bluuebunny/crossref_metadata_2025 huggingface dataset:
Soil organic carbon fractionation and metagenomics pipeline to link carbon content and stability with microbial composition – First results investigating fungal endophytes
Authors: Buss Wolfram , Sharma Raghvendra , Ferguson Scott , Borevitz Justin
Published: December 2021

Society needs to capture gigatons of carbon dioxide from the atmosphere annually and then store it long-term to limit and ultimately reverse the effects of climate change. Bringing lost carbon back into agricultural soils should be a priority as it brings the added benefit of improving soil properties. Linking soil organic carbon (SOC) fractions of different stability with soil microbial composition can help understand and subsequently manage SOC storage. Here we develop a pipeline for evaluating the effects of microbial management on SOC content using rapid and low-cost SOC fractionation and metagenomics approaches. We tested the methods in a wheat pot trial inoculated with 17 individual endophytic fungal isolates. Two fungi increased total SOC in the area under the plant stem by ~15%. The fractionation assay showed that the medium stability soil aggregate carbon fraction (AggC) was increased by one of these fungi (+21%) and the chemically recalcitrant proportion (bleach oxidation) of AggC by the other (+35%). Both fungi increased mineral-associated organic carbon (MAOC), the long-term SOC storage, by ~10%. We used rapid, portable, low-cost, whole metagenome long read sequencing to detect a shift in the microbial composition for one of the fungi-inoculated treatments. This treatment showed a more diverse microbial community and a higher quantity of DNA in soil. The results emphasise the link between composition and abundance of soil microorganisms with soil carbon formation. Our dual carbon fractional and metagenomic analysis pipeline can be used to further test the effects of microbial management and ultimately to model the soil factors that influence SOC storage, such as nutrient and water availability, starting SOC content, soil texture and aggregation.
Landscape genetics across the Andes Mountains: Environmental variation drives genetic divergence in the leaf-cutting ant Atta cephalotes
Authors: Muñoz-Valencia Vanessa , Montoya-Lerma James , Seppä Perttu , Diaz Fernando
Published: December 2021

Disentangling the mechanisms underlying spatial distribution of genetic variation into those due to environment or physical barriers from mere geographic distance is challenging in complex landscapes. The Andean uplift represents one of the most heterogeneous habitats where these questions remain unexplored as multiple mechanisms might interact, confounding their relative roles. We explore this broad question in the leaf-cutting ant Atta cephalotes, a species distributed across the Andes Mountains using nuclear microsatellite markers and mtCOI gene sequences. We investigate spatial genetic divergence across the Western range of Northern Andes in Colombia by testing the relative role of alternative scenarios of population divergence, including geographic distance (IBD), climatic conditions (IBE), and physical barriers due to the Andes Mountains (IBB). Our results reveal substantial genetic differentiation among A. cephalotes populations for both types of markers, but only nuclear divergence followed a hierarchical pattern with multiple models of genetic divergence imposed by the Western range. Model selection showed that IBD, IBE (temperature and precipitation), and IBB (Andes mountains) models, often proposed as individual drivers of genetic divergence, interact, and explain up to 33% of genetic divergence in A. cephalotes. IBE models remained significant after accounting for IBD, suggesting that environmental factors play a more prominent role when compared to IBB. These factors in combination with idiosyncratic dispersal patterns of ants appear to determine hierarchical patterns of gene flow. This study enriches our understanding of the forces shaping population divergence in complex habitat landscapes.
Health impact assessment of salt reduction in bread in Portugal: a pilot study
Authors: Santos Joana , Alves Joana , Braz Paula , Brazão Roberto , Costa Alexandra , Santos Mariana , Assunção Ricardo , Caldas de Almeida Teresa , Costa Luciana
Published: December 2021

Hypertension is a risk factor for cardiovascular diseases, which can be caused by excessive salt intake. In Portugal, one of the main foods to contribute to ingestion of salt is bread. Thus, a voluntary Protocol was signed between stakeholders with the aim to reduce salt content in bread by 2021. Herein, a retrospective HIA was carried out to assess the impact in blood pressure (BP) after this agreement. In order to find average values of salt intake and BP in Portuguese population, national surveys were used. Also, estimates of BP reduction and its size effects were calculated based upon meta-analysis data. It is expected that salt intake will be reduced mostly in individuals with low educational level, men, aged between 65-74 years old and residents in South region of Portugal. Results in hypertensive patients indicate that a higher effect on BP will occur in the same profile of individuals, except age (between 55 and 64 years old). However, the estimated effect is very low for all groups, suggesting that the Protocol will contribute to modest health gains. Complementary measures supported by HIA studies need to be adopted to actively promote salt intake reduction and effectively prevent hypertension.
Validation of Genomic and Transcriptomic Models of Homologous Recombination Deficiency in a Real-World Pan-Cancer Cohort
Authors: Leibowitz Benjamin , Dougherty Bonnie V , Bell Joshua SK , Kapilivsky Joshuah , Michuda Jackson , Sedgwick Andrew , Munson Wesley , Chandra Tushar , Dry Jonathan R , Beaubier Nike , Igartua Catherine , Taxter Timothy
Published: December 2021

Background

With the introduction of DNA-damaging therapies into standard of care cancer treatment, there is a growing need for predictive diagnostics assessing homologous recombination deficiency (HRD) status across tumor types. Following the strong clinical evidence for the utility of DNA-sequencing-based HRD testing in ovarian cancer, and growing evidence in breast cancer, we present analytical validation of the Tempus|HRD-DNA test. We further developed, validated, and explored the Tempus|HRD-RNA model, which uses gene expression data from 16,470 RNA-seq samples to predict HRD status from formalin-fixed paraffin-embedded (FFPE) tumor samples across numerous cancer types.

Methods

Genomic and transcriptomic profiling was performed using next-generation sequencing from Tempus|xT, Tempus|xO, Tempus|xE, Tempus|RS, and Tempus|RS.v2 assays on 48,843 samples. Samples were labeled based on their BRCA1, BRCA2 and selected Homologous Recombination Repair (HRR) pathway gene (CDK12, PALB2, RAD51B, RAD51C, RAD51D) mutational status to train and validate HRD-DNA, a genome-wide loss-of-heterozygosity biomarker, and HRD-RNA, a logistic regression model trained on gene expression, using several performance metrics and statistical tests.

Results

In a sample of 2,058 breast and 1,216 ovarian tumors, BRCA status was predicted by HRD-DNA with F1-scores of 0.98 and 0.96, respectively. Across an independent set of 1,363 samples across solid tumor types, the HRD-RNA model was predictive of BRCA status in prostate, pancreatic, and non-small cell lung cancer, with F1-scores of 0.88, 0.69, and 0.62, respectively.

Conclusions

We predict HRD-positive patients across many cancer types and believe both HRD models may generalize to other mechanisms of HRD outside of BRCA loss. HRD-RNA complements DNA-based HRD detection methods, especially for indications with low prevalence of BRCA alterations.
Neuropsychiatric symptoms are associated with exacerbated cognitive impairment in covert cerebral small vessel disease
Authors: Anne Arola , Tuuli Levänen , Laakso Hanna M. , Johanna Pitkänen , Juha Koikkalainen , Jyrki Lötjönen , Antti Korvenoja , Timo Erkinjuntti , Susanna Melkas , Hanna Jokinen
Published: December 2021

Background and purpose

Neuropsychiatric symptoms are related to disease progression and cognitive decline over time in cerebral small vessel disease (SVD) but their significance is poorly understood in covert SVD. We investigated neuropsychiatric symptoms and their relationships between cognitive and functional abilities in subjects with varying degrees of white matter hyperintensities (WMH), but without clinical diagnosis of stroke, dementia or significant disability.

Methods

The Helsinki Small Vessel Disease Study consisted of 152 subjects, who underwent brain magnetic resonance imaging (MRI) and comprehensive neuropsychological evaluation of global cognition, processing speed, executive functions and memory. Neuropsychiatric symptoms were evaluated with the Neuropsychiatric Inventory Questionnaire (NPI-Q, n=134) and functional abilities with the Amsterdam Instrumental Activities of Daily Living questionnaire (A-IADL, n=132), both filled in by a close informant.

Results

NPI-Q total score correlated significantly with WMH volume (rs=0.20, p=0.019) and inversely with A-IADL score (rs=-0.41, p<0.001). In total, 38% of the subjects had one or more informant evaluated neuropsychiatric symptoms. Linear regressions adjusted for age, sex and education revealed no direct associations between neuropsychiatric symptoms and cognitive performance. However, there were significant synergistic interactions between neuropsychiatric symptoms and WMH volume on cognitive outcomes. Neuropsychiatric symptoms were also associated with A-IADL score irrespective of WMH volume.

Conclusions

Neuropsychiatric symptoms are associated with an accelerated relationship between WMH and cognitive impairment. Furthermore, the presence of neuropsychiatric symptoms is related to worse functional abilities. Neuropsychiatric symptoms should be routinely assessed in covert SVD as they are related to worse cognitive and functional outcomes.
HeartNet: Self Multi-Head Attention Mechanism via Convolutional Network with Adversarial Data Synthesis for ECG-based Arrhythmia Classification
Authors: Rafi Taki Hasan , Woong-Ko Young
Published: December 2021

Abstract

Cardiovascular disease is now one of the leading causes of morbidity and mortality in humans. Electrocardiogram (ECG) is a reliable tool for monitoring the health of the cardiovascular system. Currently, there has been a lot of focus on accurately categorizing heartbeats. There is a high demand on automatic ECG classification systems to assist medical professionals. In this paper we proposed a new deep learning method called HeartNet for developing an automatic ECG classifier. The proposed deep learning method is compressed by multi-head attention mechanism on top of CNN model. The main challenge of insufficient data label is solved by adversarial data synthesis adopting generative adversarial network (GAN) with generating additional training samples. It drastically improves the overall performance of the proposed method by 5-10% on each insufficient data label category. We evaluated our proposed method utilizing MIT-BIH dataset. Our proposed method has shown 99.67 ± 0.11 accuracy and 89.24 ± 1.71 MCC trained with adversarial data synthesized dataset. However, we have also utilized two individual datasets such as Atrial Fibrillation Detection Database and PTB Diagnostic Database to see the performance of our proposed model on ECG classification. The effectiveness and robustness of proposed method are validated by extensive experiments, comparison and analysis. Later on, we also highlighted some limitations of this work.
Field evaluation of Rapid SARS-Cov2 Antigen screening test on self-collected deep throat saliva samples in Malaysia
Authors: Noordin Noorliza Mohamad , Loon Lim Steven Chee , Lim Zhuo-zhi , Lim Teck-Onn
Published: December 2021

Low cost Rapid Antigen Tests are widely used in Malaysia and the government has also mandated worksite screening as a condition for reopening. Numerous RAT kits have been approved by the Malaysian Medical Device Authority. However, it remains uncertain how these kits would perform in the field.

We enrolled workers between June and September 2021 from 23 worksites. They were trained and experienced in performing RAT selftest by virtue of their worksite participation in routine screening program. These workers also had reverse transcriptase polymerase chain reaction tests in the course of mass screening or contact tracing. We also enrolled patients with PCR confirmed Covid19 from a quarantine centre. These patients were instructed on selftesting and then immediately perform RAT under supervision. Two manufacturers donated RAT for this study.

A total of 340 participants were enrolled, 130 were from quarantine centre and 210 from worksites. The overall sensitivity of RAT compared to PCR was 70 percent. The specificity was 91 percent. Sensitivity decreased with increasing PCR cycle threshold values. Sensitivity is also lower among untrained subjects at each level of Ct. Logistic regression analysis confirmed false negative result is associated with Ct and participants prior training and experience.

This study shows that in the real world, RAT performance were markedly lower than that reported by the manufacturers. The test sensitivity is dependent on the operator training and experience, as well as on viral load as measured by Ct. User training and repeated testing for screening purpose is necessary to mitigate the low sensitivity of RAT.
MSRCall: A Multi-scale Deep Neural Network to Basecall Oxford Nanopore Sequences
Authors: Yeh Yang-Ming , Lu Yi-Chang
Published: December 2021

Abstract

MinION, a third-generation sequencer from Oxford Nanopore Technologies, is a portable device that can provide long nucleotide read data in real-time. It primarily aims to deduce the makeup of nucleotide sequences from the ionic current signals generated when passing DNA/RNA fragments through nanopores charged with a voltage difference. To determine the nucleotides from the measured signals, a translation process known as basecalling is required. However, compared to NGS basecallers, the calling accuracy of MinION still needs to be improved.

In this work, a simple but powerful neural network architecture called MSRCall is proposed. MSRCall comprises a multi-scale structure, recurrent layers, a fusion block, and a CTC decoder. To better identify both short-range and long-range dependencies, the recurrent layer is redesigned to capture various time-scale features with a multi-scale structure. The results show that MSRCall outperforms other basecallers in terms of both read and consensus accuracies.
Urine and Fecal Microbiota in a Canine Model of Bladder Cancer
Authors: Mrofchak Ryan , Madden Christopher , Evans Morgan V. , Kisseberth William C. , Dhawan Deepika , Knapp Deborah W. , Hale Vanessa L.
Published: December 2021

Introduction

Urothelial carcinoma (UC) is the tenth most diagnosed cancer in humans worldwide. Dogs are a robust model for invasive UC as tumor development and progression is similar in humans and dogs. Recent studies on urine microbiota in humans revealed alterations in microbial diversity and composition in individuals with UC; however, the potential role of microbiota in UC has yet to be elucidated. Dogs could be valuable models for this research, but microbial alterations in dogs with UC have not been evaluated.

Objective

The objective of this this pilot study was to compare the urine and fecal microbiota of dogs with UC (n = 7) and age-, sex-, and breed-matched healthy controls (n = 7).

Methods

DNA was extracted from mid-stream free-catch urine and fecal samples using Qiagen Bacteremia and PowerFecal kits, respectively. 16S rRNA gene sequencing was performed followed by sequence processing and analyses (QIIME 2 and R).

Results

Canine urine and fecal samples were dominated by taxa similar to those found in humans. Significantly decreased microbial diversity (Kruskal-Wallis: Shannon,p= 0.048) and altered bacterial composition were observed in the urine but not feces of dogs with UC (PERMANOVA: Unweighted UniFrac,p= 0.011). The relative abundances ofFusobacteriumwas also increased, although not significantly, in the urine and feces of dogs with UC.

Conclusion

This study characterizes urine and fecal microbiota in dogs with UC, and it provides a foundation for future work exploring host-microbe dynamics in UC carcinogenesis, prognosis, and treatment.
Assessing the impact of fire on spiders through a global comparative analysiss
Authors: McLean Claire A. , Melville Jane , Schubert Joseph , Rose Rebecca , Medina Iliana
Published: December 2021

In many regions fire regimes are changing due to anthropogenic factors. Understanding the responses of species to fire can help to develop predictive models and inform fire management decisions. Spiders are a diverse and ubiquitous group and can offer important insights into the impacts of fire on invertebrates and whether these depend on environmental factors, phylogenetic history, or functional traits. We conducted phylogenetic comparative analyses of data from studies investigating the impacts of fire on spiders. We investigated whether fire affects spider abundance or presence and whether ecologically relevant traits or site-specific factors influence species’ responses to fire. Although difficult to make broad generalisations about the impacts of fire due to variation in site- and fire-specific factors, we find evidence that short fire intervals may be a threat to some spiders, and that fire affects abundance and species compositions in forests relative to other vegetation types. Orb and sheet web weavers were also more likely to be absent after fire than ambush hunters, ground hunters, and other hunters suggesting functional traits may affect responses. Finally, we show that analyses of published data can be used to detect broad scale patterns and provide an alternative to traditional meta-analytical approaches.
The African swine fever modelling challenge: objectives, model description and synthetic data generation
Authors: Picault Sébastien , Vergne Timothée , Mancini Matthieu , Bareille Servane , Ezanno Pauline
Published: December 2021

African swine fever (ASF) is an emerging disease currently spreading at the interface between wild boar and pig farms in Europe and Asia. Current disease control regulations, which involve massive culling with significant economic and animal welfare costs, need to be improved. Modelling enables relevant control measures to be explored, but conducting the exercise during an epidemic is extremely difficult. Modelling challenges enhance modellers’ ability to timely advice policy makers, improve their readiness when facing emerging threats, and promote international collaborations. The ASF-Challenge, which ran between August 2020 and January 2021, was the first modelling challenge in animal health. In this paper, we describe the objectives and rules of the challenge. We then demonstrate the mechanistic multi-host model that was used to mimic as accurately as possible an ASF-like epidemic, provide a detailed explanation of the surveillance and intervention strategies that generated the synthetic data, and describe the different management strategies that were assessed by the competing modelling teams. We then outline the different technical steps of the challenge as well as its environment. Finally, we synthesize the lessons we learnt along the way to guide future modelling challenges in animal health.

Highlights

The 1st modelling challenge in animal health mimics ASF spread with synthetic data

A mechanistic spatially-explicit stochastic model was developed to represent ASF spread and control

Synthetic data concerned date and location of detected cases in pigs and wild boar

Modelling ASF at the wildlife-livestock interface illustrates the reservoir role of wild fauna
Identification of upstream transcription factor binding sites in orthologous genes using mixed Student’s t-test statistics
Authors: Huang Tinghua , Xiao Hong , Tian Qi , He Zhen , Yuan Cheng , Lin Zezhao , Gao Xuejun , Yao Min
Published: December 2021

Background

Transcription factor (TF) regulates the transcription of DNA to messenger RNA by binding to upstream sequence motifs. Identifying the locations of known motifs in whole genomes is computationally intensive.

Methodology/Principal Findings

This study presents a computational tool, named “Grit”, for screening TF-binding sites (TFBS) by coordinating transcription factors to their promoter sequences in orthologous genes. This tool employs a newly developed mixed Student’s t-test statistical method that detects high-scoring conserved and non-conserved binding sites among species. The program performs sequence scanning at a rate of 3.2 Mb/s on a quad-core Amazon server and has been benchmarked by the well-established ChIP-Seq datasets, putting Grit amongst the top-ranked TFBS predictors. It marginally outperforms the well-known transcription factor motif scanning tools, Pscan (4.8%) and FIMO (17.8%), in analyzing well-documented ChIP-Atlas human genome Chip-Seq datasets.

Significance

Grit is a good alternative to current available motif scanning tools and is publicly available at http://www.thua45.cn/grit under an academic free license.

Author Summary

Locating transcription factor-binding (TF-binding) site in the genome and identification their function is fundamental in understanding various biological processes. Improve the performance of the prediction tools is important because accurate TF-binding site prediction can save cost and time for wet-lab experiments. Also, genome wide TF-binding site prediction can provide new insights for transcriptome regulation in system biology perspective. This study developed a new TF-binding site prediction tool based on mixed Student’s t-test statistical method. The tool is amongst the top-ranked TF-binding site predictors, as such, it can help the researchers in TF-binding site identification and transcriptional regulation mechanism interpretation of genes.
Single-Cell Atlas of Common Variable Immunodeficiency reveals germinal center-associated epigenetic dysregulation in B cell responses
Authors: Rodríguez-Ubreva Javier , Arutyunyan Anna , Bonder Marc Jan , Pino-Molina Lucía Del , Clark Stephen J. , de la Calle-Fabregat Carlos , Garcia-Alonso Luz , Handfield Louis-François , Ciudad Laura , Andrés-León Eduardo , Krueger Felix , Català-Moll Francesc , Rodríguez-Cortez Virginia C. , Polanski Krzysztof , Mamanova Lira , van Dongen Stijn , Kiselev Vladimir Yu. , Martínez-Saavedra María T. , Heyn Holger , Martín Javier , Warnatz Klaus , López-Granados Eduardo , Rodríguez-Gallego Carlos , Stegle Oliver , Kelsey Gavin , Vento-Tormo Roser , Ballestar Esteban
Published: December 2021

Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, is characterized by impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and a wide range of phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. We used single-cell epigenomics and transcriptomics to create a cell census of naïve-to-memory B cell differentiation in a pair of CVID-discordant MZ twins. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B cells that mirror defective cell-cell communication defects following activation. These findings were validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naïve-to-memory B-cell transition in CVID and reveal links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, paves the way for future diagnosis and treatments of CVID patients.
Genomes of keystone Mortierella species lead to better in silico prediction of soil mycobiome functions from Taiwan’s offshore islands
Authors: Lin Yu-fei , Liu Wei-An , Liu Yu-Ching , Lee Hsin-Han , Lin Yen-Ju , Chang Ed-Haun , Lu Meiyeh J , Chiu Chih-Yu , Tsai Isheng Jason
Published: December 2021

The ability to correlate the functional relationship between microbial communities and their environment is critical to understanding microbial ecology. There is emerging knowledge on island biogeography of microbes but how island characteristics influence functions of microbial community remain elusive. Here, we explored soil mycobiomes from nine islands adjacent to Taiwan using ITS2 amplicon sequencing. Geographical distances and island size were positively correlated to dissimilarity in mycobiomes, and we identified 56 zero-radius operational taxonomic units (zOTUs) that were ubiquitously present across all islands, and as few as five Mortierella zOTUs dominate more than half of mycobiomes. Correlation network analyses revealed that seven of the 45 hub species were part of the ubiquitous zOTUs belonging to Mortierella, Trichoderma, Aspergillus, Clonostachys and Staphylotrichum. We sequenced and annotated the genomes of seven Mortierella isolates, and comparative predictions of KEGG orthologues using PICRUSt2 database updated with new genomes increased sequence reads coverage by 62.9% at the genus level. In addition, genes associated with carbohydrate and lipid metabolisms were differentially abundant between islands which remained undetected in the original database. Predicted functional pathways were similar across islands despite their geographical separation, difference in differentially abundant genes and composition. Our approach demonstrated the incorporation of the key taxa genomic data can improve functional gene prediction results and can be readily applied to investigate other niches of interests.
Ultra-deep through-skull mouse brain imaging via the combination of skull optical clearing and three-photon microscopy
Authors: He Mubin , Li Dongyu , Zheng Zheng , Zhang Hequn , Wu Tianxiang , Geng Weihang , Hu Zhengwu , Feng Zhe , Peng Shiyi , Zhu Liang , Xi Wang , Zhu Dan , Qian Jun
Published: December 2021

Optical microscopy has enabled in vivo monitoring of brain structures and functions with high spatial resolution. However, the strong optical scattering in turbid brain tissue and skull impedes the observation of microvasculature and neuronal structures at a large depth. Herein, we proposed a strategy to overcome the influence induced by the high scattering effect of both skull and brain tissue via the combination of skull optical clearing (SOC) technique and thee-photon fluorescence microscopy (3PM). The Visible-NIR-II compatible Skull Optical Clearing Agents (VNSOCA) we applied reduced the skull scattering and water absorption in long wavelength by refractive index matching and H2O replacement to D2O respectively. 3PM with the excitation in the 1300-nm window reached 1.5 mm cerebrovascular imaging depth in cranial window. Combining the two advanced technologies together, we achieved so far the largest cerebrovascular imaging depth of 1.0 mm and neuronal imaging depth of >700 μm through intact mouse skull. Dual-channel through-skull imaging of both brain vessels and neurons was also successfully realized, giving an opportunity of non-invasively monitoring the deep brain structures and functions at single-cell level simultaneously.
Isotropic Three-Dimensional Dual-Color Super-Resolution Microscopy with Metal-Induced Energy Transfer
Authors: Thiele Jan Christoph , Jungblut Marvin , Helmerich Dominic A. , Tsukanov Roman , Chizhik Anna , Chizhik Alexey I. , Schnermann Martin , Sauer Markus , Nevskyi Oleksii , Enderlein Jörg
Published: December 2021

Over the last two decades, super-resolution microscopy has seen a tremendous development in speed and resolution, but for most of its methods, there exists a remarkable gap between lateral and axial resolution. Similar to conventional optical microscopy, the axial resolution is by a factor three to five worse than the lateral resolution. One recently developed method to close this gap is metal-induced energy transfer (MIET) imaging which achieves an axial resolution down to nanometers. It exploits the distance dependent quenching of fluorescence when a fluorescent molecule is brought close to a metal surface. In the present manuscript, we combine the extreme axial resolution of MIET imaging with the extraordinary lateral resolution of single-molecule localization microscopy, in particular with direct stochastic optical reconstruction microscopy (dSTORM). This combination allows us to achieve isotropic three-dimensional super-resolution imaging of sub-cellular structures. Moreover, we employed spectral demixing for implementing dualcolor MIET-dSTORM that allows us to image and co-localize, in three dimensions, two different cellular structures simultaneously.
Boosting annotation in nutrimetabolomics by Feature-Based Molecular Networking: analytical and computational strategies applied to human urine samples from an untargeted LC-MS/MS based bilberry-blueberry intervention study
Authors: Renai Lapo , Ulaszewska Marynka , Mattivi Fulvio , Bartoletti Riccardo , Bubba Massimo Del , van der Hooft Justin J. J.
Published: December 2021

Urine represents a challenging metabolite mixture to decipher. Yet, it contains valuable information on dietary intake patterns as typically investigated using randomized, single-blinded, intervention studies. This research demonstrates how the use of Feature-Based Molecular Networking in combination with public spectral libraries, further expanded with an “In-house” library of metabolite spectra, improved the non-trivial annotation of metabolites occurring in human urine samples following bilberry and blueberry intake. Following this approach, 65 berry-related and human endogenous metabolites were annotated, increasing the annotation coverage by 72% compared to conventional annotation approaches. Furthermore, the structures of 15 additional metabolites were hypothesized by spectral analysis. Then, by leveraging the MzMine quantitative information, several molecular families of phase II (e.g., glucuronidated phenolics) and phase I (e.g., phenylpropionic acid and hydroxybenzoic acid molecular scaffolds) metabolism were identified by correlation analysis of postprandial kinetics, and the dietary impact of endogenous and exogenous metabolites following bilberry-blueberry intake was estimated.
Transcriptome of the synganglion and characterization of the cys-loop ligand-gated ion channel gene family in the tickIxodes ricinus
Authors: Rispe Claude , Hervet Caroline , de la Cotte Nathalie , Daveu Romain , Labadie Karine , Noël Benjamin , Aury Jean-Marc , Thany Steeve , Taillebois Emiliane , Cartereau Alison , Mauff Anaïs Le , Charvet Claude L. , Auger Clément , Neveu Cédric , Plantard Olivier
Published: December 2021

Ticks represent a major health issue for humans and domesticated animals. Assessing the expression patterns of the tick’s central nervous system, known as the synganglion, is an important step in understanding tick physiology and in managing tick-borne diseases. Neuron-specific genes like the cys-loop ligand-gated ion channels (cys-loop LGICs) are important pharmacological targets of acaricides. Here, we carried out the sequencing of transcriptomes of theI. ricinussynganglion, for adult ticks in different conditions (unfed males, unfed females, and partially-fed females). Thede novoassembly of these transcriptomes allowed us to obtain a large collection of cys-loop LGICs sequences. A reference meta-transcriptome based on synganglion and whole body transcriptomes was then produced, showing high completeness and allowing differential expression analyses between synganglion and whole body. Many of the genes upregulated in the synganglion were related to biological processes or functions associated with neurotransmission and located in neurones or the synaptic membrane, including most of the cys-loop LGICs. As a first step of a functional study of cysLGICs, we cloned the predicted sequence of the resistance to dieldrin (RDL) subunit homologue, and functionally reconstituted the first GABA-gated receptor ofIxodes ricinususing a hetrologous expression approach. A phylogenetic study was performed for the nicotinic acetylcholine receptors (nAChRs) and for other cys-loop LGICs respectively, showing tick-specific expansions of some types of receptors (Histamine-gated, GluCls).
GPR55 in B cells limits atherosclerosis development and regulates plasma cell maturation
Authors: Guillamat-Prats Raquel , Hering Daniel , Rami Martina , Härdtner Carmen , Santovito Donato , Rinne Petteri , Bindila Laura , Hristov Michael , Pagano Sabrina , Vuilleumier Nicolas , Schmid Sofie , Janjic Aleksandar , Enard Wolfgang , Weber Christian , Maegdefessel Lars , Faussner Alexander , Hilgendorf Ingo , Steffens Sabine
Published: December 2021

Identifying novel pathways regulating the adaptive immune response in chronic inflammatory diseases such as atherosclerosis is of particular interest in view of developing new therapeutic drugs. Here we report that the lipid receptor GPR55 is highly expressed by splenic B cells and inversely correlates with atheroma plaque size in mice. In human carotid endarterectomy specimen, GPR55 transcript levels were significantly lower in unstable compared to stable carotid plaques. To study the impact of GPR55 deficiency in atherosclerosis, we crossedGpr55knockout mice with apolipoprotein E (ApoE) knockout mice and subjected the mice to Western diet for 4 to 16 weeks. Compared toApoE-/-controls,ApoE-/-Gpr55-/-mice developed larger plaques with increased necrotic core size, associated with elevated circulating and aortic leukocyte counts. Flow cytometry, immunofluorescence and RNA-sequencing analysis of splenic B cells in these mice revealed a hyperactivated B cell phenotype with disturbed plasma cell maturation and immunoglobulin (Ig)G antibody overproduction. The specific contribution of B cell GPR55 in atherosclerosis was further studied in mixedGpr55-/-/µMTbone marrow chimeras on low density receptor deficiency (Ldlr-/-) background, revealing that B-cell specific depletion ofGpr55was sufficient to promote plaque development. Conversely, adoptive transfer of wildtype B cells intoApoE-/-Gpr55-/-mice blunted the proatherogenic phenotype.In vitrostimulation of splenocytes with the endogenous GPR55 ligand LPI promoted plasma cell proliferation and enhanced B cell activation marker expression, which was inhibited by the GPR55 antagonist CID16020046. Collectively, these discoveries provide new evidence for GPR55 as key modulator of the adaptive immune response in atherosclerosis. Targeting GPR55 could be useful to limit inflammation and plaque progression in patients suffering from atherosclerosis.
A deep dive into VDAC1 conformational diversity using all-atom simulations provides new insights into the structural origin of the closed states
Authors: Preto Jordane , Gorny Hubert , Krimm Isabelle
Published: December 2021

The voltage-dependent anion channel 1 (VDAC1) is a crucial mitochondrial transporter which controls the flow of ions and respiratory metabolites entering or exiting mitochondria. As a voltage-gated channel, VDAC1 can switch between a high conducting “open” state and low conducting “closed” states emerging at high transmembrane potential. Although cell homeostasis depends on channel gating to regulate the transport of ions and metabolites, structural hallmarks characterizing the closed states remain unknown. Here we performed microsecond accelerated molecular dynamics to highlight a vast region of VDAC1 conformational landscape accessible at typical voltage known to promote closure. Conformers exhibiting stable subconducting properties inherent to closed states were identified. In all cases, the low conductance was due to the particular positioning of an unfolded part of the N-terminus which obstructed the channel pore. While the N-terminal tail was found to be sensitive to voltage orientation, our low-conducting models suggest that closed states predominantly take place from disordered events and do not result from the displacement of a voltage sensor or a significant change in the pore. In addition, our results were consistent with conductance jumps observed in experiments and corroborates a recent study describing entropy as a key factor for VDAC gating.
Explore synergistic and competitive miRNA regulation mechanisms in the miRNA-mRNA regulatory network from the information decomposition perspective
Authors: Pan Chu , Jiang Jing , Jing Limei , Chen Wangqing , Yang Yi , Liu Ying , Luo Jiawei , Zeng Xiangxiang
Published: December 2021

Since multiple microRNAs can target 3’ untranslated regions of the same mRNA transcript, it is likely that these endogenous microRNAs may form synergistic alliances, or compete for the same mRNA harbouring overlapping binding site matches. Synergistic and competitive microRNA regulation is an intriguing yet poorly elucidated mechanism. We here introduce a computational method based on the multivariate information measurement to quantify such implicit interaction effects between microRNAs. Our informatics method of integrating sequence and expression data is designed to establish the functional correlation between microRNAs. To demonstrate our method, we exploited TargetScan and The Cancer Genome Atlas data. As a result, we indeed observed that the microRNA pair with neighbouring binding site(s) on the mRNA is likely to trigger synergistic events, while the microRNA pair with overlapping binding site(s) on the mRNA is likely to cause competitive events, provided that the pair of microRNAs has a high functional similarity and the corresponding triplet presents a positive/negative ‘synergy-redundancy’ score.
Leveraging TOPMed Imputation Server and Constructing a Cohort-Specific Imputation Reference Panel to Enhance Genotype Imputation among Cystic Fibrosis Patients
Authors: Sun Quan , Liu Weifang , Rosen Jonathan D. , Huang Le , Pace Rhonda G. , Dang Hong , Gallins Paul J. , Blue Elizabeth E. , Ling Hua , Corvol Harriet , Strug Lisa J. , Bamshad Michael J. , Gibson Ronald L. , Pugh Elizabeth W. , Blackman Scott M. , Cutting Garry R. , O’Neal Wanda K. , Zhou Yi-Hui , Wright Fred A. , Knowles Michael R. , Wen Jia , Li Yun
Published: December 2021

Cystic fibrosis (CF) is a severe genetic disorder that can cause multiple comorbidities affecting the lungs, the pancreas, the luminal digestive system and beyond. In our previous genome-wide association studies (GWAS), we genotyped ∼8,000 CF samples using a mixture of different genotyping platforms. More recently, the Cystic Fibrosis Genome Project (CFGP) performed deep (∼30x) whole genome sequencing (WGS) of 5,095 samples to better understand the genetic mechanisms underlying clinical heterogeneity among CF patients. For mixtures of GWAS array and WGS data, genotype imputation has proven effective in increasing effective sample size. Therefore, we first performed imputation for the ∼8,000 CF samples with GWAS array genotype using the TOPMed freeze 8 reference panel. Our results demonstrate that TOPMed can provide high-quality imputation for CF patients, boosting genomic coverage from ∼0.3 - 4.2 million genotyped markers to ∼11 - 43 million well-imputed markers, and significantly improving Polygenic Risk Score (PRS) prediction accuracy. Furthermore, we built a CF-specific CFGP reference panel based on WGS data of CF patients. We demonstrate that despite having ∼3% the sample size of TOPMed, our CFGP reference panel can still outperform TOPMed when imputing some CF disease-causing variants, likely due to allele and haplotype differences between CF patients and general populations. We anticipate our imputed data for 4,656 samples without WGS data will benefit our subsequent genetic association studies, and the CFGP reference panel built from CF WGS samples will benefit other investigators studying CF.
Long-lasting germinal center responses to a priming immunization with continuous proliferation and somatic mutation
Authors: Lee Jeong Hyun , Sutton Henry , Cottrell Christopher A. , Phung Ivy , Ozorowski Gabriel , Sewall Leigh M. , Nedellec Rebecca , Nakao Catherine , Silva Murillo , Richey Sara T. , Torres Jonathan L. , Lee Wen-Hsin , Georgeson Erik , Kubitz Michael , Hodges Sam , Mullen Tina-Marie , Adachi Yumiko , Cirelli Kimberly M. , Kaur Amitinder , Allers-Hernandez Carolina , Fahlberg Marissa , Grasperge Brooke F. , Dufour Jason P. , Schiro Faith , Aye Pyone P. , Carnathan Diane G. , Silvestri Guido , Shen Xiaoying , Montefiori David C. , Veazey Ronald S. , Ward Andrew B. , Hangartner Lars , Burton Dennis R. , Irvine Darrell J. , Schief William R. , Crotty Shane
Published: December 2021

Germinal centers (GCs) are the engines of antibody evolution. Using HIV Env protein immunogen priming in rhesus monkeys (RM) followed by a long period without further immunization, we demonstrate GC B cells (BGC) lasted at least 6 months (29 weeks), all the while maintaining rapid proliferation. A 186-fold BGC cell increase was present by week 10 compared to a conventional immunization. Single cell transcriptional profiling revealed that both light zone and dark zone GC states were sustained throughout the 6 months. Antibody somatic hypermutation (SHM) of BGC cells continued to accumulate throughout the 29 week priming period, with evidence of selective pressure. Additionally, Env-binding BGC cells were still 49-fold above baseline 29 weeks after immunization, suggesting that they could be active for significantly longer periods of time. High titers of HIV neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing significant immunodominance challenges for B cells, among other difficulties. Memory B cells (BMem) generated under these long priming conditions had higher levels of SHM, and both BMem cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning the >6-month GC period were identified, demonstrating continuous GC activity and selection for at least 191 days, with no additional antigen exposure. A long prime, adjuvanted, slow delivery (12-day) immunization approach holds promise for difficult vaccine targets, and suggests that patience can have great value for tuning GCs to maximize antibody responses.
Engraftment, fate, and function of HoxB8-conditional neutrophil progenitors in the unconditioned murine host
Authors: Cohen Joshua T. , Danise Michael , Hinman Kristina D. , Neumann Brittany M. , Johnson Renita , Wilson Zachary S. , Chorzalska Anna , Dubielecka Patrycja M. , Lefort Craig T.
Published: December 2021

The development and use of murine myeloid progenitor cell lines that are conditionally immortalized through expression of HoxB8 has provided a valuable tool for studies of neutrophil biology. Recent work has extended the utility of HoxB8-conditional progenitors to thein vivosetting via their transplantation into irradiated mice. Here, we describe the isolation of HoxB8-conditional progenitor cell lines that are unique in their ability to engraft in the naïve host in the absence of conditioning of the hematopoietic niche. Our results indicate that HoxB8-conditional progenitors engraft in a β1 integrin-dependent manner and transiently generate donor-derived mature neutrophils. Furthermore, we show that neutrophils derivedin vivofrom transplanted HoxB8-conditional progenitors are mobilized to the periphery and recruited to sites of inflammation in a manner that depends on the C-X-C chemokine receptor 2 and β2 integrins, the same mechanisms that have been described for recruitment of endogenous primary neutrophils. Together, our studies advance the understanding of HoxB8-conditional neutrophil progenitors and describe an innovative tool that, by virtue of its ability to engraft in the naïve host, will facilitate mechanisticin vivoexperimentation on neutrophils.
Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information
Authors: Heather James M. , Spindler Matthew J. , Alonso Marta Herrero , Shui Yifang Ivana , Millar David G. , Johnson David S. , Cobbold Mark , Hata Aaron N.
Published: December 2021

The study and manipulation of T cell receptors (TCRs) is central to multiple fields across basic and translational immunology research. Produced by V(D)J recombination, TCRs are often only recorded in the literature and data repositories as a combination of their V and J gene symbols, plus their hypervariable CDR3 amino acid sequence. However, numerous applications require full-length coding nucleotide sequences. Here we present Stitchr, a software tool developed to specifically address this limitation. Given minimal V/J/CDR3 information, Stitchr produces complete coding sequences representing a fully spliced TCR cDNA. Due to its modular design, Stitchr can be used for TCR engineering using either published germline or novel/modified variable and constant region sequences. Sequences produced by Stitchr were validated by synthesizing and transducing TCR sequences into Jurkat cells, recapitulating the expected antigen specificity of the parental TCR. Using a companion script, Thimble, we demonstrate that Stitchr can process a million TCRs in under ten minutes using a standard desktop personal computer. By systemizing the production and modification of TCR sequences, we propose that Stitchr will increase the speed, repeatability, and reproducibility of TCR research. Stitchr is available on GitHub.
MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells
Authors: Comar Courtney E. , Otter Clayton J. , Pfannenstiel Jessica , Doerger Ethan , Renner David M. , Tan Li Hui , Perlman Stanley , Cohen Noam A. , Fehr Anthony R. , Weiss Susan R.
Published: December 2021

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be in part because MERS-CoV is adept at antagonizing early innate immune pathways – interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase ribonuclease L (OAS/RNase L) – generated in response to viral double-stranded (ds)RNA generated during genome replication. This is in contrast to SARS-CoV-2, which we recently reported activates PKR and RNase L and to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of the dsRNA-induced innate immune pathways. This resulted in ten-fold attenuation of replication in human lung derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of WT MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication.

Importance

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes highly lethal respiratory disease. MERS-CoV encodes several innate immune antagonists, accessory proteins NS4a and NS4b unique to the merbeco lineage and the nsp15 protein endoribonuclease (EndoU), conserved among all coronaviruses. While mutation of each antagonist protein alone has little effect on innate immunity, infections with recombinant MERS-CoVs with mutations of EndoU in combination with either NS4a or NS4b, activate innate signaling pathways and are attenuated for replication. Our data indicate that EndoU and accessory proteins NS4a and NS4b together suppress innate immunity during MERS-CoV infection, to optimize viral replication. This is in contrast to SARS-CoV-2 which activates these pathways and consistent with greater mortality observed during MERS-CoV infection compared to SARS-CoV-2.