bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence and outcomes of co-infection and super-infection with SARS-CoV-2 and other pathogens: A Systematic Review and Meta-analysis
Authors: Musuuza Jackson , Watson Lauren , Parmasad Vishala , Putman-Buehler Nathan , Christensen Leslie , Safdar Nasia
Published: October 2020

Introduction

The recovery of other respiratory viruses in patients with SARS-CoV-2 infection has been reported, either at the time of a SARS-CoV-2 infection diagnosis (co-infection) or subsequently (superinfection). However, data on the prevalence, microbiology and outcomes of co-infection and super infection are limited. The purpose of this study was to examine occurrence of respiratory co-infections and superinfections and their outcomes among patients with SARS-CoV-2 infection.

Patients and Methods

We searched literature databases for studies published from October 1, 2019, through June 11, 2020. We included studies that reported clinical features and outcomes of co-infection or super-infection of SARS-CoV-2 and other pathogens in hospitalized and non-hospitalized patients. We followed PRISMA guidelines and we registered the protocol with PROSPERO as: CRD42020189763.

Results

Of 1310 articles screened, 48 were included in the random effects meta-analysis. The pooled prevalence of co-infection was 12% (95% confidence interval (CI): 6%-18%, n=29, I2=98%) and that of super-infection was 14% (95% CI: 9%-21%, n=18, I2=97%). Pooled prevalence of pathogen type stratified by co- or super-infection: viral co-infections, 4% (95% CI: 2%-7%); viral super-infections, 2% (95% CI: 0%-7%); bacterial co-infections, 4% (95% CI: 1%-8%); bacterial super-infections, 6% (95% CI: 2%-11%); fungal co-infections, 4% (95% CI: 1%-8%); and fungal super-infections, 4% (95% CI: 0%-11%). Compared to those with co-infections, patients with super-infections had a higher prevalence of mechanical ventilation [21% (95% CI: 13%-31%) vs. 7% (95% CI: 2%-15%)] and greater average length of hospital stay [mean=12.5 days, standard deviation (SD) =5.3 vs. mean=10.2 days, SD= 6.7].

Conclusions

Our study showed that as many as 14% of patients with COVID-19 have super-infections and 12% have co-infections. Poor outcomes were associated with super-infections. Our findings have implications for diagnostic testing and therapeutics, particularly in the upcoming respiratory virus season in the Northern Hemisphere.
Genome-wide Association Study of Non-syndromic Orofacial Clefts in a Multiethnic Sample of Families and Controls Identifies Novel Regions
Authors: Mukhopadhyay Nandita , Feingold Eleanor , Moreno-Uribe Lina , Wehby George , Valencia-Ramirez Luz Consuelo , Restrepo Muñeton Claudia P. , Padilla Carmencita , Deleyiannis Frederic , Christensen Kaare , Poletta Fernando A. , Orioli Ieda M , Hecht Jacqueline T. , Buxó Carmen J. , Butali Azeez , Adeyemo Wasiu L. , Vieira Alexandre R. , Shaffer John R. , Murray Jeffrey C. , Weinberg Seth M. , Leslie Elizabeth J. , Marazita Mary L.
Published: October 2020

Orofacial clefts (OFCs) are among the most prevalent craniofacial birth defects worldwide and create a significant public health burden. The majority of OFCs are non-syndromic and vary in prevalence by ethnicity. Africans have the lowest prevalence of OFCs (∼ 1/2,500), Asians have the highest prevalence (∼1/500), European and Latin Americans lie somewhere in the middle (∼1/800 and 1/900 respectively). Thus, ethnicity appears to be a major determinant of the risk of developing OFC. The Pittsburgh Orofacial Clefts Multiethnic study was designed to explore this ethnic variance, comprising a large number of families and individuals (∼12,000 individuals) from multiple populations worldwide: US and Europe, Asians, mixed Native American/Caucasians, and Africans. In this current study, we analyzed 2,915 OFC cases, 6,044 unaffected individuals related to the OFC cases, and 2,685 controls with no personal or family history of OFC. Participants were grouped by their ancestry into African, Asian, European, and Central and South American subsets, and genome-wide association run on the combined sample as well as the four ancestry-based groups. We observed 22 associations to cleft lip with or without cleft palate at 18 distinct loci with p-values < 1e-06, including 10 with genome-wide significance (< 5e-08), in the combined sample and within ancestry groups. Three loci - 2p12 (rs62164740, p=6.27e-07), 10q22.2 (rs150952246, p=3.14e-07), and 10q24.32 (rs118107597, p=8.21e-07) are novel. Nine were in or near known OFC loci - PAX7, IRF6, FAM49A, DCAF4L2, 8q24.21, NTN1, WNT3-WNT9B, TANC2, and RHPN2. The majority of the associations were observed only in the combined sample, European, and Central and South American groups. We investigated whether the observed differences in association strength were a) purely due to sample sizes, b) due to systematic allele frequency difference at the population level, or (c) due to the fact certain OFC-causing variants confer different amounts of risk depending on ancestral origin, by comparing effect sizes to observed allele frequencies of the effect allele in our ancestry-based groups. While some of the associations differ due to systematic differences in allele frequencies between groups, others show variation in effect size despite similar frequencies across ancestry groups.
ACE2 Netlas: In-silico functional characterization and drug-gene interactions of ACE2 gene network to understand its potential involvement in COVID-19 susceptibility
Authors: Pathak Gita A , Wendt Frank R , Goswami Aranyak , De Angelis Flavio ,   COVID-19 Human Genetics Initiative, Polimanti Renato
Published: October 2020

Angiotensin-converting enzyme-2 (ACE2) receptor has been identified as the key adhesion molecule for the transmission of the SARS-CoV-2. However, there is no evidence that human genetic variation in ACE2 is singularly responsible for COVID-19 susceptibility. Therefore, we performed a multi-level characterization of genes that interact with ACE2 (ACE2-gene network) for their over-represented biological properties in the context of COVID-19.

The phenome-wide association of 51 genes including ACE2 with 4,756 traits categorized into 26 phenotype categories, showed enrichment of immunological, respiratory, environmental, skeletal, dermatological, and metabolic domains (p<4e-4). Transcriptomic regulation of ACE2-gene network was enriched for tissue-specificity in kidney, small intestine, and colon (p<4.7e-4). Leveraging the drug-gene interaction database we identified 47 drugs, including dexamethasone and spironolactone, among others.

Considering genetic variants within ± 10 kb of ACE2-network genes we characterized functional consequences (among others) using miRNA binding-site targets. MiRNAs affected by ACE2-network variants revealed statistical over-representation of inflammation, aging, diabetes, and heart conditions. With respect to variants mapped to the ACE2-network, we observed COVID-19 related associations in RORA, SLC12A6 and SLC6A19 genes.

Overall, functional characterization of ACE2-gene network highlights several potential mechanisms in COVID-19 susceptibility. The data can also be accessed at https://gpwhiz.github.io/ACE2Netlas/
simplifyEnrichment: an R/Bioconductor package for Clustering and Visualizing Functional Enrichment Results
Authors: Gu Zuguang , Hübschmann Daniel
Published: October 2020

Functional enrichment analysis or gene set enrichment analysis is a basic bioinformatics method that evaluates biological importance of a list of genes of interest. However, it may produce a long list of significant terms with highly redundant information that is difficult to summarize. Current tools to simplify enrichment results by clustering them into groups either still produce redundancy between clusters or do not retain consistent term similarities within clusters. We propose a new method namedbinary cutfor clustering similarity matrices of functional terms. Through comprehensive benchmarks on both simulated and real-world datasets, we demonstrated that binary cut can efficiently cluster functional terms into groups where terms showed more consistent similarities within groups and were more mutually exclusive between groups. We compared binary cut clustering on the similarity matrices obtained from different similarity measures and found that the semantic similarity worked well with binary cut while similarity matrices based on gene overlap showed less consistent patterns. We implemented the binary cut algorithm in the R packagesimplifyEnrichmentwhich additionally provides functionalities for visualizing, summarizing and comparing the clusterings.
In-vitroandin-vivocharacterization of CRANAD-2 for multi-spectral optoacoustic tomography and fluorescence imaging of amyloid-beta deposits in Alzheimer mice
Authors: Ni Ruiqing , Villois Alessia , Dean-Ben Xose Luis , Chen Zhenyue , Vaas Markus , Stavrakis Stavros , Shi Gloria , deMello Andrew , Ran Chongzhao , Razansky Daniel , Arosio Paolo , Klohs Jan
Published: October 2020

The abnormal deposition of fibrillar beta-amyloid (Aβ) deposits in the brain is one of the major histopathological hallmarks of Alzheimer’s disease (AD). Here we characterize curcumin-derivative CRANAD-2 for multi-spectral optoacoustic tomography (MSOT) and fluorescence imaging of brain Aβ deposits in the arcAβ mouse model of AD cerebral amyloidosis. CRANAD-2 shows a specific and quantitative detection of A fibrilsin vitro,even in complex mixtures, and it is capable to distinguish between monomeric and fibrillar forms of A.In vivoepifluorescence and MSOT after intravenous CRANAD-2 administration demonstrated higher retention in arcAβ compared to non-transgenic littermate mice. Immunohistochemistry showed co-localization of CRANAD-2 and Aβ deposits in arcAβ mouse brain sections, thus verifying the specificity of the probe. In conclusion, we demonstrate suitability of CRANAD-2 for fluorescence- and MSOT-based detection of Aβ deposits in animal models of AD pathology, which facilitates mechanistic studies and the monitoring of putative treatments targeting Aβ deposits.
Applications of machine learning to solve genetics problems
Authors: Sowunmi Kehinde , Nweze Victor Nnanna , Abiola Soyebo Titilayo , Ajibike Okosesi Ebunoluwa , Lawal Adesiyan Ayobami , Adejoke Olademeji Kafayat , Adekunle Ajibola Oluwaseun , Ogunlana Yusuf Olayinka , Wasiu Agboola Olamilekan , Kaur Gurpreet , Hameed Atoromola , Abidemi Oladipupo Temitope
Published: October 2020

The development of precise DNA editing nucleases that induce double-strand breaks (DSBs) - including zinc finger nucleases, TALENs, and CRISPR/Cas systems - has revolutionized gene editing and genome engineering. Endogenous DNA DSB repair mechanisms are often leveraged to enhance editing efficiency and precision. While the non-homologous end joining (NHEJ) and homologous recombination (HR) DNA DSB repair pathways have already been the topic of an excellent deal of investigation, an alternate pathway, microhomology-mediated end joining (MMEJ), remains relatively unexplored. However, the MMEJ pathway’s ability to supply reproducible and efficient deletions within the course of repair makes it a perfect pathway to be used in gene knockouts. (Microhomology Evoked Deletion Judication EluciDation) may be a random forest machine learning-based method for predicting the extent to which the location of a targeted DNA DSB are going to be repaired using the MMEJ repair pathway. On an independent test set of 24 HeLa cell DSB sites, MEDJED achieved a Pearson coefficient of correlation (PCC) of 81.36%, Mean Absolute Error (MAE) of 10.96%, and Root Mean Square Error (RMSE) of13.09%. This performance demonstrates MEDJED’s value as a tool for researchers who wish to leverage MMEJ to supply efficient and precise gene knock outs.
Cell size is a determinant of stem cell potential during aging
Authors: Lengefeld Jette , Cheng Chia-Wei , Maretich Pema , Blair Marguerite , Hagen Hannah , McReynolds Melanie R. , Sullivan Emily , Majors Kyra , Roberts Christina , Kang Joon Ho , Steiner Joachim D. , Miettinen Teemu P. , Manalis Scott R. , Antebi Adam , Morrison Sean J. , Lees Jacqueline A. , Boyer Laurie A. , Yilmaz Ömer H. , Amon Angelika
Published: October 2020

Stem cells are remarkably small in size. Whether small size is important for stem cell function is unknown. We find that murine hematopoietic stem cells (HSCs) enlarge under conditions known to decrease stem cell function. This decreased fitness of large HSCs is due to reduced proliferative potential. Preventing HSC enlargement by inhibiting macromolecule biosynthesis or reducing large HSCs size by shortening G1averts the loss of stem cell potential under conditions causing stem cell exhaustion. Finally, we show that a fraction of murine and human HSCs enlarge during aging. Preventing this age-dependent enlargement improves HSC function. We conclude that small cell size is important for stem cell functionin vivoand propose that stem cell enlargement contributes to their functional decline during aging.

One Sentence Summary

Size increase drives stem cell aging.
The PfAP2-G2 transcription factor is a critical regulator of gametocyte maturation
Authors: Singh Suprita , Santos Joana M. , Orchard Lindsey M. , Yamada Naomi , van Biljon Riëtte , Painter Heather J. , Mahony Shaun , Llinás Manuel
Published: October 2020

Differentiation from asexual blood stages to sexual gametocytes is required for transmission of malaria parasites from the human to the mosquito host. Preventing gametocyte commitment and development would block parasite transmission, but the underlying molecular mechanisms behind these processes remain poorly understood. Here, we report that the ApiAP2 transcription factor, PfAP2-G2 (PF3D7_1408200) plays a critical role in the maturation ofPlasmodium falciparumgametocytes. PfAP2-G2 binds to the promoters of a wide array of genes that are expressed at many stages of the parasite life cycle. Interestingly, we also find binding of PfAP2-G2 within the gene body of almost 3000 genes, which strongly correlates with the location of H3K36me3 and several other histone modifications as well as Heterochromatin Protein 1 (HP1), suggesting that occupancy of PfAP2-G2 in gene bodies may serve as an alternative regulatory mechanism. Disruption ofpfap2-g2does not impact asexual development, parasite multiplication rate, or commitment to sexual development but the majority of sexual parasites are unable to mature beyond stage III gametocytes. The absence ofpfap2-g2leads to overexpression of 28% of the genes bound by PfAP2-G2 and none of the PfAP2-g2 bound are downregulated, suggesting that it is a repressor. We also find that PfAP2-G2 interacts with chromatin remodeling proteins, a microrchidia (MORC) protein, and another ApiAP2 protein (PF3D7_1139300). Overall our data demonstrate that PfAP2-G2 is an important transcription factor that establishes an essential gametocyte maturation program in association with other chromatin-related proteins.
Annual phytoplankton dynamics in coastal waters from Fildes Bay, Western Antarctic Peninsula
Authors: Trefault Nicole , De la Iglesia Rodrigo , Moreno-Pino Mario , dos Santos Adriana Lopes , Ribeiro Catherine Gérikas , Cristi Antonia , Marie Dominique , Vaulot Daniel
Published: October 2020

Year-round reports of phytoplankton dynamics in the West Antarctic Peninsula are rare and mainly limited to microscopy and/or pigment-based studies. We analyzed the phytoplankton community from coastal waters of Fildes Bay in the West Antarctic Peninsula between January 2014 and 2015 using metabarcoding of the nuclear and plastidial 18/16S rRNA gene from both size-fractionated and flow cytometry sorted samples. Each metabarcoding approach yielded a different image of the phytoplankton community with for example Prymnesiophyceae more prevalent in plastidial metabarcodes and Mamiellophyceae in nuclear ones. Overall 14 classes of photosynthetic eukaryotes were present in our samples with the following dominating: Bacillariophyta (diatoms), Pelagophyceae and Dictyochophyceae for division Ochrophyta, Mamiellophyceae and Pyramimonadophyceae for division Chlorophyta, Prymnesiophyceae and Cryptophyceae. Diatoms were dominant in the larger size fractions and during summer, while Prymnesiophyceae and Cryptophyceae were dominant in colder seasons. Pelagophyceae were particularly abundant towards the end of autumn (May). In addition ofMicromonas polarisandMicromonassp. clade B3, both previously reported in Arctic waters, we detected a newMicromonas18S rRNA sequence signature, close to but clearly distinct fromM. polaris, which potentially represent a new clade specific of the Antarctic. These results highlight the need for complementary strategies as well as the importance of year-round monitoring for a comprehensive description of phytoplankton communities in Antarctic coastal waters.
CRISPR-based knockin mutagenesis of the pioneer transcription factor FOXA1; optimization of strategies for multi-allelic proteins in cancer cells
Authors: Li Shen , Garay Joseph P. , Tubbs Colby A. , Franco Hector L.
Published: October 2020

Precise genome engineering of living cells has been revolutionized by the introduction of the highly specific and easily programmable properties of CRISPR-Cas9 technology. This has greatly accelerated research into human health and has facilitated the discovery of novel therapeutics. CRISPR-Cas9 is most widely employed for its ability to inactivate, or knockout, specific genes, but can be also used to introduce subtle site-specific substitutions of DNA sequences that can lead to changes in the amino acid composition of proteins. Despite the proven success of CRISPR-based knockin strategies of genes in typical diploid cells (i.e. cells containing two sets of chromosomes), precise editing of cancer cells, that typically have unstable genomes and multiple copies of chromosomes, is more challenging and not adequately addressed in the literature. Herein we detail our methodology for replacing endogenous proteins with intended knockin mutants in polyploid cancer cells and discuss our experimental design, screening strategy, and facile allele-frequency estimation methodology. As proof of principle, we performed genome editing of specific amino acids within the pioneer transcription factor FOXA1, a critical component of estrogen and androgen receptor signaling, in MCF-7 breast cancer cells. We confirm proper levels of mutant FOXA1 protein expression and intended amino acids substitutions via western blotting and mass spectrometry. In addition, we show that mutant allele-frequency estimation is easily achieved by TOPO cloning combined with allele-specific PCR, which we later confirmed by next-generation RNA-sequencing. Typically, there are 4 - 5 copies (alleles) of FOXA1 in breast cancer cells making the editing of this protein inherently challenging. As a result, most studies that focus on FOXA1 mutants rely on ectopic overexpression of FOXA1 from a plasmid. Therefore, we provide an optimized methodology for replacing endogenous wildtype FOXA1 with precise knockin mutants to enable the systematic analysis of its molecular mechanisms within the appropriate physiological context.
Winter wheat adapts to environmental pH by changing H+net flux in roots at the seedling stage
Authors: Wang Guangtao , Feng Suwei , Ding Weihua , Hu Tiezhu , Ru Zhengang
Published: October 2020

Changes in rhizosphere pH play an important role in wheat growth. To investigate the relationship between changes in rhizosphere pH and the growth of winter wheat roots and to explore the regulatory mechanism of acid and alkali resistance in winter wheat roots, the semi-winter wheat varieties Aikang 58 (AK58) and Bainong 4199 (BN4199) were used as materials for hydroponic experiments. Three pH levels (4.0, 6.5, and 9.0, with 6.5 as control) were applied during the wheat seedling stage. The results showed that the shoot and root biomass of the plants significantly decreased compared with the control under acid-base stress, with a more significant decrease with acid stress than alkali stress. Compared with the control, the root/shoot ratio increased under alkali stress and decreased under acid stress. The wheat root system showed H+net efflux at pH 6.5 and 9.0, and the H+net efflux rate at pH 4.0 was significantly lower than the control. The root activity of wheat was higher than the control at pH 9.0 and lower at pH4.0. The change of root pH was showed pH 4.0 < pH 6.5 < pH 9.0. Correlation analysis showed that changes in H+net flux were significantly positively correlated to root activity and root pH. The H+efflux rate and root activity of BN4199 were highe r than AK58 under acid and alkali stress, and the root/shoot ratio was relatively high, indicating strong acid and alkali resistance. We conclude that wheat could adapt to poor acid-base environments by adjusting root H+net flux, and in practice, the root/shoot ratio could be used as index for the rapid determination of acid-base tolerance in wheat at the seedling stage.
Genomic structural variation in ‘Nebbiolo’ grapevines at the individual, clonal and cultivar levels
Authors: Maestri Simone , Gambino Giorgio , Minio Andrea , Perrone Irene , Cosentino Emanuela , Giovannone Barbara , Lopatriello Giulia , Marcolungo Luca , Cantu Dario , Rossato Marzia , Delledonne Massimo , Calderón Luciano
Published: October 2020

Structural Variants (SVs) are a widely unexplored source of genetic variation, both due to methodological limitations and because they are generally associated to deleterious effects. However, with the advent of long-range genomic platforms, it has become easier to directly detect SVs. In the same direction, clonally propagated crops provide a unique opportunity to study SVs, offering a suitable genomic environment for their accumulation in heterozygosis. In particular, it has been reported that SVs generate drastic levels of heterozygosity in grapevines. ‘Nebbiolo’ (Vitis viniferaL.) is a grapevine cultivar typical of north-western Italy, appreciated for its use in producing high-quality red wines. Here, we aimed to analyze the frequency of SVs in ‘Nebbiolo’, at three different organizational levels. For this purpose, we generated genomic data based on long-reads, linked-reads and optical mapping. We assembled a reference genome for this cultivar and compared two different clones, includingV. viniferareference genome (PN40024) in our comparisons. Our results indicate that SVs differentially occurring between ‘Nebbiolo’ clones might be rare, while SVs differentiating haplotypes of the same individual are as abundant as those that occur differentially between cultivars.
Poly-ADP-ribosylation drives loss of protein homeostasis in ATM and Mre11 deficiency
Authors: Lee Ji-Hoon , Ryu Seung W. , Ender Nicolette A. , Paull Tanya T.
Published: October 2020

Loss of the ataxia-telangiectasia mutated (ATM) kinase causes cerebellum-specific neurodegeneration in humans. We previously demonstrated that deficiency in ATM activation via oxidative stress generates high levels of insoluble protein aggregates in human cells, reminiscent of protein dysfunction in common neurodegenerative disorders. Here we show that this process is driven by poly-ADP-ribose polymerases (PARPs) and that the insoluble protein species arise from intrinsically disordered proteins associating with PAR-associated genomic sites in ATM-deficient cells. The lesions implicated in this process are single-strand DNA breaks dependent on reactive oxygen species, transcription, and R-loops. Human cells expressing Mre11 A-T-like disorder (ATLD) mutants also show PARP-dependent aggregation identical to that of ATM deficiency. Lastly, analysis of A-T patient cerebellum samples shows widespread protein aggregation as well as loss of proteins known to be critical in human spinocerebellar ataxias. These results provide a new hypothesis for loss of protein integrity and cerebellum function in A-T.
Real-Time Linear Prediction of Simultaneous and Independent Movements of Two Finger Groups Using an Intracortical Brain-Machine Interface
Authors: Nason Samuel R. , Mender Matthew J. , Vaskov Alex K. , Willsey Matthew S. , Patil Parag G. , Chestek Cynthia A.
Published: October 2020

Modern brain-machine interfaces can return function to people with paralysis, but current hand neural prostheses are unable to reproduce control of individuated finger movements. Here, for the first time, we present a real-time, high-speed, linear brain-machine interface in nonhuman primates that utilizes intracortical neural signals to bridge this gap. We created a novel task that systematically individuates two finger groups, the index finger and the middle-ring-small fingers combined, presenting separate targets for each group. During online brain control, the ReFIT Kalman filter demonstrated the capability of individuating movements of each finger group with high performance, enabling a nonhuman primate to acquire two targets simultaneously at 1.95 targets per second, resulting in an average information throughput of 2.1 bits per second. To understand this result, we performed single unit tuning analyses. Cortical neurons were active for movements of an individual finger group, combined movements of both finger groups, or both. Linear combinations of neural activity representing individual finger group movements predicted the neural activity during combined finger group movements with high accuracy, and vice versa. Hence, a linear model was able to explain how cortical neurons encode information about multiple dimensions of movement simultaneously. Additionally, training ridge regressing decoders with independent component movements was sufficient to predict untrained higher-complexity movements. Our results suggest that linear decoders for brain-machine interfaces may be sufficient to execute high-dimensional tasks with the performance levels required for naturalistic neural prostheses.
Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development
Authors: Zhou Haipeng , He Ying , Yang Yinxiang , Wang Zhaoyan , Wang Qian , Hu Caiyan , Wang Xiaohua , Lu Siliang , Li Ke , Luan Zuo
Published: October 2020

NG2 and A2B5 are important biological markers of human oligodendrocyte progenitor cells. To study their functional differences during the development of human oligodendrocyte progenitor cells to oligodendrocytes, we used cell sorting technology and obtained a large number of sterile, high-purity NG2+/- and A2B5+/- cells with high viability. Further research was then conducted viain vitrocell proliferation and migration assays, single-cell sequencing, mRNA sequencing, and cell transplantation into shiverer mice. The results showed that the migration ability of the cells was inversely proportional to the myelination ability. NG2 may be a marker of early oligodendrocyte progenitor cells and is conducive to cell migration and proliferation, while A2B5 may be a marker of slightly mature oligodendrocyte progenitor cells and is conducive to cell differentiation. Further, cell migration, proliferation, and myelination capacity of the negative cell population were stronger than those of the positive cell population. In summary, these results suggest that oligodendrocyte progenitor cells in the mid-stage may be more suitable for clinical cell transplantation to treat demyelinating diseases.

Summary statement

This research found that oligodendrocyte progenitor cells in the middle developmental stages may be more suitable for cell transplantation to treat demyelinating diseases.
Chromosome-level assembly of the Atlantic silverside genome reveals extreme levels of sequence diversity and structural genetic variation
Authors: Tigano Anna , Jacobs Arne , Wilder Aryn P. , Nand Ankita , Zhan Ye , Dekker Job , Therkildsen Nina O.
Published: October 2020

The levels and distribution of standing genetic variation in a genome can provide a wealth of insights about the adaptive potential, demographic history, and genome structure of a population or species. As structural variants are increasingly associated with traits important for adaptation and speciation, investigating both sequence and structural variation is essential for wholly tapping this potential. Using a combination of shotgun sequencing, 10X Genomics linked reads and proximity-ligation data (Chicago and Hi-C), we produced and annotated a chromosome-level genome assembly for the Atlantic silverside (Menidia menidia) - an established ecological model for studying the phenotypic effects of natural and artificial selection - and examined patterns of genomic variation across two individuals sampled from different populations with divergent local adaptations. Levels of diversity varied substantially across each chromosome, consistently being highly elevated near the ends (presumably near telomeric regions) and dipping to near zero around putative centromeres. Overall, our estimate of the genome-wide average heterozygosity in the Atlantic silverside is the highest reported for a fish, or any vertebrate, to date (1.32-1.76% depending on inference method and sample). Furthermore, we also found extreme levels of structural variation, affecting ~23% of the total genome sequence, including multiple large inversions (> 1 Mb and up to 12.6 Mb) associated with previously identified haploblocks showing strong differentiation between locally adapted populations. These extreme levels of standing genetic variation are likely associated with large effective population sizes and may help explain the remarkable adaptive divergence among populations of the Atlantic silverside.
Binding Mode of SARS-CoV2 Fusion Peptide to Human Cellular Membrane
Authors: Gorgun Defne , Lihan Muyun , Kapoor Karan , Tajkhorshid Emad
Published: October 2020

Infection of human cells by the SARS-CoV2 relies on its binding to a specific receptor and subsequent fusion of the viral and host cell membranes. The fusion peptide (FP), a short peptide segment in the spike protein, plays a central role in the initial penetration of the virus into the host cell membrane, followed by the fusion of the two membranes. Here, we use an array of molecular dynamics (MD) simulations taking advantage of the Highly Mobile Membrane Mimetic (HMMM) model, to investigate the interaction of the SARS-CoV2 FP with a lipid bilayer representing mammalian cellular membranes at an atomic level, and to characterize the membrane-bound form of the peptide. Six independent systems were generated by changing the initial positioning and orientation of the FP with respect to the membrane, and each system was simulated in five independent replicas, each for 300 ns. In 73% of the simulations, the FP reaches a stable, membrane-bound configuration where the peptide deeply penetrated into the membrane. Clustering of the results reveals three major membrane binding modes (binding modes 1-3) where binding mode 1 populates over half of the data points. Taking into account the sequence conservation among the viral FPs and the results of mutagenesis studies establishing the role of specific residues in the helical portion of the FP in membrane association, the significant depth of penetration of the whole peptide, and the dense population of the respective cluster, we propose that the most deeply inserted membrane-bound form (binding mode 1) represents more closely the biologically relevant form. Analysis of FP-lipid interactions shows the involvement of specific residues, previously described as the “fusion active core residues”, in membrane binding. Taken together, the results shed light on a key step involved in SARS-CoV2 infection with potential implications in designing novel inhibitors.

Significance

A key step in cellular infection by the SARS-CoV2 virus is its attachment to and penetration into the plasma membrane of human cells. These processes hinge upon the membrane interaction of the viral fusion peptide, a segment exposed by the spike protein upon its conformational changes after encountering the host cell. In this study, using molecular dynamics simulations, we describe how the fusion peptide from the SARS-CoV2 virus binds human cellular membranes and characterize, at an atomic level, lipid-protein interactions important for the stability of its membrane-bound state.
Integrity of the short arm of nuclear pore Y-complex is required for mouse embryonic stem cell growth and differentiation
Authors: Gonzalez-Estevez Alba , Verrico Annalisa , Orniacki Clarisse , Reina-San-Martin Bernardo , Doye Valérie
Published: October 2020

Many cellular processes, ranging from cell division to differentiation, are controlled by nuclear pore complexes (NPCs). However studying contributions of individual NPC subunits to these processes in vertebrates has long been impeded by their complexity and the lack of efficient genetic tools. Here we use genome editing in mouse embryonic stem cells (mESCs) to characterize the role of NPC structural components, focusing on the short arm of the Y-complex that comprises Nup85, Seh1 and Nup43. We show that Seh1 and Nup43, although dispensable in pluripotent mESCs, are required for their normal cell growth rates, their viability upon differentiation, and for the maintenance of proper NPC density. mESCs with an N-terminally truncated Nup85 mutation (in which interaction with Seh1 is greatly impaired) feature a similar reduction of NPC density. However, their proliferation and differentiation are unaltered, indicating that it is the integrity of the Y-complex, rather than the number of NPCs, that is critical to ensure these processes.

Summary statement

Seh1 and Nup43, although dispensable in pluripotent mouse embryonic stem cells, are required for normal cell growth, viability upon differentiation, and maintenance of proper NPC density.
High-throughput fluorescent assay for inhibitor screening of proteases from RNA viruses
Authors: Cihlova Bara , Huskova Andrea , Böserle Jiri , Nencka Radim , Boura Evzen , Silhan Jan
Published: October 2020

Spanish flu and other influenza outbreaks, the recent Zika epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases that are caused by RNA viruses. Perhaps less well known yet dangerous RNA viruses cause deadly diseases such as polio, Ebola, measles, rubella, yellow fever, dengue fever and many others. To combat a particular viral disease by diminishing its spread and number of fatal cases, effective vaccines and antivirals are indispensable. Therefore, quick access to the means of discovery of new treatments for any epidemic outbreak is of great interest and in vitro biochemical assays are the basis of drug discovery. The recent outbreak of the coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands an affordable and reliable assay for testing antivirals. Here, we developed a quick and inexpensive high-throughput fluorescent assay to test inhibitors of viral proteases. Accordingly, we employed this assay to sample inhibitors for papain-like protease from SARS-CoV-2. In addition, we validated this assay for screening inhibitors of flaviviral protease from the tick-borne encephalitis virus to emphasize a broad range of applications of our approach. This fluorescent high-throughput assay is based on fluorescent energy transfer (FRET) between two distinct fluorescent proteins (eGFP and mCherry) connected via a substrate polypeptide. When the substrate is cleaved, FRET is abolished and the change in fluorescence corresponds to reaction progress. Our data show that this assay can be used for testing the inhibitors in the 96 or 384 well plates format with robust and reproducible outcomes.
Rag GTPases and Phosphatidylinositol 3-Phosphate Mediate Recruitment of the AP-5/SPG11/SPG15 Complex
Authors: Hirst Jennifer , Hesketh Geoffrey G. , Gingras Anne-Claude , Robinson Margaret S.
Published: October 2020

Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells, and occurs by coincidence detection, requiring both phosphatidylinositol 3-phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which on its own localises to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15 while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway, and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation.

The AP-5/SPG11/SPG15 complex is recruited onto late endosomes/lysosomes, and contributes to lysosomal homeostasis and autophagic lysosome reformation. Hirst et al. show that recruitment is by coincidence detection, requiring both phosphatidylinositol 3-phosphate and Rag GTPases, thus uncovering a link between AP-5/SPG11/SPG15 and the mTORC1 pathway.
SARS-CoV-2 viroporin triggers the NLRP3 inflammatory pathway
Authors: Xu Huanzhou , Chitre Siddhi A. , Akinyemi Ibukun A. , Loeb Julia C. , Lednicky John A. , McIntosh Michael T. , Bhaduri-McIntosh Sumita
Published: October 2020

Cytokine storm resulting from a heightened inflammatory response is a prominent feature of severe COVID-19 disease. This inflammatory response results from assembly/activation of a cell-intrinsic defense platform known as the inflammasome. We report that the SARS-CoV-2 viroporin encoded by ORF3a activates the NLRP3 inflammasome, the most promiscuous of known inflammasomes. ORF3a triggers IL-1β expression via NFκB, thus priming the inflammasome while also activating it via ASC-dependent and -independent modes. ORF3a-mediated inflammasome activation requires efflux of potassium ions and oligomerization between NEK7 and NLRP3. With the selective NLRP3 inhibitor MCC950 able to block ORF3a-mediated inflammasome activation and key ORF3a residues needed for virus release and inflammasome activation conserved in SARS-CoV-2 isolates across continents, ORF3a and NLRP3 present prime targets for intervention.

Development of anti-SARS-CoV-2 therapies is aimed predominantly at blocking infection or halting virus replication. Yet, the inflammatory response is a significant contributor towards disease, especially in those severely affected. In a pared-down system, we investigate the influence of ORF3a, an essential SARS-CoV-2 protein, on the inflammatory machinery and find that it activates NLRP3, the most prominent inflammasome by causing potassium loss across the cell membrane. We also define key amino acid residues on ORF3a needed to activate the inflammatory response, and likely to facilitate virus release, and find that they are conserved in virus isolates across continents. These findings reveal ORF3a and NLRP3 to be attractive targets for therapy.
Base editing strategy allows insertion of the A673T mutation in APP gene to prevent the development of Alzheimer’s disease
Authors: Guyon Antoine , Rousseau Joël , Bégin Francis-Gabriel , Bertin Tom , Lamothe Gabriel , Tremblay Jacques P.
Published: October 2020

Amyloid precursor protein (APP), a membrane protein mostly found in neurons, is preferentially cut by the α-secretase enzyme, however, abnormal cleavage by β-secretase leads to the formation of β-amyloid peptide plaque in the brains of Alzheimer’s patients. Genome analysis of an Icelandic population that did not appear to show symptoms of Alzheimer’s at advanced age led to the discovery of the A673T mutation, reducing β-secretase cleavage by 40%. We hypothesized that the insertion of this mutation in a patient’s genome could be an effective and sustainable method to slow down or prevent the progression of familial and sporadic forms of Alzheimer’s disease. We have thus modified the APP gene in HEK293T cells and in SH-SY5Y neuroblastoma using a Cas9n-deaminase enzyme, which changes a cytosine into a thymine, thus converting the alanine codon to a threonine. Several Cas9n-deaminase variants were tested to compare their efficiency of conversion. The results were characterized and quantified by deep sequencing. We successfully modified the APP gene in up to 56.7% of the HEK293T cells. Our approach aimed to attest to the efficiency of base editing in the development of treatments against genetic diseases as well as provide a new strategy for the treatment of Alzheimer’s.
High catalytic rate of the cold-activeVibrioalkaline phosphatase depends on a hydrogen bonding network involving a large interface loop
Authors: Hjörleifsson Jens Guðmundur , Helland Ronny , Magnúsdóttir Manuela , Ásgeirsson Bjarni
Published: October 2020

The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part of cold-adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacteriumVibrio splendidus(VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large-loop, that hovers over the active site of the other monomer. It presumably has a role in VAP high catalytic efficiency that accompanies extremely low thermal stability. We designed several different mutagenic variants of VAP with the aim of removing inter-subunit interactions at the dimer interface. Breaking the inter-subunit contacts from one residue in particular (Arg336) caused diminished temperature stability of the catalytically potent conformation and a drop in catalytic rate by a half. The relative B-factors of the R336L crystal structure, compared to the wild-type, confirmed increased surface flexibility in a loop on the opposite monomer, but not in the large-loop. Contrary to expectations, the observed reduction in stability with an expected increase in dynamic mobility resulted in reduced catalytic rate. This contradicts common theories explaining high catalytic rates of enzyme from cold-adapted organisms as being due to reduced internal cohesion bringing increased dynamic flexibility to catalytic groups. The large-loop increases the area of the interface between the subunits through its contacts and may facilitate an alternating structural cycle demanded by a half-of-sites reaction mechanism through stronger ties, as the dimer oscillates between high affinity (active) or low phosphoryl-group affinity (inactive).
Targeting C5aR1 Increases the Therapeutic Window of Radiotherapy
Authors: Olcina Monica M. , Stavros Melemenidis , Nambiar Dhanya K. , Kim Ryan K. , Casey Kerriann M. , Rie von Eyben , Woodruff Trent M. , Graves Edward G. , Quynh-Thu Le , Manuel Stucki , Giaccia Amato J.
Published: October 2020

Engaging innate immune pathways is emerging as a productive way of achieving durable anti-tumor responses. However, systemic administration of these therapies can result in toxicity, deemed to be particularly problematic when combined with current standard-of-care cytotoxic treatments such as radiotherapy. Increasing the therapeutic window of radiotherapy may be achieved by using targeted therapies, however, few pre-clinical studies investigate both tumor and normal tissue responses in detail. Here we show that targeting innate immune receptor C5aR1 improves tumor radiation response while reducing radiation-induced normal tissue toxicity, thereby increasing the therapeutic window. Genetically or pharmacologically targeting C5aR1 increases both IL-10 expression in the small intestine and IL-10 secretion by tumor cells. Increased IL-10 attenuates RelA phosphorylation and increases apoptosis in tumor cells, leading to improved radiation responses in murine models. Of note, these radiosensitizing effects are tumor-specific since, in the gastrointestinal tract, targeting C5aR1 instead results in decreased crypt cell apoptosis reduced signs of histological damage and improved survival following total abdominal irradiation in mice. Furthermore, the potent and orally active C5aR1 inhibitor, PMX205, improves tumor radiation responses even in a context of reduced/absent CD8+ T cell infiltration. These data indicate that PMX205 can modulate cancer-cell intrinsic functions to potentiate anti-tumor radiation responses even in tumors displaying features of T-cell deficiency or exclusion. Finally, using a preclinical murine model allowing the simultaneous assessment of tumor and normal tissue radiation responses, we show that PMX205 treatment reduces histological and functional markers of small-bowel toxicity while affording a positive tumor response. Our data, therefore, suggest that targeting C5aR1 could be a promising approach for increasing the therapeutic window of radiotherapy.
Polymorphic SINEC_Cf Retrotransposons in the Genome of the Dog (Canis familiaris)
Authors: Kalla Sara E. , Moghadam Hooman K. , Tomlinson Max , Seebald Allison , Allen Jeremy J. , Whitney Jordan , Choi Jessica D. , Sutter Nathan B.
Published: October 2020

The dog is an exciting genetic system in which many simple and complex traits have now been mapped. For many traits the causal mutation is a polymorphic SINE. To investigate the genome-wide pattern of young SINEC_Cf insertions, we sampled 62 dogs representing 59 breeds and sequenced libraries enriched for SINE flanks. In each dog we detect an average of 10,423 polymorphic loci and all together the libraries identify 81,747 putative polymorphic SINEs. We validated 184 SINEs inserted in protein-coding exons, untranslated regions, introns and intergenic sequence. In dogs both SINEC_Cf and LINEs exhibit a strand bias in introns where antisense copies are more frequent. Antisense polymorphic SINEs also have a higher density in introns. Both SINEs and LINEs drop to very low density near exons. Both sense and antisense polymorphic SINEs also drop to low density upstream of coding exons but not downstream. Antisense polymorphic SINEC_Cfs upstream of coding exons are known to cause narcolepsy, merle, and progressive retinal atrophy in dogs. In other mammals SINE pairs in inverted orientation disrupt gene expression. We find inverted pairs of SINEC_Cf are rare in both introns and intergenic sequence when the two SINEs are separated by less than 100 bp. The lack of inverted pairs is even more pronounced when the SINEs have high sequence identity. Intronic and intergenic LINE pairs show similar patterns. Polymorphic SINEs rarely pair with either SINEC_Cf or SINEC_Cf2. Overall, the high insertion rate of SINEC_Cf provides a natural mutagenesis screen in the dog genome.
Single cell transcriptomic analyses reveal the impact of bHLH factors ATOH7 and Neurog2 on human retinal organoid development
Authors: Zhang Xiangmei , Mandric Igor , Nguyen Kevin H. , Nguyen Thao T. T. , Pellegrini Matteo , Grove James C. R. , Barnes Steven , Yang Xian-Jie
Published: October 2020

The developing retina expresses multiple bHLH transcription factors. Their precise functions and interactions in uncommitted retinal progenitors remain to be fully elucidated. Here, we investigate the roles of bHLH factors ATOH7 and Neurog2 in developing human ES cell-derived 3D retinal organoids. Single cell transcriptome analyses identify three states of proliferating retinal progenitors: pre-neurogenic, neurogenic, and cell cycle-exiting progenitors. Each shows different expression profile of bHLH factors. The distinct cell cycle-exiting progenitors feed into a postmitotic heterozygous neuroblast pool that gives rise to early born neuronal lineages. Elevating ATOH7 or Neurog2 expression accelerates the transition from the pre-neurogenic to the neurogenic state, and expands the exiting progenitor and neuroblast populations. In addition, ATOH7 and Neurog2 significantly, yet differentially, enhance retinal ganglion cell and cone photoreceptor production. Moreover, single cell transcriptome analyses reveal that ATOH7 and Neurog2 assert positive autoregulation, suppress key bHLH factors associated with the neurogenic progenitors, and elevate bHLH factors expressed by exiting progenitors and differentiating neuroblasts. This study thus provides novel insight regarding how ATOH7 and Neurog2 impact human retinal progenitor behaviors and neuroblast fate choices.