplexan Amino acid Transporters (ApiATs). The family is found in apicomplexans and their closest free-living relatives. We show that TgApiAT5-3, a member of the family in the apicomplexan Toxoplasma gondii, is an exchanger for aromatic and large neutral amino acids. In particular, it is critical for uptake of tyrosine, and for parasite virulence in a mouse infection model. We conclude that ApiATs are a family of plasma membrane transporters that play crucial roles in amino acid scavenging by apicomplexan parasites.

microbiology↗

bioRxiv2018

Characterization of genomic DNA of lactic acid bacteria for activation of plasmacytoid dendritic cells .

Lactococcus lactis strain Plasma (LC-Plasma) possesses strong activity of stimulating plasmacytoid dendritic cells (pDCs) via the TLR9-Myd88 pathway. To reveal the effective genome structure for pDCs stimulatory activity, we performed an in vitro screening, using randomly selected DNA fragments from the LC-Plasma genome. The results showed that CpG motifs are necessary factor for active DNA fragment, but the copy number of CpG motifs did not show strong correlation to the pDCs stimulatory activity of DNA fragment. We also found that the G+C contents of DNA fragments have significant negative effects on pDCs stimulatory activity. We also performed bioinformatics analysis of genome of lactic acid bacteria (LAB) and investigated the relation between CpG copy number in the genome and pDCs stimulatory activity. We found that strains of lactic acid bacteria (LAB) with high copy number of CpG motifs in the low-G+C region of the genome had higher probability of having high pDCs stimulatory activity. Three species, L.lactis subsp. lactis, Leuconostoc mesenteroides, and Pediococcus pentosaceus were the typical examples of high pDCs stimulatory activity LAB.\n\nImportanceThis study provides a new perspective on the structure of DNA fragments that are able to activate pDCs via the TLR9-Myd88 pathway. The information from this study should be useful for designing new DNA fragments, including phosphodiesterbond-DNA oligomers containing CpG motifs and DNA-containing vaccines. This work also presented an in silico screening method for identifying bacterial species that are able to activate pDCs. Therefore, this study should be useful for providing data for the development of vaccine adjuvants and therapeutics for infectious and allergic diseases.

microbiology↗

bioRxiv2018

Interspecies DNA acquisition by a naturally competent Acinetobacter baumannii strain

Acinetobacter baumannii is a human pathogen that frequently acquires antibiotic resistance genes leading to the emergence of multi-drug-resistant (MDR) strains. To investigate the role of transformation in the acquisition of resistance determinants by this species, the susceptible strain A118 was exposed to genomic DNA of carbapenem-resistant Klebsiella pneumoniae (CRKp). Resistant transformants were obtained and an increase in the resistance level to all {beta}-lactam antibiotics was observed. Whole genome analysis of transformant clones demonstrated the acquisition of CRKp DNA. The most frequently acquired genes correspond to mobile elements, antibiotic resistance genes, and operons involved in metabolism. Bioinformatic analyses and in silico gene flow prediction strengthen our findings, showing that a continuing exchange of genetic material between A. baumannii and K. pneumoniae occurs when they share the same niche. Our results reinforce the idea that natural transformation may play a key role in the increasing emergence of A. baumannii MDR.\n\nIMPORTANCESince the characterization of antibiotic resistance in the late 50s, antibiotic resistance propagation was classically associated with horizontal gene transfer (HGT) mediated by plasmids bearing multiple resistance genes. Here we show that, at least in the human pathogen A. baumannii, transformation also plays a major role in the acquisition of antibiotic resistance determinants. This study unravels that at least for certain pathogens the propagation of resistance genes occurs by alternative HGT mechanisms which in the past have been unappreciated.

microbiology↗

bioRxiv2018

A consistent picture of TRPV1 activation emerges from molecular simulations and experiments

Although the structure of TRPV1 has been experimentally determined in both the closed and open states, very little is known about its activation mechanism. In particular, the conformational changes occurring in the pore domain and resulting in ionic conduction have not been identified yet. Here, we suggest a hypothetical molecular mechanism for TRPV1 activation, which involves the rotation of a conserved asparagine in S6 from the S4-S5 linker toward the pore. This rotation is correlated with the dehydration of four peripheral cavities located between S6 and the S4-S5 linker and the hydration of the pore. In light of our hypothesis, we perform bioinformatics analyses of TRP and other evolutionary related ion channels, analyze newly available structures and re-examine previously reported water accessibility and mutagenesis experiments. Overall, we provide several independent lines of evidence that corroborate our hypothesis. Finally, we show that the proposed molecular mechanism is compatible with the currently existing idea that in TRPV1 the selectivity filter acts as a secondary gate.

biophysics↗