bluuebunny/crossref_metadata_2025 huggingface dataset:
Gargle-Direct: Extraction-Free Detection of SARS-CoV-2 using Real-time PCR (RT-qPCR) of Saline Gargle Rinse Samples
Authors: Gadkar Vijay J. , Goldfarb David M. , Young Virginia , Watson Nicole , Hoang Linda , Lee Tracy , Prystajecky Natalie , Al-Rawahi Ghada N. , Srigley Jocelyn A , Tilley Peter
Published: October 2020

Background

Saline mouth rinse/gargle samples have recently been shown to be a suitable option for swab-independent self-collection for SARS-CoV-2 diagnosis. We sought to evaluate a simplified process for direct reverse transcriptase PCR (RT-qPCR) testing of this novel sample type and to compare performance with routine RT-qPCR using automated nucleic acid extraction.

Methods

Clinical saline mouth rinse/gargle samples were subjected to automated nucleic acid extraction (“standard method”), followed by RT-qPCR using three assays including the FDA authorized US-CDC’s N1/N2 assay, which was the reference standard for determining sensitivity/specificity. For extraction-free workflow, an aliquot of each gargle sample underwent viral heat inactivation at 65 °C for 20 minutes followed by RT-qPCR testing, without an intermediate extraction step. An in-house validated RT-qPCR lab developed test (LDT), targeting the SARS-CoV-2’s S/ORF8 genes (SORP triplex assay) and the N1/N2 US-CDC assay was used to evaluate the extraction-free protocol. To improve the analytical sensitivity, we developed a single-tube hemi-nested (STHN) version of the SORP triplex assay.

Results

A total of 38 SARS-CoV-2 positive and 75 negative saline mouth rinse/gargle samples were included in this evaluation. A 100% concordance in detection rate was obtained between the standard method and the extraction-free approach for the SORP assay. An average increase of +2.63 to +5.74 of the cycle threshold (CT) values was observed for both the SORP and N1/N2 assay when extraction-free was compared between the standard method. The average ΔCT [ΔCT=CT(Direct PCR)-CT(Extracted RNA)], for each of the gene targets were: S (ΔCT= +4.24), ORF8 (ΔCT=+2.63), N1 (ΔCT=+2.74) and N2 (ΔCT=+5.74). The ΔCT for the STHN SORP assay was +1.51 and −2.05 for the S and ORF8 targets respectively, when extracted method was compared to the standard method.

Conclusion

Our Gargle-Direct SARS-CoV-2 method is operationally simple, minimizes pre-analytical sample processing and is potentially implementable by most molecular diagnostic laboratories. The empirical demonstration of single-tube hemi-nested RT-qPCR, to specifically address and alleviate the widely-acknowledged problem of reduced analytical sensitivity of detection of extraction-free templates, should help diagnostic laboratories in choosing Gargle-Direct protocol for high-throughput testing.
Two modes of inhibitory neuronal shutdown distinctly amplify seizures in humans
Authors: Ahmed Omar J. , John Tibin T. , Sudhakar Shyam K. , Brennan Ellen K.W. , Gonzalez Alcides Lorenzo , Naftulin Jason S. , Eskandar Emad , Madsen Joseph R. , Cosgrove G. Rees , Blum Andrew S. , Potter N. Stevenson , Mashour George A. , Hochberg Leigh R. , Cash Sydney S.
Published: October 2020

Inhibitory neurons are critical for normal brain function but dysregulated in disorders such as epilepsy. At least two theories exist for how inhibition may acutely decrease during a seizure: hyperpolarization of fast-spiking (FS) inhibitory neurons by other inhibitory neurons, or depolarization block (DB) of FS neurons resulting in an inability to fire action potentials. Firing rate alone is unable to disambiguate these alternatives. Here, we show that human FS neurons can stop firing due to both hyperpolarization and DB within the same seizure. However, only DB of FS cells is associated with dramatic increases in local seizure amplitude, unobstructed traveling waves, and transient increases in excitatory neuronal firing. This result is independent of seizure etiology or focus. Computational models of DB reproduce thein vivohuman biophysics. These methods enable intracellular decoding using only extracellular recordings in humans and explain the otherwise ambiguous inhibitory neuronal control of human seizures.
Heme induces mRNA expression and activation of tissue factor by TLR4 dependent mechanisms
Authors: Hounkpe B.W. , Moraes C.R.P. , do Santos M.N.N. , Costa F. F. , De Paula E.V.
Published: October 2020

Introduction

Hemolytic diseases such as Sickle Cell Disease (SCD) are characterized by a natural propensity for both arterial and venous thrombosis. Evidence showing that heme can induce tissue factor (TF) expression in endothelial cells and TF-dependent coagulation activation in animal models of SCD suggest that heme can contribute to hypercoagulability in this condition. We recently demonstrated that heme can induce coagulation activation in whole blood of healthy volunteers in a TF-dependent fashion.

Methods

Herein, we aimed to evaluate whether this heme-induced coagulation activity was dependent on the expression and/or activation of hematopoietic TF in human mononuclear cells. TF mRNA expression was evaluated by qPCR and TF procoagulant activity was evaluated using a 2-stage assay based on the generation of FXa.

Results

Heme was capable of inducing TF expression and activation in a TLR4-dependent pathway. This activity was further amplified after TNF-α-priming.

Conclusion

Our results provide additional evidences on the mechanisms by which heme is involved in the pathogenesis of hypercoagulability in hemolytic diseases.
Estimating COVID-19 cases and outbreaks on-stream through phone-calls
Authors: Alvarez Ezequiel , Obando Daniela , Crespo Sebastian , Garcia Enio , Kreplak Nicolas , Marsico Franco
Published: October 2020

One of the main problems in controlling COVID-19 epidemic spread is the delay in confirming cases. Having information on changes in the epidemic evolution or outbreaks rise before lab-confirmation is crucial in decision making for Public Health policies. We present an algorithm to estimate on-stream the number of COVID-19 cases using the data from telephone calls to a COVID-line. By modeling the calls as background (proportional to population) plus signal (proportional to infected), we fit the calls in Province of Buenos Aires (Argentina) with coefficient of determination R2 > 0.85. This result allows us to estimate the number of cases given the number of calls from a specific district, days before the lab results are available. We validate the algorithm with real data. We show how to use the algorithm to track on-stream the epidemic, and present the Early Outbreak Alarm to detect outbreaks in advance to lab results. One key point in the developed algorithm is a detailed track of the uncertainties in the estimations, since the alarm uses the significance of the observables as a main indicator to detect an anomaly. We present the details of the explicit example in Villa Azul (Quilmes) where this tool resulted crucial to control an outbreak on time. The presented tools have been designed in urgency with the available data at the time of the development, and therefore have their limitations which we describe and discuss. We consider possible improvements on the tools, many of which are currently under development.
Extracorporeal Blood Purification in moderate and severe COVID-19 patients: a prospective cohort study
Authors: Rosalia Rodney Alexander , Ugurov Petar , Neziri Dashurie , Despotovska Simona , Kostoska Emilja , Veljanovska-Kiridjievska Lidija , Kuzmanov Dimche , Trifunovski Aleksandar , Villa Gianluca , Popevski Dijana , Mitrev Zan
Published: October 2020

Introduction

COVID-19 is characterised by hyperinflammation and coagulopathy. Severe cases often develop respiratory distress, requiring mechanical ventilation and critical cases progressing to ARDS. Control of hyperinflammation has been proposed as a possible therapeutic avenue for COVID-19; extracorporeal blood purification (EBP) modalities offer an attractive mean to ameliorate maladaptive inflammation.

With this work, we describe the longitudinal variation of parameters of systemic inflammation in critically ill COVID-19 patients treated with blood purification using AN69ST (oXiris®) hemodiafilter.

Methods

We performed a time-series analysis of 44 consecutive COVID-19 cases treated with the AN69ST (oXiris®) cytokine adsorbing hemodiafilter; we visualise longitudinal results of biochemical, inflammatory, blood gas- and vital sign parameters.

Results

Blood purification was indicated for suspected hyperinflammation or hypercoagulation, (= CRP ≥ 100 mg/L and/or IL-6 ≥ 40 pg/mL and/or Ferritin ≥ 500 ng/mL and/or Lactate Dehydrogenase > 365 U/L or D-dimers > 2000 ng/mL). All patients were treated with ≥ 1 cycle extracorporeal continuous venovenous hemofiltration (CVVHF) with cytokine adsorbing hemodiafilter (CAH); of these, 30 severe patients received CVVHF-CAH within 4 – 12 hours of hospitalisation. Another 14 patients admitted with mild-to-moderate symptoms progressed to severe disease and placed on EBP during the course of hospitalisation. The treatment was associated with a reduction of Ferritin, C-reactive protein, Fibrinogen, several inflammatory markers and a resolution of numerous cytopenias. The observed mortality across the cohort was 36.3% across the cohort.

Conclusion

Extracorporeal blood purification with cytokine adsorbing hemofilter was associated with a decrease in the acute phase proteins CRP, Ferritin, and resolution of numerous cytopenias. Repetitive hemofiltration has been associated with lower levels of IL-6 in COVID-19 patients.
Predicting mortality of individual COVID-19 patients: A multicenter Dutch cohort
Authors: Ottenhoff Maarten C. , Ramos Lucas L. , Potters Wouter , Janssen Marcus L.F. , Hubers Deborah , Piña-Fuentes Dan , Thomas Rajat , van der Horst Iwan C.C. , Herff Christian , Kubben Pieter , Elbers Paul W.G. , Marquering Henk A. , Welling Max , Hu Shi , Simsek Suat , de Kruif Martijn D. , Dormans Tom , Fleuren Lucas M. , Schinkel Michiel , Noordzij Peter G. , van den Bergh Joop P. , Wyers Caroline E. , Buis David T. B. , Wiersinga Joost , van den Hout Ella H.C. , Reidinga Auke C. , Rusch Daisy , Sigaloff Kim C.E. , Douma Renée , de Haan Lianne , Fridgeirsson Egill A. , van den Oever Niels C. Gritters , Rennenberg Roger J.M.W. , van Wingen Guido A. , Aries Marcel J.H. , Beudel Martijn
Published: October 2020

Objective

Develop and validate models that predict mortality of SARS-CoV-2 infected patients admitted to the hospital.

Design

Retrospective cohort study

Setting

A multicenter cohort across ten Dutch hospitals including patients from February 27 to June 8 2020.

Participants

SARS-CoV-2 positive patients (age ≥ 18) admitted to the hospital.

Main Outcome Measures

21-day mortality evaluated by the area under the receiver operatory curve (AUC), sensitivity, specificity, positive predictive value and negative predictive value. The predictive value of age was explored by comparison with age-based rules used in practice and by excluding age from analysis.

Results

2273 patients were included, of whom 516 had died or discharged to palliative care within 21 days after admission. Five feature sets, including premorbid, clinical presentation and laboratory & radiology values, were derived from 80 features. Additionally, an ANOVA-based data-driven feature selection selected the ten features with the highest F-values: age, number of home medications, urea nitrogen, lactate dehydrogenase, albumin, oxygen saturation (%), oxygen saturation is measured on room air, oxygen saturation is measured on oxygen therapy, blood gas pH and history of chronic cardiac disease. A linear logistic regression (LR) and non-linear tree-based gradient boosting (XGB) algorithm fitted the data with an AUC of 0.81 (95% confidence interval 0.77 to 0.85) and 0.82 (0.79 to 0.85), respectively, using the ten selected features. Both models outperformed age-based decision rules used in practice (AUC of 0.69, 0.65 to 0.74 for age > 70). Furthermore, performance remained stable when excluding age as predictor (AUC of 0.78, 0.75 to 0.81)

Conclusion

Both models showed excellent performance and had better test characteristics than age-based decision rules, using ten admission features readily available in Dutch hospitals. The models hold promise to aid decision making during a hospital bed shortage.
Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress
Authors: Jobava Raul , Mao Yuanhui , Guan Bo-Jhih , Krokowski Dawid , Shu Erica , Hu Di , Chukwurah Evelyn , Wu Jing , Gao Zhaofeng , Zagore Leah L. , Merrick William C. , Zhang Youwei , Qi Xin , Jankowsky Eckhard , Topisirovic Ivan , Licatalosi Donny D. , Qian Shu-Bing , Hatzoglou Maria
Published: October 2020

Mammalian cells have to adapt to environmental challenges that range from mild to severe stress. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe stress conditions, cells induce a transient hibernation-like mechanism that anticipates recovery. We demonstrate that thisAdaptivePausingResponse (APR) is a coordinated cellular response that limits ATP supply and consumption though mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery from severe stress. We further show that recovery from severe stress involves adaptive ISR (Integrated Stress Response) signaling that in turn permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress through the APR, a mechanism that preserves vital elements of cellular function under harsh environmental conditions.
SARS-CoV-2 infects carotid arteries: implications for vascular disease and organ injury in COVID-19
Authors: Pfefferle Susanne , Günther Thomas , Puelles Victor G. , Heinrich Fabian , Nörz Dominik , Czech-Sioli Manja , Carsten Alexander , Krasemann Susanne , Wong Milagros N. , Oestereich Lisa , Magnus Tim , Allweiss Lena , Edler Carolin , Schröder Ann Sophie , Dandri Maura , Huber Tobias B. , Glatzel Markus , Püschel Klaus , Grundhoff Adam , Lütgehetmann Marc , Aepfelbacher Martin , Fischer Nicole
Published: October 2020

Stroke and central nervous system dysfunction are cardinal symptoms in critically ill corona virus disease 19 (COVID-19) patients. In an autopsy series of 32 COVID-19 patients, we investigated whether carotid arteries were infected with SARS-CoV-2 by employing genomic, virologic, histochemical and transcriptomic analyses. We show that SARS-CoV-2 productively infects and modulates vascular responses in carotid arteries. This finding has far reaching implications for the understanding and clinical treatment of COVID-19.
Mental health service activity during COVID-19 lockdown among individuals with potential neurodevelopmental disorders: South London and Maudsley data on services and mortality from January to July 2020
Authors: Martin Evangelia , Nuzum Eleanor , Broadbent Matthew , Stewart Robert
Published: October 2020

The lockdown and social distancing policy imposed due to the COVID-19 pandemic is likely to have had a widespread impact on mental healthcare service provision and use. Previous reports from the South London and Maudsley NHS Trust (SLaM; a large mental health service provider for 1.2m residents in South London) highlighted a shift to virtual contacts among those accessing community mental health and home treatment teams and an increase in deaths over the pandemic’s first wave. However, there is a need to quantify this for individuals with particular vulnerabilities, including those with learning disabilities and other neurodevelopmental disorders. Taking advantage of the Clinical Record Interactive Search (CRIS) data resource with 24-hourly updates of electronic mental health records data, this paper describes daily caseloads and contact numbers (face-to-face and virtual) for individuals with potential neurodevelopmental disorders across community, specialist, crisis and inpatient services. The report focussed on the period 1st January to 31st July 2020. We also report on daily accepted and discharged trust referrals, total trust caseloads and daily inpatient admissions and discharges for individuals with potential neurodevelopmental disorders. In addition, daily deaths are described for all current and previous SLaM service users with potential neurodevelopmental disorders over this period. In summary, comparing periods before and after 16th March 2020 there was a shift from face-to-face contacts to virtual contacts across all teams. The largest declines in caseloads and total contacts were seen in Home Treatment Team, Liaison/A&E and Older Adult teams. Reduced accepted referrals and inpatient admissions were observed and there was an 103% increase in average daily deaths in the period after 16th March, compared to the period 1st January to 15th March (or a 282% increase if the 2-month period from 16th March to 15th May was considered alone).
Findings from serological surveys (in August 2020) to assess the exposure of adult population to SARS Cov-2 infection in three cities of Odisha, India
Authors: Kshatri Jaya Singh , Bhattacharya Debdutta , Kanungo Srikanta , Giri Sidhartha , Palo Subrata Kumar , Parai Debaprasad , Turuk Jyotirmayee , Mansingh Asit , Choudhary Hariram , Dash Girish Chandra , Mishra Niranjan , Satapathy D.M. , Sahoo Sanjaya Ku , Pati Sanghamitra
Published: October 2020

Background

There is always an uncertainty of epidemiological, serological infectivity and virulence of the emerging novel coronavirus. Antibody test can be used for assessing whether immunity has developed in the infected person after 5-7 days of illness and understand cumulative exposure levels to the infection, make inferences on the actual burden of infection, its geographical spread, effect on specific demographic/risk groups, gaps in testing and infection fatality rates.

Objective

To estimate and compare the sero-prevalence, hidden prevalence and determine the demographic risk factors associated with SARS-CoV-2 infection among adults in three largest cities of Odisha, India.

Methodology

This was a population based cross sectional serological survey carried out in August 2020 in the three largest cities of the state of Odisha. Sample size per city was estimated to be 1500 and participants were enrolled from the community using multi-stage random sampling from 25 clusters from each city. Data was collected using ODK based tools by household visits and 3-4 ml of blood samples were collected after informed consent. Samples were transported to testing lab where Serum was separated and tested for anti-SARS CoV-2 antibodies using automated CLIA platform. Statistical analysis was done using R-software packages.

Results

A total of 4146 participants from the 3 cities of Bhubaneswar (BBS), Berhampur (BAM) and Rourkela (RKL) participated. A total of 5635 households were approached and the average non response rate in the community was 17.4%. The gender weighted seroprevalence across the three cities was 20.78% (95% CI: 19.56%-22.05%). Seroprevalence was highest in BAM at 31.14% (95% CI: 28.69-33.66%) followed by 24.59% (95% CI: 22.39-26.88%) in RKL and 5.24% (95% CI: 4.10-6.58%) in BBS. While females reported a higher seroprevalence (22.8%) as compared to males (18.8%), there was no significant difference in seroprevalence across age groups. A majority of the seropositive participants were asymptomatic (93.87%). Among those who reported symptoms, the most common symptom was fever (68.89%) followed by cough (46.06%) and myalgia (32.67%). The case to infection ratio on the date of serosurvey was 1: 6.6 in BBS, 1:61 in BAM and 1:29.8 in RKL.

Conclusion

The study found a high seroprevalence against COVID-19 in urban Odisha as well as high numbers of asymptomatic infections.
Are the SIR and SEIR models suitable to estimate the basic reproduction number for the CoViD-19 epidemic?
Authors: Yang Hyun Mo , Lombardi Junior Luis Pedro , Yang Ariana Campos
Published: October 2020

The transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) becomes pandemic but presents different incidences in the world. Mathematical models were formulated to describe the coronavirus disease 2019 (CoViD-19) epidemic in each country or region. At the beginning of the pandemic, many authors used the SIR (susceptible, infectious, and recovered compartments) and SEIR (including exposed compartment) models to estimate the basic reproduction numberR0for the CoViD-19 epidemic. These simple deterministic models assumed that the only available collection of the severe CoViD-19 cases transmitted the SARS-CoV-2 and estimated lower values forR0, ranging from 1.5 to 3.0. However, the major flaw in the estimation ofR0provided by the SIR and SEIR models was that the severe CoViD-19 patients were hospitalized, and, consequently, not transmitting. Hence, we proposed a more elaborate model considering the natural history of CoViD-19: the inclusion of asymptomatic, pre-symptomatic, mild and severe CoViD-19 compartments. The model also encompassed the fatality rate depending on age. This SEAPMDR model estimatedR0using the severe CoViD-19 data from São Paulo State (Brazil) and Spain, yielding higher values forR0, that is, 6.54 and 5.88, respectively. It is worth stressing that this model assumed that severe CoViD-19 cases were not participating in the SARS-CoV-2 transmission chain. Therefore, the SIR and SEIR models are not suitable to estimateR0at the beginning of the epidemic by considering the isolated severe CoViD-19 data as transmitters.
Determining subpopulation methylation profiles from bisulfite sequencing data of heterogeneous samples using DXM
Authors: Fong Jerry , Gardner Jacob R. , Andrews Jared M. , Cashen Amanda F. , Payton Jacqueline E. , Weinberger Kilian Q. , Edwards John R.
Published: October 2020

Epigenetic changes, such as aberrant DNA methylation, contribute to cancer clonal expansion and disease progression. However, identifying subpopulation-level changes in a heterogeneous sample remains challenging. Thus, we have developed a computational approach, DXM, to deconvolve the methylation profiles of major allelic subpopulations from the bisulfite sequencing data of a heterogeneous sample. DXM does not require prior knowledge of the number of subpopulations or types of cells to expect. We benchmark DXM’s performance and demonstrate improvement over existing methods. We further experimentally validate DXM predicted allelic subpopulation-methylation profiles in four Diffuse Large B-Cell Lymphomas (DLBCLs). Lastly, as proof-of-concept, we apply DXM to a cohort of 31 DLBCLs and relate allelic subpopulation methylation profiles to relapse. We thus demonstrate that DXM can robustly find allelic subpopulation methylation profiles that may contribute to disease progression using bisulfite sequencing data of any heterogeneous sample.
LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency
Authors: O’Connor Marie N. , Kallenberg David M. , Jackstadt Rene , Watson Angharad H. , Alatsatianos Markella , Ohme Julia , Camilli Carlotta , Pilotti Camilla , Dritsoula Athina , Bowers Chantelle E. , Dowsett Laura , George Jestin , Wang Xiaomeng , Ager Ann , Sansom Owen J. , Moss Stephen E. , Greenwood John
Published: October 2020

Vascular dysfunction contributes to the pro-oncogenic tumor microenvironment and impedes the delivery of therapeutics. Normalizing of the tumor vasculature has therefore become a potential therapeutic objective. We previously reported that the secreted glycoprotein, leucine-rich α-2-glycoprotein 1 (LRG1), contributes to the formation of pathogenic neovascularization. Here we show that in mouse models of cancer,Lrg1is induced in tumor endothelial cells. We demonstrate that the expression of LRG1 impacts on tumor progression asLrg1deletion or treatment with a LRG1 function-blocking antibody inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function resulting in significantly enhanced efficacy of cisplatin chemotherapy, adoptive T-cell therapy and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent (cold) to immune active (hot). LRG1 therefore drives vascular abnormalization and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics.
Integrated analysis of multimodal single-cell data
Authors: Hao Yuhan , Hao Stephanie , Andersen-Nissen Erica , Mauck William M. , Zheng Shiwei , Butler Andrew , Lee Maddie J. , Wilk Aaron J. , Darby Charlotte , Zagar Michael , Hoffman Paul , Stoeckius Marlon , Papalexi Efthymia , Mimitou Eleni P. , Jain Jaison , Srivastava Avi , Stuart Tim , Fleming Lamar B. , Yeung Bertrand , Rogers Angela J. , McElrath Juliana M. , Blish Catherine A. , Gottardo Raphael , Smibert Peter , Satija Rahul
Published: October 2020

The simultaneous measurement of multiple modalities, known as multimodal analysis, represents an exciting frontier for single-cell genomics and necessitates new computational methods that can define cellular states based on multiple data types. Here, we introduce ‘weighted-nearest neighbor’ analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of hundreds of thousands of human white blood cells alongside a panel of 228 antibodies to construct a multimodal reference atlas of the circulating immune system. We demonstrate that integrative analysis substantially improves our ability to resolve cell states and validate the presence of previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets, and to interpret immune responses to vaccination and COVID-19. Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets, including paired measurements of RNA and chromatin state, and to look beyond the transcriptome towards a unified and multimodal definition of cellular identity.

Availability

Installation instructions, documentation, tutorials, and CITE-seq datasets are available athttp://www.satijalab.org/seurat
A CRISPR interference platform for selective downregulation of gene expression inBorrelia burgdorferi
Authors: Takacs Constantin N. , Scott Molly , Chang Yunjie , Kloos Zachary A. , Irnov Irnov , Rosa Patricia A. , Liu Jun , Jacobs-Wagner Christine
Published: October 2020

The spirocheteBorrelia burgdorfericauses Lyme disease, an increasingly prevalent infection. While previous studies have provided important insight intoB. burgdorferibiology, many aspects, including basic cellular processes, remain underexplored. To help speed up the discovery process, we adapted a CRISPR interference (CRISPRi) platform for use inB. burgdorferi. For efficiency and flexibility of use, we generated various CRISPRi template constructs that produce different basal and induced levels ofdcas9and carry different antibiotic resistance markers. We characterized the effectiveness of our CRISPRi platform by targeting the motility and cell morphogenesis genesflaB, mreB, rodA,andftsI,whose native expression levels span two orders of magnitude. For all four genes, we obtained gene repression efficiencies of at least 95%. We showed by darkfield microscopy and cryo-electron tomography that flagellin (FlaB) depletion reduced the length and number of periplasmic flagella, which impaired cellular motility and resulted in cell straightening. Depletion of FtsI caused cell filamentation, implicating this protein in cell division inB. burgdorferi. Finally, localized cell bulging in MreB- and RodA-depleted cells matched the locations of new peptidoglycan insertion specific to spirochetes of theBorreliagenus. These results therefore implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. Collectively, our results demonstrate the efficiency and ease of use of ourB. burgdorferiCRISPRi platform, which should facilitate future genetic studies of this important pathogen.

IMPORTANCE

Gene function studies are facilitated by the availability of rapid and easy-to-use genetic tools. Homologous recombination-based methods traditionally used to genetically investigate gene function remain cumbersome to perform inB. burgdorferi, as they often are relatively inefficient. In comparison, our CRISPRi platform offers an easy and fast method to implement as it only requires a single plasmid transformation step and IPTG addition to obtain potent (>95%) downregulation of gene expression. To facilitate studies of various genes in wild-type and genetically modified strains, we provide over 30 CRISPRi plasmids that produce distinct levels ofdcas9expression and carry different antibiotic resistance markers. Our CRISPRi platform represents a useful and efficient complement to traditional genetic and chemical methods to study gene function inB. burgdorferi.
Codon usage pattern reveals SARS-CoV-2 as a monomorphic pathogen of hybrid origin with role of silent mutations in rapid evolutionary success
Authors: Bansal Kanika , Kumar Sanjeet , Patil Prabhu B.
Published: October 2020

Viruses are dependent on the host tRNA pool, and an optimum codon usage pattern (CUP) is the driving force in its evolution. Systematic analysis of CUP of the coding sequences (CDS) of representative major pangolin lineages A and B of SARS-CoV-2 indicate a single transmission event of a codon-optimized virus from its source into humans. Here, no direct congruence could be detected in CUP of all CDS of SARS-CoV-2 with the non-human natural SARS viruses further reiterating its novelty. Several CDS show similar CUP with bat or pangolin, while others have distinct CUP pointing towards a possible hybrid nature of the virus. At the same time, phylogenetic diversity suggests the role of even silent mutations in its success by adapting to host tRNA pool. However, genomes of SARS-CoV-2 from primary infections are required to investigate the origins amongst the competing natural or lab leak theories.
A Systematic Review and Meta-analysis for Association of Helicobacter pylori Colonization and Celiac Disease
Authors: Amlashi Fazel Isapanah , Norouzi Zahra , Sohrabi Ahmad , Shirzad-Aski Hesamaddin , Norouzi Alireza , Ashkbari Ali , Gilani Naghme , Fatemi Seyed Alireza , Besharat Sima
Published: October 2020

Background and objectives

Based on some previous observational studies there is a theory that suggests a potential relationship between Helicobacter pylori (H. pylori) colonization and celiac disease (CD), however, the type of this relationship is still controversial. Therefore, we aimed to conduct a systematic review and meta-analysis to explore all related primary studies to find any possible association between CD and human H. pylori colonization.

Data sources

Studies were systematically searched and collected from four databases and different types of gray literature to cover all available evidence. After screening, the quality and risk of bias assessment of the selected articles were evaluated.

Synthesis methods

Meta-analysis calculated pooled odds ratio (OR) on the extracted data. Furthermore, heterogeneity, sensitivity, subgroups, and publication bias analyses were assessed.

Results

Twenty-four studies were included in this systematic review, with a total of 5241 cases and 132947 control people. The results of meta-analysis on 24 studies showed a significant and negative association between H. pylori colonization and CD (pooled OR= 0.58; 95% CI = 0.45 - 0.76; P < 0.001), with no publication bias (P = 0.407). The L’Abbé plots also showed a trend of having more H. pylori colonization in the control group. Among subgroups, ORs were notably different only when the data were stratified by continents or risk of bias; however, subgroup analysis could not determine the source of heterogeneity.

Conclusions

According to the meta-analysis, H. pylori has a mild protective role toward CD. Although this negative association is not strong, it is statistically significant and should be further considered. Further investigations in both molecular and clinic fields with proper methodology and more detailed information are needed to discover more evidence and underlying mechanisms to clear the interactive aspects of H. pylori colonization in CD patients.

Systematic review registration number (PROSPERO)

CRD42020167730
Targeted-Antibacterial-Plasmids (TAPs) combining conjugation and CRISPR/Cas systems achieve strain-specific antibacterial activity
Authors: Reuter Audrey , Hilpert Cécile , Dedieu-Berne Annick , Lematre Sophie , Gueguen Erwan , Launay Guillaume , Bigot Sarah , Lesterlin Christian
Published: October 2020

The global emergence of drug-resistant bacteria leads to the loss of efficacy of our antibiotics arsenal and severely limits the success of currently available treatments. Here, we developed an innovative strategy based on Targeted-Antibacterial-Plasmids (TAPs) that use bacterial conjugation to deliver CRISPR/Cas systems exerting a strain-specific antibacterial activity. TAPs are highly versatile as they can be directed against any specific genomic or plasmid DNA using the custom algorithm (CSTB) that identifies appropriate targeting spacer sequences. We demonstrate the ability of TAPs to induce strain-selective killing by introducing lethal double strand breaks (DSBs) into the targeted genomes. TAPs directed against a plasmid-born carbapenem resistance gene efficiently resensitise the strain to the drug. This work represents an essential step towards the development of an alternative to antibiotic treatments, which could be used forin situmicrobiota modification to eradicate targeted resistant and/or pathogenic bacteria without affecting other non-targeted bacterial species.
The Himalayan Uplift and the Evolution of Aquatic Biodiversity Across Asia: Snowtrout (Cyprininae: Schizothorax) as a Test Case
Authors: Regmi Binod , Douglas Marlis R. , Edds David R. , Wangchuk Karma , Lu Chang , Khanal Gopal Prasad , Norbu Pema , Norbu Sangay , Dorji Sonam , Tshering Singye , Angel Zachary , Chafin Tyler K. , Zbinden Zachery D. , Douglas Michael E.
Published: October 2020

The Himalayan uplift, a tectonic event of global importance, seemingly disseminated aquatic biodiversity broadly across Asia. But surprisingly, this hypothesis has yet to be tested. We do so herein by sequencing 1,140 base-pair of mtDNA cytochrome-b for 72 tetraploid Nepalese/Bhutanese Snowtrout (Schizothorax spp.), combining those data with 67 GENBANK® sequences (59 ingroup/8 outgroup), then reconstructing phylogenetic relationships using maximum likelihood/ Bayesian analyses. Results indicate Snowtrout originated in Central Asia, dispersed across the Qinghai-Tibetan Plateau (QTP), then into Bhutan via south-flowing tributaries of the east-flowing Yarlung-Tsangpo River (YLTR). The headwaters of five large Asian rivers provided dispersal corridors into southeast Asia. South of the Himalaya, the YLTR transitions into a westward-flowing Brahmaputra River that facilitated successive colonization of Himalayan drainages: First Bhutan, then Nepal, followed by far-western drainages subsequently captured by the Indus River. We found greater species-divergences across rather than within-basins, implicating vicariant evolution as a driver. The Himalaya is a component of the “third-pole” [Earth’s largest (but rapidly shrinking) glacial reservoir outside the Arctic/Antarctic]. Its unique aquatic biodiversity must not only be recognized (as herein) but also conserved through broad, trans-national collaborations. Our results effectively contrast phylogeography with taxonomy as a necessary first step in this process.

The Himalaya is the most extensive and recently evolved mountain system on Earth (length=2400km; width=240km; elevation=75-8800m), with a global significance underscored by its large-scale lithospheric, cryospheric, and atmospheric interactions [1]. These have not only driven global climate, but also defined the cultural and biological endemism of the region [2]. Massive, tectonically derived mountain chains such as the Alps and the Himalaya are hypothesized as being fundamental to the formation of global biodiversity gradients via vicariance and local adaptation, with a significantly stronger signal in terrestrial rather than aquatic systems [3]. Here we test how orogeny (the deformation and folding of Earth’s crust by lateral compression) has contributed to the diversification of freshwater fishes broadly across Asia. We do so by evaluating the phylogeography of an endemic high-elevation fish, the Snowtrout (Schizothorax: Cyprinidae).
Conserved and divergent aspects of Robo receptor signaling and regulation betweenDrosophilaRobo1 andC. elegansSAX-3
Authors: Daiber Trent , VanderZwan-Butler Christine J. , Bashaw Greg J. , Evans Timothy A.
Published: October 2020

The evolutionarily conserved Roundabout (Robo) family of axon guidance receptors control midline crossing of axons in response to the midline repellant ligand Slit in bilaterian animals including insects, nematodes, and vertebrates. Despite this strong evolutionary conservation, it is unclear whether the signaling mechanism(s) downstream of Robo receptors are similarly conserved. To directly compare midline repulsive signaling in Robo family members from different species, here we use a transgenic approach to express the Robo family receptor SAX-3 from the nematodeCaenorhabditis elegansin neurons of the fruit fly,Drosophila melanogaster. We examine SAX-3’s ability to repelDrosophilaaxons from the Slit-expressing midline in gain of function assays, and test SAX-3’s ability to substitute forDrosophilaRobo1 during fly embryonic development in genetic rescue experiments. We show thatC. elegansSAX-3 is properly translated and localized to neuronal axons when expressed in theDrosophilaembryonic CNS, and that SAX-3 can signal midline repulsion inDrosophilaembryonic neurons, although not as efficiently asDrosophilaRobo1. Using a series of Robo1/SAX-3 chimeras, we show that the SAX-3 cytoplasmic domain can signal midline repulsion to the same extent as Robo1 when combined with the Robo1 ectodomain. We show that SAX-3 is not subject to endosomal sorting by the negative regulator Commissureless (Comm) inDrosophilaneuronsin vivo, and that peri-membrane and ectodomain sequences are both required for Comm sorting ofDrosophilaRobo1.
Metabolic Reprogramming byIn UteroMaternal Benzene Exposure
Authors: Koshko Lisa , Debarba Lucas K. , Sacla Mikaela , de Lima Juliana M.B. , Didyuk Olesya , Fakhoury Patrick , Sadagurski Marianna
Published: October 2020

Environmental chemicals play a significant role in the development of metabolic disorders, especially when exposure occurs early in life. We have recently demonstrated that benzene exposure, at concentrations relevant to a cigarette smoke, induces a severe metabolic imbalance in a sex-specific manner affecting male but not female mice. However, the roles of benzene in the development of aberrant metabolic outcomes following gestational exposure, remain largely unexplored. In this study, we exposed pregnant C57BL/6JB dams to benzene at 50 ppm or filtered air for 5 days/week (6h/day from gestational day 1 to birth) and studied male and female offspring metabolic phenotypes in their adult life. While no changes in body weight or body composition were observed between groups, 4-month-old male and female offspring exhibited reduced parameters of energy homeostasis (VO2, VCO2, and heat production). However, only male offspring from benzene-exposed dams were glucose intolerant and insulin resistant at this age. By six months of age, both male and female offspring displayed glucose and insulin intolerance, associated with elevated expression of hepatic gluconeogenesis and inflammatory genes. Additionally, this effect was accompanied by elevated insulin secretion and increased beta-cell mass only in male offspring. Thus, gestational benzene exposure can reprogram offspring for increased susceptibility to the metabolic imbalance in adulthood with differential sensitivity between sexes.
Genomic Similarity of Nucleotides in SARS CoronaVirus using K-Means Unsupervised Learning Algorithm
Authors: Singh Jairaj
Published: October 2020

The drastic increase in the number of coronaviruses discovered and coronavirus genomes being sequenced have given us a great opportunity to perform genomics and bioinformatics analysis on this family of viruses. Coronaviruses possess the largest genomes (26.4 to 31.7 kb) among all known RNA viruses, with G + C contents varying from 32% to 43%. Phylogenetically, three genera, Alphacoronavirus, Betacoronavirus and Gammacoronavirus, with Betacoronavirus consisting of subgroups A, B, C were known to exist but now a new genus D also exists,namely the Deltacoronavirus. In such a situation, it becomes highly important for efficient classification of all virus data so that it helps us in suitable planning,containment and treatment. The objective of this paper is to classify SARS corona-virus nucleotide sequences based on parameters such assequence length,percentage similarity between the sequence information,open and closed gaps in the sequence due to multiple mutationsand many others.By doing this,we will be able to predict accurately the similarity ofSARS CoV-2virus with respect to other corona-viruses like the Wuhan corona-virus,the bat corona-virus and the pneumonia virus and would help us better understand about thetaxonomyof the corona-virus family.

In addition to the guidelines provided in the abstract above,the following points summarizes the article below:

The article discusses an application of Machine Learning in the field of virology.

It aims to classify the SARS CoV2 virus as per the already known sequences of the bat-coronavirus, the Wuhan Sea Food Market pneumonia virus and the Wuhan coronavirus.

To solve and predict the similarity of the SARS CoV2 coronavirus w.r.t other viruses discussed above,K-Means Unsupervised LearningAlgorithm has been chosen.

The data-set used isMN997409.1-4NY0T82X016-Alignment-HitTable.csvfound onwww.kaggle.com.(Complete link shared in the references section).[17]

The results have been validated by using a simple data-correlation technique namelySpearman’s Rank Correlation Coeffecient.

I have also discussed my future work usingDeep Neural Netsthat can help predict new virus sequences and effectively find similarity if any with already discovered viruses.
Host-pathogen co-existence incurs reproductive costs
Authors: Strubbe Diederik , Haesendonck Roel , Verbrugghe Elin , Lens Luc , Ducatelle Richard , Croubels Siska , Haesebrouck Freddy , Martel An , Pasmans Frank
Published: October 2020

Widespread endemism of host-adapted pathogens poses a heavy burden on animal and human health. Mechanisms underpinning long-term host pathogen co-existence and concurrent costs are poorly understood. We use infections in pigeons with pathogenic, pigeon adaptedSalmonellaTyphimurium to explain how host and pathogen trade-offs and benefits sustain long-term pathogen endemism. An experimentally infected group of pigeons that was studied for 15 months showed that pathogen persistence decreased host condition and reproductive success, but conferred protection againstSalmonella-induced clinical disease. The relevance of these findings was confirmed in nature, where this pathogen was shown to widely occur in feral pigeons (Columba livia), yet without clinical disease. Pathogen transmission and long-term persistence were associated with intermittent faecal shedding, which markedly increased during crop feeding and natural stress periods. Exploiting host specific traits in the presence of protective host population immunity thus facilitates long-term co-existence, be it at a significant reproductive cost.
A novel and conserved cell wall enzyme that can substitute for the Lipid II synthase MurG
Authors: Zhang L. , Ramijan K. , Carrión V.J. , van der Aart L.T , Willemse J. , van Wezel G.P. , Claessen D.
Published: October 2020

The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule Lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here we present the discovery of a novel cell wall enzyme that can substitute for MurG. A mutant ofKitasatospora viridifacienslacking a significant part of thedcwcluster includingmurGsurprisingly produced Lipid II and wild-type peptidoglycan. Genomic analysis identified a distantmurGparalogue, which encodes a putative enzyme that shares only around 31% aa sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MurG2. Orthologues ofmurG2are present in 38% of all genomes ofKitasatosporaeand members of the sister genusStreptomyces. CRISPRi experiments showed thatK. viridifaciens murG2can also functionally replacemurGinStreptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. Altogether, these results identify MurG2 as abona fideLipid II synthase, thus demonstrating plasticity in cell wall synthesis.
SEX DIFFERENCES IN μ-OPIOID REGULATION OF COERULEAR-CORTICAL TRANSMISSION
Authors: Guajardo Herminio M , Valentino Rita J
Published: October 2020

Stress-induced activation of locus coeruleus (LC)-norepinephrine (NE) projections to the prefrontal cortex is thought to promote cognitive responses to stressors. LC activation by stressors is modulated by endogenous opioids that serve to restrain LC activation and to facilitate a return to baseline activity upon stress termination. Sex differences in this opioid influence could be a basis for sex differences in stress vulnerability. Consistent with this, we recently demonstrated that μ-opioid receptor (MOR) expression is decreased in the female rat LC compared to the male LC and this was associated with sexually distinct consequences of activating MOR in the LC on cognitive flexibility. Given that the LC-NE system affects cognitive flexibility through its projections to the medial prefrontal cortex (mPFC), the present study quantified and compared the effects of LC-MOR activation on mPFC neural activity in male and female rats. Local field potential (LFPs) were recorded from the mPFC of freely behaving male and female rats before and following local LC microinjection of the MOR agonist, DAMGO or vehicle. Intra-LC DAMGO altered the LFP power spectrum selectively in male, but not female rats, resulting in a time-dependent increase in the power in delta and alpha frequency bands. LC microinfusion of ACSF had no effect in either sex. Together, the results are consistent with previous evidence for decreased MOR function in the female rat LC and demonstrate that this translates to a diminished effect on cortical activity that can account for sex differences in cognitive consequences. Decreased LC-MOR function in females could contribute to greater stress-induced activation of the LC, and increased vulnerability of females to hyperarousal symptoms of stress-related neuropsychiatric pathologies.