N-to-S Acyl Transfer as an Enabling Strategy in Asymmetric and Chemoenzymatic Synthesis.

The observation of thioester-mediated acyl transfer processes in nature has inspired the development of novel protein synthesis and functionalization methodologies. The chemoselective transfer of an acyl group from S-to-N is the basis of several powerful ligation strategies. In this work, we sought to apply the reverse process, the transfer of an acyl group from N-to-S, as a method to convert stable chiral amides into more reactive thioesters. To this end, we developed a novel cysteine-derived oxazolidinone that serves as both a chiral imide auxiliary and an acyl transfer agent. This auxiliary combines the desirable features of rigid chiral imides as templates for asymmetric transformations with the synthetic applicability of thioesters. We demonstrate that the auxiliary can be applied in a range of highly selective asymmetric transformations. Subsequent intramolecular N-to-S acyl transfer of the chiral product and in situ trapping of the resulting thioester provides access to diverse carboxylic acid derivatives under mild conditions. The oxazolidinone thioester products can also be isolated and used in Pd-mediated transformations to furnish highly valuable chiral scaffolds, such as noncanonical amino acids, cyclic ketones, tetrahydropyrones, and dihydroquinolinones. Finally, we demonstrate that the oxazolidinone thioesters can also serve as a surrogate for SNAC-thioesters, enabling their seamless use as non-native substrates in biocatalytic transformations.

Sleepy without stimulation: subjective and objective sleepiness in actigraphy-verified natural short sleepers.

Natural short sleepers (NSS)-individuals who report minimal sleepiness or daytime dysfunction despite habitually sleeping less than the recommended amount (i.e., <7 h)-are a focus of growing interest in sleep research. Yet, the predominance of research on NSS has relied on subjective reports of functionality. The present study examined subjective and objective sleepiness among actigraphy-verified NSS in comparison with recommended (7-9 h/day) length sleepers (RLS) who reported similarly minimal daytime dysfunction. The study tested the hypothesis that under conditions of low environmental stimulation, NSS have increased risk of drowsiness and sleep onset, regardless of perceived alertness. The NSS and RLS groups were identified via screening and verified with a 14 day assessment with actigraphy, sleep diaries, and morning ratings of sleep restoration. In-laboratory resting electroencephalography (EEG) data were analysed using a computerised EEG-based algorithm (Vigilance Algorithm Leipzig; VIGALL) to classify second-by-second changes in objective sleepiness ranging from cognitively active alertness to sleep onset. Results demonstrated that NSS exhibited significantly higher drowsiness and sleep onset ('microsleeps') across 15 min of resting EEG despite perceptions of lower subjective sleepiness compared to RLS. Findings suggest that irrespective of perceived sleep restoration and alertness, NSS appear to be at high risk of objective sleepiness that is rapidly unmasked under conditions of low environmental stimulation. Such apparent discrepancy between subjective and objective sleepiness has potentially important public health implications. Future research directions, including tests of mechanisms and tailored sleep extension intervention, are discussed.

Evolutionarily related host and microbial pathways regulate fat desaturation in C. elegans.

Fatty acid desaturation is central to metazoan lipid metabolism and provides building blocks of membrane lipids and precursors of diverse signaling molecules. Nutritional conditions and associated microbiota regulate desaturase expression, but the underlying mechanisms have remained unclear. Here, we show that endogenous and microbiota-dependent small molecule signals promote lipid desaturation via the nuclear receptor NHR-49/PPARα in C. elegans. Untargeted metabolomics of a ß-oxidation mutant, acdh-11, in which expression of the stearoyl-CoA desaturase FAT-7/SCD1 is constitutively increased, revealed accumulation of a ß-cyclopropyl fatty acid, becyp#1, that potently activates fat-7 expression via NHR-49. Biosynthesis of becyp#1 is strictly dependent on expression of cyclopropane synthase by associated bacteria, e.g., E. coli. Screening for structurally related endogenous metabolites revealed a ß-methyl fatty acid, bemeth#1, which mimics the activity of microbiota-dependent becyp#1 but is derived from a methyltransferase, fcmt-1, that is conserved across Nematoda and likely originates from bacterial cyclopropane synthase via ancient horizontal gene transfer. Activation of fat-7 expression by these structurally similar metabolites is controlled by distinct mechanisms, as microbiota-dependent becyp#1 is metabolized by a dedicated ß-oxidation pathway, while the endogenous bemeth#1 is metabolized via α-oxidation. Collectively, we demonstrate that evolutionarily related biosynthetic pathways in metazoan host and associated microbiota converge on NHR-49/PPARα to regulate fat desaturation.

Posterolateral dislocation and incarceration of the long head of the biceps tendon: a rare cause of irreducible anterior glenohumeral joint dislocation-a case report and brief review of the literature.

Failure of closed reduction of anterior glenohumeral joint dislocation is infrequent. It can be secondary to osseous (e.g., fracture fragments or Hill-Sachs lesion) or soft tissue (e.g., labrum or rotator cuff tendon) impediments. Herein, we present a case of a prolonged irreducible glenohumeral joint secondary to a posterolaterally dislocated and incarcerated long head of the biceps tendon after an episode of anterior instability, highlight the utility of MR imaging for guiding management, and review the literature of this sporadic diagnosis.

O-linked sialic acid residues on platelet membrane glycoprotein IIb mask the human HPA-9b alloepitope.

Sialic acids occupy the terminal position of glycan chains and have the potential to influence the antigenicity of glycoproteins (GP). The polymorphisms of human platelet alloantigens (HPA)-3 and HPA-9, located near the C-terminus of the extracellular domain of platelet membrane GPIIb, are adjacent to sialyl-core 1 O-glycans emanating from serines 845 and 847. Whether the nearby O-glycans affect the antigenicity of HPA-9b or influence the binding of anti-HPA-9b alloantibodies in clinically significant cases of neonatal alloimmune thrombocytopenia is unknown. To address this issue, we generated a series of O-glycan mutant HPA-9 allele-specific induced pluripotent stem cell lines, differentiated them to megakaryocytes (MKs), and examined their ability to bind HPA-9b-specific alloantibodies. We found that both wild-type MKs treated with neuraminidase and those genetically modified to lack the sialidases ST3GAL1 and ST3GAL2 dramatically increased anti-HPA-9b alloantibody binding, indicating that the HPA-9b epitope is partially masked by terminal sialic acids on nearby O-glycans of GPIIb. Interestingly, mutating the serine residues that carry these glycan chains to alanine actually reduced the binding of anti-HPA-9b alloantibodies, indicating that these 2 O-glycan chains contribute to the presentation of the HPA-9b epitope-perhaps by stabilizing the conformation of the GP in this region. Collectively, our data suggest that detection of anti-HPA-9b alloantibodies may be enhanced through the use of HPA-9b-specific MKs that have been genetically altered to lack nearby terminal sialic acid residues but retain the glycan chains to which they are attached.

Evolutionarily related host and microbial pathways regulate fat desaturation.

Fatty acid desaturation is central to metazoan lipid metabolism and provides building blocks of membrane lipids and precursors of diverse signaling molecules. Nutritional conditions and associated microbiota regulate desaturase expression1-4, but the underlying mechanisms have remained unclear. Here, we show that endogenous and microbiota-dependent small molecule signals promote lipid desaturation via the nuclear receptor NHR-49/PPARα in C. elegans. Untargeted metabolomics of a ß-oxidation mutant, acdh-11, in which expression of the stearoyl-CoA desaturase FAT-7/SCD1 is constitutively increased, revealed accumulation of a ß-cyclopropyl fatty acid, becyp#1, that potently activates fat-7 expression via NHR-49. Biosynthesis of becyp#1 is strictly dependent on expression of cyclopropane synthase by associated bacteria, e.g., E. coli. Screening for structurally related endogenous metabolites revealed a ß-methyl fatty acid, bemeth#1, whose activity mimics that of microbiota-dependent becyp#1, but is derived from a methyltransferase, fcmt-1, that is conserved across Nematoda and likely originates from bacterial cyclopropane synthase via ancient horizontal gene transfer. Activation of fat-7 expression by these structurally similar metabolites is controlled by distinct mechanisms, as microbiota-dependent becyp#1 is metabolized by a dedicated ß-oxidation pathway, while the endogenous bemeth#1 is metabolized via α-oxidation. Collectively, we demonstrate that evolutionarily related biosynthetic pathways in metazoan host and associated microbiota converge on NHR-49/PPARα to regulate fat desaturation.

World human neutrophil antigens investigation survey.

BACKGROUND AND OBJECTIVES: Human neutrophil antigens (HNAs) are categorized into five systems: HNA-1 to HNA-5. Given the importance of neutrophils in immunity, we sought to create awareness of the role of HNA diagnostic services in managing immune neutropenia and transfusion-related acute lung injury. To provide health communities all around the world with access to these services, we conducted a survey to create a directory of these HNA diagnostic services. MATERIALS AND METHODS: An Excel table-based survey was created to capture information on the laboratory's location and was emailed to 55 individuals with known or possible HNA investigation activity. The collected data were then summarized and analysed. RESULTS: Of contacted laboratories, the surveys were returned from 23 (38.2%) laboratories; 17 have already established HNA diagnostic (of them 12 were regular participants of the International Granulocyte Immunobiology Workshop [ISBT-IGIW]), 4 laboratories were in the process of establishing their HNA investigation and the remaining 2 responder laboratories, did not conduct HNA investigations. In established laboratories, investigation for autoimmune neutropenia (infancies and adults) was the most frequently requested, and antibodies against HNA-1a and HNA-1b were the most commonly detected. CONCLUSION: The directory of survey respondents provides a resource for health professionals wanting to access HNA diagnostic services. The present study offers a comprehensive picture of HNA diagnostics (typing and serology), identifying weak points and areas for improvement for the first time. Identifying more laboratories involved in HNA diagnostics with limited access to international societies in the field will globally improve HNA diagnostics.

Oligonucleotide Catabolism-Derived Gluconucleosides in Caenorhabditis elegans.

Nucleosides are essential cornerstones of life, and nucleoside derivatives and synthetic analogues have important biomedical applications. Correspondingly, production of non-canonical nucleoside derivatives in animal model systems is of particular interest. Here, we report the discovery of diverse glucose-based nucleosides in Caenorhabditis elegans and related nematodes. Using a mass spectrometric screen based on all-ion fragmentation in combination with total synthesis, we show that C. elegans selectively glucosylates a series of modified purines but not the canonical purine and pyrimidine bases. Analogous to ribonucleosides, the resulting gluconucleosides exist as phosphorylated and non-phosphorylated forms. The phosphorylated gluconucleosides can be additionally decorated with diverse acyl moieties from amino acid catabolism. Syntheses of representative variants, facilitated by a novel 2'-O- to 3'-O-dibenzyl phosphoryl transesterification reaction, demonstrated selective incorporation of different nucleobases and acyl moieties. Using stable-isotope labeling, we further show that gluconucleosides incorporate modified nucleobases derived from RNA and possibly DNA breakdown, revealing extensive recycling of oligonucleotide catabolites. Gluconucleosides are conserved in other nematodes, and biosynthesis of specific subsets is increased in germline mutants and during aging. Bioassays indicate that gluconucleosides may function in stress response pathways.

Complement C3d/C4d Deposition on Platelets Correlates with 2'-O-Methoxyethyl Antisense Oligonucleotide-Induced Thrombocytopenia in Monkeys.

2'-O-Methoxyethyl antisense oligonucleotide (2'-MOE ASO)-induced severe thrombocytopenia (TCP) [platelet (PLT) count <50 K/µL] was observed in the Asian-sourced cynomolgus monkeys with low incidence (2%-4% at doses >5 mg/kg/week). The potential mechanisms for TCP were studied using the Mauritian-sourced cynomolgus monkeys, which were shown to be more susceptible to ASO-induced TCP, along with the Asian-sourced animals. ISIS 405879, a 2'-MOE ASO, induced severe TCP (PLT <50 K/µL) in seven of nine Mauritian-sourced monkeys but not in the Asian-sourced monkeys after 16 weeks of treatment at 40 mg/kg/week. Marked increases in PLT-bound C3d/C4d were detected in all thrombocytopenic Mauritian-sourced monkeys but not in the unaffected Mauritian- or Asian-sourced monkeys, suggesting increased PLT clearance due to complement deposition on the PLTs. However, this effect was independent of the ASO-mediated fluid-phase alternative complement activation. A correlation was also observed between serum antiglycoprotein (GP) IIb/IIIa immunoglobulin G (IgG) and PLT reduction. In addition, increases in total serum IgM, anti-PLT IgM, and anti-PLT factor 4 IgM levels were observed in monkeys from both sources but were more evident in the Mauritian-sourced monkeys. These data suggest an enhanced innate immune cell activation to ISIS 405879, leading to increased PLT destruction through complement fixation on the PLTs or PLT crossreacting polyclonal antibody production.

HLA antibodies in fetal and neonatal alloimmune thrombocytopenia.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by antibodies against human platelet antigens (HPA). However, in many cases that meet clinical criteria for the condition, maternal sera do not have HPA antibodies. In studies examining whether human leukocyte antigen (HLA) antibodies cause FNAIT, the results are limited and inconclusive. This study sought to examine whether clinically suspected FNAIT cases with absent maternal HPA antibodies had different HLA antibody strength and specificity compared to controls. STUDY DESIGN AND METHODS: A retrospective case-control study assessed class I HLA antibody strength and specificity in cases submitted for testing to Versiti, Wisconsin. There were 813 cases that met initial screening criteria, but written consent could only be obtained for 50. After review of medical records and expert panel review, 31 cases with clinical criteria of FNAIT and maternal HLA but not HPA antibodies were included. Each case was matched for maternal age, gestational age at delivery, parity, and race/ethnicity to two controls from unaffected pregnancies that had maternal serum HLA antibodies. RESULTS: FNAIT cases were found to have both significantly higher HLA antibody strength, measured by mean fluorescence index (MFI), and broader HLA antibody specificity at antigen epitope level, compared to matched controls (p < .001). p-values remained significant after controlling for parity and gestational age at delivery. DISCUSSION: Additional studies are needed to further examine whether the strong HLA antibodies identified in HPA-antibody-negative cases directly cause neonatal thrombocytopenia and whether prenatal treatment may be warranted in select cases to prevent recurrence.

Sex-specificity of the C. elegans metabolome.

Recent studies of animal metabolism have revealed large numbers of novel metabolites that are involved in all aspects of organismal biology, but it is unclear to what extent metabolomes differ between sexes. Here, using untargeted comparative metabolomics for the analysis of wildtype animals and sex determination mutants, we show that C. elegans hermaphrodites and males exhibit pervasive metabolomic differences. Several hundred small molecules are produced exclusively or in much larger amounts in one sex, including a host of previously unreported metabolites that incorporate building blocks from nucleoside, carbohydrate, lipid, and amino acid metabolism. A subset of male-enriched metabolites is specifically associated with the presence of a male germline, whereas enrichment of other compounds requires a male soma. Further, we show that one of the male germline-dependent metabolites, an unusual dipeptide incorporating N,N-dimethyltryptophan, increases food consumption, reduces lifespan, and accelerates the last stage of larval development in hermaphrodites. Our results serve as a foundation for mechanistic studies of how the genetic sex of soma and germline shape the C. elegans metabolome and provide a blueprint for the discovery of sex-dependent metabolites in other animals.

Parallel pathways for serotonin biosynthesis and metabolism in C. elegans.

The neurotransmitter serotonin plays a central role in animal behavior and physiology, and many of its functions are regulated via evolutionarily conserved biosynthesis and degradation pathways. Here we show that in Caenorhabditis elegans, serotonin is abundantly produced in nonneuronal tissues via phenylalanine hydroxylase, in addition to canonical biosynthesis via tryptophan hydroxylase in neurons. Combining CRISPR-Cas9 genome editing, comparative metabolomics and synthesis, we demonstrate that most serotonin in C. elegans is incorporated into N-acetylserotonin-derived glucosides, which are retained in the worm body and further modified via the carboxylesterase CEST-4. Expression patterns of CEST-4 suggest that serotonin or serotonin derivatives are transported between different tissues. Last, we show that bacterial indole production interacts with serotonin metabolism via CEST-4. Our results reveal a parallel pathway for serotonin biosynthesis in nonneuronal cell types and further indicate that serotonin-derived metabolites may serve distinct signaling functions and contribute to previously described serotonin-dependent phenotypes.

C. elegans as a model for inter-individual variation in metabolism.

Individuals can exhibit differences in metabolism that are caused by the interplay of genetic background, nutritional input, microbiota and other environmental factors1-4. It is difficult to connect differences in metabolism to genomic variation and derive underlying molecular mechanisms in humans, owing to differences in diet and lifestyle, among others. Here we use the nematode Caenorhabditis elegans as a model to study inter-individual variation in metabolism. By comparing three wild strains and the commonly used N2 laboratory strain, we find differences in the abundances of both known metabolites and those that have not to our knowledge been previously described. The latter metabolites include conjugates between 3-hydroxypropionate (3HP) and several amino acids (3HP-AAs), which are much higher in abundance in one of the wild strains. 3HP is an intermediate in the propionate shunt pathway, which is activated when flux through the canonical, vitamin-B12-dependent propionate breakdown pathway is perturbed5. We show that increased accumulation of 3HP-AAs is caused by genetic variation in HPHD-1, for which 3HP is a substrate. Our results suggest that the production of 3HP-AAs represents a 'shunt-within-a-shunt' pathway to accommodate a reduction-of-function allele in hphd-1. This study provides a step towards the development of metabolic network models that capture individual-specific differences of metabolism and more closely represent the diversity that is found in entire species.

Anchoring IgG-degrading enzymes to the surface of platelets selectively neutralizes antiplatelet antibodies.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by immunoglobulin G (IgG)-mediated platelet destruction. Current therapies primarily focus on reducing antiplatelet antibodies using immunosuppression or increasing platelet production with thrombopoietin mimetics. However, there are no universally safe and effective treatments for patients presenting with severe life-threatening bleeding. The IgG-degrading enzyme of Streptococcus pyogenes (IdeS), a protease with strict specificity for IgG, prevents IgG-driven immune disorders in murine models, including ITP. In clinical trials, IdeS prevented IgG-mediated kidney transplant rejection; however, the concentration of IdeS used to remove pathogenic antibodies causes profound hypogammaglobulinemia, and IdeS is immunogenic, which limits its use. Therefore, this study sought to determine whether targeting IdeS to FcγRIIA, a low-affinity IgG receptor on the surface of platelets, neutrophils, and monocytes, would be a viable strategy to decrease the pathogenesis of antiplatelet IgG and reduce treatment-related complications of nontargeted IdeS. We generated a recombinant protein conjugate by site-specifically linking the C-terminus of a single-chain variable fragment from an FcγRIIA antibody, clone IV.3, to the N-terminus of IdeS (scIV.3-IdeS). Platelets treated with scIV.3-IdeS had reduced binding of antiplatelet IgG from patients with ITP and decreased platelet phagocytosis in vitro, with no decrease in normal IgG. Treatment of mice expressing human FcγRIIA with scIV.3-IdeS reduced thrombocytopenia in a model of ITP and significantly improved the half-life of transfused platelets expressing human FcγRIIA. Together, these data suggest that scIV.3-IdeS can selectively remove pathogenic antiplatelet IgG and may be a potential treatment for patients with ITP and severe bleeding.

Resilience to stress-related sleep disturbance: Examination of early pandemic coping and affect.

OBJECTIVE: Stress associated with global health threats such as the coronavirus disease 2019 (COVID-19) pandemic and related containment efforts may be associated with significant sleep disruption. Stress-related sleep disturbance is an established transdiagnostic risk factor; thus, identifying associations with coping strategies may inform future intervention efforts. The current study examined secondary control-oriented coping strategies, including positive reappraisal, which may be particularly effective in the context of stressors characterized by high uncertainty and low controllability such as a pandemic. METHOD: The current study (total N = 227 undergraduate students, predominantly female) examined the associations among primary and secondary control-oriented coping strategies, positive and negative affect (PA, NA), and the development of acute sleep disturbance in the month after the declaration of the COVID-19 pandemic. Control of prepandemic reported sleep disturbance allowed for prospective analyses of pandemic-related change. RESULTS: Participants reported high levels of stress due to the pandemic onset, including difficulties with time management, difficulties with work or school, and worry about the future. Reappraisal and acceptance were both associated with higher concurrent PA, lower NA, and less increase in sleep disturbance; however, positive reappraisal was the only coping strategy that predicted unique variance in increased sleep disturbance. CONCLUSIONS: Current findings add to our understanding of stress adaptation in response to stressors characterized by high severity, high uncertainty, and low controllability, such as the COVID-19 pandemic, and suggest that positive reappraisal and PA may foster resilience to stress-related sleep disturbance. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Multicentre evaluation of 5B9, a monoclonal anti-PF4/heparin IgG mimicking human HIT antibodies, as an internal quality control in HIT functional assays: Communication from the ISTH SSC Subcommittee on Platelet Immunology.

BACKGROUND: Functional tests for the diagnosis of heparin-induced thrombocytopenia (HIT) exhibit variable performance. OBJECTIVES: We evaluated in a multicenter study whether 5B9, a monoclonal anti-PF4/heparin IgG mimicking human HIT antibodies, could be used as an internal quality control. METHODS: 5B9 was sent to 11 laboratories in seven countries, and six initial concentrations ranging from 10 to 400 µg/mL were tested by heparin-induced platelet activation assay (HIPA), serotonin release assay (SRA), platelet aggregation test (PAT), flow cytometry (FC), or heparin-induced multiple-electrode aggregometry (HIMEA). Each method was evaluated in three different laboratories using experimental procedures identical to those usually applied for the diagnosis of HIT by testing platelets from 10 different healthy donors. RESULTS: The procedures used varied among the laboratories, particularly when platelet-rich plasma and whole blood were used. Nevertheless, positive results were obtained with at least 100 µg/ml of 5B9 for most donors tested by all centers (except one) performing HIPA, SRA, or HIMEA. FC and PAT results were more heterogeneous. FC results from one center that used washed platelets preincubated with PF4 were positive with all donors at 50 µg/ml 5B9, but at least 200 µg/ml of 5B9 were required to activate cells with most donors tested using PAT. CONCLUSION: This study confirms that HIT functional tests are not well standardized and exhibit variable sensitivity for the detection of platelet-activating antibodies. However, 5B9 is a potentially useful tool to standardize functional tests, to select responding platelet donors, and consequently to improve the performance of these assays and comparability between laboratories.

Individual differences in habitual short sleep duration and dysfunction: Subjective health versus objective cardiovascular disease risk.

OBJECTIVE: The adverse health effects of short sleep duration (i.e., six or fewer hours per night) are well established, including an increased risk of cardiovascular disease (CVD) and related mortality. However, there is heterogeneity in perceived sleep need among habitual short sleepers (HSS), with a sizable minority reporting no sleep-related daytime dysfunction. It has not been determined whether health risk associated with short sleep duration is consistent across individuals with and without reported dysfunction. The current study examined self-rated health (SRH), previously demonstrated to predict CVD risk, and objective CVD risk among HSS with and without reported dysfunction in the National Health and Nutrition Examination Surveys (NHANES). METHOD: Participants were adults age 40-79 in the 2005-2006 and 2007-2008 NHANES cycles. Assessments included the single item SRH (poor to excellent), self-reported average sleep duration, and self-reported daytime sleep-related dysfunction. Ten-year atherosclerotic CVD and high lifetime CVD risk (≥39%) were calculated using previously validated algorithms. RESULTS: HSS with no reported dysfunction rated their overall health significantly better than those with reported dysfunction; however, the "no dysfunction" HSS group evidenced modestly, though significantly, higher 10-year CVD risk compared with their dysfunction-reporting counterparts. High lifetime CVD risk, including younger adults age 20-39, was slightly higher for persons not reporting dysfunction, with the exception of short sleepers at the highest level of dysfunction who had the highest prevalence of high lifetime risk. CONCLUSIONS: Findings suggest that the absence of perceived sleep-related dysfunction does not confer lower CVD risk, despite higher SRH. (PsycInfo Database Record (c) 2021 APA, all rights reserved).