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The eukaryotic genome is packaged around histone proteins, which are subject to a myriad of post-translational modifications. By controlling DNA accessibility and the recruitment of protein complexes that mediate chromatin-related processes, these modifications constitute a key mechanism of epigenetic regulation. Since mass spectrometry can easily distinguish between these different modifications, it has become an essential technique in deciphering the histone code. Although robust LC-MS/MS methods are available to analyze modifications on the histone N-terminal tails, routine methods for characterizing ubiquitin marks on histone C-terminal regions, especially H2AK119ub, are less robust. Here, we report the development of a simple workflow for the detection and improved quantification of the canonical histone ubiquitination marks H2AK119ub and H2BK120ub. The method entails a fully tryptic digestion of acid-extracted histones, followed by derivatization with heavy or light propionic anhydride. A pooled sample is then spiked into oppositely labeled single samples as a reference channel for relative quantification, and data is acquired using PRM-based nano-LC-MS/MS. We validated our approach with synthetic peptides as well as treatments known to modulate the levels of H2AK119ub and H2BK120ub. This new method complements existing histone workflows, largely focused on the lysine-rich N-terminal regions, by extending modification analysis to other sequence contexts.

Pancreatic cancer is the third leading cause of cancer death in the United States, and while conventional chemotherapy remains the standard treatment, responses are poor. Safe and alternative therapeutic strategies are urgently needed 1 . A ketogenic diet has been shown to have anti-tumor effects across diverse cancer types but will unlikely have a significant effect alone. However, the diet shifts metabolism in tumors to create new vulnerabilities that can be targeted (1). Modulators of glutamine metabolism have shown promise in pre-clinical models but have failed to have a marked impact against cancer in the clinic. We show that a ketogenic diet increases TCA and glutamine-associated metabolites in murine pancreatic cancer models and under metabolic conditions that simulate a ketogenic diet in vitro. The metabolic shift leads to increased reliance on glutamine-mediated anaplerosis to compensate for low glucose abundance associated with a ketogenic diet. As a result, glutamine metabolism inhibitors, such as DON and CB839 in combination with a ketogenic diet had robust anti-cancer effects. These findings provide rationale to study the use of a ketogenic diet with glutamine targeted therapies in a clinical context.

The eukaryotic genome is packaged around histone proteins, which are subject to a myriad of post-translational modifications. By controlling DNA accessibility and the recruitment of protein complexes that mediate chromatin-related processes, these modifications constitute a key mechanism of epigenetic regulation. Since mass spectrometry can easily distinguish between these different modifications, it has become an essential technique in deciphering the histone code. Although robust LC-MS/MS methods are available to analyze modifications on the histone N-terminal tails, routine methods for characterizing ubiquitin marks on histone C-terminal regions, especially H2AK119ub, are less robust. Here we report the development of a simple workflow for the detection and improved quantification of the canonical histone ubiquitination marks H2AK119ub and H2BK120ub. The method entails a fully tryptic digestion of acid-extracted histones followed by derivatization with heavy or light propionic anhydride. A pooled sample is then spiked into oppositely labeled single samples as a reference channel for relative quantification, and data is acquired using PRM-based nanoLC-MS/MS. We validated our approach with synthetic peptides as well as treatments known to modulate the levels of H2AK119ub and H2BK120ub. This new method complements existing histone workflows, largely focused on the lysine-rich N-terminal regions, by extending modification analysis to other sequence contexts., Competing Interests: CONFLICTS OF INTEREST The authors have no financial conflicts of interest to disclose.

Post-translational modifications (PTMs) on histones are a key source of regulation on chromatin through impacting cellular processes, including gene expression 1 . These PTMs often arise from metabolites and are thus impacted by metabolism and environmental cues 2-7 . One class of metabolically regulated PTMs are histone acylations, which include histone acetylation, butyrylation, crotonylation and propionylation 3,8 . As these PTMs can be derived from short-chain fatty acids, which are generated by the commensal microbiota in the intestinal lumen 9-11 , we aimed to define how microbes impact the host intestinal chromatin landscape, mainly in female mice. Here we show that in addition to acetylation, intestinal epithelial cells from the caecum and distal mouse intestine also harbour high levels of butyrylation and propionylation on lysines 9 and 27 of histone H3. We demonstrate that these acylations are regulated by the microbiota and that histone butyrylation is additionally regulated by the metabolite tributyrin. Tributyrin-regulated gene programmes are correlated with histone butyrylation, which is associated with active gene-regulatory elements and levels of gene expression. Together, our study uncovers a regulatory layer of how the microbiota and metabolites influence the intestinal epithelium through chromatin, demonstrating a physiological setting in which histone acylations are dynamically regulated and associated with gene regulation., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated H3.3G34R/V/W knock-in mice and identified strikingly distinct developmental defects for each mutation. H3.3G34R-mutants exhibited progressive microcephaly and neurodegeneration, with abnormal accumulation of disease-associated microglia and concurrent neuronal depletion. G34R severely decreased H3K36me2 on the mutant H3.3 tail, impairing recruitment of DNA methyltransferase DNMT3A and its redistribution on chromatin. These changes were concurrent with sustained expression of complement and other innate immune genes possibly through loss of non-CG (CH) methylation and silencing of neuronal gene promoters through aberrant CG methylation. Complement expression in G34R brains may lead to neuroinflammation possibly accounting for progressive neurodegeneration. Our study reveals that H3.3G34-substitutions have differential impact on the epigenome, which underlie the diverse phenotypes observed, and uncovers potential roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating synaptic pruning and neuroinflammation in post-natal brains., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The gut microbiota influences acetylation on host histones by fermenting dietary fiber into butyrate. Although butyrate could promote histone acetylation by inhibiting histone deacetylases, it may also undergo oxidation to acetyl-coenzyme A (CoA), a necessary cofactor for histone acetyltransferases. Here, we find that epithelial cells from germ-free mice harbor a loss of histone H4 acetylation across the genome except at promoter regions. Using stable isotope tracing in vivo with 13 C-labeled fiber, we demonstrate that the microbiota supplies carbon for histone acetylation. Subsequent metabolomic profiling revealed hundreds of labeled molecules and supported a microbial contribution to host fatty acid metabolism, which declined in response to colitis and correlated with reduced expression of genes involved in fatty acid oxidation. These results illuminate the flow of carbon from the diet to the host via the microbiota, disruptions to which may affect energy homeostasis in the distal gut and contribute to the development of colitis., Competing Interests: Declarations of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Histone variants, such as histone H3.3, replace canonical histones within the nucleosome to alter chromatin accessibility and gene expression. Although the biological roles of selected histone post-translational modifications (PTMs) have been extensively characterized, the potential differences in the function of a given PTM on different histone variants is almost always elusive. By applying proteomics and genomics techniques, we investigate the role of lysine 27 tri-methylation specifically on the histone variant H3.3 (H3.3K27me3) in the context of mouse embryonic stem cell pluripotency and differentiation as a model system for development. We demonstrate that while the steady state overall levels of methylation on both H3K27 and H3.3K27 decrease during differentiation, methylation dynamics studies indicate that methylation on H3.3K27 is maintained more than on H3K27. Using a custom-made antibody, we identify a unique enrichment of H3.3K27me3 at lineage-specific genes, such as olfactory receptor genes, and at binding motifs for the transcription factors FOXJ2/3. REST, a predicted FOXJ2/3 target that acts as a transcriptional repressor of terminal neuronal genes, was identified with H3.3K27me3 at its promoter region. H3.3K27A mutant cells confirmed an upregulation of FOXJ2/3 targets upon the loss of methylation at H3.3K27. Thus, while canonical H3K27me3 has been characterized to regulate the expression of transcription factors that play a general role in differentiation, our work suggests H3.3K27me3 is essential for regulating distinct terminal differentiation genes. This work highlights the importance of understanding the effects of PTMs not only on canonical histones but also on specific histone variants, as they may exhibit distinct roles.

Background and Aims: Although germ-free mice are an indispensable tool in studying the gut microbiome and its effects on host physiology, they are phenotypically different than their conventional counterparts. While antibiotic-mediated microbiota depletion in conventional mice leads to physiologic alterations that often mimic the germ-free state, the degree to which the effects of microbial colonization on the host are reversible is unclear. The gut microbiota produce abundant short chain fatty acids (SCFAs), and previous studies have demonstrated a link between microbial-derived SCFAs and global hepatic histone acetylation in germ-free mice., Approach and Results: We demonstrate that global hepatic histone acetylation states measured by mass spectrometry remained largely unchanged despite loss of luminal and portal vein SCFAs after antibiotic-mediated microbiota depletion. In contrast to stable hepatic histone acetylation states, we see robust hepatic transcriptomic alterations after microbiota depletion. Additionally, neither dietary supplementation with supraphysiologic levels of SCFA nor the induction of hepatocyte proliferation in the absence of microbiota-derived SCFAs led to alterations in global hepatic histone acetylation., Conclusions: These results suggest that microbiota-dependent landscaping of the hepatic epigenome through global histone acetylation is static in nature, while the hepatic transcriptome is responsive to alterations in the gut microbiota., (© 2021 American Association for the Study of Liver Diseases.)

Paradoxically, oncogenes that drive cell cycle progression may also trigger pathways leading to senescence, thereby inhibiting the growth of tumorigenic cells. Knowledge of how these pathways operate, and how tumor cells may evade these pathways, is important for understanding tumorigenesis. The Y1 cell line, which harbors an amplification of the proto-oncogene Ras, rapidly senesces in response to the mitogen fibroblast growth factor-2 (FGF-2). To gain a more complete picture of how FGF-2 promotes senescence, we employed a multi-omics approach to analyze histone modifications, mRNA and protein expression, and protein phosphorylation in Y1 cells treated with FGF-2. Compared to control cells treated with serum alone, FGF-2 caused a delayed accumulation of acetylation on histone H4 and higher levels of H3K27me3. Sequencing analysis revealed decreased expression of cell cycle-related genes with concomitant loss of H3K27ac. At the same time, FGF-2 promoted the expression of p21, various cytokines, and MAPK-related genes. Nuclear envelope proteins, particularly lamin B1, displayed increased phosphorylation in response to FGF-2. Proteome analysis suggested alterations in cellular metabolism, as evident by modulated expression of enzymes involved in purine biosynthesis, tRNA aminoacylation, and the TCA cycle. We propose that Y1 cells senesce due to an inability to progress through the cell cycle, which may stem from DNA damage or TGFb signaling. Altogether, the phenotype of Y1 cells is consistent with rapidly established oncogene-induced senescence, demonstrating the synergy between growth factors and oncogenes in driving senescence and bringing additional insight into this tumor suppressor mechanism.

Proteoforms containing post-translational modifications (PTMs) represent a degree of functional diversity only harnessed through analytically precise simultaneous quantification of multiple PTMs. Here we present a method to accurately differentiate an unmodified peptide from its PTM-containing counterpart through data-independent acquisition-mass spectrometry, leveraging small precursor mass windows to physically separate modified peptidoforms from each other during MS2 acquisition. We utilize a lysine and arginine PTM-enriched peptide assay library and site localization algorithm to simultaneously localize and quantify seven PTMs including mono-, di-, and trimethylation, acetylation, and succinylation in addition to total protein quantification in a single MS run without the need to enrich experimental samples. To evaluate biological relevance, this method was applied to liver lysate from differentially methylated nonalcoholic steatohepatitis (NASH) mouse models. We report that altered methylation and acetylation together with total protein changes drive the novel hypothesis of a regulatory function of PTMs in protein synthesis and mRNA stability in NASH.

Histone post-translational modifications (PTMs) contribute to chromatin accessibility due to their chemical properties and their ability to recruit enzymes responsible for DNA readout and chromatin remodeling. To date, more than 400 different histone PTMs and thousands of combinations of PTMs have been identified, the vast majority with still unknown biological function. Identification and quantification of histone PTMs has become routine in mass spectrometry (MS) but, since raising antibodies for each PTM in a study can be prohibitive, lots of potential is lost from MS datasets when uncharacterized PTMs are found to be significantly regulated. We developed an assay that uses metabolic labeling and MS to associate chromatin accessibility with histone PTMs and their combinations. The labeling is achieved by spiking in the cell media a 5x concentration of stable isotope labeled arginine and allow cells to grow for at least one cell cycle. We quantified the labeling incorporation of about 200 histone peptides with a proteomics workflow, and we confirmed that peptides carrying PTMs with extensively characterized roles in active transcription or gene silencing were in highly or poorly labeled forms, respectively. Data were further validated using next-generation sequencing to assess the transcription rate of chromatin regions modified with five selected PTMs. Furthermore, we quantified the labeling rate of peptides carrying co-existing PTMs, proving that this method is suitable for combinatorial PTMs. We focus on the abundant bivalent mark H3K27me3K36me2, showing that H3K27me3 dominantly represses histone swapping rate even in the presence of the more permissive PTM H3K36me2. Together, we envision this method will help to generate hypotheses regarding histone PTM functions and, potentially, elucidate the role of combinatorial histone codes.

The impact of glucose metabolism on muscle regeneration remains unresolved. We identify glucose metabolism as a crucial driver of histone acetylation and myogenic cell fate. We use single-cell mass cytometry (CyTOF) and flow cytometry to characterize the histone acetylation and metabolic states of quiescent, activated, and differentiating muscle stem cells (MuSCs). We find glucose is dispensable for mitochondrial respiration in proliferating MuSCs, so that glucose becomes available for maintaining high histone acetylation via acetyl-CoA. Conversely, quiescent and differentiating MuSCs increase glucose utilization for respiration and have consequently reduced acetylation. Pyruvate dehydrogenase (PDH) activity serves as a rheostat for histone acetylation and must be controlled for muscle regeneration. Increased PDH activity in proliferation increases histone acetylation and chromatin accessibility at genes that must be silenced for differentiation to proceed, and thus promotes self-renewal. These results highlight metabolism as a determinant of MuSC histone acetylation, fate, and function during muscle regeneration., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Posterior fossa type A (PFA) ependymomas exhibit very low H3K27 methylation and express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). Here we find that a conserved sequence in EZHIP is necessary and sufficient to inhibit PRC2 catalytic activity in vitro and in vivo. EZHIP directly contacts the active site of the EZH2 subunit in a mechanism similar to the H3 K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promotes similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at CpG islands. We find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a mechanism to explain the observed loss of H3K27me3 spreading in tumors. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors, the K27M oncohistone and the EZHIP 'oncohistone-mimic', that dysregulate gene silencing to promote tumorigenesis.

Since protein activity is often regulated by posttranslational modifications, the qualitative and quantitative analysis of modification sites is critical for understanding the regulation of biological pathways that control cell function and phenotype. Methylation constitutes one of the many types of posttranslational modifications that target lysine residues. Although lysine methylation is perhaps most commonly associated with histone proteins and the epigenetic regulation of processes involving chromatin, methylation has also been observed as an important regulatory modification on other proteins, which has spurred the development of methods to profile lysine methylation sites more globally. As with many posttranslational modifications, tandem mass spectrometry represents an ideal platform for the high-throughput analysis of lysine methylation due to its high sensitivity and resolving power. The following protocol outlines a general method to assay lysine methylation across the proteome using SILAC and quantitative proteomics. First, cells are labeled by SILAC to allow for relative quantitation across different experimental conditions, such as cells with or without ectopic expression of a methyltransferase. Next, cells are lysed and proteins are digested into peptides. Methylated peptides are then enriched by immunoprecipitation with pan-specific antibodies against methylated lysine. Finally, the enriched peptides are analyzed by LC-MS/MS to identify methylated peptides and their modification sites and to compare the relative abundance of methylation events between different conditions. This approach should yield detection of a couple hundred lysine methylation sites, and those showing differential abundance may then be prioritized for further study., (© 2019 Elsevier Inc. All rights reserved.)

Lysine acetylation is an important posttranslational modification (PTM) that regulates the function of proteins by affecting their localization, stability, binding, and enzymatic activity. Aberrant acetylation patterns have been observed in numerous diseases, most notably cancer, which has spurred the development of potential therapeutics that target acetylation pathways. Mass spectrometry (MS) has become the most adopted tool not only for the qualitative identification of acetylation sites but also for their large-scale quantification. By using heavy isotope labeling in cell culture combined with MS, it is now possible to accurately quantify newly synthesized acetyl groups and other PTMs, allowing differentiation between dynamically regulated and steady-state modifications. Here, we describe MS-based protocols to identify acetylation sites and quantify acetylation rates on both proteins in general and in the special case of histones. In the experimental approach for the former, 13 C-glucose and D 3 -acetate are used to metabolically label protein acetylation in cells with stable isotopes, thus allowing isotope incorporation to be tracked over time. After protein extraction and digestion, acetylated peptides are enriched via immunoprecipitation and then analyzed by MS. For histones, a similar metabolic labeling approach is performed, followed by acid extraction, derivatization with propionic anhydride, and trypsin digestion prior to MS analysis. The procedures presented may be adapted to investigate acetylation dynamics in a broad range of experimental contexts, including different cell types and stimulation conditions.

Acetate is a major nutrient that supports acetyl-coenzyme A (Ac-CoA) metabolism and thus lipogenesis and protein acetylation. However, its source is unclear. Here, we report that pyruvate, the end product of glycolysis and key node in central carbon metabolism, quantitatively generates acetate in mammals. This phenomenon becomes more pronounced in the context of nutritional excess, such as during hyperactive glucose metabolism. Conversion of pyruvate to acetate occurs through two mechanisms: (1) coupling to reactive oxygen species (ROS) and (2) neomorphic enzyme activity from keto acid dehydrogenases that enable function as pyruvate decarboxylases. Further, we demonstrate that de novo acetate production sustains Ac-CoA pools and cell proliferation in limited metabolic environments, such as during mitochondrial dysfunction or ATP citrate lyase (ACLY) deficiency. By virtue of de novo acetate production being coupled to mitochondrial metabolism, there are numerous possible regulatory mechanisms and links to pathophysiology., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Millions of neurons drive the activity of hundreds of muscles, meaning many different neural population activity patterns could generate the same movement. Studies have suggested that these redundant (i.e. behaviorally equivalent) activity patterns may be beneficial for neural computation. However, it is unknown what constraints may limit the selection of different redundant activity patterns. We leveraged a brain-computer interface, allowing us to define precisely which neural activity patterns were redundant. Rhesus monkeys made cursor movements by modulating neural activity in primary motor cortex. We attempted to predict the observed distribution of redundant neural activity. Principles inspired by work on muscular redundancy did not accurately predict these distributions. Surprisingly, the distributions of redundant neural activity and task-relevant activity were coupled, which enabled accurate predictions of the distributions of redundant activity. This suggests limits on the extent to which redundancy may be exploited by the brain for computation., Competing Interests: JH, MG, PL, PS, EO, KQ, SR, ET, AB, BY, SC No competing interests declared, (© 2018, Hennig et al.)

The histone H3 variant H3.3 is a highly conserved and dynamic regulator of chromatin organization. Therefore, fully elucidating its nucleosome incorporation mechanisms is essential to understanding its functions in epigenetic inheritance. We previously identified the RNase P protein subunit, Rpp29, as a repressor of H3.3 chromatin assembly. Here, we use a biochemical assay to show that Rpp29 interacts with H3.3 through a sequence element in its own N terminus, and we identify a novel interaction with histone H2B at an adjacent site. The fact that archaeal Rpp29 does not include this N-terminal region suggests that it evolved to regulate eukaryote-specific functions. Oncogenic H3.3 mutations alter the H3.3-Rpp29 interaction, which suggests that they could dysregulate Rpp29 function in chromatin assembly. We also used KNS42 cells, an H3.3(G34V) pediatric high-grade glioma cell line, to show that Rpp29 1) represses H3.3 incorporation into transcriptionally active protein-coding, rRNA, and tRNA genes; 2) represses mRNA, protein expression, and antisense RNA; and 3) represses euchromatic post-translational modifications (PTMs) and promotes heterochromatic PTM deposition ( i.e. histone H3 Lys-9 trimethylation (H3K9me3) and H3.1/2/3K27me3). Notably, we also found that K27me2 is increased and K36me1 decreased on H3.3(G34V), which suggests that Gly-34 mutations dysregulate Lys-27 and Lys-36 methylation in cis The fact that Rpp29 represses H3.3 chromatin assembly and sense and antisense RNA and promotes H3K9me3 and H3K27me3 suggests that Rpp29 regulates H3.3-mediated epigenetic mechanisms by processing a transcribed signal that recruits H3.3 to its incorporation sites., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2018 Krishna Shastrula et al.)

The succession from aerobic and facultative anaerobic bacteria to obligate anaerobes in the infant gut along with the differences between the compositions of the mucosally adherent vs. luminal microbiota suggests that the gut microbes consume oxygen, which diffuses into the lumen from the intestinal tissue, maintaining the lumen in a deeply anaerobic state. Remarkably, measurements of luminal oxygen levels show nearly identical pO 2 (partial pressure of oxygen) profiles in conventional and germ-free mice, pointing to the existence of oxygen consumption mechanisms other than microbial respiration. In vitro experiments confirmed that the luminal contents of germ-free mice are able to chemically consume oxygen (e.g., via lipid oxidation reactions), although at rates significantly lower than those observed in the case of conventionally housed mice. For conventional mice, we also show that the taxonomic composition of the gut microbiota adherent to the gut mucosa and in the lumen throughout the length of the gut correlates with oxygen levels. At the same time, an increase in the biomass of the gut microbiota provides an explanation for the reduction of luminal oxygen in the distal vs. proximal gut. These results demonstrate how oxygen from the mammalian host is used by the gut microbiota, while both the microbes and the oxidative chemical reactions regulate luminal oxygen levels, shaping the composition of the microbial community throughout different regions of the gut., Competing Interests: The authors declare no conflict of interest.

Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example. Mounting evidence indicates that the modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation. Yet, a mechanistic understanding of how O-GlcNAc functions in T cell activation remains elusive, partly because of the difficulties in mapping and quantifying O-GlcNAc sites. Thus, to advance insight into the role of O-GlcNAc in T cell activation, we performed glycosite mapping studies via direct glycopeptide measurement on resting and activated primary human T cells with a technique termed Isotope Targeted Glycoproteomics. This approach led to the identification of 2219 intact O-linked glycopeptides across 1045 glycoproteins. A significant proportion (>45%) of the identified O-GlcNAc sites lie near or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk. Consistent with other studies, we find that O-GlcNAc sites in T cells lack a strict consensus sequence. To validate our results, we employed gel shift assays based on conjugating mass tags to O-GlcNAc groups. Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-GlcNAc glycosylation and higher levels of expression in activated T cells. Overall, our findings provide a quantitative characterization of O-GlcNAc glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein acetylation plays a critical role in biological processes by regulating the functions and properties of proteins. Thus, the study of protein acetylation dynamics is critical for understanding of how this modification influences protein stability, localization, and function. Here we performed a comprehensive characterization of protein acetylation dynamics using mass spectrometry (MS) based proteomics through utilization of 13 C-glucose or D 3 -acetate, which are metabolized into acetyl-coA, labeling acetyl groups through subsequent incorporation into proteins. Samples were collected at eight time points to monitor rates and trends of heavy acetyl incorporation. Through this platform, we characterized around 1,000 sites with significantly increasing acetylation trends, which we clustered based on their rates of acetylation. Faster rates were enriched on proteins associated with chromatin and RNA metabolism, while slower rates were more typical on proteins involved with lipid metabolism. Among others, we identified sites catalyzed at faster rates with potential critical roles in protein activation, including the histone acetyltransferase p300 acetylated in its activation loop, which could explain self-acetylation as an important feedback mechanism to regulate acetyltransferases. Overall, our studies highlight the dynamic nature of protein acetylation, and how metabolism plays a central role in this regulation.

T cell activation in response to Ag is largely regulated by protein posttranslational modifications. Although phosphorylation has been extensively characterized in T cells, much less is known about the glycosylation of serine/threonine residues by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate cell signaling pathways and protein activity similarly to phosphorylation, we performed a comprehensive analysis of O-GlcNAc during T cell activation to address the functional importance of this modification and to identify the modified proteins. Activation of T cells through the TCR resulted in a global elevation of O-GlcNAc levels and in the absence of O-GlcNAc, IL-2 production and proliferation were compromised. T cell activation also led to changes in the relative expression of O-GlcNAc transferase (OGT) isoforms and accumulation of OGT at the immunological synapse of murine T cells. Using a glycoproteomics approach, we identified >200 O-GlcNAc proteins in human T cells. Many of the identified proteins had a functional relationship to RNA metabolism, and consistent with a connection between O-GlcNAc and RNA, inhibition of OGT impaired nascent RNA synthesis upon T cell activation. Overall, our studies provide a global analysis of O-GlcNAc dynamics during T cell activation and the first characterization, to our knowledge, of the O-GlcNAc glycoproteome in human T cells., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Here we report a peptide-MHC (pMHC) dodecamer as a "next generation" technology that is a significantly more sensitive and versatile alternative to pMHC tetramers for the detection, isolation, and phenotypic analysis of antigen-specific T cells. In particular, dodecamers are able to detect two- to fivefold more antigen-specific T cells in both human and murine CD4(+)and CD8(+)αβ T-cell compartments compared with the equivalent tetramers. The low-affinity, tetramer-negative, dodecamer-positive T cells showed comparable effector cytokine responses as those of high-affinity, tetramer-positive T cells. Dodecamers are able to detect early stage CD4(+)CD8(+)double-positive thymocytes on which T-cell receptors are 10- to 30-fold less dense than mature T cells. Dodecamers also show utility in the analysis of γδ T cells and in cytometry by time-of-flight applications. This construct has a simple structure with a central scaffold protein linked to four streptavidin molecules, each having three pMHC ligands or other molecules. The dodecamer is straightforward and inexpensive to produce and is compatible with current tetramer technology and commercially available streptavidin conjugates.

Background: A new scaffold design combined with a peptide growth factor was tested prospectively for safety and for improved tendon healing in sheep., Methods: The infraspinatus tendon was detached and then surgically repaired to the humerus using sutures and anchors in 50 adult sheep. The repairs in 40 of these sheep were reinforced with a scaffold containing F2A, a peptide mimetic of basic fibroblast growth factor. The sheep were examined after 8 or 26 weeks with magnetic resonance imaging, full necropsy, and histopathologic analysis. A second cohort of 30 sheep underwent surgical repair--20 with scaffolds containing F2A. The 30 shoulders were tested mechanically after 8 weeks., Results: The scaffold and F2A showed no toxicity. Scaffold-repaired tendons were 31% thicker than surgically repaired controls (P = .037) at 8 weeks. There was more new bone formed at the tendon footprint in sheep treated with F2A. Surgically repaired tendons delaminated from the humerus across 14% of the footprint area. The extent of delamination decreased to 1.3% with increasing doses of F2A (P = .004). More of the repair tissue at the footprint was tendon-like in the peptide-treated sheep. On mechanical testing, only 7 shoulders tore at the repair site. The repairs in the other 23 shoulders were already stronger than the midsubstance tendon at 8 weeks., Conclusions: The new scaffold and peptide safely improved tendon healing., (Copyright © 2015 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

It has long been thought that clonal deletion efficiently removes almost all self-specific T cells from the peripheral repertoire. We found that self-peptide MHC-specific CD8(+) T cells in the blood of healthy humans were present in frequencies similar to those specific for non-self antigens. For the Y chromosome-encoded SMCY antigen, self-specific T cells exhibited only a 3-fold lower average frequency in males versus females and were anergic with respect to peptide activation, although this inhibition could be overcome by a stronger stimulus. We conclude that clonal deletion prunes but does not eliminate self-specific T cells and suggest that to do so would create holes in the repertoire that pathogens could readily exploit. In support of this hypothesis, we detected T cells specific for all 20 amino acid variants at the p5 position of a hepatitis C virus epitope in a random group of blood donors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To identify Toxoplasma gondii genes important in the establishment of a persistent infection, we previously used signature-tagged mutagenesis to identify mutants with reduced cyst numbers in the brains of mice. One of the mutants, 95C5, has an insertion within a predicted six transmembrane domain protein, which localizes to the parasite pellicle, thus we named it transmembrane pellicle protein 1 (TgTPP1). Although the 95C5 mutant was found be reduced in its ability to form brain cysts, it is defective during acute infection. Addition of TgTPP1 expressed from its endogenous promoter restored the acute lethality of the 95C5 mutant to parental levels. The 95C5 mutant does not have a growth defect in standard tissue culture conditions; however, we found a significant defect in host cell penetration after extracellular stress. Overall, TgTPP1 may function during acute infection by enhancing the parasites ability to invade after extracellular stress., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Global biodiversity loss and disease emergence are two of the most challenging issues confronting science and society. Recently, observed linkages between species-loss and vector-borne infections suggest that biodiversity may help reduce pathogenic infections in humans and wildlife, but the mechanisms underlying this relationship and its applicability to a broader range of pathogens have remained speculative. Here, we experimentally evaluated the effects of host community structure on transmission of the human pathogen, Schistosoma mansoni, which alternates between snail intermediate hosts and vertebrate definitive hosts. By manipulating parasite exposure and community diversity, we show that heterospecific communities cause a 25-50 per cent reduction in infection among snail hosts (Biomphalaria glabrata). Infected snails raised alongside non-host snails (Lymnaea or Helisoma sp.) also produced 60-80 per cent fewer cercariae, suggesting that diverse communities could reduce human infection risk. Because focal host density was held constant during experiments, decreases in transmission resulted entirely from diversity-mediated pathways. Finally, the decrease in infection in mixed-species communities led to an increase in reproductive output by hosts, representing a novel example of parasite-mediated facilitation. Our results underscore the significance of community structure on transmission of complex life-cycle pathogens, and we emphasize enhanced integration between ecological and parasitological research on the diversity-disease relationship.

The physician William Carlos Williams (1883-1963) was one of the most important poets in USA in the 20th century. This article provides some biographical data, examples of his poems and extracts from his autobiography, in which he discusses the relationship between medical practice and poetry.

Emil Aarestrup (1800-56) worked as a general physician, later as a public health officer in Denmark. He married his cousin and wrote love poems to her and to other women. His erotic poetry is regarded as the most elegant in Danish literature. His poems have precise words and colourful details and show a special sense of the condensed mood in the erotic situation. He writes upon the complex nature of love: union, separation and death.

In 1901 the Russian physician V.V. Veresajev published his book Zapiski vratsja (A doctor's notes). A Norwegian translation was out the next year. The book treats ethical problems in medicine and is critical of several aspects of medical practice. The article contains passages from the book, with comments.

Mikhail Bulgakov (1891-1940) studied at Kiev University and qualified in medicine in 1916. He worked as a doctor for a few years, then chose a literary career. All his life he was sceptical to the Soviet system and used his satire against the regime. Because of that he met difficulties as an author and could not publish anything after 1927. He worked on his main work, The Master and Margarita, from 1928 until his death. The novel was not published in his lifetime. In 1966-67 an abridged and censored edition appeared. Not until 1973 was a complete edition published in Moscow, and it soon became a cult book in Russia. In the major part of The Master and Margarita, the scene is in Moscow between the two world wars, with Bulgakov using his own experience. Through subtle irony and wild humour he exposes the imperfections of this society. To obtain this, he employs the Devil with his suite of strange figures. The book is also a love story between the Master and Margarita. The Master is writing a novel about Pontius Pilate, and this narrative runs parallel to the Moscow story, so that the book puts an eternal perspective on human frailty.

The Russian author and doctor Anton Chekhov (1860-1904) died from tuberculosis in 1904. He had his first haemoptysis in 1884. In spite of continuing symptoms he did not let himself be examined by colleagues until he had a severe haemorrhage in 1897. He was then hospitalized, and extensive bilateral pulmonary tuberculosis was diagnosed. Even after that, he did not take his disease seriously. His attitude has been taken as an example of a doctor's self-deception. Almost 4,500 of Chekhov's letters has been published. In this article, the letters and contemporary memorial literature are used to illustrate the development of his disease and his ambivalence towards it. His self-deception was not that massive; there is a strain of uneasiness in his reports of his symptoms.

Early experience using a new laser guidance device to assist CT-guided percutaneous musculoskeletal procedures is presented. We describe six cases, which demonstrate typical musculoskeletal applications of laser guidance. In our experience laser guidance for these procedures resulted in improved accuracy with no significant increase in biopsy time when a short learning period is considered. Other musculoskeletal procedures may benefit from laser guidance in preference to current standard CT-guided techniques, particularly when precision and accuracy are essential.

Anton Pavlovich Chekhov (1860-1904) was a doctor and a writer. He practised medicine in a rather sporadic manner throughout his life, and his main occupation was his authorship. His collected works comprise 581 items of a narrative type, 17 plays, a narrative and a treatise of his voyage to Sakhalin, a number of articles and about 4,000 letters. This paper describes the doctors who appear in 83 of his short stories and associated material, and shows how Chekhov's medical background influenced his writing.

An electrophysiological study was carried out in sixteen decerebrate and paralyzed New Zealand rabbits to determine how a bolus injection of a nociceptor stimulant (hypertonic saline, 5%) into the masseter muscle influences the activity of the trigeminal motor circuitry during fictive jaw movements. Hypodermic needles connected to a syringe held in a computer-controlled infusion pump were inserted into the anterior deep layer of either the right or the left masseter. Twenty-three infusions of 50, 70 or 80 microl saline were made in fourteen animals at constant rates over 1 min. Eight control infusions of normal saline (0.9%) were made in a subpopulation of five animals in an identical manner. Fictive jaw movements were evoked before and after the infusions by repetitive electrical stimulation of the corticobulbar tract. Effects were assessed by extracellular microelectrode recordings made from the digastric motoneuron pool and from putative last-order interneurons in the oral subnucleus of the spinal trigeminal tract and adjacent structures. In comparison with pre-infusion control cycles, nociceptor stimulation caused significant slowing of the rhythm and a reduction of the area of the digastric motoneuron bursts in the majority of the animals (12/14). The decrease in cycle frequency was due almost entirely to a lengthening of the time between the digastric bursts. Changes usually began 1-2 min after the infusion and returned to pre-infusion values within 10-15 min. No significant effects were seen when isotonic saline was applied. Recordings were obtained from nine interneurons, eight of which had low threshold mechanosensitive receptive fields. One neuron was, in addition, excited by pinch. Eight were not active in the absence of motor activity and this did not change when hypertonic saline was applied. However, once fictive movements began, all started to fire rhythmic bursts of spikes. In five cases, there was a significant post-infusion increase in spike frequency, and three showed decreases. Seven showed significant post-infusion changes in mean phase and/or concentration of their firing within the movement cycle. Changes in the preferred phase of interneuronal firing were significantly correlated to changes in the phase of offset of the digastric burst. The present results provide evidence that the stimulation of nociceptors in a muscle slows the frequency of rhythmical movements in the absence of sensory feedback. They confirm that infusions into one muscle affect the output of its antagonist. The results also suggest that neurons in the oral subnucleus of the spinal trigeminal tract and adjacent reticular formation appear to participate in programming these changes in motor output.

Early experience using a new laser guidance device to assist CT-guided percutaneous musculoskeletal procedures is presented. We describe six cases which demonstrate typical musculoskeletal applications of laser guidance. In our experience laser guidance for these procedures resulted in improved accuracy with no significant increase in biopsy time when a short learning period is considered. Other musculoskeletal procedures may benefit from laser guidance compared with current standard CT-guided techniques, particularly when precision and accuracy are essential.

Rationale and Objectives: Reader performance and image quality wee assessed for standard film, computed film, and computer monitor radiography viewing formats in the evaluation of skeletal extremity trauma., Materials and Methods: Three radiologists and three orthopedic surgeons interpreted 27 skeletal radiographs obtained with equivalent technical parameters. Readers evaluated standard film, computed film, and computer monitor formats randomly for fracture and soft-tissue abnormalities. Sessions were videotaped, and eye motion was recorded., Results: No statistically significant differences were found between image formats for true-positive or false-positive findings of trauma indicators. Findings were classified as false-negative based on eye position fixation times. Search errors (lesion not fixated) accounted for 21.7%, 20.6% and 17.1% of false-negative errors with the computer monitor, computed film, and standard film formats, respectively. Combined recognition errors and decision errors were 78.3%, 79.4%, and 82.9%, respectively. Viewing times were longest for the computer monitor images (P < .001). Image quality, contrast, and sharpness were rated highest for computed radiographs (P = .001). Radiologists had a higher true-positive decision rate than orthopedic surgeons (P = .03)., Conclusion: No statistically significant differences were seen in reader performance among viewing formats. The computed film format received the highest quality rating, and workstation viewing times were longest.

Four knee-scoring systems were used to evaluate 200 adult subjects who had no history of injury, abnormality, or treatment of the knees, hips, lower extremities, or spine. All subjects were in the age-range (fifty to 100 years; average, 65.5 years) typical of candidates for total knee replacement. In addition to a physical examination, complete demographic data were collected for each subject. The knee scores were normalized by dividing the observed score by the maximum possible score. The average normalized total knee score was 91 per cent (range, 22 to 100 per cent) according to the knee score of The Hospital for Special Surgery, 95 per cent (range, 10 to 100 per cent) according to the system of Hungerford and Kenna, 89 per cent (range, -7.75 to 100 per cent) according to a modification of the scoring system of The Knee Society, and 95 per cent (range, 26.5 to 100 per cent) according to the system of Hofmann et al. Demographic variables that had a significant negative correlation with the knee scores included advanced age (particularly of eighty-five years or more), a family income below the poverty level, and two major medical conditions or more. Observed differences in knee scores between different study groups that have not been matched for various clinically relevant factors are at least as likely to represent differences in the patient populations as they are to represent differences in the operative technique or the design of the implant.

Objective: To review imaging patterns and injury mechanisms in patients with thoracolumbar facet instability (TFI)., Design: Imaging studies for thoracolumbar osseous injuries over a 2-year period were reviewed. Imaging findings, injury pattern and reported mechanism of injury were established for patients with TFI., Patients: One hundred and ten patients with thoracolumbar acute, osseous injuries were studied., Results: Eleven of 68 (16.2%) unstable thoracolumbar injuries demonstrated TFI. Seven (64%) of the eleven TFI patients were unrestrained occupants in a motor vehicle accident (MVA) and the remainder were involved in injuries dominated by blunt impact to the back. Only two (18%) had serious, permanent neurological deficits., Conclusion: TFI is a common injury pattern, particularly for unrestrained occupants in MVAs. Characteristic radiographic, CT and MRI findings are presented and correlated with the injury mechanism and clinical findings.

We studied six adult male dogs to determine whether free patellar tendon grafts show evidence of reinnervation when used for anterior cruciate ligament reconstruction. Histologic return of neural elements and return of a somatosensory-evoked potential defined evidence of reinnervation. Before removal, the native anterior cruciate ligament was electrically stimulated with a bipolar electrode and a somatosensory-evoked potential was recorded from a scalp electrode. The ligament was excised and reconstructed using an autogenous patellar tendon graft. Somatosensory-evoked potential was attempted immediately after reconstruction. Histology for nerve endings was performed on the native ligaments. Each animal underwent repeat arthrotomy 6 months later. The grafts were isolated and somatosensory-evoked potentials were attempted. An evoked potential was seen in all six dogs before reconstruction. No graft demonstrated a somatosensory-evoked potential acutely; however, 6 months postoperatively, the somatosensory-evoked potential returned in two cases. Histology of native ligaments showed that 25% of the 100 sections evaluated contained neural elements. Of the receptors present, 89% were mechanoreceptors and 11% were free nerve endings. Histologic examination of the graft tissue 6 months postoperatively revealed that all six grafts also contained neural elements. Mechanoreceptors and free nerve endings were present in approximately equal numbers in the grafts. The results of histology and somatosensory-evoked potential demonstrate that in at least some cases, free patellar tendon grafts show evidence of reinnervation when used for anterior cruciate ligament reconstruction.

Rationale and Objectives: The authors evaluated how observers search hard-copy versus soft-copy images to determine why viewing times are longer for images displayed on a monitor., Methods: Twenty-seven nonconsecutive bone-trauma computed radiographs were collected from the routine emergency practice. Eye positions of three bone radiologists and three orthopedic surgeons were recorded as they searched images on a view box and digital images at a workstation., Results: Overall viewing time was longer for images displayed on a monitor. Time to first fixate a lesion and true-negative dwell times were significantly longer with the monitor than with the film. Absolute numbers of clusters and dwell times were greater for diagnostic image areas on the monitor than on the film. Twenty percent of the clusters for images viewed on the monitor were on the image-processing menu., Conclusion: The amount and type of information that is processed during search is different when images are viewed on a monitor rather than on film.

Objective: The authors assessed the MRI findings of appendicular coccidioidal arthritis., Design: T1- and T2-weighted MR images of affected joints, both with and without intravenous gadopentetate dimeglumine, were performed in nine adult patients (ten studies) and evaluated by three masted readers, using a four-point certainty scale for: synovial abnormality, articular cartilage loss, subarticular bone loss, abnormal marrow signal, enhancement of osseous and articular structures, and assessment of disease activity. Findings were correlated with biopsy results or clinical course., Results: Eight patients had active and one had inactive arthritis, involving the knee (five patients), ankle (two patients), and elbow (one patient). Synovial complex was the most common finding in active arthritis (P < 0.025). Cartilage and subarticular bone loss were seen 56% and 89% of patients with active disease, respectively. Abnormal marrow signal was uncommon (two patients). All cases showed synovial and/or osseus enhancement., Conclusions: MRI findings in coccidiodal arthritis are described. Enhancement of thickened synovium and erosions was seen after intravenous gadopentetate.

Four hip scoring systems were used in evaluating 200 adult subjects who had no prior history of injury, pathologic condition, or treatment of the hips, knees, lower extremities, or spine. All subjects were in the age range typical of a total hip arthroplasty candidate (average age, 65 years; range, 50-100 years). In addition to a physical examination, complete demographic data were collected on each subject. Data were recorded on standardized flow sheets so that hip scores could be calculated; scores were normalized by dividing the observed scores by the maximum possible score. The average normalized total hip scores were Harris hip score, 90.8%; modified Harris hip score, 91.9%; Merle D'Aubigne score, 93.9%; and Hospital for Special Surgery hip rating 87.5%. Demographic variables that had a significant negative correlation with hip scores included advanced age (particularly past age 85), an income below the poverty level, and the presence of two or more major medical conditions. Differences in hip scores between different study groups that have not been matched for various clinically relevant factors ("case mix") are at least as likely to represent differences in the patient populations as differences in surgical technique or implant design. Hip scores may decline over the course of a 10- to 20-year follow-up period due to the change in a patient's age and/or medical condition rather than any factor relating to the hip arthroplasty.