bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondria Controlled mTORC1 Activation Compartmentalizes Translation Initiation Factor eIF4E to Augment Intracellular Trafficking and Extracellular Export of miRNA in Mammalian Cells
Authors: Chatterjee Susanta , Chakrabarty Yogaditya , Banerjee Saikat , Ghosh Souvik , Bhattacharyya Suvendra N.
Published: May 2020

Defective intracellular trafficking and export of miRNAs has been observed in senescent mammalian cells having impaired mitochondrial potential. Similar to what happens in senescent cells, Uncoupling Protein 2 mediated depolarization of mitochondrial membrane potential results in progressive sequestration of miRNAs with polysomes and lowered release of miRNAs through extracellular vesicles. Supporting importance of mitochondrial membrane potential on miRNAs’ fate determination, impaired miRNA-trafficking process in growth retarded human cells has been found to be reversed in presence of Genipin an inhibitor of Uncoupling Protein 2. Mitochondrial detethering of endoplasmic reticulum in mitochondria depolarized cells, found to be responsible for defective compartmentalization of translation initiation factor eIF4E to ER attached polysomes. It causes retarded translation process of target mRNAs with rER attached polysomes to ensure reduced intracellular trafficking and extracellular export of miRNAs. We have identified a reduced activity of mTORC1 complex in mitochondria defective cells to cause reduced phosphorylation of eIF4E-BP1 to cause retarded eIF-4E targeting to ER attached polysome. Cumulatively, these data suggest intricate involvement of mitochondrial membrane potential and dynamics to determine stability of miRNAs in mammalian cells by affecting sub-cellular locations and export of miRNPs by affecting mTORC1 complex, the regulator of the protein translational machinery.

Significance statement

How the reduced mitochondrial activity in growth retarded cells causes defective miRNA export is an open question. Mitochondrial defects induces a retarded subcellular miRNP trafficking in human cells to cause an upregulation in cellular miRNA content by reducing extracellular vesicle-mediated export of miRNA. We have identified a defective compartmentalization of translation initiation factor eIF4E in mitochondria-ER detethered mammalian cells to cause the retarded intracellular miRNA movement and export Activity of mTORC1 complex, a key regulator of protein translation in mammalian cells, is found to be responsible for ER-compartmentalization of eIF4E. mTORC1 activity reduction in growth retarded and mitochondria detethered cells influences the cell fate by acting on miRNA-mRNA axis. This is a unique way how mitochondrial activity is linked with protein translation and gene repression control in mammalian cells.
The RAC1 activator Tiam1 regulates centriole duplication through controlling PLK4 levels
Authors: Porter Andrew P. , White Gavin R. M. , Ogg Erinn-Lee , Whalley Helen J. , Malliri Angeliki
Published: May 2020

Centriole duplication is tightly controlled to maintain correct centriole number through the cell cycle. A key component of this control is the regulated degradation of PLK4, the master regulator of centriole duplication. Here we show that the Rac1 guanine nucleotide exchange factor (GEF) Tiam1 localises to centrosomes during S-phase, where it is required for maintenance of normal centriole number. Depletion of Tiam1 leads to an increase in centrosomal PLK4, centriole overduplication and ultimately to lagging chromosomes at anaphase and aneuploidy. The effects of Tiam1 depletion can be rescued by re-expression of wild-type Tiam1 and catalytically inactive (GEF*) Tiam1, but not by Tiam1 mutants unable to bind to the F-box protein βTRCP, implying that Tiam1 regulates PLK4 levels through promoting βTRCP-mediated degradation.
Combining auxin-induced degradation and RNAi screening identifies novel genes involved in lipid bilayer stress sensing inCaenorhabditis elegans
Authors: Venz Richard , Korosteleva Anastasiia , Ewald Collin Y.
Published: May 2020

Alteration of the lipid composition of biological membranes interferes with their function and can cause tissue damage by triggering apoptosis. Upon lipid bilayer stress, the endoplasmic reticulum mounts a stress response that is similar to the unfolded protein response. However, only a few genes are known to regulate lipid bilayer stress. Here, we performed a suppressor screen that combined the auxin-inducible degradation (AID) system with conventional RNAi inC. elegansto identify members of the lipid bilayer stress response. AID-mediated knockdown of the mediator MDT-15, a protein required for the upregulation of fatty acid desaturases, caused activation of a lipid bilayer stress sensitive reporters. We screened through almost allC. eleganskinases and transcription factors using RNAi by feeding. We report the identification of 8 genes that have not been implicated previously with lipid bilayer stress before inC. elegans. These suppressor genes includeskn-1/NRF1,2,3 andlet-607/CREB3. Our candidate suppressor genes suggest a network of transcription factors and the integration of multiple tissues for a centralized lipotoxicity response in the intestine. Additionally, we propose and demonstrate the proof-of-concept for combining AID and RNAi as a new screening strategy.
Effects of cellular stress on pigment production inRhodotorula mucilaginosa/alborubescensAJB01 strain from the Caribbean region of Colombia
Authors: Cortes Alejandra Garcia , Vásquez Julián Andres Garcia , Díaz Yani Cristina Aranguren , Castrillón Mauricio Ramirez
Published: May 2020

Considering that a large proportion of industrially used pigments are toxic and potentially mutagenic, with some having carcinogenic effects, it is essential to replace them as well as eliminate them from industrial effluents. The discovery of biopigments has received considerable attention from the industry sector. The main objective of this study was to increase the production of carotenoid pigments in naturally pigmented yeast by subjecting the yeast to various cellular stresses. The fungal strainRhodotorula mucilaginosa/alborubescensAJB01 was isolated from a food sample collected in Barranquilla, Colombia, and one of the pigments produced was β-carotene. This strain was subjected to various stress conditions, including osmotic stress using different salts, physical stress by ultraviolet (UV) light, and light stress using different photoperiods. The optimal growth conditions for carotenoid production were determined to be 1 min of UV light, 0.5 mg/L of magnesium sulfate, and 18:6 h light/dark period, which resulted in a carotenoid yield of 118.3 μg of carotenoid per gram of yeast.
METATRYP v 2.0: Metaproteomic Least Common Ancestor Analysis for Taxonomic Inference Using Specialized Sequence Assemblies - Standalone Software and Web Servers for Marine Microorganisms and Coronaviruses
Authors: Saunders Jaclyn K. , Gaylord David , Held Noelle , Symmonds Nick , Dupont Chris , Shepherd Adam , Kinkade Danie , Saito Mak A.
Published: May 2020

We present METATRYP version-2 software that identifies shared peptides across organisms within environmental metaproteomics studies to enable accurate taxonomic attribution of peptides during protein inference. Improvements include: ingestion of complex sequence assembly data categories (metagenomic and metatranscriptomic assemblies, single cell amplified genomes, and metagenome assembled genomes), prediction of the Least Common Ancestor (LCA) for a peptide shared across multiple organisms, increased performance through updates to the backend architecture, and development of a web portal (https://metatryp.whoi.edu). Major expansion of the marine database confirms low occurrence of shared tryptic peptides among disparate marine microorganisms, implying tractability for targeted metaproteomics. METATRYP was designed for ocean metaproteomics and has been integrated into the Ocean Protein Portal (https://oceanproteinportal.org); however, it can be readily applied to other domains. We describe the rapid deployment of a coronavirus-specific web portal (https://metatryp-coronavirus.whoi.edu/) to aid in use of proteomics on coronavirus research during the ongoing pandemic. A Coronavirus-focused METATRYP database identified potential SARS-CoV-2 peptide biomarkers and indicated very few shared tryptic peptides between SARS-CoV-2 and other disparate taxa, sharing 0.1% peptides or less (1 peptide) with the Influenza A & B pan-proteomes, establishing that taxonomic specificity is achievable using tryptic peptide-based proteomic diagnostic approaches.

Statement of significance

When assigning taxonomic attribution in bottom-up metaproteomics, the potential for shared tryptic peptides among organisms in mixed communities should be considered. The software program METATRYP v 2 and associated interactive web portals enables users to identify the frequency of shared tryptic peptides among taxonomic groups and evaluate the occurrence of specific tryptic peptides within complex communities. METATRYP facilitates phyloproteomic studies of taxonomic groups and supports the identification and evaluation of potential metaproteomic biomarkers.
Antibody recognition of the Pneumovirus fusion protein trimer interface
Authors: Huang Jiachen , Diaz Darren , Mousa Jarrod J.
Published: May 2020

Human metapneumovirus is a leading cause of viral respiratory infection in children, and can cause severe lower respiratory infection in infants, the elderly, and immunocompromised patients. However, there remain no licensed vaccines or specific treatments for hMPV infection. Although the hMPV fusion (F) protein is the sole target of neutralizing antibodies, the immunological properties of hMPV F are still poorly understood. To further define the humoral immune response to the hMPV F protein, we isolated two new human monoclonal antibodies (mAbs), MPV458 and MPV465. Both mAbs are neutralizingin vitroand target a unique antigenic site harbored within the trimeric interface of the hMPV F protein. We determined both MPV458 and MPV465 have higher affinity for monomeric hMPV F than trimeric hMPV F. MPV458 was co-crystallized with hMPV F, and the mAb primarily interacts with an alpha helix on the F2 region of the hMPV F protein. Surprisingly, the major epitope for MPV458 lies within the trimeric interface of the hMPV F protein, suggesting significant breathing of the hMPV F protein must occur for hMPV F protein recognition of the novel epitope. In addition, significant glycan interactions were observed with a somatically mutated light chain framework residue. The data presented identifies a novel epitope on the hMPV F protein for structure-based vaccine design, and provides a new mechanism for human antibody neutralization of viral glycoproteins.
Identification of a persisting bacterial contamination of cell culture asBrevibacterium
Authors: Mahesh P.P , Retnakumar R. J , Mundayoor Sathish
Published: May 2020

Cell culture is an important prerequisite for many of the basic and applied researches in life sciences and contamination is a major problem faced by the researchers in maintaining healthy cell lines. We noted a dot like contamination in our THP1 cell line, which does not make the culture medium turbid as fast as other common contaminants and remains in the culture for many days before it makes the culture unusable. The contaminant was identified as a member of the genusBrevibacteriumand we could find that the antibiotic rifampicin effectively suppresses the growth of the bacterium.
MosMedData: Chest CT Scans with COVID-19 Related Findings Dataset
Authors: Morozov S.P. , Andreychenko A.E. , Pavlov N.A. , Vladzymyrskyy A.V. , Ledikhova N.V. , Gombolevskiy V.A. , Blokhin I.A. , Gelezhe P.B. , Gonchar A.V. , Chernina V.Yu.
Published: May 2020

This dataset contains anonymised human lung computed tomography (CT) scans with COVID-19 related findings, as well as without such findings. A small subset of studies has been annotated with binary pixel masks depicting regions of interests (ground-glass opacifications and consolidations). CT scans were obtained between 1st of March, 2020 and 25th of April, 2020, and provided by municipal hospitals in Moscow, Russia. Permanent link:https://mosmed.ai/datasets/covid19_1110. This dataset is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC-ND 3.0) License.
The use of social media and online dating among individuals with unipolar depression and bipolar disorder
Authors: Rydahl Klara F. K. , Brund Renè B. K. , Medici Clara R. , Hansen Vibeke , Straarup Krista M. N. Straarup M. N. , Straszek Sune P. V. , Østergaard Søren Dinesen
Published: May 2020

Background: Studies on how individuals with affective disorders use and perceive their use of social media are lacking from the literature.
Methods: A questionnaire focusing on affective disorders and the use of social media and online dating was handed out to outpatients with unipolar depression or bipolar disorder and general practice patients with or without affective disorders (latter as controls). The association between affective disorders and the use of social media and online dating was visualized using network graphs and analyzed using linear/logistic regression.
Results: A total of 194 individuals with unipolar depression, 124 individuals with bipolar disorder and 196 controls were included in the analysis. Having unipolar depression or bipolar disorder was not associated with the time spent on social media compared with controls. Using the controls as reference, having bipolar disorder was associated with use of online dating (adjusted odds ratio: 2.2 (95% CI: 1.3; 3.7)). The use of social media and online dating had a mood-congruent pattern with decreased and more passive use during depressive episodes, and increased and more active use during hypomanic/manic episodes. Among the respondents with affective disorder, 51% reported that social media use had an aggravating effect on symptoms during mood episodes, while 10% reported a beneficial effect. For online dating, the equivalent proportions were 49% (aggravation) and 20% (benefit), respectively.

Limitations: Selection and report biases may have affected the results of the survey.

Conclusions: The use of social media and online dating seems related to symptom deterioration among individuals with affective disorders.
Comorbidities, clinical signs and symptoms, laboratory findings, imaging features, treatment strategies, and outcomes in adult and pediatric patients with COVID-19: A systematic review and meta-analysis
Authors: Jutzeler Catherine Ruth , Bourguignon Lucie , Weis Caroline V. , Tong Bobo , Wong Cyrus , Rieck Bastian , Pargger Hans , Tschudin-Sutter Sarah , Egli Adrian , Borgwardt Karsten , Walter Matthias
Published: May 2020

Introduction
Since December 2019, a novel coronavirus (SARS-CoV-2) has triggered a world-wide pandemic with an enormous medical, societal, and economic toll. Thus, our aim was to gather all available information regarding comorbidities, clinical signs and symptoms, outcomes, laboratory findings, imaging features, and treatments in patients with coronavirus disease 2019 (COVID-19).

Methods
EMBASE, PubMed/ Medline, Scopus, and Web of Science were searched for studies published in any language between December 1st, 2019 and March 28th. Original studies were included if the exposure of interest was an infection with SARS-CoV-2 or confirmed COVID-19. The primary outcome was the risk ratio of comorbidities, clinical signs and symptoms, imaging features, treatments, outcomes, and complications associated with COVID-19 morbidity and mortality. We performed random-effects pairwise meta-analyses for proportions and relative risks, I2, Tau2, and Cochrane Q, sensitivity analyses, and assessed publication bias.

Results:
148 met the inclusion criteria for the systematic review and meta-analysis with 12149 patients (5739 female) and a median age was 47.0 [35.0-64.6]. 617 patients died from COVID-19 and its complication, while 297 patients were reported as asymptomatic. Older age (SMD: 1.25 [0.78- 1.72]; p < 0.001), being male (RR = 1.32 [1.13-1.54], p = 0.005) and pre-existing comorbidity (RR = 1.69 [1.48-1.94]; p < 0.001) were identified as risk factors of in-hospital mortality. The heterogeneity between studies varied substantially (I2; range: 1.5-98.2%). Publication bias was only found in eight studies (Eggers test: p < 0.05).

Conclusions:
Our meta-analyses revealed important risk factors that are associated with severity and mortality of COVID-19.
Erythrocytes Reveal Complement Activation in Patients with COVID-19
Authors: Lam LK Metthew , Murphy Sophie J. , Kuri-Cervantes Leticia , Weisman Ariel R. , Ittner Caroline A. G. , Reilly John P. , Pampena M. Betina , Betts Michael R. , Wherry E. John , Song Wen-Chao , Lambris John D. , Cines Douglas B. , Meyer Nuala J. , Mangalmurti Nilam S.
Published: May 2020

COVID-19, the disease caused by the SARS-CoV-2 virus, can progress to multi-organ failure characterized by respiratory insufficiency, arrhythmias, thromboembolic complications and shock. The mortality of patients hospitalized with COVID-19 is unacceptably high and new strategies are urgently needed to rapidly identify and treat patients at risk for organ failure. Clinical epidemiologic studies demonstrate that vulnerability to organ failure is greatest after viral clearance from the upper airway, which suggests that dysregulation of the host immune response is a critical mediator of clinical deterioration and death. Autopsy and pre-clinical evidence implicate aberrant complement activation in endothelial injury and organ failure. A potential therapeutic strategy warranting investigation is to inhibit complement, with case reports of successful treatment of COVID-19 with inhibitors of complement. However, this approach requires careful balance between the host protective and potential injurious effects of complement activation, and biomarkers to identify the optimal timing and candidates for therapy are lacking. Here we report the presence of complement activation products on circulating erythrocytes from hospitalized COVID-19 patients using flow cytometry. These findings suggest that novel erythrocyte-based diagnostics provide a method to identify patients with dysregulated complement activation.
Serious Workplace Violence against healthcare providers in China in the past 15 years
Authors: Ma Jing , Chen Xi , zheng qiongjuan , Zhang Yun , Ming Zhi , Wang Dongxin , Wu Hua , Ye Haisen , Zhou Xiaoxuan , Xu Yunxuan , Li Renjiao , Sheng Xia , Fan Fangxiu , Yang Zuiwen , Luo Ting , Lu Yajun , Deng Ye , Yang Fen , Liu Chuntao , Liu Chunyu , Li Xiaosong
Published: May 2020

Introduction: Workplace violence (WPV) against healthcare providers has severe consequences and underreported worldwide. The aim of this study was to present the features, causes, and outcomes of serious WPV against healthcare providers in China.
Method: We searched serious WPV events reported online and collected information about time, location, people involved, methods used, motivations, and outcomes related to the incident, and analyzed their summary statistics.
Result: Serious WPV reported online (n=379) in China were mainly physical (97%) and often involved the use of weapons (34.5%). Doctors were victims in most instances (81.1%). WPV mostly happened in cities (90.2%), teaching hospitals (87.4%), and tertiary hospitals (67.9%), frequently in ED, OB-GYN, and pediatrics, in the months of June, May, and February. WPV Rates increased dramatically in 2014 and decreased after 2015. Death (12.8%), severe injury (6%), and hospitalization (24.2%) were the major outcomes.
Conclusion: Serious WPV in China may stem from poor patient-doctor relationships, overstressed health providers in the highly demanded hospitals, poorly educated/informed patients, insufficient legal protection and poor communications. A law protecting healthcare providers implemented in 2015 may have helped curb the violence.
Amplification of human β-glucoronidase gene for appraising the accuracy of negative SARS-CoV-2 RT-PCR results in upper respiratory tract specimens
Authors: Albert Eliseo , Ferrer Blanca , Torres Ignacio , Serrano Alicia , Alcaraz Maria Jesus , Buesa Javier , Solnao Carlos , Colomina Javier , Bueno Felipe , Huntley Dixie , olea beatriz , valdivia Arantxa , Navarro David
Published: May 2020

Real-time reverse transcription polymerase-chain reaction (RT-PCR) is the mainstay of Covid-19 diagnosis. False-negative RT-PCR results may hamper clinical management of patients and hinder the adoption of epidemiological measures to control the pandemic. The current study was aimed at assessing whether amplification of β-glucoronidase (GUSB) gene would help estimate the accuracy of SARS-CoV-2 RT-PCR negative results in upper respiratory tract (URT) specimens. URT specimens that tested negative by SARS-CoV-2 RT-PCR displayed higher GUSB RT-PCR cycle thresholds (CT) (P=0.070) than those testing positive (median, 30.7; range, 27.0-40.0, and median 29.7; range 25.5-36.8, respectively), this reflecting poorer cellularity. Receiver operating characteristic (roc) curve analysis indicated that a CT threshold of 31.2 discriminated best between positive and negative SARS CoV-2 RT-PCRs (area under a curve, 0.66; 95% CI, 0.50-0.81; P=0.08). This cut-off yielded a true negative ratio of 89% and accuracy of 70%. The data suggested that amplification of the GUSB gene by RT-PCR may help to appraise the accuracy of negative SARS-CoV-2 RT-PCR results in patients in whom Covid-19 is eventually diagnosed.
Regulation of SETD2 stability is important for the fidelity of H3K36me3 deposition
Authors: Bhattacharya Saikat , Workman Jerry L.
Published: May 2020

The histone H3K36me3 mark regulates transcription elongation, pre-mRNA splicing, DNA methylation, and DNA damage repair. However, knowledge of the regulation of the enzyme SETD2, which deposits this functionally important mark, is very limited. Here we show that the poorly characterized N-terminal region of SETD2 plays a determining role in regulating the stability of SETD2. This stretch of 1-1403 amino acids contributes to the robust degradation of SETD2 by the proteasome. Besides, the SETD2 protein is aggregate-prone and forms insoluble bodies in nuclei especially upon proteasome inhibition. Removal of the N-terminal segment results in the stabilization of SETD2 and leads to a marked increase in global H3K36me3 which, uncharacteristically, happens in a Pol II-independent manner. Thus, the regulation of SETD2 levels through proteasomal mediated decay is important to maintain the fidelity of H3K36me3 deposition.
A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression
Authors: Workman Rachael E. , Pammi Teja , Nguyen Binh T. K. , Graeff Leonardo W. , Smith Erika , Sebald Suzanne M. , Stoltzfus Marie J. , Modell Joshua W.
Published: May 2020

CRISPR-Cas systems provide their prokaryotic hosts with acquired immunity against viruses and other foreign genetic elements, but how these systems are regulated to prevent auto-immunity is poorly understood. In type II CRISPR-Cas systems, a transactivating CRISPR RNA (tracrRNA) scaffold functions together with a CRISPR RNA (crRNA) guide to program Cas9 for the recognition and cleavage of foreign DNA targets. Here, we show that a long-form tracrRNA performs an unexpected second function by folding into a natural single guide that directs Cas9 to transcriptionally repress its own promoter. Further, we demonstrate that Pcas9serves as a critical regulatory node; de-repression causes a dramatic induction of Cas genes, crRNAs and tracrRNAs resulting in a 3,000-fold increase in immunization rates against unrecognized viruses. Heightened immunity comes at the cost of increased auto-immune toxicity, demonstrating the critical importance of the controller. Using a bioinformatic analysis, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.
Glutathione S-transferase Pi (Gstp) Proteins Regulate Neuritogenesis in the Developing Cerebral Cortex
Authors: Liu Xiaonan , Blazejewski Sara M. , Bennison Sarah A. , Toyo-oka Kazuhito
Published: May 2020

GSTP proteins are metabolic enzymes involved in removal of oxidative stress and intracellular signaling and also have inhibitory effects on JNK activity. However, the functions of Gstp proteins in the developing brain are unknown. In mice, there are three Gstp proteins, Gstp1, 2 and 3, while there is only one GSTP in humans. By RT-PCR analysis, we found that Gstp1 was expressed beginning at E15.5 in the cortex, but Gstp2 and 3 started expressing at E18.5. Gstp 1 and 2 knockdown caused decreased neurite number in cortical neurons, implicating them in neurite initiation. Usingin uteroelectroporation to knockdown Gstp1 and 2 in layer 2/3 pyramidal neuronsin vivo, we found abnormal swelling of the apical dendrite at P3 and reduced neurite number at P15. Using time-lapse live imaging, we found that the apical dendrite orientation was skewed compared to the control, but these defects were ameliorated. Overexpression of Gstp 1 or 2 resulted in changes in neurite length, suggesting a role in neurite elongation. We explored the molecular mechanism and found that JNK inhibition rescued reduced neurite number caused by Gstp knockdown, indicating that Gstp regulates neurite formation through JNK signaling. Thus, we found novel functions of Gstp proteins in neurite initiation during cortical development. Furthermore, the overexpression experiments suggest different functions of Gstp1 and 2 in neurite elongation. Since previous studies have shown the potential implication of Gstp in Autism Spectrum Disorder, our findings will attract more clinical interests in Gstp proteins in neurodevelopmental disorders.

Significance

Neurite formation, including neurite initiation and elongation, is the first step of generating polarized neuronal morphology in developing neurons, and thus is essential for establishing a neuronal network. Therefore, it is crucial to understand the mechanisms of neurite formation. Limited studies have been performed to clarify the mechanisms of neurite formation, especially neurite initiation. In this present study, we report a novel, essential role of Gstp in neurite initiation in mouse cortical neuronsin vitroandin vivo. We also found that Gstp regulates neurite formation via JNK signaling pathways. These findings not only provide novel functions of Gstp proteins in neuritogenesis during cortical development but also help us to understand the complexity of neurite formation.
Computational Analysis of Dynamic Allostery and Control in the SARS-CoV-2 Main Protease
Authors: Dubanevics Igors , McLeish Tom C.B.
Published: May 2020

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has generated a global pandemic and no vaccine or antiviral drugs exist at the moment of writing. An attractive coronavirus drug target is the main protease (Mpro, also known as 3CLpro) because of its vital role in the viral cycle. A significant body of work has been focused on finding inhibitors which bind and block the active site of the main protease, but little has been done to address potential non-competitive inhibition which targets regions beyond the active site, partly because the fundamental biophysics of such allosteric control is still poorly understood. In this work, we construct an Elastic Network Model (ENM) of the SARS-CoV-2 Mprohomodimer protein and analyse the dynamics and thermodynamics of the main protease’s ENM. We found a rich and heterogeneous dynamical structure in the correlated motions, including allosterically correlated motions between the homodimeric protease’s active sites. Exhaustive 1-point and 2-point mutation scans of the ENM and their effect on fluctuation free energies confirm previously experimentally identified bioactive residues, but also suggest several new candidate regions that are distant from the active site for control of the protease function. Our results suggest new dynamically-driven control regions as possible candidates for non-competitive inhibiting binding sites in the protease, which may assist the development of current fragmentbased binding screens. The results also provide new insight into the protein physics of fluctuation allostery and its underpinning dynamical structure.
Analyzing the impact of SARS CoV-2 on the human proteome
Authors: Estrada Ernesto
Published: May 2020

The COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2, which uses the enzyme ACE2 to entry human cells. This disease is characterized by important damages at multi-organ level, partially due to the abundant expression of ACE2 in practically all human tissues. However, not every organ in which ACE2 is abundant is affected by SARS CoV-2, which suggests the existence of other multi-organ routes for transmitting the perturbations produced by the virus. We consider here diffusive processes through the protein-protein interaction (PPI) network of proteins targeted by SARS CoV-2 as such alternative route. We found a subdiffusive regime that allows the propagation of virus perturbations through the PPI network at a significant rate. By following the main subdiffusive routes across the PPI network we identify proteins mainly expressed in the heart, cerebral cortex, thymus, testis, lymph node, kidney, among others of the organs reported to be affected by COVID-19.
Full-field optical coherence tomography for the diagnosis of giant cell arteritis
Authors: Maldiney Thomas , Greigert Hélène , Martin Laurent , Benoit Emilie , Creuzot-Garcher Catherine , Gabrielle Pierre-Henry , Chassot Jean-Marie , Boccara Claude , Balvay Daniel , Tavitian Bertrand , Clément Olivier , Audia Sylvain , Bonnotte Bernard , Samson Maxime
Published: May 2020

Histopathological examination of temporal artery biopsy (TAB) remains the gold standard for the diagnosis of giant cell arteritis (GCA) but is associated with essential limitations that emphasize the need for an upgraded pathological process. This study pioneered the use of full-field optical coherence tomography (FF-OCT) for rapid and automated on-site pathological diagnosis of GCA. Sixteen TABs (12 negative and 4 positive for GCA) were selected according to major histopathological criteria of GCA following hematoxylin-eosin-saffron-staining for subsequent acquisition with FF-OCT to compare structural modifications of the artery cell wall and thickness of each tunica. Gabor filtering of FF-OCT images was then used to compute TAB orientation maps and validate a potential automated analysis of TAB sections. FF-OCT allowed both qualitative and quantitative visualization of the main structures of the temporal artery wall, from the internal elastic lamina to thevasa vasorumand red blood cells, unveiling a significant correlation with conventional histology. FF-OCT imaging of GCA TABs revealed destruction of the media with distinct remodeling of the whole arterial wall into a denser reticular fibrous neo-intima, which is distinctive of GCA pathogenesis and accessible through automated Gabor filtering. Rapid on-site FF-OCT TAB acquisition makes it possible to identify some characteristic pathological lesions of GCA within a few minutes, paving the way for potential machine intelligence-based or even non-invasive diagnosis of GCA.
IgY antibodies against Ebola virus possess post-exposure protection and excellent thermostability
Authors: Zhang Yuan , Wei Yanqiu , Li Yunlong , Wang Xuan , Liu Yang , Tian Deyu , Jia Xiaojuan , Gong Rui , Liu Wenjun , Yang Limin
Published: May 2020

Ebola virus (EBOV) is the most virulent pathogens that cause hemorrhagic fever with high mortality rates in humans and nonhuman primates. The postexposure antibody therapies to prevent EBOV infection are considered efficient. However, due to the poor thermal stability of mammalian antibody, their application in the tropics has been limited. Here, we developed a thermostable therapeutic antibody against EBOV based on chicken immunoglobulin Y (IgY). The IgY antibodies demonstrated excellent thermal stability, which retained their neutralizing activity at 25°C for one year, in contrast to conventional polyclonal or monoclonal antibodies (MAbs). We immunized laying hens with a variety of EBOV vaccine candidates and confirmed that VSV Δ G/EBOVGP encoding the EBOV glycoprotein could induce high titer neutralizing antibodies against EBOV. The therapeutic efficacy of immune IgY antibodiesin vivowas evaluated in the newborn Balb/c mice model. Lethal dose of virus challenged mice were treated 2 or 24 h post-infection with different doses of anti-EBOV IgY. The group receiving a high dose of 106NAU/kg (neutralizing antibody units/kilogram) achieved complete protection with no signs of disease, while the low-dose group was only partially protected. In contrast, all mice receiving naïve IgY died within 10 days. In conclusion, the anti-EBOV IgY exhibits excellent thermostability and protective efficacy, and it is very promising to be developed as alternative therapeutic entities.

Author Summary

Although several Ebola virus therapeutic antibodies have been reported in recent years, however, due to the poor thermal stability of mammalian antibody, their application in tropical endemic areas has been limited. We developed a highly thermostable therapeutic antibody against EBOV based on chicken immunoglobulin Y (IgY). The IgY antibodies demonstrated excellent thermal stability, which retained their neutralizing activity at 25°C for one year. The newborn mice receiving passive transfer of IgY achieved complete protection against a lethal dose of virus challenge indicating that the anti-EBOV IgY provides a promising countermeasure to solve the current clinical application problems of Ebola antibody-based treatments in Africa.
The NHEJ Repair of DNA Double Strand Breaks inPhyscomitrella patensDepends on the Kleisin NSE4 of the SMC5/6 Complex
Authors: Vágnerová Radka , Holá Marcela , Angelis Karel J.
Published: May 2020

Structural maintenance of chromosomes (SMC) complexes are involve in cohesion, condensation and maintenance of genome stability. Based on the sensitivity of mutants to genotoxic stress the SMC5/6 complex is thought to play imminent role in DNA stabilization during repair by encircling DNA at the site of lesion by bridging the heteroduplex of SMC5 and SMC6 by non SMC kleisin components NSE1, 3 and 4. In this study, we tested how formation of the SMC5/6 circular structure affects mutant sensitivity to genotoxic stress, kinetics of DSB repair and insertion mutagenesis. In the mossPhyscomitrella patens SMC6andNSE4are essential single copy genes and this is why we used blocking of transcription to reveal their mutated phenotype. Even slight attenuation of transcription by dCas9 binding was enough to obtain stable lines with DSB repair defect and specific bleomycin sensitivity. Whereas survival after bleomycin or MMS treatment fully depends on active SMC6, NSE4 has little or negligible effect. We conclude that whereas circularization of SMC5/6 provided by the kleisin NSE4 is indispensable for the immediate NHEJ DSB repair response, other functions associated with SMC5/6 complex are critical to survive DNA damage.
Low-density lipoprotein receptor-related protein 1 (LRP1) is a negative regulator of oligodendrocyte progenitor cell differentiation in the adult mouse brain
Authors: Auderset Loic , Pitman Kimberley A , Cullen Carlie L , Pepper Renee E , Taylor Bruce V , Foa Lisa , Young Kaylene M
Published: May 2020

Low-density lipoprotein receptor-related protein 1 (LRP1) is a large, endocytic cell surface receptor that is highly expressed by oligodendrocyte progenitor cells (OPCs), and LRP1 expression is rapidly downregulated as OPCs differentiate into oligodendrocytes (OLs). We report that the conditional deletion ofLrp1from adult mouse OPCs (Pdgfrα-CreER :: Lrp1fl/fl) increases the number of new myelinating OLs added to brain, but that each new cell elaborates a normal quantity of myelin. OPC proliferation is also elevated followingLrp1deletionin vivo, however, this is likely to be a secondary, homeostatic response to increased OPC differentiation, as ourin vitroexperiments show that LRP1 is a direct negative regulator of OPC differentiation, not proliferation. DeletingLrp1from adult OPCs also enhances remyelination, as cuprizone-induced lesions are smaller inLrp1-deleted mice, and parenchymal OPCs produce a larger number of mature OLs. These data suggest that the selective blockade of LRP1 function on adult OPCs may enhance myelin repair in demyelinating diseases, such as multiple sclerosis.
Mechanistic insights into ventricular arrhythmogenesis of hydroxychloroquine and azithromycin for the treatment of COVID-19
Authors: Wang Gongxin , Lu Chieh-Ju , Trafford Andrew W. , Tian Xiaohui , Flores Hannali M , Maj Piotr , Zhang Kevin , Niu Yanhong , Wang Luxi , Du Yimei , Ji Xinying , Xu Yanfang , Wu Lin , Li Dan , Herring Neil , Paterson David , Huang Christopher L.-H. , Zhang Henggui , Lei Ming , Hao Guoliang
Published: May 2020

Aims

We investigate mechanisms for potential pro-arrhythmic effects of hydroxychloroquine (HCQ) alone, or combined with azithromycin (AZM), in Covid-19 management supplementing the limited available experimental cardiac safety data.

Methods

We integrated patch-clamp studies utilizing In Vitro ProArrhythmia Assay (CiPA) Schema IC50paradigms, molecular modelling, cardiac multi-electrode array and voltage (RH237) mapping, ECG studies, and Ca2+(Rhod-2 AM) mapping in isolated Langendorff-perfused guinea-pig hearts with human in-silico ion current modelling.

Results

HCQ blocked IKrand IK1with IC50s (10±0.6 and 34±5.0 μM) within clinical therapeutic ranges, INaand ICaLat higher IC50s, leaving Itoand IKsunaffected. AZM produced minor inhibition of INa, ICaL, IKs, and IKr,, sparing IK1and Ito. HCQ+AZM combined inhibited IKrand IK1with IC50s of 7.7±0.8 μM and 30.4±3.0 μM, sparing INa, ICaLand Ito. Molecular modelling confirmed potential HCQ binding to hERG. HCQ slowed heart rate and ventricular conduction. It prolonged PR, QRS and QT intervals, and caused prolonged, more heterogeneous, action potential durations and intracellular Ca2+transients. These effects were accentuated with combined HCQ+AZM treatment, which then elicited electrical alternans, re-entrant circuits and wave break. Modelling studies attributed these to integrated HCQ and AZM actions reducing IKrand IK1, thence altering cell Ca2+homeostasis.

Conclusions

Combined HCQ+AZM treatment exerts pro-arrhythmic ventricular events by synergetically inhibiting IKr, IKswith resulting effects on cellular Ca2+signalling, and action potential propagation and duration. These findings provide an electrophysiological basis for recent FDA cardiac safety guidelines cautioning against combining HCQ/AZM when treating Covid-19.
Intrinsic disorder in the T cell receptor creates cooperativity and controls ZAP70 binding
Authors: Clemens Lara , Dushek Omer , Allard Jun
Published: May 2020

Many immunoreceptors have cytoplasmic domains that are intrinsically disordered (i.e., have high configurational entropy), have multiple sites of post-translational modification (e.g., tyrosine phosphorylation), and participate in nonlinear signaling pathways (e.g., exhibiting switch-like behavior). Several hypotheses to explain the origin of these nonlinearities fall under the broad hypothesis that modification at one site changes the immunoreceptor’s entropy, which in turn changes further modification dynamics. Here we use coarse-grain simulation to study three scenarios, all related to the chains that comprise the T Cell Receptor. We find that, first, if phosphorylation induces local changes in the flexibility of TCRζ-chain, this naturally leads to rate enhancements and cooperativity. Second, we find that TCR CD3ϵcan provide a switch by modulating its residence in the plasma membrane. By constraining our model to be consistent with the previous observation that both basic residues and phosphorylation control membrane residence, we find that there is only a moderate rate enhancement of 10% between first and subsequent phosphorylation events. And third, we find that volume constraints do not limit the number of ZAP70s that can bind the TCR, but that entropic penalties lead to a 200-fold decrease in binding rate by the seventh ZAP70, potentially explaining the observation that each TCR has around six ZAP70 molecules bound following receptor triggering. In all three scenarios, our results demonstrate that phenomena that change an immunoreceptor chain’s entropy (stiffening, confinement to a membrane, and multiple simultaneous binding) can lead to nonlinearities (rate enhancement, switching, and negative cooperativity) in how the receptor participates in signaling. These polymer-entropy-driven nonlinearities may augment the nonlinearities that arise from, e.g., kinetic proofreading and cluster formation. They also suggest different design strategies for engineered receptors, e.g., whether or not to put signaling modules on one chain or multiple clustered chains.

STATEMENT OF SIGNIFICANCE

Many of the proteins involved in signal processing are both mechanically flexible and have multiple sites of interaction, leading to a combinatorial complexity making them challenging to study. One example is the T Cell Receptor, a key player in immunological decision making. It consists of 6 flexible chains with 20 interaction sites, and exhibits nonlinear responses to signal inputs, although the mechanisms are elusive. By using polymer physics to simulate the T Cell Receptor’s chains, this work demonstrates that several of the nonlinear responses observed experimentally emerge naturally due to constraints on the chains that change their entropy. This work points to new avenues to modulate signaling proteins for therapeutics by modulating their mechanical flexibility and spatial extent.
Redox proteomics of stomatal immunity reveals role of a lipid transfer protein in plant defense
Authors: Balmant Kelly M , Lawrence Sheldon R , Duong Benjamin V , Zhu Fanzhao , Zhu Ning , Nicklay Joshua , Chen Sixue
Published: May 2020

Redox-based post-translational modifications (PTMs) involving protein cysteine residues as redox sensors are important to various physiological processes. However, little is known about redox-sensitive proteins in guard cells and their functions in stomatal immunity. In this study, we applied an integrative protein labeling method cysTMTRAQ and identified guard cell proteins that were altered by thiol redox PTMs in response to a bacterial flagellin peptide flg22. In total, eight, seven and 20 potential redox-responsive proteins were identified in guard cells treated with flg22 for 15, 30 and 60 min, respectively. The proteins fall into several functional groups including photosynthesis, lipid binding, oxidation-reduction, and defense. Among the proteins, a lipid transfer protein (LTP)-II was confirmed to be redox-responsive and involved in plant resistance toPseudomonas syringe pv. tomatoDC3000. This study not only creates an inventory of potential redox-sensitive proteins in flg22 signal transduction in guard cells, but also highlights the relevance of the lipid transfer protein in plant defense against the bacterial pathogens.

Sentence summary

Thiol-redox proteomics identified potential redox sensors important in stomatal immunity, and a lipid transfer protein was characterized to function as a redox sensor in plant immune response.
First passage time study of DNA strand displacement
Authors: Broadwater D. W. Bo , Cook Alexander W. , Kim Harold D.
Published: May 2020

DNA strand displacement, where a single-stranded nucleic acid invades a DNA duplex, is pervasive in genomic processes and DNA engineering applications. The kinetics of strand displacement have been studied in bulk; however, the kinetics of the underlying strand exchange were obfuscated by a slow bimolecular association step. Here, we use a novel single-molecule Fluorescence Resonance Energy Transfer (smFRET) approach termed the “fission” assay to obtain the full distribution of first passage times of unimolecular strand displacement. At a frame time of 4.4 ms, the first passage time distribution for a 14-nt displacement domain exhibited a nearly monotonic decay with little delay. Among the eight different sequences we tested, the mean displacement time was on average 35 ms and varied by up to a factor of 13. The measured displacement kinetics also varied between complementary invaders and between RNA and DNA invaders of the same base sequence except for T→U substitution. However, displacement times were largely insensitive to the monovalent salt concentration in the range of 0.25 M to 1 M. Using a one-dimensional random walk model, we infer that the single-step displacement time is in the range of ∼30 µs to ∼300 µs depending on the base identity. The framework presented here is broadly applicable to the kinetic analysis of multistep processes investigated at the single-molecule level.
Structure and reconstitution of a hydrolase complex that releases peptidoglycan from the membrane after polymerization
Authors: Schaefer Kaitlin , Owens Tristan W. , Page Julia E. , Santiago Marina , Kahne Daniel , Walker Suzanne
Published: May 2020

Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan. As Lipid II is polymerized, nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix2–4. A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan. Here we show that aStaphylococcus aureuscell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that acts as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A 2.6 Å resolution structure of the complex shows that the membrane protein scaffolds the hydrolase to orient its active site for cleavage of the glycan strand. We propose that this complex serves to detach newly-synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix.