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Abstract In cancer genome analysis, identifying pathogenic alterations and assessing their effects on oncogenic processes is important. Although whole exome sequencing (WES) can effectively detect such changes, driver alterations could not be identified in 27.8% of the cases, according to a previous study. The objectives of the present study were to evaluate the utility of whole genome sequencing (WGS) and clarify its differences with WES in terms of driver alteration detection. For this purpose, WGS analysis was conducted on 177 driverless WES samples, selected from 5,480 fresh frozen samples derived from 5,140 Japanese patients with cancer. These samples were selected as primary tumor, both WES and transcriptome profiling were performed, estimated tumor content of ≥ 30%, and no driver alterations were identified by WES. WGS identified driver and likely driver alterations in 68.4 and 22.6% of the samples, respectively. The most frequent alteration type was oncogene amplification, followed by tumor suppressor gene deletion and small variants located outside the coding region. In the remaining 9.0% of samples, no such signals were identified; therefore, further investigations are required. The current study clearly demonstrated the role and utility of WGS in identifying genomic alterations that contribute to tumorigenesis.

Abstract While certain human hepatitis B virus-targeting nucleoside analogs (NAs) serve as crucial anti-HBV drugs, HBV yet remains to be a major global health threat. E-CFCP is a 4′-modified and fluoromethylenated NA that exhibits potent antiviral activity against both wild-type and drug-resistant HBVs but less potent against human immunodeficiency virus type-1 (HIV-1). Here, we show that HIV-1 with HBV-associated amino acid substitutions introduced into the RT’s dNTP-binding site (N-site) is highly susceptible to E-CFCP. We determined the X-ray structures of HBV-associated HIV-1 RT mutants complexed with DNA:E-CFCP-triphosphate (E-CFCP-TP). The structures revealed that exocyclic fluoromethylene pushes the Met184 sidechain backward, and the resultant enlarged hydrophobic pocket accommodates both the fluoromethylene and 4′-cyano moiety of E-CFCP. Structural comparison with the DNA:dGTP/entecavir-triphosphate complex also indicated that the cyclopentene moiety of the bound E-CFCP-TP is slightly skewed and deviated. This positioning partly corresponds to that of the bound dNTP observed in the HIV-1 RT mutant with drug-resistant mutations F160M/M184V, resulting in the attenuation of the structural effects of F160M/M184V substitutions. These results expand our knowledge of the interactions between NAs and the RT N-site and should help further design antiviral NAs against both HIV-1 and HBV.

Abstract The incidence of vulvar carcinoma varies by race; however, it is a rare disease, and its genomic profiles remain largely unknown. This study examined the characteristics of vulvar squamous cell carcinoma (VSCC) in Japanese patients, focusing on genomic profiles and potential racial disparities. The study included two Japanese groups: the National Cancer Center Hospital (NCCH) group comprised 19 patients diagnosed between 2015 and 2023, and the Center for Cancer Genomics and Advanced Therapeutics group comprised 29 patients diagnosed between 2019 and 2022. Somatic mutations were identified by targeted or panel sequencing, and TP53 was identified as the most common mutation (52–81%), followed by HRAS (7–26%), CDKN2A (21–24%), and PIK3CA (5–10%). The mutation frequencies, except for TP53, were similar to those of Caucasian cohorts. In the NCCH group, 16 patients of HPV-independent tumors were identified by immunohistochemistry and genotyping. Univariate analysis revealed that TP53-mutated patients were associated with a poor prognosis (log-rank test, P = 0.089). Japanese VSCC mutations resembled those of Caucasian vulvar carcinomas, and TP53 mutations predicted prognosis regardless of ethnicity. The present findings suggest potential molecular-targeted therapies for select VSCC patients.

Abstract SARS-CoV-2-BA.4/5-adapted-bivalent-BNT162b2-vaccine (bvBNT), developed in response to the recent emergence of immune-evasive Omicron-variants, has been given to individuals who completed at least 2-doses of the monovalent-BNT162b2-vaccine (mvBNT). In the present cohort study, we evaluated neutralization-titers (NT50s) against Wuhan-strain (SCoV2Wuhan) and Omicron-sublineages including BA.2/BA.5/BQ.1.1/XBB/XBB.1.5, and vaccine-elicited S1-binding-IgG in sera from participants-vaccinated with 5th-bvBNT following 4th-mvBNT. The 5th-bvBNT-dose elicited good protective-activity against SCoV2Wuhan with geometric-mean (gMean)-NT50 of 1966–2091, higher than the peak-values post-4th-mvBNT with no statistical significance, and favorable neutralization-activity against not only BA.5 but also BA.2, with ~ 3.2-/~ 2.2-fold greater gMean-NT50 compared to the peak-values post-4th-mvBNT-dose, in participants with or without risk factors. However, neutralization-activity of sera post-5th-bvBNT-dose was low against BQ.1.1/XBB/XBB.1.5. Interestingly, participants receiving bvBNT following breakthrough (BT) infection during Omicron-wave had significantly enhanced neutralization-activity against SCoV2Wuhan/BA.2/BA.5 with ~ 4.6-/~ 6.3-/~ 8.1-fold greater gMean-NT50, respectively, compared to uninfected participants receiving bvBNT. Sera from BT-infected-participants receiving bvBNT had enhanced neutralization-activity against BQ.1.1/XBB/XBB.1.5 by ~ 3.8-fold compared to those from the same participants post-4th-mvBNT-dose, and had enhanced gMean-NT50 ~ 5.4-fold greater compared to those of uninfected-participants’ sera post-bvBNT. These results suggest that repeated stimulation brought about by exposure to BA.5’s-Spike elicit favorable cross-neutralization-activity against various SARS-CoV-2-variants.

Abstract Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the anti-SARS-CoV-2 antibody levels, anti-spike (S)-immunoglobulin G (IgG) and anti-nucleocapsid (N)-IgG, and the neutralization activity of IgG antibody in COVID‑19‑convalescent plasma against variants of SARS-CoV-2, alpha, beta, gamma, delta, kappa, omicron and R.1 strains. The study included 30 patients with clinically diagnosed COVID-19. The anti-S-IgG and anti-N-IgG levels ranged from 30.0 to 555.1 and from 10.1 to 752.6, respectively. The neutralization activity (50% inhibition concentration: IC50) for the wild-type Wuhan strain ranged from 100 µg/ml in 18 of 30 (60%) subjects infected with the beta variant. The IC50 values for wild-type and beta variants correlated inversely with anti-S-IgG levels (p

Abstract The humoral and cellular immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) upon the coronavirus disease 2019 (COVID-19) vaccination remain to be clarified. Hence, we aimed to investigate the long-term chronological changes in SARS-CoV-2 specific IgG antibody, neutralizing antibody, and T cell responses during and after receiving the BNT162b2 vaccine. We performed serological, neutralization, and T cell assays among 100 hospital workers aged 22–73 years who received the vaccine. We conducted seven surveys up to 8 months after the second vaccination dose. SARS-CoV-2 spike protein-specific IgG (IgG-S) titers and T cell responses increased significantly following the first vaccination dose. The highest titers were observed on day 29 and decreased gradually until the end of the follow-up period. There was no correlation between IgG-S and T cell responses. Notably, T cell responses were detected on day 15, earlier than the onset of neutralizing activity. This study demonstrated that both IgG-S and T cell responses were detected before acquiring sufficient levels of SARS-CoV-2 neutralizing antibodies. These immune responses are sustained for approximately 6 to 10 weeks but not for 7 months or later following the second vaccination, indicating the need for the booster dose (i.e., third vaccination).

Abstract In the present prospective study, 225 individuals in Kumamoto General Hospital, Japan, who received two-doses of BNT162b2 vaccine were enrolled/followed up over 150 days and neutralizing activity (NT50) of their sera and antiviral activity (EC50) of IgG purified from sera on day-60 post-1st-dose were determined against wild-type SARS-CoV-2 (SARS-CoV-2Wuhan) (n = 211) and 9 variants (Alpha, Beta, Gamma, Delta, and Kappa) (n = 45). Time-dependent changes of IgG-activity (n = 25) against SARS-CoV-2Wuhan and variants were also examined. Day-60 sera showed reduced NT50 by more than 50% against all variants examined, and greatest reduction was seen with Beta. IgG fractions of high-responders and moderate-responders showed similar fold-changes in EC50 against each variant compared to SARS-CoV-2Wuhan. Evaluation of EC50 of IgG obtained at different time-points (day-28 to -150) revealed time-dependent reduction of activity against all variants. However, against Delta, relatively long-lasting favorable antiviral activity (at least 150 days) was observed. Our data strongly suggest that the successful antecedent scale-up of mRNA-based vaccine administrations in Japan was the primary contributor to the lessening of the otherwise more devastating SARS-CoV-2 pandemic wave caused by the Delta variant. The present data that the effectiveness of vaccine against the then-dominant SARS-CoV-2 variant was likely associated with the moderation of the COVID-19 pandemic wave suggest that as in the case of influenza vaccines, the development of multivalent mRNA-based vaccines represent a generalizable approach to pre-emptively respond pandemic with mutable pathogens.

Abstract This study aimed to identify factors associated with engaging in sports and exercise volunteering among older adults. We used cross-sectional data from the Japan Gerontological Evaluation Study (JAGES), a nationwide mail survey of 20,877 older adults from 60 municipalities. Multilevel mixed-effects logistic regression analysis was used to investigate the correlation between engaging in sports and exercise volunteering and 39 variables classified into five factors: (1) demographic and biological, (2) behavioral, (3) psychological, cognitive, and emotional, (4) social and cultural, and (5) environmental factors. Among the analyzed samples, 1580 (7.6%) participants volunteered a few times/year or more often. Factors that showed positive association with the volunteering were older age, a current drinking habit, excellent self-rated health, high proportion of sports group participants in a living area, low municipal population density, and rich social and cultural features (i.e., social cohesion, support, network, and participation). Meanwhile, those that had a negative association were women, low level of education, deteriorated instrumental activities of daily living, having a past or current smoking habit, poor self-rated health, and depressive symptoms. We clarified the characteristics of the population that is more likely to participate in sports and exercise volunteering as well as those of the population that is less likely to participate and requires support.

Abstract The persistence of covalently closed circular DNA (cccDNA) poses a major obstacle to curing chronic hepatitis B (CHB). Here, we used droplet digital PCR (ddPCR) for cccDNA quantitation. The cccDNA-specific ddPCR showed high accuracy with the dynamic range of cccDNA detection from 101 to 105 copies/assay. The ddPCR had higher sensitivity, specificity and precisely than qPCR. The results of ddPCR correlated closely with serum HB core-related antigen and HB surface antigen (HBsAg) in 24 HBV-infected human-liver-chimeric mice (PXB-mice). We demonstrated that in 2 PXB-mice after entecavir treatment, the total cccDNA content did not change during liver repopulation, although the cccDNA content per hepatocyte was reduced after the treatment. In the 6 patients with HBV-related hepatocellular carcinoma, ddPCR detected cccDNA in both tumor and non-tumor tissues. In 13 HBeAg-negative CHB patients with pegylated interferon alpha-2a, cccDNA contents from paired biopsies were more significantly reduced in virological response (VR) than in non-VR at week 48 (p = 0.0051). Interestingly, cccDNA levels were the lowest in VR with HBsAg clearance but remained detectable after the treatment. Collectively, ddPCR revealed that cccDNA content is stable during hepatocyte proliferation and persists at quantifiable levels, even after serum HBsAg clearance.

Abstract Mutations in superoxide dismutase 1 gene (SOD1) are linked to amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder predominantly affecting upper and lower motor neurons. The clinical phenotype of ALS shows inter- and intrafamilial heterogeneity. The aim of the study was to analyze the relations between individual SOD1 mutations and the clinical presentation using in silico methods to assess the SOD1 mutations severity. We identified SOD1 causative variants in a group of 915 prospectively tested consecutive Polish ALS patients from a neuromuscular clinical center, performed molecular modeling of mutated SOD1 proteins and in silico analysis of mutation impact on clinical phenotype and survival analysis of associations between mutations and hazard of clinical end-points. Fifteen SOD1 mutations were identified in 21.1% familial and 2.3% sporadic ALS cases. Their effects on SOD1 protein structure and functioning inferred from molecular modeling and in silico analyses correlate well with the clinical data. Molecular modeling results support the hypothesis that folding intermediates rather than mature SOD1 protein give rise to the source of cytotoxic conformations in ALS. Significant associations between type of mutation and clinical end-points were found.

Abstract While mRNA vaccines against SARS-CoV-2 are exceedingly effective in preventing symptomatic infection, their immune response features remain to be clarified. In the present prospective study, 225 healthy individuals in Japan, who received two BNT162b2 doses, were enrolled. Correlates of BNT162b2-elicited SARS-CoV-2-neutralizing activity (50% neutralization titer: NT50; assessed using infectious virions) with various determinants were examined and the potency of sera against variants of concerns was determined. Significant rise in NT50s was seen in sera on day 28 post-1st dose. A moderate inverse correlation was seen between NT50s and ages, but no correlation seen between NT50s and adverse effects. NT50s and SARS-CoV-2-S1-binding-IgG levels on day 28 post-1st dose and pain scores following the 2nd dose were greater in women than in men. The average half-life of NT50s was ~ 68 days, and 23.6% (49 out of 208 individuals) failed to show detectable neutralizing activity on day 150. While sera from elite-responders (NT50s > 1,500: the top 4% among the participants) potently to moderately blocked all variants of concerns examined, some sera with low NT50s failed to block the B.1.351-beta strain. Since BNT162b2-elicited immunity against SARS-CoV-2 is short, an additional vaccine or other protective measures are needed.

Abstract Although several prognostic factors in nivolumab therapy have been reported in recurrent or metastatic head and neck cancer (RM-HNC) patients, these factors remain controversial. Here, we conducted a multicenter retrospective cohort study to investigate the impact of clinico-hematological factors on survival in RM-HNC patients treated with nivolumab. We reviewed 126 RM-HNC patients from seven institutes. We evaluated the prognostic effects of clinico-hematological factors on survival. The median overall survival (OS) was 12.3 months, and the 1 year-OS rate was 51.2%. Patients without immune-related adverse events, lower relative eosinophil count, worse best overall response, higher performance status, and higher modified Glasgow Prognostic Score had worse survival. The score, generated by combining these factors, was associated with survival. Patients with score of 4–5 had worse survival than those with score of 2–3 and 0–1 [adjusted HR for PFS: score of 4–5, 7.77 (3.98–15.15); score of 2–3, 3.44 (1.95–6.06), compared to score of 0–1], [adjusted HR for OS: score of 4–5, 14.66 (4.28–50.22); score of 2–3, 7.63 (2.29–25.37), compared to score of 0–1]. Our novel prognostic score utilizing clinico-hematological factors might be useful to establish an individual treatment strategy in RM-HNC patients treated with nivolumab therapy.

Abstract The quantitative range and reproducibility of current serological tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are not optimized. Herein, we developed a diagnostic test that detects SARS-CoV-2 IgG and IgM with high quantitativeness and reproducibility and low interference. The system was based on the high-sensitivity chemiluminescence enzyme immunoassay (HISCL) platform and detects IgG and IgM specific to SARS-CoV-2 spike and nucleocapsid proteins. Quantification accuracy and reproducibility were evaluated using serially diluted samples from 60 SARS-CoV-2-infected patients. Assay performance was evaluated using serum samples from the SARS-CoV-2-infected patients and 500 SARS-CoV-2-negative serum samples collected before the emergence of SARS-CoV-2. The system showed high quantification accuracy (range, 102), high reproducibility (within 5%), and no cross-reaction between SARS1- and MERS-S proteins. Detection accuracy was 98.3% and 93.3% for IgG and IgM against spike proteins and 100% and 71.7% for IgG and IgM against nucleocapsid proteins, respectively. Mean antibody levels were > 10 times that in negative samples upon admission and > 100 times that at convalescent periods. Clinical severity upon admission was not correlated with IgG or IgM levels. This highly quantitative, reproducible assay system with high clinical performance may help analyze temporal serological/immunological profiles of SARS-CoV-2 infection and SARS-CoV-2 vaccine effectiveness.

Abstract While there are various attempts to administer COVID-19-convalescent plasmas to SARS-CoV-2-infected patients, neither appropriate approach nor clinical utility has been established. We examined the presence and temporal changes of the neutralizing activity of IgG fractions from 43 COVID-19-convalescent plasmas using cell-based assays with multiple endpoints. IgG fractions from 27 cases (62.8%) had significant neutralizing activity and moderately to potently inhibited SARS-CoV-2 infection in cell-based assays; however, no detectable neutralizing activity was found in 16 cases (37.2%). Approximately half of the patients (~ 41%), who had significant neutralizing activity, lost the neutralization activity within ~ 1 month. Despite the rapid decline of neutralizing activity in plasmas, good amounts of SARS-CoV-2-S1-binding antibodies were persistently seen. The longer exposure of COVID-19 patients to greater amounts of SARS-CoV-2 elicits potent immune response to SARS-CoV-2, producing greater neutralization activity and SARS-CoV-2-S1-binding antibody amounts. The dilution of highly-neutralizing plasmas with poorly-neutralizing plasmas relatively readily reduced neutralizing activity. The presence of good amounts of SARS-CoV-2-S1-binding antibodies does not serve as a surrogate ensuring the presence of good neutralizing activity. In selecting good COVID-19-convalescent plasmas, quantification of neutralizing activity in each plasma sample before collection and use is required.

Abstract The central nervous system is involved in regulation of defaecation. It is generally considered that supraspinal regions control the spinal defaecation centre. However, signal transmission from supraspinal regions to the spinal defaecation centre is still unclear. In this study, we investigated the regulatory role of an anorexigenic neuropeptide, α-MSH, in the spinal defaecation centre in rats. Intrathecal administration of α-MSH to the L6-S1 spinal cord enhanced colorectal motility. The prokinetic effect of α-MSH was abolished by severing the pelvic nerves. In contrast, severing the colonic nerves or thoracic cord transection at the T4 level had no impact on the effect of α-MSH. RT-PCR analysis revealed MC1R mRNA and MC4R mRNA expression in the L6-S1 spinal cord. Intrathecally administered MC1R agonists, BMS470539 and SHU9119, mimicked the α-MSH effect, but a MC4R agonist, THIQ, had no effect. These results demonstrate that α-MSH binds to MC1R in the spinal defaecation centre and activates pelvic nerves, leading to enhancement of colorectal motility. This is, to our knowledge, the first report showing the functional role of α-MSH in the spinal cord. In conclusion, our findings suggest that α-MSH is a candidate for a neurotransmitter from supraspinal regions to the spinal defaecation centre.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the anti-SARS-CoV-2 antibody levels, anti-spike (S)-immunoglobulin G (IgG) and anti-nucleocapsid (N)-IgG, and the neutralization activity of IgG antibody in COVID‑19‑convalescent plasma against variants of SARS-CoV-2, alpha, beta, gamma, delta, kappa, omicron and R.1 strains. The study included 30 patients with clinically diagnosed COVID-19. The anti-S-IgG and anti-N-IgG levels ranged from 30.0 to 555.1 and from 10.1 to 752.6, respectively. The neutralization activity (50% inhibition concentration: IC50) for the wild-type Wuhan strain ranged from < 6.3 to 81.5 µg/ml. IgG antibodies were > 100 µg/ml in 18 of 30 (60%) subjects infected with the beta variant. The IC50 values for wild-type and beta variants correlated inversely with anti-S-IgG levels (p < 0.05), but no such correlation was noted with anti-N-IgG. IgG antibodies prevented infectivity and cytopathic effects of six different variants of concern in the cell-based assays of wild-type, alpha, gamma, delta, kappa and R.1 strains, but not that of the beta and omicron strains. IgG is considered the main neutralizing activity in the blood, although other factors may be important in other body tissues. [ABSTRACT FROM AUTHOR]