SEARCH · Search bioRxiv

Results for “Bioinformatics”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 703 records · Page 39Linked to original sources

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↗

bioRxiv2018

Precision Identification of Diverse Bloodstream Pathogens from the Gut Microbiome

Bloodstream infection is the most common infectious complication in hematopoietic cell transplantation recipients. To evaluate the genomic concordance of bloodstream pathogens and bacterial strains within the intestinal microbiome using whole genome sequencing, we developed StrainSifter, a bioinformatic pipeline to compare nucleotide variation between bacterial isolate strains and stool metagenomes. We applied StrainSifter to bloodstream isolates and stool metagenome samples from hematopoietic stem cell transplant recipients with bloodstream infections. StrainSifter is designed to identify single nucleotide variants between isolate and metagenomic short reads using stringent alignment, coverage, and variant frequency criteria for strain comparison. We identified enteric BSI isolates that were highly concordant with those in the gut microbiota, as well as highly concordant strains of typically non-enteric bacteria. These findings demonstrate the utility of StrainSifter in strain matching and provide a more precise investigation of the intestine as a reservoir of diverse pathogens capable of causing bloodstream infections.

genomics↗

bioRxiv2018

Identification of the interaction of 2-aminosteroids with G-quadruplex in BCR/ABL gene promoter: An emerging potential target for treatment of human chronic myelogenous leukemia

Our preliminary studies have verified that the small molecules aminosteroid could inhibit the mRNA expression of bcr/abl fusion gene in CML (Chronic Myelogenous Leukemia) cells, which may be effective in treating CML and that may have dramatically different mechanism underlying the effects by tyrosine kinase inhibitors (TKI) binding to the BCR/ABL protein. Therefore, the exact mechanism of how the aminosteroid inhibited the CML growth should be clarified and we pay our attention to the promoter domain of BCR/ABL gene to see if any interaction between aminosteroid and the promoter. First, it should be verified if G-quadruplex could be formed in BCR/ABL promoter region. Secondly, it is highly desirable to verify if aminoteroid could be interacted with the G-quadruplex structure. Here, we reported a novel therapeutic strategy based on the targeting of the G-quadruplex which formed in the promoter regions of BCR/ABL gene by aminosteroid compounds KH and BH, verified by using bioinformatics and computer simulation, UV-Vis absorption spectra, circular dichrosism(CD), fluorescence absorption spectra, fluorescence emission lifetime expenditure experiments and polymerase chain stop assays. It showed that G-quadruplex structures can be folded in BCR/ABL promoter regions and aminosteroid inhibits the mRNA expression of BCR/ABL fusion gene by stabilizing the structure of G-quadruplex, and then inhibiting the DNA replication and transcription. This demonstrates the theory that is the so-called \"chemical gene therapy\" by aminosteroid in the interaction with G-quadruplex is an emerging therapeutic protocol in treatment of chronic myeloid leukemia.

cancer biology↗

bioRxiv2018

Structural basis for transfer RNA mimicry by a bacterial Y RNA

Noncoding Y RNAs are present in both animal cells and many bacteria. In all species examined, Y RNAs tether the Ro60 protein to an effector protein to perform various cellular functions. For example, in the bacterium Deinococcus radiodurans, Y RNA tethers Ro60 to the exoribonuclease polynucleotide phosphorylase, specializing this nuclease for structured RNA degradation. Recently, a new Y RNA subfamily was identified in bacteria. Bioinformatic analyses of these YrlA (Y RNA-like A) RNAs predict that the effector-binding domain resembles tRNA. We present the structure of this domain, the overall folding of which is strikingly similar to canonical tRNAs. The tertiary interactions that are responsible for stabilizing tRNA are present in YrlA, making it a close tRNA mimic. However, YrlA lacks a free CCA end and contains a kink in the stem corresponding to the anticodon stem. Since nucleotides in the D and T stems are conserved among YrlAs, they may be an interaction site for an unknown factor. Our experiments identify YrlA RNAs as a new class of tRNA mimics.

biochemistry↗

bioRxiv2018

Analysis of 19 Highly Conserved Vibrio cholerae Bacteriophages Isolated from Environmental and Patient Sources Over a Twelve-Year Period

The Vibrio cholerae biotype El Tor is responsible for all current epidemic and endemic cholera outbreaks worldwide. These outbreaks are clonal and are hypothesized to originate from the coastal areas near the Bay of Bengal where the lytic bacteriophage ICP1 specifically preys upon these pathogenic outbreak strains. ICP1 has also been the dominant bacteriophage found in cholera patient stool since 2001. However, little is known about its genomic differences between ICP1 strains collected over time. Here we elucidate the pan-genome and phylogeny of ICP1 strains by aligning, annotating and analyzing the genomes of 19 distinct isolates collected between 2001 and 2012. Our results reveal that ICP1 isolates are highly conserved and possess a large core-genome as well as a smaller, somewhat flexible accessory-genome. Despite its overall conservation, ICP1 strains have managed to acquire a number of unknown genes as well as a CRISPR-Cas system, which is known to be critical for its ongoing struggle for co-evolutionary dominance over its host. This study describes a foundation on which to construct future molecular and bioinformatic studies of this V. cholerae-associated bacteriophages.

microbiology↗

bioRxiv2018

Specific Oxylipins Enhance Vertebrate Hematopoiesis via the Receptor GPR132

Epoxyeicosatrienoic acids (EETs) are endogenous lipid signaling molecules with cardioprotective and vasodilatory actions. We recently showed that exogenous addition of 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via a G-protein coupled receptor(s), and significant research supports the existence of a specific high-affinity receptor. Identification of a hematopoietic specific EET receptor would enable genetic interrogation of the EET signaling pathway and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify the EET receptor based on the expression of GPCRs in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are commonly expressed in three EET-responsive human cell lines, but not expressed in an EET-unresponsive line. Of these candidates, only GPR132 showed EET-responsiveness in vitro using a luminescence-based assay for {beta}-arrestin recruitment. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to the putative high-affinity EET receptor, GPR132 is reported to have affinity for additional fatty acids in vitro, and we found that these same fatty acids enhance hematopoietic stem cell specification in the zebrafish. We conducted structure-activity relationship analyses using both in vitro and in vivo assays on diverse medium chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, while unoxygenated or saturated fatty acids had lower activity. Absence of the carboxylic acid moiety prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of polyunsaturated, oxygenated fatty acids to enhance both embryonic and adult hematopoiesis.

developmental biology↗

bioRxiv2018

Methylation patterns reveal cryptic structure and a pathway for adaptation in a panmictic carnivore

Determining the molecular signatures of adaptive differentiation is a fundamental component of evolutionary biology. A key challenge remains for identifying such signatures in wild organisms, particularly between populations of highly mobile species that undergo substantial gene flow. The Canada lynx (Lynx canadensis) is one species where mainland populations appear largely undifferentiated at traditional genetic markers, despite inhabiting diverse environments and displaying phenotypic variation. Here, we used high-throughput sequencing to investigate both neutral genetic structure and epigenetic differentiation across the distributional range of Canada lynx. Using a customized bioinformatics pipeline we scored both neutral SNPs and methylated nucleotides across the lynx genome. Newfoundland lynx were identified as the most differentiated population at neutral genetic markers, with diffusion approximations of allele frequencies indicating that divergence from the panmictic mainland occurred at the end of the last glaciation, with minimal contemporary admixture. In contrast, epigenetic structure revealed hidden levels of differentiation across the range coincident with environmental determinants including winter conditions, particularly in the peripheral Newfoundland and Alaskan populations. Several biological pathways related to morphology were differentially methylated between populations, with Newfoundland being disproportionately methylated for genes that could explain the observed island dwarfism. Our results indicate that epigenetic modifications, specifically DNA methylation, are powerful markers to investigate population differentiation and functional plasticity in wild and non-model systems.\n\nSIGNIFICANCEPopulations experiencing high rates of gene flow often appear undifferentiated at neutral genetic markers, despite often extensive environmental and phenotypic variation. We examined genome-wide genetic differentiation and DNA methylation between three interconnected regions and one insular population of Canada lynx (Lynx canadensis) to determine if epigenetic modifications characterized climatic associations and functional molecular plasticity. Demographic approximations indicated divergence of Newfoundland during the last glaciation, while cryptic epigenetic structure identified putatively functional differentiation that might explain island dwarfism. Our study suggests that DNA methylation is a useful marker for differentiating wild populations, particularly when faced with functional plasticity and low genetic differentiation.

genomics↗

bioRxiv2018

Sensitive detection of pre-integration intermediates of LTR retrotransposons in crop plants

Retrotransposons have played an important role in the evolution of host genomes1,2. Their impact on host chromosomes is mainly deduced from the composition of DNA sequences, which have been fixed over evolutionary time. These studies provide important \"snapshots\" reflecting historical activities of transposons but do not predict current transposition potential. We previously reported Sequence-Independent Retrotransposon Trapping (SIRT) as a methodology that, by identification of extrachromosomal linear DNA (eclDNA), revealed the presence of active LTR retrotransposons in Arabidopsis9. Unfortunately, SIRT cannot be applied to large and transposon-rich genomes of crop plants. We have since developed an alternative approach named ALE-seq (amplification of LTR of eclDNAs followed by sequencing). ALE-seq reveals sequences of 5 LTRs of eclDNAs after two-step amplification: in vitro transcription and subsequent reverse transcription. Using ALE-seq in rice, we detected eclDNAs for a novel Copia family LTR retrotransposon, Go-on, which is activated by heat stress. Sequencing of rice accessions revealed that Go-on has preferentially accumulated in indica rice grown at higher temperatures. Furthermore, ALE-seq applied to tomato fruits identified a developmentally regulated Gypsy family of retrotransposons. Importantly, a bioinformatic pipeline adapted for ALE-seq data analyses allows the direct and reference-free annotation of new active retroelements. This pipeline allows assessment of LTR retrotransposon activities in organisms for which genomic sequences and/or reference genomes are unavailable or are of low quality.

plant biology↗

bioRxiv2018

TMEM106B, a risk factor for FTLD and aging, has an intrinsically disordered cytoplasmic domain

TMEM106B was initially identified as a risk factor for FTLD, but recent studies highlighted its general role in neurodegenerative diseases. Very recently TMEM106B has also been characterized to regulate aging phenotypes. TMEM106B is a 274-residue lysosomal protein whose cytoplasmic domain functions in the endosomal/autophagy pathway by dynamically and transiently interacting with diverse categories of proteins but the underlying structural basis remains completely unknown. Here we conducted bioinformatics analysis and biophysical characterization by CD and NMR spectroscopy, and obtained results reveal that the TMEM106B cytoplasmic domain is intrinsically disordered with no well-defined three-dimensional structure. Nevertheless, detailed analysis of various multi-dimensional NMR spectra allowed defining residue-specific conformations and dynamics. Overall, the TMEM106B cytoplasmic domain is lacking of any tight tertiary packing and relatively flexible. However, several segments are populated with dynamic/nascent secondary structures and have relatively restricted backbone motions. In particular, the fragment Ser12-Met36 is highly populated with - helix conformation. Our study thus decodes that being intrinsically disordered allows the TMEM106B cytoplasmic domain to dynamically and transiently interact with a variety of distinct partners.

biophysics↗

bioRxiv2018

Dissection of protein cargo of citrus fruit juice sac cells-derived vesicles reveals heterogeneous transport and extracellular vesicles subpopulations

Cellular vesicles are membrane-enclosed organelles that transport material inside and outside the cell. Plant-derived vesicles are receiving more and more attention due to their potential as nanovectors for the delivery of biologically active substances. We aimed to expand our understanding about the heterogeneity and the protein biocargo of citrus fruit juice sac cell-derived vesicles. Micro- and nano-sized vesicle fractions were isolated from four citrus species, C. sinensis, C. limon, C. paradisi and C. aurantium, characterized using physicochemical methods and protein cargos were compared using label-free quantitative shotgun proteomics. In each sample approximately 600-800 proteins were identified. Orthologues of most of the top-ranking proteins have previously been reported in extracellular vesicles of mammalian origin. High expression of patellin-3-like, clathrin heavy chain, HSPs, 14-3-3 protein, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase 6 could be observed in all the vesicle samples while Aquaporin was highly expressed only in nanovesicles. Bioinformatics revealed more than hundred protein orthologues potentially implicated in vesicular trafficking. In particular, the presence of CCV, COPI and COPII coat proteins indicates the presence of highly heterogeneous populations of intracellular transport vesicles. Moreover, the differential expressions of hydrolases and oxidoreductases enabled us to gain insights into their functions in citrus fruit sac cells.

plant biology↗