bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic and molecular characterization of extended spectrum β-lactamase producingEscherichia coliandKlebsiella pneumoniaeisolates from various samples of animal origin from Assam, India
Authors: Das Leena , Borah Probodh , Sharma R.K. , Malakar Dipika , Saikia G.K. , Sharma K. , Tamuly S. , Dutta Rupam
Published: May 2020

Extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae has become a major threat globally. Here we have characterized ESBL producingE. coliandK. pneumoniaefrom various sources, studied antibiogram and resistance gene profiles. Out of 385 samples, 31 (8.05%) were positive for ESBL producingE. coli. Such isolates could be recovered from 10.05, 8.33, 15.63, 6.67 and 4.35 per cent of cattle milk, curd, chicken, pork and cattle faeces samples, respectively. A total of 59 (15.32%) samples were positive for ESBL producingK. pneumoniae, which were isolated from 14.35, 6.25, 21.43 and 34.78 per cent cattle milk, chicken, beef and cattle faeces, respectively. All the 90 isolates were confirmed as ESBL producers by CDT and ESBL-E strip tests. Antibiogram revealed that 74.19% and 69.49% of the ESBL producingE. coliandK. pneumoniaeisolates, respectively showed resistance to ceftizoxime, 25.81% and 23.73% to both co-trimoxazole and tetracycline, 19.35% and 25.42% to ciprofloxacin, 9.68% and 16.95% to chloramphenicol, 3.23% and 5.08% to pipercillin-tazobactam, and 3.23% and 3.39% to gentamicin. Resistance gene profiling showedblaCTX-M gene as most predominant (100%). TheblaTEM gene was found in 54.84% and 55.93%,blaSHV gene in 90.32% and 77.97%,Sul1 gene in 90.32% and 86.44% of ESBL producingE. coliandK. pneumoniaeisolates, respectively. TheInt1 gene was detected in 70.97% and 62.71% isolates, whileqnrB gene was found in 3.23% and 10.17% ofE. coliand K. pneumoniaeisolates, respectively.
Medical students perceptions and motivations in time of COVID-19 pandemic
Authors: Tempski Patricia , Arantes-Costa Fernanda M , Kobayasi Renata , Siqueira Marina AM , Torsani Matheus M , Amaro Bianca QRC , Nascimento Maria Eduarda FM , Siqueira Saulo L , Santos Itamar S , Martins Milton A
Published: May 2020

Background: There has been a rapid increase in the number of cases of COVID19 in Latin America, Africa and Asia, in many countries that have an insufficient number of physicians and other health care personnel, and the need for the inclusion of medical students as part of the health teams is a very important issue. It has been recommended that medical students work as volunteers, have appropriate training, do not undertake any activity beyond their level of competence, have continuous supervision and adequate personal protective equipment. However, motivation of medical students must be evaluated in order to make volunteering a more evidence-based initiative. The aim of our study was to evaluate motivation of medical students to be part of the health team to help in the COVID19 pandemic.

Methods and Findings: We developed a questionnaire specifically to evaluate medical students perceptions about participating in care of patients with suspected infection due to coronavirus during the COVID19 pandemic. The questionnaire had two parts: a) individual characteristics, year and geographic location of medical school; b) twenty eight statements responded on a 5 point Likert scale (totally agree, agree, neither agree nor disagree, disagree and totally disagree). To develop the questionnaire, we performed consensus meetings of a group of faculty and medical students. The questionnaire was sent to student organizations of 257 medical schools in Brazil and answered by 10,433 students. We used multinomial logistic regression models to analyse the data.

Statements with greater odds ratios for participation of medical students in COVID19 pandemic were related to sense of purpose or duty (It is the duty of the medical student to put himself at the service of the population in the pandemic), altruism (I am willing to take risks by participating in practical in the context of pandemic), perception of good performance and professional identity (I will be a better health professional for having experienced the pandemic). Males had higher odds ratios than females (1.36 [95% CI: 1.24 to 1.49] to 1.68 [95% CI: 1.47 to 1.91]).

Conclusions: Medical students are motivated by sense of purpose or duty, altruism, perception of good performance and values of professionalism more than their interest in learning. These results have implications in the developing of programs of volunteering and in the design of health force policies in the present pandemic and in future health emergencies.
Mortality rate and estimate of fraction of undiagnosed COVID-19 cases in the US in March and April 2020
Authors: Gao RuShan , Rosenlof Karen H.
Published: May 2020

We use a simple model to derive a mortality probability distribution for a patient as a function of days since diagnosis (considering diagnoses made between 25 February and 29 March 2020). The peak of the mortality probability is the 13th day after diagnosis. The overall shape and peak location of this probability curve are similar to the onset-to-death probability distribution in a case study using Chinese data.

The total mortality probability of a COVID-19 patient in the US diagnosed between 25 February and 29 March is about 21%. We speculate that this high value is caused by severe under-testing of the population to identify all COVID-19 patients. With this probability, and an assumption that the true probability is 2.4%, we estimate that 89% of all SARS-CoV-2 infection cases were not diagnosed during this period.

When the same method is applied to data extended to 25 April, we found that the total mortality probability of a patient diagnosed in the US after 1 April is about 6.4%, significantly lower than for the earlier period. We attribute this drop to increasingly available tests. Given the assumption that the true mortality probability is 2.4%, we estimate that 63% of all SARS-CoV-2 infection cases were not diagnosed during this period (1 - 25 April).
Predicting the end-stage of the COVID-19 epidemic in Brazil
Authors: Fitzgibbon W.E. , Morgan J.J. , Webb G.F. , Wu Y.
Published: May 2020

We develop a dynamic model of a COVID-19 epidemic as a system of differential equations. The model incorporates an asymptomatic infectious stage and a symptomatic infectious stage. We apply the model to the current COVID-19 epidemic in Brazil. We compare the model output to current epidemic data, and project forward in time possible end-stages of the epidemic in Brazil. The model emphasizes the importance of reducing asymptomatic infections in controlling the epidemic.
Worsening of pre-existing psychiatric conditions during the COVID-19 pandemic
Authors: Gobbi Susanna , Płomecka Martyna Beata , Ashraf Zainab , Radziński Piotr , Neckels Rachael , Lazzeri Samuel , Dedić Alisa , Bakalović Asja , Hrustić Lejla , Skórko Beata , Es haghi Sarvin , Almazidou Kristina , Rodríguez-Pino Luis , Beyza Alp A. , Jabeen Hafsa , Waller Verena , Shibli Dana , A Behnam Mehdi , Arshad Ahmed Hussain , Barańczuk-Turska Zofia , Haq Zeeshan , Qureshi Salah U , Jawaid Ali
Published: May 2020

This study anonymously examined 2,734 psychiatric patients worldwide for worsening of their pre-existing psychiatric condition during the COVID-19 pandemic. Valid responses mainly from 12 featured countries indicated self-reported worsening of psychiatric conditions in 2/3rd of the patients assessed that was validated through their significantly higher scores on scales for general psychological disturbance, post-traumatic stress disorder, and depression. Female gender, feeling no control of the situation and reporting dissatisfaction with the response of the state during the COVID-19 pandemic, and reduced interaction with family and friends increased the worsening of pre-existing psychiatric conditions, whereas optimism, ability to share concerns with family and friends and using social media like usual were associated with less worsening. An independent clinical investigation from the USA confirmed worsening of psychiatric conditions during the COVID-19 pandemic based on identification of new symptoms that necessiated clinical interventions such as dose adjustment or starting new medications in more than half of the patients.
A Survey of Electronic Health Record (EHR) Variables Associated with Postoperative Delirium: A Systematic Review
Authors: Ruppert Matthew M , Hashemighouchani Haleh , Bihorac Emel , Williams Seth , Velez Laura , Cupka Julie S , Ozrazgat-Baslanti Tezcan , Rashidi Parisa , Bihorac Azra
Published: May 2020

Introduction: Delirium is a common post-operative complication in critically ill patients, displaying transient changes in consciousness, inattention, awareness, and organized thought. Not a lot is known about the specific causes of this condition, as it is a complex physiologic state that is currently being unraveled to determine any correlation with imbalances in homeostasis.
Objective: The aim of this systematic review is to report on and summarize risk factors associated with the development of postoperative delirium in critically ill adult patients.
Methods: This systematic review assessed studies reporting on risk factors for postoperative delirium in critically ill patients. PubMed, PsycINFO, and CINAHL databases were searched for studies. Observational or interventional studies reviewing predictors for postoperative delirium in delirious versus non-delirious patients were included.
Results: Fifty-one potential risk factors were identified and divided into eight subgroups. The significance of a specific risk factor for postoperative delirium was found to depend on the patient population in question, but consistently cited significant risk factors across cohorts included high mortality risk, abnormal laboratory values, and use of vasopressors, analgesics, thiopentones, propofol, and benzodiazepines. There was not enough evidence found to definitively state the significance of type of surgery being performed or cognitive impairment on development of this condition.
Conclusion: Several risk factors were found to be significantly associated with the development of postoperative delirium, including high risk of mortality, specific medication use, and abnormal laboratory values. Further research needs to be performed to fully define the importance of these individual risk factors across broad critical care population.
Early Impact of COVID-19 on Individuals with Eating Disorders: A survey of ~1000 Individuals in the United States and the Netherlands
Authors: Termorshuizen Jet D. , Watson Hunna J. , Thornton Laura M. , Borg Stina , Flatt Rachael E. , MacDermod Casey M. , Harper Lauren E. , van Furth Eric F. , Peat Christine M. , Bulik Cynthia M.
Published: May 2020

We received rapid ethical permission to evaluate the early impact of COVID-19 on people with eating disorders. Participants in the United States (US, N=511) and the Netherlands (NL, N=510), recruited through ongoing studies and social media, completed an online baseline survey that included both quantitative measures and free-text responses assessing the impact of COVID-19 on situational circumstances, eating disorder symptoms, eating disorder treatment, and general well-being. Results revealed strong and wide-ranging effects on eating disorder concerns and illness behaviors that were consistent with diagnoses. Participants with anorexia nervosa (US 62% of sample; NL 69%) reported increased restriction and fears about being able to find foods consistent with their meal plan. Individuals with bulimia nervosa and binge-eating disorder (US 30% of sample; NL 15%) reported increases in their binge-eating episodes and urges to binge. Respondents noted marked increases in anxiety since 2019 and reported greater concerns about the impact of COVID-19 on their mental health than physical health. Although many participants acknowledged and appreciated the transition to telehealth, limitations of this treatment modality for this population were raised. Individuals with past histories of eating disorders noted concerns about relapse related to COVID-19 circumstances. Encouragingly, respondents also noted positive effects including greater connection with family, more time for self-care, and motivation to recover.
Changes in intake of dairy product subgroups and risk of type 2 diabetes: modelling specified food substitutions in the Danish Diet, Cancer and Health cohort
Authors: Ibsen Daniel B. , Overvad Kim , Laursen Anne Sofie D. , Halkjær Jytte , Tjønneland Anne , Kilpeläinen Tuomas O. , Parner Erik T. , Jakobsen Marianne U.
Published: May 2020

We investigated the association between an increased intake of one dairy product subgroup at the expense of another within a 5-year period and the subsequent 10-year risk of developing type 2 diabetes. Effect modification by the initial level of the dairy product intake was also examined. The cohort included 39,438 men and women with two measurements of diet assessed using food frequency questionnaires (FFQ) administered in 1993-1997 and 1999-2003. Dairy products were skimmed milk, semi-skimmed milk, whole-fat milk, buttermilk, low-fat yogurt, whole-fat yogurt, cheese and butter. Type 2 diabetes cases were ascertained from the Danish National Diabetes Register. The pseudo-observation method was used to calculate risk differences (RD) with 95% confidence intervals (CI). Among participants aged 56-59 years at hand-in of the follow-up FFQ, increased intake of whole-fat yogurt in place of skimmed, semi-skimmed or whole-fat milk was associated with a reduced risk (RD% [95% CI]: −0.8% [−1.3, −0.2]; −0.6% [−1,1, −0.1]; −0.7 [−1.2, −0.1]; per 50g/d, respectively). Among participants aged 60-64 and 65-72, substitution of skimmed milk for semi-skimmed milk was associated with an increased risk of type 2 diabetes (0.5% [0.2, 0.7]; 0.4% [0.1, 0.7]; per 50g/d, respectively). No effect modification by the initial intake of milk products was observed. Our results suggest that substitution of whole-fat yogurt for milk among those aged 56-59 decreases the risk of type 2 diabetes and substitution of skimmed milk for semi-skimmed milk was associated with an increased risk of type 2 diabetes among those aged 60-64 and 65-72, regardless of the initial intake level.
A searchable image resource ofDrosophilaGAL4-driver expression patterns with single neuron resolution
Authors: Meissner Geoffrey W. , Nern Aljoscha , Dorman Zachary , DePasquale Gina M. , Forster Kaitlyn , Gibney Theresa , Hausenfluck Joanna H. , He Yisheng , Iyer Nirmala , Jeter Jennifer , Johnson Lauren , Johnston Rebecca M. , Lee Kelley , Melton Brian , Yarbrough Brianna , Zugates Christopher T. , Clements Jody , Goina Cristian , Otsuna Hideo , Rokicki Konrad , Svirskas Robert R. , Aso Yoshinori , Card Gwyneth M. , Dickson Barry J. , Ehrhardt Erica , Goldammer Jens , Ito Masayoshi , Kainmueller Dagmar , Korff Wyatt , Mais Lisa , Minegishi Ryo , Namiki Shigehiro , Rubin Gerald M. , Sterne Gabriella R. , Wolff Tanya , Malkesman Oz ,   FlyLight Project Team
Published: May 2020

Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage ofDrosophilaneuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which generally lack the single-cell resolution required for reliable cell type identification. Here we use stochastic GAL4 labeling with the MultiColor FlpOut approach to generate cellular resolution confocal images at large scale. We are releasing aligned images of 74,000 such adult central nervous systems. An anticipated use of this resource is to bridge the gap between neurons identified by electron or light microscopy. Identifying individual neurons that make up each GAL4 expression pattern improves the prediction of split-GAL4 combinations targeting particular neurons. To this end we have made the images searchable on the NeuronBridge website. We demonstrate the potential of NeuronBridge to rapidly and effectively identify neuron matches based on morphology across imaging modalities and datasets.
Design and Performance Evaluation of a Wearable Passive Cable-driven Shoulder Exoskeleton
Authors: Asgari Morteza , Phillips Elizabeth A. , Dalton Britt M. , Rudl Jennifer L. , Crouch Dustin L.
Published: May 2020

The mechanical assistance provided by exoskeletons could potentially replace, assist, or rehabilitate upper extremity function in patients with mild to moderate shoulder disability to perform activities of daily living. While many exoskeletons are “active” (e.g. motorized), mechanically passive exoskeletons may be a more practical and affordable solution to meet a growing clinical need for continuous, home-based movement assistance. In the current study, we designed, fabricated, and evaluated the performance of a wearable, passive, cable-driven shoulder exoskeleton (WPCSE) prototype. An innovative feature of the WPCSE is a modular spring-cam-wheel system that can be custom designed to compensate for any proportion of the shoulder elevation moment due to gravity over a large range of shoulder motion. The force produced by the spring-cam-wheel system is transmitted over the superior aspect of the shoulder to an arm cuff through a Bowden cable. The results from mechanical evaluation revealed that the modular spring-cam-wheel system could successfully produce an assistive positive shoulder elevation moment that matched the desired, theoretical moment. However, when measured from the physical WPCSE prototype, the moment was lower (up to 30%) during positive shoulder elevation and higher (up to 120%) during negative shoulder elevation due primarily to friction. Even so, our biomechanical evaluation showed that the WPCSE prototype reduced the root mean square (up to 35%) and peak (up to 33%) muscular activity, as measured by electromyography, of several muscles crossing the shoulder during shoulder elevation and horizontal adduction/abduction movements. These preliminary results suggest that our WPCSE may be suitable for providing movement assistance to people with shoulder disability.
Shared PKS modules in biosynthesis of synergistic laxaphycins
Authors: Heinilä LMP , Fewer DP , Jokela J , Wahlsten M , Jortikka A , Sivonen K
Published: May 2020

Cyanobacteria produce a wide range of lipopeptides that exhibit potent membrane-disrupting activities. Laxaphycins consist of two families of structurally distinct macrocyclic lipopeptides that act in a synergistic manner to produce antifungal and antiproliferative activities. Laxaphycins are produced by range of cyanobacteria but their biosynthetic origins remain unclear. Here, we identified the biosynthetic pathways responsible for the biosynthesis of the laxaphycins produced byScytonema hofmanniiPCC 7110. We show that these laxaphycins, called scytocyclamides, are produced by this cyanobacterium and are encoded in a single biosynthetic gene cluster with shared polyketide synthase enzymes initiating two distinct non-ribosomal peptide synthetase pathways. To our knowledge, laxaphycins are the first clearly distinct polyketide synthase and non-ribosomal peptide synthetase hybrid natural products with shared branched biosynthesis. The unusual mechanism of shared enzymes synthesizing two distinct types of products may aid future research in identifying and expressing natural product biosynthetic pathways and in expanding the known biosynthetic logic of this important family of natural products.
Gene Expression Changes Occurring at Bolting Time are Associated with Leaf Senescence in Arabidopsis
Authors: Hinckley Will E , Brusslan Judy A.
Published: May 2020

In plants, the vegetative to reproductive phase transition (termed bolting in Arabidopsis) generally precedes age-dependent leaf senescence (LS). Many studies describe a temporal link between bolting time and LS, as plants that bolt early, senesce early, and plants that bolt late, senesce late. However, the molecular mechanisms underlying this relationship are unknown and are potentially agriculturally important, as they may allow for the development of crops that can overcome early LS caused by stress-related early phase transition. We hypothesized that gene expression changes associated with bolting time were regulating LS. We used a mutant that displays both early bolting and early LS as a model to test this hypothesis. An RNA-seq time series experiment was completed to compare the early bolting mutant to vegetative WT plants of the same age. This allowed us to identify bolting time-associated genes (BAGs) expressed in an older rosette leaf at the time of inflorescence emergence. The BAG list contains many well characterized LS regulators (ORE1, WRKY45, NAP, WRKY28), and GO analysis revealed enrichment for LS and LS-related processes. These bolting associated LS regulators likely contribute to the temporal coupling of bolting time to LS.
Oxidative switch drives mitophagy defects in dopaminergicparkinmutant patient neurons
Authors: Schwartzentruber Aurelie , Boschian Camilla , Lopes Fernanda Martins , Myszczynska Monika A , New Elizabeth J , Beyrath Julien , Smeitink Jan , Ferraiuolo Laura , Mortiboys Heather
Published: May 2020

Background Mutations in parkin are the most common cause of early onset Parkinson’s disease. Parkin is an E3 ubiquitin ligase, functioning in mitophagy. Mitochondrial abnormalities are present in parkin mutant models. Patient derived neurons are a promising model in which to study pathogenic mechanisms and therapeutic targets. Here we generate induced neuronal progenitor cells from parkin mutant patient fibroblasts with a high dopaminergic neuron yield. We reveal changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Methods Fibroblasts from 4 controls and 4 parkin mutant patients were transformed into induced neuronal progenitor cells and subsequently differentiated into dopaminergic neurons. Mitochondrial morphology, function and mitophagy were evaluated using live cell fluorescent imaging, cellular ATP and reactive oxygen species production quantification. Results Direct conversion of control and parkin mutant patient fibroblasts results in induced neuronal progenitor and their differentiation yields high percentage of dopaminergic neurons. We were able to observe changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Our results show that when pre-neurons are glycolytic early in differentiation mitophagy is unimpaired by PRKN deficiency. However as neurons become oxidative phosphorylation dependent, mitophagy is severely impaired in the PRKN mutant patient neurons. These changes correlate with changes in mitochondrial function and morphology; resulting in lower neuron yield and altered neuronal morphology. Conclusions Induced neuronal progenitor cell conversion can produce a high yield of dopaminergic neurons. The mitochondrial phenotype, including mitophagy status, is highly dependent on the metabolic status of the cell. Only when neurons are oxidative phosphorylation reliant the extent of mitochondrial abnormalities are identified. These data provide insight into cell specific effects of PRKN mutations, in particular in relation to mitophagy dependent disease phenotypes and provide avenues for alternative therapeutic approaches.
The variant call format provides efficient and robust storage of GWAS summary statistics
Authors: Lyon Matthew , Andrews Shea J , Elsworth Ben , Gaunt Tom R , Hemani Gibran , Marcora Edoardo
Published: May 2020

Genome-wide association study (GWAS) summary statistics are a fundamental resource for a variety of research applications1–6. Yet despite their widespread utility, no common storage format has been widely adopted, hindering tool development and data sharing, analysis and integration. Existing tabular formats7,8often ambiguously or incompletely store information about genetic variants and their associations, and also lack essential metadata increasing the possibility of errors in data interpretation and post-GWAS analyses. Additionally, data in these formats are typically not indexed, requiring the whole file to be read which is computationally inefficient. To address these issues, we propose an adaptation of the variant call format9(GWAS-VCF) and have produced a suite of open-source tools for using this format in downstream analyses. Simulation studies determine GWAS-VCF is 9-46x faster than tabular alternatives when extracting variant(s) by genomic position. Our results demonstrate the GWAS-VCF provides a robust and performant solution for sharing, analysis and integration of GWAS data. We provide open access to over 10,000 complete GWAS summary datasets converted to this format (available from:https://gwas.mrcieu.ac.uk).
Decision-making in a synthetic cell: the limits of biological computation
Authors: Greiss Ferdinand , Daube Shirley S. , Noireaux Vincent , Bar-Ziv Roy
Published: May 2020

We measured the dynamics of decision-making by a minimal bistable gene network integrated in a synthetic cell model, free of external perturbations. Reducing the number of gene copies from 105to about 10 per cell revealed a transition from deterministic and slow computation to a fuzzy and rapid regime dominated by singleprotein fluctuations. Fuzzy computation appeared at DNA and protein concentrations 100-fold lower than necessary in equilibrium, suggesting rate enhancement by co-expressional localization. Whereas the high-copy regime was characterized by a sharp transition, hysteresis and robust memory, the low-copy limit showed incipient strong fluctuations, switching between states, and a signature of cellular individuality across the decision-making point. Our work establishes synthetic cells operating rapidly at the single molecule level to integrate gene regulatory networks with metabolic pathways for sustained survival with low energetic cost.

One Sentence Summary

Decision-making in a synthetic cell can be slow and precise or rapid and probabilistic by reducing the number of computing molecules by five decades down to single-molecule fluctuations.
Understanding the diversity of DNA methylation in Mycobacterium tuberculosis
Authors: Ndhlovu Victor , Kiran Anmol , Sloan Derek , Mandala Wilson , Nliwasa Marriot , Everett Dean B , Mwapasa Mphatso , Kontogianni Konstantina , Kamdolozi Mercy , Corbett Elizabeth L , Caws Maxine , Davies Gerry
Published: May 2020

AlthoughMycobacterium tuberculosis (Mtb)strains exhibit genomic homology of >99%, there is considerable variation in the phenotype. The underlying mechanisms of phenotypic heterogeneity inMtbare not well understood but epigenetic variation is thought to contribute. At present the methylome ofMtbhas not been completely characterized. We completed methylomes of 18Mycobacterium tuberculosis(Mtb) clinical isolates from Malawi representing the largest number ofMtbgenomes to be completed in a single study using Single Molecule Real Time (SMRT) sequencing to date. We replicate and confirm four methylation disrupting mutations in lineages ofMtb. For the first time we report complete loss of methylation courtesy of C758T (S253L) mutation in theMamBgene of Indo-oceanic lineage ofMtb. We also conducted a genomic and methylome comparison of the Malawian samples against a global sample. We confirm that methylation inMtbis lineage specific although some unresolved issues still remain.
The MNK1/2-eIF4E axis drives melanoma plasticity, progression, and resistance to immunotherapy
Authors: Huang Fan , Gonçalves Christophe , Bartish Margarita , Rémy-Sarrazin Joelle , Guo Qianyu , Emond Audrey , Yang William , Plourde Dany , Su Jie , Gimeno Marina Godoy , Zhan Yao , Attias Mikhael , Galán Alba , Rzymski Tomasz , Mazan Milena , Masiejczyk Magdalena , Faber Jacek , Khoury Elie , Benoit Alexandre , Gagnon Natascha , Dankort David , Piccirillo Ciriaco A. , Journe Fabrice , Ghanem Ghanem , Saragovi H. Uri , Sonenberg Nahum , Topisirovic Ivan , Miller Wilson H. , del Rincón Sonia V.
Published: May 2020

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Melanoma plasticity exhibited as phenotype switching contributes to immunotherapy resistance, however the mechanisms are not completely understood and thus therapeutically unexploited. Here, using a transgenic melanoma mouse model, we demonstrated a critical role of the MNK1/2-eIF4E axis in melanoma plasticity and resistance to immunotherapy. We showed that phospho-eIF4E deficient murine melanomas express high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified that phospho-eIF4E controls the translation of NGFR, a critical effector of phenotype switching. In patients with melanoma, the expression of MKNK1, the kinase for eIF4E, positively correlated with markers of immune exhaustion. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors and increased CD8+ T cell infiltrates. Blocking phospho-eIF4E, using MNK1/2 inhibitors, offers a new strategy to inhibit melanoma plasticity and improve the survival response to anti-PD-1 immunotherapy.
Epigenetic heterogeneity afterde novoassembly of native full-length Hepatitis B Virus genomes
Authors: Goldsmith Chloe , Cohen Damien , Dubois Anaëlle , Martinez Maria-Guadalupe , Petitjean Kilian , Corlu Anne , Testoni Barbara , Hernandez-Vargas Hector , Chemin Isabelle
Published: May 2020

Hepatitis B Virus (HBV) is a 3.2KB DNA virus that causes acute and chronic hepatitis. HBV infection is a world health problem, with 350 million chronically infected people at increased risk of developing liver disease and hepatocellular carcinoma (HCC). Methylation of HBV DNA in a CpG context (5mCpG) can alter the expression patterns of viral genes related to infection and cellular transformation. Moreover, it may also provide clues to why certain infections are cleared, or persist with or without progression to cancer. The detection of 5mCpG often requires techniques that damage DNA or introduce bias through a myriad of limitations. Therefore, we developed a method for the detection of 5mCpG on the HBV genome that does not rely on bisulfite conversion or PCR. With cas9 guided RNPs to specifically target the HBV genome, we enriched in HBV DNA from Primary Human Hepatocytes (PHH) infected with different HBV genotypes, as well as enriching in HBV from infected patient liver tissue; followed by sequencing with Oxford Nanopore Technologies MinION. Detection of 5mCpG by Nanopore sequencing was benchmarked with Bisulfite-quantitative Methyl Specific PCR (BS-qMSP). 5mCpG levels in HBV determined by BS-qMSP and Nanopore sequencing were highly correlated. Our Nanopore sequencing approach achieved a coverage of ∼2000x of HBV depending on infection efficacy, sufficient coverage to perform ade novoassembly and detect small fluctuations in HBV methylation, providing the firstde novoassembly of native HBV DNA, as well as the first landscape of 5mCpG from native HBV sequences. Moreover, by capturing entire HBV genomes, we explored the epigenetic heterogeneity of HBV in infected patients and identified 4 epigenetically distinct clusters based on methylation profiles. This method is a novel approach that enables the enrichment of viral DNA in a mixture of nucleic acid material from different species and will serve as a valuable tool for infectious disease monitoring.
Comparison of Different Kits for SARS-CoV-2 RNA Extraction Marketed in Brazil
Authors: Eisen Ana Karolina Antunes , Demoliner Meriane , Gularte Juliana Schons , Hansen Alana Witt , Schallenberger Karoline , Mallmann Larissa , Hermann Bruna Saraiva , Heldt Fágner Henrique , de Almeida Paula Rodrigues , Fleck Juliane Deise , Spilki Fernando Rosado
Published: May 2020

December 2019 marked the begining of the greatest pandemic since Spanish Flu, the disease named Covid-19 that cause severe pneumonia. Until May 19, 2020 more than 4 million and 700 thousand cases were oficially notified with about 316 thousand deaths. Etiological agent of the disease was identified as being a new coronavirus,Severe acute respiratory syndrome-related coronavirus(SARS-CoV-2). In this study we compared four different manual methods for RNA isolation and purification for detection of SARS-CoV-2 through qRT-PCR, as well as the extraction quality itself through detection of RNAse P. Magnetic beads-based (MagMax™) and silica column-based (Biopur®) methods presented the better performances. Concerning to the mean delay in CT values when compared to MagMax™, TRIzol™, Biopur®and EasyExtract presented 0,39, 0,95 and 5,23 respectively. Agreement between positive and negative results of different methods when compared with the one with better performance MagMax™ was 94,44% for silica column-based method (Biopur®), 88,89% for phenol-chroloform-based method (TRIzol™) and 77,78% for EasyExtract. We aimed to evaluate how reliable each method is for diagnostic purposes and to propose alternatives when usual methods are not available. In this regard, magnectic beads and silica column-based methods are convenient and reliable choices and phenol-chloroform-based method could also be chosen as an alternative.
Spatiotemporal chloride dynamics in hypothalamic CRHPVNneurons
Authors: Lanz Aaron , Gordon Grant , Bains Jaideep
Published: May 2020

Chloride (Cl-) dynamics shape inhibitory neurotransmission in the brain. In the hypothalamus, GABA signalling onto corticotropin-releasing hormone (CRHPVN) neurons can switch from inhibitory to excitatory during stress. Although Cl-fluctuations mediate this stress-dependent shift in GABA signalling, the underlying Cl-dynamics are poorly understood. Here, using a novel optogenetic strategy to load intracellular Cl-using halorhodopsin, we demonstrate that KCC2 rapidly restores Cl-setpoints in CRHPVNneurons from naïve animals, but that this process is slowed following stress. Further, we report that somatic Cl-homeostasis remains intact after stress. Our results strongly support the idea that KCC2 functions primarily to maintain Cl-setpoints and that inhibitory synapses onto dendritic and somatic compartments of CRHPVNneurons are differentially regulated during stress.
A minimally disruptive method for measuring water potential in-planta using hydrogel nanoreporters
Authors: Jain Piyush , Liu Weizhen , Zhu Siyu , Melkonian Jeff , Pauli Duke , Riha Susan Jean , Gore Michael A. , Stroock Abraham D.
Published: May 2020

Leaf water potential is a critical indicator of plant water status, integrating soil moisture status, plant physiology, and environmental conditions. There are few tools for measuring plant water status (water potential) in situ, presenting a critical barrier for the development of appropriate phenotyping (measurement) methods for crop development and modeling efforts aimed at understanding water transport in plants. Here, we present the development of an in situ, minimally-disruptive hydrogel nanoreporter (AquaDust) for measuring leaf water potential. The gel matrix responds to changes in water potential in its local environment by swelling; the distance between covalently linked dyes changes with the reconfiguration of the polymer, leading to changes in the emission spectrum via Fluorescence Resonance Energy Transfer (FRET). Upon infiltration into leaves, the nanoparticles localize within the apoplastic space in the mesophyll; they do not enter the cytoplast or the xylem. We characterize the physical basis for AquaDust’s response and demonstrate its function in intact maize (Zea maysL.) leaves as a reporter of leaf water potential. We use AquaDust to measure gradients of water potential along intact, actively transpiring leaves as a function of water status; the localized nature of the reporters allows us to define a hydraulic model that distinguishes resistances inside and outside the xylem. We also present field measurements with AquaDust through a full diurnal cycle to confirm the robustness of the technique and of our model. We conclude that AquaDust offers potential opportunities for high-throughput, field measurements and spatially resolved studies of water relations within plant tissues.
GermlineERBB3mutation in familial non-small cell lung carcinoma: expanding ErbB’s role in oncogenesis
Authors: McInerney-Leo Aideen M. , Chew Hui Yi , Inglis Po-Ling , Leo Paul J. , Joseph Shannon R. , Cooper Caroline L. , Okano Satomi , Hassall Tim , Anderson Lisa , Bowman Rayleen V. , Gattas Michael , Harris Jessica E. , Marshall Mhairi S. , Shaw Janet G. , Wheeler Lawrie , Yang Ian A. , Brown Matthew A. , Fong Kwun M. , Simpson Fiona , Duncan Emma L.
Published: May 2020

Background

Lung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations inEGFR/ErbB1andERBB2.

Methods

Germline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and sub-cellular localisation were assessed; and cellular proliferation, pAkt/Akt, and pERK levels were determined.

Results

A novel germline variant inERBB3(c.1946T>G: p.Iso649Arg), coding for receptor tyrosineprotein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80kD form (which enhances proliferation) compared to the full-length (180kD) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localisation pre- and post-EGF stimulation; however, EGFR levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared to wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 minutes post-stimulation compared to wtErbB3 transfection, demonstrating altered signalling pathway activation by mutErbB3. Cumulatively, these results support this mutation as tumorogenic.

Conclusions

This is the first reported family with a germlineERBB3mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.
Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding
Authors: Yperman Klaas , Papageorgiou Anna C. , Merceron Romain , De Munck Steven , Bloch Yehudi , Eeckhout Dominique , Tack Pieter , Evangelidis Thomas , Van Leene Jelle , Vincze Laszlo , Vandenabeele Peter , Potocký Martin , De Jaeger Geert , Savvides Savvas N. , Tripsianes Konstantinos , Pleskot Roman , Van Damme Daniel
Published: May 2020

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. The mechanistic contribution of the individual TPC subunits to plant CME remains however elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Whereas one EH domain binds negatively charged PI(4,5)P2lipids, unbiased peptidome profiling by mass-spectrometry revealed that the other EH domain interacts with the double N-terminal NPF motif of a novel TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 have in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.
Phenological, Physiological and yield markers as efficient tools to identify drought tolerant rice genotypes in Eastern India
Authors: Sahoo Soumya Kumar , Dash Goutam Kumar , Guhey Arti , Baig Mirza Jaynul , Barik Madhusmita , Parida Selukash , Swain Padmini
Published: May 2020

Rice production is severely threatened by drought stress in Eastern India. To develop drought tolerant varieties, selection of donors for breeding programme is crucial. Twenty one selected rice genotypes including both tolerant and sensitive to drought were grown under well-watered and drought stress conditions in dry seasons of two successive years of 2017 and 2018. Leaf water potential, relative water content displayed significant difference among the genotypes during vegetative screening. At reproductive stage drought screening, days to 50% flowering was delayed in all genotypes except N22 and Anjali (showed early flowering) however grain yield and other yield related traits decreased significantly compared to well watered condition. Correlation analysis of phenological and yield related traits with grain yield revealed that tiller numbers and panicle numbers are highly correlated with grain yield both under well-watered and water stress conditions and contributes maximum towards grain yield. The dendrogram grouped Mahamaya, Sahabhagidhan, Poornima, IBD 1, Hazaridhan, Samleshwari and Danteshwari into one cluster which performed better under water stress conditions and had grain yield more than 1.69 tha−1. Sahabhagidhan, Poornima, Vandana, and N22 displayed tolerance to drought both under vegetative and reproductive conditions which could be a good selection for the breeders to develop drought tolerant rice cultivars for eastern region of India.
drICE restrains DIAP2-mediated inflammatory signalling and intestinal inflammation
Authors: Kietz Christa , Pollari Vilma , Tuominen Ida-Emma , Ribeiro Paulo S , Meier Pascal , Meinander Annika
Published: May 2020

TheDrosophilaIAP protein, DIAP2, is a key mediator of NF-κB signalling and is required for immune activation, both locally in the intestinal epithelia and during systemic, fat body-induced responses. We have found that transgenic expression of DIAP2 induces inflammation in the intestine, but not in the fat body, indicating a need for regulating DIAP2 in microbiotic environments. We describe theDrosophilacaspase drICE, a known interaction partner of DIAP2, as a regulator of DIAP2 and NF-κB signalling in the intestinal epithelium. drICE acts by cleaving, and interfering with DIAP2’s ability to ubiquitinate and, thereby, activate mediators of the NF-κB pathway. The drICE-cleaved form of DIAP2 is, moreover, unable to induce inflammation during basal conditions in the intestine. Interestingly, cleavage of DIAP2 does not interfere with pathogen-induced signalling, suggesting that drICE protects from immune responses induced by resident microbes. Accordingly, the negative regulatory effect of drICE is lost when rearing flies axenic. Hence, we show that drICE halts unwanted inflammatory signalling in the intestine by forming an inhibitory complex with DIAP2, interfering with the ability of DIAP2 to induce downstream signalling and NF-κB target gene activation.
Risk factors associated with mortality in hospitalized patients with SARS-CoV-2 infection. A prospective, longitudinal, unicenter study in Reus, Spain
Authors: Iftimie Simona , López-Azcona Ana F. , Vicente-Miralles Manuel , Descarrega-Reina Ramon , Hernández-Aguilera Anna , Riu Francesc , Simó Josep M. , Garrido Pedro , Joven Jorge , Camps Jordi , Castro Antoni
Published: May 2020

Spain is one of the countries that has suffered the most from the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the strain that causes coronavirus disease 2019 (COVID-19). However, there is a lack of information on the characteristics of this disease in the Spanish population. The objective of this study has been to characterize our patients from an epidemiological point of view and to identify the risk factors associated with mortality in our geographical area. We performed a prospective, longitudinal study on 188 hospitalized cases of SARS-Cov-2 infection inHospital Universitari de Sant Joan, in Reus, Spain, admitted between 15thMarch 2020 and 30thApril 2020. We recorded demographic data, signs and symptoms and comorbidities. We also calculated the Charlson and McCabe indices. A total of 43 deaths occurred during the study period. Deceased patients were older than the survivors (77.7 ± 13.1vs. 62.8 ± 18.4 years;p< 0.001). Logistic regression analyses showed that fever, pneumonia, acute respiratory distress syndrome, diabetes mellitus and cancer were the variables that showed independent and statistically significant associations with mortality. The Charlson index was more efficient than the McCabe index in discriminating between deceased and survivors. This is one of the first studies to describe the factors associated with mortality in patients infected with SARS-CoV-2 in Spain, and one of the few in the Mediterranean area. We identified the main factors independently associated with mortality in our population. Further studies in are needed to complete and confirm our findings.
In vivo recellularization of xenogeneic vascular grafts decellularized with high hydrostatic pressure method in a porcine carotid arterial interpose model
Authors: Kurokawa Shunji , Hashimoto Yoshihide , Funamoto Seiichi , Yamashita Akitatsu , Yamazaki Kazuhiro , Ikeda Tadashi , Minatoya Kenji , Kishida Akio , Masumoto Hidetoshi
Published: May 2020

Autologous vascular grafts are widely used in revascularization surgeries for small caliber targets. However, the availability of autologous conduits might be limited due to prior surgeries or the quality of vessels. Xenogeneic decellularized vascular grafts from animals potentially substitute for autologous vascular grafts. Decellularization with high hydrostatic pressure (HHP) is reported to highly preserve extracellular matrix (ECM) which would be feasible for recellularization and vascular remodeling after implantation. In the present study, we conducted xenogeneic implantation of HHP-decellularized bovine vascular grafts from dorsalis pedis arteries to porcine carotid arteries then evaluated graft patency, ECM preservation and recellularization. Surgical procedure not to damage luminal surface of the grafts from drying significantly increased the graft patency at 4 weeks after implantation (P = 0.0079). After the technical improvement, all grafts (N = 5) were patent with mild stenosis due to intimal hyperplasia at 4 weeks after implantation. Neither aneurysmal change nor massive thrombosis was observed even without administration of anticoagulants nor anti-platelet agents. Elastica van Gieson and Sirius-red stainings revealed fair preservation of ECM proteins including elastin and collagen after implantation. Luminal surface of grafts was thoroughly covered with von Willebrand factor-positive endothelium. Scanning electron microscopy on luminal surface of implanted grafts exhibited cobblestone-like endothelial cell layer which is similar to native vascular endothelium. Recellularization of tunica media with alpha-smooth muscle actin-positive smooth muscle cells was partly observed. Thus, we confirmed that HHP-decellularized grafts are feasible for xenogeneic implantation accompanied by recellularization by recipient cells.