Recent ucr_som_cmtm_oapolicydeposits items

ThIEF: Finding Genome-wide Trajectories of Epigenetics Marks

Thu, 21 May 2026 00:00:00 +0000

We address the problem of comparing multiple genome-wide maps representing nucleosome positions or specific histone marks. These maps can originate from the comparative analysis of ChIP-Seq/MNase-Seq/FAIRE-Seq data for different cell types/tissues or multiple time points. The input to the problem is a set of maps, each of which is a list of genomics locations for nucleosomes or histone marks. The output is an alignment of nucleosomes/histone marks across time points (that we call trajectories), allowing small movements and gaps in some of the maps. We present a tool called ThIEF (TrackIng of Epigenetic Features) that can efficiently compute these trajectories. ThIEF comes into two "flavors": ThIEF:Iterative finds the trajectories progressively using bipartite matching, while ThIEF:LP solves a k-partite matching problem on a hyper graph using linear programming. ThIEF:LP is guaranteed to find the optimal solution, but it is slower than ThIEF:Iterative. We demonstrate the utility...

Evaluating Imatinib's Affinities and Specificities for Tyrosine Kinases Using Molecular Dynamics Simulations

Thu, 21 May 2026 00:00:00 +0000

Computational chemistry lets us model intermolecular interactions in ways assays cannot. My project focuses on the multi-kinase interactions of the cancer drug, imatinib. Most cancer drugs target one kinase, but some affect multiple kinases. Imatinib treats chronic myeloid leukemia by targeting ABL kinase. Proteomics data reveals it can interact with other kinases, such as KIT to treat gastrointestinal stromal tumors, but the mechanisms are unknown. Imatinib has different affinities for similar kinases, such as a 3000x difference between ABL and SRC, despite sharing 50% structural homology. Here, I investigate the conformational differences between free and imatinib-bound ABL, KIT, and SRC using Molecular Dynamics simulations to understand the key imatinib-kinase interactions. The alignment analysis shows the docked conformations are similar to co-crystal structures in the Protein Data Bank. Root-mean-square-deviation and fluctuation (RMSD and RMSF) analysis show that all simulations...

Heterogeneous CPU+GPU-Enabled Simulations for DFTB Molecular Dynamics of Large Chemical and Biological Systems

Thu, 21 May 2026 00:00:00 +0000

We introduce a new heterogeneous CPU+GPU-enhanced DFTB approach for the routine and efficient simulation of large chemical and biological systems. Compared to homogenous computing with conventional CPUs, heterogeneous computing approaches exhibit substantial performance with only a modest increase in power consumption, both of which are essential to upcoming exascale computing initiatives. We show that DFTB-based molecular dynamics is a natural candidate for heterogeneous computing since the computational bottleneck in these simulations is the diagonalization of the Hamiltonian matrix, which is performed several times during a single molecular dynamics trajectory. To thoroughly test and understand the performance of our heterogeneous CPU+GPU approach, we examine a variety of algorithmic implementations, benchmarks of different hardware configurations, and applications of this methodology on several large chemical and biological systems. Finally, to demonstrate the capability of...

systemPipeR: a multipurpose workflow management system for reproducible data analysis

Tue, 21 Apr 2026 00:00:00 +0000

Workflow management systems (WMS) are essential for creating and automating multi-step data analyses and ensuring the reproducibility of biological insights. Although numerous WMS solutions exist, few provide deep integration of command-line software with the R and Bioconductor ecosystems, where a substantial portion of statistical modeling and downstream scientific analysis is performed by a large user base. systemPipeR addresses this gap by offering a unified environment that links R-based analytical steps with command-line tools through a standardized workflow specification. It enables the design and execution of reproducible workflows on both local and high-performance computing systems, while allowing users to select the most appropriate R or command-line tool for each analysis step. The latest version introduces a fully redesigned architecture that streamlines workflow construction, execution, monitoring, and reporting. Key enhancements include a flexible workflow management...

Babesia hegotelforum sp. nov., a zoonotic Babesia species previously referred to as Babesia sp. MO1.

Thu, 12 Mar 2026 00:00:00 +0000

A zoonotic Babesia species previously referred to as Babesia sp. MO1 is formally described and named here as Babesia hegotelforum sp. nov. This taxon is distinct from Babesia divergens based on genome-wide sequence divergence, phylogenetic placement, host associations, and clinical presentation. The parasite infects erythrocytes of humans, and eastern cottontail rabbits (Sylvilagus floridanus), and is transmitted by Ixodes dentatus. The holotype consists of a Giemsa-stained thin blood smear and cryopreserved infected erythrocytes from the cloned isolate BML-Bh-B12 at ≤10 passages in continuous in vitro culture. Paratype material includes five additional clones (BML-Bh-H1, BML-Bh-F12, BML-Bh-H6, BML-Bh-A3, and BML-Bh-F1) derived from BEI Resources strain NR-50441, along with the original mixed isolate NR-50441. This species description meets the requirements of the International Code of Zoological...

Proposed Key Characteristics of Neurotoxic Chemicals

Wed, 11 Mar 2026 00:00:00 +0000

A critical component of evaluating whether a chemical can cause human neurotoxicity is hazard identification, which typically involves a comprehensive literature search to identify and synthesize epidemiological, animal, and mechanistic data for the chemical of interest. The key characteristics (KCs) concept has proven to be a useful tool for searching, organizing, and evaluating mechanistic data for hazard identification. KCs are the established chemical and biological properties of known human neurotoxic agents based on understanding of their mechanisms of neurotoxicity. KCs were originally developed for carcinogens but have now also been published for endocrine- and metabolism-disruptors and various organ-selective toxic chemicals. To identify KCs associated with neurotoxic chemicals, an expert committee was convened to consider current mechanistic understanding of chemicals known to be neurotoxic in humans with the goal of identifying established molecular and cellular actions...

Fuel-driven filamentous phage nanomotors

Thu, 26 Feb 2026 00:00:00 +0000

Virus-based nanocarriers have shown great potential for noninvasive delivery of drugs, diagnostics, and imaging agents to hard-to-reach anatomical locations. Yet, they largely depend on diffusion for transport, often lacking the force to actively penetrate biological barriers, and navigation to guide therapeutic agents. In these studies, the M13 bacteriophage, a linearly shaped virus, was converted from passive nanocarrier to actively propelled, fuel-driven nanomotor. Using the distinctive low symmetry of its capsid, a single Pt nanoparticle was added to one end of the M13 virus to form a tadpole-like structure. The Pt/M13 head/tail nanomotors exhibited notably enhanced diffusion in the presence of hydrogen peroxide fuel, and significantly improved uptake by SVOK3 ovarian cancer cells in vitro. Given the successes of the M13 bacteriophage as a nanocarrier, the demonstration of this simple, but comparatively mobile M13-based nanomotor platform represents an important step...

A cholesterol-responsive hepatic tRNA-derived small RNA regulates cholesterol homeostasis and atherosclerosis development

Mon, 22 Dec 2025 00:00:00 +0000

Transfer RNA-derived small RNAs (tsRNAs) have emerged as crucial players in diverse biological processes. Yet, their involvement in lipid metabolism and cardiovascular disease remains elusive. Using an advanced PANDORA-seq method, we identify tsRNA-Glu-CTC as the most abundant tsRNA in mouse liver. Intriguingly, tsRNA-Glu-CTC is cholesterol responsive. Overexpression of tsRNA-Glu-CTC elicits hypercholesterolemia and hepatic steatosis, whereas its knockdown protects against diet-induced hypercholesterolemia and atherosclerosis in mice. Mechanistically, tsRNA-Glu-CTC regulates key hepatic lipogenic genes including Srebp2, a master regulator of lipid metabolism. tsRNA-Glu-CTC interacts with SREBP2 to regulate its own transcription through an E-box motif. We further identify site-specific RNA modifications of endogenous tsRNA-Glu-CTC by a mass spectrometry-based MLC-seq and demonstrate the modified tsRNA-Glu-CTC as a more potent regulator of cholesterol homeostasis compared to its...

Durable humoral immunity and long-term protection induced by a Crimean-Congo hemorrhagic fever virus replicon particle vaccine in mice

Mon, 8 Dec 2025 00:00:00 +0000

A Crimean-Congo hemorrhagic fever virus replicon particle vaccine was evaluated for long-term immunity and efficacy in mice. IgG responses persisted up to 18 months, with similar titers across dosing strategies through 12 months. Protective efficacy reached ≥75% at 6 months (prime-only) and up to 12 months (prime-boost). Booster dosing enhanced antibody avidity, effector function, and improved long-term protection. These findings support durable immunity from single or boosted vaccination.

Rapid, sensitive, and species-independent detection of Crimean Congo hemorrhagic fever virus nucleoprotein and GP38 antibodies

Thu, 14 Aug 2025 00:00:00 +0000

BACKGROUND: Crimean-Congo hemorrhagic fever virus (CCHFV), a zoonotic agent in the Nairoviridae family (genus Orthonairovirus), is a high-priority pathogen. CCHFV infection causes Crimean-Congo hemorrhagic fever (CCHF), a human disease with case fatality rates of up to 40%. Serological surveillance of CCHFV in animals and humans is crucial for ecological studies and public health. METHODS: We developed CCHFV mix-and-read assays utilizing split-NanoLuc technology (NanoBiT) to detect anti-CCHFV antibodies against the nucleoprotein (NP) stalk region and the GP38 glycoprotein. These species- and isotype-agnostic assays provide results in ∼30 min. Using serum samples from RT-PCR-confirmed CCHF cases collected during and after hospitalization, we investigated anti-NP and anti-GP38 antibody development. The performance of the mix-and-read assays was compared to the NP-based IDScreen® CCHF commercial assay using human sera, and cross-reactivity potential was evaluated using a diverse...

PTPN2 Regulates Iron Handling Protein Expression in Inflammatory Bowel Disease Patients and Prevents Iron Deficiency in Mice

Sat, 26 Apr 2025 00:00:00 +0000

Anemia is the most common extraintestinal manifestation of inflammatory bowel disease (IBD). Iron deficiency is the most frequent cause of anemia in IBD; however, the mechanisms involved are still poorly understood. Here, we investigated the role of the IBD risk gene, protein tyrosine phosphatase non-receptor type 2 (PTPN2), in regulating iron homeostasis. Proteomic analyses were performed on serum from IBD patients genotyped for the IBD-associated loss-of-function rs1893217 PTPN2 variant. Constitutive Ptpn2 wild type (WT), heterozygous (Het), and knockout (KO) mice were analyzed for iron content, blood parameters, and expression of iron handling proteins. Iron absorption was assessed through radiotracer assays. Serum proteomic analyses revealed that the "iron homeostasis signaling pathway" was the main pathway downregulated in Crohn's disease (CD) patients carrying the PTPN2 risk allele, independent of disease activity. Ptpn2-KO mice showed...

Tofacitinib Mitigates the Increased SARS-CoV-2 Infection Susceptibility Caused by an IBD Risk Variant in the PTPN2 Gene

Fri, 11 Apr 2025 00:00:00 +0000

BACKGROUND & AIMS: Coronavirus disease (COVID-19), caused by severe acquired respiratory syndrome-Coronavirus-2 (SARS-CoV-2), triggered a global pandemic with severe medical and socioeconomic consequences. Although fatality rates are higher among the elderly and those with underlying comorbidities, host factors that promote susceptibility to SARS-CoV-2 infection and severe disease are poorly understood. Although individuals with certain autoimmune/inflammatory disorders show increased susceptibility to viral infections, there is incomplete knowledge of SARS-CoV-2 susceptibility in these diseases. The aim of our study was to investigate whether the autoimmunity risk gene, PTPN2, which also confers elevated risk to develop inflammatory bowel disease, affects susceptibility to SARS-CoV-2 viral uptake. METHODS: Using samples from PTPN2 genotyped patients with inflammatory bowel disease, PTPN2-deficient mice, and human intestinal and lung epithelial cell lines, we investigated...

Mixed-Methods Approach: Impact of Clinical Consenter Diversity on Clinical Trials Enrollment

Fri, 11 Apr 2025 00:00:00 +0000

BACKGROUND: Clinical trials should benefit all people. Consequently, the National Cancer Institute expects cancer centers to accrue individuals to clinical trials in proportion to the cancer burden experienced by populations that live in their respective catchment areas; unfortunately, many cancer centers fail to meet this expectation. The person who gives consent for individuals in clinical trials frequently has significant contact with potential trial participants. We hypothesized that the race, ethnicity, and language of the consenter may have an important bearing on whether an individual chooses to participate in a clinical trial. METHODS: We used mixed methods to investigate the impact of the socio-cultural background of the consenter on the decision of a potential research subject to participate in a clinical trial. Between 01/2018 and 02/2020, 205 women were approached in the sequential order they appeared in our breast clinic; of the 181 participants who agreed to complete...

PROTAC-induced protein structural dynamics in targeted protein degradation

Thu, 3 Apr 2025 00:00:00 +0000

PROteolysis TArgeting Chimeras (PROTACs) are small molecules that induce target protein degradation via the ubiquitin-proteasome system. PROTACs recruit the target protein and E3 ligase; a critical first step is forming a ternary complex. However, while the formation of a ternary complex is crucial, it may not always guarantee successful protein degradation. The dynamics of the PROTAC-induced degradation complex play a key role in ubiquitination and subsequent degradation. In this study, we computationally modelled protein complex structures and dynamics associated with a series of PROTACs featuring different linkers to investigate why these PROTACs, all of which formed ternary complexes with Cereblon (CRBN) E3 ligase and the target protein bromodomain-containing protein 4 (BRD4^BD1), exhibited varying degrees of degradation potency. We constructed the degradation machinery complexes with Culling-Ring Ligase 4A (CRL4A) E3 ligase scaffolds. Through atomistic molecular...

Demyelination and neurodegeneration early in experimental autoimmune encephalomyelitis contribute to functional deficits in the anterior visual pathway

Tue, 1 Apr 2025 00:00:00 +0000

Impaired visual function is a prevalent feature of optic neuritis (ON) in multiple sclerosis (MS). Abnormal visual evoked potential (VEP) findings of increased latencies, reduced amplitudes and abnormal waveforms as well as decreased retinal nerve fiber layer (RNFL) assessed by optical coherence tomography (OCT) are hallmarks of ON-induced visual dysfunction. Here we utilized the experimental autoimmune encephalomyelitis (EAE) mouse model of MS to investigate the functional and pathological progression during early (before any clinical symptoms), peak (initial maximal clinical symptoms), and late (chronic disease for > 3 weeks) disease stages. Demyelination and initial stages of axon damage were observed in early EAE. Significant demyelination, inflammation, increased axon damage and impaired P1/N2 amplitudes and latencies by VEP were seen in middle and late EAE groups. A decrease in RNFL thickness by OCT was observed only during late EAE. NanoString analysis of optic...

Pathway Specific Unbinding Free Energy Profiles of Ritonavir Dissociation from HIV‑1 Protease

Thu, 27 Feb 2025 00:00:00 +0000

Investigation of protein-drug recognition is key to understanding drug selectivity and binding affinity. In combination, the binding/unbinding free energy landscape and intermolecular interactions can be used to understand drug binding/unbinding mechanisms. This information is vital for the development of drugs with improved efficacy and explanation of mutation effects. This study investigated the dissociation processes of ritonavir unbinding from HIV protease (HIVp). Analyzing unbinding trajectories modeled by accelerated molecular dynamics (MD) simulations, three distinct pathways, pathways A-C, were characterized. Using a reduced dimensionality strategy with the principal component analysis, we carried out short classical MD runs with explicit water to sample local fluctuation during ritonavir dissociation and applied the milestoning theory to construct an unbinding free energy landscape. We found that each pathway showed similar values of binding free energy, albeit pathway...

The Toxoplasma rhoptry protein ROP55 is a major virulence factor that prevents lytic host cell death

Thu, 27 Feb 2025 00:00:00 +0000

Programmed-cell death is an antimicrobial defense mechanism that promotes clearance of intracellular pathogens. Toxoplasma counteracts host immune defenses by secreting effector proteins into host cells; however, how the parasite evades lytic cell death and the effectors involved remain poorly characterized. We identified ROP55, a rhoptry protein that promotes parasite survival by preventing lytic cell death in absence of IFN-γ stimulation. RNA-Seq analysis revealed that ROP55 acts as a repressor of host pro-inflammatory responses. In THP-1 monocytes ΔROP55 infection increased NF-κB p65 nuclear translocation, IL-1β production, and GSDMD cleavage compared to wild type or complemented parasites. ΔROP55 infection also induced RIPK3-dependent necroptosis in human and mouse primary macrophages. Moreover, ΔROP55 parasites were significantly impaired in virulence in female mice and prevented NF-κB activation and parasite clearance in mBMDM. These findings place ROP55 as a major virulence...

Global atlas of predicted functional domains in Legionella pneumophila Dot/Icm translocated effectors

Mon, 20 Jan 2025 00:00:00 +0000

Legionella pneumophila utilizes the Dot/Icm type IVB secretion system to deliver hundreds of effector proteins inside eukaryotic cells to ensure intracellular replication. Our understanding of the molecular functions of the largest pathogenic arsenal known to the bacterial world remains incomplete. By leveraging advancements in 3D protein structure prediction, we provide a comprehensive structural analysis of 368 L. pneumophila effectors, representing a global atlas of predicted functional domains summarized in a database (https://pathogens3d.org/legionella-pneumophila). Our analysis identified 157 types of diverse functional domains in 287 effectors, including 159 effectors with no prior functional annotations. Furthermore, we identified 35 cryptic domains in 30 effector models that have no similarity with experimentally structurally characterized proteins, thus, hinting at novel functionalities. Using this analysis, we demonstrate the activity of thirteen functional domains,...

Alleviation of extensive visual pathway dysfunction by a remyelinating drug in a chronic mouse model of multiple sclerosis

Fri, 17 Jan 2025 00:00:00 +0000

Visual deficits are among the most prevalent symptoms in patients with multiple sclerosis (MS). To understand deficits in the visual pathway during MS and potential treatment effects, we used experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS. The afferent visual pathway was assessed in vivo using optical coherence tomography (OCT), electroretinography (ERG), and visually evoked cortical potentials (VEPs). Inflammation, demyelination, and neurodegeneration were examined by immunohistochemistry ex vivo. In addition, an immunomodulatory, remyelinating agent, the estrogen receptor β ligand chloroindazole (IndCl), was tested for its therapeutic potential in the visual pathway. EAE produced functional deficits in visual system electrophysiology, including suppression of ERG and VEP waveform amplitudes and increased signal latencies. Therapeutic IndCl rescued overall visual system latency by VEP but had little impact on amplitude or ERG findings...

The role of long noncoding RNAs in malaria parasites

Wed, 8 Jan 2025 00:00:00 +0000

The human malaria parasites, including Plasmodium falciparum, persist as a major cause of global morbidity and mortality. The recent stalling of progress toward malaria elimination substantiates a need for novel interventions. Controlled gene expression is central to the parasite's numerous life cycle transformations and adaptation. With few specific transcription factors (TFs) identified, crucial roles for chromatin states and epigenetics in parasite transcription have become evident. Although many chromatin-modifying enzymes are known, less is known about which factors mediate their impacts on transcriptional variation. Like those of higher eukaryotes, long noncoding RNAs (lncRNAs) have recently been shown to have integral roles in parasite gene regulation. This review aims to summarize recent developments and key findings on the role of lncRNAs in P. falciparum.

What Strengthens Protein-Protein Interactions: Analysis and Applications of Residue Correlation Networks

Wed, 25 Dec 2024 00:00:00 +0000

Identifying residues critical to protein-protein binding and efficient design of stable and specific protein binders are challenging tasks. Extending beyond the direct contacts in a protein-protein binding interface, our study employs computational modeling to reveal the essential network of residue interactions and dihedral angle correlations critical in protein-protein recognition. We hypothesized that mutating residues exhibiting highly correlated dynamic motion within the interaction network could efficiently optimize protein-protein interactions to create tight and selective protein binders. We tested this hypothesis using the ubiquitin (Ub) and MERS coronaviral papain-like protease (PLpro) complex, since Ub is a central player in multiple cellular functions and PLpro is an antiviral drug target. Our designed ubiquitin variant (UbV) hosting three mutated residues displayed a ∼3,500-fold increase in functional inhibition relative to wild-type Ub. Further optimization of two...

Synthesis and Molecular Properties of Nerve Agent Reactivator HLö‑7 Dimethanesulfonate

Tue, 24 Dec 2024 00:00:00 +0000

The threat of a deliberate release of chemical nerve agents has underscored the need to continually improve field effective treatments for these types of poisonings. The oxime containing HLö-7 is a potential second-generation therapeutic reactivator. A synthetic process for HLö-7 is detailed with improvements to the DIBAL reduction and ion exchange steps. HLö-7 was visualized for the first time within the active site of human acetylcholinesterase and its relative ex vivo potency confirmed against various nerve agents using a phrenic nerve hemidiaphragm assay.

Replicon particle vaccination induces non-neutralizing anti-nucleoprotein antibody-mediated control of Crimean-Congo hemorrhagic fever virus

Mon, 9 Dec 2024 00:00:00 +0000

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe human disease and is considered a WHO priority pathogen due to the lack of efficacious vaccines and antivirals. A CCHF virus replicon particle (VRP) has previously shown protective efficacy in a lethal Ifnar-/- mouse model when administered as a single dose at least 3 days prior to challenge. Here, we determine that non-specific immune responses are not sufficient to confer short-term protection, since Lassa virus VRP vaccination 3 days prior to CCHFV challenge was not protective. We also investigate how CCHF VRP vaccination confers protective efficacy by examining viral kinetics, histopathology, clinical analytes and immunity early after challenge (3 and 6 days post infection) and compare to unvaccinated controls. We characterize how these effects differ based on vaccination period and correspond to previously reported CCHF VRP-mediated protection. Vaccinating Ifnar-/- mice with CCHF VRP 28, 14, 7, or 3 days prior...

Structural characterization of protective non-neutralizing antibodies targeting Crimean-Congo hemorrhagic fever virus

Mon, 9 Dec 2024 00:00:00 +0000

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) causes a life-threatening disease with up to a 40% mortality rate. With no approved medical countermeasures, CCHFV is considered a public health priority agent. The non-neutralizing mouse monoclonal antibody (mAb) 13G8 targets CCHFV glycoprotein GP38 and protects mice from lethal CCHFV challenge when administered prophylactically or therapeutically. Here, we reveal the structures of GP38 bound with a human chimeric 13G8 mAb and a newly isolated CC5-17 mAb from a human survivor. These mAbs bind overlapping epitopes with a shifted angle. The broad-spectrum potential of c13G8 and CC5-17 and the practicality of using them against Aigai virus, a closely related nairovirus were examined. Binding studies demonstrate that the presence of non-conserved amino acids in Aigai virus corresponding region prevent CCHFV mAbs from binding Aigai virus GP38. This information, coupled with in vivo efficacy, paves the way for future mAb therapeutics effective...

Exploring Noncovalent Protease Inhibitors for the Treatment of Severe Acute Respiratory Syndrome and Severe Acute Respiratory Syndrome-Like Coronaviruses

Sat, 7 Dec 2024 00:00:00 +0000

Over the last 20 years, both severe acute respiratory syndrome coronavirus-1 and severe acute respiratory syndrome coronavirus-2 have transmitted from animal hosts to humans causing zoonotic outbreaks of severe disease. Both viruses originate from a group of betacoronaviruses known as subgroup 2b. The emergence of two dangerous human pathogens from this group along with previous studies illustrating the potential of other subgroup 2b members to transmit to humans has underscored the need for antiviral development against them. Coronaviruses modify the host innate immune response in part through the reversal of ubiquitination and ISGylation with their papain-like protease (PLpro). To identify unique or overarching subgroup 2b structural features or enzymatic biases, the PLpro from a subgroup 2b bat coronavirus, BtSCoV-Rf1.2004, was biochemically and structurally evaluated. This evaluation revealed that PLpros from subgroup 2b coronaviruses have narrow substrate specificity for...

Polyphenols as alternative treatments of COVID-19

Sat, 7 Dec 2024 00:00:00 +0000

Although scientists around the world have put lots of effort into the development of new treatments for COVID-19 since the outbreak, no drugs except Veklury (remdesivir) have been approved by FDA. There is an urgent need to discover some alternative antiviral treatment for COVID-19. Because polyphenols have been shown to possess antiviral activities, here we conducted a large-scale virtual screening for more than 400 polyphenols. Several lead compounds such as Petunidin 3-O-(6″-p-coumaroyl-glucoside) were identified to have promising binding affinities and convincing binding mechanisms. Analyzing the docking results and ADME properties sheds light on the potential efficacy of the top-ranked drug candidates and pinpoints the key residues on the target proteins for the future of drug development.

The structural and biochemical impacts of monomerizing human acetylcholinesterase

Fri, 6 Dec 2024 00:00:00 +0000

Serving a critical role in neurotransmission, human acetylcholinesterase (hAChE) is the target of organophosphate nerve agents. Hence, there is an active interest in studying the mechanism of inhibition and recovery of enzymatic activity, which could lead to better countermeasures against nerve agents. As hAChE is found in different oligomeric assemblies, certain approaches to studying it have been problematic. Herein, we examine the biochemical and structural impact of monomerizing hAChE by using two mutations: L380R/F535K. The activities of monomeric hAChE L380R/F535K and dimeric hAChE were determined to be comparable utilizing a modified Ellman's assay. To investigate the influence of subunit-subunit interactions on the structure of hAChE, a 2.1 Å X-ray crystallographic structure was determined. Apart from minor shifts along the dimer interface, the overall structure of the hAChE L380R/F535K mutant is similar to that of dimeric hAChE. To probe whether the plasticity of the...

Identifying Drug Candidates for COVID-19 with Large-Scale Drug Screening

Fri, 6 Dec 2024 00:00:00 +0000

Papain-like protease (PL^pro) is critical to COVID-19 infection. Therefore, it is a significant target protein for drug development. We virtually screened a 26,193 compound library against the PL^pro of SARS-CoV-2 and identified several drug candidates with convincing binding affinities. The three best compounds all had better estimated binding energy than those of the drug candidates proposed in previous studies. By analyzing the docking results for the drug candidates identified in this and previous studies, we demonstrate that the critical interactions between the compounds and PL^pro proposed by the computational approaches are consistent with those proposed by the biological experiments. In addition, the predicted binding energies of the compounds in the dataset showed a similar trend as their IC₅₀ values. The predicted ADME and drug-likeness properties also suggested that these identified compounds can be used for COVID-19 treatment.

The SARS-CoV‑2 SSHHPS Recognized by the Papain-like Protease

Fri, 6 Dec 2024 00:00:00 +0000

Viral proteases are highly specific and recognize conserved cleavage site sequences of ∼6-8 amino acids. Short stretches of homologous host-pathogen sequences (SSHHPS) can be found spanning the viral protease cleavage sites. We hypothesized that these sequences corresponded to specific host protein targets since >40 host proteins have been shown to be cleaved by Group IV viral proteases and one Group VI viral protease. Using PHI-BLAST and the viral protease cleavage site sequences, we searched the human proteome for host targets and analyzed the hit results. Although the polyprotein and host proteins related to the suppression of the innate immune responses may be the primary targets of these viral proteases, we identified other cleavable host proteins. These proteins appear to be related to the virus-induced phenotype associated with Group IV viruses, suggesting that information about viral pathogenesis may be extractable directly from the viral genome sequence. Here we identify...

Inflammation associated with monocyte/macrophage activation and recruitment corresponds with lethal outcome in a mouse model of Crimean-Congo haemorrhagic fever

Fri, 6 Dec 2024 00:00:00 +0000

Crimean-Congo haemorrhagic fever virus (CCHFV) causes human disease ranging from subclinical to a fatal haemorrhagic syndrome. Determinants of CCHF pathogenesis are largely unknown and animal models that recapitulate human disease are limited. A recently described mouse model uses a monoclonal antibody (mAb 5A3) targeting the interferon (IFN) alpha/beta receptor to suppress type I IFN responses, making animals transiently susceptible to infection. To advance utility of this model, we investigated effects of challenge route, timing of 5A3 delivery, mouse sex and age, and virus strain on clinical course and outcome. C57BL/6J mice received mAb 5A3 -1, 0, or -1/+1 days post-infection (dpi). Subsets were challenged with CCHFV strain Turkey04 or IbAr10200 subcutaneously or intraperitoneally, and serially euthanized 3- and 7-dpi, when meeting euthanasia criteria or at study completion (14 dpi). CCHFV-IbAr10200-infected mice almost uniformly succumbed to infection, whereas CCHFV-Turkey04-infected...

The structure of DNA methyltransferase DNMT3C reveals an activity-tuning mechanism for DNA methylation

Mon, 25 Nov 2024 00:00:00 +0000

DNA methylation is one of the major epigenetic mechanisms crucial for gene regulation and genome stability. De novo DNA methyltransferase DNMT3C is required for silencing evolutionarily young transposons during mice spermatogenesis. Mutation of DNMT3C led to a sterility phenotype that cannot be rescued by its homologs DNMT3A and DNMT3B. However, the structural basis of DNMT3C-mediated DNA methylation remains unknown. Here, we report the structure and mechanism of DNMT3C-mediated DNA methylation. The DNMT3C methyltransferase domain recognizes CpG-containing DNA in a manner similar to that of DNMT3A and DNMT3B, in line with their high sequence similarity. However, two evolutionary covariation sites, C543 and E590, diversify the substrate interaction among DNMT3C, DNMT3A, and DNMT3B, resulting in distinct DNA methylation activity and specificity between DNMT3C, DNMT3A, and DNMT3B in vitro. In addition, our combined structural and biochemical analysis reveals that the disease-causing...

Substrate specificity and protein stability drive the divergence of plant-specific DNA methyltransferases

Fri, 22 Nov 2024 00:00:00 +0000

DNA methylation is an important epigenetic mechanism essential for transposon silencing and genome integrity. Across evolution, the substrates of DNA methylation have diversified between kingdoms. In plants, chromomethylase3 (CMT3) and CMT2 mediate CHG and CHH methylation, respectively. However, how these two methyltransferases diverge on substrate specificities during evolution remains unknown. Here, we reveal that CMT2 originates from a duplication of an evolutionarily ancient CMT3 in flowering plants. Lacking a key arginine residue recognizing CHG in CMT2 impairs its CHG methylation activity in most flowering plants. An engineered V1200R mutation empowers CMT2 to restore CHG and CHH methylations in Arabidopsis cmt2cmt3 mutant, testifying a loss-of-function effect for CMT2 during evolution. CMT2 has evolved a long and unstructured amino terminus critical for protein stability, especially under heat stress, and is plastic to tolerate various natural mutations. Together,...

Protocol for the longitudinal study of neuroinflammation and reactive astrocytes in Lcn2CreERT2 mice

Mon, 11 Nov 2024 00:00:00 +0000

During brain disease, astrocytes can reprogram into a reactive state that alters many of their functions. Here, we present a protocol for studying neuroinflammation and reactive astrogliosis in mice using lipopolysaccharide (LPS) from E. coli. We describe steps for employing the Lcn2CreERT2 mouse crossed into a fluorescent Cre reporter line to label a subset of reactive astrocytes during and after inflammation. We then detail procedures for the longitudinal study of reactive astrocytes during the induction, progression, and/or resolution of astrogliosis. For complete details on the use and execution of this protocol, please refer to Agnew-Svoboda et al.¹.

A Divergent Synthesis of Numerous Pyrroloiminoquinone Alkaloids Identifies Promising Antiprotozoal Agents

Fri, 8 Nov 2024 00:00:00 +0000

On the basis of a streamlined route to the pyrroloiminoquinone (PIQ) core, we made 16 natural products spread across four classes of biosynthetically related alkaloid natural products, and multiple structural analogs, all in ≤8 steps longest linear sequence (LLS). The strategy features a Larock indole synthesis as the key operation in a five-step synthesis of a key methoxy-PIQ intermediate. Critically, this compound was readily diverged via selective methylation of either (or both) of the imine-like or pyrrole nitrogens, which then permitted further divergence by either O-demethylation to o-quinone natural products or displacement of the methoxy group with a range of amine nucleophiles. Based on a single, early report of their potential utility against the malaria parasite, we assayed these compounds against several strains of Plasmodium falciparum, as well as two species of the related protozoan parasite Babesia. In combination with evaluations of...

EphB2 Signaling Is Implicated in Astrocyte-Mediated Parvalbumin Inhibitory Synapse Development

Thu, 7 Nov 2024 00:00:00 +0000

Impaired inhibitory synapse development is suggested to drive neuronal hyperactivity in autism spectrum disorders (ASD) and epilepsy. We propose a novel mechanism by which astrocytes control the development of parvalbumin (PV)-specific inhibitory synapses in the hippocampus, implicating ephrin-B/EphB signaling. Here, we utilize genetic approaches to assess functional and structural connectivity between PV and pyramidal cells (PCs) through whole-cell patch-clamp electrophysiology, optogenetics, immunohistochemical analysis, and behaviors in male and female mice. While inhibitory synapse development is adversely affected by PV-specific expression of EphB2, a strong candidate ASD risk gene, astrocytic ephrin-B1 facilitates PV→PC connectivity through a mechanism involving EphB signaling in PV boutons. In contrast, the loss of astrocytic ephrin-B1 reduces PV→PC connectivity and inhibition, resulting in increased seizure susceptibility and an ASD-like phenotype. Our findings underscore...

Functional consequences of postnatal interventions in a mouse model of Fragile X syndrome

Thu, 7 Nov 2024 00:00:00 +0000

BACKGROUND: Fragile X syndrome (FXS) is a leading genetic cause of autism and intellectual disability with cortical hyperexcitability and sensory hypersensitivity attributed to loss and hypofunction of inhibitory parvalbumin-expressing (PV) cells. Our studies provide novel insights into the role of excitatory neurons in abnormal development of PV cells during a postnatal period of inhibitory circuit refinement. METHODS: To achieve Fragile X mental retardation gene (Fmr1) deletion and re-expression in excitatory neurons during the postnatal day (P)14-P21 period, we generated Cre^CaMKIIa/Fmr1^Flox/y (cOFF) and Cre^CaMKIIa/Fmr1^FloxNeo/y (cON) mice, respectively. Cortical phenotypes were evaluated in adult mice using biochemical, cellular, clinically relevant electroencephalogram (EEG) and behavioral tests. RESULTS: We found that similar to global Fmr1 KO mice, the density of PV-expressing cells, their activation, and sound-evoked gamma synchronization...

Urokinase plasminogen activator mediates changes in human astrocytes modeling fragile X syndrome

Thu, 7 Nov 2024 00:00:00 +0000

The function of astrocytes intertwines with the extracellular matrix, whose neuron and glial cell-derived components shape neuronal plasticity. Astrocyte abnormalities have been reported in the brain of the mouse model for fragile X syndrome (FXS), the most common cause of inherited intellectual disability, and a monogenic cause of autism spectrum disorder. We compared human FXS and control astrocytes generated from human induced pluripotent stem cells and we found increased expression of urokinase plasminogen activator (uPA), which modulates degradation of extracellular matrix. Several pathways associated with uPA and its receptor function were activated in FXS astrocytes. Levels of uPA were also increased in conditioned medium collected from FXS hiPSC-derived astrocyte cultures and correlated inversely with intracellular Ca²⁺ responses to activation of L-type voltage-gated calcium channels in human astrocytes. Increased uPA augmented neuronal phosphorylation of TrkB...

Editorial: Neural markers of sensory processing in development

Thu, 7 Nov 2024 00:00:00 +0000

Editorial: Neural markers of sensory processing in development

Astrocytic Ephrin-B1 Regulates Oligodendrocyte Development and Myelination

Thu, 7 Nov 2024 00:00:00 +0000

Astrocytes have been implicated in oligodendrocyte development and myelination, however, the mechanisms by which astrocytes regulate oligodendrocytes remain unclear. Our findings suggest a new mechanism that regulates astrocyte-mediated oligodendrocyte development through ephrin-B1 signaling in astrocytes. Using a mouse model, we examined the role of astrocytic ephrin-B1 signaling in oligodendrocyte development by deleting ephrin-B1 specifically in astrocytes during the postnatal days (P)14-P28 period and used mRNA analysis, immunohistochemistry, and mouse behaviors to study its effects on oligodendrocytes and myelination. We found that deletion of astrocytic ephrin-B1 downregulated many genes associated with oligodendrocyte development, myelination, and lipid metabolism in the hippocampus and the corpus callosum. Additionally, we observed a reduced number of oligodendrocytes and impaired myelination in the corpus callosum of astrocyte-specific ephrin-B1 KO mice. Finally, our...

HNF4α isoforms: the fraternal twin master regulators of liver function

Fri, 25 Oct 2024 00:00:00 +0000

In the more than 30 years since the purification and cloning of Hepatocyte Nuclear Factor 4 (HNF4α), considerable insight into its role in liver function has been gleaned from its target genes and mouse experiments. HNF4α plays a key role in lipid and glucose metabolism and intersects with not just diabetes and circadian rhythms but also with liver cancer, although much remains to be elucidated about those interactions. Similarly, while we are beginning to elucidate the role of the isoforms expressed from its two promoters, we know little about the alternatively spliced variants in other portions of the protein and their impact on the 1000-plus HNF4α target genes. This review will address how HNF4α came to be called the master regulator of liver-specific gene expression with a focus on its role in basic metabolism, the contributions of the various isoforms and the intriguing intersection with the circadian clock.

Epigenetic Regulation and Chromatin Remodeling in Malaria Parasites

Fri, 27 Sep 2024 00:00:00 +0000

Plasmodium falciparum, the human malaria parasite, infects two hosts and various cell types, inducing distinct morphological and physiological changes in the parasite in response to different environmental conditions. These variations required the parasite to adapt and develop elaborate molecular mechanisms to ensure its spread and transmission. Recent findings have significantly improved our understanding of the regulation of gene expression in P. falciparum. Here, we provide an up-to-date overview of technologies used to highlight the transcriptomic adjustments occurring in the parasite throughout its life cycle. We also emphasize the complementary and complex epigenetic mechanisms regulating gene expression in malaria parasites. This review concludes with an outlook on the chromatin architecture, the remodeling systems, and how this 3D genome organization is critical in various biological processes.

Crimean Congo hemorrhagic fever virus nucleoprotein and GP38 subunit vaccine combination prevents morbidity in mice

Thu, 19 Sep 2024 00:00:00 +0000

Immunizing mice with Crimean-Congo hemorrhagic fever virus (CCHFV) nucleoprotein (NP), glycoprotein precursor (GPC), or with the GP38 domain of GPC, can be protective when the proteins are delivered with viral vectors or as a DNA or RNA vaccine. Subunit vaccines are a safe and cost-effective alternative to some vaccine platforms, but Gc and Gn glycoprotein subunit vaccines for CCHFV fail to protect despite eliciting high levels of neutralizing antibodies. Here, we investigated humoral and cellular immune responses and the protective efficacy of recombinant NP, GP38, and GP38 forms (GP85 and GP160) associated with the highly glycosylated mucin-like (MLD) domain, as well as the NP + GP38 combination. Vaccination with GP160, GP85, or GP38 did not confer protection, and vaccination with the MLD-associated GP38 forms blunted the humoral immune responses to GP38, worsened clinical chemistry, and increased viral RNA in the blood compared to the GP38 vaccination. In contrast, NP vaccination...

Insights into the evolution, virulence and speciation of Babesia MO1 and Babesia divergens through multiomics analyses

Thu, 12 Sep 2024 00:00:00 +0000

Babesiosis, caused by protozoan parasites of the genus Babesia, is an emerging tick-borne disease of significance for both human and animal health. Babesia parasites infect erythrocytes of vertebrate hosts where they develop and multiply rapidly to cause the pathological symptoms associated with the disease. The identification of new Babesia species underscores the ongoing risk of zoonotic pathogens capable of infecting humans, a concern amplified by anthropogenic activities and environmental changes. One such pathogen, Babesia MO1, previously implicated in severe cases of human babesiosis in the United States, was initially considered a subspecies of B. divergens, the predominant agent of human babesiosis in Europe. Here we report comparative multiomics analyses of B. divergens and B. MO1 that offer insight into their biology and evolution. Our analysis shows that despite their highly similar genomic sequences, substantial genetic...

Erythrocyte nano-ghosts with dual optical and magnetic resonance characteristics

Mon, 9 Sep 2024 00:00:00 +0000

Significance: Fluorescent organic dyes provide imaging capabilities at cellular and sub-cellular levels. However, a common problem associated with some of the existing dyes such as the US FDA-approved indocyanine green (ICG) is their weak fluorescence emission. Alternative dyes with greater emission characteristics would be useful in various imaging applications. Complementing optical imaging, magnetic resonance (MR) imaging enables deep tissue imaging. Nano-sized delivery systems containing dyes with greater fluorescence emission as well as MR contrast agents present a promising dual-mode platform with high optical sensitivity and deep tissue imaging for image-guided surgical applications. Aim: We have engineered a nano-sized platform, derived from erythrocyte ghosts (EGs), with dual near-infrared fluorescence and MR characteristics by co-encapsulation of a brominated carbocyanine dye and gadobenate dimeglumine (Gd-BOPTA). Approach: We have investigated the use of three brominated...

Adipocyte-Derived PXR Signaling Is Dispensable for Diet-Induced Obesity and Metabolic Disorders in Mice

Wed, 4 Sep 2024 00:00:00 +0000

Pregnane X receptor (PXR) is a xenobiotic receptor that can be activated by numerous chemicals including endogenous hormones, dietary steroids, pharmaceutical agents, and environmental chemicals. PXR has been established to function as a xenobiotic sensor to coordinately regulate xenobiotic metabolism by regulating the expression of many enzymes and transporters required for xenobiotic metabolism. Recent studies have implicated a potentially important role for PXR in obesity and metabolic disease beyond xenobiotic metabolism, but how PXR action in different tissues or cell types contributes to obesity and metabolic disorders remains elusive. To investigate the role of adipocyte PXR in obesity, we generated a novel adipocyte-specific PXR deficient mouse model (PXR^ΔAd). Notably, we found that loss of adipocyte PXR did not affect food intake, energy expenditure, and obesity in high-fat diet-fed male mice. PXR^ΔAd mice also had similar obesity-associated metabolic...

Insulin Resistance in Women Correlates with Chromatin Histone Lysine Acetylation, Inflammatory Signaling, and Accelerated Aging

Tue, 27 Aug 2024 00:00:00 +0000

BACKGROUND: Epigenetic changes link medical, social, and environmental factors with cardiovascular and kidney disease and, more recently, with cancer. The mechanistic link between metabolic health and epigenetic changes is only starting to be investigated. In our in vitro and in vivo studies, we performed a broad analysis of the link between hyperinsulinemia and chromatin acetylation; our top "hit" was chromatin opening at H3K9ac. METHODS: Building on our published preclinical studies, here, we performed a detailed analysis of the link between insulin resistance, chromatin acetylation, and inflammation using an initial test set of 28 women and validation sets of 245, 22, and 53 women. RESULTS: ChIP-seq identified chromatin acetylation and opening at the genes coding for TNFα and IL6 in insulin-resistant women. Pathway analysis identified inflammatory response genes, NFκB/TNFα-signaling, reactome cytokine signaling, innate immunity, and senescence. Consistent with this finding,...

Multi-layered heterochromatin interaction as a switch for DIM2-mediated DNA methylation

Wed, 14 Aug 2024 00:00:00 +0000

Functional crosstalk between DNA methylation, histone H3 lysine-9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1) is essential for proper heterochromatin assembly and genome stability. However, how repressive chromatin cues guide DNA methyltransferases for region-specific DNA methylation remains largely unknown. Here, we report structure-function characterizations of DNA methyltransferase Defective-In-Methylation-2 (DIM2) in Neurospora. The DNA methylation activity of DIM2 requires the presence of both H3K9me3 and HP1. Our structural study reveals a bipartite DIM2-HP1 interaction, leading to a disorder-to-order transition of the DIM2 target-recognition domain that is essential for substrate binding. Furthermore, the structure of DIM2-HP1-H3K9me3-DNA complex reveals a substrate-binding mechanism distinct from that for its mammalian orthologue DNMT1. In addition, the dual recognition of H3K9me3 peptide by the DIM2 RFTS and BAH1 domains allosterically impacts the DIM2-substrate...

Structural basis for the H2AK119ub1-specific DNMT3A-nucleosome interaction

Tue, 13 Aug 2024 00:00:00 +0000

Isoform 1 of DNA methyltransferase DNMT3A (DNMT3A1) specifically recognizes nucleosome monoubiquitylated at histone H2A lysine-119 (H2AK119ub1) for establishment of DNA methylation. Mis-regulation of this process may cause aberrant DNA methylation and pathogenesis. However, the molecular basis underlying DNMT3A1−nucleosome interaction remains elusive. Here we report the cryo-EM structure of DNMT3A1’s ubiquitin-dependent recruitment (UDR) fragment complexed with H2AK119ub1-modified nucleosome. DNMT3A1 UDR occupies an extensive nucleosome surface, involving the H2A-H2B acidic patch, a surface groove formed by H2A and H3, nucleosomal DNA, and H2AK119ub1. The DNMT3A1 UDR’s interaction with H2AK119ub1 affects the functionality of DNMT3A1 in cells in a context-dependent manner. Our structural and biochemical analysis also reveals competition between DNMT3A1 and JARID2, a cofactor of polycomb repression complex 2 (PRC2), for nucleosome binding, suggesting the interplay between different...

Toxoplasma infection induces an aged neutrophil population in the CNS that is associated with neuronal protection

Mon, 12 Aug 2024 00:00:00 +0000

BackgroundInfection with the protozoan parasite Toxoplasma gondii leads to the formation of lifelong cysts in neurons that can have devastating consequences in the immunocompromised. In the immunocompetent individual, anti-parasitic effector mechanisms and a balanced immune response characterized by pro- and anti-inflammatory cytokine production establishes an asymptomatic infection that rarely leads to neurological symptoms. Several mechanisms are known to play a role in this successful immune response in the brain including T cell production of IFNγ and IL-10 and the involvement of CNS resident cells. This limitation of clinical neuropathology during chronic infection suggests a balance between immune response and neuroprotective mechanisms that collectively prevent clinical manifestations of disease. However, how these two vital mechanisms of protection interact during chronic Toxoplasma infection remains poorly understood.Main textThis study demonstrates a previously undescribed...

Multiscale simulation-guided design of enzyme bioconjugates with enhanced catalysis

Fri, 2 Aug 2024 00:00:00 +0000

Biopolymer-scaffold modification is widely used to enhance enzyme catalysis. A central challenge is predicting the effects of scaffold position on enzyme properties. Here, we use a computational-experimental approach to develop a model for the effects of DNA scaffold position on enzyme kinetics. Using phosphotriesterase modified with a 20bp dsDNA, we demonstrate that conjugation position is as important as the scaffold's chemistry and structure. Multiscale simulations predict the effective substrate concentration increases close to the scaffold, which has μM-strength binding to the substrate. Kinetic analysis shows that the effective concentration that the scaffold provides is best utilized when positioned next to, but not blocking, the active site. At ~5Å distance between scaffold and active site a 7-fold increase in k _cat /K _M was achieved. A model that accounts for the substrate concentration as well PTE-DNA geometry accurately...

Human neutrophil‐like cells demonstrate antimicrobial responses to the chronic cyst form of Toxoplasma gondii

Fri, 19 Jul 2024 00:00:00 +0000

The protozoan parasite Toxoplasma gondii infects approximately 2.5 billion people worldwide. Infection induces a rapid dissemination of parasites throughout the body followed by the formation of lifelong cysts within neurons of the host brain. Both stages require a dynamic immune response comprised of both innate and adaptive cells. Neutrophils are a primary responding cell to acute infection and have been observed in the brain during murine chronic infection. Previous studies investigating human neutrophils found that invasion by Toxoplasma tachyzoites inhibits apoptosis of neutrophils, prolonging their survival under inflammatory conditions. Here, we demonstrate the differentiation of two distinct subsets following exposure of human neutrophil-like-cells (HNLC) to Toxoplasma cysts. In vitro stimulation and imaging studies show cyst-specific induction of cytokines and cyst clearance by HNLCs. Further testing demonstrates that aged HNLCs perform less phagocytosis of cysts compared...

Spatiotemporal patterns of GFAP upregulation in rat brain following acute intoxication with diisopropylfluorophosphate (DFP).

Wed, 17 Jul 2024 00:00:00 +0000

We previously demonstrated that the organophosphate diisopropylfluorophosphate (DFP) induced region-specific delayed neuronal injury in rat brain. The goal of this study was to examine the spatiotemporal pattern of the astroglial response after acute intoxication of rats with DFP. Rats were euthanized at varying times between 1 h to 7 days after DFP administration (9mg/kg, i.p.). The distribution of activated astrocytes in different brain regions was determined by immunostaining for glial fibrillary acidic acid (GFAP). GFAP immunoreactivity increased in the hippocampus, and piriform/entorhinal cortex at 1 h, peaked between 4-8 h then decreased from 16 to 24 h. GFAP labeling in the amygdala gradually increased over the 24 h period. A large increase in GFAP labeling was seen at 3-7 days following DFP administration in the hippocampus, piriform/entorhinal cortex and dorsolateral thalamus. At 3 days post-DFP exposure, GFAP immunoreactivity was totally absent in central areas of the...

Neuregulin-1 is neuroprotective in a rat model of organophosphate-induced delayed neuronal injury

Wed, 17 Jul 2024 00:00:00 +0000

Current medical countermeasures against organophosphate (OP) nerve agents are effective in reducing mortality, but do not sufficiently protect the CNS from delayed brain damage and persistent neurological symptoms. In this study, we examined the efficacy of neuregulin-1 (NRG-1) in protecting against delayed neuronal cell death following acute intoxication with the OP diisopropylflurophosphate (DFP). Adult male Sprague-Dawley rats were pretreated with pyridostigmine (0.1 mg/kg BW, i.m.) and atropine methylnitrate (20 mg/kg BW, i.m.) prior to DFP (9 mg/kg BW, i.p.) intoxication to increase survival and reduce peripheral signs of cholinergic toxicity but not prevent DFP-induced seizures or delayed neuronal injury. Pretreatment with NRG-1 did not protect against seizures in rats exposed to DFP. However, neuronal injury was significantly reduced in most brain regions by pretreatment with NRG-1 isoforms NRG-EGF (3.2 μg/kg BW, i.a) or NRG-GGF2 (48 μg/kg BW, i.a.) as determined by FluroJade-B...

Deciphering Abnormal Platelet Subpopulations in COVID-19, Sepsis and Systemic Lupus Erythematosus through Machine Learning and Single-Cell Transcriptomics

Sat, 6 Jul 2024 00:00:00 +0000

This study focuses on understanding the transcriptional heterogeneity of activated platelets and its impact on diseases such as sepsis, COVID-19, and systemic lupus erythematosus (SLE). Recognizing the limited knowledge in this area, our research aims to dissect the complex transcriptional profiles of activated platelets to aid in developing targeted therapies for abnormal and pathogenic platelet subtypes. We analyzed single-cell transcriptional profiles from 47,977 platelets derived from 413 samples of patients with these diseases, utilizing Deep Neural Network (DNN) and eXtreme Gradient Boosting (XGB) to distinguish transcriptomic signatures predictive of fatal or survival outcomes. Our approach included source data annotations and platelet markers, along with SingleR and Seurat for comprehensive profiling. Additionally, we employed Uniform Manifold Approximation and Projection (UMAP) for effective dimensionality reduction and visualization, aiding in the identification of various...

Stable Occupancy of the Crimean-Congo Hemorrhagic Fever Virus-Encoded Deubiquitinase Blocks Viral Infection

Mon, 24 Jun 2024 00:00:00 +0000

Crimean-Congo hemorrhagic fever virus (CCHFV) infection can result in a severe hemorrhagic syndrome for which there are no antiviral interventions available to date. Certain RNA viruses, such as CCHFV, encode cysteine proteases of the ovarian tumor (OTU) family that antagonize interferon (IFN) production by deconjugating ubiquitin (Ub). The OTU of CCHFV, a negative-strand RNA virus, is dispensable for replication of the viral genome, despite being part of the large viral RNA polymerase. Here, we show that mutations that prevent binding of the OTU to cellular ubiquitin are required for the generation of recombinant CCHFV containing a mutated catalytic cysteine. Similarly, the high-affinity binding of a synthetic ubiquitin variant (UbV-CC4) to CCHFV OTU strongly inhibits viral growth. UbV-CC4 inhibits CCHFV infection even in the absence of intact IFN signaling, suggesting that its antiviral activity is not due to blocking the OTU's immunosuppressive function. Instead, the prolonged...

Structural Insights into the Interaction of Coronavirus Papain-Like Proteases and Interferon-Stimulated Gene Product 15 from Different Species

Mon, 24 Jun 2024 00:00:00 +0000

Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) encode multifunctional papain-like proteases (PLPs) that have the ability to process the viral polyprotein to facilitate RNA replication and antagonize the host innate immune response. The latter function involves reversing the post-translational modification of cellular proteins conjugated with either ubiquitin (Ub) or Ub-like interferon-stimulated gene product 15 (ISG15). Ub is known to be highly conserved among eukaryotes, but surprisingly, ISG15 is highly divergent among animals. The ramifications of this sequence divergence to the recognition of ISG15 by coronavirus PLPs at a structural and biochemical level are poorly understood. Therefore, the activity of PLPs from SARS-CoV, MERS-CoV, and mouse hepatitis virus was evaluated against seven ISG15s originating from an assortment of animal species susceptible, and not, to certain coronavirus infections. Excitingly,...

Structural insights into the interaction of papain-like protease 2 from the alphacoronavirus porcine epidemic diarrhea virus and ubiquitin

Mon, 24 Jun 2024 00:00:00 +0000

Porcine epidemic diarrhea is a devastating porcine disease that is caused by the alphacoronavirus porcine epidemic diarrhea virus (PEDV). Like other members of the Coronaviridae family, PEDV encodes a multifunctional papain-like protease 2 (PLP2) that has the ability to process the coronavirus viral polyprotein to aid in RNA replication and antagonize the host innate immune response through cleavage of the regulatory proteins ubiquitin (Ub) and/or interferon-stimulated gene product 15 (ISG15) (deubiquitination and deISGylation, respectively). Because Betacoronavirus PLPs have been well characterized, it was sought to determine how PLP2 from the alphacoronavirus PEDV differentiates itself from its related counterparts. PEDV PLP2 was first biochemically characterized, and a 3.1 Å resolution crystal structure of PEDV PLP2 bound to Ub was then solved, providing insight into how Alphacoronavirus PLPs bind to their preferred substrate, Ub. It was found that PEDV PLP2 is a deubiquitinase...

Crimean-Congo Hemorrhagic Fever Virus Suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease

Mon, 24 Jun 2024 00:00:00 +0000

Antiviral responses are regulated by conjugation of ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) to proteins. Certain classes of viruses encode Ub- or ISG15-specific proteases belonging to the ovarian tumor (OTU) superfamily. Their activity is thought to suppress cellular immune responses, but studies demonstrating the function of viral OTU proteases during infection are lacking. Crimean-Congo hemorrhagic fever virus (CCHFV, family Nairoviridae) is a highly pathogenic human virus that encodes an OTU with both deubiquitinase and deISGylase activity as part of the viral RNA polymerase. We investigated CCHFV OTU function by inactivating protease catalytic activity or by selectively disrupting its deubiquitinase and deISGylase activity using reverse genetics. CCHFV OTU inactivation blocked viral replication independently of its RNA polymerase activity, while deubiquitinase activity proved critical for suppressing the interferon responses. Our findings provide insights...

ISG15: It's Complicated

Mon, 24 Jun 2024 00:00:00 +0000

Interferon-stimulated gene product 15 (ISG15) is a key component of host responses to microbial infection. Despite having been known for four decades, grasping the functions and features of ISG15 has been a slow and elusive process. Substantial work over the past two decades has greatly enhanced this understanding, revealing the complex and variable nature of this protein. This has unveiled multiple mechanisms of action that are only now beginning to be understood. In addition, it has uncovered diversity not only between how ISG15 affects different pathogens but also between the function and structure of ISG15 itself between different host species. Here we review the complexity of ISG15 within the context of viral infection, focusing primarily on its antiviral function and the mechanisms viruses employ to thwart its effects. We highlight what is known regarding the impact of ISG15 sequence and structural diversity on these interactions and discuss the aspects presenting the next...

Probing the impact of nairovirus genomic diversity on viral ovarian tumor domain protease (vOTU) structure and deubiquitinase activity

Mon, 24 Jun 2024 00:00:00 +0000

Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely...

Determining the molecular drivers of species-specific interferon-stimulated gene product 15 interactions with nairovirus ovarian tumor domain proteases

Mon, 24 Jun 2024 00:00:00 +0000

Tick-borne nairoviruses (order Bunyavirales) encode an ovarian tumor domain protease (OTU) that suppresses the innate immune response by reversing the post-translational modification of proteins by ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15). Ub is highly conserved across eukaryotes, whereas ISG15 is only present in vertebrates and shows substantial sequence diversity. Prior attempts to address the effect of ISG15 diversity on viral protein-ISG15 interactions have focused on only a single species' ISG15 or a limited selection of nairovirus OTUs. To gain a more complete perspective of OTU-ISG15 interactions, we biochemically assessed the relative activities of 14 diverse nairovirus OTUs for 12 species' ISG15 and found that ISG15 activity is predominantly restricted to particular nairovirus lineages reflecting, in general, known virus-host associations. To uncover the underlying molecular factors driving OTUs affinity for ISG15, X-ray crystal structures of Kupe...

Structure of interferon-stimulated gene product 15 (ISG15) from the bat species Myotis davidii and the impact of interdomain ISG15 interactions on viral protein engagement

Mon, 24 Jun 2024 00:00:00 +0000

Bats have long been observed to be the hosts and the origin of numerous human diseases. Bats, like all mammals, rely on a number of innate immune mechanisms to combat invading pathogens, including the interferon type I, II and III responses. Ubiquitin-like interferon-stimulated gene product 15 (ISG15) is a key modulator of these interferon responses. Within these pathways, ISG15 can serve to stabilize host proteins modulating innate immune responses and act as a cytokine. Post-translational modifications of viral proteins introduced by ISG15 have also been observed to directly affect the function of numerous viral proteins. Unlike ubiquitin, which is virtually identical across all animals, comparison of ISG15s across species reveals that they are relatively divergent, with sequence identity dropping to as low as ∼58% among mammals. In addition to serving as an obstacle to the zoonotic transmission of influenza, these ISG15 species-species differences have also long been shown...

Biochemical and Structural Insights into the Preference of Nairoviral DeISGylases for Interferon-Stimulated Gene Product 15 Originating from Certain Species

Mon, 24 Jun 2024 00:00:00 +0000

The regulation of the interferon type I (IFN-I) response has been shown to rely on posttranslational modification by ubiquitin (Ub) and Ub-like interferon-stimulated gene product 15 (ISG15) to stabilize, or activate, a variety of IFN-I signaling and downstream effector proteins. Unlike Ub, which is almost perfectly conserved among eukaryotes, ISG15 is highly divergent, even among mammals. Since zoonotic viruses rely on viral proteins to recognize, or cleave, ISG15 conjugates in order to evade, or suppress, innate immunity, the impact of ISG15 biodiversity on deISGylating proteases of the ovarian tumor family (vOTU) from nairoviruses was evaluated. The enzymatic activities of vOTUs originating from the Crimean-Congo hemorrhagic fever virus, Erve virus, and Nairobi sheep disease virus were tested against ISG15s from humans, mice, shrews, sheep, bats, and camels, which are mammalian species known to be infected by nairoviruses. This along with investigation of binding by isothermal...

Polysome profiling reveals translational control of gene expression in the human malaria parasite Plasmodium falciparum

Mon, 17 Jun 2024 00:00:00 +0000

BackgroundIn eukaryotic organisms, gene expression is regulated at multiple levels during the processes of transcription and translation. The absence of a tight regulatory network for transcription in the human malaria parasite suggests that gene expression may largely be controlled at post-transcriptional and translational levels.ResultsIn this study, we compare steady-state mRNA and polysome-associated mRNA levels of Plasmodium falciparum at different time points during its asexual cell cycle. For more than 30% of its genes, we observe a delay in peak transcript abundance in the polysomal fraction as compared to the steady-state mRNA fraction, suggestive of strong translational control. Our data show that key regulatory mechanisms could include inhibitory activity of upstream open reading frames and translational repression of the major virulence gene family by intronic transcripts. In addition, we observe polysomal mRNA-specific alternative splicing events and widespread transcription...

Live-attenuated virus vaccine defective in RNAi suppression induces rapid protection in neonatal and adult mice lacking mature B and T cells

Thu, 6 Jun 2024 00:00:00 +0000

Global control of infectious diseases depends on the continuous development and deployment of diverse vaccination strategies. Currently available live-attenuated and killed virus vaccines typically take a week or longer to activate specific protection by the adaptive immunity. The mosquito-transmitted Nodamura virus (NoV) is attenuated in mice by mutations that prevent expression of the B2 viral suppressor of RNA interference (VSR) and consequently, drastically enhance in vivo production of the virus-targeting small-interfering RNAs. We reported recently that 2 d after immunization with live-attenuated VSR-disabled NoV (NoVΔB2), neonatal mice become fully protected against lethal NoV challenge and develop no detectable infection. Using Rag1^-/- mice that produce no mature B and T lymphocytes as a model, here we examined the hypothesis that adaptive immunity is dispensable for the RNAi-based protective immunity activated by NoVΔB2 immunization. We show that immunization...

Intravital Multiphoton Examination of Implant-Associated Staphylococcus aureus Biofilm Infection

Mon, 27 May 2024 00:00:00 +0000

Bacterial infections associated with implanted medical devices represents a healthcare crisis due to their persistence, antibiotic tolerance, and immune avoidance. Indwelling devices are rapidly coated with host plasma and extracellular matrix proteins which can then be exploited by bacterial pathogens for adherence and subsequent biofilm development. Our understanding of the host-pathogen interface that determines the fate of biofilm-mediated infections is limited to the experimental models employed by laboratories studying these organisms. Current in vivo models of biofilm-mediated infection, while certainly useful, are typically limited to end-point analyses of bacterial burden enumeration, immune cell profiling, and cytokine/chemokine analysis. Thus, with these models, the complex, real-time assessment of biofilm development and innate immune cell activity remains imperceptible. Here, we describe a novel murine biofilm infection model employing time-lapse intravital...

Novel Mechanical Strain Characterization of Ventilated ex vivo Porcine and Murine Lung using Digital Image Correlation

Mon, 27 May 2024 00:00:00 +0000

Respiratory illnesses, such as bronchitis, emphysema, asthma, and COVID-19, substantially remodel lung tissue, deteriorate function, and culminate in a compromised breathing ability. Yet, the structural mechanics of the lung is significantly understudied. Classical pressure-volume air or saline inflation studies of the lung have attempted to characterize the organ's elasticity and compliance, measuring deviatory responses in diseased states; however, these investigations are exclusively limited to the bulk composite or global response of the entire lung and disregard local expansion and stretch phenomena within the lung lobes, overlooking potentially valuable physiological insights, as particularly related to mechanical ventilation. Here, we present a method to collect the first non-contact, full-field deformation measures of ex vivo porcine and murine lungs and interface with a pressure-volume ventilation system to investigate lung behavior in real time. We share preliminary...

CD137 signaling enhances tight junction resistance in intestinal epithelial cells

Mon, 27 May 2024 00:00:00 +0000

Treatment of Caco-2-BBe intestinal epithelial cells (BBe) with TNF-α and lymphotoxin-β (LT-β) receptor agonists induced the expression of the TNF receptor superfamily gene TNFRSF9/CD137. In the gut, these cytokines are known to be involved in both inflammatory responses and development of organized lymphoid tissues; thus, it was notable that in CD137-deficient mice Peyer's patch M cells lacked transcytosis function. To examine the direct effect of CD137 expression on epithelial cell function independent of other cytokine effects including CD137L triggering, we stably transfected BBe cells to express CD137. CD137 was found at the cell surface as well as the cytoplasm, and confocal microscopy suggested that aggregates of CD137 at the lateral and basolateral surface may be associated with cytoplasmic actin filament termini. Many of the CD137 clusters were colocalized with extracellular fibronectin providing a possible alternative ligand for CD137. Interestingly, we found that CD137-expressing...

Inducible Colonic M Cells Are Dependent on TNFR2 but Not Ltβr, Identifying Distinct Signalling Requirements for Constitutive Versus Inducible M Cells

Mon, 27 May 2024 00:00:00 +0000

BACKGROUND AND AIMS: M cells associated with organised lymphoid tissues such as intestinal Peyer's patches provide surveillance of the intestinal lumen. Inflammation or infection in the colon can induce an M cell population associated with lymphoid infiltrates; paradoxically, induction is dependent on the inflammatory cytokine tumour necrosis factor [TNF]-α. Anti-TNFα blockade is an important therapeutic in inflammatory bowel disease, so understanding the effects of TNFα signalling is important in refining therapeutics. METHODS: To dissect pro-inflammatory signals from M cell inductive signals, we used confocal microscopy image analysis to assess requirements for specific cytokine receptor signals using TNF receptor 1 [TNFR1] and 2 [TNFR2] knockouts [ko] back-crossed to the PGRP-S-dsRed transgene; separate groups were treated with soluble lymphotoxin β receptor [sLTβR] to block LTβR signalling. All groups were treated with dextran sodium sulphate [DSS] to induce colitis. RESULTS:...

Staphylococcus aureus Fibronectin Binding Protein A Mediates Biofilm Development and Infection

Mon, 27 May 2024 00:00:00 +0000

Implanted medical device-associated infections pose significant health risks, as they are often the result of bacterial biofilm formation. Staphylococcus aureus is a leading cause of biofilm-associated infections which persist due to mechanisms of device surface adhesion, biofilm accumulation, and reprogramming of host innate immune responses. We found that the S. aureus fibronectin binding protein A (FnBPA) is required for normal biofilm development in mammalian serum and that the SaeRS two-component system is required for functional FnBPA activity in serum. Furthermore, serum-developed biofilms deficient in FnBPA were more susceptible to macrophage invasion, and in a model of biofilm-associated implant infection, we found that FnBPA is crucial for the establishment of infection. Together, these findings show that S. aureus FnBPA plays an important role in physical biofilm development and represents a potential therapeutic target for the prevention and treatment...

M Cells: Intelligent Engineering of Mucosal Immune Surveillance

Mon, 27 May 2024 00:00:00 +0000

M cells are specialized intestinal epithelial cells that provide the main machinery for sampling luminal microbes for mucosal immune surveillance. M cells are usually found in the epithelium overlying organized mucosal lymphoid tissues, but studies have identified multiple distinct lineages of M cells that are produced under different conditions, including intestinal inflammation. Among these lineages there is a common morphology that helps explain the efficiency of M cells in capturing luminal bacteria and viruses; in addition, M cells recruit novel cellular mechanisms to transport the particles across the mucosal barrier into the lamina propria, a process known as transcytosis. These specializations used by M cells point to a novel engineering of cellular machinery to selectively capture and transport microbial particles of interest. Because of the ability of M cells to effectively violate the mucosal barrier, the circumstances of M cell induction have important consequences....

Route of administration significantly affects particle deposition and cellular recruitment

Mon, 27 May 2024 00:00:00 +0000

Lung exposures to dusts, pollutants, and other aerosol particulates are known to be associated with pulmonary diseases such as asthma and Chronic Obstructive Pulmonary Disease. These health impacts are attributed to the ability of aerosol components to induce pulmonary inflammation, which promotes tissue remodeling, including fibrosis, tissue degradation, and smooth muscle proliferation. Consequently, the distribution of these effects can have a significant impact on the physiologic function of the lung. In order to study the impact of distribution of inhaled particulates on lung pathogenesis, we compared the effect of different methods of particle delivery. By comparing intranasal versus aerosol delivery of fluorescent microspheres, we observed strikingly distinct patterns of particle deposition; intranasal delivery provided focused deposition concentrated on larger airways, while aerosol delivery showed unform deposition throughout the lung parenchyma. Recognizing that the impacts...

Selective Targeting of Tumour Necrosis Factor Receptor 1 Induces Stable Protection from Crohn’s-Like Ileitis in TNFΔARE Mice

Mon, 27 May 2024 00:00:00 +0000

BACKGROUND AND AIMS: Crohn's disease is a debilitating chronic inflammatory disorder of the mammalian gastrointestinal tract. Current interventions using anti-tumour necrosis factor [anti-TNF] biologics show long-term benefit in only half of patients. This study focused on the role of the TNF receptor 1 [TNFR1] in pathogenesis in a TNF-driven model of ileitis. METHODS: We studied TNFΔAU-rich element [ARE]/+ [TNFdARE] mice, which develop progressive ileitis similar to Crohn's ileitis. Histopathological analysis and gene expression profiling were used to characterize disease progression from 5 to 16 weeks. Mice with TNFR1 hemizygosity [TNFdARE/R1het] allowed us to assess gene dosage effects. Transcriptional profiling established inflection points in disease progression; inflammatory gene expression increased at 8 weeks with a plateau by 10 weeks, so these were selected as endpoints of treatment using the TNF biologic infliximab and the TNFR1-specific XPro1595. Differences in recruitment...

The lethal and sublethal impacts of two tire rubber-derived chemicals on brook trout (Salvelinus fontinalis) fry and fingerlings