bluuebunny/crossref_metadata_2025 huggingface dataset:
Predominance of the SARS-CoV-2 lineage P.1 and its sublineage P.1.2 in patients from the metropolitan region of Porto Alegre, Southern Brazil in March 2021: a phylogenomic analysis
Authors: Franceschi Vinícius Bonetti , Caldana Gabriel Dickin , Perin Christiano , Horn Alexandre , Peter Camila , Cybis Gabriela Bettella , Ferrareze Patrícia Aline Gröhs , Rotta Liane Nanci , Cadegiani Flávio Adsuara , Zimerman Ricardo Ariel , Thompson Claudia Elizabeth
Published: May 2021

Almost a year after the COVID-19 pandemic had begun, The United Kingdom, South Africa, and Brazil became the epicenter of new lineages, the Variant of Concern (VOCs), B.1.1.7, B.1.351, and P.1, respectively. These VOCs are increasingly associated with enhanced transmissibility, immunity evasion, and mortality. The previous most prevalent lineages in the state of Rio Grande do South (Brazil), B.1.1.28 and B.1.1.33 were rapidly replaced by P.1 and P.2, two B.1.1.28-derived lineages harboring the E484K mutation. To perform a genomic characterization of SARS-CoV-2 samples from COVID-19 patients from the metropolitan region of Porto Alegre (Rio Grande do Sul, Southern Brazil), in this second pandemic wave, we sequenced viral samples from patients of this region to: (i) identify the prevalence of SARS-CoV-2 lineages in the region, the state and bordering countries/states, (ii) characterize the mutation spectra, and (iii) hypothesize possible viral dispersal routes by using phylogenetic and phylogeographic approaches. As results, we not only confirmed that 96.4% of the samples belonged to the P.1 lineage but also that approximately 20% of which could be assigned as the newer P.1.2 (a P.1 derived new sublineage harboring new signature substitutions recently described and present in other Brazilian states and foreign countries). Moreover, P.1 sequences from this study were allocated in several distinct branches (four clades and five clusters) of the P.1 phylogeny, suggesting multiple introductions of P.1 in Rio Grande do Sul still in 2020 and placing this state as a potential core of diffusion and emergence of P.1-derived clades. It is still uncertain if the emergence of P.1.2 and other P.1 clades are related to further virological, clinical, or epidemiological consequences. However, the clear signs of viral molecular diversification from recently introduced P.1 warrant further genomic surveillance.
Evaluation of Tumor Response to Adjuvant Treatments using an Ex Vivo Culture of Breast Carcinoma Spheroids in a Microfluidic Device
Authors: Aboulkheyr Es Hamidreza , Aref Amir Reza , Granpayeh Lobat , Ebrahimi Marzieh , Baharvand Hossein
Published: May 2021

Abstract
Purpose Breast cancer is the leading cause of cancer-related death among women worldwide. Conventional chemotherapy is considered a clinical state of the art treatment; however, resistance or recurrence occurs among a considerable portion of these patients. Besides understanding the genomic alterations pattern of tumor cells and their association with drug resistance or response, the development of a reliable tumor models that reflect the major cellular and molecular features of tumors may aid with screening of candidate drugs and identification of appropriate treatment regimens. Here, we developed a simple and low-cost tumor model of breast cancer to screen library of chemotherapy agents in a pre-clinical setting. Methods we generated and cultured ex-vivo 3D culture of patient-derived tumor spheroids from both pre-treated primary and metastatic tumors using a partial digestion approach in a microfluidic device. We assessed chemotherapy response of the seven patient-derived breast tumor spheroids and expanded evaluation of drug sensitivity through molecular analysis of a small panel of genes. Results We observed various chemotherapy responses across primary and metastasis tumor samples. Interestingly, we demonstrated response to paclitaxel and doxorubicin and resistance to cisplatin in 2/3 metastatic tumor samples while most of the primary tumor were responsive to chemotherapy. Additionally, the expression of PIK3CA and loss of PTEN were associated to treatment resistance. Conclusion Our study suggests potential application of microfluidic-based cell culture technology coupled with patient derived tumor spheroids in prediction of treatment response in a personalized manner.
Neutrophil extracellular traps arm DC vaccination against NPM-mutant myeloproliferation
Authors: Tripodo Claudio , Bassani Barbara , Jachetti Elena , Cancila Valeria , Chiodoni Claudia , Portararo Paola , Botti Laura , Valenti Cesare , Perrone Milena , Ponzoni Maurilio , Comoli Patrizia , Curti Antonio , Colombo Mario Paolo , Sangaletti Sabina
Published: May 2021

Neutrophil extracellular traps (NET) are web-like chromatin structures composed by dsDNA and histones, decorated with anti-microbial proteins. Their interaction with dendritic cells (DC) allows DC activation and maturation toward presentation of NET-associated antigens. Differently from other types of cell death that imply protein denaturation, NETosis preserves the proteins localized onto the DNA threads for proper enzymatic activity and conformational status, including immunogenic epitopes. Besides neutrophils, leukemic cells can release extracellular traps displaying leukemia-associated antigens, prototypically mutant nucleophosmin (NPMc+) that upon mutation translocates from nucleolus to the cytoplasm localizing onto NET threads. We tested NPMc+ immunogenicity through a NET/DC vaccine to treat NPMc-driven myeloproliferation in transgenic and transplantable models. Vaccination with DC loaded with NPMc+ NET (NPMc+ NET/DC) reduced myeloproliferation in transgenic mice, favoring the development of antibodies to mutant NPMc and and the induction of a CD8+ T cell response. The efficacy of this vaccine was also tested in mixed NPMc/WT bone marrow chimeras in a competitive bone marrow transplantation setting, where the NPMc+ NET/DC vaccination impaired the expansion of NPMc+ in favor of WT myeloid compartment. NPMc+ NET/DC vaccination also achieved control of an aggressive leukemia transduced with mutant NPMc, effectively inducing an anti-leukemia CD8 T cell memory response.

eTOC Summary

NPMc is an AML-triggering mutation inducing translocation of NPM into the cytoplasm, allowing its association to NET threads. On NET, NPMc becomes immunogenic acting as an alarmin and exposing immunogenic peptides. We demonstrate that NET can be exploited as anti-tumor weapons in DC-based vaccination strategies.
Application of transposon-insertion sequencing to determine gene essentiality in the acetogen Clostridium autoethanogenum
Authors: Woods Craig , Humphreys Christopher M. , Tomi-Andrino Claudio , Henstra Anne M. , Köpke Michael , Simpson Sean D. , Winzer Klaus , Minton Nigel P.
Published: May 2021

The majority of the genes present in bacterial genomes remain poorly characterised with up to one third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to easily transformable species leaving those with low DNA-transfer rates out of reach. Here we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as a sole carbon and energy source.

IMPORTANCE

Although microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterised, leaving the function of many as either hypothetical or entirely unknown. High-throughput, transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA-transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here we developed approaches to facilitate transposon-insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases, CO and CO2, into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth providing insights into the utilisation of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon-insertion sequencing to other microbes where DNA-transfer has until now represented a barrier to progress.
Implementation of a qPCR assay coupled with genomic surveillance for real-time monitoring of SARS-CoV-2 variants of concern
Authors: Rego Natalia , Costábile Alicia , Paz Mercedes , Salazar Cecilia , Perbolianachis Paula , Spangenberg Lucía , Ferrés Ignacio , Arce Rodrigo , Fajardo Alvaro , Arleo Mailen , Possi Tania , Bellini Inés , Bilbao Lucia , Reyes Natalia , Bentancor Ma Noel , Lizasoain Andrés , Benítez María José , Castells Matías , Victoria Matías , Maya Leticia , Bortagaray Viviana , Moller Ana , Bello Gonzalo , Arantes Ighor , Brandes Mariana , Smircich Pablo , Chappos Odhille , Duquía Melissa , González Belén , Griffero Luciana , Méndez Mauricio , Techera Ma Pía , Zanetti Juan , Rivera Bernardina , Maidana Matías , Alonso Martina , Alonso Cecilia , Medina Julio , Albornoz Henry , Colina Rodney , Noya Veronica , Iraola Gregorio , Fernández-Calero Tamara , Moratorio Gonzalo , Moreno Pilar
Published: May 2021

We developed a genomic surveillance program for real-time monitoring of SARS-CoV-2 variants of concern in Uruguay. Here, we present the first results, including the proposed qPCR-VOC method, the general workflow and the report of the introduction and community transmission of the VOC P.1 in Uruguay in multiple independent events.
Clinical and experimental factors that affect the reported performance characteristics of rapid testing for SARS-CoV-2
Authors: Parvu Valentin , Gary Devin S. , Mann Joseph , Lin Yu-Chih , Mills Dorsey , Cooper Lauren , Andrews Jeffrey C. , Manabe Yukari C. , Pekosz Andrew , Cooper Charles K.
Published: May 2021

Tests that detect the presence of SARS-CoV-2 antigen in clinical specimens from the upper respiratory tract can provide a rapid means of COVID-19 disease diagnosis and help identify individuals that may be infectious and should isolate to prevent SARS-CoV-2 transmission. This systematic review assesses the diagnostic accuracy of SARS-CoV-2 antigen detection in COVID-19 symptomatic and asymptomatic individuals compared to RT-qPCR, and summarizes antigen test sensitivity using meta-regression. In total, 83 studies were included that compared SARS-CoV-2 rapid antigen lateral flow testing (RALFT) to RT-qPCR for SARS-CoV-2. Generally, the quality of the evaluated studies was inconsistent, nevertheless, the overall sensitivity for RALFT was determined to be 75.0% (95% confidence interval [CI]: 71.0-78.0). Additionally, RALFT sensitivity was found to be higher for symptomatic versus asymptomatic individuals and was higher for a symptomatic population within 7 days from symptom onset (DSO) compared to a population with extended days of symptoms. Viral load was found to be the most important factor for determining SARS-CoV-2 antigen test sensitivity. Other design factors, such as specimen storage and anatomical collection type, also affect the performance of RAFLT. RALFT and RT-qPCR testing both achieve high sensitivity when compared to SARS-CoV-2 viral culture.
Exploring Patients’ Needs & Desires for Quality Prenatal Care in Florida
Authors: Fryer Kimberly , Reid Chinyere N. , Cabral Naciely , Marshall Jennifer , Menon Usha
Published: May 2021

Introduction

Early and adequate prenatal care is important for patients to obtain health education, risk reduction, psychosocial support, and necessary medical interventions during the prenatal period. High-quality prenatal care encourages adequate care throughout pregnancy by increasing patients’ desires to return for subsequent visits. This study aims to investigate the prenatal care experiences, desires, and needs of women in Florida.

Methods

A mixed-methods study was conducted from April to December 2019 with postpartum women (n=55) who received no or late prenatal care and delivered in [City], Florida. Eligible women completed a survey and a semi-structured interview. The interview contextualized conditions shaping perceptions of the quality of prenatal care. Univariate analysis was conducted on the demographic characteristics and prenatal care locations. Qualitative analysis was performed using MAXQDA software and modified grounded theory. The analysis was based on Donabedian’s quality of care model.

Results

The participants self-identified as Hispanic (52%), White (48%), and Black (21%). Sixty-nine percent had Medicaid while 22% were self-pay. In the qualitative analysis, three core themes emerged. Clinical care processes included provision of health education and medical assessments. Structural conditions included language barriers, clinic availability, and ancillary staff presence. Lastly, interpersonal communication included impersonal care and multiple providers. The overarching conclusion from this analysis was the desire for patient-centered care. Participants wanted the care they received to be informative, tailored to their needs, and to work within the realities of their everyday lives.

Discussion

Investigating women’s experiences with seeking and receiving prenatal care are necessary to inform future interventions. Fostering a positive and patient-centered healthcare environment is necessary to improve the quality of care.

Quick points

What is already known on this subject?

Racial and ethnic disparities exist in prenatal care access, utilization and both maternal and infant mortality and morbidity.

Timely and adequate prenatal care is an important determinant for maternal and infant birth outcomes.

What this study adds

This study identified process, structure, and interpersonal factors associated with prenatal care access and utilization from the perspectives of a diverse group of postpartum women.

Specific recommendations for patient-centered prenatal care for diverse women in the context of patient expectations and perceptions of quality are provided.
Release of infectious virus and cytokines in nasopharyngeal swabs from individuals infected with non-B.1.1.7 or B.1.1.7 SARS-CoV-2 variants
Authors: Monel Blandine , Planas Delphine , Grzelak Ludivine , Smith Nikaïa , Robillard Nicolas , Staropoli Isabelle , Goncalves Pedro , Porrot Françoise , Guivel-Benhassine Florence , Guinet Nathalie Demory , Rodary Julien , Puech Julien , Euzen Victor , Bélec Laurent , Orvoen Galdric , Nunes Léa , Moulin Véronique , Fourgeaud Jacques , Wack Maxime , Imbeaud Sandrine , Campagne Pascal , Duffy Darragh , Di Santo James P. , Bruel Timothée , Péré Hélène , Veyer David , Schwartz Olivier
Published: May 2021

The mechanisms that allowed for the SARS-CoV-2 B.1.1.7 variant to rapidly outcompete pre-existing variants in many countries remain poorly characterized. Here, we analyzed viral release, anti-SARS-CoV-2 antibodies and cytokine production in a retrospective series of 427 RT–qPCR+ nasopharyngeal swabs collected in COVID-19 patients harbouring either non-B.1.1.7 or B.1.17 variants. We utilized a novel rapid assay, based on S-Fuse-T reporter cells, to quantify infectious SARS-CoV-2. With both non-B.1.1.7 and B.1.1.7 variants, viral titers were highly variable, ranging from 0 to >106 infectious units, and correlated with viral RNA levels. Lateral flow antigenic rapid diagnostic tests (RDTs) were positive in 96% of the samples harbouring infectious virus. About 67 % of individuals carried detectable infectious virus within the first two days after onset of symptoms. This proportion decreased overtime, and viable virus was detected up to 14 days. Samples containing anti-SARS-CoV-2 IgG or IgA did not generally harbour infectious virus. The proportion of individuals displaying viable virus or being RDT-positive was not higher with B.1.1.7 than with non-B.1.1.7 variants. Ct values were slightly but not significantly lower with B.1.1.7. The variant was characterized by a fast decrease of infectivity overtime and a marked release of 17 cytokines (including IFN-β, IP-10, IL-10 and TRAIL). Our results highlight differences between non-B.1.1.7 and B.1.1.7 variants. B.1.1.7 is associated with modified viral decays and cytokine profiles at the nasopharyngeal mucosae during symptomatic infection.
Public Health and Health Systems Impacts of SARS-CoV-2 Variants of Concern: A Rapid Scoping Review
Authors: Curran J , Dol J , Boulos L , Somerville M , McCulloch H
Published: May 2021

Background

As of April 2021, three SARS-CoV-2 variants of concern (VOC: B.1.1.7, B.1.351 and P.1) have been detected in over 132 countries. Increased transmissibility of VOC has implications for public health measures and health system arrangements. This rapid scoping review aims to provide a synthesis of current evidence related to public health measures and health system arrangements associated with VOC.

Methods

Rapid scoping review. Seven databases were searched up to April 7, 2021 for terms related to VOC, transmission, public health and health systems. A grey literature search was conducted up to April 14, 2021. Title, abstracts and full text were screened independently by two reviewers. Data were double extracted using a standardized form. Studies were included if they reported on at least one of the VOC and public health or health system outcomes.

Results

Of the 2487 articles and 59 grey literature sources retrieved, 37 studies and 21 guidance documents were included. Included studies used a wide range of designs and methods. Most of the studies and guidance documents reported on B.1.1.7, and 18 studies and 4 reports provided data for consideration in relation to public health measures. Public health measures, including lockdowns, physical distancing, testing and contact tracing, were identified as critical adjuncts to a comprehensive vaccination campaign. No studies reported on handwashing or masking procedures related to VOC. For health system arrangements, 17 studies were identified. Some studies found an increase in hospitalization due to B.1.1.7 but no difference in length of stay or ICU admission. Six studies found an increased risk of death ranging from 15-67% with B.1.1.7 compared non-B.1.1.7, but three studies reported no change. One study reported on the effectiveness of personal protective equipment in reducing VOC transmission in the hospital. No studies reported on screening staff and visitors, adjusting service provisions, or adjusting patient accommodations and shared spaces, which is a significant gap in the literature. Guidance documents did not tend to cite any evidence and were thus assumed to be based on expert opinion.

Conclusion

While the findings should be interpreted with caution as most of the sources identified were preprints, findings suggest a combination of non-pharmaceutical interventions (e.g., masking, physical distancing, lockdowns, testing) should be employed alongside a vaccine strategy to improve population and health system outcomes. While the findings are mixed on the impact of VOC on health system arrangements, the evidence is trending towards increased hospitalization and death.
Cryptic diversity within thePoecilochirus carabimite species complex phoretic onNicrophorus buryingbeetles: phylogeny, biogeography, and host specificity
Authors: Canitz Julia , Sikes Derek S. , Knee Wayne , Baumann Julia , Haftaro Petra , Steinmetz Nadine , Nave Martin , Eggert Anne-Katrin , Hwang Wenbe , Nehring Volker
Published: May 2021

Coevolution is often considered a major driver of speciation, but evidence for this claim is not always found because diversity might be cryptic. When morphological divergence is low, molecular data are needed to uncover diversity. A taxon for which this holds true are the mites, which are known for their extensive and often cryptic diversity. We studied mites of the genusPoecilochirusthat are phoretic on burying beetles (Silphidae:Nicrophorus).Poecilochirustaxonomy is poorly understood. Most studies on this genus focus on the evolutionary ecology ofPoecilochirus carabi sensu lato, a complex of at least two biological species. Based on molecular data of 230 specimens from 43 locations worldwide, we identified 24 genetic clusters that may represent species. We estimate that these mites began to diversify during the Paleogene, when the clade containingP. subterraneusbranched off and the remaining mites diverged into two further clades. One clade resemblesP. monospinosusandP. austroasiaticus. The other clade contains 17 genetic clusters resemblingP. carabi s.l.. Among these areP. carabi sensu stricto, P. necrophori, and potentially many additional cryptic species. Our analyses suggest that these clades were formed in the miocene by large-scale geographic separation. Diversification also seems to have happened on a smaller scale, potentially due to adaptation to specific hosts or local abiotic conditions, causing some clusters to specialize on certain beetle species. Our results suggest that biodiversity in this genus was generated by multiple interacting forces shaping the tangled webs of life.
SOD2 in Skeletal Muscle: New Insights from an Inducible Deletion Model
Authors: Zhuang Aowen , Yang Christine , Liu Yingying , Tan Yanie , Bond Simon T. , Walker Shannen , Sikora Tim , Sharma Arpeeta , de Haan Judy B. , Meikle Peter J. , Coughlan Melinda T. , Calkin Anna C. , Drew Brian G.
Published: May 2021

Metabolic conditions such as obesity, insulin resistance and glucose intolerance are frequently associated with impairments in skeletal muscle function and metabolism. This is often linked to dysregulation of homeostatic pathways including an increase in reactive oxygen species (ROS) and oxidative stress. One of the main sites of ROS production is the mitochondria, where the flux of substrates through the electron transport chain (ETC) can result in the generation of oxygen free radicals. Fortunately, several mechanisms exist to buffer bursts of intracellular ROS and peroxide production, including the enzymes Catalase, Glutathione Peroxidase and Superoxide Dismutase (SOD). Of the latter there are two intracellular isoforms; SOD1 which is mostly cytoplasmic, and SOD2 which is found exclusively in the mitochondria. Developmental and chronic loss of these enzymes has been linked to disease in several studies, however the temporal effects of these disturbances remain largely unexplored. Here, we induced a post-developmental (8-week old mice) deletion of SOD2 in skeletal muscle (SOD2-iMKO) and demonstrate that 16 weeks of SOD2 deletion leads to no major impairment in whole body metabolism, despite these mice displaying alterations in aspects of mitochondrial abundance and voluntary ambulatory movement. Furthermore, we demonstrated that SOD2 deletion impacts on specific aspects of muscle lipid metabolism, including the abundance of phospholipids and phosphatidic acid (PA), the latter being a key intermediate in several cellular signaling pathways. Thus, our findings suggest that post-developmental deletion of SOD2 induces a more subtle phenotype than previous embryonic models have shown, allowing us to highlight a previously unrecognized link between SOD2, mitochondrial function and bioactive lipid species including PA.
An optimized protocol for dual RNA-Seq of human macrophages infected with Mycobacterium avium
Authors: Schildkraut Jodie A , Koeken Valerie ACM , Coolen Jordy PM , van Crevel Reinout , van Ingen Jakko
Published: May 2021

Recently, dual RNA-sequencing (RNA-Seq) has been identified as a powerful tool to study host-pathogen interactions, which is particularly interesting for intracellular pathogens such as mycobacteria. However, due to the complexity of obtaining human host cells, many models rely on the usage of host cells derived from animals or cell lines, which does not accurately mimic the situation in the patient. Furthermore, due to the severe disbalance in host and pathogen RNA content, it is difficult to obtain sufficient sequencing depth for the infecting pathogen. Here, we present an optimized method to perform dual RNA-sequencing on human monocyte-derived macrophages (hMDMs) infected with Mycobacterium avium (M. avium). It is likely that, with slight modifications in multiplicity of infection (MOI) to account for differences in virulence, this methodology will be applicable for other difficult-to-lyse intracellular mycobacteria.
Universal allometry from empirical parameters
Authors: McKerral Jody C. , Kleshnina Maria , Bartle Louise , Mitchell James G. , Filar Jerzy A.
Published: May 2021

Allometric settings of population dynamics models are appealing due to their parsimonious nature and broad utility when studying system level effects. Here, we parameterise the size-scaled Rosenzweig-Macarthur ODEs to eliminate prey-mass dependency. We define the functional response term to match experiments, and examine situations where metabolic theory derivations and observation diverge. We produce dynamics consistent with observation. Our parameterisation of the Rosenzweig-Macarthur system is an accurate minimal model across 15+ orders of mass magnitude.
Breeding with Major and Minor Genes: Genomic Selection for Quantitative Disease Resistance
Authors: Merrick Lance F. , Burke Adrienne B. , Chen Xianming , Carter Arron H.
Published: May 2021

Most disease resistance in plants is quantitative, with both major and minor genes controlling resistance. This research aimed to optimize genomic selection (GS) models for use in breeding programs needing to select both major and minor genes for resistance. In this experiment, stripe rust (Puccinia striiformis Westend. f. sp. tritici Erikss.) of wheat (Triticum aestivum L.) was used as a model for quantitative disease resistance. The quantitative nature of stripe rust is usually phenotyped with two disease traits, infection type and disease severity. We compared two types of training populations composed of 2,630 breeding lines phenotyped in single plot trials from four years (2016-2020) and 475 diversity panel lines from four years (2013-2016), both across two locations. We also compared the accuracy of models with four different major gene markers and genome-wide association (GWAS) markers as fixed effects. The prediction models used 31,975 markers replicated 50 times using 5-fold cross-validation. We then compared the GS models with marker-assisted selection to compare the prediction accuracy of the markers alone and in combination. The GS models had higher accuracies than marker-assisted selection and reached an accuracy of 0.72 for disease severity. The major gene and GWAS markers had only a small to zero increase in prediction accuracy over the base GS model, with the highest accuracy increase of 0.03 for major markers and 0.06 for GWAS markers. There was a statistical increase in accuracy by using the disease severity trait, the breeding lines, population type, and by combing years. There was also a statistical increase in accuracy using major markers within the validation sets as the mean accuracy decreased. The inclusion of fixed effects in low prediction scenarios increased accuracy up to 0.06 for GS models using significant GWAS markers. Our results indicate that GS can accurately predict quantitative disease resistance in the presence of major and minor genes.
Structure and dynamics of RNA guanine quadruplexes in SARS-CoV-2 genome. Original strategies against emerging viruses
Authors: Miclot Tom , Hognon Cécilia , Bignon Emmanuelle , Terenzi Alessio , Marazzi Marco , Barone Giampaolo , Monari Antonio
Published: May 2021

Guanine quadruplexes (G4) structures in viral genome have a key role in modulating viruses’ biological activity. While several DNA G4 structures have been experimentally resolved, RNA G4s are definitely less explored. We report the first calculated G4 structure of the RG-1 RNA sequence of SARS-CoV-2 genome, obtained by using a multiscale approach combining quantum and classical molecular modelling and corroborated by the excellent agreement between the corresponding calculated and experimental circular dichroism spectra. We prove the stability of RG-1 G4 arrangement as well as its interaction with G4 ligands potentially inhibiting viral protein translation.
Syntaxin-1 is necessary for UNC5/Netrin-1-dependent macropinocytosis and chemorepulsion
Authors: Martínez-Mármol Ramón , Muhaisen Ashraf , Cotrufo Tiziana , Roselló-Busquets Cristina , Hernaiz-Llorens Marc , Pérez-Branguli Francesc , Andrés Rosa Maria , Ros Oriol , Pascual Marta , Ulloa Fausto , Soriano Eduardo
Published: May 2021

Brain connectivity requires correct axonal guidance to drive axons to their appropriate targets. This process is orchestrated by guidance cues that exert attraction or repulsion to developing axons. However, the intricacies of the cellular machinery responsible for the correct response of growth cones are just being unveiled. Netrin-1 is a bifunctional molecule involved in axon pathfinding and cell migration that induces repulsion during postnatal cerebellar development. This process is mediated by Uncoordinated locomotion 5 (UNC5) receptors located on external granule layer (EGL) tracts. Here, we demonstrate that this response is characterized by enhanced membrane internalization through macropinocytosis, but not clathrin-mediated endocytosis. We show that UNC5 receptors form a protein complex with the t-SNARE syntaxin-1 (Stx1). By combining botulinum neurotoxins, a shRNA knock-down strategy and Stx1 knock-out mice, we demonstrate that this SNARE protein is required for Netrin-1-induced macropinocytosis and chemorepulsion, suggesting that Stx1 is crucial in regulating Netrin-1-mediated axonal guidance.
Tumor-secreted versican co-opts myeloid IKKβ during metastasis
Authors: Spella Magda , Lamort Anne-Sophie , Vreka Malamati , Marazioti Antonia , Lilis Ioannis , Ntaliarda Giannoula , Skiadas Georgios , Bouloukou Eleni , Giotopoulou Georgia A. , Pepe Mario A.A. , Weiss Stefanie A.I. , Petrera Agnese , Hauck Stefanie M. , Koch Ina , Lindner Michael , Hatz Rudolph A. , Behr Juergen , Arendt Kristina A.M. , Giopanou Ioanna , Brunn David , Savai Rajkumar , Jenne Dieter E. , Yull Fiona E. , Blackwell Timothy S. , Stathopoulos Georgios T.
Published: May 2021

The mechanisms tumor cells use to hijack the immune system are largely uncharted. Here we used bioluminescent nuclear factor (NF)-κB reporter mice and macrophages to discover that metastatic tumors trigger NF-κB activation in host macrophages, dependent on mutant KRAS signaling and delivered via secretory versican. Versican activates NF-κB in tumor-associated macrophages via inhibitor of NF-κB kinase (IKK) β, resulting in release of interleukin (IL)-1β into the tumor microenvironment. Versican silencing in cancer cells or conditional IKKβ deletion in macrophages prevents myeloid NF-κB activation and metastasis. Versican is overexpressed and/or mutated in human cancers and metastatic effusions with KRAS mutations, predicts poor survival, can aid in the development of diagnostic platforms for pleural metastasis, and is druggable via toll-like receptor (TLR) 1/2 inhibition. The data indicate a cardinal role for tumor-derived versican in establishing cross-talk with macrophage IKKβ during metastasis and may foster the development of new therapies and diagnostic tools.
Genomic comparison of eight closed genomes of multidrug resistant Salmonella enterica strains isolated from broiler farms and processing plants in Trinidad and Tobago
Authors: Maguire Meghan , Khan Anisa S. , Adesiyun Abiodun A. , Georges Karla , Gonzalez-Escalona Narjol
Published: May 2021

Salmonella enterica is an important foodborne pathogen worldwide. We used long and short read sequencing to close genomes of eight multidrug resistant (MDR) Salmonella enterica strains, belonging to serovars Infantis (2), Albany, Oranienburg, I 4,[5],12:i:-, Javiana, Schwarzengrund, and Kentucky from broiler chicken farms and processing plants in Trinidad and Tobago. They also belonged to seven different sequence types (STs- 32, 292, 1510, 19, 24, and 96). Seven of the strains demonstrated multi-drug resistance with the presence of at least three AMR genes. Three isolates contained the quinolone resistance gene qnrB19 in plasmids (CFSAN103840, CFSAN103854, and CFSAN103872). The extended spectrum β-lactamase genes blaCTX-M-65 (CFSAN103796) and blaTEM-1 (CFSAN103852) were detected in this study. The genomes closed in this study will be useful for future source tracking and outbreak investigations in Trinidad and Tobago and worldwide.
Superscan: Supervised Single-Cell Annotation
Authors: Shasha Carolyn , Tian Yuan , Mair Florian , Miller Helen E.R. , Gottardo Raphael
Published: May 2021

Automated cell type annotation of single-cell RNA-seq data has the potential to significantly improve and streamline single cell data analysis, facilitating comparisons and meta-analyses. However, many of the current state-of-the-art techniques suffer from limitations, such as reliance on a single reference dataset or marker gene set, or excessive run times for large datasets. Acquiring high-quality labeled data to use as a reference can be challenging. With CITE-seq, surface protein expression of cells can be directly measured in addition to the RNA expression, facilitating cell type annotation. Here, we compiled and annotated a collection of 16 publicly available CITE-seq datasets. This data was then used as training data to develop Superscan, a supervised machine learning-based prediction model. Using our 16 reference datasets, we benchmarked Superscan and showed that it performs better in terms of both accuracy and speed when compared to other state-of-the-art cell annotation methods. Superscan is pre-trained on a collection of primarily PBMC immune datasets; however, additional data and cell types can be easily added to the training data for further improvement. Finally, we used Superscan to reanalyze a previously published dataset, demonstrating its applicability even when the dataset includes cell types that are missing from the training set.
Decoding the effects of synonymous variants
Authors: Zeng Zishuo , Aptekmann Ariel A. , Bromberg Yana
Published: May 2021

Synonymous single nucleotide variants (sSNVs) are common in the human genome but are often overlooked. However, sSNVs can have significant biological impact and may lead to disease. Existing computational methods for evaluating the effect of sSNVs suffer from the lack of gold-standard training/evaluation data and exhibit over-reliance on sequence conservation signals. We developed synVep (synonymous Variant effect predictor), a machine learning-based method that overcomes both of these limitations. Our training data was a combination of variants reported by gnomAD (observed) and those unreported, but possible in the human genome (generated). We used positive-unlabeled learning to purify the generated variant set of any likely unobservable variants. We then trained two sequential extreme gradient boosting models to identify subsets of the remaining variants putatively enriched and depleted in effect. Our method attained 90% precision/recall on a previously unseen set of variants. Furthermore, although synVep does not explicitly use conservation, its scores correlated with evolutionary distances between orthologs in cross-species variation analysis. synVep was also able to differentiate pathogenic vs. benign variants, as well as splice-site disrupting variants (SDV) vs. non-SDVs. Thus, synVep provides an important improvement in annotation of sSNVs, allowing users to focus on variants that most likely harbor effects.
Challenges of γ9δ2TCR affinity maturation when using phage display
Authors: Kramer Lovro , Demuysere Margaux , van Diest Eline , Beringer Dennis X. , Kuball Jürgen
Published: May 2021

Background

Over the past years we showed that the efficacy of αβT cells engineered to express a defined γδTCR (TEG) depends on the functional avidity of the γ9δ2TCR. We hypothesized that functional avidity mediated through γ9δ2TCR in the TEG format could be further enhanced by increasing affinity of the γ9δ2TCR.

Methods

We attempted to overcome limited affinity of natural occurring γ9δ2TCRs through affinity maturation by phage display using a library containing mutations in CDR1 and CDR2 of both TCR chains.

Conclusion

Affinity maturation of γ9δ2TCR by using phage display was not successful. The largest hurdle was the periplasmic expression of γ9δ2TCR constructs in E.coli which is a prerequisite for successful phage display. The underlying reason for this lack of expression was the instability of the single chain (sc)TCR format. Expression of scTCR formats in HEK293F cells yielded only 15-20% correctly folded scTCR.
SC3s - efficient scaling of single cell consensus clustering to millions of cells
Authors: Quah Fu Xiang , Hemberg Martin
Published: May 2021

Technological advances have paved the way for single cell RNAseq (scRNAseq) datasets containing several million cells 1. Such large datasets require highly efficient algorithms to enable analyses at reasonable times and hardware requirements 2. A crucial step in single cell workflows is unsupervised clustering, which aims to delineate putative cell types or cell states based on transcriptional similarity 3. Here, we present a highly efficient k-means based approach, and we demonstrate that it scales linearly with the number of cells with regards to time and memory.
The evolution of size-dependent competitive interactions promotes species coexistence
Authors: Anaya-Rojas Jaime M. , Bassar Ronald D. , Potter Tomos , Blanchette Allison , Callahan Shay , Framstead Nick , Reznick David , Travis Joseph
Published: May 2021

Theory indicates that competing species coexist in a community when intraspecific competition is stronger than interspecific competition. When body size determines the outcome of competitive interactions between individuals, coexistence depends also on how resource use and the ability to compete for these resources change with body size. Testing coexistence theory in size-structured communities, therefore, requires disentangling the effects of size-dependent competitive abilities and niche shifts.

Here, we tested the hypothesis that the evolution of species and size-dependent competitive asymmetries increased the likelihood of coexistence between interacting species.

We experimentally estimated the effects of size-dependent competitive interactions on somatic growth rates of two interacting fish species, Trinidadian guppies (Poecilia reticulata) and killifish (Rivulus hartii). We controlled for the effects of size-dependent changes in the niche at two competitive settings representing the early (allopatric) and late (sympatric) evolutionary stages of a killifish-guppy community. We fitted the growth data to a model that incorporates species and size-dependent competitive asymmetries to test whether changes in the competitive interactions across stages increased the likelihood of species coexistence from allopatry to sympatry.

We found that guppies are competitively superior to killifish but were less so in sympatric populations. The decrease in the effects of interspecific competition on the fitness of killifish and increase in the interspecific effect on guppies’ fitness increased the likelihood that sympatric guppies and killifish will coexist. However, while the competitive asymmetries between the species changed consistently between allopatry and sympatry between drainages, the size-dependent competitive asymmetries varied between drainages.

These results demonstrate the importance of integrating evolution and trait-based interactions into the research of how species coexistence.
Unique molecular features and cellular responses differentiate two populations of motor cortical layer 5b neurons in a preclinical model of ALS
Authors: Moya Maria V. , Kim Rachel D. , Rao Meghana N. , Cotto Bianca A. , Pickett Sarah B. , Sferrazza Caroline E. , Heintz Nathaniel , Schmidt Eric F.
Published: May 2021

Many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), lead to the selective degeneration of discrete cell types in the CNS despite the ubiquitous expression of many genes linked to disease. Therapeutic advancement depends on understanding unique cellular adaptations that underlie pathology of vulnerable cells in the context of disease-causing mutations. Here, we employ bacTRAP molecular profiling to elucidate cell type specific molecular responses of cortical upper motor neurons in a preclinical ALS model. Using two bacTRAP mouse lines that label distinct vulnerable or resilient projection neuron populations in motor cortex, we show that the regulation of oxidative phosphorylation (Oxphos) pathways is a common response in both cell types. However, differences in the baseline expression of genes involved in Oxphos and the handling of reactive oxygen species likely lead to the selective degeneration of the vulnerable cells. These results provide a framework to identify cell type-specific processes in neurodegenerative disease.
Deep Neural Networks for Genomic Prediction Do Not Estimate Marker Effects
Authors: Ubbens Jordan , Parkin Isobel , Eynck Christina , Stavness Ian , Sharpe Andrew
Published: May 2021

Genomic prediction is a promising technology for advancing both plant and animal breeding, with many different prediction models evaluated in the literature. It has been suggested that the ability of powerful nonlinear models such as deep neural networks to capture complex epistatic effects between markers offers advantages for genomic prediction. However, these methods tend not to outperform classical linear methods, leaving it an open question why this capacity to model nonlinear effects does not seem to result in better predictive capability. In this work, we propose the theory that, due to a principle called shortcut learning, deep neural networks tend to base their predictions on overall genetic relatedness, rather than on the effects of particular markers, such as epistatic effects. Using several datasets of crop plants (lentil, wheat, and Brassica carinata), we demonstrate the network’s indifference to the values of the markers by showing that the same network, provided with only the locations of matches between markers for two individuals, is able to perform prediction to the same level of accuracy.
MOFA: Multi-Objective Flux Analysis for the COBRA Toolbox
Authors: Griesemer Marc , Navid Ali
Published: May 2021

Multi-objective Optimization (MO) is an important tool for quantitative examination of the trade-offs faced by biological organisms. Using genome-scale constraint-based models of metabolism (GSMs), Multi-Objective Flux Analysis (MOFA) allows MO analyses of trade-offs among key biological tasks. The leading software package for conducting a plethora of different types of constraint-based analyses using GSMs is the COBRA Toolbox for MATLAB. We have developed a new add-on tool for this toolbox using Normalized Normal Constraint (NNC) that performs MOFA for a number of objectives only limited by computation power (n ≤ 10). This development will facilitate MOFA analyses by COBRA’s large user base and allow greater multi-faceted examination of metabolic trade-offs in complicated biological systems.

Availability and Implementation

The MOFA software is freely available for download from https://bbs.llnl.gov under the GPL v2 license. The program runs on MATLAB with the COBRA software on Windows, Linux, and MacOS. It includes a detailed manual explaining the input and output of a simulation, a listing of the code’s functions, and an example MOFA run using a well-curated GSM model of E. coli.

Contact

griesemer1@llnl.gov or navid1@llnl.gov
Validated machine learning algorithm with sub-clonal sensitivity reveals widespread pan-cancer human leukocyte antigen loss of heterozygosity
Authors: Pyke Rachel Marty , Mellacheruvu Dattatreya , Dea Steven , Abbott Charles W. , Zhang Simo V. , McDaniel Lee , Levy Eric , Bartha Gabor , West John , Snyder Michael P. , Chen Richard , Boyle Sean Michael
Published: May 2021

Human leukocyte antigen loss of heterozygosity (HLA LOH) allows cancer cells to escape immune recognition by deleting HLA alleles, causing the suppressed presentation of tumor neoantigens that would otherwise bind to them. Despite its importance in immunotherapy response, few methods exist to detect HLA LOH, and their accuracy is not well understood. Here, we develop DASH (Deletion of Allele-Specific HLAs), a novel machine learning-based algorithm to detect HLA LOH from paired tumor-normal sequencing data. Through validation with cell line mixtures and patient-specific digital PCR, we demonstrate increased sensitivity compared to previously published tools and pave the way for clinical utility. Using DASH on 611 patients across 15 tumor types, we found that 18% of patients had HLA LOH. Moreover, we show inflated HLA LOH rates compared to genome-wide LOH and correlations between CD274 (PD-L1) expression and MSI status, suggesting the HLA LOH is a key immune resistance strategy.
KIM-1-mediated anti-inflammatory activity is preserved by MUC1 induction in the proximal tubule during ischemia-reperfusion injury
Authors: Al-bataineh Mohammad M. , Kinlough Carol L. , Mi Zaichuan , Jackson Edwin K. , Mutchler Stephanie M. , Emlet David R. , Kellum John A. , Hughey Rebecca P.
Published: May 2021

Cell-associated kidney injury molecule-1 (KIM-1) exerts an anti-inflammatory role following kidney injury by mediating efferocytosis and downregulating the NF-κB pathway. KIM-1 cleavage blunts its anti-inflammatory activities. We reported that Mucin 1 (MUC1) is protective in a mouse model of ischemia-reperfusion injury (IRI). As both KIM-1 and MUC1 are induced in the proximal tubule (PT) during IRI and are ADAM17 substrates, we tested the hypothesis that MUC1 protects KIM-1 activity. Muc1 KO mice and wild-type (WT) littermates were subjected to IRI. KIM-1, MUC1 and ADAM17 levels (and signaling pathways) were assessed by immunoblotting. PT localization was assessed by confocal microscopy and in situ proximity ligation assay. Findings were extended using human kidneys and urine, and KIM-1-mediated efferocytosis assays in mouse PT cultures. In response to tubular injury in mouse and human kidneys, we observed induction and co-expression of KIM-1 and MUC1 in the PT. Compared to WT, Muc1 KO mice had higher urinary KIM-1 and lower kidney KIM-1. KIM-1 was apical in PT of WT kidneys, but predominately with luminal debris in Muc1 KO mice. Efferocytosis was reduced in Muc1 KO PT cultures when compared to WT cells, while inflammation was increased in Muc1 KO kidneys when compared to WT mice. MUC1 was cleaved by ADAM17 in PT cultures, and blocked KIM-1 shedding in MDCK cells. We conclude that KIM-1-mediated efferocytosis and thus anti-inflammatory activity during IRI is preserved in the injured kidney by MUC1 inhibition of KIM-1 shedding.

NEW & NOTEWORTHY

KIM-1 plays a key role in the recovery of the tubule epithelium during renal IRI by mediating efferocytosis and associated signaling that suppresses inflammation. Excessive cleavage of KIM-1 by ADAM17 provides decoy receptor that aggravates efferocytosis and subsequent signaling. Our data from studies in mice, patients and cultured cells show that MUC1 is also induced during IRI and competes with KIM-1 for cleavage by ADAM17. Consequently, MUC1 protects KIM-1 anti-inflammatory activity in the damaged kidney.