Histone H3 monoaminylations at Gln5 represent an important family of epigenetic marks in brain that have critical roles in permissive gene expression1-3. We previously demonstrated that serotonylation4-10 and dopaminylation9,11-13 of Gln5 of histone H3 (H3Q5ser and H3Q5dop, respectively) are catalysed by transglutaminase 2 (TG2), and alter both local and global chromatin states. Here we found that TG2 additionally functions as an eraser and exchanger of H3 monoaminylations, including H3Q5 histaminylation (H3Q5his), which displays diurnally rhythmic expression in brain and contributes to circadian gene expression and behaviour. We found that H3Q5his, in contrast to H3Q5ser, inhibits the binding of WDR5, a core member of histone H3 Lys4 (H3K4) methyltransferase complexes, thereby antagonizing methyltransferase activities on H3K4. Taken together, these data elucidate a mechanism through which a single chromatin regulatory enzyme has the ability to sense chemical microenvironments to affect the epigenetic states of cells, the dynamics of which have critical roles in the regulation of neural rhythmicity.

Long COVID is a major public health consequence of COVID-19 and is characterized by multiple neurological and neuropsychatric symptoms. SARS-CoV-2 antigens (e.g., spike S1 subunit) are found in the circulation of Long COVID patients, have been detected in post-mortem brain of COVID patients, and exhibit neuroinflammatory properties. Considering recent observations of chronic neuroinflammation in Long COVID patients, the present study explores the idea that antigens derived from SARS-CoV-2 might produce a long-term priming or sensitization of neuroinflammatory processes, thereby potentiating the magnitude and/or duration of the neuroinflammatory response to future inflammatory insults. Rats were administered S1 or vehicle intra-cisterna magna and 7d later challenged with vehicle or LPS. The neuroinflammatory, physiological, and behavioral responses to LPS were measured at various time points post-LPS. We found that prior S1 treatment potentiated many of these responses to LPS suggesting that S1 produces a protracted priming of these processes. Further, S1 produced a protracted reduction in basal brain corticosteroids. Considering the anti-inflammatory properties of corticosteroids, these findings suggest that S1 might disinhibit innate immune processes in brain by reducing anti-inflammatory drive, thereby priming neuroinflammatory processes. Given that hypocortisolism is observed in Long COVID, we propose that similar S1-induced innate immune priming processes might play role in the pathophysiology of Long COVID.

Protein monoaminylation is a class of posttranslational modification (PTM) that contributes to transcription, physiology and behavior. While recent analyses have focused on histones as critical substrates of monoaminylation, the broader repertoire of monoaminylated proteins in brain remains unclear. Here, we report the development/implementation of a chemical probe for the bioorthogonal labeling, enrichment and proteomics-based detection of dopaminylated proteins in brain. We identified 1,557 dopaminylated proteins - many synaptic - including γCaMKII, which mediates Ca2+-dependent cellular signaling and hippocampal-dependent memory. We found that γCaMKII dopaminylation is largely synaptic and mediates synaptic-to-nuclear signaling, neuronal gene expression and intrinsic excitability, and contextual memory. These results indicate a critical role for synaptic dopaminylation in adaptive brain plasticity, and may suggest roles for these phenomena in pathologies associated with altered monoaminergic signaling.

Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processes in vitro. In general, this strategy necessitates a protein folding step following ligation of the unstructured fragments, a requirement that constrains the types of systems amenable to the approach. Here, we report an in vitro strategy that allows internal regions of target proteins to be replaced in a single operation. Conceptually, our system is analogous to a DNA transposition reaction, but employs orthogonal pairs of split inteins to swap out a designated region of a host protein with an exogenous molecular cassette. We show using isotopic labeling experiments that this 'protein transposition' reaction is concerted when the kinetics for the embedded intein pairs are suitably matched. Critically, this feature allows for efficient manipulation of protein primary structure in the context of a native fold. The utility of this method is illustrated using several protein systems including the multisubunit chromatin remodeling complex, ACF, where we also show protein transposition can occur in situ within the cell nucleus. By carrying out a molecular 'cut and paste' on a protein or protein complex under native folding conditions, our approach dramatically expands the scope of protein semisynthesis.

Despite substantial evidence supporting the efficacy of prebiotics for promoting host health and stress resilience, few experiments present evidence documenting the dynamic changes in microbial ecology and fecal microbially modified metabolites over time. Furthermore, the literature reports a lack of reproducible effects of prebiotics on specific bacteria and bacterial-modified metabolites. The current experiments examined whether consumption of diets enriched in prebiotics (galactooligosaccharides (GOS) and polydextrose (PDX)), compared to a control diet, would consistently impact the gut microbiome and microbially modified bile acids over time and between two research sites. Male Sprague Dawley rats were fed control or prebiotic diets for several weeks, and their gut microbiomes and metabolomes were examined using 16S rRNA gene sequencing and untargeted LC–MS/MS analysis. Dietary prebiotics altered the beta diversity, relative abundance of bacterial genera, and microbially modified bile acids over time. PICRUSt2 analyses identified four inferred functional metabolic pathways modified by the prebiotic diet. Correlational network analyses between inferred metabolic pathways and microbially modified bile acids revealed deoxycholic acid as a potential network hub. All these reported effects were consistent between the two research sites, supporting the conclusion that dietary prebiotics robustly changed the gut microbial ecosystem. Consistent with our previous work demonstrating that GOS/PDX reduces the negative impacts of stressor exposure, we propose that ingesting a diet enriched in prebiotics facilitates the development of a health-promoting gut microbial ecosystem.

Thoracic outlet syndrome (TOS) is a controversial and uncommon syndrome. Three different diagnoses can be made based on the compressed structure: arterial TOS, venous TOS, and neurogenic TOS. Diagnosing TOS, especially neurogenic TOS, remains difficult since a single diagnostic tool does not exist. Although this resulted in a lot of confusion, standardization of care and outcome improved daily care practice measures in the last decade. Current treatment algorithms consist of both conservative and surgical treatment approaches, which should be chosen depending on the type of TOS and extend of the complaints. Surgical treatment of TOS is performed via thoracic outlet decompression (TOD). TOD surgery includes complete resection of the first rib (cartilage to cartilage), transection of the scalene muscles and complete neurolysis/venolysis or arteriolysis. Four different approaches can be chosen for TOD surgery: the transaxillary (TA), supraclavicular (SC), paraclavicular (PC), and infraclavicular (IC) approach. The TA, SC, and PC approach can be used for every form of TOS. However, the PC approach is mostly used for treating venous TOS. The IC approach has no role in treating neurogenic or arterial TOS and is only used for venous TOS. Every approach has its own benefits and limitations and literature does not agree on what approach is best. Therefore, the used surgical approach for TOD should be based on the surgeon's preference and experience. The aim of this review is to present an overview of the diagnostic pathway and provide an in-depth description of the surgical approach in each form of TOS.

Introduction: Cannabidiol (CBD) extract from the cannabis plant has biomedical and nutraceutical potential. Unlike tetrahydrocannabinol (THC), CBD products produce few psychoactive effects and pose little risk for abuse. There is emerging preclinical and clinical evidence that CBD is stress modulatory and may have anti-inflammatory properties. People across the United States legally ingest CBD-rich hemp extracts to manage mental and physical health problems, including stress and inflammation. Preclinical studies have revealed potential mechanisms for these effects; however, the impact of this prior work is diminished because many studies: 1) tested synthetic CBD rather than CBD-rich hemp extracts containing terpenes and/or other cannabinoids thought to enhance therapeutic benefits; 2) administered CBD via injection into the peritoneal cavity or the brain instead of oral ingestion; and 3) failed to examine potential sex differences. To address these gaps in the literature, the following study tested the hypothesis that the voluntary oral ingestion of CBD-rich hemp extract will attenuate the impact of stressor exposure on plasma and tissue inflammatory and stress proteins in females and males.Methods: Adult male and female Sprague Dawley rats (10–15/group) were randomly assigned to be given cereal coated with either vehicle (coconut oil) or CBD-rich hemp extract (L-M0717, CBDrx/Functional Remedies, 20.0 mg/kg). After 7 days, rats were exposed to a well-established acute model of stress (100, 1.5 mA, 5-s, intermittent tail shocks, 90 min total duration) or remained in home cages as non-stressed controls.Results: Stressor exposure induced a robust stress response, i.e., increased plasma corticosterone and blood glucose, and decreased spleen weight (a surrogate measure of sympathetic nervous system activation). Overall, stress-induced increases in inflammatory and stress proteins were lower in females than males, and oral CBD-rich hemp extract constrained these responses in adipose tissue (AT) and mesenteric lymph nodes (MLN). Consistent with previous reports, females had higher levels of stress-evoked corticosterone compared to males, which may have contributed to the constrained inflammatory response measured in females.Discussion: Results from this study suggest that features of the acute stress response are impacted by oral ingestion of CBD-rich hemp extract in female and male rats, and the pattern of changes may be sex and tissue dependent.

The MAX phases are a group of ternary carbides and nitrides with potential for use in advanced high temperature applications. Numerous studies have shown their main deformation mechanism to be basal plane slip, even in extreme orientations, yet the fundamentals of this mechanism and dependencies on size and applied stress state remain inconclusive. Based on similar studies in Ti3SiC2, Ti3AlC2 and Ti2AlC, the current work investigated the onset of basal plane slip as a function of loading orientation by compressing single crystal micropillars of Cr2AlC. The results suggest clear changes in the critical resolved shear stress with loading orientation (non-Schmid effects), and attempts were made to rationalize this behavior by comparison with models of dislocation activity. On this basis, it is proposed that external influences on dislocation mobility are likely the governing factor in the observed non-Schmid effects in the MAX phases.

Diurnal rhythms of physiology and behavior are driven by both circadian and non-circadian mechanism. Those effects that are circadian occur in the absence of photic or non-photic diurnal cues and are generated by a core set of clock genes that regulate their own expression in an approximately 24-hour transcriptional-translational feedback loop. In stark contrast, rats exposed to stress have a dampened diurnal rhythm due to an elevation in the trough body temperature during light. Research in humans and animals indicates that the experience of choice can itself improve mental health and produce stress resistance. Diurnal rhythms of physiology and behavior are driven by both circadian and non-circadian mechanism. Those effects that are circadian occur in the absence of photic or non-photic diurnal cues and are generated by a core set of clock genes that regulate their own expression in an approximately 24-hour transcriptional-translational feedback loop.

The frontal pole (Brodmann area 10, BA10) is the largest cytoarchitectonic region of the human cortex, performing complex integrative functions. BA10 undergoes intensive adolescent grey matter pruning prior to the age of onset for bipolar disorder (BP) and schizophrenia (SCHIZ), and its dysfunction is likely to underly aspects of their shared symptomology. In this study, we investigated the role of BA10 neurotransmission-related gene expression in BP and SCHIZ. We performed qPCR to measure the expression of 115 neurotransmission-related targets in control, BP, and SCHIZ postmortem samples ( n = 72). We chose this method for its high sensitivity to detect low-level expression. We then strengthened our findings by performing a meta-analysis of publicly released BA10 microarray data ( n = 101) and identified sources of convergence with our qPCR results. To improve interpretation, we leveraged the unusually large database of clinical metadata accompanying our samples to explore the relationship between BA10 gene expression, therapeutics, substances of abuse, and symptom profiles, and validated these findings with publicly available datasets. Using these convergent sources of evidence, we identified 20 neurotransmission-related genes that were differentially expressed in BP and SCHIZ in BA10. These results included a large diagnosis-related decrease in two important therapeutic targets with low levels of expression, HTR2B and DRD4, as well as other findings related to dopaminergic, GABAergic and astrocytic function. We also observed that therapeutics may produce a differential expression that opposes diagnosis effects. In contrast, substances of abuse showed similar effects on BA10 gene expression as BP and SCHIZ, potentially amplifying diagnosis-related dysregulation.

ABSTRACT Histone H3 monoaminylations at glutamine(Q) 5 represent an important family of epigenetic markers in neurons that play critical roles in the mediation of permissive gene expression (1, 2). We previously demonstrated that H3Q5 serotonylation(ser) and dopaminylation(dop) are catalyzed by the Transglutaminase 2 (TGM2) enzyme and alter both local and global chromatin states (3, 4). Here, we found that TGM2 additionally functions as an “eraser” of H3 monoaminylations that is capable of “re-writing” these epigenetic marks in cells, including a new class of this modification, H3Q5 histaminylation(his), which displays dynamic diurnal expression in brain and contributes to neural rhythmicity. We found that H3Q5his inhibits binding of the MLL1 complex to the H3 N-terminus and attenuates its methyltransferase activity on H3 lysine(K) 4. We determined that H3Q5 monoaminylation dynamics are dictated by local monoamine concentrations, which are utilized by TGM2. Taken together, we present here a novel mechanism through which a single chromatin regulatory enzyme is capable of sensing chemical microenvironments to affect the epigenetic states of cells. One sentence summary A single enzyme, TGM2, bidirectionally controls H3 monoaminylation dynamics, which, in turn, facilitate neural rhythmicity.

Sleep disruption is a challenging and exceedingly common physiological state that contributes to a wide range of biochemical and molecular perturbations and has been linked to numerous adverse health outcomes. Modern society exerts significant pressure on the sleep/wake cycle via myriad factors, including exposure to electric light, psychological stressors, technological interconnection, jet travel, shift work, and widespread use of sleep-affecting compounds. Interestingly, recent research has identified a link between the microbiome and the regulation of sleep, suggesting that interventions targeting the microbiome may offer unique therapeutic approaches to challenges posed by sleep disruption. In this study, we test the hypothesis that administration of a prebiotic diet containing galactooligosaccharides (GOS) and polydextrose (PDX) in adult male rats improves sleep in response to repeated sleep disruption and during recovery sleep. We found that animals fed the GOS/PDX prebiotic diet for 4 weeks exhibit increased non-rapid eye movement (NREM) and rapid eye movement (REM) sleep during 5 days of sleep disruption and increased total sleep time during 24 h of recovery from sleep disruption compared to animals fed a control diet, despite similar baseline sleep characteristics. Further, the GOS/PDX prebiotic diet led to significant changes in the fecal microbiome. Consistent with previous reports, the prebiotic diet increased the relative abundance of the species Parabacteroides distasonis, which positively correlated with sleep parameters during recovery sleep. Taken together, these findings suggest that the GOS/PDX prebiotic diet may offer an approach to improve resilience to the physiologic challenge of sleep disruption, in part through impacts on the microbiome.

BACKGROUND:The clinical outcomes of reoperations for recurrent neurogenic thoracic outlet syndrome (NTOS) remain undefined.METHODS:From 2009 to 2019, 90 patients with recurrent NTOS underwent anatomically complete supraclavicular reoperation after previous operation(s) performed at other institutions using either supraclavicular (Prev-SC = 48), transaxillary (Prev-TA = 31), or multiple/combination (Prev-MC = 11) approaches. Prospectively maintained data were analyzed retrospectively.RESULTS:The mean patient age was 39.9 ± 1.4 years, 72% were female, and the mean interval after previous operation was 4.1 ± 0.6 years. The mean Disabilities of the Arm, Shoulder, and Hand (QuickDASH) score was 62 ± 2, reflecting substantial preoperative disability. Residual scalene muscle was present in 100% Prev-TA, 79% Prev-SC, and 55% Prev-MC (P < .05). Retained/residual first rib was present in 90% Prev-TA, 75% Prev-SC, and 55% Prev-MC (P < .05). There were no differences in operative time (overall 210 ± 5 minutes), length of hospital stay (4.7 ± 0.2 days), or 30-day readmissions (7%). During follow-up of 5.6 ± 0.3 years, the improvement in QuickDASH scores was 21 ± 2 (36% ± 3%) (P < .01) and patient-rated outcomes were excellent in 10%, good in 36%, fair in 43%, and poor in 11%.CONCLUSIONS:Anatomically complete decompression for recurrent NTOS can be safely and effectively accomplished by supraclavicular reoperation, regardless of the type of previous operation. Residual scalene muscle and retained/residual first rib are more frequently encountered after transaxillary operations than after supraclavicular or multiple/combined operations. Supraclavicular reoperation can achieve significant symptom reduction and functional improvement for approximately 90% of patients with recurrent NTOS.

Nucleosomes frequently exist as asymmetric species in native chromatin contexts. Current methods for the traceless generation of these heterotypic chromatin substrates are inefficient and/or difficult to implement. Here, we report an application of the SpyCatcher/SpyTag system as a convenient route to assemble desymmetrized nucleoprotein complexes. This genetically encoded covalent tethering system serves as an internal chaperone, maintained through the assembly process, affording traceless asymmetric nucleosomes following proteolytic removal of the tethers. The strategy allows for generation of nucleosomes containing asymmetric modifications on single or multiple histones, thereby providing facile access to a range of substrates. Herein, we use such constructs to interrogate how nucleosome desymmetrization caused by the incorporation of cancer-associated histone mutations alters chromatin remodeling processes. We also establish that our system provides access to asymmetric dinucleosomes, which allowed us to query the geometric/symmetry constraints of the unmodified histone H3 tail in stimulating the activity of the histone lysine demethylase, KDM5B. By providing a streamlined approach to generate these sophisticated substrates, our method expands the chemical biology toolbox available for interrogating the consequences of asymmetry on chromatin structure and function.

Objectives: Determine the performance and return to play (RTP) outcomes for professional baseball players that undergo surgical treatment for thoracic outlet syndrome. Methods: All professional baseball players who underwent surgical treatment of TOS between 2010-2017 were identified using the major league baseball (MLB) HITS (Health and Injury Tracking System) database. Demographic and performance data (pre and post-surgery) for each player was recorded. Standard demographic and return to play outcomes were measured for all players and compared between different types of TOS and league status. Performance variables were analyzed for pitchers and compared to a 1:1 matched set of control pitchers. Results: Overall, 52 players underwent surgery for TOS of which 46 (88%) were pitchers. Neurogenic TOS was the most common and seen in 69% of players. Venous TOS was seen in 29% of players, and a single player had arterial TOS. Following TOS surgery, 79% of players returned to play at the same level (RTSP) and played at least 3 years after surgery. There was no differences in RTSP rate based on the type of TOS. There was no statistically significant difference in RTP and RTSP rates between major and minor league players. In pitchers only, there was a 76% RTSP, which was not different from the natural attrition of control pitchers (p=0.874). Following TOS surgery, pitchers saw a decline in several performance metrics, but not statistically different when compared to the control pitchers. This indicated that the decline in performance following TOS surgery was similar to the decline seen in healthy professional pitchers as they age. Conclusions: The most common type of TOS seen in professional baseball players is neurogenic. The RTSP rate following surgery for TOS in all professional baseball players is 79%. There was no statistically significant difference in ability to RTP based on the type of TOS or league status. Pitchers who underwent surgery for TOS had no significant difference in pitching performance metrics following surgery compared to matched controls. Overall, surgery for thoracic outlet syndrome can help prolong a professional baseball players career and help pitchers maintain their performance over time similar to their peers who have not had surgery for TOS.

OBJECTIVES:The objective of this retrospective analysis of prospectively collected data was to assess the test-retest reliability and validity of the elevated arm stress test (EAST) as measured by the duration in a cohort of patients with suspected neurogenic thoracic outlet syndrome (NTOS).METHODS:Patients evaluated for NTOS between January 2017 and September 2018 were identified. Test-retest reliability by the intraclass correlation coefficient was determined for duration of the EAST. For the validity analysis, patients were classified in a proven NTOS group or a symptomatic control group without NTOS using the Society for Vascular Surgery reporting standards and the outcome of thoracic outlet decompression surgery. A receiver operating characteristic curve was made for the duration of EAST. The area under the curve, and positive and negative predictive values were calculated for the EAST.RESULTS:In total, 428 patients with suspected NTOS were retrospectively analyzed. Of these patients, 61 were excluded because no EAST data was available. Another 101 patients were excluded because of inconclusive reporting standards, arterial or venous TOS, or because thoracic outlet decompression surgery was not performed or had a negative result. The validity analysis in the remaining 266 patients showed an area under the curve for the duration of the EAST of 0.62 (95% confidence interval, 0.55-0.69). The positive predictive value of the duration ranged between 65% and 66%, and the negative predictive value between 53% and 58%. For the test-retest reliability analysis, 118 patients were excluded because they performed only one measurement in a 100-day time period. Analysis in the remaining 148 patients showed an intraclass correlation coefficient value of 0.65 (95% confidence interval, 0.55-0.74) for duration.CONCLUSIONS:The EAST measured by the duration showed a moderate test-retest reliability, but the discriminative value was low in the diagnosis of NTOS. The outcome of the EAST measured by the duration should be used with caution.

Prenatal testosterone (T)-treated female sheep manifest peripheral insulin resistance, ectopic lipid accumulation, and insulin signaling disruption in liver and muscle. This study investigated transcriptional changes and transcriptome signature of prenatal T excess-induced hepatic and muscle-specific metabolic disruptions. Genome-wide coding and noncoding (nc) RNA expression in liver and muscle from 21-month-old prenatal T-treated (T propionate 100 mg intramuscular twice weekly from days 30-90 of gestation; term: 147 days) and control females were compared. Prenatal T (1) induced differential expression of messenger RNAs (mRNAs) in liver (15 down, 17 up) and muscle (66 down, 176 up) (false discovery rate < 0.05, absolute log2 fold change > 0.5); (2) downregulated mitochondrial pathway genes in liver and muscle; (3) downregulated hepatic lipid catabolism and peroxisome proliferator-activated receptor (PPAR) signaling gene pathways; (4) modulated noncoding RNA (ncRNA) metabolic processes gene pathway in muscle; and (5) downregulated 5 uncharacterized long noncoding RNA (lncRNA) in the muscle but no ncRNA changes in the liver. Correlation analysis showed downregulation of lncRNAs LOC114112974 and LOC105607806 was associated with decreased TPK1, and LOC114113790 with increased ZNF470 expression. Orthogonal projections to latent structures discriminant analysis identified mRNAs HADHA and SLC25A45, and microRNAs MIR154A, MIR25, and MIR487B in the liver and ARIH1 and ITCH and miRNAs MIR369, MIR10A, and MIR10B in muscle as potential biomarkers of prenatal T excess. These findings suggest downregulation of mitochondria, lipid catabolism, and PPAR signaling genes in the liver and dysregulation of mitochondrial and ncRNA gene pathways in muscle are contributors of lipotoxic and insulin-resistant hepatic and muscle phenotype. Gestational T excess programming of metabolic dysfunctions involve tissue-specific ncRNA-modulated transcriptional changes.

Serotonylation of glutamine 5 on histone H3 (H3Q5ser) was recently identified as a permissive posttranslational modification that coexists with adjacent lysine 4 trimethylation (H3K4me3). While the resulting dual modification, H3K4me3Q5ser, is enriched at regions of active gene expression in serotonergic neurons, the molecular outcome underlying H3K4me3-H3Q5ser crosstalk remains largely unexplored. Herein, we examine the impact of H3Q5ser on the readers, writers, and erasers of H3K4me3. All tested H3K4me3 readers retain binding to the H3K4me3Q5ser dual modification. Of note, the PHD finger of TAF3 favors H3K4me3Q5ser, and this binding preference is dependent on the Q5ser modification regardless of H3K4 methylation states. While the activity of the H3K4 methyltransferase, MLL1, is unaffected by H3Q5ser, the corresponding H3K4me3/2 erasers, KDM5B/C and LSD1, are profoundly inhibited by the presence of the mark. Collectively, this work suggests that adjacent H3Q5ser potentiates H3K4me3 function by either stabilizing H3K4me3 from dynamic turnover or enhancing its physical readout by downstream effectors, thereby potentially providing a mechanism for fine-tuning critical gene expression programs.