bluuebunny/crossref_metadata_2025_split huggingface dataset:
An economic and robust TMT labeling approach for high throughput proteomic and metaproteomic analysis
Authors: Creskey Marybeth , Li Leyuan , Ning Zhibin , Brown Emily EF , Mayne Janice , Walker Krystal , Ampaw Anna , Ben Robert , Zhang Xu , Figeys Daniel 
Published: August 2022

AbstractMultiplexed quantitative proteomics using tandem mass tag (TMT) is increasingly used in –omic study of complex samples. While TMT-based proteomics has the advantages of the higher quantitative accuracy, fewer missing values, and reduced instrument analysis time, it is limited by the increased cost due to the use of labeling reagents. In addition, current TMT labeling workflows involve repeated small volume pipetting of reagents in volatile organic solvents, which may increase the sample-to-sample variations and is not readily suitable for high throughput applications. In this study, we demonstrated that the TMT labeling procedures could be streamlined by using pre-aliquoted dry TMT reagents in a 96 well plate or 12-tube strip. As little as 50 μg dry TMT per channel effectively labels 6-12 μg peptides, yielding efficient TMT labeling efficiency (∼99%) in both microbiome and mammalian cell line samples. This streamlined workflow decreases reagent loss and reduces inter-sample variations. We applied this workflow to analyze 97 samples in a study to evaluate whether ice recrystallization inhibitors improve the cultivability and activity of frozen microbiota. The results demonstrated tight sample clustering corresponding to groups and consistent microbiome responses to prebiotic treatments. This study supports the use of TMT reagents that are pre-aliquoted, dried, and stored for streamlined and robust quantitative proteomics and metaproteomics in high throughput applications.
The microRNA processing subunit DGCR8 is required for a T cell-dependent germinal center response
Authors: Daum Patrick , Meinzinger Julia , Schulz Sebastian R. , Côrte-Real Joana , Hauke Manuela , Roth Edith , Schuh Wolfgang , Mielenz Dirk , Wittmann Jürgen , Jäck Hans-Martin , Pracht Katharina 
Published: August 2022

ABSTRACTWe have previously shown that the microRNA (miRNA) processor complex consisting of the RNAse Drosha and the DiGeorge Critical Region (DGCR) 8 protein is essential for central B cell maturation. To determine whether miRNA processing is required to initiate T cell-mediated antibody responses, we deleted DGCR8 in maturing B-2 cells by crossing a mouse with loxP-flanked DGCR8 alleles with a CD23-Cre mouse. As expected, non-immunized mice showed reduced numbers of mature B-2 cells and IgG-secreting cells and diminished serum IgG titers. In accordance, germinal centers and antigen-specific IgG-secreting cells were absent in mice immunized with T cell-dependent antigens. Therefore, DGCR8 is required to mount an efficient T cell-dependent antibody response. However, DGCR8 deletion in B-1 cells was incomplete, which explains relatively unaffected B-1 cell numbers and adequate IgM and IgA titers in DGCR8-knock out mice and suggests that this mouse model could be used to analyze B-1 responses in the absence of functional B-2 cells.
Necrosis reduction efficacy of subdermal biomaterial mediated oxygen delivery in ischemic skin flaps
Authors: Rastinfard Arghavan , Dalisson Benjamin , Gilardino Mirko , Watters Kevin , Job Dario , Merle Geraldine , Lasagabaster Arturo Vela , Ouhaddi Yassine , Barralet Jake 
Published: August 2022

AbstractInadequate tissue blood supply (e.g., in a wound or a poorly vascularised graft) can result in tissue ischemia and necrosis. As revascularization is a slow process relative to the proliferation of bacteria and the onset and spread of tissue necrosis, extensive tissue damage and loss can occur. Necrosis can spread rapidly, and treatment options are limited such that loss of tissue in ischemic tissue following necrosis onset is considered unavoidable and irreversible.Oxygen delivery from biomaterials exploiting aqueous decomposition of peroxy-compounds has shown some potential in overcoming the supply limitations caused by quite short oxygen diffusion distances in tissues by creating higher concentration gradients than can be attained by air saturated solutions or by distributing oxygen supply throughout a scaffold or construct by using particulate formulations. These have found application in tissue preservation, bioinks, creation of 3D tissue analogues etc. In preclinical models among the more exciting reports was a single study demonstrating reduction of ischemic skin necrosis albeit only short term using short term sub dermal delivery of oxygen below ischemic skin flaps. To explore this effect further, we developed an implantable solid peroxide-biomaterial based system with reduced hydrogen peroxide release by virtue of incorporation of minerals to catalytically decompose it in a much longer flap than examined previously. Blood flow in this flap reduced from essentially normal to essentially zero, along its 9cm length. Without treatment ∼50% of the total flap was necrotic in 2-4 days. In both groups, complete necrosis in the distal third of the flap with no observable flood flow was observed. But in the middle low blood flow region of the flap, treatment did prevent necrosis. This study indicated that subdermal oxygen delivery alone cannot completely mitigate dermal necrosis if no blood flow is present, but it could improve the survival of partially tissue at least in the short term which could find application to augment conventional treatments or to gain time until surgical intervention.
Quantifying the Effect of an Acute Stressor in Laying Hens using Thermographic Imaging and Vocalisations
Authors: van den Heuvel Huib , Youssef Ali , Grat Lisette M , Neethirajan Suresh 
Published: August 2022

AbstractThe laying hen sector has multiple issues concerning the animal’s welfare. One crucial factor negatively impacts chicken welfare is stress. The conventional way of measuring and assessing chicken stress is time-consuming and subjective to the assessor. On the other hand, sensing and sensor technologies can be used to obtain objective, continuous and non-invasive/contactless measures of animal behavioural and physiological welfare indicators. The present study aims to investigate the use of thermographic imaging and microphones (sound) in obtaining objective indicators for acute stress in laying hens. During this study, 40 laying hens were stressed by opening an umbrella as a stressor starting from one day age until nine-weeks old. The birds were stressed every other day. Another 12 birds were housed in another cage. These birds were not stressed and served as a control group. The surface temperatures of the bird’s comb and beak decreased (1°C and 2.5°C respectively) in response to the applied stressor. This effect was only seen in the treatment group and not in the control birds. The number of vocalisations the birds produced significantly decreased shortly after stress. The number of calls in the stressed group decreased from 39.5 to 12.1 calls/minute, where the control group decreased from 27.8 to 22.5 calls/minute. It was hypothesized that the number of vocalisations would increase after stress. This difference could be due to the daily behavioural rhythm performed by the birds. The birds might naturally produce more calls at certain hours of the day because of certain behaviours they perform. Three different neural network algorithms were employed to differentiate between the vocalisation of stressed and the control group. This was done by converting the audio files to images and feeding them to the pretrained convolutional neural networks (CNN). The Resnet CNN had the highest categorising accuracy with an overall accuracy of 86 percent. The changes in surface temperature of the beak, comb, eye, and head, as well as the results from the audio analysis could serve as potential indicator for acute stress in laying hens. Future research is warranted to validate the methodologies and findings under different environmental conditions and stressors.
Gene Birth in a Model of Non-genic Adaptation
Authors: Mani Somya , Tlusty Tsvi 
Published: August 2022

AbstractBackgroundOver evolutionary timescales, genomic loci can switch between functional and non-functional states through processes such as pseudogenization andde novogene birth. Particularly,de novogene birth is a widespread process, and many examples continue to be discovered across diverse evolutionary lineages. However, the general mechanisms that lead to functionalization are poorly understood, and estimated rates ofde novogene birth remain contentious. Here, we address this problem within a model that takes into account mutations and structural variation, allowing us to estimate the likelihood of emergence of new functions at non-functional loci.ResultsAssuming biologically reasonable mutation rates and mutational effects, we find that functionalization of non-genic loci requires the realization of strict conditions. This is in line with the observation that mostde novogenes are localized to the vicinity of established genes. Our model also provides an explanation for the empirical observation that emerging proto-genes are often lost despite showing signs of adaptation.ConclusionsOur work elucidates the properties of non-genic loci that make them fertile for adaptation, and our results offer mechanistic insights into the process ofde novogene birth.
SARS-CoV-2-specific CD4+and CD8+T cell responses can originate from cross-reactive CMV-specific T cells
Authors: Pothast Cilia R , Dijkland Romy C , Thaler Melissa , Hagedoorn Renate S , Kester Michel GD , Wouters Anne K , Hiemstra Pieter S , van Hemert Martijn J , Gras Stephanie , Falkenburg JH Frederik , Heemskerk Mirjam HM 
Published: August 2022

AbstractDetection of SARS-coronavirus-2 (SARS-CoV-2) specific CD4+and CD8+T cells in SARS-CoV-2-unexposed donors has been explained by the presence of T cells primed by other coronaviruses. However, based on the relative high frequency and prevalence of cross-reactive T cells, we hypothesized CMV may induce these cross-reactive T cells. Stimulation of pre-pandemic cryo-preserved PBMCs with SARS-CoV-2 peptides revealed that frequencies of SARS-CoV-2-specific T cells were higher in CMV-seropositive donors. Characterization of these T cells demonstrated that membrane-specific CD4+and spike-specific CD8+T cells originate from cross-reactive CMV-specific T cells. Spike-specific CD8+T cells recognize SARS-CoV-2 spike peptide FVSNGTHWF (FVS) and dissimilar CMV pp65 peptide IPSINVHHY (IPS) presented by HLA-B*35:01. These dual IPS/FVS-reactive CD8+T cells were found in multiple donors as well as severe COVID-19 patients and shared a common T cell receptor (TCR), illustrating that IPS/FVS-cross-reactivity is caused by a public TCR. In conclusion, CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-existing immunity against SARS-CoV-2.
Deep-SMOLM: Deep Learning Resolves the 3D Orientations and 2D Positions of Overlapping Single Molecules with Optimal Nanoscale Resolution
Authors: Wu Tingting , Lu Peng , Rahman Md Ashequr , Li Xiao , Lew Matthew D. 
Published: August 2022

AbstractDipole-spread function (DSF) engineering reshapes the images of a microscope to maximize the sensitivity of measuring the 3D orientations of dipole-like emitters. However, severe Poisson shot noise, overlapping images, and simultaneously fitting high-dimensional information–both orientation and position–greatly complicates image analysis in single-molecule orientation-localization microscopy (SMOLM). Here, we report a deep-learning based estimator, termed Deep-SMOLM, that archives superior 3D orientation and 2D position measurement precision within 3% of the theoretical limit (3.8◦ orientation, 0.32 sr wobble angle, and 8.5 nm lateral position using 1000 detected photons). Deep-SMOLM also achieves state-of-art estimation performance on overlapping images of emitters, e.g., a 0.95 Jaccard index for emitters separated by 139 nm, corresponding to a 43% image overlap. Deep-SMOLM accurately and precisely reconstructs 5D information of both simulated biological fibers and experimental amyloid fibrils from images containing highly overlapped DSFs, at a speed ∼10 times faster than iterative estimators.
Structural basis for assembly and lipid-mediated gating of LRRC8A:C volume-regulated anion channels
Authors: Kern David M. , Bleier Julia , Mukherjee Somnath , Hill Jennifer M. , Kossiakoff Anthony A. , Isacoff Ehud Y. , Brohawn Stephen G. 
Published: August 2022

AbstractLeucine-rich repeat-containing protein 8 (LRRC8) family members form volume regulated anion channels activated by hypoosmotic cell swelling. LRRC8 channels are ubiquitously expressed in vertebrate cells as heteromeric assemblies of LRRC8A (Swell1) and LRRC8B-E subunits. Channels of different subunit composition have distinct properties that explain the functional diversity of LRRC8 currents implicated in a broad range of physiology. However, the basis for heteromeric LRRC8 channel assembly and function is unknown. Here, we leverage a fiducial-tagging strategy to determine single-particle cryo-electron microscopy structures of heterohexameric LRRC8A:C channels in detergent micelles and lipid nanodiscs in three conformations. LRRC8A:C channels show pronounced changes in channel architecture compared to homomeric channels due to heterotypic cytoplasmic LRR interactions that displace LRRs and the LRRC8C subunit away from the conduction axis and poise the channel for activation. The structures and associated functional studies further reveal that lipids embedded in the channel pore block ion conduction in the closed state. Together, our results provide insight into determinants for heteromeric LRRC8 channel assembly, activity, and gating by lipids.
Viral and Symptom Rebound in Untreated COVID-19 Infection
Authors: Deo Rinki , Choudhary Manish C. , Moser Carlee , Ritz Justin , Daar Eric S. , Wohl David A. , Greninger Alexander L. , Eron Joseph J. , Currier Judith S. , Hughes Michael D. , Smith Davey M. , Chew Kara W. , Li Jonathan Z. ,   the ACTIV-2/A5401 Study Team
Published: August 2022

SummaryBackgroundThere are reports of viral RNA and symptom rebound in people with COVID-19 treated with nirmatrelvir/ritonavir. Since the natural course of viral and symptom trajectories of COVID-19 has not been well described, we evaluated the incidence of viral and symptom rebound in untreated outpatients with mild-moderate COVID-19.MethodsThe study population included 568 participants enrolled in the ACTIV-2/A5401 platform trial who received placebo. Anterior nasal swabs were collected for SARS-CoV-2 RNA testing on days 0-14, 21 and 28. Participants recorded the severity of 13 targeted symptoms daily from day 0 to 28. Viral rebound was defined as ≥0.5 log10 viral RNA copies/mL increase and symptom rebound was defined as a 4-point total symptom score increase from baseline. Baseline was defined as study day 4 (primary analysis) or 8 days from symptom onset (secondary analysis).FindingsIn both the primary and secondary analyses, 12% of participants had viral rebound. Viral rebounders were older than non-rebounders (median 54 vs 47 years, P=0.04). Symptom rebound occurred in 27% of participants after initial symptom improvement and in 10% of participants after initial symptom resolution. The combination of high-level viral rebound to ≥5.0 log10 RNA copies/mL and symptom rebound after initial improvement was observed in 1-2% of participants.InterpretationViral RNA rebound or symptom relapse in the absence of antiviral treatment is common, but the combination of high-level viral and symptom rebound is rare.FundingThis study was supported by the National Institute of Allergy and Infectious Diseases; ACTIV-2/A5401 ClinicalTrials.gov number NCT04518410.
Mutational screens highlight glycosylation as a modulator of CSF3R activity
Authors: Hollander Michael J. , Malaker Stacy A. , Perez Idalia , Maxson Julia E. , Cochran Jennifer R. , Bertozzi Carolyn R. 
Published: August 2022

AbstractThe colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia (CNL). We investigated why this threonine to isoleucine substitution is the predominant mutation in CNL and how it leads to uncontrolled neutrophil growth. Using protein domain mapping, we demonstrated that the single CSF3R domain containing residue 618 is sufficient for ligand-independent activity. We then applied an unbiased mutational screening strategy focused on this domain and found that activating mutations are enriched at sites normally occupied by asparagine, threonine, and serine residues – the three amino acids which are commonly glycosylated. We confirmed glycosylation at multiple CSF3R residues by mass spectrometry, including the presence of GalNAc and Gal-GalNAc glycans at wild-type threonine 618. Using the same approach applied to other cell surface receptors, we identified an activating mutation, S489F, in the interleukin-31 receptor alpha chain (IL-31Rα). Combined, these results suggest a role for glycosylated hotspot residues in regulating receptor signaling, mutation of which can lead to ligand-independent, uncontrolled activity.
SARS-CoV-2 ORF8 is a viral cytokine regulating immune responses
Authors: Kohyama Masako , Suzuki Tatsuya , Nakai Wataru , Ono Chikako , Matsuoka Sumiko , Iwatani Koichi , Liu Yafei , Sakai Yusuke , Nakagawa Atsushi , Tomii Keisuke , Ohmura Koichiro , Okada Masato , Matsuura Yoshiharu , Ohshima Shiro , Maeda Yusuke , Okamoto Toru , Arase Hisashi 
Published: August 2022

AbstractMany patients with severe COVID-19 suffer from pneumonia, and thus elucidation of the mechanisms underlying the development of such severe pneumonia is important. The ORF8 protein is a secreted protein of SARS-CoV-2, whose in vivo function is not well understood. Here, we analyzed the function of ORF8 protein by generating ORF8-knockout SARS-CoV-2. We found that the lung inflammation observed in wild-type SARS-CoV-2-infected hamsters was decreased in ORF8-knockout SARS-CoV-2-infected hamsters. Administration of recombinant ORF8 protein to hamsters also induced lymphocyte infiltration into the lungs. Similar pro-inflammatory cytokine production was observed in primary human monocytes treated with recombinant ORF8 protein. Furthermore, we demonstrate that the serum ORF8 protein levels are correlated well with clinical markers of inflammation. These results demonstrated that the ORF8 protein is a viral cytokine of SARS-CoV-2 involved in the in the immune dysregulation observed in COVID-19 patients, and that the ORF8 protein could be a novel therapeutic target in severe COVID-19 patients.
Free energy perturbation calculations of mutation effects on SARS-CoV-2 RBD::ACE2 binding affinity
Authors: Sergeeva Alina P. , Katsamba Phinikoula S. , Sampson Jared M. , Bahna Fabiana , Mannepalli Seetha , Morano Nicholas C. , Shapiro Lawrence , Friesner Richard A. , Honig Barry 
Published: August 2022

AbstractThe strength of binding between human angiotensin converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of viral spike protein plays a role in the transmissibility of the SARS-CoV-2 virus. In this study we focus on a subset of RBD mutations that have been frequently observed in infected individuals and probe binding affinity changes to ACE2 using surface plasmon resonance (SPR) measurements and free energy perturbation (FEP) calculations. Our SPR results are largely in accord with previous studies but discrepancies do arise due to differences in experimental methods and to protocol differences even when a single method is used. Overall, we find that FEP performance is superior to that of other computational approaches examined as determined by agreement with experiment and, in particular, by its ability to identify stabilizing mutations. Moreover, the calculations successfully predict the observed cooperative stabilization of binding by the Q498R N501Y double mutant present in Omicron variants and offer a physical explanation for the underlying mechanism. Overall, our results suggest that despite the significant computational cost, FEP calculations may offer an effective strategy to understand the effects of interfacial mutations on protein-protein binding affinities and in practical applications such as the optimization of neutralizing antibodies.
Epitranscriptomic N6-methyladenosine profile of SARS-CoV-2-infected human lung epithelial cells
Authors: Phillips Stacia , Khadka Shaubhagya , Bohan Dana , Espada Constanza E. , Maury Wendy , Wu Li 
Published: August 2022

AbstractN6-methyladenosine (m6A) is a dynamic post-transcriptional RNA modification that plays an important role in determining transcript fate. Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused the global pandemic of coronavirus disease 2019 (COVID-19) and the virus has been extensively studied. However, how m6A modification of host cell RNAs change during SARS-CoV-2 infection has not been reported. Here we define the epitranscriptomic m6A profile of SARS-CoV-2-infected human lung epithelial cells compared to uninfected controls. Biological pathway analyses revealed that differentially methylated transcripts were significantly associated with cancer-related pathways, protein processing in the endoplasmic reticulum, cell death and proliferation. Upstream regulators predicted to be associated with the proteins encoded by differentially methylated mRNAs include proteins involved in the type I interferon response, inflammation, and cytokine signaling. These data suggest that m6A modification of cellular RNA is an important mechanism of regulating host gene expression during SARS-CoV-2 infection of lung epithelial cells.
Discovery of RNA polymerase α-subunit protein as a novel quorum sensing reprograming factor inPseudomonas aeruginosa
Authors: Cai Wenjie , Liao Huimin , Lu Mingqi , Zhou Xiangting , Cheng Xiaoyan , Staehelin Christian , Dai Weijun 
Published: August 2022

AbstractLasR is a master regulator of quorum-sensing (QS) inPseudomonas aeruginosa.LasR-null mutants commonly appear in lung isolates from chronically infected cystic fibrosis (CF) patients. However, numerous such CF isolates have a QS-active phenotype, but factors underlying QS-reprogramming in LasR-null mutants remain largely unknown. Mutations in the transcriptional regulator genemexTare well known to be responsible for QS-reprogramming in a laboratory LasR-null mutant strain, however, simultaneous occurrence oflasRandmexTmutations is rare in CF isolates. To identify QS-reprogramming determinants, we developed an experimental evolution approach, for which a QS-inactive LasR null mutant with an extra copy ofmexTwas engineered. In such a strain, spontaneous singlemexTmutations are expected to have no or little phenotypic consequences. This novel method, named “targeted gene duplication followed by mutant screening” (TGD-MS), resulted in the identification of QS-active revertants with mutations in genes other thanmexT. We characterized a QS-active revertant with a point mutation inrpoA,a gene encoding the α-subunit of RNA polymerase. QS activation in this mutant was found to be associated with the down-regulated expression ofmexEF-oprNefflux pump genes. Our study therefore uncovers a new functional role for RpoA in regulating QS activity. Furthermore, our results suggest that a regulatory circuit controlling the expression of themexEF-oprNoperon is critical for QS-reprogramming. In conclusion, our study reports on the identification of non-MexT proteins associated with QS-reprogramming in a laboratory strain, shedding light on possible QS activation mechanisms in clinicalP. aeruginosaisolates.