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Histone post-translational modifications (PTMs) alter chromatin dynamics and contribute to the regulation of gene expression in health and disease. Mass spectrometry-based analysis is the gold-standard for histone PTM analysis, but it remains constrained by inefficient sample preparation workflows requiring multiple days. Here, we develop RIPUP ( R apid I dentification of histone P TMs in U nderivatized P eptides), a streamlined multi-protease workflow that reduces sample preparation from days to hours while improving PTM coverage and quantitative accuracy. Through systematic evaluation of the Arg-C Ultra protease and a prototype recombinant (r)-Chymotrypsin protease under varied conditions, such as chemical derivatization using propionic anhydride and tandem mass tags (TMT), we demonstrated that Arg-C Ultra with TMT labeling achieves a detection of total PTM comparable to conventional Trypsin-based approaches. Using the HiP-Frag computational framework for unrestrictive PTM identification, we discovered that TMT's tertiary amine provides charge compensation that rescues the ionization of negatively charged acylations revealing 50 succinylation and 27 glutarylation sites - a 'dark epigenome' largely undetected by propionylation-based methods. We demonstrated that complementary digestion with Arg-C Ultra and r-Chymotrypsin provides orthogonal sequence coverage, enabling detection of PTMs in H2A variants, linker histones, and regions poorly represented by arginine-specific cleavage alone. Application of RIPUP to frozen-thawed rat hippocampal sections within a 3-hour workflow identifies >200 PTMs including biologically critical PTM sites H3 K27/K36/K37 methylation, H4 N-terminal acetylation patterns, and H2A ubiquitination at K118/K119. This rapid, high-efficiency platform enables timely discovery of epigenetic mechanisms and accelerates the path from PTM identification to therapeutic target validation.

Background: Serotype 3 (ST3) Streptococcus pneumoniae remains a major cause of invasive pneumococcal disease and pneumonia despite PCV13 introduction, in part due to potent immune evasion properties. The contribution of the pyruvate metabolic node (SpxB/LctO pathways) to ST3 pathogenesis is poorly defined.

Methods: We investigated oxygen-dependent fitness, virulence, and lung pathology in the ST3 strain WU2 and isogenic Δ spxB , Δl ctO , and Δ spxB Δ lctO mutants. In vitro growth was assessed under nasopharyngeal (21% O₂) and alveolar (14% O₂) conditions. Murine pneumonia models evaluated survival, bacterial burdens, histopathology (H&E, confocal microscopy), and lung transcriptomics (RNA-seq).

Results: ST3-strain exhibited a unique oxygen-sensitive growth defect at 21% O₂, alleviated by spxB deletion, indicating metabolic burden from pyruvate flux. In mice, wild-type WU2 caused high mortality with severe suppurative bronchopneumonia, alveolar consolidation, hemorrhage, and perivascular inflammation. The Δ spxB mutant accelerated lethality with enhanced distal lung damage, uncontrolled dissemination, and amplified inflammation. Wild-type infection uniquely induced targeted reorganization of bronchial epithelial membranes, forming prominent bacterium-laden blebs-host-derived membrane protrusions encapsulating intact pneumococci. These novel structures facilitated organized bacterial translocation into tissue without overt cytotoxicity and were largely absent in spxB -deficient mutants despite comparable lung burdens. RNA-seq analysis revealed SpxB-dependent suppression of T cell activation (e.g., Rag1 and Themis ) and acute inflammatory pathways, consistent with immune sequestration via blebs.

Conclusions: The SpxB-dependent pathway orchestrates ST3 virulence by enabling metabolic adaptation and driving bleb-mediated epithelial invasion and immune evasion in the lung. These bacterium-laden blebs represent a novel hallmark mechanism in pneumococcal pathogenesis, offering new insights and potential therapeutic targets.

The brain's complex network relies on both electrical and chemical signaling to support its physiological and cognitive functions. To fully understand neural circuit dynamics and their dysfunctions, it is crucial to simultaneously detect neurotransmitters and modulators alongside electrophysiological signals. The striatal dopamine circuits are integral to neurological processes such as movement, reward, learning, and circadian rhythm regulation, making it highly desirable to monitor both neural activity and dopamine (DA) levels in freely behaving animals. One promising approach involves the implantation of multimodal microelectrode arrays (MEAs). However, chronic electrochemical sensing of DA in freely moving animals faces significant challenges, including biofouling of sensing electrodes and the instability of Ag/AgCl reference electrodes. In this study, we developed two complementary strategies-surface grafting and photo crosslinking-to coat the MEA and implanted Ag/AgCl reference electrodes, respectively, with zwitterionic poly(sulfobetaine methacrylate) (PSB). The surface-grafted thin PSB coating effectively inhibits protein fouling and inflammatory responses to the MEA, while the PSB hydrogel protects the Ag/AgCl electrodes from delamination in vivo, ensuring a stable reference potential. By coating both the Ag/AgCl reference electrodes and flexible polyimide MEAs with PSB and PEDOT/CNT, we achieved stable DA detection and electrophysiological recordings in freely moving mice over a four-week period. Weekly electrochemical impedance spectroscopy confirmed the long-term stability of the implanted electrodes. Our method enables multidimensional analysis of behavioral patterns, electrophysiological activity, and DA dynamics, providing a comprehensive approach for neuroscience research. This work advances neurochemical and electrophysiological methodologies by offering reliable tools for longitudinal investigations of brain function in freely behaving animals.

Aging is associated with cognitive decline and increased vulnerability to neurodegeneration driven by an array of molecular and cellular changes like impaired vascular integrity, demyelination, reduced neurogenesis, and chronic inflammation. Recent studies implicate the gut microbiome as a modulator of brain aging, but the underlying mechanisms remain elusive. Here, we show that depleting the gut microbiome by administering antibiotics to aged mice induces widespread molecular and structural rejuvenation in the brain. Our transcriptomic analyses by single-nucleus RNA sequencing revealed pronounced transcriptional shifts across multiple brain cell types. We confirmed that antibiotic treatment improves vascular density, promotes myelination, enhances neurogenesis, and reduces microglial reactivity. Functionally, microbiome-depleted mice showed improved hippocampal memory performance. Analyses of brain and plasma cytokine levels showed a decrease in several pro-inflammatory factors post-treatment and identified candidate factors, including the chemokine eotaxin-1. Inhibiting eotaxin-1 alone can reverse several aspects of brain aging. Our findings demonstrate that age-associated microbial inflammation contributes to brain aging and that its attenuation can restore youthful features at the molecular, cellular, and functional levels. Targeting the gut microbiome or its circulating mediators may therefore represent a non-invasive approach to promote brain health and cognitive resilience in aging.

Dogma suggests protein quantification is a pre-requisite to LC-MS/MS based proteomics studies. Such quantification allows a standardized ratio of sample to digestion enzyme and enables physical normalization of protein digest loaded onto the mass spectrometer for analysis. Most proteomics studies include these steps. However, there are significant costs in time, money and experimental complexity, associated with performing protein quantification and physical normalization for every sample, especially for larger studies. Proteomics data analysis pipelines typically include computational normalization strategies to compensate for unavoidable systematic biases. These strategies also have the potential to compensate for avoidable variation such as omitting sample amount normalization. Here we investigate the effects of either physically normalizing the amount of protein for each individual sample or leaving it unnormalized. Our results show the relationship between increased protein amount variation in sample input, and the variance of quantified relative abundances of peptides and proteins output after data analysis. The experiments presented here suggest that protein quantification and physical normalization steps can be omitted from some quantitative proteomic experiments without incurring an unacceptable increase in measurement variability after computational normalization has been applied. This work will enable important time and cost saving optimizations to be made to many proteomics workflows.

Alcohol use disorder (AUD) affects over 28 million people in the U.S and is associated with neurobiological alterations, including in the basal ganglia. Within the basal ganglia, the caudate nucleus (caudate) and putamen are implicated in AUD due to their roles in ethanol reinforcement, with the caudate receiving inputs from cortico-associative areas and the putamen receiving inputs from somatosensory areas, supporting goal-directed and habitual behaviors respectively. These distinct behavioral roles are supported by dopamine signaling, including phasic dopamine, involved in assessing action-outcome associations, and tonic dopamine, which reflects ongoing dopaminergic tone that biases action initiation. Intrastriatal dopamine release is modulated by cholinergic interneurons via nicotinic acetylcholine receptors. Dysregulation of these mechanisms can contribute to the transition from occasional to habitual ethanol drinking. Here, we used in-vitro fast-scan cyclic voltammetry to measure dopamine signaling in male (n=6) and female (n=6) rhesus macaques following six months of ethanol self-administration. In putamen, ethanol increased tonic dopamine in both sexes, with females exhibiting greater release and faster dopamine uptake rates than males. In the caudate, ethanol self-administration enhanced dopamine uptake rates only in males. Phasic dopamine release was enhanced in caudate of both sexes but only putamen in males. nAChR blockade revealed that phasic dopamine release in males, but not females, was dependent on cholinergic modulation. These results demonstrate basal and sex-specific dopamine release and uptake are uniquely altered in rhesus macaque caudate and putamen in conjunction with chronic ethanol drinking.

Commensal Neisseria species are major reservoirs of adaptive genetic variation, including antimicrobial resistance, for their pathogenic relatives, yet they remain poorly characterized. This gap limits our ability to anticipate resistance mechanisms that may ultimately emerge Neisseria gonorrhoeae and N. meningitidis . Here, we analyzed 166 novel commensal Neisseria isolates collected from 31 study participants and measured minimum inhibitory concentrations (MICs) for seven antimicrobials: azithromycin, cefixime, ceftriaxone, ciprofloxacin, doxycycline, and gentamicin. Resistance, defined using the Clinical and Laboratory Standards Institute (CLSI) guidelines, was highly prevalent for azithromycin (76%) and doxycycline (52%), while no resistance to gentamicin was observed. High-level doxycycline resistance was always associated with inheritance of tetM . Reduced susceptibility to azithromycin was linked to an MtrD K823E substitution, and reduced susceptibility to ciprofloxacin was associated with GyrA T91I ( N. subflava ) or S91V ( N. mucosa ). The PenA 312M mutation was associated with significantly elevated ceftriaxone and cefixime MICs. Across all antimicrobials, MICs varied widely, indicating the presence of additional modulating mutations. Finally, the genetic determinants underlying low-level doxycycline resistance and reduced penicillin susceptibility remain unresolved. Overall, here we continue to build on the foundation of surveillance efforts in the commensal Neisseria , and continue to flesh out what is known and unknown about this early warning system - or canary in the coal mine - for emerging resistance and clinically consequential evolution in pathogenic Neisseria .

Pain remains a major clinical challenge, with current therapies often limited by low efficacy and adverse effects. While excitability of sensory neurons in the dorsal root ganglia (DRG) is central to pain signaling, accumulating evidence highlights the importance of non-neuronal cells in modulating neuronal activity. Here, we identify satellite glial cells (SGCs) as a source of Fibulin-2, an extracellular matrix glycoprotein with diverse roles in nervous system development and repair. Using SGC primary cultures and mass spectrometry, we demonstrate that Fibulin-2 is secreted by SGCs in part via extracellular vesicles. Electrophysiological functional assays reveal that application of recombinant Fibulin-2 to cultured sensory neurons reduces neuronal excitability by modulating Kv4-mediated currents. In vivo , loss of Fibulin-2 leads to reduced Kv4.2 and Kv4.3 expression and heightened mechanical, heat and cold sensitivity in mice. Our findings uncover a novel SGC-sensory neuron signaling mechanism modulating pain sensitivity, suggesting Fibulin-2 as a potential therapeutic target for pain management.

Highlights: Fibulin-2 is secreted by cultured SGCsFibulin-2 reduces sensory neuron excitability and firing frequencyFibulin-2 acts by modulating Kv4-mediated potassium currentsLoss of Fibulin-2 heightens mechanical, heat and cold sensitivity in mice.

While a history of TBI is associated with an increased risk of neurodegenerative disease, associated mechanisms remain largely unknown. Neuroinflammation is commonly implicated as playing a role in progressive neurodegeneration in general, yet little is known about the adaptive response of neuroinflammation in TBI or how it may contribute to progressive pathologies. To parse out components of the adaptive response, we assessed for intraparenchymal T-cell infiltration in two different translational large animal (swine) models of TBI, inertial injury and focal contusion. We characterized the extent and distribution of T cells post-injury and their association with blood-brain barrier disruption and axonal pathology. T-cell infiltration following focal TBI followed a spatiotemporal progression from gray matter at 72 hours to both gray and white matter at 6 months post-injury, consistent with recruitment into the parenchyma and then white matter. Inertial injury did not lead to substantial T-cell infiltration despite BBB breakdown and axonal pathology. We did not find a spatial correlation between blood-brain barrier breakdown or axonal pathology and T-cell infiltration in focal TBI. These data suggest that there is an active adaptive response to TBI, particularly in tissue proximal to contusions. A large animal model that reproducibly demonstrates chronic T-cell infiltration may allow for examination of the downstream effects of the adaptive response to TBI, and whether targeting this adaptive response may reduce chronic inflammation and improve recovery.

Background & aims: Intrahepatic biliary epithelial cell (BEC) heterogeneity remains challenging to define. Here, we sought to identify BEC subpopulations and biomarkers in mouse liver.

Methods: We performed scRNA-seq on Sox9EGFP+ liver epithelium from mice subjected to bile duct ligation (BDL) and sham controls. A machine learning algorithm, NSForest, identified minimal, multi-gene signatures for BEC subpopulations. These metagenes and were validated using hybridization chain reaction (HCR) FISH in tissue sections from wild-type mice and on primary BECs expanded in vitro. Metagenes were used to match BDL subpopulations to their corresponding sham subpopulations for differential gene expression (DGE) analysis.

Results: We identified 4 BEC subpopulations in sham controls, each associated with 1-2 gene metagenes. Spatial localization of metagene-defined BEC subpopulations by HCR FISH revealed heterogeneous cellular composition of intrahepatic bile ducts. BECs belonging to a given subpopulation were most likely to have neighbors of the same identity, forming homogenous cellular compartments within ducts. BDL downregulated subpopulation-specific genes and upregulated a damage-associated gene set. BDL samples also included a proliferative subpopulation not found in sham controls, which contained populations enriched for three of the four metagenes. All BEC subpopulations were also found in monolayers in vitro, where they clustered spatially with BECs of the same subtype.

Conclusions: Novel metagene biomarkers of BEC subpopulations facilitated spatial localization of BECs in situ, identified subpopulation specific injury responses, and confirmed that BEC heterogeneity is preserved in vitro. The presence of locally homogenous BEC neighborhoods in vitro suggests some degree of BEC organization may be epithelial-autonomous.