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We estimated the seroprevalence of anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) immunoglobulin G (IgG) in different island groups in Okinawa. A cross-sectional serosurvey was conducted over three periods between July 2020 and February 2021. A total of 2,683 serum samples were collected from six referral medical centers, each covering a separate region in Okinawa. In the main island, the seroprevalence was 0.0% (0/392; 95% confidence interval (CI), 0.0- 0.9%), 0.6% (8/1,448, 0.2-1.1%), and 1.4% (8/582, 0.6-2.7%) during the 1st, 2nd, and 3rd serosurvey, respectively. In remote islands, the seroprevalence was 0.0% (0/144; 95% CI, 0.0-2.5%) and 1.6% (2/123, 0.2-5.8%) during the 2nd and 3rd survey, respectively. The case detection ratio was 2.7 (95% CI, 1.3-5.3) in the main island and 2.8 (0.7-11.1) in remote islands during the 3rd survey. The case detection ratio was the highest in individuals aged 20-29 years (8.3; 95% CI, 3.3-21.4) in the main island and in those aged 50-59 years (14.1; 2.1-92.7) in remote islands, suggesting underreporting of clinical cases by the surveillance system in these subgroups. A serosurvey conducted during an emerging infectious disease epidemic can be useful for validating the reliability of the surveillance system by providing case detection ratio.

Initial studies on the immunogenicity of SARS-CoV-2 (CoV-2) S glycoprotein ("spike") as a protein subunit vaccine suggested sub-optimal efficacy in mammals. Although protein engineering efforts have produced CoV-2 spike protein sequences with greatly improved immunogenicity, additional strategies for improving the immunogenicity of CoV-2 protein subunit vaccines are scaffolding and the use of adjuvants. Comparisons of the effectiveness of engineered protein-only and engineered protein-nanoparticles vaccines have been rare. To explore this knowledge gap, we inoculated mice with two doses of either sequence-optimized trimeric spike protein or one of several sequence-optimized spike nanoparticles. We measured their immune response up to two months after the first dose. We also measured the immune response and protection against live virus in hamsters inoculated with either sequence-optimized trimeric spike protein or a liposome-based sequence-optimized spike nanoparticle. We found that in the presence of adjuvant, the antibody and neutralization titers elicited by spike-nanoparticles were not significantly greater than those elicited by spike-only in mice, even at doses as low as 0.1 µg/animal. Hamsters vaccinated with spike-only or spike-nanoparticles were equally protected from live virus one month after their first inoculation. These results suggest that sequence-optimized protein subunit vaccines in the form of individual prefusion-stabilized trimers can be as effective in improving immunogenicity as scaffolded forms., (© 2024. The Author(s).)

COVID-19 mRNA vaccines induce protective adaptive immunity against SARS-CoV-2 in most individuals, but there is wide variation in levels of vaccine-induced antibody and T-cell responses. However, the mechanisms underlying this inter-individual variation remain unclear. Here, using a systems biology approach based on multi-omics analyses of human blood and stool samples, we identified several factors that are associated with COVID-19 vaccine-induced adaptive immune responses. BNT162b2-induced T cell response is positively associated with late monocyte responses and inversely associated with baseline mRNA expression of activation protein 1 (AP-1) transcription factors. Interestingly, the gut microbial fucose/rhamnose degradation pathway is positively correlated with mRNA expression of AP-1, as well as a gene encoding an enzyme producing prostaglandin E2 (PGE2), which promotes AP-1 expression, and inversely correlated with BNT162b2-induced T-cell responses. These results suggest that baseline AP-1 expression, which is affected by commensal microbial activity, is a negative correlate of BNT162b2-induced T-cell responses., (© 2023. The Author(s).)

Sensing of midgut internal contents is important for ensuring appropriate hormonal response and digestion following the ingestion of dietary components. Studies in mammals have demonstrated that taste receptors (TRs), a subgroup of G protein-coupled receptors (GPCRs), are expressed in gut enteroendocrine cells (EECs) to sense dietary compounds and regulate the production and/or secretion of peptide hormones. Although progress has been made in identifying expression patterns of gustatory receptors (GRs) in gut EECs, it is currently unknown whether these receptors, which act as ligand-gated ion channels, serve similar functions as mammalian GPCR TRs to elicit hormone production and/or secretion. A Bombyx mori Gr, BmGr6, has been demonstrated to express in cells by oral sensory organs, midgut and nervous system; and to sense isoquercitrin and chlorogenic acid, which are non-nutritional secondary metabolites of host mulberry. Here, we show that BmGr6 co-expresses with Bommo-myosuppressin (BMS) in midgut EECs, responds to dietary compounds and is involved in regulation of BMS secretion. The presence of dietary compounds in midgut lumen after food intake resulted in an increase of BMS secretions in hemolymph of both wild-type and BmGr9 knockout larvae, but BMS secretions in BmGr6 knockout larvae decreased relative to wild-type. In addition, loss of BmGr6 led to a significant decrease in weight gain, excrement, hemolymph carbohydrates levels and hemolymph lipid levels. Interestingly, although BMS is produced in both midgut EECs and brain neurosecretory cells (NSCs), BMS levels in tissue extracts suggested that the increase in hemolymph BMS during feeding conditions is primarily due to secretion from midgut EECs. Our studies indicate that BmGr6 expressed in midgut EECs responds to the presence of dietary compounds in the lumen by eliciting BMS secretion in B. mori larvae., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

This article is the lead piece in a special report that presents the results of a bioethical investigation into chimeric research, which involves the insertion of human cells into nonhuman animals and nonhuman animal embryos, including into their brains. Rapid scientific developments in this field may advance knowledge and could lead to new therapies for humans. They also reveal the conceptual, ethical, and procedural limitations of existing ethics guidance for human-nonhuman chimeric research. Led by bioethics researchers working closely with an interdisciplinary work group, the investigation focused on generating conceptual clarity and identifying improvements to governance approaches, with the goal of helping scholars, funders, scientists, institutional leaders, and oversight bodies (embryonic stem cell research oversight [ESCRO] committees and institutional animal care and use committees [IACUCs]) deliver principled and trustworthy oversight of this area of science. The article, which focuses on human-nonhuman animal chimeric research that is stem cell based, identifies key ethical issues in and offers ten recommendations regarding the ethics and oversight of this research. Turning from bioethics' previous focus on human-centered questions about the ethics of "humanization" and this research's potential impact on concepts like human dignity, this article emphasizes the importance of nonhuman animal welfare concerns in chimeric research and argues for less-siloed governance and oversight and more-comprehensive public communication., (© 2022 The Hastings Center.)

During August 2020, we carried out a serological survey among students and employees at the Okinawa Institute of Science and Technology Graduate University (OIST), Japan, testing for the presence of antibodies against SARS-CoV-2, the causative agent of COVID-19. We used a FDA-authorized 2-step ELISA protocol in combination with at-home self-collection of blood samples using a custom low-cost finger prick-based capillary blood collection kit. Although our survey did not find any COVID-19 seropositive individuals among the OIST cohort, it reliably detected all positive control samples obtained from a local hospital and excluded all negatives controls. We found that high serum antibody titers can persist for more than 9 months post infection. Among our controls, we found strong cross-reactivity of antibodies in samples from a serum pool from two MERS patients in the anti-SARS-CoV-2-S ELISA. Here we show that a centralized ELISA in combination with patient-based capillary blood collection using as little as one drop of blood can reliably assess the seroprevalence among communities. Anonymous sample tracking and an integrated website created a stream-lined procedure. Major parts of the workflow were automated on a liquid handler, demonstrating scalability. We anticipate this concept to serve as a prototype for reliable serological testing among larger populations.

Cytidine 5'-monophosphate (CMP)-sialic acid synthetase (CSS) is an essential enzyme involved in the biosynthesis of carbohydrates and glycoconjugates containing sialic acids, a class of α-keto acids that are generally terminal key recognition residues by many proteins that play important biological and pathological roles. The CSS from Neisseria meningitidis (NmCSS) has been commonly used with other enzymes such as sialic acid aldolase and/or sialyltransferase in synthesizing a diverse array of compounds containing sialic acid or its naturally occurring and non-natural derivatives. To better understand its catalytic mechanism and substrate promiscuity, four NmCSS crystal structures trapped at various stages of the catalytic cycle with bound substrates, substrate analogues, and products have been obtained and are presented here. These structures suggest a mechanism for an "open" and "closed" conformational transition that occurs as sialic acid binds to the NmCSS/cytidine-5'-triphosphate (CTP) complex. The closed conformation positions critical residues to help facilitate the nucleophilic attack of sialic acid C2-OH to the α-phosphate of CTP, which is also aided by two observed divalent cations. Product formation drives the active site opening, promoting the release of products.

Bacterial adhesion is a general strategy for host-microbe and microbe-microbe interactions. Adhesive pili are essential for colonization, biofilm formation, virulence and pathogenesis of many environmental and pathogenic bacteria 1,2 . Members of the class Bacteroidia have unique type V pili, assembled by protease-mediated polymerization 3 . Porphyromonas gingivalis is the main contributor to periodontal disease and its type V pili are a key factor for its virulence 4 . However, the structure of the polymerized pilus and its assembly mechanism are unknown. Here we show structures of polymerized and monomeric states of FimA stalk pilin from P. gingivalis, determined by cryo-electron microscopy and crystallography. The atomic model of assembled FimA shows that the C-terminal strand of a donor subunit is inserted into a groove in the β-sheet of an acceptor subunit after N-terminal cleavage by the protease RgpB. The C terminus of the donor strand is essential for polymerization. We propose that type V pili assemble via a sequential polar assembly mechanism at the cell surface, involving protease-mediated strand exchange, employed by various Gram-negative species belonging to the class Bacteroidia. Our results reveal functional surfaces related to pathogenic properties of polymerized FimA. These insights may facilitate development of antibacterial drugs.

Molecules capable of directing changes to nucleic acid sequences are powerful tools for molecular biology and promising candidates for the therapeutic correction of disease-causing mutations. However, unwanted reactions at off-target sites complicate their use. Here we report selective combinations of mutant editing enzyme and directing oligonucleotide. Mutations in human ADAR2 (adenosine deaminase acting on RNA 2) that introduce aromatic amino acids at position 488 reduce background RNA editing. This residue is juxtaposed to the nucleobase that pairs with the editing site adenine, suggesting a steric clash for the bulky mutants. Replacing this nucleobase with a hydrogen atom removes the clash and restores editing activity. A crystal structure of the E488Y mutant bound to abasic site-containing RNA shows the accommodation of the tyrosine side chain. Finally, we demonstrate directed RNA editing in vitro and in human cells using mutant ADAR2 proteins and modified guide RNAs with reduced off-target activity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The population of Zulu sheep is reported to have declined by 7.4% between 2007 and 2011 due to crossbreeding. There is insufficient information on the genetic diversity of the Zulu sheep populations in the different area of KwaZulu Natal where they are reared. The study investigated genetic variation and genetic structure within and among eight Zulu sheep populations using 26 microsatellite markers. In addition, Damara, Dorper and South African Merino breeds were included to assess the genetic relationship between these breeds and the Zulu sheep. The results showed that there is considerable genetic diversity among the Zulu sheep populations (expected heterozygosity ranging from 0.57 to 0.69) and the level of inbreeding was not remarkable. The structure analysis results revealed that Makhathini Research Station and UNIZULU research station share common genetic structure, while three populations (Nongoma, Ulundi and Nquthu) had some admixture with the exotic Dorper breed. Thus, there is a need for sustainable breeding and conservation programmes to control the gene flow, in order to stop possible genetic dilution of the Zulu sheep.

Adenosine deaminases acting on RNA (ADARs) are editing enzymes that convert adenosine to inosine in duplex RNA, a modification reaction with wide-ranging consequences in RNA function. Understanding of the ADAR reaction mechanism, the origin of editing-site selectivity, and the effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. Here we describe four crystal structures of the human ADAR2 deaminase domain bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis of the ADAR deaminase domain's dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. In addition, we identified an ADAR2-specific RNA-binding loop near the enzyme active site, thus rationalizing differences in selectivity observed between different ADARs. Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.

Objectives: To determine whether the presence of high depressive symptoms (Center for Epidemiologic Studies Depression Scale (CES-D) score ≥ 14) diminished physical performance benefits after a comprehensive physical activity intervention in older adults., Design: A post hoc analysis of data from the Lifestyle Interventions and Independence for Elders Pilot (LIFE-P) study, a single-blind randomized controlled trial comparing a moderate-intensity physical activity intervention (PA) with a successful aging control (SA)., Setting: Multicenter U.S. institutions participating in the LIFE-P trial., Participants: Four hundred twenty-four sedentary, noninstitutionalized adults aged 70 to 89., Measurements: Depressive symptoms were assessed using the CES-D. Physical performance tests included the Short Physical Performance Battery (SPPB) and 400-m walk time (400 mw) at baseline and 6 and 12 months., Results: Of the participants, 15.8% had high depressive symptom scores (CES-D ≥ 14). For participants with low depressive symptoms, SPPB scores improved more in the PA than the SA group over 12 months (adjusted score difference +0.70; P < .001 at 6 months and +0.58; P = .004 at 12 months), and 400 mw times improved in the PA group at 6 months (adjusted score difference -0.41 minutes; P = .02). For participants with high depressive symptoms, the difference in improvement fell short of statistical significance on the SPPB between the PA and SA groups (adjusted score difference +0.76 (P = .18) at 6 months and +0.94 (P = .12) at 12 months)., Conclusion: The presence of high depressive symptoms did not substantially diminish physical performance benefits realized after a PA intervention in sedentary older adults., (© 2011, Copyright the Authors. Journal compilation © 2011, The American Geriatrics Society.)

We explored tasks of cognitive and physical performance in 20 older adult subjects after a 10 week Tai chi program in older adults using a pre-to-post test design. Improvement post intervention was seen in two cognitive measures of executive function and several physical performance measures. Preliminary findings in this non-controlled study suggest the possibility of beneficial effects of Tai chi on cognitive executive function in older adults and support the need to pursue this hypothesis in a randomized controlled trial.

An overview of issues associated with the design and development of low molecular weight inhibitors of protein-protein interactions is presented. Areas discussed include information on the nature of protein-protein interfaces, methods to characterize those interfaces and methods by which that information is applied towards ligand identification and design. Specific examples of the strategy for the identification of inhibitors of protein-protein interactions involving the proteins p56lck kinase, ERK2 and the calcium-binding protein S100B are presented. Physical characterization of the inhibitors identified in those studies shows them to have drug-like and lead-like properties, indicating their potential to be developed into therapeutic agents.

gamma-Hydroxybutyrate (GHB), a therapeutic for narcolepsy and a drug of abuse, has several mechanisms of action that involve GHB and GABA(B) receptors, metabolism to GABA, and modulation of dopaminergic signaling. The aim of these studies was to examine the role of GHB and GABA(B) receptors in the behavioral effects of GHB. Three approaches were used to synthesize GHB analogs that bind selectively to GHB receptors and are not metabolized to GABA-active compounds. Radioligand binding assays identified UMB86 (4-hydroxy-4-napthylbutanoic acid, sodium salt), UMB72 [4-(3-phenylpropyloxy)butyric acid, sodium salt], UMB73 (4-benzyloxybutyric acid, sodium salt), 2-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid (3-HPA), and 4-hydroxy-4-phenylbutyric acid as compounds that displace [(3)H]NCS-382 [5-[(3)H]-(2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7] annulen-6-ylidene) ethanoic acid] from GHB receptors at concentrations that do not markedly affect [(3)H]GABA binding to GABA(B) receptors. In rats and pigeons, GHB discriminative stimulus effects were not mimicked or attenuated by UMB86, UMB72, or 3-HPA up to doses that decreased responding. In mice, GHB, GHB precursors (gamma-butyrolactone and 1,4-butanediol) and GABA(B) receptor agonists [SKF97541 [3-aminopropyl(methyl)phosphinic acid hydrochloride] and baclofen] dose-dependently produced hypolocomotion, catalepsy, ataxia, and loss of righting. The GABA(B) receptor antagonist CGP35348 (3-aminopropyl(diethoxymethyl)phosphinic acid) attenuated catalepsy and ataxia that was observed after GHB and GABA(B) receptor agonists SKF97541 and baclofen. UMB86, UMB72, UMB73, and 3-HPA, like GHB, produced hypolocomotion, ataxia, and loss of righting; however, catalepsy was never observed with these compounds, which is consistent with the cataleptic effects of GHB being mediated by GABA(B) receptors. Ataxia that was observed with UMB86, UMB72, UMB73, and 3-HPA was not antagonized by CGP35348, suggesting that ataxia induced by these analogs is not mediated by GABA(B) receptors and might involve GHB receptors.

beta-Alanine synthase has been purified greater than 1000-fold to homogeneity from rat liver. The enzyme has a subunit molecular weight of 42,000 and a native size of hexamer. The enzyme undergoes ligand-induced changes in polymerization: association in response to the substrate, N-carbamoyl-beta-alanine, and the inhibitor, propionate; and dissociation in response to the product, beta-alanine. The ability of the substrate to associate the pure native enzyme to a larger polymeric species was exploited in the final purification step. The purified enzyme had a pI of 6.7, a Km of 8 microM, and a kcat/Km of 7.9 x 10(4) M-1 s-1. Positive cooperativity was observed toward the substrate N-carbamoyl-beta-alanine, with nH = 1.9. Such cooperativity occurred at substrate concentrations below 12 nM, so that this activation most likely occurs at a regulatory site, with a significantly stronger affinity for N-carbamoyl-beta-alanine than that shown by the catalytic site. The enzyme was sensitive to denaturation, which could be minimized by avoiding heat steps during the purification and by the presence of reducing agents. Such denatured enzyme had little change in Vmax, but had much higher Km, and had also lost the ability to associate or dissociate in response to effectors. After purification, enzyme stability was achieved by the addition of glycerol and detergent.

To help answer questions regarding the percentage of black patients catheterized for suspected cardiac ischemia who are found to have negative coronary arteriograms, and how this percentage compares with that of similarly classified white patients, the coronary arteriographic and left ventriculographic findings of 100 consecutive blacks were reviewed and compared with those of 226 contemporaneously catheterized whites. After excluding patients with prior histories of a clinical cardiac ischemic event, invasive therapeutic intervention for coronary artery disease (CAD), and nonatherosclerotic cardiac disease, a subgroup of 50 black and 104 white patients remained with undiagnosed chest discomfort that was suspicious for cardiac ischemia. Of this subgroup, 68 percent of the black patients showed no or insignificant CAD by arteriography, as compared with 21 percent of the white patients (P < .001). An incidental but note-worthy finding pertaining to this same subgroup was that 18 percent of the black patients who underwent arteriography and were found to have no CAD exhibited a subnormal left ventricular ejection fraction; in contrast, none of the white patients in the same category did so (P < .05).

N-Carbamoyl-beta-alanine (NC beta A) amidohydrolase (EC 3.5.1.6) is regulated in opposing fashion by the substrate, NC beta A and the product, beta-alanine. The native enzyme from rat liver has a molecular weight of 235,000 in the absence of ligands. NC beta A and substrate analogs (N-amidino-beta-alanine, N-carbamoyl-glycine) produced association of the enzyme. beta-Alanine and its analog gamma-aminobutyrate caused dissociation of the enzyme and produced inhibition. Negative cooperativity was observed for the binding of all ligands as measured by the change in polymerization of the enzyme, with an average Hill coefficient (napp) of 0.5. Enzyme that had been dissociated by preincubation with beta-alanine had little or no initial activity; only after a lag of 9 s was a steady state progress curve evident. The existence of a regulatory site is proposed as a model to explain physical and kinetic data. The enzyme activity was highest in rat liver and detectable in kidney; activity was not detected in brain, lung, muscle, or spleen of rat, nor in mouse Ehrlich ascites tumor cells. The rat liver enzyme has a pH optimum of 6.8, with a Km of 6.5 microM for NC beta A and a Ki of 1.08 mM for beta-alanine at this pH.