bluuebunny/crossref_metadata_2025 huggingface dataset:
TheRhinolophus affinisbat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2
Authors: Li Pei , Guo Ruixuan , Liu Yan , Zhang Yintgtao , Hu Jiaxin , Ou Xiuyuan , Mi Dan , Chen Ting , Mu Zhixia , Han Yelin , Cui Zhewei , Zhang Leiliang , Wang Xinquan , Wu Zhiqiang , Wang Jianwei , Jin Qi , Qian Zhaohui
Published: November 2020

Bat coronavirus (CoV) RaTG13 shares the highest genome sequence identity with SARS-CoV-2 among all known coronaviruses, and also uses human angiotensin converting enzyme 2 (hACE2) for virus entry. Thus, SARS-CoV-2 is thought to have originated from bat. However, whether SARS-CoV-2 emerged from bats directly or through an intermediate host remains elusive. Here, we found thatRhinolophus affinisbat ACE2 (RaACE2) is an entry receptor for both SARS-CoV-2 and RaTG13, although RaACE2 binding to the receptor binding domain (RBD) of SARS-CoV-2 is markedly weaker than that of hACE2. We further evaluated the receptor activities of ACE2s from additional 16 diverse animal species for RaTG13, SARS-CoV, and SARS-CoV-2 in terms of S protein binding, membrane fusion, and pseudovirus entry. We found that the RaTG13 spike (S) protein is significantly less fusogenic than SARS-CoV and SARS-CoV-2, and seven out of sixteen different ACE2s function as entry receptors for all three viruses, indicating that all three viruses might have broad host rages. Of note, RaTG13 S pseudovirions can use mouse, but not pangolin ACE2, for virus entry, whereas SARS-CoV-2 S pseudovirions can use pangolin, but limited for mouse, ACE2s enter cells. Mutagenesis analysis revealed that residues 484 and 498 in RaTG13 and SARS-CoV-2 S proteins play critical roles in recognition of mouse and human ACE2. Finally, two polymorphousRhinolophous sinicusbat ACE2s showed different susceptibilities to virus entry by RaTG13 and SARS-CoV-2 S pseudovirions, suggesting possible coevolution. Our results offer better understanding of the mechanism of coronavirus entry, host range, and virus-host coevolution.
Elephant endotheliotropic herpesvirus is omnipresent in elephants in European zoos and an Asian elephant range country
Authors: Hoornweg Tabitha E. , Schaftenaar Willem , Maurer Gilles , van den Doel Petra B. , Molenaar Fieke M. , Chamouard-Galante Alexandre , Vercammen Francis , Rutten Victor P.M.G. , de Haan Cornelis A.M.
Published: November 2020

Elephant endotheliotropic herpesviruses (EEHVs) are a group of evolutionary divergent herpesviruses that may cause acute, often lethal, hemorrhagic disease (EEHV-HD) in young elephants. Although EEHV was first discovered over 20 years ago, its prevalence in different elephant populations is still largely unknown, partially due to the lack of readily available, sensitive serological assays. In order to improve diagnostic tools for the detection of EEHV infections and to obtain insight in its spread among elephants, we developed novel ELISAs focusing on EEHV1A gB and gH/gL as antigens. Performance of the ELISAs was assessed using sera taken from 41 European zoo elephants and 69 semi-captive elephants from Laos, one of the Asian elephant range countries. Sera from all (sub)adult animals tested (≥5 years of age) showed high reactivity with both gB and gH/gL, whereas reactivity towards the antigens was generally lower for sera of juvenile animals (1 > 5 years). Only one (juvenile) animal, which was sampled directly after succumbing to EEHV-HD, was found to be seronegative for EEHV. The two other EEHV-HD cases tested showed low antibody levels, suggesting that all three cases died upon a primary EEHV infection. Direct comparison with another EEHV-specific ELISA previously used in two large serosurveys, showed that EEHV prevalence was underestimated before, likely due to aberrant folding of the antigen used. In conclusion, our study suggests that essentially all (semi-)captive (sub)adult elephants in European zoos and in Laos carry EEHV, and that young elephants with low antibody levels are at risk of dying from EEHV-HD.

Importance

Over the last 30 years, nearly 20% of all Asian elephants born in Western zoos succumbed to acute hemorrhagic disease caused by elephant endotheliotropic herpesvirus (EEHV-HD). Yet, the prevalence of EEHV in captive and wild elephant populations is still largely unknown, mainly due to the lack of readily available, sensitive serological assays. For this study two highly sensitive EEHV-specific ELISAs were developed. Using these assays, it was shown that nearly all elephants tested were seropositive for EEHV, with highest antibody levels detected in (sub)adult elephants. In contrast, antibody levels in EEHV-HD cases were very low or non-detectable. Lack of antibodies may thus be a risk factor for developing severe disease. As the novel ELISAs are low-tech in nature, these assays may easily be disseminated to local laboratories in zoos and elephant range countries in order to determine EEHV serostatus of individual animals or complete herds and (wild) populations.
A LAMP-based microfluidic chip for rapid detection of pathogen in Cryptococcal meningitis
Authors: Tian Yueru , Zhang Tong , Guo Jian , Lu Huijun , Yao Yuhan , Chen Xi , Zhang Xinlian , Sui Guodong , Guan Ming
Published: November 2020

Cryptococcal meningitis (CM) is a global threat with significant attributable morbidity and mortality. Information on integrated detection for CM diagnosis is still limited. This is mainly due to the presence of a large polysaccharide capsule and the tough cell wall ofCryptococcus, which makes it difficult to extract nucleic acids on the chip. In this study, we developed a LAMP-based microfluidic chip for rapid detection of pathogen in CM. We adopted 4 duplicate filtration membrane structures to improve target capture and simplify the enrichment process, and combined lyticase digestion and thermal alkaline lysisto optimize the nucleic acid extraction ofCryptococcuson the chip, and selected a portable UVA flashlight to shine the LAMP products to obtain the visual detection results which could be observed by the naked eye. This microfluidic chip, integrating sampleCryptococcusenrichment, nucleic acid extraction and LAMP detection unit, streamlined the operation process and reduced the exposure risk of directly handling cryptococcal samples. It did not require any additional instruments and demonstrated a rapid, reliable, as well as high-efficiency approach. It truly realized the “sample-to-answer” application and could be easily used for clinical cryptococcal prediagnosis.
Projections and fractional dynamics of COVID-19 with optimal control analysis
Authors: Nabi Khondoker Nazmoon , Kumar Pushpendra , Erturk Vedat Suat
Published: November 2020

When the entire world is eagerly waiting for a safe, effective and widely available COVID-19 vaccine, un-precedented spikes of new cases are evident in numerous countries. To gain a deeper understanding about the future dynamics of COVID-19, a compartmental mathematical model has been proposed in this paper incorporating all possible non-pharmaceutical intervention policies. Model parameters have been calibrated using sophisticated trust-region-reflective algorithm and short-term projection results have been illustrated for Argentina, Bangladesh, Brazil, Colombia and India. Control reproduction numbers (ℛc) have been calculated in order to get insights about the current epidemic scenario in the above-mentioned countries. Forecasting results depict that the aforesaid countries are having downward trends in daily COVID-19 cases. However, it is highly recommended to use efficacious face coverings and maintain strict physical distancing, as the pandemic is not over in any country. Global sensitivity analysis enlightens the fact that efficacy of face coverings is the most significant parameter, which could significantly control the transmission dynamics of the novel coronavirus compared to other non-pharmaceutical measures. In addition, reduction in effective contact rate with isolated patients is also essential in bringing down the epidemic threshold (ℛc) below unity. All necessary graphical simulations have been performed with the help of Caputo-Fabrizio fractional derivatives. In addition, optimal control problem for fractional system has been designed and the existence of unique solution has also been showed by using Picard-Lindelof technique. Finally, the unconditionally stability of the given fractional numerical technique has been proved.
High content Image Analysis to study phenotypic heterogeneity in endothelial cell monolayers
Authors: Chesnais Francois , Hue Jonas , Roy Errin , Branco Marco , Stokes Ruby , Pellon Aize , Le Caillec Juliette , Elbahtety Eyad , Battilocchi Matteo , Danovi Davide , Veschini Lorenzo
Published: November 2020

Endothelial cells (EC) are heterogeneous across and within tissues, reflecting distinct, specialised functions. EC heterogeneity has been proposed to underpin EC plasticity independently from vessel microenvironments. However, heterogeneity driven by contact-dependent or short-range cell-cell crosstalk cannot be evaluated with single cell transcriptomic approaches as spatial and contextual information is lost. Nonetheless, quantification of EC heterogeneity and understanding of its molecular drivers is key to developing novel therapeutics for cancer, cardiovascular diseases and for revascularisation in regenerative medicine.

Here, we developed an EC profiling tool (ECPT) to examine individual cells within intact monolayers. We used ECPT to characterise different phenotypes in arterial, venous and microvascular EC populations. In line with other studies, we measured heterogeneity in terms of cell cycle, proliferation, and junction organisation. ECPT uncovered a previously under-appreciated single-cell heterogeneity in NOTCH activation. We correlated cell proliferation with different NOTCH activation states at the single cell and population levels. The positional and relational information extracted with our novel approach is key to elucidating the molecular mechanisms underpinning EC heterogeneity.

Summary statement

Endothelial cells heterogeneity is key to complex collective functions and cell behaviour. We developed a novel image based endothelial cell profiling tool and quantified heterogeneity in NOTCH signalling in monolayers.
Immunogenicity and protective efficacy of one- and two-dose regimens of the Ad26.COV2.S COVID-19 vaccine candidate in adult and aged rhesus macaques
Authors: Solforosi Laura , Kuipers Harmjan , Huber Sietske K. Rosendahl , van der Lubbe Joan E.M. , Dekking Liesbeth , Czapska-Casey Dominika N. , Gil Ana Izquierdo , Baert Miranda R.M. , Drijver Joke , Vaneman Joost , van Huizen Ella , Choi Ying , Vreugdenhil Jessica , Kroos Sanne , de Wilde Adriaan H. , Kourkouta Eleni , Custers Jerome , Dalebout Tim J. , Myeni Sebenzile K. , Kikkert Marjolein , Snijder Eric J. , Barouch Dan H. , Böszörményi Kinga P. , Stammes Marieke A. , Kondova Ivanela , Verschoor Ernst J. , Verstrepen Babs E. , Koopman Gerrit , Mooij Petra , Bogers Willy M.J.M. , van Heerden Marjolein , Muchene Leacky , Tolboom Jeroen T.B.M. , Roozendaal Ramon , Schuitemaker Hanneke , Wegmann Frank , Zahn Roland C.
Published: November 2020

Safe and effective coronavirus disease (COVID)-19 vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one- and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged non-human primates (NHP). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared to a single dose. In one-dose regimens neutralizing antibody responses were stable for at least 14 weeks, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and Th1 skewed cellular responses in aged NHP that were comparable to adult animals. Importantly, aged Ad26.COV2.S-vaccinated animals challenged 3 months post -dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. These are the first NHP data showing COVID-19 vaccine protection against the SARS-CoV-2 spike G614 variant and support ongoing clinical Ad26.COV2.S development.

COVID-19 vaccines are urgently needed and while single-dose vaccines are preferred, two-dose regimens may improve efficacy. We show improved Ad26.COV2.S immunogenicity in non-human primates after a second vaccine dose, while both regimens protected aged animals against SARS-CoV-2 disease.
USP28 deletion and small molecule inhibition destabilises c-Myc and elicits regression of squamous cell lung carcinoma
Authors: Ruiz E. Josue , Pinto-Fernandez Adan , Turnbull Andrew P. , Lan Linxiang , Charlton Thomas M. , Scott Hannah Claire , Damianou Andreas , Vere George , Riising Eva M. , Da Costa Clive , Krajewski Wojciech W. , Guerin David , Kearns Jeffrey , Ioannidis Stephanos , Katz Marie , McKinnon Crystal , O’Connell Jonathan C. , Moncaut Natalia , Rosewell Ian , Nye Emma , Jones Neil , Heride Claire , Gersch Malte , Wu Min , Dinsmore Christopher J. , Hammonds Tim R. , Kim Sunkyu , Komander David , Urbé Sylvie , Clague Michael J. , Kessler Benedikt M. , Behrens Axel
Published: November 2020

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient 5-year survival rate is less than 5%. The ubiquitin specific protease 28 (USP28) has been implicated in tumorigenesis through its stabilization of the oncoprotein c-MYC. Here, we show that genetic inactivation ofUsp28induced regression of established murine LSCC lung tumors. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-Myc proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumors and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.
Development of a mass-spectrometry based method for the identification of thein vivowhole blood and plasma ADP-ribosylomes
Authors: Lüthi Stephanie C. , Howald Anna , Nowak Kathrin , Graage Robert , Bartolomei Giody , Neupert Christine , Sidler Xaver , Pedrioli Deena M. Leslie , Hottiger Michael O.
Published: November 2020

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modification states of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove these modification after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we discovered that plasma phosphodiesterases and/or ADP-ribosylhydrolases rapidly demodify in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a novel mass-spectrometry based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.
Shifting gradients of macroscale cortical organization mark the transition from childhood to adolescence
Authors: Dong Hao-Ming , Margulies Daniel S. , Zuo Xi-Nian , Holmes Avram J.
Published: November 2020

The transition from childhood to adolescence is marked by pronounced shifts in brain structure and function that coincide with the development of physical, cognitive, and social abilities. Prior work in adult populations has characterized the topographical organization of cortex, revealing macroscale functional gradients that extend from unimodal (somato/motor and visual) regions through the cortical association areas that underpin complex cognition in humans. However, the presence of these core functional gradients across development as well as their maturational course have yet to be established. Here, leveraging 378 resting-state fMRI scans from 190 healthy individuals aged 6-17 years, we demonstrate that the transition from childhood to adolescence is reflected in the gradual maturation of gradient patterns across the cortical sheet. In children, the overarching organizational gradient is anchored within unimodal cortex, between somato/motor and visual territories. Conversely, in adolescence the principal gradient of connectivity transitions into an adult-like spatial framework, with the default network at the opposite end of a spectrum from primary sensory and motor regions. The observed gradient transitions are gradually refined with age, reaching a sharp inflection point in 13- and 14-year-olds. Functional maturation was nonuniformly distributed across cortical networks. Unimodal networks reached their mature positions early in development, while association regions, in particular medial prefrontal cortex, reached a later peak during adolescence. These data reveal age-dependent changes in the macroscale organization of cortex and suggest the scheduled maturation of functional gradient patterns may be critically important for understanding how cognitive and behavioral capabilities are refined across development.

Significance

Human abilities and behavior change dramatically across development, emerging from a cascade of hierarchical changes in brain circuitry. Here, we describe age-dependent shifts in the macroscale functional organization of cortex in childhood and adolescence. The characterization of functional connectivity patterns in children revealed an overarching organizational framework anchored within unimodal cortex, between somato/motor and visual regions. Conversely, in adolescents we observed a transition into an adult-like gradient that situates the default network at the opposite end of a spectrum from primary sensory and motor regions. This spatial framework emerged gradually with age, reaching a sharp inflection point at the transition from childhood to adolescence. These data reveal the presence of a developmental change from a functional motif first dominated by the distinction between sensory and motor systems, and then balanced through interactions with later-maturing aspects of association cortex that support more abstract cognitive functions.
Multiplexed single-cell profiling of chromatin states at genomic loci by expansion microscopy
Authors: Woodworth Marcus A. , Ng Kenneth K.H. , Halpern Aaron R. , Pease Nicholas A. , Nguyen Phuc H.B. , Kueh Hao Yuan , Vaughan Joshua C.
Published: November 2020

Proper regulation of genome architecture and activity is essential for the development and function of multicellular organisms. Histone modifications, acting in combination, specify these activity states at individual genomic loci. However, the methods used to study these modifications often require either a large number of cells or are limited to targeting one histone mark at a time. Here, we developed a new method called Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE) that uses Expansion Microscopy (ExM) to visualize and quantify multiple histone modifications at non-repetitive genomic regions in single cells at a spatial resolution of ~75 nm. Using SCEPTRE, we distinguished multiple histone modifications at a single housekeeping gene, quantified histone modification levels at multiple developmentally-regulated genes in individual cells, and identified a relationship between histone H3K4 trimethylation and the loading of paused RNA polymerase II at individual loci. Thus, SCEPTRE enables multiplexed detection of combinatorial chromatin states at single genomic loci in single cells.
Neuroglian regulatesDrosophilaintestinal stem cell proliferation through enhanced signaling via the Epidermal Growth Factor Receptor
Authors: Resnik-Docampo Martin , Cunningham Kathleen M. , Ruvalcaba S. Mateo , Choi Charles , Sauer Vivien , Jones D. Leanne
Published: November 2020

TheDrosophila melanogasterintestine is an excellent system for elucidating mechanisms regulating stem cell behavior under homeostatic conditions or in response to injury, stress, or ageing. Here we show that the septate junction (SJ) protein Neuroglian (Nrg) is expressed in intestinal stem cells (ISCs) and daughter enteroblasts (EBs) within the fly midgut, the equivalent of the mammalian small intestine. Although Nrg localizes to the plasma membrane, SJs are not present between ISC/EBs, suggesting Nrg plays a different role in this tissue. Generation of ISCs homozygous for a null allele ofNrgrevealed that Nrg is required for ISC proliferation in young flies, and depletion of Nrg from ISCs/EBs was able to suppress the increase in ISC proliferation with age. Conversely, overexpression ofNrgin ISC/EBs was sufficient to drive ISC proliferation, leading to an increase in cells expressing ISC/EB markers. In addition, we observed an increase in EGFR activation. Genetic epistasis experiments revealed that Nrg acts upstream of EGFR in the midgut to regulate ISC proliferation. As Nrg function is highly conserved in mammalian systems, our work characterizing the role of Nrg in the intestine has implications for the etiology and treatment of intestinal disorders due to altered ISC behavior.
The RAS GTPase RIT1 compromises mitotic fidelity through spindle assembly checkpoint suppression
Authors: Cuevas-Navarro Antonio , Van Richard , Cheng Alice , Urisman Anatoly , Castel Pau , McCormick Frank
Published: November 2020

The spindle assembly checkpoint (SAC) is an evolutionarily conserved safety mechanism that maintains genomic stability. However, despite the understanding of the fundamental mechanisms that control the SAC, it remains unknown how signaling pathways directly interact with and regulate the mitotic checkpoint activity. In response to extracellular stimuli, a diverse network of signaling pathways involved in cell growth, survival, and differentiation are activated and this process is prominently regulated by the Ras family of GTPases. Here we show that RIT1, a Ras-related GTPase, is essential for timely progression through mitosis and proper chromosome segregation. Furthermore, pathogenic levels of RIT1 silence the SAC, accelerate transit through mitosis, and promote chromosome segregation errors through direct association with SAC proteins MAD2 and p31comet. Our results highlight a unique function of RIT1 compared to other Ras GTPases and elucidate a direct link between a signaling pathway and the SAC through a novel regulatory mechanism.

Graphical Abstract
Random Forest Regression models for Lactation and Successful Insemination in Holstein Friesian cows
Authors: Oluoch Lillian , Stachó László , Viharos László , Viharos Andor , Mikó Edit
Published: November 2020

To overcome well-known difficulties in establishing reliable models based on large data sets, the Random Forest Regression (RFR) method is applied to study economical breeding and milk production of dairy cows. As for the features of RFR, there are several positive experiences in various areas of applications supporting that with RFR one can achieve reliable model predictions for industrial production of any product providing a useful base for decisions. In this study, a data set of a period of ten years including about eighty thousand cows was analysed by means of RFR. Ranking of production control parameters is obtained, the most important explanatory variables are found by computing the variances of the target variable on the sets created during the training phases of the RFR. Predictions are made for the milk production and the conception of the calves with high accuracy on given data and simulations are used to investigate prediction accuracy. This paper is primarily concerned with the mathematical aspects of a forthcoming work focused on the agricultural viewpoints. As for future mathematical research plans, the results will be compared with models based on factor analysis and linear regression.
Newborns’ sensitivity to speed changes as a building block for animacy perception
Authors: Di Giorgio Elisa , Lunghi Marco , Vallortigara Giorgio , Simion Francesca
Published: November 2020

The human visual system can discriminate between animate beingsvs. inanimate objects on the basis of some kinematic cues, such as starting from rest and speed changes by self-propulsion. The ontogenetic origin of such capability is still under debate. Here we investigate for the first time whether newborns manifest an attentional bias toward objects that abruptly change their speed along a trajectory as contrasted with objects that move at a constant speed. To this end, we systematically manipulated the motion speed of two objects. An object that moves with a constant speed was contrasted with an object that suddenly increases (Experiment 1) or with one that suddenly decreases its speed (Experiment 2). When presented with a single speed change, newborns did not show any visual preference. However, newborns preferred an object that abruptly increases and then decreases its speed (Experiment 3), but they did not show any visual preference for the reverse sequence pattern (Experiment 4). Overall, results are discussed in line with the hypothesis of the existence of attentional biases in newborns that trigger their attention towards some visual cues of motion that characterized animate perception in adults.
The histone deacetylase HDA15 interacts with MAC3A and MAC3B to regulate intron retention of ABA-responsive genes
Authors: Tu Yi-Tsung , Chen Chia-Yang , Huang Yi-Sui , Yen Ming-Ren , Hsieh Jo-Wei Allison , Chen Pao-Yang , Wu Keqiang
Published: November 2020

Histone deacetylases (HDAs) play an important role in transcriptional regulation involved in multiple biological processes. In this study, we investigate the function of HDA15 in abscisic acid (ABA) responses. Immunopurification coupled with mass spectrometry-based proteomics was used to identify the HDA15 interacting proteins. We found that HDA15 can interact with the core subunits of MOS4-Associated Complex (MAC), MAC3A and MAC3B. In addition, ABA enhances the interaction of HDA15 with MAC3B.hda15andmac3a/mac3bmutants are ABA-insensitive in seed germination and hyposensitive to salinity. RNA sequencing (RNA-seq) analysis demonstrate that HDA15 and MAC3A/MAC3B not only affect the expression of ABA-related genes, but also regulate ABA-responsive intron retention (IR). Furthermore, HDA15 and MAC3A/MAC3B reduce the histone acetylation level of the genomic regions near ABA-responsive IRs. Our studies uncovered the role of histone deacetylation in ABA-mediated splicing regulation and identified that HDA15-MAC3A/MAC3B acts as an important regulation module to mediate splicing of introns in ABA responses.

One Sentence Summary

HDA15 and MAC3A/MAC3B coregulate intron retention and reduce the histone acetylation level of the genomic regions near ABA-responsive retained introns.
Targeting G protein-coupled receptor-17 (GPR17) upregulation in paediatric diffuse midbrain gliomas leads to altered phenotype and susceptibility to therapies
Authors: Loveson Katie F. , Fillmore Helen L.
Published: November 2020

Paediatric diffuse midline glioma (pDMG) also known as Diffuse intrinsic pontine gliomas (DIPG) is an incurable, aggressive childhood brain malignancy, that arises in a region- and age-specific nature. The underlying pathophysiology suggests dysregulation of postnatal neurodevelopmental processes causing aborted cell differentiation. The cell of origin is unclear, but data suggests an oligodendrocytic lineage (OPC), supported by the over-expression of transcription factors such as Olig1 and Olig2 in 80% of DIPG cases.

In-depth bioinformatics and principal component analyses (PCA) of genes involved in brain development and pDMG support reports of OPC gene dysregulation and led to the identification of the G-protein coupled receptor 17 (GPR17) and its association with pDMG. GPR17 mRNA and protein expression was confirmed in all pDMG cell lines tested. Using a well-characterised agonist (MDL 299,51) and antagonist (HAMI3379) to modulate GPR17 function in pDMG cell lines resulted in phenotypic and genomic changes as well as in cell growth and migration. HAMI3379, a GPR17 specific antagonist resulted in a significant reduction in GPR17 mRNA and protein expression (p<0.006) and a significant reduction in migration (p<0.0025). When pDMG cells were pre-treatment with HAMI3379 in combination with known cytotoxic agents (Bleomycin, a radiation mimic, Panobinostat or Vincristine), there was a decrease in cell viability compare to cytotoxic agent alone. There are no current effective therapies for pDMG patients and the ability of blocking GPR17 function to enhance sensitivity to standard therapies is appealing and warrants further investigation.
Population structure and transmission ofMycobacterium bovisin Ethiopia
Authors: Almaw Gizat , Mekonnen Getnet Abie , Mihret Adane , Aseffa Abraham , Taye Hawult , Conlan Andrew JK , Gumi Balako , Zewude Aboma , Aliy Abde , Tamiru Mekdes , Olani Abebe , Lakew Matios , Sombo Melaku , Gebre Solomon , Diguimbaye Colette , Hilty Markus , Fané Adama , Müller Borna , Hewinson R Glyn , Ellis Richard J , Nunez-Garcia Javier , Palkopoulou Eleftheria ,   the ETHICOBOTS consortium, Abebe Tamrat , Ameni Gobena , Parkhill Julian , Wood James LN , Berg Stefan , van Tonder Andries J
Published: November 2020

Bovine tuberculosis (bTB) is endemic in cattle in Ethiopia, a country that hosts the largest national cattle herd in Africa. The intensive dairy sector, most of which is peri-urban, has the highest prevalence of disease. Previous studies in Ethiopia have demonstrated that the main cause isMycobacterium bovis(M. bovis), which has been investigated using conventional molecular tools including deletion typing, spoligotyping and Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). Here we use whole genome sequencing (WGS) to examine the population structure ofM. bovisin Ethiopia. A total of 134M. bovisisolates were sequenced including 128 genomes from 85 mainly dairy cattle and six genomes isolated from humans, originating from 12 study sites across Ethiopia. These genomes provided a good representation of the previously described population structure ofM. bovis, based on spoligotyping and demonstrated that the population is dominated by the clonal complexes African 2 (Af2) and European 3 (Eu3). A range of within-host diversity was observed amongst the isolates and evidence was found for both short- and long-distance transmission. Detailed analysis of available genomes from the Eu3 clonal complex combined with previously published genomes revealed two distinct introductions of this clonal complex into Ethiopia between 1950 and 1987, likely from Europe. This work is important to help better understand bTB transmission in cattle in Ethiopia and can potentially inform national strategies for bTB control in Ethiopia and beyond.
TC10 regulates breast cancer invasion and metastasis by controlling membrane type-1 matrix metalloproteinase at invadopodia
Authors: Hülsemann M. , Donnelly S.K. , Verkhusha P.V. , Mao S.P.H. , Segall J.E. , Hodgson L.
Published: November 2020

During breast cancer metastasis, cancer cell invasion is driven by actin-rich protrusions called invadopodia, which mediate the extracellular matrix degradation required for the success of the invasive cascade. In this study, we demonstrated that TC10, a member of a Cdc42 subfamily of p21 small GTPases, regulates the membrane type 1 matrix metalloproteinase (MT1-MMP)-driven extracellular matrix degradation at invadopodia. We show that TC10 is required for the plasma membrane surface exposure of MT1-MMP at invadopodia. By utilizing our new Förster resonance energy transfer (FRET) biosensor, we demonstrated the p190RhoGAP-dependent regulation of spatiotemporal TC10 activity at invadopodia. We identified a pathway that regulates TC10 activity and function at invadopodia through the activation of p190RhoGAP and the downstream interacting effector Exo70 at the invadopodia sites. Our findings reveal the role of a previously unknown regulator of vesicular fusion at invadopodia, TC10, on the invasive potential of breast cancer cells during invasion and metastasis.
Quantitative analysis of Histone modifications in gene silencing
Authors: Wu Kenneth , Dhillon Namrita , Du Kelvin , Kamakaka Rohinton T.
Published: November 2020

Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. This transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown and we developed a method to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state. We show that loss of silencing required between 50% and 75% of the unacetylated histones to be replaced with acetylated histone mimics. The precise levels of unacetylated nucleosomes required varied from locus to locus and was influenced by both silencer strength and UAS enhancer/promoter strength. Simple calculations suggest that an approximately 50% reduction in the ability of acetylases to acetylate individual nucleosomes across a large domain may be sufficient to generate a transcriptionally silent region in the nucleus.
Preclinical evaluation of Imatinib does not support its use as an antiviral drug against SARS-CoV-2
Authors: Touret Franck , Driouich Jean-Sélim , Cochin Maxime , Petit Paul Rémi , Gilles Magali , Barthélémy Karine , Moureau Grégory , Mahon Francois-Xavier , Malvy Denis , Solas Caroline , de Lamballerie Xavier , Nougairède Antoine
Published: November 2020

Following the emergence of SARS-CoV-2, the search for an effective and rapidly available treatment was initiated worldwide based on repurposing of available drugs. Previous reports described the antiviral activity of certain tyrosine kinase inhibitors (TKIs) targeting the Abelson kinase 2 against pathogenic coronaviruses. Imatinib, one of them, has more than twenty years of safe utilization for the treatment of hematological malignancies. In this context, Imatinib was rapidly evaluated in clinical trials against Covid-19. Here, we present the pre-clinical evaluation of Imatinib in multiple models. Our results indicated that Imatinib and another TKI, the Masitinib, exhibit an antiviral activity in VeroE6 cells. However, Imatinib was inactive in a reconstructed bronchial human airway epithelium model.In vivo, Imatinib therapy failed to impair SARS-CoV-2 replication in a golden Syrian hamster model despite high concentrations in plasma and in the lung. Overall, these results do not support the use of Imatinib and similar TKIs as antivirals in the treatment of Covid-19.
“Multiplex” rheostat positions cluster around allosterically critical regions of the lactose repressor protein
Authors: Bantis Leonidas E. , Parente Daniel J. , Fenton Aron W. , Swint-Kruse Liskin
Published: November 2020

Amino acid variation at “rheostat” positions provides opportunity to modulate various aspects of protein function – such as binding affinity or allosteric coupling – across a wide range. Previously a subclass of “multiplex” rheostat positions was identified at which substitutions simultaneously modulated more than one functional parameter. Using the Miller laboratory’s dataset of ∼4000 variants of lactose repressor protein (LacI), we compared the structural properties of multiplex rheostat positions with (i) “single” rheostat positions that modulate only one functional parameter, (ii) “toggle” positions that follow textbook substitution rules, and (iii) “neutral” positions that tolerate any substitution without changing function. The combined rheostat classes comprised >40% of LacI positions, more than either toggle or neutral positions. Single rheostat positions were broadly distributed over the structure. Multiplex rheostat positions structurally overlapped with positions involved in allosteric regulation. When their phenotypic outcomes were interpreted within a thermodynamic framework, functional changes at multiplex positions were uncorrelated. This suggests that substitutions lead to complex changes in the underlying molecular biophysics. Bivariable and multivariable analyses of evolutionary signals within multiple sequence alignments could not differentiate single and multiplex rheostat positions. Phylogenetic analyses – such as ConSurf – could distinguish rheostats from toggle and neutral positions. Multivariable analyses could also identify a subset of neutral positions with high probability. Taken together, these results suggest that detailed understanding of the underlying molecular biophysics, likely including protein dynamics, will be required to discriminate single and multiplex rheostat positions from each other and to predict substitution outcomes at these sites.
Single-colony sequencing reveals phylosymbiosis, co-phylogeny, and horizontal gene transfer between the cyanobacteriumMicrocystisand its microbiome
Authors: Pérez-Carrascal Olga M. , Tromas Nicolas , Terrat Yves , Moreno Elisa , Giani Alessandra , Marques Laisa Corrêa Braga , Fortin Nathalie , Shapiro B. Jesse
Published: November 2020

Cyanobacteria from the genusMicrocystiscan form large mucilaginous colonies with attached heterotrophic bacteria – their microbiome. However, the nature of the relationship betweenMicrocystisand its microbiome remains unclear. Is it a long-term, evolutionarily stable association? Which partners benefit? Here we report the genomic diversity of 109 individualMicrocystiscolonies – including cyanobacteria and associated bacterial genomes – isolatedin situand without culture from Lake Champlain, Canada and Pampulha Reservoir, Brazil. We found 14 distinctMicrocystisgenotypes from Canada, of which only two have been previously reported, and four genotypes specific to Brazil.Microcystisgenetic diversity was much greater between than within colonies, consistent with colony growth by clonal expansion rather than aggregation ofMicrocystiscells. We also identified 72 bacterial species in the microbiome. EachMicrocystisgenotype had a distinct microbiome composition, and more closely-related genotypes had more similar microbiomes. This pattern of phylosymbiosis could be explained by co-phylogeny in two out of the nine most prevalent associated bacterial genera,RoseomonasandRhodobacter, suggesting long-term evolutionary associations.RoseomonasandRhodobactergenomes encode functions which could complement the metabolic repertoire ofMicrocystis, such as cobalamin and carotenoid biosynthesis, and nitrogen fixation. In contrast, other colony-associated bacteria showed weaker signals of co-phylogeny, but stronger evidence of horizontal gene transfer withMicrocystis. These observations suggest that acquired genes are more likely to be retained in both partners (Microcystisand members of its microbiome) when they are loosely associated, whereas one gene copy is sufficient when the association is physically tight and evolutionarily long-lasting.
Assessment of protein-protein interfaces in cryo-EM derived assemblies
Authors: Malhotra Sony , Joseph Agnel Praveen , Thiyagalingam Jeyan , Topf Maya
Published: November 2020

Structures of macromolecular assemblies derived from cryo-EM maps often contain errors that become more abundant with decreasing resolution. Despite efforts in the cryo-EM community to develop metrics for the map and atomistic model validation, thus far, no specific scoring metrics have been applied systematically to assess the interface between the assembly subunits. Here, we have assessed protein-protein interfaces in macromolecular assemblies derived by cryo-EM. To this end, we developed PI-score, a density-independent machine learning-based metric, trained using protein-protein interfaces’ features in high-resolution crystal structures. Using PI-score, we were able to identify errors at interfaces in the PDB-deposited cryo-EM structures (including SARS-CoV-2 complexes) and in the models submitted for cryo-EM targets in CASP13 and the EM model challenge. Some of the identified errors, especially at medium-to-low resolution structures, were not captured by density-based assessment scores. Our method can therefore provide a powerful complementary assessment tool for the increasing number of complexes solved by cryo-EM.
Fibrillar α-synuclein induces neurotoxic astrocyte activation via RIP kinase signaling and NF-κB
Authors: Chou Tsui-Wen , Chang Nydia P , Krishnagiri Medha , Patel Aisha P , Atkins Colm , Daniels Brian P.
Published: November 2020

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by death of midbrain dopamine neurons. The pathogenesis of PD is poorly understood, though misfolded and/or aggregated forms of the protein α-synuclein have been implicated in several neurodegenerative disease processes, including neuroinflammation and astrocyte activation. Astrocytes in the midbrain play complex roles during PD, initiating both harmful and protective processes that vary over the course of disease. However, despite their significant regulatory roles during neurodegeneration, the cellular and molecular mechanisms that promote pathogenic astrocyte activity remain mysterious. Here, we show that α-synuclein preformed fibrils (PFFs) induce pathogenic activation of human midbrain astrocytes, marked by inflammatory transcriptional responses, downregulation of phagocytic function, and conferral of neurotoxic activity. These effects required the necroptotic kinases RIPK1 and RIPK3, but were independent of MLKL and necroptosis. Instead, both transcriptional and functional markers of astrocyte activation occurred via RIPK-dependent activation of NF-κB signaling. Our study identifies a previously unknown function for α-synuclein in promoting neurotoxic astrocyte activation, as well as new cell death-independent roles for RIP kinase signaling in the regulation of glial cell biology and neuroinflammation. Together, these findings highlight previously unappreciated molecular mechanisms of pathologic astrocyte activation and neuronal cell death with implications for Parkinsonian neurodegeneration.
Palmitoylation Couples DLK to JNK3 to Facilitate Pro-degenerative Axon-to-Soma Signaling
Authors: Niu Jingwen , Holland Sabrina M. , Ketschek Andrea , Collura Kaitlin M. , Hayashi Takashi , Gallo Gianluca , Thomas Gareth M.
Published: November 2020

Dual Leucine-zipper Kinase (DLK, a MAP3K) mediates neuronal responses to diverse injuries and insults via c-Jun N-terminal Kinase (JNK) family Mitogen-activated Protein Kinases (MAPKs). It is unclear why DLK couples to JNKs in mammalian neurons versus other MAPKs, especially because some invertebrate DLK orthologs couple instead to the related p38 family MAPKs. Here we identify two mechanisms that potentially explain this DLK-JNK coupling. First, neural-specific JNK3, but not p38-MAPK, catalyzes positive feedback phosphorylation of DLK that further activates DLK and locks the DLK-JNK3 module in a highly active state. Furthermore, the pro-degenerative JNK2 and JNK3, but not the related JNK1, are endogenously palmitoylated. Moreover, palmitoylation targets both DLK and JNK3 to the same axonal vesicles and JNK3 palmitoylation is essential for pro-degenerative axonal retrograde signalingin vivo. These findings provide insights into DLK-JNK signaling relevant to multiple neuropathological conditions and answer long-standing questions regarding the selective pro-degenerative roles of JNK2/3.
Rab27 GTPases regulate alpha-synuclein uptake, cell-to-cell transmission, and toxicity
Authors: Underwood Rachel , Wang Bing , Pathak Aneesh , Volpicelli-Daley Laura , Yacoubian Talene A.
Published: November 2020

Parkinson’s disease and Dementia with Lewy Bodies are two common neurodegenerative disorders marked by proteinaceous aggregates composed primarily of the protein α-synuclein. α-Synuclein is hypothesized to have prion-like properties, by which misfolded α-synuclein induces the pathological aggregation of endogenous α-synuclein and neuronal loss. Rab27a and Rab27b are two highly homologous Rab GTPases that regulate α-synuclein secretion, clearance, and toxicityin vitro. In this study, we tested the impact of Rab27a/b on the transmission of pathogenic α-synuclein. Double knockout of both Rab27 isoforms eliminated α-synuclein aggregation and neuronal toxicity in primary cultured neurons exposed to fibrillary α-synuclein.In vivo, Rab27 double knockout mice lacked fibril-induced α-synuclein inclusions, dopaminergic neuron loss, and behavioral deficits seen in wildtype mice with fibril-induced inclusions. Studies using AlexaFluor488-labeled α-synuclein fibrils revealed that Rab27a/b knockout prevented α-synuclein internalization without affecting bulk endocytosis. Rab27a/b knockout also blocked the cell-to-cell spread of α-synuclein pathology in multifluidic, multichambered devices. This study provides critical insight into the role of Rab GTPases in Parkinson’s disease and identifies Rab27s as key players in the progression of synucleinopathies.
Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome
Authors: Enwerem Isioma I.I. , Elrod Nathan D. , Chang Chung-Te , Lin Ai , Ji Ping , Bohn Jennifer A. , Levdansky Yevgen , Wagner Eric J. , Valkov Eugene , Goldstrohm Aaron C.
Published: November 2020

Pumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad co-regulation of target mRNAs through the PUM-CNOT repression mechanism.

Functional dissection of the domains of PUM1&2 identified a conserved N-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the mRNA decapping enzyme, DCP2, has an important role in repression by PUM1&2 N-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent mRNA decay pathway.