bluuebunny/crossref_metadata_2025 huggingface dataset:
Robust CRISPR/dCas13 RNA blockers specifically perturb miRNA-target interactions and rescue type 1 myotonic dystrophy pathology
Authors: Hanifi Muhammad , Ates-Kalkan Perihan Seda , Wen Sean , Fischer Mathieu , Kroesen Amanda , Yu Zulin , Wood Matthew , Thongjuea Supat , Mead Adam , Fulga Tudor Alexandru , Rinaldi Carlo , Sauka-Spengler Tatjana
Published: September 2024

While RNA-targeting strategies are powerful tools for disease therapy, challenges, including low target engagement and off-target collateral effects, currently limit their efficacy. Here, we report the engineering and optimisation of a CRISPR/dCas13 RNA steric blocker (CRISPR-Lock) that prevents mRNA translation, shields mRNAs from miRNA-mediated silencing, and blocks RNA-protein interactions. By tuning the spatial resolution and mismatch tolerance of CRISPR-Lock, we develop a high-resolution perturbation approach that employs genetically encoded CRISPR-Lock as a miRNA target protector. This system enables precise spatiotemporal control of miRNA:mRNA interactions, offering broader applicability compared to phosphorodiamidate mor-pholino (PMO) target protectors. Moreover, we demonstrate the potential therapeutic application of CRISPR-Lock for blocking pathological RNA-protein interactions in type 1 myotonic dystro-phy (DM1). Optimising CRISPR-Lock to target expanded repeat RNAs corrects approximately 85% of clinically relevant splicing biomarkers in patient-derived myotubes and significantly out-performs third-generation PMO antisense oligonucleotides. Finally, by delivering a miniaturised AAV-encoded CRISPR-Lock system into an established DM1 mouse model, we demonstrate the dose-dependent correction of intranuclear foci and splicing dysregulation, underscoring the potential therapeutic application of this technology.
Optimal control of immune checkpoint inhibitor therapy in a heart-tumour model
Authors: van der Vegt Solveig A. , Baker Ruth E. , Waters Sarah L.
Published: September 2024

Autoimmune myocarditis, or cardiac muscle inflammation, is a rare but frequently fatal side–effect of immune checkpoint inhibitors (ICIs), a class of cancer therapies. Despite the dangers that side-effects such as these pose to patients, they are rarely, if ever, included explicitly when mechanistic mathematical modelling of cancer therapy is used for optimization of treatment. In this paper, we develop a two-compartment mathematical model which incorporates the impact of ICIs on both the heart and the tumour. Such a model can be used to inform the conditions under which autoimmune myocarditis may develop as a consequence of treatment. We use this model in an optimal control framework to design optimized dosing schedules for three types of ICI therapy that balance the positive and negative effects of treatment. We show that including the negative side-effects of ICI treatment explicitly within the mathematical framework significantly impacts the predictions for the optimized dosing schedule, thus stressing the importance of a holistic approach to optimizing cancer therapy regimens.
Orchestrated long-distance gene activation by a ParB-like BisD-CTP DNA clamp in low-frequency transfer competence development inPseudomonas putida
Authors: Antar Hammam , Carraro Nicolas , Gruber Stephan , van der Meer Jan Roelof
Published: September 2024

Integrative conjugative elements (ICEs) are mobile DNA that remain integrated within the host bacterial genome until they activate their excision and transfer to recipient cells via conjugation. ICE transfer is initiated in a small subpopulation of cells that undergo a hierarchical gene expression cascade leading to transfer competence formation. In this study, we demonstrate that transfer competence formation of the ICEclcelement in stationary phasePseudomonas putidacells is regulated by the coordinated activity of a two-component transcriptional activator, BisC and BisD. Chromatin immunoprecipitation of tagged BisC or BisD followed by high throughput sequencing showed that both proteins accumulate at similar sites around ICEclctransfer competence promoters inP. putidacells. Genetic dissection and single cell microscopy showed that BisD is a dual domain protein, with a C-terminal gene activator domain and an N-terminal ParB-like domain, forming dimeric DNA clamps that self-load at distant sites and reach target promoters after extensive one-dimensional DNA sliding. Expressed mCherry-BisD inP. putidaICEclccells form discrete fluorescent foci, dependent onparS-like sequences on the ICE. This focus formation is similar as what is seen with canonical ParB proteins accumulating on chromosomal DNA, but in case of BisD corresponds to both chromosomal and excised ICE-molecules. Sliding of BisD over ∼50 kb of ICE-DNA is asymmetric and follows the direction of ICE gene transcription. This may help to establish a temporal order of activation of transfer competence formation, optimizing ICE transfer. Given that ICEclcis activated in stationary phase cells, we hypothesize that BisD is not involved in segregating excised ICE-DNA among daughter cells, but rather in actively directing ICE-DNA molecules towards the (multiple) conjugative systems that are produced in transfer competent cells. BisD thus serves as a twin function protein, integrating gene activation and DNA segregation functions.
Functional analyses and integrated mechanisms of cellular destruction by L-amino acid oxidase
Authors: Prak Krisna , Luft Christin , Tsefou Eliona , Chávez-Olórtegui Carlos , Kriston-Vizi Janos , Kettler Robin , Braga Vania M. M.
Published: September 2024

Snakebite accidents are prevalent worldwide and cause a spectrum of severe clinical manifestations and reduction of patient quality of life and economic income. L-amino acid oxidase (LAAO) is a highly toxic enzyme present in various venoms that causes tissue necrosis, oedema, coagulopathies, and organ failure. Here we investigate the mechanisms of LAAO cytotoxicity preceding cell death using recombinant LAAO and a catalytic inactive LAAO mutant. Wild-type LAAO uptake by cells leads to a decrease in lysosome number and size and inhibition of autophagy flux. Mitochondria function is impaired by severe proton leakage and mitochondrial fission is stimulated. Despite engulfment by autophagosomes, mitochondrial clearance is prevented by the lysosomal defects. The coordinate multi-organelle dysfunction strongly perturbs energy production, cell metabolism and clearance of defective organelles by autophagy, thereby triggering an irreversible destructive path. Considering the fast organelle impairment, strategies to reduce multi-organelle injury after LAAO exposure may be effective to maintain critical cell functions and strengthen adaptive responses against cytotoxicity.
Rebalance the inhibitory system in the elderly brain: Influence of balance learning on GABAergic inhibition and functional connectivity
Authors: Liu Xinyu , Scherrer Selin , Egger Sven , Lim Song-I , Lauber Benedikt , Jelescu Ileana , Griffa Alessandra , Gambarota Giulio , Taube Wolfgang , Xin Lijing
Published: September 2024

Aging involves complex processes that impact the structure, function, and metabolism of the human brain. Declines in both structural and functional integrity along with reduced local inhibitory tone in the motor areas, as indicated by reduced γ-Aminobutyric acid (GABA) levels, are often associated with compromised motor performance in elderly adults. Using multi-modal neuroimaging techniques including magnetic resonance spectroscopy (MRS), diffusion magnetic resonance imaging (MRI), functional MRI as well as transcranial magnetic stimulation to assess short interval intracortical inhibition (SICI), this study explores whether these age-related changes can be mitigated by motor learning. The investigation focused on the effects of long-term balance learning (3 months) on intracortical inhibition, metabolism, structural and functional connectivity in the cortical sensorimotor network among an elderly cohort. We found that after three months of balance learning, subjects significantly improved balance performance, upregulated sensorimotor cortical GABA levels and ventral sensorimotor network functional connectivity (VSN-FC) compared to a passive control group. Furthermore, correlation analysis suggested a positive association between baseline VSN-FC and balance performance, between baseline VSN-FC and SICI, and between improvements in balance performance and upregulation in SICI in the training group, though these correlations did not survive the false discovery rate correction. These findings demonstrate that balance learning has the potential to counteract aging-related decline in functional connectivity and cortical inhibition on the ‘tonic’ (MRS) and ‘functional’ (SICI) level and shed new light on the close interplay between the GABAergic system, functional connectivity, and behavior.
Expansion microscopy allows quantitative characterisation of structural organisation of platelet aggregates
Authors: Faulkner Emma L. , Pike Jeremy A. , Garlick Evelyn , Neely Robert K. , Styles Iain B. , Watson Stephen P. , Poulter Natalie S. , Thomas Steven G.
Published: September 2024

1

Current microscopy approaches applied to platelet aggregates in both haemostatic and thrombotic settings indicate their structure has important implications in efficient haemostasis and in clinical treatment of thrombosis. However, current fluorescence microscopy approaches are not amenable to volumetric imaging of platelet aggregate structures. This is largely due to the small size of individual platelets and the tight packing of platelets within aggregates, resulting in optical opacity.

Here we demonstrate that expansion microscopy, applied to platelet aggregates, can reveal multi-scale information about the structure of platelet aggregates. We produced volumetric images at nanoscale resolution of >700 platelet aggregates under normal and perturbed conditions, stained for cytoskeletal and membrane components. We demonstrate our custom analysis workflow provides quantitative description of platelet numbers, volumes and morphology within entire platelet aggregates. Additionally, we quantitatively describe subcellular organisation of F-actin. By comparing these measurements following treatment with the actin inhibitors, cytochalasin D and latrunculin A, we can robustly detect structural disruptions in platelet aggregates. Together these data provide a workflow to qualitatively and quantitatively describe the architecture of platelet aggregates at a range of scales (whole aggregates down to sub-cellular features within individual platelets).
Complete Genomic Characterization of Global Pathogens, Respiratory Syncytial Virus (RSV), and Human Norovirus (HuNoV) Using Probe-based Capture Enrichment
Authors: Bhamidipati Sravya V , Surathu Anil , Chao Hsu , Agustinho Daniel P , Xiang Qin , Kottapalli Kavya , Santhanam Abirami , Momin Zeineen , Walker Kimberly , Menon Vipin K , Weissenberger George , Emerick Nathanael , Mahjabeen Faria , Meng Qingchang , Hu Jianhong , Sucgang Richard , Henke David , Sedlazeck Fritz J , Khan Ziad , Metcalf Ginger A , Avadhanula Vasanthi , Piedra Pedro A , Ramani Sasirekha , Atmar Robert L , Estes Mary K , Petrosino Joseph F , Gibbs Richard A , Muzny Donna M , Cregeen Sara Javornik , Doddapaneni Harsha
Published: September 2024

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, while human noroviruses (HuNoV) are a leading cause of epidemic and sporadic acute gastroenteritis. Generating full-length genome sequences for these viruses is crucial for understanding viral diversity and tracking emerging variants. However, obtaining high-quality sequencing data is often challenging due to viral strain variability, quality, and low titers. Here, we present a set of comprehensive oligonucleotide probe sets designed from 1,570 RSV and 1,376 HuNoV isolate sequences in GenBank. Using these probe sets and a capture enrichment sequencing workflow, 85 RSV positive nasal swab samples and 55 (49 stool and six human intestinal enteroids) HuNoV positive samples encompassing major subtypes and genotypes were characterized. The Ct values of these samples ranged from 17.0-29.9 for RSV, and from 20.2-34.8 for HuNoV, with some HuNoV having below the detection limit. The mean percentage of post-processing reads mapped to viral genomes was 85.1% for RSV and 40.8% for HuNoV post-capture, compared to 0.08% and 1.15% in pre-capture libraries, respectively. Full-length genomes were>99% complete in all RSV positive samples and >96% complete in 47/55 HuNoV positive samples—a significant improvement over genome recovery from pre-capture libraries. RSV transcriptome (subgenomic mRNAs) sequences were also characterized from this data. Probe-based capture enrichment offers a comprehensive approach for RSV and HuNoV genome sequencing and monitoring emerging variants.

IMPORTANCE

Respiratory syncytial virus (RSV) and human noroviruses (HuNoV) are NIAID category C and category B priority pathogens, respectively, that inflict significant health consequences on children, adults, immunocompromised patients, and the elderly. Due to the high strain diversity of RSV and HuNoV genomes, obtaining complete genomes to monitor viral evolution and pathogenesis is challenging. In this paper, we present the design, optimization, and benchmarking of a comprehensive oligonucleotide target capture method for these pathogens. All 85 RSV samples and 49/55 HuNoV samples were patient-derived with six human intestinal enteroids. The methodology described here results has a higher success rate in obtaining full-length RSV and HuNoV genomes, enhancing the efficiency of studying these viruses and mutations directly from patient-derived samples.
Epigenomic analysis of hepatocellular carcinoma reveals aberrantcis-regulatory changes and dysregulated retrotransposons with prognostic potentials
Authors: Cheng Clooney C. Y. , Cheung Ming Fung , Lee Ah Young , Wu Qiong , Chow Savio Ho-Chit , Ang Julie Y. J. , Medina Ignacio Riquelme , Lo Grace , Wu Haoran , Yang Weiqin , Lai Paul B. S. , Yip Kevin Y. , Cheng Alfred S. , Leung Danny
Published: September 2024

Hepatocellular carcinoma (HCC) exhibits widespread epigenetic alterations, yet their impact oncis-regulatory elements (CREs) and retrotransposons remains poorly understood. Here, we present an integrated epigenomic and transcriptomic analysis of HCC tumors and matched tumor-adjacent normal tissues. We identified extensive DNA hypomethylation coupled with changes in histone modifications at partially methylated domains, CREs, and retrotransposons. These epigenetic aberrations were associated with dysregulated expression of genes involved in cell cycle regulation, immune response, and extracellular matrix organization. Notably, our findings revealed a novel mechanism for the transcriptional dysregulation ofGPC3, a key HCC biomarker and immunotherapeutic target. We observed thatGPC3upregulation is driven by both the reactivation of a fetal liver super enhancer and hypomethylation ofGPC3-associated CpG islands. Moreover, we found that DNA hypomethylation-driven aberrant expression of retrotransposons carries prognostic significance in HCC. Patients with high expression of a long non-coding RNA driven by a HERVE-int element exhibited more aggressive tumors, poorer clinical outcomes, and molecular features associated with favorable immunotherapy response. Together, our study provides a comprehensive resource for understanding the role of epigenetic dysregulation in HCC and identifies retrotransposon-associated transcripts as potential biomarkers.
Peripheral opioid receptor antagonism alleviates fentanyl-induced cardiorespiratory depression and is devoid of aversive behavior
Authors: Ruyle Brian C. , Masud Sarah , Kesaraju Rohith , Tahirkheli Mubariz , Modh Juhi , Roth Caroline G. , Angulo-Lopera Sofia , Lintz Tania , Higginbotham Jessica A. , Massaly Nicolas , Moron Jose A.
Published: September 2024

Millions of Americans suffering from Opioid Use Disorders (OUD) face a high risk of fatal overdose due to opioid-induced respiratory depression (OIRD). Fentanyl, a powerful synthetic opioid, is a major contributor to the rising rates of overdose deaths. Reversing fentanyl overdoses has proved challenging due to its high potency and the rapid onset of OIRD. We assessed the contributions of central and peripheral mu opioid receptors (MORs) in mediating fentanyl-induced physiological responses. The peripherally restricted MOR antagonist naloxone methiodide (NLXM) both prevented and reversed OIRD to a degree comparable to that of naloxone (NLX), indicating substantial involvement of peripheral MORs to OIRD. Interestingly, NLXM-mediated OIRD reversal did not produce aversive behaviors observed after NLX. We show that neurons in the nucleus of the solitary tract (nTS), the first central synapse of peripheral afferents, exhibit a biphasic activity profile following fentanyl exposure. NLXM pretreatment attenuates this activity, suggesting that these responses are mediated by peripheral MORs. Together, these findings establish a critical role for peripheral MORs, including ascending inputs to the nTS, as sites of dysfunction during OIRD. Furthermore, selective peripheral MOR antagonism could be a promising therapeutic strategy for managing OIRD by sparing CNS-driven acute opioid-associated withdrawal and aversion observed after NLX.

Significance Statement

In this study, we compare the central versus peripheral components underlying fentanyl-induced cardiorespiratory depression to prevent overdose deaths. Our data indicate that these effects are, at least partially, due to the activation of mu opioid receptors present in peripheral sites. These findings provide insight into peripheral contributions to fentanyl-induced overdoses and could potentially lead to the development of treatments selectively targeting the peripheral system, sparing individuals from the CNS-driven acute opioid withdrawal generally observed with the use of naloxone.
The Pattern and Staging of Brain Atrophy in Spinocerebellar Ataxia Type 2 (SCA2): MRI Volumetrics from ENIGMA-Ataxia
Authors: Robertson Jason W. , Adanyeguh Isaac , Bender Benjamin , Boesch Sylvia , Brunetti Arturo , Cocozza Sirio , Coutinho Léo , Deistung Andreas , Diciotti Stefano , Dogan Imis , Durr Alexandra , Fernandez-Ruiz Juan , Göricke Sophia L. , Grisoli Marina , Han Shuo , Mariotti Caterina , Marzi Chiara , Mascalchi Mario , Mochel Fanny , Nachbauer Wolfgang , Nanetti Lorenzo , Nigri Anna , Ono Sergio E. , Onyike Chiadi U. , Prince Jerry L. , Reetz Kathrin , Romanzetti Sandro , Saccà Francesco , Synofzik Matthis , Teive Hélio A. Ghizoni , Thomopoulos Sophia I. , Thompson Paul M. , Timmann Dagmar , Ying Sarah H. , Harding Ian H. , Hernandez-Castillo Carlos R.
Published: September 2024

Objective

Spinocerebellar ataxia type 2 (SCA2) is a rare, inherited neurodegenerative disease characterised by progressive deterioration in both motor coordination and cognitive function. Atrophy of the cerebellum, brainstem, and spinal cord are core features of SCA2, however the evolution and pattern of whole-brain atrophy in SCA2 remain unclear. We undertook a multi-site, structural magnetic resonance imaging (MRI) study to comprehensively characterize the neurodegeneration profile of SCA2.

Methods

Voxel-based morphometry analyses of 110 participants with SCA2 and 128 controls were undertaken to assess groupwise differences in whole-brain volume. Correlations with clinical severity and genotype, and cross-sectional profiling of atrophy patterns at different disease stages, were also performed.

Results

Atrophy in SCA2 relative to controls was greatest (Cohen’sd>2.5) in the cerebellar white matter (WM), middle cerebellar peduncle, pons, and corticospinal tract. Very large effects (d>1.5) were also evident in the superior cerebellar, inferior cerebellar, and cerebral peduncles. In cerebellar grey matter (GM), large effects (d>0.8) mapped to areas related to both motor coordination and cognitive tasks. Strong correlations (|r|>0.4) between volume and disease severity largely mirrored these groupwise outcomes. Stratification by disease severity showed a degeneration pattern beginning in cerebellar and pontine WM in pre-clinical subjects; spreading to the cerebellar GM and cerebro-cerebellar/corticospinal WM tracts; then finally involving the thalamus, striatum, and cortex in severe stages.

Interpretation

The magnitude and pattern of brain atrophy evolves over the course of SCA2, with widespread, non-uniform involvement across the brainstem, cerebellar tracts, and cerebellar cortex; and late involvement of the cerebral cortex and striatum.
NR2F2 regulation of interstitial to fetal Leydig cell differentiation in the testis: insights into differences of sex development
Authors: Estermann Martín Andrés , Grimm Sara , Kitakule Abigail , Rodriguez Karina , Brown Paula , McClelland Kathryn , Amato Ciro , Yao Humphrey Hung-Chang
Published: September 2024

Testicular fetal Leydig cells are a specialized cell type responsible for embryo masculinization. Fetal Leydig cells produce androgens, that induce the differentiation of male reproductive system and sexual characteristics. Deficiencies in Leydig cell differentiation leads to various disorders of sex development and male reproductive defects such as ambiguous genitalia, hypospadias, cryptorchidism, and infertility. Fetal Leydig cells are thought to originate from proliferating progenitor cells in the testis interstitium, marked by genes likeArx,Pdgfra,Tcf21andWnt5a. However, the precise mechanisms governing the transition from interstitial cells to fetal Leydig cells remain elusive. Through integrated approaches involving mouse models and single-nucleus multiomic analyses, we discovered that fetal Leydig cells originate from aNr2f2-positive non-steroidogenic interstitial cell population. Embryonic deletion ofNr2f2in mouse testes resulted in disorders of sex development, including dysgenic testes, Leydig cell hypoplasia, cryptorchidism, and hypospadias. We found that NR2F2 promotes the progenitor cell fate while suppresses Leydig cell differentiation by directly and indirectly controlling a cohort of transcription factors and downstream genes. Bioinformatic analyses of single-nucleus ATAC-seq and NR2F2 ChIP-seq data revealed putative transcription factors co-regulating the process of interstitial to Leydig cell differentiation. Collectively, our findings not only highlight the critical role ofNr2f2in orchestrating the transition from interstitial cells to fetal Leydig cells, but also provide molecular insight into the disorders of sex development as a result ofNr2f2mutations.
Biological volume EM with focused Ga ion beam depends on formation of radiation-resistant Ga-rich layer at block face
Authors: Yang Zijie , Kim Joshua , Zhang Guofeng , Aronova Maria A. , Leapman Richard D.
Published: September 2024

Volume electron microscopy (vEM) enables biologists to visualize nanoscale 3D ultrastructure of entire eukaryotic cells and tissues prepared by heavy atom staining and plastic embedding. The highest resolution vEM technique is focused ion-beam scanning electron microscopy (FIB-SEM), which provides nearly isotropic (~5-10 nm) spatial resolution at fluences of > 10,000 e−/nm2. However, it is not clear how such high resolution is achievable because serial block-face (SBF) SEM, which incorporates an in-situ ultramicrotome instead of a Ga+FIB beam, results in radiation-induced collapse of similar specimen blocks at fluences of only ~20 e−/nm2. Moreover, the z-resolution (perpendicular to block-face) in FIB-SEM is substantially better than predicted by Monte Carlo modeling of SBF-SEM samples. Here, we show that FIB-SEM implants a thin highly concentrated layer of Ga+ions at the block surface, which greatly reduces the effect of beam-damage, reduces the depth from which backscattered electrons are detected, and prevents specimen charging and collapse.
Diphthamide formation in Arabidopsis requires DPH1-interacting DPH2 for light and oxidative stress resistance
Authors: Zhang Hongliang , Janina Nadežda , Ütkür Koray , Manivannan Thirishika , Zhang Lei , Wang Lizhen , Grefen Christopher , Schaffrath Raffael , Krämer Ute
Published: September 2024

Diphthamide is a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR 2 (eEF2) and the target of diphtheria toxin in human cells. In yeast and mammals, the 4Fe-4S cluster-containing proteins Dph1 and Dph2 catalyze the first biosynthetic step of diphthamide formation. Here we identifyArabidopsis thalianaDPH2 and show that it is required for diphthamide biosynthesis, localizes to the cytosol and interacts physically with AtDPH1. Arabidopsisdph2mutants form shorter primary roots and smaller rosettes than the wild type, similar todph1mutants which we characterized previously. Additionally, increased ribosomal -1 frameshifting error rates and attenuated TARGET OF RAPAMYCIN (TOR) kinase activity indph2mutants also phenocopy thedph1mutant. Beyond the known heavy-metal hypersensitivity and heat shock tolerance ofdph1, we newly show here that bothdph1anddph2mutants are hypersensitive to elevated light intensities and oxidative stress, and that wild-type Arabidopsis seedlings accumulate diphthamide-unmodified eEF2 under oxidative stress. Both mutants share the deregulation of 1,186 transcripts in numerous environmental and hormone responses. AtDPH1 and AtDPH2 do not complement the corresponding mutants ofSaccharomyces cerevisiae. In summary, DPH2 and DPH1 interact to function inter-dependently in diphthamide formation, the maintenance of translational fidelity, wild-type growth rates and TOR kinase activation, and they contribute to mitigating damage from elevated light intensities and oxidative stress. Under oxidative stress, a dose-dependent loss of diphthamide could potentiate downstream effects in a feed-forward loop. This work advances our understanding of translation and its interactions with growth regulation and stress responses in plants.
Conformational flexibility is a critical factor in designing broad-spectrum human norovirus protease inhibitors
Authors: Pham Son , Zhao Boyang , Neetu Neetu , Sankaran Banumathi , Patil Ketki , Ramani Sasirekha , Song Yongcheng , Estes Mary K. , Palzkill Timothy , Prasad B.V. Venkataram
Published: September 2024

Human norovirus (HuNoV) infection is a global health and economic burden. Currently, there are no licensed HuNoV vaccines or antiviral drugs available. The protease encoded by the HuNoV genome plays a critical role in virus replication by cleaving the polyprotein and is, therefore, an excellent target for developing small molecule inhibitors. While rupintrivir, a potent small-molecule inhibitor of several picornavirus proteases, effectively inhibits GI.1 protease, it is an order of magnitude less effective against GII protease. Other GI.1 protease inhibitors also tend to be less effective against GII proteases. To understand the structural basis for the potency difference, we determined the crystal structures of proteases of GI.1, pandemic GII.4 (Houston and Sydney), and GII.3 in complex with rupintrivir. These structures show that the open substrate pocket in GI protease binds rupintrivir without requiring significant conformational changes, whereas, in GII proteases, the closed pocket flexibly extends, reorienting arginine-112 in the BII-CII loop to accommodate rupintrivir. Structures of R112A protease mutants with rupintrivir, coupled with enzymatic and inhibition studies, suggest R112 is involved in displacing both substrate and ligands from the active site, implying a role in the release of cleaved products during polyprotein processing. Thus, the primary determinant for differential inhibitor potency between the GI and GII proteases is the increased flexibility in the BII-CII loop of the GII proteases caused by H-G mutation in this loop. Therefore, the inherent flexibility of the BII-CII loop in GII proteases is a critical factor to consider when developing broad-spectrum inhibitors for HuNoV proteases.

IMPORTANCE

Human noroviruses are a significant cause of sporadic and epidemic gastroenteritis worldwide. There are no vaccines or antiviral drugs currently available to treat infections. Our work elucidates the structural differences between GI.1 and GII proteases in response to inhibitor binding and will inform the future development of broad-spectrum norovirus protease inhibitors.
Exploring putative enteric methanogenesis inhibitors using molecular simulations and a graph neural network
Authors: Aryee Randy , Mohammed Noor S. , Dey Supantha , Arunraj B. , Nadendla Swathi , Sajeevan Karuna Anna , Beck Matthew R. , Frazier A. Nathan , Koziel Jacek A. , Mansell Thomas J. , Chowdhury Ratul
Published: September 2024

Atmospheric methane (CH4) acts as a key contributor to global warming. As CH4is a short-lived climate forcer (12 years atmospheric lifespan), its mitigation represents the most promising means to address climate change in the short term. Enteric CH4(the biosynthesized CH4from the rumen of ruminants) represents 5.1% of total global greenhouse gas (GHG) emissions, 23% of emissions from agriculture, and 27.2% of global CH4emissions. Therefore, it is imperative to investigate methanogenesis inhibitors and their underlying modes of action. We hereby elucidate the detailed biophysical and thermodynamic interplay between anti-methanogenic molecules and cofactor F430of methyl coenzyme M reductase and interpret the stoichiometric ratios and binding affinities of sixteen inhibitor molecules. We leverage this as prior in a graph neural network to first functionally cluster these sixteen known inhibitors among ∼54,000 bovine metabolites. We subsequently demonstrate a protocol to identify precursors to and putative inhibitors for methanogenesis, based on Tanimoto chemical similarity and membrane permeability predictions. This work lays the foundation for computational andde novodesign of inhibitor molecules that retain/ reject one or more biochemical properties of known inhibitors discussed in this study.

COVER ABSTRACT
Yatakemycin biosynthesis requires two deoxyribonucleases for toxin self-resistance
Authors: Dorival Jonathan , Yuan Hua , Walker Allison S. , Tang Gong-Li , Eichman Brandt F.
Published: September 2024

The highly active natural product yatakemycin (YTM) fromStreptomycessp. TP-A0356 is a potent DNA damaging agent with antimicrobial and antitumor properties. The YTM biosynthesis gene cluster (ytk) contains several toxin self-resistance genes. Of these,ytkR2encodes a DNA glycosylase that is important for YTM production and host survival by excising lethal YTM-adenine lesions from the genome, presumably initiating a base excision repair (BER) pathway. However, the genes involved in repair of the resulting apurinic/apyrimidinic (AP) site as the second BER step have not been identified. Here, we show thatytkR4andytkR5are essential for YTM production and encode deoxyribonucleases related to other known DNA repair nucleases. Purified YtkR4 and YtkR5 exhibit AP endonuclease activity specific for YtkR2-generated AP sites, providing a basis for BER of the toxic AP intermediate produced from YTM-adenine excision and consistent with co-evolution ofytkR2, ytkR4, andytkR5. YtkR4 and YtkR5 also exhibit 3′-5′ exonuclease activity with differing substrate specificities. The YtkR5 exonuclease is capable of digesting through a YTM-DNA lesion and may represent an alternative repair mechanism to BER. We also show thatytkR4andytkR5homologs are often clustered together in putative gene clusters related to natural product production, consistent with non-redundant roles in repair of other DNA adducts derived from genotoxic natural products.
Fish environmental RNA sequencing sensitively captures accumulative stress responses through short-term aquarium sampling
Authors: Miyata Kaede , Inoue Yasuaki , Yamane Masayuki , Honda Hiroshi
Published: September 2024

The utility of environmental RNA (eRNA) in capturing biological responses to stresses has been discussed previously; however, the limited number of genes detected remains a significant hindrance to its widespread implementation. Here, we investigated the potential of eRNA to assess the health status of Japanese medaka fish exposed to linear alkylbenzene sulfonate. Analyzing eRNA and organismal RNA (oRNA) in aquarium water for > 12 h, we achieved high mapping rates and 10 times more differentially expressed genes than previously reported. This advancement has facilitated the previously unattainable capability of gene ontology (GO) analysis. In GO analysis, it was found that eRNA could detect nuclear genes in cellular component, and oRNA provided gene expression signatures in the short term, whereas eRNA demonstrated the potential to reflect cumulative gene expression signatures over a period of time in biological process. Moreover, eRNA exhibited high sensitivity in responding to genes associated with sphingolipid and ceramide biosynthesis, which are involved in inflammatory responses possibly originating from impaired cells. This finding aligns with the observations made in oRNA. Conclusively, eRNA-sequencing (eRNA-seq) using aquarium water emerges as a valuable high sensitivity tool for analyzing physiological stress. The findings of this study lay the foundation for further development of eRNA-seq technologies.
A high-resolution microscopy system for biological studies of cold-adapted species under physiological conditions
Authors: Marty Anne-Pia M. , Ward Edward N. , Lamb Jacob R. , van Tartwijk Francesca W. , Peck Lloyd S. , Clark Melody S. , Kaminski Clemens F.
Published: September 2024

The fundamental processes governing life are sensitively dependent on temperature. Whilst much is known about the constraints on how proteins operate at 37°C, little knowledge exists about how biological function is maintained sub-zero temperature conditions, where proteins are less stable and oxidative damage is high. However, almost 90% of habitable environments on Earth are permanently below 5°C (i.e. the deep sea and polar regions). This means that we do not understand how a large and diverse proportion of the global biome functions. To address this question at the cellular level, tools are required for imaging biological systems at high resolution under physiological conditions. This poses severe technical challenges that cannot be addressed with traditional optical microscopy techniques. High-resolution imaging objectives require short working distances and the use of immersion media, which lead to rapid heat transfer from the microscope to the sample. This affects the viability of live specimens and the interpretability of the results when the sample function optimally at low temperatures. Condensation and temperature-induced shrinking of components pose further challenges, reducing image resolution and contrast. Here, we address these issues and provide a method for high-fidelity imaging of live biological samples at temperatures of around, or below, 0°C. Our method is compatible with different microscopy modalities, including super-resolution imaging. It relies on hardware additions to traditional microscopy systems that can be straightforwardly implemented, namely, a cooling collar, 10% ethanol as an immersion medium, and nitrogen flow to mitigate condensation. We demonstrate the method in live cell cultures derived from Antarctic fish species and highlight the need to maintain physiological conditions for these fragile biological samples. Future applications are diverse and include evolutionary biology and the study of cold-adapted organisms, as well as cellular biophysics and several applications in biotechnology.
Expression of XCR1, CX3CR1, CSF1R and ADGRE1 on subsets of bovine blood dendritic cells and monocytes
Authors: Talker Stephanie C , Wu Zhiguang , Dry Inga , Summerfield Artur , Hope Jayne C
Published: September 2024

Among dendritic cells (DC) and monocytes in blood of cattle we can distinguish conventional and plasmacytoid DC (cDC1, cDC2, pDC) and classical, intermediate and nonclassical monocytes (cM, intM, ncM), respectively. Phenotypic definitions of subsets rely on combinations of only a few markers such as CD13 and CD4 for Flt3+dendritic cells, and CD14 and CD16 for CD172ahighmonocytes. Additional reagents for flow cytometry are urgently needed to refine these phenotypic classifications and account for heterogeneity of these cells, in particular cDC2 and intM.

In the current study we have investigated expression of CX3CR1 and ADGRE1 on bovine blood DC and monocyte subsets, and have tested two newly generated bovine recombinant proteins (XCL1 and CSF1) for staining of XCR1 and CSF1R.

Staining patterns obtained by multicolor flow cytometry of peripheral blood mononuclear cells from two cows were largely in line with gene expression data available from a previous study (bulk RNA-seq of sorted DC and monocyte subsets from bovine blood).
Highly accurate assembly polishing with DeepPolisher
Authors: Mastoras Mira , Asri Mobin , Brambrink Lucas , Hebbar Prajna , Kolesnikov Alexey , Cook Daniel E. , Nattestad Maria , Lucas Julian , Won Taylor S. , Chang Pi-Chuan , Carroll Andrew , Paten Benedict , Shafin Kishwar
Published: September 2024

Accurate genome assemblies are essential for biological research, but even the highest quality assemblies retain errors caused by the technologies used to construct them. Base-level errors are typically fixed with an additional polishing step that uses reads aligned to the draft assembly to identify necessary edits. However, current methods struggle to find a balance between over- and under-polishing. Here, we present an encoder-only transformer model for assembly polishing called DeepPolisher, which predicts corrections to the underlying sequence using Pacbio HiFi read alignments to a diploid assembly. Our pipeline introduces a method, PHARAOH (Phasing Reads in Areas Of Homozygosity), which uses ultra-long ONT data to ensure alignments are accurately phased and to correctly introduce heterozygous edits in falsely homozygous regions. We demonstrate that the DeepPolisher pipeline can reduce assembly errors by half, with a greater than 70% reduction in indel errors. We have applied our DeepPolisher-based pipeline to 180 assemblies from the next Human Pangenome Reference Consortium (HPRC) data release, producing an average predicted Quality Value (QV) improvement of 3.4 (54% error reduction) for the majority of the genome.
A new highly specific and soluble protease for precise removal of N-terminal purification tags
Authors: Shams Raef , Regan Lynne
Published: September 2024

The affinity purification tags have significantly enhanced the ease of purification of many proteins. After purification, it is necessary to remove the affinity purification tag for many applications. Several specific proteases have been developed for tag removal, but none yet has both, a desirable cleavage specificity and high solubility. Here we describe the design and characterization of a novel protease, which we have named Con1, which exhibits desirable cleavage specificity and high solubility. We present a detailed analysis of the kinetic properties of Con1, along with demonstrating its ability to remove purification tags from different proteins.
Integrating Clinical Research with Electronic Health Records to Improve Diversity in Research: Findings from an Urban Hospital System
Authors: Brown Edward H. , Peiper Nicholas C. , Furmanek Stephen , McCants Kelly
Published: September 2024

Background

The under-representation of racial, ethnic, and other minority populations in clinical research has threatened the validity of novel therapeutic interventions and exacerbated the longstanding inequities in health outcomes. Despite attention and mandates across institutions and federal agencies to address these disparities, effective and actionable strategies have remained a subject of debate in the existing literature. Thus, the need for comprehensive and rigorous methods to assess diversity in study population samples as well as strategies for improved recruitment and retention has been made clear.

Objective

Examine the distribution of research participant demographics at Norton Healthcare (NHC) and compare to applicable benchmarks from the overall NHC patient population and local census.

Design

Successive-independent samples comparison of EPIC electronic health records (EHR) and census data from Jefferson County.

Participants

A total of 2,053 adult research participants enrolled at NHC from 2020 – 2024.

Main Measures

Demographic data were reported as frequency and percentage across the three benchmarks. The z-test for independent proportions was used to compare the research participant demographics to the NHC patients and Jefferson County during a consistent time period. Temporal trends in research participant demographics were also examined.

Key Results

Apart from being relatively older, the NHC research participant population closely resembled the NHC patient population. A similar age-related pattern emerged as well as an over-representation of White individuals in the research participant group when compared to the census data. When looking at research participant demographic trends overtime, increases in White, Black and older cohorts were noted while decreases were observed in Hispanic/Latinx and younger cohorts. Trends related to participant sex remained stable.

Conclusions

The findings from this study will inform future strategies for setting enrollment goals and facilitate the creation of tools and metrics to evaluate appropriate standards for diversity in clinical research study population samples.
Behavioural rhythms of two gammarid speciesEchinogammarus marinusandGammarus pulexunder increasing levels of light at night
Authors: Underwood Charlotte N. , Ford Alex T. , Robson Samuel C. , Wijnen Herman
Published: September 2024

Artificial light at night (ALAN) is proliferating at an alarming rate across the globe, particularly around aquatic habitats. Natural and predictable light cycles dictate much of an individual organism’s life by acting as a major signal for their circadian clock, driving rhythmic behaviours and physiological changes throughout the body. Light cycles also help populations coordinate group behaviour and greatly impact the interspecies dynamics of a community. Research into the ecological impacts of ALAN has highlighted numerous effects on these biological processes, including higher predation rates, impaired growth and development, and diminished reproductive success. Invertebrates play an undeniable role in ecosystem functioning and show robust daily rhythms. As such, it is vital to understand how ALAN may disrupt their behavioural patterns. The aim of this study was to monitor the impacts of increasing levels of light at night (0 lux – 80 lux), as well as constant light and constant darkness, on the behavioural rhythms of the intertidal amphipod,Echinogammarus marinus, and the freshwater species,Gammarus pulex. G. pulexactivity was not strongly synchronised to any of the light at night treatments.E. marinus, however, exhibited strong behavioural rhythmicity in diurnal cycles with dark night periods. All the ALAN treatments resulted in a significant decrease inE. marinusrhythmicity and overall activity. Moreover, ALAN between 1-50 lux disrupted nocturnality in this species. These results indicate that while some gammarids show some adaptive plasticity when it comes to light pollution, others may experience strong direct impacts on their activity. This may be relevant to individual and population level fitness of vulnerable species in more heavily urbanised areas.
The Role of Purine Interactions in Biogenic Crystal Shape Determination
Authors: Rothkegel Jannik , Kaufmann Sylvia , Wilsch-Braeuninger Michaela , Mateus Rita
Published: September 2024

Widespread through phyla, purine crystals are intracellular inclusions serving a myriad of organismal functions. In zebrafish, iridophores concentrate purines in membrane-bound organelles, the iridosomes, for controlled crystallization. These crystals assemble into large, flat, and thin hexagons following unknown mechanisms that evolve against thermodynamically favorable interactions. Here, we investigate the initial development of zebrafish iridosomal crystals. By performing in vivo confocal reflection imaging, cryoFIB-SEM, and establishing novel 2D and 3D analysis pipelines, we show that these crystals grow four times faster along the b-crystallographic axis, leading to their characteristic hexagonal shape. By analyzing zebrafish with impaired guanine production, while conducting crystal growth simulations, we find that crystal shape is directed by bond type, number, and interaction strength between purines. Mechanistically, the macroscopic shape of zebrafish crystals is controlled by the relative concentration of purines present in the iridosome. This process impacts crystal growth along the b-axis, by disrupting the crystal's in-plane hydrogen bond structure, without alteration of the other axes. Our work uncovers a layer of biogenic crystal growth regulation occurring in vertebrate biocrystallization processes.
Computational Analysis of Human Cysteine Redox Proteoforms Reveals Novel Insights
Authors: Cobley James N. , Chatzinikolaou Panagiotis N. , Schmidt Cameron
Published: September 2024

Since cysteine redox proteoforms (i) are virtually unstudied, we derived novel insights by computationally analysing the human proteome. Our analysis revealed a vast, effectively infinite, theoreticalispace housing 3.02 x 10169unique cysteine redox proteoforms. For >80% and 99% of the human proteome, theispace comprises 6.83 x 108and 1.76 x 1031unique proteoforms, respectively. The heterogenous distribution of theispace by gene ontology terms, suggests, but does not prove, functional speciation. To theoretically limit the number of cysteine redox proteoforms that can be “downloaded” from the abstracti“cloud”, we implement novel equations. Protein copy numbers limit theispace by 161-logs to 4.04 x 107unique cysteine redox proteoforms per HeLa cell. An immutable law: the number of cysteine redox proteoform molecules (Ni) must equal the number of cysteine-containing protein molecules. We compute anNivalue of 1.70 x 109per HeLa cell. WhileNiwill be displaced from thermodynamic equilibrium towards the reduced state (e.g., ≈90%-reduced), it is possible that the number of partially oxidised cysteine redox proteoform molecules is in the order of 106-8per HeLa cell. Consistent with this, 100%-oxidised forms were observed in 60% of the proteins studied to date. Our analysis advances understanding of redox biology at the proteoform level.
An Inflammation Centered Perspective to the Mechanisms and Interactions Related to Vascular Cognitive Impairment
Authors: Zeylan Melisa Ece , Senyuz Simge , Keskin Ozlem , Gursoy Attila
Published: September 2024

A major health burden for the elderly, vascular cognitive impairment (VCI) is a disease that combines cognitive (CD) and cardiovascular (CVD) components. The molecular mechanisms underlying this disease are poorly understood, and our work attempts to bridge this knowledge gap by building protein-protein interaction (PPI) networks of CD and CVD. Our earlier research not only showed how well these two primary components work together, but also hinted at the potential role of inflammation in the development of VCI. For this reason, we decided to examine the relationship between inflammation and VCI in further detail.We identified the top three most connected clusters, which could represent significant modules, enriched these clusters with alternative conformations, and used PRISM to predict the interactions between the conformations. We proposed putative VCI-related interactions, such as NFKBIA-RELA and the proteasome complex, as well as their effects. The five interactions that we discovered have a higher predicted binding affinity when one of the conformations is mutated: LTF-SNCA, FGA-LTF, UBE2D1-VCP, ERBB4-INS, and NFE2L2-VCP. Additionally, since VCP has a conformational mutation linked to dementia, we proposed that the cancer-related protein BRCA1 may have implications for VCI. BRCA1’s interaction with both wild-type and mutant XRCC4 and LIG4 suggests the significance of the DNA damage response pathway which can be shared between VCI and cancer.Altogether, our results suggest various pathways and interactions that can act as targets for therapeutic interventions or early diagnosis of VCI.