Biology subjects

Shi, W.

Publications and source records attributed to Shi, W..

At least 199 records · Page 11Linked to original sources

bioRxiv2019

Chikungunya virus infection impairs osteogenic differentiation of bone marrow-derived mesenchymal stem cells

Chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus, belonging to the genus alphavirus in the family Togaviridae. The virus is spread by the Aedes species (sp.) mosquitoes in tropical and subtropical regions of the world. CHIKV causes Chikungunya fever (CHIKF), where the acute stage of infection is characterized by high fever, headache, rash, and polyarthralgia. In 30-40% of cases, patients develop a chronic stage with debilitating joint pain persisting for months to years imposing a burden on the population in terms of disability adjusted life years (DALY). Presently, no vaccines or treatment options are available for this infection. Prior investigations reveal that CHIKV infection is associated with bone pathology; however, the molecular mechanism underlying CHIKV-induced bone pathology remains poorly defined. Studies show that disruption of osteogenic differentiation and function of bone marrow-derived mesenchymal stem cells (BMMSCs) can lead to bone pathologies. However, to date pathogenesis of CHIKV infection in this context has not been studied. In the current study, we investigated the susceptibility of BMMSCs to CHIKV and studied the effect of infection on BMMSCs-derived osteogenic cells. To our knowledge, for the first time we report that CHIKV can productively infect BMMSCs. We observed a decrease in the intracellular and extracellular alkaline phosphatase (ALP) activity and reduction in calcium phosphate deposition in infected cells compared to mock-infected control. Thus, we conclude that CHIKV infects BMMSCs and disrupts function of osteogenic cells.\n\nImportanceAlthough studies have shown association of bone pathology and CHIKV infection, the pathogenesis of infection causing altered bone homeostasis is not fully understood. Here, we demonstrate for the first time that BMMSCs are susceptible to CHIKV infection. Furthermore, we observe that infection causes disruption in the function of BMMSC- derived osteogenic cells. Impaired function of these osteogenic cells will likely lead to a disruption in bone homeostasis and in part, provides a mechanism for the observed bone pathology associated with CHIKV pathogenesis.

microbiology↗

bioRxiv2019

TRANSCUP: a scalable workflow for predicting cancer of unknown primary based on next-generation transcriptome profiling

SummaryCancer of unknown primary site (CUP) accounts for 5% of all cancer diagnoses. These patients may benefit from more precise treatment when primary cancer site was identified. Advances in high-throughput sequencing have enabled cost-effective sequencing the transcriptome for clinical application. Here, we present a free, scalable and extendable software for CUP predication called TRANSCUP, which enables (1) raw data processing, (2) read mapping, (3) quality re-port, (4) gene expression quantification, (5) random forest machine learning model building for cancer type classification. TRANSCUP achieved high accuracy, sensitivity and specificity for tumor type classification based on external RNA-seq datasets. It has potential for broad clinical application for solving the CUP problem.\n\nAvailabilityTRANSCUP is open-source and freely available at https://github.com/plsysu/TRANSCUP\n\nContactpeng-li@outlook.com

bioinformatics↗

bioRxiv2019

Identification of essential oils with activity against stationary phase Staphylococcus aureus

Staphylococcus aureus is the most dominant human pathogen, responsible for a variety of chronic and severe infections. There is mounting evidence that persisters are associated with treatment failure and relapse of persistent infections. While some essential oils were reported to have antimicrobial activity against growing S. aureus, activity of essential oils against the non-growing stationary phase S. aureus enriched in persisters has not been investigated. In this study, we evaluated the activity of 143 essential oils against stationary phase S. aureus and identified 39 essential oils (Cinnamon bark, Oregano, Thyme white, Bandit \"Thieves\", Lemongrass (Cymbopogon flexuosus), Sandalwood oil, Health shield, Allspice, Amyris, Palmarosa, Cinnamon leaf, Clove bud, Citronella, Geranium bourbon, Marjoram, Peppermint, Lemongrass (Cymbopogon citratus), Cornmint, Elemi, Ho wood, Head ease, Lemon eucalyptus, Litsea cubeba, Myrrh, Parsley seed, Coriander oil, Dillweed, Hyssop, Neroli, Rosewood oil, Tea tree, Cajeput, Glove bud, Lavender, Sleep tight, Vetiver, Palo santo, Sage oil, Yarrow) at 0.5% concentration, 10 essential oils (Cinnamon bark, Oregano, Thyme white, Bandit \"Thieves\", Lemongrass (Cymbopogon flexuosus), Sandalwood oil, Health shield, Allspice, Amyris, Palmarosa) at 0.25% concentration, and 7 essential oils (Cinnamon bark, Oregano, Thyme white, Lemongrass (Cymbopogon flexuosus), Allspice, Amyris, Palmarosa) at 0.125% concentration to have high activity against stationary phase S. aureus with no visible growth on agar plates after five-day exposure. Among the 10 essential oils which showed high activity at 0.25% concentration, 9 (Cinnamon bark, Oregano, Thyme white, Bandit \"Thieves\", Lemongrass (Cymbopogon flexuosus), Health shield, Allspice, Palmarosa, Amyris) showed higher activity than the known persister drug tosufloxacin, while the other one (Sandalwood oil) was found to be active at a higher concentration. In Oregano essential oil drug combination studies with clinical antibiotics, Oregano plus quinolone drugs (tosufloxacin, levofloxacin, ciprofloxacin) and rifampin completely eradicated all stationary phase S. aureus cells, but had no apparent enhancement for linezolid, vancomycin, sulfamethoxazole, trimethoprim, azithromycin and gentamicin. Our findings may facilitate development of more effective treatment for persistent S. aureus infections.

microbiology↗

bioRxiv2019

Identification of essential oils with strong activity against stationary phase uropathogenic Escherichia coli

Escherichia coli is the most dominant pathogen causing urinary tract infections (UTIs), but the current most frequently prescribed antibiotics do not always effectively cure the infection due to quiescent persister bacteria. While it has been reported that some essential oils have antimicrobial activity against growing E. coli, the activity of essential oils against the non-growing stationary phase E. coli which is enriched in persisters has not been investigated. We evaluated the activity of 140 essential oils against stationary phase uropathogenic E. coli UTI89 and identified 39, 8 and 3 essential oils at 0.5%, 0.25% and 0.125% concentrations to have high activity against stationary phase E. coli. Among the top eight essential oils, Oregano showed higher activity than the known persister drug tosufloxacin. The other top seven hits included Allspice, Bandit \"Thieves\", Cinnamon bark, Syzygium aromaticum, Health shield, Cinnamon leaf and Clove bud. In Oregano essential oil drug combination studies with common UTI antibiotics, Oregano plus quinolone drugs (tosufloxacin, levofloxacin, ciprofloxacin) completely eradicated all stationary phase E. coli cells, partially enhanced the activity of nitrofurantoin, but had no apparent enhancement for fosfomycin, meropenem and cefdinir. Our findings may facilitate development of more effective treatments for persistent UTIs.

microbiology↗

bioRxiv2019

Ranking of Major Classes of Antibiotics for Activity against Stationary Phase Pseudomonas aeruginosa and Identification of Clinafloxacin + Cefuroxime + Gentamicin Drug Combination that Eradicates Persistent P. aeruginosa Infection in a Murine Cystic Fibrosis Model

Pseudomonas aeruginosa can cause serious persistent infections such as ventilator-associated pneumonia, sepsis, biofilm-related infections as in cystic fibrosis (CF) patients. Although CF lung infections can be treated with antibiotics, full clearance is difficult due to P. aeruginosa persistence. While antibiotic activity against growing P. aeruginosa is well documented, their activity against the non-growing persisters enriched in stationary phase cultures has not been well studied. Here, we systematically evaluated and ranked the six major classes of antibiotics, cell wall and cell membrane inhibitors, protein synthesis inhibitors, DNA synthesis inhibitors, RNA synthesis inhibitors, sulfa drugs, and nitrofurantoin, for their activity against both growing and persister forms of P. aeruginosa using colony forming count (CFU) and SYBR Green I/Propidium Iodide (PI) viability assay. Among the six major classes of antibiotics, cell wall and cell membrane inhibitors (Cefuroxime and Colistin), DNA synthesis inhibitors (Clinafloxacin) and sulfa drugs (Sulfamethoxazole) had good activity against stationary phase cells. In contrast, protein synthesis inhibitors (Gentamicin), RNA synthesis inhibitor (Rifampicin) and Nitrofurantoin had relatively poor activity against the stationary phase P. aeruginosa but relatively high activity against log phase P. aeruginosa. Clinafloxacin is the only single drug that could completely kill all (109 CFU) stationary phase cells in a 4 day drug exposure. The Cefuroxime + Gentamicin+ Clinafloxacin combination could kill all biofilm bacteria in 2 days whereas the clinically used drug combination Cefuroxime + Gentamicin + Colistin only partially killed the biofilm bacteria with 103 CFU remaining. In a murine persistent CF lung infection model, only Cefuroxime + Gentamicin+ Clinafloxacin cleared all bacteria in the infected lungs, whereas Clinafloxacin alone, or Cefuroxime + Clinafloxacin, or the current recommended drug combination Cefuroxime + Gentamicin, all failed to completely clear the bacterial load in the lungs. The complete sterilization of the bacterial load is a property of Clinafloxacin combination, as Cefuroxime + Gentamicin+ Levofloxacin combination was unable to clear the bacterial load in the lungs. Our findings demonstrate the importance of persister drug clinafloxacin, offer new therapeutic approaches for more effective treatment of persistent P. aeruginosa infections, and may have implications for treating other persistent infections.

microbiology↗

bioRxiv2019

A Drug Combination Approach Targeting Both Growing Bacteria and Dormant Persisters Eradicate Persistent Staphylococcus aureus Biofilm Infection

Staphylococcus aureus can cause a variety of infections, many of which involve biofilm infections. Inside biofilms, growing and non-growing bacteria such as persisters co-exist, making it challenging to completely eradicate a persistent and recurrent infection with current treatments. Despite the clinical relevance, most of the current antibiotic treatments mainly kill the growing bacteria and have poor activity against non-growing persister bacteria and thus have limited effect on treating persistent infections including biofilm infections. We previously proposed a Yin-Yang model using a drug combination approach targeting both growing bacteria and persister bacteria for more effective clearance of persistent infections. Here, as a proof of principle, we showed that combining drugs that have high activity against growing forms, such as vancomycin or meropenem, with drugs that have robust anti-persister activity, such as clinafloxacin and oritavancin, could completely eradicate S. aureus biofilm bacteria in vitro. In contrast, single or two drugs including the current treatment for persistent S. aureus infection doxycycline plus rifampin failed to kill all biofilm bacteria in vitro. We then developed a chronic persistent skin infection mouse model with biofilm-seeded bacterial inocula demonstrating that biofilm bacteria caused more severe and persistent skin lesions than log phase S. aureus bacteria. More importantly, we found that the drug combination which eradicated biofilm bacteria in vitro is more efficacious than current treatments and completely eradicated S. aureus biofilm infection in mice. The complete eradication of biofilm bacteria is attributed to the unique high anti-persister activity of clinafloxacin, which could not be replaced by other fluoroquinolones such as moxifloxacin, levofloxacin or ciprofloxacin. Our study is the first to demonstrate that the combination of meropenem, daptomycin, plus clinafloxacin completely cleared the persistent infection, healed the lesions, and had less inflammation, while mice treated with doxycycline plus rifampin, the current clinically recommended treatment for chronic tissue infection, failed to do so. We also compared our persister drug combination with other approaches for treating persistent infections including gentamicin+fructose and ADEP4+rifampin in the S. aureus biofilm infection mouse model. Neither gentamicin+fructose nor ADEP4+rifampin could eradicate or cure the persistent biofilm infection in mice. In contrast, our drug combination regimen with persister drug clinafloxacin plus meropenem and daptomycin completely eradicated and cured the persistent biofilm infection in 7 days. An unexpected observation is that ADEP4 treatment group developed worsened skin lesions and caused more extensive pathology than the untreated control mice. Our study demonstrates an important treatment principle for persistent infections by targeting both growing and non-growing heterogeneous bacterial populations utilizing persister drugs for more effective eradication of persistent and biofilm infections. Our findings may have implications for improved treatment of many other persistent infections in general.

microbiology↗

bioRxiv2019

Introducing RpsA Point Mutation Δ438A or D123A into the Chromosome of M. tuberculosis Confirms its Role in Causing Resistance to Pyrazinamide

Pyrazinamide (PZA) is a unique frontiline drug for shortening tuberculosis treatment, but its mechanisms of action are elusive. We previously identified RpsA as a target of PZA and found an alanine deletion at position 438 ({Delta}438A) in RpsA associated with PZA resistance, but its role in PZA resistance is controversial. Here, we introduced RpsA mutation {Delta}438A or D123A into M. tuberculosis chromosome and demonstrated that the introduced RspA mutations are indeed responsible for PZA resistance.

microbiology↗

bioRxiv2018

Design of nanoparticulate group 2 influenza hemagglutinin stem antigens that activate unmutated ancestor B cell receptors of broadly neutralizing antibody lineages

Influenza vaccines targeting the highly-conserved stem of the hemagglutinin (HA) surface glycoprotein have the potential to protect against pandemic and drifted seasonal influenza viruses not covered by current vaccines. While HA stem-based immunogens derived from group 1 influenza A have been shown to induce intra-group heterosubtypic protection, HA stem-specific antibody lineages originating from group 2 may be more likely to possess broad cross-group reactivity. We report the structure-guided development of mammalian cell-expressed candidate vaccine immunogens based on influenza A group 2 H3 and H7 HA stem trimers displayed on self-assembling ferritin nanoparticles using an iterative, multipronged approach involving helix stabilization, loop optimization, disulfide bond addition, and side chain repacking. These immunogens were thermostable, formed uniform and symmetric nanoparticles, were recognized by cross-group-reactive broadly neutralizing antibodies (bNAbs) with nanomolar affinity, and elicited protective, homosubtypic antibodies in mice. Importantly, several immunogens were able to activate B cells expressing inferred unmutated common ancestor (UCA) versions of cross-group-reactive human bNAbs from two multi-donor classes, suggesting they could initiate elicitation of these bNAbs in humans.

immunology↗

bioRxiv2018

Infection with Persister Forms of Staphylococcus aureus Causes a Chronic Persistent Skin Infection with More Severe Lesion that Takes Longer to Heal and is not Eradicated by the Current Recommended Treatment in Mice

Staphylococcus aureus is an opportunistic pathogen that can cause persistent infections clinically. Treatment for chronic S. aureus infections ranges from at least one week to several months and such infections are prone to relapse likely due to the presence of persistent forms of bacteria such as persister cells. Persister cells, which are bacterial cells that become dormant under stress conditions, can be isolated in vitro but their clinical significance in in vivo infections are largely unclear. Here, we evaluated S. aureus persistent forms using stationary phase cultures and biofilm bacteria (enriched in persisters) in comparison with log phase cultures in terms of their ability to cause disease in a mouse skin infection model. Surprisingly, we found that infection of mice with stationary phase cultures and biofilm bacteria produced a more severe chronic skin infection with more pronounced lesions which took longer to heal than log phase (actively growing) cultures. After two week infection, the bacterial load and skin tissue pathology, as determined by hyperplasia, immune cell infiltration, and crust/lesion formation, of mice infected with the more persistent forms (e.g. stationary phase bacteria and biofilm bacteria) were greater than mice infected with log phase bacteria. Using our persistent infection mouse model, we showed that the clinically recommended treatment for recurrent S. aureus skin infection, doxycycline + rifampin, was not effective in eradicating the bacteria in the treatment study, despite reducing lesion sizes and pathology in infected mice. Analogous findings were also observed in a Caenorhabditis elegans model, where S.aureus stationary phase cultures caused a greater mortality than log phase culture as early as two days post-infection. Thus, we established a new model for chronic persistent infections using persister bacteria that could serve as a relevant model to evaluate therapeutic options for persistent infections in general. Our findings connect persisters with persistent infections, have implications for understanding disease pathogenesis, and are likely to be broadly valid for other pathogens.

microbiology↗

bioRxiv2018

The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads

The first steps in the analysis of RNA sequencing (RNA-seq) data are usually to map the reads to a reference genome and then to count reads by gene, by exon or by exon-exon junction. These two steps are at once the most common and also typically the most expensive computational steps in an RNA-seq analysis. These steps are typically undertaken using Unix command-line or Python software tools, even when downstream analysis is to be undertaken using R.\n\nWe present Rsubread, a Bioconductor software package that provides high-performance alignment and counting functions for RNA-seq reads. Rsubread provides the ease-of-use of the R programming environment, creating a matrix of read counts directly as an R object ready for downstream analysis. It has no software dependencies other than R itself. Using SEQC data and simulations, we compare Rsubread to the popular non-R tools TopHat2, STAR and HTSeq. We also compare to counting functions provided in the Bioconductor infrastructure packages. We show that Rsubread is faster, uses less memory and produces read count summaries that more accurately correlate with true values. The results show that users can adopt the R environment for alignment and quantification without suffering any loss of performance.

bioinformatics↗

bioRxiv2018

Viral etiology of Acute Respiratory Infections in Hospitalized Children in Novosibirsk City, Russia (2013 - 2017)

Introduction Introduction Materials and methods Results Discussion References Acute respiratory infections (ARIs) pose a significant public health problem worldwide, causing considerable morbidity and mortality among people of all age groups [1]. Children are on average infected two to three times more frequently than adults. [2]. There are more than 200 respiratory viruses that can cause ARIs. Respiratory syncytial virus (RSV), human rhinovirus (HRV), human metapneumovirus (HMPV), human parainfluenza virus (PIV), human enterovirus (EV), influenza virus (IFV), human coronavirus (CoV), adenovirus (ADV), and human bocavirus (BoV) are the most common viral agents associated with ARIs, accounting for around 70 % of ARIs [3, 4]. The frequency of mixed respiratory viral ...

microbiology↗

bioRxiv2018

The synchronization and adaptation of Neurospora crassa circadian and conidiation rhythms to short light-dark cycles

Circadian clocks control the physiological and behavioral daily rhythms to adapt to the changing environment with a period of ~24 h. However, the influence and mechanism of extreme light-dark cycles on the circadian clock remain unclear. We show that, in the fungus Neurospora crassa under short LD cycles, both the growth rate and the ratio of microconidia production contributes to adaptation in LD12:12 (light for 12 h and dark for 12 h, periodically). Mathematical modeling and experiments demonstrate that in short LD cycles, the expression of the core clock protein FREQUENCY is entrained to the LD cycles when LD>3:3 while it free runs when T[≤] LD3:3. We investigated the changes in circadian/diurnal rhythms under a series of different LD conditions, and the results showed that conidial rhythmicity can be adapted to the short LD cycles. We further demonstrate that the existence of unknown blue light photoreceptor(s) and the circadian clock might promote the conidiation rhythms that resonate with the environment. A high-intensity light induced the expression of a set of downstream genes involved in various metabolic pathways. The ubiquitin E3 ligase FWD-1 and the previously described CRY-dependent oscillator system were implicated in regulating conidiation under short LD conditions.

microbiology↗

bioRxiv2018

Metastable contacts and structural disorder in the estrogen receptor transactivation domain

The N-terminal transactivation domain (NTD) of estrogen receptor alpha, a well-known member of the family of intrinsically disordered proteins (IDPs), mediates the receptors transactivation function to regulate gene expression. However, an accurate molecular dissection of NTDs structure-function relationships remains elusive. Here, using small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), circular dichroism, and hydrogen exchange mass spectrometry, we show that NTD adopts a mostly disordered, unexpectedly compact conformation that undergoes structural expansion upon chemical denaturation. By combining SAXS, hydroxyl radical protein footprinting and computational modeling, we derive the ensemble-structures of the NTD and determine its ensemble-contact map that reveals metastable regional and long-range contacts, including interactions between residues I33 and S118. We show that mutation at S118, a known phosphorylation site, promotes conformational changes and increases coactivator binding. We further demonstrate via fluorine-19 (19F) NMR that mutations near residue I33 alter 19F chemical shifts at residue S118, confirming the proposed I33-S118 contact in the ensemble of structural disorder. These findings extend our understanding of IDPs structure-function relationship, and how specific metastable contacts mediate critical functions of disordered proteins.\n\nHighlightsO_LIA compact disorder is observed for the N-terminal domain (NTD) of estrogen receptor\nC_LIO_LIMulti-technique modeling elucidates the NTD ensemble structures\nC_LIO_LIEnsemble-based contact map reveals metastable contacts between I33 and S118\nC_LIO_LI19F-NMR data validate the proposed I33-S118 contact in the IDP\nC_LI

biophysics↗

bioRxiv2018

A novel histone H4 variant regulates rDNA transcription in breast cancer

Histone variants, present in various cell types and tissues, are known to exhibit different functions. For example, histone H3.3 and H2A.Z are both involved in gene expression regulation, whereas H2A.X is a specific variant that responds to DNA double-strand breaks. In this study, we characterized H4G, a novel hominidae-specific histone H4 variant. H4G expression was found in a variety of cell lines and was particularly overexpressed in the tissues of breast cancer patients. H4G was found to localize primarily to the nucleoli of the cell nucleus. This localization was controlled by the interaction of the alpha helix 3 of the histone fold motif with the histone chaperone, nucleophosphomin 1. In addition, we found that H4G nucleolar localization increased rRNA levels, protein synthesis rates, and cell cycle progression. Furthermore, micrococcal nuclease digestion of H4G-containing nucleosomes reconstituted in vitro indicated that H4G destabilizes the nucleosome, which may serve to alter nucleolar chromatin in a way that enhances rDNA transcription in breast cancer tissues.

molecular biology↗

bioRxiv2018

The effector of Hippo signaling, Taz, is required for formation of the micropyle and fertilization in zebrafish

The mechanisms that ensure fertilization of eggs by a single sperm are not fully understood. In all teleosts, a channel called the micropyle is the only route of entry for sperm to enter and fertilize the egg. The micropyle forms by penetration of the developing vitelline envelope by a single specialized follicle cell, the micropylar cell, which subsequently degenerates. The mechanisms underlying micropylar cell specification and micropyle formation are poorly understood. Here, we show that an effector of the Hippo signaling pathway, the Transcriptional co-activator with a PDZ-binding domain (Taz), plays crucial roles in micropyle formation and fertilization in zebrafish. Genome editing mutants affecting taz can grow to adults, however, eggs from homozygous taz females are not fertilized even though oocytes in mutant females are histologically normal with intact animal-vegetal polarity, complete meiosis and proper ovulation. However, taz mutant eggs have no micropyle. We show that Taz protein is specifically enriched from mid-oogenesis onwards in two follicle cells located at the animal pole of the oocyte, and co-localizes with the actin and tubulin cytoskeleton. Taz protein and micropylar cell are not detected in taz mutant ovaries. Our work identifies a novel role for the Hippo/Taz pathway in micropylar cell specification in zebrafish, and uncovers the molecular basis of micropyle formation in teleosts.

developmental biology↗

bioRxiv2018

Additional Essential Oils with High Activity against Stationary Phase Borrelia burgdorferi

Lyme disease is the most common vector borne-disease in the US. While the majority of the Lyme disease patients can be cured with 2-4 week antibiotic treatment, about 10-20% of patients continue to suffer from persisting symptoms. While the cause of this condition is unclear, persistent infection was proposed as one possibility. It has recently been shown that B. burgdorferi develops dormant persisters in stationary phase cultures that are not killed by the current Lyme antibiotics, and there is interest to identify novel drug candidates that more effectively kill such forms. We previously evaluated 34 essential oils and identified some highly active candidates with excellent activity against biofilm and stationary phase B. burgdorferi. Here we screened another 35 essential oils and found 10 essential oils (garlic, allspice, cumin, palmarosa, myrrh, hedycheim, amyris, thyme white, litsea cubeba, lemon eucalyptus) and the active component of cinnamon bark cinnamaldehyde (CA) at a low concentration of 0.1% to have high activity against stationary phase B. burgdorferi. At a very low 0.05% concentration, garlic, allspice, palmarosa and CA still exhibited strong activity against the stationary phase B. burgdorferi. CA also showed strong activity against replicating B. burgdorferi, with a MIC of 0.02% (or 0.2 g/mL). In subculture studies, the top 5 hits garlic, allspice, myrrh, hedycheim, and litsea cubeba completely eradicated all B. burgdorferi stationary phase cells at 0.1%, while palmarosa, lemon eucalyptus, amyris, cumin, and thyme white failed to do so as shown by visible spirochetal growth after 21-day subculture. At 0.05% concentration, only garlic essential oil and CA sterilized the B. burgdorferi stationary phase culture as shown by no regrowth during subculture, while allspice, myrrh, hedycheim and litsea cubeba all had visible growth during subculture. Future studies are needed to determine if these highly active essential oils could eradicate persistent B. burgdorferi infection in vivo.

microbiology↗

bioRxiv2018

Evidence of independent acquisition and adaption of ultra-small bacteria to human hosts across the highly diverse yet reduced genomes of the phylum Saccharibacteria

Recently, we discovered that a member of the Saccharibacteria/TM7 phylum (strain TM7x) isolated from the human oral cavity, has an ultra-small cell size (200-300nm), a highly reduced genome (705 Kbp) with limited de novo biosynthetic capabilities, and a very novel lifestyle as an obligate epibiont on the surface of another bacterium 1. There has been considerable interest in uncultivated phyla, particularly those that are now classified as the proposed candidate phyla radiation (CPR) reported to include 35 or more phyla and are estimated to make up nearly 15% of the domain Bacteria. Most members of the larger CPR group share genomic properties with Saccharibacteria including reduced genomes (<1Mbp) and lack of biosynthetic capabilities, yet to date, strain TM7x represents the only member of the CPR that has been cultivated and is one of only three CPR routinely detected in the human body. Through small subunit ribosomal RNA (SSU rRNA) gene surveys, members of the Saccharibacteria phylum are reported in many environments as well as within a diversity of host species and have been shown to increase dramatically in human oral and gut diseases. With a single copy of the 16S rRNA gene resolved on a few limited genomes, their absolute abundance is most often underestimated and their potential role in disease pathogenesis is therefore underappreciated. Despite being an obligate parasite dependent on other bacteria, six groups (G1-G6) are recognized using SSU rRNA gene phylogeny in the oral cavity alone. At present, only genomes from the G1 group, which includes related and remarkably syntenic environmental and human oral associated representatives1, have been uncovered to date. In this study we systematically captured the spectrum of known diversity in this phylum by reconstructing completely novel Class level genomes belonging to groups G3, G6 and G5 through cultivation enrichment and/or metagenomic binning from humans and mammalian rumen. Additional genomes for representatives of G1 were also obtained from modern oral plaque and ancient dental calculus. Comparative analysis revealed remarkable divergence in the host-associated members across this phylum. Within the human oral cavity alone, variation in as much as 70% of the genes from nearest oral clade (AAI 50%) as well as wide GC content variation is evident in these newly captured divergent members (G3, G5 and G6) with no environmental relatives. Comparative analyses suggest independent episodes of transmission of these TM7 groups into humans and convergent evolution of several key functions during adaptation within hosts. In addition, we provide evidence from in vivo collected samples that each of these major groups are ultra-small in size and are found attached to larger cells.

microbiology↗

bioRxiv2018

Reliability of Whole-Exome Sequencing for Assessing Intratumor Genetic Heterogeneity

Multi-region sequencing is used to detect intratumor genetic heterogeneity (ITGH) in tumors. To assess whether genuine ITGH can be distinguished from sequencing artifacts, we whole-exome sequenced (WES) three anatomically distinct regions of the same tumor with technical replicates to estimate technical noise. Somatic variants were detected with three different WES pipelines and subsequently validated by high-depth amplicon sequencing. The cancer-only pipeline was unreliable, with about 69% of the identified somatic variants being false positive. Even with matched normal DNA where 82% of the somatic variants were detected reliably, only 36%-78% were found consistently in technical replicate pairs. Overall 34%-80% of the discordant somatic variants, which could be interpreted as ITGH, were found to constitute technical noise. Excluding mutations affecting low mappability regions or occurring in certain mutational contexts was found to reduce artifacts, yet detection of subclonal mutations by WES in the absence of orthogonal validation remains unreliable.

genomics↗