bluuebunny/crossref_metadata_2025_split huggingface dataset:
FUS binding to RNA prevents R-loops
Authors: Thompson Valery F. , Wieland Daniel R. , Mendoza-Leon Vivian , Janis Helen I. , Lay Michelle A. , Harrell Lucas M. , Schwartz Jacob C. 
Published: August 2022

ABSTRACTThe protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. We explored the role of FUS binding interactions for activity during transcription.In vitrorun-off transcription assays revealed FUS enhanced RNA produced by a non-eukaryote polymerase. Activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.
Cellular, Molecular, and Enzymatic Signatures of Thrombi are Vascular Bed-Dependent
Authors: Bender Matthew , Aggarwal Anu , Mix Doran , Godwin Matthew , Guntupalli Suman , Nanjundappa Aravinda , Tefera Leben , Hadadin Ihab , Bhandari Rohan , Tong Michael , Baldwin William M. , Fairchild Robert L , Gomes Marcelo , Campbell Joseph , Schumick David , Cameron Scott J. 
Published: August 2022

AbstractBackgroundThe contribution of arterial and venous thrombi to vascular remodeling is unclear. While catheter-extraction of thrombus in cerebrovascular accident (CVA) is time-sensitive, similar urgency is rare in managing venous thromboembolism (VTE).ObjectivesOur goal was to determine molecular cellular signatures of thrombus extracted by catheter from various vascular beds to gain insight into vascular remodeling.MethodsTwenty-five patients underwent catheter-directed thrombectomy (CDT), 13 for acute CVA, 8 for pulmonary embolism (PE), and 4 for deep vein thrombosis (DVT). Protein and RNA extracted from thrombus was evaluated by immunoblotting and sequencing, respectively. Thrombus-derived enzymes for which substrate is present in the blood vessel wall were examined for enzymatic activity.ResultsTime from symptom onset to thrombus extraction was 7.7 ± 1.9 hours for CVA and 109 ± 55 hours for VTE. Protein concentration, white blood cell and red blood cell content were all greater in venous compared with arterial thrombus while platelet content was similar. Both venous and arterial thrombus contained multiple Matrix Metalloproteinase (MMP) isoforms. MMP9 specific activity was greater in venous than in arterial thrombus (57 ± 6 ng/mL.μg protein-1 vs. 24 ± 8 ng/mL.μg protein-1, P=0.0051).ConclusionsArterial and venous thrombus have dissimilar phenotypes, each with biologically-active enzymes known to remodel blood vessels, and enzymatic activity proportional to the white blood cell content which increases with thrombus age. These data suggest a mechanistically-important role for early CDT to avoid the consequences of irreversible vascular remodeling.Condensed AbstractEmergent extraction of acute thrombus from arterial vascular beds restores limb and end-organ perfusion and is widely-accepted to be the standard of care. Extraction of thrombus from venous vascular beds, however, is rarely considered urgent, even though many patients subsequently develop debilitating symptoms. By capitalizing on privileged thrombus extracted from multiple vascular beds, we gained mechanistic insight regarding the cellular composition and cell-derived enzymes secreted from thrombus that may remodel the vessel wall. This study shows thrombi are biologically-active entities, continuously recruiting circulating cells that secrete enzymes both proportional to thrombus age and the time of patient presentation.
Characterization of RNA Polymerase II Trigger Loop Mutations using Molecular Dynamics Simulations and Machine Learning
Authors: Dutagaci Bercem , Duan Bingbing , Qiu Chenxi , Kaplan Craig D. , Feig Michael 
Published: August 2022

ABSTRACTCatalysis and fidelity of multisubunit RNA polymerases rely on a highly conserved active site domain called the trigger loop (TL), which achieves roles in transcription through conformational changes and interaction with NTP substrates. The mutations of TL residues cause distinct effects on catalysis including hypo- and hyperactivity and altered fidelity. We applied molecular dynamics simulation (MD) and machine learning (ML) techniques to characterize TL mutations in the Saccharomyces cerevisiae RNA Polymerase II (Pol II) system. We did so to determine relationships between individual mutations and phenotypes and to associate phenotypes with MD simulated structural alterations. Using fitness values of mutants under various stress conditions, we modeled phenotypes along a spectrum of continual values. We found that ML could predict the phenotypes with 0.68 R2 correlation from amino acid sequences alone. It was more difficult to incorporate MD data to improve predictions from machine learning, presumably because MD data is too noisy and possibly incomplete to directly infer functional phenotypes. However, a variational auto-encoder model based on the MD data allowed the clustering of mutants with different phenotypes based on structural details. Overall, we found that lethal mutations tended to increase distances of TL residues to the NTP substrate, while viable loss-of-function (LOF) substitutions tended to confer an increase in distances between TL and bridge helix (BH). In contrast, GOF mutants generally have a disrupting effect on hydrophobic contacts among TL and nearby helices.AUTHOR SUMMARYRNA polymerase II (Pol II) synthesizes RNA with the help of an active site domain called trigger loop (TL). The mutations of TL cause changes in the activity of Pol II that could range from gain-of function (GOF) to loss-of-function (LOF) or lethal. This study provides a systematic characterization of the structural and functional outcomes of the TL mutations using molecular dynamics (MD) simulations and machine learning (ML). We obtained functional phenotypes of mutants by ML using the genetic fitness scores as the input. We revealed that mutant TL sequences could predict the functional outcomes at a relatively high correlation. Then, we performed MD simulations to relate the structural information to the phenotypes. The analysis of the MD data suggested that the lethal mutants had increased distances between the TL and the substrate, while a subset of LOF mutants showed increased distances between TL and another active site domain called bridge helix (BH). On the other hand, GOF mutants had effects on the hydrophobic interactions around the active site. Overall, this study enhances our understanding of the effects of TL mutations to the Pol II function.
Polynuclear ruthenium complexes are effective antibiotics against Pseudomonas aeruginosa
Authors: Weber Brent S. , Carfrae Lindsey A. , Woods Joshua J. , Klobucar Kristina , Bigham Nicholas P. , MacNair Craig R. , Raivio Tracy L. , Wilson Justin J. , Brown Eric D. 
Published: August 2022

AbstractThere is an urgent need to develop new antibiotics for the treatment of infections caused by drug-resistant Gram-negative bacteria. In particular, new and diverse chemical classes of antibiotics are needed, as most antibiotics in clinical development are derivatives of existing drugs. Despite a history of use as antimicrobials, metals and metal-based compounds have largely been overlooked as a source of new chemical matter for antibacterial drug discovery. In this work, we identify several ruthenium complexes, ruthenium red, Ru265, and Ru360’, that possess potent antibacterial activity against both laboratory and clinical isolates of Pseudomonas aeruginosa. Suppressors with increased resistance were sequenced and found to contain mutations in the mechanosensitive ion channel mscS-1 or the colRS two component system. The antibacterial activity of these compounds translated in vivo to Galleria mellonella larvae and mouse infection models. Finally, we identify strong synergy between these compounds and the antibiotic rifampicin, with a dose-sparing combination therapy showing efficacy in both infection models. Our findings provide clear evidence that these ruthenium complexes are effective antibacterial compounds against a critical priority pathogen and show promise for the development of future therapeutics.
Comparison of antibody responses to SARS-CoV-2 variants in Australian children
Authors: Toh Zheng Quan , Mazarakis Nadia , Nguyen Jill , Higgins Rachel A. , Anderson Jeremy , Ha Do Lien Anh , Burgner David P. , Curtis Nigel , Steer Andrew C. , Mulholland Kim , Crawford Nigel W. , Tosif Shidan , Licciardi Paul V 
Published: August 2022

AbstractThere is limited understanding of antibody responses in children across different SARS-CoV-2 variants. As part of an ongoing household cohort study, we assessed the antibody response among unvaccinated children infected with Wuhan, Delta or Omicron variants, as well as vaccinated children with breakthrough Omicron infection, using a SARS-CoV-2 S1-specific IgG assay and surrogate virus neutralisation test (sVNT). Most children infected with Delta (100%, 35/35) or Omicron (81.3%, 13/16) variants seroconverted by one month following infection. In contrast, 37.5% (21/56) children infected with Wuhan seroconverted, as previously reported. However, Omicron-infected children (GMC 46.4 BAU/ml; sVNT % inhibition: 16.3%) mounted a significantly lower antibody response than Delta (435.5 BAU/mL, sVNT=76.9%) or Wuhan (359.0 BAU/mL, sVNT=74.0%). Vaccinated children with breakthrough Omicron infection mounted the highest antibody response (2856 BAU/mL, sVNT=96.5%). Our findings suggest that despite a high seroconversion rate, Omicron infection in children results in lower antibody levels and function compared with Wuhan or Delta infection or with vaccinated children with breakthrough Omicron infection. Our data have important implications for public health measures and vaccination strategies to protect children.
Estimation of the Total Number of Infected Cases in the 5th Wave of COVID-19 in Hong Kong
Authors: Chow W.K. , Chow C.L. , Cheng C.H. 
Published: August 2022

AbstractThe total number of infected cases in a region in an epidemic is an important measure of the severity of the disease. With the increase in the number of infected people, the number of susceptible people will be reduced, and the recovery number is increased. The present study attempts to estimate the total number of infected cases in the 5th wave of COVID-19 in Hong Kong based on the daily additional cases supplied by the government based on two test schemes. Scheme 1 covers citizens suspected to be infected as referred by medical professionals, or requested or reported by citizens themselves, those returning from overseas, and those in close contact with the infected persons. Scheme 2 covers residents in buildings with a high concentration of virus in sewage. Polymerase chain reaction (PCR) test and then rapid antigen test (RAT) after 26 February 2022 were accepted by the Hong Kong Special Administrative Government in confirming infected cases. The number of infected cases in these two schemes were compared.A prediction model on infection case was proposed based on the transient daily infection curves. The averaged recovery number was estimated by assuming a 10-day infection period, including an incubation period of 5 days, and another 5 days for recovery. The transient number of infected, susceptible, recovered people were then presented. An adjustment factor to extend the scenario to the whole population of 7 million in Hong Kong was estimated and applied to study infection number in Hong Kong. Further, it appears that the infection number at the later stage of the 5th wave is weak around end July 2022. However, the number stayed at a constant value in comparing with rapid rise at the early stage in February 2022, even though the gathering activities were kept normal.
Cell surface excess is essential for protrusions and motility in 3D matrix
Authors: Kapustina Maryna , Li Donna , Zhu James , Wall Brittany , Weinreb Violetta , Cheney Richard E. 
Published: August 2022

ABSTRACTTo facilitate rapid changes in morphology without endangering cell integrity, each cell possesses a substantial amount of cell surface excess (CSE) that can be promptly deployed to cover cell extensions. CSE can be stored in different types of small surface projections such as filopodia, microvilli, and ridges, with rounded bleb-like projections being the most common and rapidly achieved form of storage. We demonstrate in this paper that cells migrating in 3D collagen use CSE to cover the developing protrusions. After retraction of a protrusion, the CSE this produces is stored over the cell body similar to the CSE produced by cell rounding. For the coordinated process of CSE storage and release, all cells should have specific mechanisms of regulation, and we hypothesize that microtubules (MT) play an important role in this mechanism. We show here that different effects of MT depolymerization on cell motility such as inhibiting mesenchymal motility and enhancing amoeboid, can be explained by the essential role of MT in CSE regulation and dynamics.
Metabolic adaptations underpin resistance to histone acetyltransferase inhibition
Authors: Bishop Timothy R. , Subramanian Chitra , Bilotta Eric M. , Garnar-Wortzel Leopold , Ramos Anissa R. , Zhang Yuxiang , Asiaban Joshua N. , Ott Christopher J. , Rock Charles O. , Erb Michael A. 
Published: August 2022

AbstractHistone acetyltransferases (HAT) catalyze the acylation of lysine side chains and are implicated in diverse human cancers as both oncogenes and non-oncogene dependencies1. Acetyl-CoA-competitive HAT inhibitors have garnered attention as potential cancer therapeutics and the first clinical trial for this class is ongoing (NCT04606446). Despite broad enthusiasm for these targets, notably including CBP/p300 and KAT6A/B2–5, the potential mechanisms of therapeutic response and evolved drug resistance remain poorly understood. Using comparative transcriptional genomics, we found that the direct gene regulatory consequences of CBP/p300 HAT inhibition are indistinguishable in models of intrinsically hypersensitive and insensitive acute myeloid leukemia (AML). We therefore modelled acquired drug resistance using a forward genetic selection and identified dysregulation of coenzyme A (CoA) metabolism as a facile driver of resistance to HAT inhibitors. Specifically, drug resistance selected for mutations in PANK3, a pantothenate kinase that controls the rate limiting step in CoA biosynthesis6. These mutations prevent negative feedback inhibition, resulting in drastically elevated concentrations of intracellular acetyl-CoA, which directly outcompetes drug-target engagement. This not only impacts the activity of structurally diverse CBP/p300 HAT inhibitors, but also agents related to an investigational KAT6A/B inhibitor that is currently in Phase-1 clinical trials. We further validated these results using a genome-scale CRISPR/Cas9 loss-of-function genetic modifier screen, which identified additional gene-drug interactions between HAT inhibitors and the CoA biosynthetic pathway. Top hits from the screen included the phosphatase, PANK4, which negatively regulates CoA production and therefore suppresses sensitivity to HAT inhibition upon knockout7, as well as the pantothenate transporter, SLC5A68, which enhances sensitivity. Altogether, this work uncovers CoA plasticity as an unexpected but potentially class-wide liability of anti-cancer HAT inhibitors and will therefore buoy future efforts to optimize the efficacy of this new form of targeted therapy.
APE1 recruits ATRIP to ssDNA in an RPA-independent manner to promote the ATR DNA damage response
Authors: Lin Yunfeng , Li Jia , Zhao Haichao , McMahon Anne , Yan Shan 
Published: August 2022

AbstractCells have evolved the DNA damage response (DDR) pathways in response to DNA replication stress or DNA damage. In the ATR-Chk1 DDR pathway, it has been proposed that ATR is recruited to RPA-coated single-strand DNA (ssDNA) by direct ATRIP-RPA interaction. However, it remains elusive whether and how ATRIP is recruited to ssDNA in an RPA-independent manner. Here, we provide evidence that APE1 directly associates ssDNA to recruit ATRIP onto ssDNA in an RPA-independent fashion. The N-terminal motif within APE1 is required and sufficient for the APE1-ATRIP interaction in vitro and the distinct APE1-ATRIP interaction is required for ATRIP recruitment to ssDNA and the ATR-Chk1 DDR pathway activation in Xenopus egg extracts. In addition, APE1 directly associates with RPA70 and RPA32 via two distinct motifs. Taken together, our evidence identifies APE1 as a direct recruiter of ATRIP onto ssDNA independent of RPA in the activation of ATR DDR pathway.SummaryAPE1 associates with ssDNA and ATRIP directly via distinct motifs and thereby recruits ATRIP onto ssDNA independent of RPA to promote the ATR DDR.APE1 interacts with RPA via distinct two motifs in vitro but such RPA-APE1 interaction is dispensable for ATRIP recruitment onto ssDNA.
Attachment/detachment of cortical myosin regulates cell junction exchange during cell rearrangement
Authors: Ikawa Keisuke , Ishihara Shuji , Tamori Yoichiro , Sugimura Kaoru 
Published: August 2022

AbstractEpithelial cells remodel cell adhesion and change their neighbors to shape a tissue. This cell rearrangement proceeds in three steps: the shrinkage of a junction, exchange of junctions, and elongation of the newly generated junction. Herein, by combining live imaging and physical modeling, we showed that the formation of myosin-II (myo-II) cables around the cell vertices underlies the exchange of junctions. The local and transient detachment of myo-II from the cell cortex is coupled with the junction shrinkage and elongation via an interplay between the LIM domain-containing protein Jub and the tricellular junction protein M6. Furthermore, we developed a mechanical model based on the wetting theory and clarified the way by which the physical properties of myo-II cables are integrated with the junction geometry to induce the transition between the attached and detached states and support the unidirectionality of cell rearrangement. Collectively, the present study elucidates the orchestration of geometry, mechanics and signaling for exchanging junctions.
Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies
Authors: Hartmann Fabian S. F. , Weiß Tamara , Kastberg Louise L. B. , Workman Christopher T. , Seibold Gerd M. 
Published: August 2022

AbstractGenetically encoded fluorescent biosensors have emerged as a powerful tool to support phenotypic screenings of microbes. Optical analyses of fluorescent sensor signals from colonies grown on solid media can be challenging as imaging devices need to be equipped with appropriate filters matching the properties of fluorescent biosensors. Towards versatile fluorescence analyses of different types of biosensor signals derived from arrayed colonies, we investigate here the use of monochromator equipped microplate readers as an alternative to imaging approaches. Indeed, for analyses of the LacI-controlled expression of the reporter mCherry inCorynebacterium glutamicum, or promoter activity using GFP as reporter inSaccharomyces cerevisiae, an improved sensitivity and dynamic range was observed for a microplate reader-based analyses compared to their analysesviaimaging. The microplate reader allowed us to capture signals of ratiometric fluorescent reporter proteins (FRPs) with a high sensitivity and thereby to further improve the analysis of internal pH via the pH-sensitive FRP mCherryEA inEscherichia colicolonies. Applicability of this novel technique was further demonstrated by assessing redox states inC. glutamicumcolonies using the FRP Mrx1-roGFP2. By the use of a microplate reader, oxidative redox shifts were measured in a mutant strain lacking the non-enzymatic antioxidant mycothiol (MSH), indicating its major role for maintaining a reduced redox state also in colonies on agar plates. Taken together, analyses of biosensor signals from microbial colonies using a microplate reader allows comprehensive phenotypic screenings and thus facilitates further development of new strains for metabolic engineering and systems biology.
Inhibition of Talin-induced Integrin Activation by a Double-hit Stapled Peptide
Authors: Gao Tong , Cho Eun-ah , Zhang Pingfeng , Wu Jinhua 
Published: August 2022

AbstractIntegrins are ubiquitously expressed cell-adhesion proteins. Talin is required for integrin activation through an inside-out signaling pathway, during which talin is recruited to the plasma membrane (PM) by RAP1 directly or through its effector, RAP1-Interacting Adaptor Molecule (RIAM). RIAM also activates talin from autoinhibition by binding to talin head domain. A helical talin-binding segment (TBS) in RIAM mediates both talin activation and recruitment by binding to two distinct sites in talin head and rod domains respectively. The bi-specificity of the TBS fragment allows us to develop a new strategy to suppress talin-induced integrin activation through a “double-hit” approach. We designed an experimental peptidomimetic inhibitor by engineering a hydrocarbon “staple” in the helical TBS fragment to mask the integrin binding site in the talin head. The stapled peptide (S-TBS) exhibits a stronger binding affinity with talin and inhibits talin:integrin interaction. Crystal structure of S-TBS in complex with talin rod domains reveals an interface that overlaps with the TBS-binding site in talin. Consistently, S-TBS also exhibits an inhibitory effect on TBS:talin-rod interaction during the PM recruitment of talin. Importantly, S-TBS possesses excellent cell permeability and inhibits integrin activation in a talin-dependent manner. Hence, our results present a novel approach to design a new class of intracellular inhibitors targeting integrin functions.
Comparative analysis of actin visualization by genetically encoded probes in cultured neurons
Authors: Ignácz Attila , Nagy-Herczeg Domonkos , Hausser Angelika , Schlett Katalin 
Published: August 2022

AbstractActin cytoskeleton predominantly regulates the formation and maintenance of synapses by controlling dendritic spine morphology and motility. To visualize actin dynamics, actin molecules can be labelled by genetically fusing fluorescent proteins to actin monomers or using fluorescently tagged actin-binding proteins or single-chain anti-actin antibodies. However, the effects of these labels on the morphology of neurons have not been quantitatively compared yet. In the present study, we analysed Actin-Chromobody-GFP, LifeAct-GFP and EGFP-actin with respect to their effects on actin-related features in mouse cultured hippocampal neurons.The actin-binding probes LifeAct and Actin-Chromobody showed similar affinity to F-actin, and along with EGFP-actin, were enriched in dendritic protrusions. In contrast to EGFP-actin, neither of these constructs was able to detect subtle changes in actin remodelling between mature mushroom shaped spine and less developed filopodia. None of the compared probes altered filopodial motility compared to control EGFP expression, however, within 24 hours expression, minor changes in dendritic spine morphology and density were visible. Furthermore, while EGFP-actin and LifeAct-GFP expression did not alter dendritic arborization, AC-GFP expressing neurons displayed a reduced dendritic arborization. We therefore conclude that careful consideration of cellular consequences is required before performing experiments with a particular actin labelling probe in primary neurons.