bluuebunny/crossref_metadata_2025 huggingface dataset:
Vertical Stratification in Urban Green Space Aerobiomes
Authors: Robinson Jake M. , Cando-Dumancela Christian , Liddicoat Craig , Weinstein Philip , Cameron Ross , Breed Martin F.
Published: June 2020

Exposure to a diverse environmental microbiome is thought to play an important role in ‘educating’ the immune system and facilitating competitive exclusion of pathogens to maintain human health. Vegetation and soil are known to be key sources of airborne microbiota––theaerobiome. Only a limited number of studies have attempted to characterise the dynamics of the aerobiome, and no studies to date have investigated these dynamics from a vertical perspective simulating human exposure. Studies of pollution and allergenic pollen show vertical stratification at various scales, and present an expectation that such vertical stratification may also be present in the aerobiome. Such stratification could have important implications for public health and for the design, engineering and management of urban green spaces. For example, do children receive the same exposure to airborne microbiota as taller adults, and what are the downstream implications for health? In this study, we combine an innovative columnar sampling method at soil level, 0.0, 0.5, 1.0, and 2.0 m together with high-throughput sequencing of the bacterial 16S rRNA gene to assess whether significant vertical stratification of the aerobiome occurred in a parkland habitat in Adelaide, South Australia. Our results provide evidence of vertical stratification in both alpha and beta (compositional) diversity of airborne bacterial communities, with diversity increasing roughly with height. We also found significant vertical stratification in known pathogenic and beneficial bacterial taxa, suggesting potentially different exposure attributes between adults and children. These results could have important implications for public health and urban planning, potentially informing ways to optimise the design and management of health-promoting urban green spaces.
Dynamic changes in β-cell electrical activity and [Ca2+] regulates NFATc3 activation and downstream gene transcription
Authors: Miranda Jose G. , Schleicher Wolfgang E , Ramirez David G. , Landgrave Samantha P , Benninger Richard KP
Published: June 2020

Diabetes results from insufficient insulin secretion as a result of dysfunction to β-cells within the islet of Langerhans. Elevated glucose causes β-cell membrane depolarization and action potential generation, voltage gated Ca2+channel activation and oscillations in free-Ca2+activity ([Ca2+]), triggering insulin release. Nuclear Factor of Activated T-cell (NFAT) is a transcription factor that is regulated by increases in [Ca2+] and calceineurin (CaN) activation. NFAT regulation links cell activity with gene transcription in many systems, and within the β-cell regulates proliferation and insulin granule biogenesis. However the link between the regulation of β-cell electrical activity and oscillatory [Ca2+], with NFAT activation and downstream transcription is poorly understood. In this study we tested whether dynamic changes to β-cell electrical activity and [Ca2+] regulates NFAT activation and downstream transcription. In cell lines, mouse islets and human islets, including those from donors with type2 diabetes, we applied both agonists/antagonists of ion channels together with optogenetics to modulate β-cell electrical activity. Both glucose-induced membrane depolarization and optogenetic-stimulation triggered NFAT activation, and increased transcription of NFAT targets and intermediate early genes (IEGs). Importantly only conditions in which slow sustained [Ca2+] oscillations were generated led to NFAT activation and downstream transcription. In contrast in human islets from donors with type2 diabetes NFAT activation by glucose was diminished, but rescued upon pharmacological stimulation of electrical activity. Thus, we gain insight into the specific patterns of electrical activity that regulate NFAT activation and gene transcription and how this is disrupted in diabetes.
EVALUATION OF THE ABBOTT SARS-COV-2 IG-G ASSAY
Authors: Lau CS , Hoo SP , Liang YL , Aw TC
Published: June 2020

Introduction

Antibodies to the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can increase as soon as 10-13 days after infection. We describe our evaluation of the Abbott SARS-CoV-2 IgG assay on the Architect immunoassay analyser.

Methods

We assessed the precision, sensitivity, and specificity of the Abbott SARS-CoV-2 IgG assay in samples from polymerase chain reaction (PCR) positive patients and healthy healthcare workers. The manufacturer cut-off index (COI) of 1.4 was adopted to identify positive results. We examined the assay cross-reactivity with other viral antibodies (influenza/dengue/hepatitis C/hepatitis B) and rheumatoid factor (RF). The sample throughput of the Abbott assay was also assessed.

Results

The Abbott assay showed excellent precision, with a CV of 3.4% for the negative control (COI = 0.06) and 1.6% for a high positive serum sample (COI = 8.6). Residual serum was available from 57 inpatients not initially suspected of having COVID-19, 29 of whom tested positive for SARS-CoV-2 IgG. The Abbott assay has a sensitivity of 90.9-100% when tested in 54 subjects ≥14 days post PCR positive, and a specificity of 100% (N = 358). There was no cross-reactivity with other viral antibodies (influenza/dengue/hepatitis C/hepatitis B) and RF. The Architect Abbott assay has a throughput of 100 samples in 70 minutes.

Conclusion

The Abbott SARS-CoV-2 IgG assay shows excellent performance that is well within FDA and CDC guidelines when testing patients ≥14 days POS with little cross-reactivity from other viral antibodies. There is some evidence that SARS-CoV-2 IgG develops early in the disease process.

IMPACT STATEMENT

With the current SARS-CoV-2 pandemic still ongoing, laboratories are hard pressed to introduce SARS-CoV-2 antibody testing to help as an indirect marker for infection to identify patients with prior infection/exposure. The Abbott SARS-CoV-2 IgG assay has excellent performance with good precision, specificity, and sensitivity ≥14 days after a positive SARS-CoV-2 PCR test, with a competitive throughput of 100 samples in 70 minutes. It shows no cross-reactivity with other viral antibodies (influenza/dengue/hepatitis B/hepatitis C) and rheumatoid factor. We have also found evidence of early antibody development in some patients before they tested positive on the SARS-CoV-2 PCR test.
Discovery of a Cellular Mechanism Regulating Transcriptional Noise
Authors: Desai Ravi V. , Hansen Maike M.K. , Martin Benjamin , Yu Chen , Ding Sheng , Thomson Matt , Weinberger Leor S.
Published: June 2020

Stochastic fluctuations in gene expression (‘noise’) are often considered detrimental but, in other fields, fluctuations are harnessed for benefit (e.g., ‘dither’ or amplification of thermal fluctuations to accelerate chemical reactions). Here, we find that DNA base-excision repair amplifies transcriptional noise, generating increased cellular plasticity and facilitating reprogramming. The DNA-repair protein Apex1 recognizes modified nucleoside substrates to amplify expression noise—while homeostatically maintaining mean levels of expression— for virtually all genes across the transcriptome. This noise amplification occurs for both naturally occurring base modifications and unnatural base analogs. Single-molecule imaging shows amplified noise originates from shorter, but more intense, transcriptional bursts that occur via increased DNA supercoiling which first impedes and then accelerates transcription, thereby maintaining mean levels. Strikingly, homeostatic noise amplification potentiates fate-conversion signals during cellular reprogramming. These data suggest a functional role for the observed occurrence of modified bases within DNA in embryonic development and disease.
Genome-wide association studies identify 137 loci for DNA methylation biomarkers of ageing
Authors: McCartney Daniel L. , Min Josine L. , Richmond Rebecca C. , Lu Ake T. , Sobczyk Maria K. , Davies Gail , Broer Linda , Guo Xiuqing , Jeong Ayoung , Jung Jeesun , Kasela Silva , Katrinli Seyma , Kuo Pei-Lun , Matias-Garcia Pamela R. , Mishra Pashupati P. , Nygaard Marianne , Palviainen Teemu , Patki Amit , Raffield Laura M. , Ratliff Scott M. , Richardson Tom G. , Robinson Oliver , Soerensen Mette , Sun Dianjianyi , Tsai Pei-Chien , van der Zee Matthijs D. , Walker Rosie M. , Wang Xiaochuan , Wang Yunzhang , Xia Rui , Xu Zongli , Yao Jie , Zhao Wei , Correa Adolfo , Boerwinkle Eric , Dugué Pierre-Antoine , Durda Peter , Elliott Hannah R , Gieger Christian ,   The Genetics of DNA Methylation Consortium, de Geus Eco J.C. , Harris Sarah E. , Hemani Gibran , Imboden Medea , Kähönen Mika , Kardia Sharon L.R. , Kresovich Jacob K. , Li Shengxu , Lunetta Kathryn L. , Mangino Massimo , Mason Dan , McIntosh Andrew M. , Mengel-From Jonas , Moore Ann Zenobia , Murabito Joanne M. ,   NHLBI Trans-Omics for Precision Medicine (TOPMed) Consortium, Ollikainen Miina , Pankow James S. , Pedersen Nancy L. , Peters Annette , Polidoro Silvia , Porteous David J. , Raitakari Olli , Rich Stephen S. , Sandler Dale P. , Sillanpää Elina , Smith Alicia K , Southey Melissa C. , Strauch Konstantin , Tiwari Hemant , Tanaka Toshiko , Tillin Therese , Uitterlinden Andre G , Van Den Berg David J. , van Dongen Jenny , Wilson James G. , Wright John , Yet Idil , Arnett Donna , Bandinelli Stefania , Bell Jordana T. , Binder Alexandra M , Boomsma Dorret I , Chen Wei , Christensen Kaare , Conneely Karen N. , Elliott Paul , Ferrucci Luigi , Fornage Myriam , Hägg Sara , Hayward Caroline , Irvin Marguerite , Kaprio Jaakko , Lawlor Deborah A. , Lehtimäki Terho , Lohoff Falk W. , Milani Lili , Milne Roger L. , Probst-Hensch Nicole , Reiner Alex P. , Ritz Beate , Rotter Jerome I. , Smith Jennifer A. , Taylor Jack A. , van Meurs Joyce B.J. , Vineis Paolo , Waldenberger Melanie , Deary Ian J. , Relton Caroline L. , Horvath Steve , Marioni Riccardo E.
Published: June 2020

Biological ageing estimators derived from DNA methylation (DNAm) data are heritable and correlate with morbidity and mortality. Leveraging DNAm and SNP data from >41,000 individuals, we identify 137 genome-wide significant loci (113 novel) from meta-analyses of four epigenetic clocks and epigenetic surrogate markers for granulocyte proportions and plasminogen activator inhibitor 1 levels, respectively. We report strong genetic correlations with longevity and lifestyle factors such as smoking, education, and obesity. Significant associations are observed in polygenic risk score analysis and to a lesser extent in Mendelian randomization analyses. This study illuminates the genetic architecture underlying epigenetic ageing and its shared genetic contributions with lifestyle factors and longevity.
Iron-meditated fungal starvation by lupine rhizosphere-associated and extremotolerantStreptomycessp. S29 desferrioxamine production
Authors: Jarmusch Scott A. , Lagos-Susaeta Diego , Diab Emtinan , Salazar Oriana , Asenjo Juan A. , Ebel Rainer , Jaspars Marcel
Published: June 2020

Siderophores are iron-chelating compounds that aid iron uptake, one of the key strategies for microorganisms to carve out ecological niches in microbially diverse environments. Desferrioxamines are the principal siderophores produced byStreptomycesspp. Their biosynthesis has been well studied and as a consequence, the chemical potential of the pathway continues to expand. With all of this in mind, our study aimed to explore extremotolerant and Lupine rhizosphere-derivedStreptomycessp. S29 for its potential antifungal capabilities. Cocultivation of isolate S29 was carried out withAspergillus nigerandBotrytis cinerea, both costly fungal phytopathogens in the wine industry, to simulate their interaction within the rhizosphere. The results indicate that not only isStreptomycessp. S29 extraordinary at producing hydroxamate siderophores but uses siderophore production as a means to ‘starve’ the fungi of iron. High resolution LC-MS/MS followed by GNPS molecular networking was used to observe the datasets for desferrioxamines and guided structure elucidation of new desferrioxamine analogues. Comparing the new chemistry, using tools like molecular networking and MS2LDA, with the known biosynthesis, we show that the chemical potential of the desferrioxamine pathway has further room for exploration.
Single molecule occupancy patterns of transcription factors reveal determinants of cooperative bindingin vivo
Authors: Sönmezer Can , Kleinendorst Rozemarijn , Imanci Dilek , Villacorta Laura , Schübeler Dirk , Benes Vladimir , Krebs Arnaud R
Published: June 2020

Gene activation requires the cooperative activity of multiple transcription factors at cis-regulatory elements. Yet, most transcription factors have short residence time, questioning the requirement of their physical co-occupancy on DNA to achieve cooperativity. Here, we advance Single Molecule Footprinting to detect individual molecular interactions of transcription factors and nucleosomes with DNA at mouse cis-regulatory elements. We apply this strategy to quantify the simultaneous binding of multiple transcription factors on single DNA molecules. Analysis of the binary occupancy patterns at thousands of motif combinations reveals that for most types of transcription factors high DNA co-occupancy can occur in absence of direct physical interaction, at sites of competition with nucleosomes. Perturbation of pairwise interactions demonstrates the function of molecular co-occupancy for binding cooperativity. These findings elucidate the binding cooperativity mechanism used by transcription factors in absence of strict organisation of their binding motifs, a characteristic feature of most of enhancers.
A universal RT-qPCR assay for “One Health” detection of influenza A viruses
Authors: Nagy Alexander , Černíková Lenka , Kunteová Kateřina , Dirbáková Zuzana , Thomas Saumya S , Slomka Marek J , Dán Ádám , Varga Tünde , Máté Martina , Jiřincová Helena , Brown Ian H
Published: June 2020

The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and establish host-specific lineages. The four main host reservoirs are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV MP-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93-97% and LOD95%of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensivein silicoevaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.
Mapping genetic effects on cellular phenotypes with “cell villages”
Authors: Mitchell Jana M. , Nemesh James , Ghosh Sulagna , Handsaker Robert E. , Mello Curtis J. , Meyer Daniel , Raghunathan Kavya , de Rivera Heather , Tegtmeyer Matt , Hawes Derek , Neumann Anna , Nehme Ralda , Eggan Kevin , McCarroll Steven A.
Published: June 2020

Tens of thousands of genetic variants shape human phenotypes, mostly by unknown cellular mechanisms. Here we describe Census-seq, a way to measure cellular phenotypes in cells from many people simultaneously. Analogous to pooled CRISPR screens but for natural variation, Census-seq associates cellular phenotypes to donors’ genotypes by quantifying the presence of each donor’s DNA in cell “villages” before and after sorting or selection for cellular traits of interest. Census-seq enables population-scale cell-biological phenotyping with low cost and high internal control. We demonstrate Census-seq through investigation of genetic effects on the SMN protein whose deficiency underlies spinal muscular atrophy (SMA). Census-seq quantified and mapped effects of many common alleles on SMN protein levels and response to SMN-targeted therapeutics, including a common, cryptic non-responder allele. We provide tools enabling population-scale cell experiments and explain how Census-seq can be used to map genetic effects on diverse cell phenotypes.

Abstract Figure

Highlights

Census-seq reveals how inherited genetic variation affects cell phenotypes

Genetic analysis of cellular traits in cell villages of >100 donors

Characterizing human alleles that shape SMN protein expression and drug responses

Development of protocols and software to enable cellular population genetics
Hierarchical sparse coding of objects in deep convolutional neural networks
Authors: Liu Xingyu , Zhen Zonglei , Liu Jia
Published: June 2020

Recently, deep convolutional neural networks (DCNNs) have attained human-level performances on challenging object recognition tasks owing to their complex internal representation. However, it remains unclear how objects are represented in DCNNs with an overwhelming number of features and non-linear operations. In parallel, the same question has been extensively studied in primates’ brain, and three types of coding schemes have been found: one object is coded by entire neuronal population (distributed coding), or by one single neuron (local coding), or by a subset of neuronal population (sparse coding). Here we asked whether DCNNs adopted any of these coding schemes to represent objects. Specifically, we used the population sparseness index, which is widely-used in neurophysiological studies on primates’ brain, to characterize the degree of sparseness at each layer in two representative DCNNs pretrained for object categorization, AlexNet and VGG11. We found that the sparse coding scheme was adopted at all layers of the DCNNs, and the degree of sparseness increased along the hierarchy. That is, the coding scheme shifted from distributed-like coding at lower layers to local-like coding at higher layers. Further, the degree of sparseness was positively correlated with DCNNs’ performance in object categorization, suggesting that the coding scheme was related to behavioral performance. Finally, with the lesion approach, we demonstrated that both external learning experiences and built-in gating operations were necessary to construct such a hierarchical coding scheme. In sum, our study provides direct evidence that DCNNs adopted a hierarchically-evolved sparse coding scheme as the biological brain does, suggesting an implementation-independent principle of representing a myriad of objects efficiently.
NGS-PrimerPlex: high-throughput primer design for multiplex polymerase chain reactions
Authors: Kechin Andrey , Borobova Viktoria , Boyarskikh Ulyana , Khrapov Evgeniy , Subbotin Sergey , Filipenko Maxim
Published: June 2020

Multiplex PCR has multiple applications in molecular biology, including developing new targeted NGS-panels. We present NGS-PrimerPlex, an efficient and versatile command-line application that designs primers for different refined types of amplicon-based genome target enrichment. It supports nested and anchored multiplex PCR, redistribution among multiplex reactions of primers constructed earlier and extension of existing NGS-panels. The primer design process takes into consideration formation of secondary structures, non-target amplicons between all primers of a pool, primers and high-frequent genome SNPs overlapping. Moreover, users of NGS-PrimerPlex are free from manually defining input genome regions, because it can be done automatically from a list of genes or their parts like exon or codon numbers. Using the program, NGS-panel for sequencing theLRRK2gene coding regions was created, and 354 DNA samples were studied successfully with median coverage of 97.4% of target regions by at least 30 reads.

Availability and implementation

NGS-PrimerPlex is open-source and freely available athttps://github.com/aakechin/NGS-PrimerPlex/.

Contact

aa_kechin@niboch.nsc.ru

Supplementary information

Supplementary data
Morpho-molecular diversity and evolutionary analyses suggest hidden life styles in Spumellaria (Radiolaria)
Authors: Sandin Miguel M. , Biard Tristan , Romac Sarah , O’Dogherty Luis , Suzuki Noritoshi , Not Fabrice
Published: June 2020

Spumellaria (Radiolaria, Rhizaria) are holoplanktonic amoeboid protists, ubiquitous and abundant in the global ocean. Their silicified skeleton preserves very well in sediments displaying an excellent fossil record, from the early middle Cambrian (ca. 509-521 Ma), extremely valuable for paleo-environmental reconstruction studies. Spumellaria are tedious to maintain in laboratory conditions preventing an accurate perception of their extant diversity and ecology in today’s oceans, most of which being inferred from sediment records. This study represents an integrated and comprehensive classification of Spumellaria based on the combination of ribosomal taxonomic marker genes (rDNA) and morphological characteristics. In contrast to established taxonomic knowledge, we demonstrate that symmetry of the skeleton takes more importance than internal structures at high taxonomic rank classification. Such reconsideration allows gathering different morphologies with concentric structure and a spherical or radial symmetry believed to belong to other Radiolaria orders from the fossil record, as for some Entactinaria families. Results obtained in this study suggest the existence of new Spumellaria diversity at early diverging positions, in which a non-bearing skeleton organism lives within shelled ones. Using fossil calibrated molecular clock we estimated the origin of Spumellaria in the middle Cambrian (ca. 515 Ma), in agreement with the appearance of the first radiolarian representatives in the fossil record. The morpho-molecular and evolutionary framework established herein allows a direct connection between living specimens and fossil morphologies from the Cambrian, bringing both a standpoint for future molecular environmental surveys and a better understanding for paleo-environmental reconstruction studies.
RNA Atlas of Human Bacterial Pathogens Uncovers Stress Dynamics Linked to Infection
Authors: Avican Kemal , Aldahdooh Jehad , Togninalli Matteo , Tang Jing , Borgwardt Karsten M. , Rhen Mikael , Fällman Maria
Published: June 2020

Despite being genetically diverse, bacterial pathogens can adapt to similar stressful environments in human host, but how this diversity allows them to achive this is yet not fully understood. Knowledge gained through comparative genomics is insufficient as it lacks the level of gene expression reflecting gene usage. To fill this gap, we investigated the transcriptome of 32 diverse bacterial pathogens under 11 host related stress conditions. We revealed that diverse bacterial pathogens have common responses to similar stresses to a certain extent but mostly employ their unique repertoire with intersections between different stress responses. We also identified universal stress responders which shed light on the nature of antimicrobial targets. In addition, we found that known and unknown putative novel ncRNAs comprised a significant proportion of the responses. All the data is collected in PATHOgenex atlas, providing ample opportunities to discover novel players critical for virulence and maintenance of infection.
AutoSpill: A method for calculating spillover coefficients to compensate or unmix high-parameter flow cytometry data
Authors: Roca Carlos P. , Burton Oliver T. , Prezzemolo Teresa , Whyte Carly E. , Halpert Richard , Kreft Łukasz , Collier James , Botzki Alexander , Spidlen Josef , Humblet-Baron Stéphanie , Liston Adrian
Published: June 2020

Compensating in classical flow cytometry or unmixing in spectral systems is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. In both cases, spillover coefficients are estimated for each fluorophore using single-color controls. This approach has remained essentially unchanged since its inception, and is increasingly limited in its ability to deal with high-parameter flow cytometry. Here, we present AutoSpill, a novel approach for calculating spillover coefficients or spectral signatures of fluorophores. The approach combines automated gating of cells, calculation of an initial spillover matrix based on robust linear regression, and iterative refinement to reduce error. Moreover, autofluorescence can be compensated out, by processing it as an endogenous dye in an unstained control. AutoSpill uses single-color controls and is compatible with common flow cytometry software, but it differs in two key aspects from current methods: (1) it is much less demanding in the preparation of controls, as it does not require the presence of well-defined positive and negative populations, and (2) it does not require manual tuning of the spillover matrix, as the algorithm iteratively computes the tuning, producing an optimal compensation matrix. Another algorithm, AutoSpread, complements this approach, providing a robust estimate of the Spillover Spreading Matrix (SSM), while avoiding the need for well-defined positive and negative populations. Together, AutoSpill and AutoSpread provide a superior solution to the problem of fluorophore spillover, allowing simpler and more robust workflows in high-parameter flow cytometry.
Perversely expressed long noncoding RNAs can alter host response and viral proliferation in SARS-CoV-2 infection
Authors: Turjya Rafeed Rahman , Khan Md. Abdullah-Al-Kamran , Islam Abul Bashar Mir Md. Khademul
Published: June 2020

Background

Since December 2019, the world is experiencing an unprecedented crisis due to a novel coronavirus, SARS-CoV-2. Owing to poor understanding of pathogenicity, the virus is eluding treatment and complicating recovery. Regulatory roles of long non-coding RNAs (lncRNAs) during viral infection and associated antagonism of host antiviral immune responses has become more evident in last decade. To elucidate possible functions of lncRNAs in the COVID-19 pathobiology, we have utilized RNA-seq dataset of SARS-CoV-2 infected lung epithelial cells.

Results

Our analyses uncover 21 differentially expressed lncRNAs whose functions are broadly involved in cell survival and regulation of gene expression. By network enrichment analysis we find that these lncRNAs can directly interact with differentially expressed protein-coding genesADAR, EDN1, KYNU, MALL, TLR2andYWHAG; and alsoAKAP8L, EXOSC5, GDF15, HECTD1, LARP4B, LARP7, MIPOL1, UPF1, MOV10andPRKAR2A, host genes that interact with SARS-CoV-2 proteins. These genes are involved in cellular signaling, metabolism, immune response and RNA homeostasis. Since lncRNAs have been known to sponge microRNAs and protect expression of upregulated genes, we also identified 9 microRNAs that are induced in viral infections; however, some lncRNAs are able to block their usual suppressive effect on overexpressed genes and consequently contribute to host defense and cell survival.

Conclusions

Our investigation determines that deregulated lncRNAs in SARS-CoV-2 infection are involved in viral proliferation, cellular survival, and immune response, ultimately determining disease outcome and this information could drive the search for novel RNA therapeutics as a treatment option.
Comprehensive annotations of the mutational spectra of SARS-CoV-2 spike protein: a fast and accurate pipeline
Authors: Rahman M. Shaminur , Islam M. Rafiul , Hoque M. Nazmul , Alam A. S. M. Rubayet Ul , Akther Masuda , Puspo J. Akter , Akter Salma , Anwar Azraf , Sultana Munawar , Hossain M. Anwar
Published: June 2020

In order to explore nonsynonymous mutations and deletions in the spike (S) protein of SARS-CoV-2, we comprehensively analyzed 35,750 complete S protein gene sequences from across six continents and five climate zones around the world, as documented in the GISAID database as of June 24th, 2020. Through a custom Python-based pipeline for analyzing mutations, we identified 27,801 (77.77 % of spike sequences) mutated strains compared to Wuhan-Hu-1 strain. 84.40% of these strains had only single amino-acid (aa) substitution mutations, but an outlier strain from Bosnia and Herzegovina (EPI_ISL_463893) was found to possess six aa substitutions. The D614G variant of the major G clade was found to be predominant across circulating strains in all climates. We also identified 988 unique aa substitution mutations distributed across 660 positions within the spike protein, with eleven sites showing high variability – these sites had four types of aa variations at each position. Besides, 17 in-frame deletions at four major regions (three in N-terminal domain and one just downstream of the RBD) may have possible impact on attenuation. Moreover, the mutational frequency differed significantly (p= 0.003, Kruskal–Wallis test) among the SARS-CoV-2 strains worldwide. This study presents a fast and accurate pipeline for identifying nonsynonymous mutations and deletions from large dataset for any particular protein coding sequence and presents this S protein data as representative analysis. By using separate multi-sequence alignment with MAFFT, removing ambiguous sequences and in-frame stop codons, and utilizing pairwise alignment, this method can derive nonsynonymus mutations (Reference:Position:Strain). We believe this will aid in the surveillance of any proteins encoded by SARS-CoV-2, and will prove to be crucial in tracking the ever-increasing variation of many other divergent RNA viruses in the future.
Structure of human RNA Polymerase III
Authors: Ramsay Ewan Phillip , Abascal-Palacios Guillermo , Daiß Julia L. , King Helen , Gouge Jerome , Pilsl Michael , Beuron Fabienne , Morris Edward , Gunkel Philip , Engel Christoph , Vannini Alessandro
Published: June 2020

In eukaryotes, RNA Polymerase (Pol) III is the enzyme specialised for the transcription of the entire pool of tRNAs and several other short, essential, untranslated RNAs. Pol III is a critical determinant of cellular growth and lifespan across the eukaryotic kingdom. Upregulation of Pol III transcription is often observed in cancer cells and causative Pol III mutations have been described in patients affected by severe neurodevelopmental disorders and hypersensitivity to viral infection.

Harnessing CRISPR-Cas9 genome editing in HeLa cells, we isolated endogenous human Pol III and obtained a cryo-EM reconstruction at 4.0 Å. The structure of human Pol III allowed us to map the reported genetic mutations and rationalise them. Mutations causing neurodevelopmental defects cluster in hotspots that affect the stability and/or biogenesis of Pol III, thereby resulting in loss-of-function of the enzyme. Mutations affecting viral sensing are located in the periphery of the enzyme in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing activity.

Furthermore, integrating x-ray crystallography and SAXS data, we describe the structure of the RPC5 C-terminal extension, which is absent in lower eukaryotes and not visible in our EM map. Surprisingly, experiments in living cells highlight a role for the RPC5 C-terminal extension in the correct assembly and stability of the human Pol III enzyme, thus suggesting an added layer of regulation during the biogenesis of Pol III in higher eukaryotes.
Interplay between UNG and AID governs intratumoral heterogeneity in mature B cell lymphoma
Authors: Delgado Pilar , Álvarez-Prado Ángel F , Marina-Zárate Ester , Sernandez Isora V , Mur Sonia M , de la Barrera Jorge , Sanchez-Cabo Fátima , Cañamero Marta , de Molina Antonio , Belver Laura , de Yebenes Virginia G , Ramiro Almudena R
Published: June 2020

Most B cell lymphomas originate from B cells that have germinal center (GC) experience and bear chromosome translocations and numerous point mutations. GCs B cells remodel their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR) in their immunoglobulin (Ig) genes. Activation Induced Deaminase (AID) initiates CSR and SHM by generating U:G mismatches on Ig DNA that can then be processed by Uracyl-N-glycosylase (UNG). AID promotes collateral damage in the form of chromosome translocations and off-target SHM, however, the exact contribution of AID activity to lymphoma generation and progression is not completely understood. Here we show using a conditional knock-in strategy that AID supraactivity alone is not sufficient to generate B cell transformation. In contrast, in the absence of UNG, AID supra-expression increases SHM and promotes lymphoma. Whole exome sequencing revealed that AID heavily contributes to lymphoma SHM, promoting subclonal variability and a wider range of oncogenic variants. Thus, our data provide direct evidence that UNG is a brake to AID-induced intratumoral heterogeneity and evolution of B cell lymphoma.
REV-ERB Agonism Improves Liver Pathology in a Mouse Model of NASH
Authors: Griffett Kristine , Bedia-Diaz Gonzalo , Elgendy Bahaa , Burris Thomas P.
Published: June 2020

Non-alcoholic fatty liver disease (NAFLD) affects a significant number of people worldwide and currently there are no pharmacological treatments. NAFLD often presents with obesity, insulin resistance, and in some cases cardiovascular diseases. There is a clear need for treatment options to alleviate this disease since it often progresses to much more the much more severe non-alcoholic steatohepatitis (NASH). The REV-ERB nuclear receptor is a transcriptional repressor that regulates physiological processes involved in the development of NAFLD including lipogenesis and inflammation. We hypothesized that pharmacologically activating REV-ERB would suppress the progression of fatty liver in a mouse model of NASH. Using REV-ERB agonist SR9009 in a mouse NASH model, we demonstrate the beneficial effects of REV-ERB activation that led to an overall improvement of hepatic health by suppressing hepatic fibrosis and inflammatory response.
Event-related network changes unfold the dynamics of cortical integration during face processing
Authors: Maffei Antonio , Sessa Paola
Published: June 2020

Face perception arises from a collective activation of brain regions in the occipital, parietal and temporal cortices. Despite wide acknowledgement that these regions act in an intertwined network, the network behavior itself is poorly understood. Here we present a study in which time-varying connectivity estimated from EEG activity elicited by facial expressions presentation was characterized using graph-theoretical measures of node centrality and global network topology. Results revealed that face perception results from a dynamic reshaping of the network architecture, characterized by the emergence of hubs located in the occipital and temporal regions of the scalp. The importance of these nodes can be observed from early stages of visual processing and reaches a climax in the same time-window in which the face-sensitive N170 is observed. Furthermore, using Granger causality, we found that the time-evolving centrality of these nodes is associated with ERP amplitude, providing a direct link between the network state and local neural response. Additionally, investigating global network topology by means of small-worldness and modularity, we found that face processing requires a functional network with a strong small-world organization that maximizes integration, at the cost of segregated subdivisions. Interestingly, we found that this architecture is not static, but instead it is implemented by the network from stimulus onset to ~200 msec. Altogether, this study reveals the event-related changes underlying face processing at the network level, suggesting that a distributed processing mechanism operates through dynamically weighting the contribution of the cortical regions involved.

Data Availability

Data and code related to this manuscript can be accessed through the OSF at this link https://osf.io/hc3sk/?view_only=af52bc4295c044ffbbd3be019cc083f4
Population Dynamics and Neuronal Polyploidy in the Developing Neocortex
Authors: Jungas Thomas , Joseph Mathieu , Fawal Mohamad-Ali , Davy Alice
Published: June 2020

The mammalian neocortex is composed of different subtypes of neurons which are generated during embryogenesis by sequential differentiation of neural progenitors. While molecular mechanisms that control neuronal production in the developing neocortex have been extensively studied, the dynamics and absolute numbers of the different progenitor and neuronal populations are still poorly characterized. Here we describe a medium throughput approach based on flow cytometry and well known identity markers of cortical subpopulations to collect quantitative data over the course of mouse neocortex development. We collected a complete dataset in a physiological developmental context on two progenitor and two neuron populations, including relative proportions and absolute numbers. Our study reveals unexpected numbers of progenitors. In addition, we discovered that a fraction of neurons in the developing mouse neocortex are polyploid.
Bright Near-infrared Anti-Stokes Fluorescence of ICG under Low Power CW Laser Excitation and its Applications in Bioimaging
Authors: Zhou Jing , Wu Di , Ye Zikang , Xue Dingwei , He Mubin , Qian Jun
Published: June 2020

Anti-Stokes fluorescence was observed in ICG, a molecule approved by the FDA for clinical use. The wavelengths of its fluorescence are mainly located in the near-infrared band of 800 nm~900 nm, with a high quantum yield up to 8%. In order to know its generation mechanism, based on multi-photon absorption (MPA) theory, thermally activated delayed fluorescence (TADF) theory and hot band absorption theory, its power dependence, temperature dependence of absorption spectra and fluorescence spectra, and fluorescence lifetime were measured. Its generation mechanism was finally determined to be hot band absorption process. Since ICG showed bright anti-Stokes fluorescence in near-infrared region, which offers substantially longer penetration depth in biological tissues than visible light, excellent photostability and biosafety, we applied it to in vivo imaging and compared it with upconversion nanoparticles (UCNPs). The result is that ICG exhibited much stronger fluorescence than UCNPs, providing more anatomical information of samples. This contributes to a better choice for anti-Stokes fluorescence bioimaging.
Structural basis for the HMGCR interaction with UBIAD1 mutants causing Schnyder corneal dystrophy
Authors: Zhou Fengbo , Weiss Jayne S. , Li Weikai
Published: June 2020

Schnyder corneal dystrophy (SCD) is an autosomal dominant disease characterized by abnormal deposition of cholesterol and lipid in the cornea. The molecular mechanism underlying this process, which involves the interaction between UBAID1 and HMGCR, remains unclear. Here we investigate these events within silicoapproaches. We built the homology models of UBIAD1 and HMGCR based on the existing crystal and cryo-EM structures. The UBIAD1 and HMGCR models are docked and their binding interactions are interrogated by MD simulation. We find that the transmembrane helices of UBIAD1 bind to sterol sensing domain of HMGCR. Upon binding of the GGPP substrate, UBIAD1 shows lower structural flexibility in the TM regions binding to HMGCR. The N102S and G177R mutations disrupts GGPP binding, thereby lowering the binding affinity of HMGCR. Overall, our modeling suggests that SCD mutations in UBIAD1 or lower GGPP concentration increase the structural flexibility of UBIAD1, thereby facilitating its association with HMGCR.
HLFExpression Defines the Human Hematopoietic Stem Cell State
Authors: Lehnertz Bernhard , Chagraoui Jalila , MacRae Tara , Tomellini Elisa , Corneau Sophie , Mayotte Nadine , Boivin Isabel , Sauvageau Guy
Published: June 2020

Hematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and are able to regenerate all blood lineages following transplantation.

Despite this clear functional definition, highly enriched isolation of human HSCs can currently only be achieved through combinatorial assessment of multiple surface antigens. While a number of transgenic HSC reporter mouse strains have been described, no analogous approach to prospectively isolate human HSCs has been reported.

To identify genes with the most selective expression in human HSCs, we profiled population- and single-cell transcriptomes of fresh andex vivocultured cord blood derived HSPCs as well as peripheral blood, adult bone marrow and fetal liver. Based on these analyses, we propose the master transcription factorHLF(Hepatic Leukemia Factor) as one of the most specific HSC marker genes.

To directly track its expression in human hematopoietic cells, we developed a genomicHLFreporter strategy, capable of selectively labeling the most immature blood cells on the basis of a single engineered parameter.

Most importantly,HLF-expressing cells comprise all of the stem cell activity in culture andin vivoduring serial transplantation.

Taken together, these results experimentally establishHLFas a defining gene of the human hematopoietic stem cell state and outline a new approach to continuously mark these cells with high fidelity.

Key Points

In the human blood system,HLFexpression is specific to stem cell populations in primary anatomical sites and duringex vivoexpansion.

CRISPR/rAAV6-mediated integration of a genomicHLF-reporter allows selective and stable genetic labeling of human HSCsex vivoandin vivo.
Vinculin mediated axon growth requires interaction with actin but not talin
Authors: Mandal Pranay , Belapurkar Vivek , Nair Deepak , Ramanan Narendrakumar
Published: June 2020

Axon growth requires coordination of the actin cytoskeleton by actin-binding proteins in the extending neurites. Vinculin is a major constituent of focal adhesion but its role in neuronal migration and axon growth is poorly understood. We found that vinculin deletion in mouse neocortical neurons attenuated axon growth bothin vitroandin vivo. Using different functional mutants of vinculin, we found that expression of a constitutively active vinculin significantly enhanced axon growth while the head-neck domain had a moderate inhibitory effect. Interesting, we found that vinculin-talin interaction was dispensable for axon growth and neuronal migration. Strikingly, expression of the tail domain delayed migration, increased branching and stunted axon. Inhibition of the Arp2/3 complex or abolishing the tail domain interaction with actin completely reversed the branching phenotype caused by tail domain expression without affecting axon length. Super-resolution microscopy showed increased mobile fraction of actin in tail domain expressing neurons. Our results provide novel insights into the role of vinculin and its functional domains in regulating neuronal migration and axon growth.
RING Finger Protein 11 (RNF11) modulates dopamine release inDrosophila
Authors: Champaloux Eve Privman , Donelson Nathan , Pyakurel Poojan , Wolin Danielle , Ostendorf Leah , Denno Madelaine , Borman Ryan , Burke Chris , Short-Miller Jonah C. , Yoder Maria R. , Copeland Jeffrey M. , Sanyal Subhabrata , Venton B. Jill
Published: June 2020

Recent work indicates a role for RING finger protein 11 (RNF11) in Parkinson disease (PD) pathology, which involves the loss of dopaminergic neurons. However, the role ofRNF11in regulating dopamine neurotransmission has not been studied. In this work, we tested the effect ofRNF11RNAi knockdown or overexpression on stimulated dopamine release in the larvalDrosophilacentral nervous system. Dopamine release was stimulated using optogenetics and monitored in real-time using fast-scan cyclic voltammetry at an electrode implanted in an isolated ventral nerve cord.RNF11knockdown doubled dopamine release, but there was no decrease in dopamine fromRNF11overexpression.RNF11knockdown did not significantly increase stimulated serotonin or octopamine release, indicating the effect is dopamine specific. Dopamine clearance was also changed, asRNF11RNAi flies had a higher VmaxandRNF11overexpressing flies had a lower Vmaxthan control flies.RNF11RNAi flies had increased mRNA levels of dopamine transporter (DAT) inRNF11, confirming changes in DAT. InRNF11RNAi flies, release was maintained better for stimulations repeated at short intervals, indicating increases in the recycled releasable pool of dopamine. Nisoxetine, a DAT inhibitor, and flupenthixol, a D2 antagonist, did not affectRNF11RNAi or overexpressing flies differently than control. Thus,RNF11knockdown causes early changes in dopamine neurotransmission, and this is the first work to demonstrate thatRNF11affects both dopamine release and uptake.RNF11expression decreases in human dopaminergic neurons during PD, and that decrease may be protective by increasing dopamine neurotransmission in the surviving dopaminergic neurons.
A Parallel Ratchet-Stroke Mechanism Leads to an Optimum Force for Molecular Motor Function
Authors: Mallimadugula U.L. , Galburt E.A.
Published: June 2020

Molecular motors convert chemical potential energy into mechanical work and perform a great number of critical biological functions. Examples include the polymerization and manipulation of nucleic acids, the generation of cellular motility and contractility, the formation and maintenance of cell shape, and the transport of materials within cells. The mechanisms underlying these molecular machines are routinely divided into two categories: Brownian ratchet and power stroke. While a ratchet uses chemical energy to bias thermally activated motion, a stroke depends on a direct coupling between chemical events and motion. However, the multi-dimensional nature of protein energy landscapes allows for the possibility of multiple reaction paths connecting two states. Here, we investigate the properties of a hypothetical molecular motor able to utilize parallel ratchet and stroke translocation mechanisms. We explore motor velocity and force-dependence as a function of the energy landscape of each path and reveal the potential for such a mechanism to result in an optimum force for motor function. We explore how the presence of this optimum depends on the rates of the individual paths and show that the distribution of stepping times characterized by the randomness parameter may be used to test for parallel path mechanisms. Lastly, we caution that experimental data consisting solely of measurements of velocity as a function of ATP concentration and force cannot be used to eliminate the possibility of such a parallel path mechanism.

SIGNIFICANCE

Molecular motors perform various mechanical functions in cells allowing them to move, replicate and perform various housekeeping functions required for life. Biophysical studies often aim to determine the molecular mechanism by which these motors convert chemical energy to mechanical work by fitting experimental data with kinetic models that fall into one of two classes: Brownian ratchets or power strokes. However, nothinga priorirequires that a motor function via a single mechanism. Here, we consider a theoretical construct where a motor has access to both class of mechanism in parallel. Combining stochastic simulations and analytical solutions we describe unique signatures of such a mechanism that could be observed experimentally. We also show that absence of these signatures does not formally eliminate the existence of such a parallel mechanism. These findings expand our theoretical understanding of the potential motor behaviors with which to interpret experimental results.