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Abstract Acute leukemia has rapid onset and severe complications. Anticancer peptides from natural sources have demonstrated efficacy in eliminating various cancers through apoptosis signaling pathways. Additionally, extracellular vesicles containing microRNAs play pivotal roles in promoting tumorigenesis. Therefore, this study aimed to investigate the impact of PG2, a pomegranate peptide that regulates extracellular vesicles, on the induction of acute leukemia cell apoptosis. NB4 and MOLT-4 leukemia cell lines were treated with PG2 alone or in combination with daunorubicin to assess cell viability using the MTT assay. Extracellular vesicles were extracted from PG2-treated NB4 and MOLT-4 cells. Bioinformatic tools were utilized to predict target proteins and microRNAs, following which mRNA and protein expression were determined by using RT‒qPCR and western blotting, respectively. PG2 significantly reduced the viability of NB4 and MOLT-4 cells. Furthermore, the combination of PG2 with daunorubicin had a synergistic effect on NB4 and MOLT-4 cells. Subsequent treatment with PG2 or PG2-treated extracellular vesicles decreased CDK2 expression while increasing microRNA-339-5p and caspase-3 expression in NB4 and MOLT-4 cells. Our findings revealed that the anticancer activity of PG2 through the CDK2/miR-339-5p/caspase-3 pathway is mediated by extracellular vesicles, ultimately inducing apoptosis. PG2 holds promise as a potential antileukemic drug.

Abstract Background Severe acute cholecystitis (AC) is a challenging disease because it comprises coexisting systemic infections that lead to vital organ dysfunction. This study evaluated the optimal surgical timing and efficacy of preoperative percutaneous cholecystostomy (PC) for patients with severe AC. Methods Data of 142 patients who underwent cholecystectomy for severe AC between 2011 and 2021 were retrospectively collected from the multi-institutional database of the Hiroshima Surgical Study Group of Clinical Oncology. Patients were divided into the early cholecystectomy (EC) group (within 72 h of symptom onset) and delayed cholecystectomy (DC) group. They were also subdivided into the upfront cholecystectomy group and preoperative PC before cholecystectomy group. The diagnosis and severity of AC were graded according to the Tokyo Guidelines 2018. Clinicopathological variables and outcomes were compared. Results No significant differences in age, body mass index, American Society of Anesthesiologists (ASA) classification, and Charlson comorbidity index between the EC and DC groups were observed. Preoperative drainage was more commonly performed for the DC group than for the EC group. Local severe AC features were more commonly detected in the DC group than in the EC group. The postoperative outcomes of the EC and DC groups were comparable. Compared to the PC before cholecystectomy group, the upfront cholecystectomy group included more patients with ASA physical status ≥ 3 and more patients who used oral warfarin. Warfarin usage and cardiovascular dysfunction rates of the PC after cholecystectomy group were higher than those of the upfront cholecystectomy group. PC was associated with significantly less intraoperative bleeding and shorter hospital stays. Conclusions Patients who can tolerate general anesthesia are good candidates for EC. Patients who use warfarin and those with cardiovascular dysfunction are considered to be at high risk for postoperative complications; therefore, to prevent AC recurrence during the waiting period, PC before cholecystectomy during the same admission is more appropriate than upfront cholecystectomy for these patients.

Continuous-wave electron paramagnetic resonance (EPR) spectroscopy at 35 GHz is an essential cornerstone in multi-frequency EPR studies and is crucial for differentiating multiple species in complex systems due to the improved g-tensor resolution compared to lower microwave frequencies. Especially for unstable and highly sensitive paramagnetic centers, the reliability of the measurements can be improved upon through the use of a single sample for EPR experiments at all frequencies. Besides the advantages, the lack of common availability of oversized-sample resonators at 35 GHz often limits scientists to lower frequencies or smaller sample geometries, and the latter may be non-trivial for sensitive materials. In this work, we present the design and performance of an oversized-sample 35 GHz EPR resonator with a high loaded Q value, QL, of up to 2550, well-suited for continuous-wave EPR and pulsed single-microwave-frequency experiments. The design is driven by electromagnetic field simulations, and the microwave characteristics of manufactured prototypes were found to be in agreement with the predictions. The resonator is based on a cylindrical cavity with a TE011 mode, allowing for 3 mm sample access. The design targets that we met include high sensitivity, robustness, and ease of manufacturing and maintenance. The resonator is compatible with commercial EPR spectrometers and with helium flow, as well as with cryogen-free cryostats, allowing for measurements at temperatures down to 1.8 K. To highlight the general applicability, the resonator was tested on metal centers, as well as on organic radicals featuring extremely narrow lines.

Abstract The COVID-19 pandemic has increased germ aversion, an aversive affective response to a high likelihood of pathogen transmission. While psychological factors are associated with chronic pain, the relationship between germ aversion and chronic pain remains unexplored. This study aimed to examine the relationship between germ aversion and new-onset and prognosis of chronic pain using longitudinal data collected during the COVID-19 pandemic. We conducted web-based surveys of full-time workers at baseline and after three months. Data were collected on demographic characteristics, psychological factors, and chronic pain. Germ aversion was assessed using a modified Perceived Vulnerability to Disease scale. We analyzed responses from 1265 panelists who completed the survey twice. The prevalence of chronic low back pain (CLBP) and chronic neck and shoulder pain (CNSP) was associated with sex, short sleep duration, psychological distress, loneliness, and germ aversion. Stratified analyses showed that germ aversion was a risk factor for CLBP at three months in both individuals with and without CLBP at baseline, and for CNSP at three months in those with CNSP at baseline, even after adjustment for confounders. In conclusion, this preliminary study suggests that high germ aversion is a risk factor for CLBP and CNSP in young and middle-aged workers.

Chronic lymphocytic leukemia (CLL) patients have an increased risk of secondary cancers, along with predisposition to CLL in their relatives. We have previously identified germline ATM variants as associated with CLL risk; here, we present their impact on predisposition to secondary neoplasms in CLL patients and their relatives. Patients enrolled in our tissue bank who had germline ATM status available were mailed a questionnaire between April 2022 and May 2023. Of the 333 patients who replied to the questionnaire, 283 patients (85%) reported at least one relative with a cancer history. The prevalence of family history of B-cell lymphoproliferative disorders was significantly higher (p=0.02) in patients with germline ATM variants (32%) compared to those without germline ATM variants (21%) including familial CLL (25% vs 18%) (p=0.04). No significant difference in the prevalence of secondary cancers was found between patients with and without germline ATM variants (p=0.73), although the role for individual ATM variants in other malignancies could not be excluded given the small sample size. Time to first CLL treatment (TTFT) was shorter in patients harboring somatic ATM events while no difference was observed in patients with germline ATM variants. In conclusion, we demonstrate a higher prevalence of B-cell lymphoproliferative disorders, including familial CLL, in relatives of CLL patients carrying germline ATM variants. The presence of these germline variants did not impact TTFT compared to patients harboring somatic ATM mutations.

Sota Katsube,1 Satohiro Matsumoto,1 Masahiro Misawa,2 Nao Kakizawa,3 Ryo Hashimoto,1 Taku Mizutani,1 Keita Matsumoto,1 Shuhei Yoshikawa,1 Hirosato Mashima1 1Department of Gastroenterology, Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Department of Obstetrics and Gynecology, Jichi Medical University Saitama Medical Center, Saitama, Japan; 3Department of General and Gastrointestinal Surgery, Jichi Medical University Saitama Medical Center, Saitama, JapanCorrespondence: Sota Katsube, Department of Gastroenterology, Jichi Medical University Saitama Medical Center, 1-847 Amanuma, Saitama, Omiya, 330-8503, Japan, Tel +81 48 647 2111, Fax +81 48 648 5188, Email m08029sk@jichi.ac.jpAbstract: The patient was a 50-year-old Japanese woman who was diagnosed with total-colitis-type ulcerative colitis (UC) at the age of 26 years. She was treated with mesalazine and azathioprine, and her disease activity was well controlled. At the age of 50 years, the patient was experiencing fever, abdominal pain, diarrhea, bloody stool, and anal pain, which led to a diagnosis of a relapse of UC. Although steroid therapy was administered and tended to improve her symptoms, fecaloid vaginal discharge occurred, and rectovaginal fistula (RVF) was confirmed. Colostomy was performed, and infliximab was initiated as maintenance therapy for UC. All symptoms improved, and RVF closure was confirmed 6 months after the initiation of infliximab. To date, she has been free from relapse of UC. There have been only a few reports of UC complicated by RVF, and this condition is often difficult to treat. To the best of our knowledge, no other case of UC complicated by RVF in which the fistula was closed after treatment with colostomy and infliximab has been previously reported; thus, our report of the present case is valuable to the literature.Keywords: ulcerative colitis, rectovaginal fistula, infliximab, colostomy

Abstract Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis.

Abstract This study focused on the heterogeneity in progress notes written by physicians or nurses. A total of 806 days of progress notes written by physicians or nurses from 83 randomly selected patients hospitalized in the Gastroenterology Department at Kagawa University Hospital from January to December 2021 were analyzed. We extracted symptoms as the International Classification of Diseases (ICD) Chapter 18 (R00–R99, hereinafter R codes) from each progress note using MedNER-J natural language processing software and counted the days one or more symptoms were extracted to calculate the extraction rate. The R-code extraction rate was significantly higher from progress notes by nurses than by physicians (physicians 68.5% vs. nurses 75.2%; p = 0.00112), regardless of specialty. By contrast, the R-code subcategory R10–R19 for digestive system symptoms (44.2 vs. 37.5%, respectively; p = 0.00299) and many chapters of ICD codes for disease names, as represented by Chapter 11 K00–K93 (68.4 vs. 30.9%, respectively; p

Leukemia is one of the most deadly cancers in Thailand. Natural compounds have been developed for cancer treatment. Menthol, a peppermint compound, has shown pharmacological properties such as anti-cancer activity. However, the mechanism of menthol inducing extracellular vesicles in leukemic cells is not yet understood. In this study, we investigated the effects of menthol on leukemic extracellular vesicles and their role in apoptosis. NB4 and Molt-4 leukemic cells were cultured with menthol in various concentrations and times. Bioinformatic analysis was used to investigate target proteins of extracellular vesicle and apoptosis, followed by mRNA and protein expression by RT‒PCR and western blotting, respectively. Our findings indicate that menthol inhibits leukemic cell proliferation and increases extracellular vesicles. Furthermore, menthol treated leukemic extracellular vesicles induce apoptosis and upregulate the expression of ATG3 and caspase-3 in both mRNA and protein levels. These results suggest that menthol has an antileukemic effect through ATG3 and caspase-3 in apoptosis of leukemic extracellular vesicles.

Shu Kojima,1 Satohiro Matsumoto,1 Yudai Koito,1 Takaya Miura,1 Masanari Sekine,1 Takeshi Uehara,1 Takeharu Asano,1 Yasuhiro Yamaguchi,2 Hirosato Mashima1 1Department of Gastroenterology, Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Department of Respiratory Medicine, Jichi Medical University Saitama Medical Center, Saitama, JapanCorrespondence: Shu Kojima, Department of Gastroenterology, Jichi Medical University Saitama Medical Center, 1-847 Amanuma, Omiya, Saitama, 330-8503, Japan, Tel +81 48 647 2111, Fax +81 48 648 5188, Email s.kojima@jichi.ac.jpAbstract: A 51-year-old Japanese man was diagnosed with left-sided ulcerative colitis (UC) at age 41. He was treated with mesalazine and azathioprine and maintained remission. At age 51, the patient developed bloody stools, abdominal pain, scleritis, arthritis, cough, bloody sputum, and pericardial effusion. Considering that pericardial effusion is an atypical extraintestinal complication of UC, and the patient met the diagnostic criteria for relapsing polychondritis (RP), a diagnosis of RP complicating a relapse of UC was made. Steroid therapy was administered, and both diseases improved. Golimumab, an anti-tumor necrosis factor-α inhibitor, was introduced as maintenance therapy for UC. All symptoms, including pericardial effusion, improved. Subsequently, no relapse of UC or RP was observed. As only a few cases of RP overlapping with UC have been reported and no treatment protocol has been established, we considered this case valuable and worthy of publication.Keywords: ulcerative colitis, relapsing polychondritis, anti-tumor necrosis factor-α inhibitor, golimumab

Abstract Rab7 is known to function in the autophagy and endocytosis pathways in eukaryocytes and is related to various diseases. We recently reported that Rab7 plays a protective role against acute pancreatitis. However, its physiological function in exocytic cells remains unclear. Therefore, we investigated the role of Rab7 in pancreas-specific Rab7 knockout mice (Rab7Δpan). Immunofluorescence microscopy revealed that Rab7 colocalized with amylase in pancreatic acinar cells of wild-type mice, but not in Rab7Δpan mice. Western blotting confirmed Rab7 localization in the zymogen granule (ZG) membranes of wild-type mice. Cholecystokinin (CCK)-stimulated amylase secretion examined using isolated pancreatic acini was similar in Rab7Δpan and wild-type mice. In contrast, electron microscopy revealed that the diameters of ZGs were shorter and the number of ZGs was larger in the pancreatic acinar cells of Rab7Δpan mice than in those of wild-type mice. However, the number of ZGs decreased in both Rab7Δpan and wild-type mice after 24 h of starvation. In addition, the amount of amylase in the pancreas was decreased in both Rab7Δpan and wild-type mice. These data indicate that Rab7 localized on ZGs plays a crucial role in the maturation of ZGs but not in their autophagy or regulated exocytosis in pancreatic acinar cells.

Abstract Overexpression of antibody light chains in small plasma cell clones can lead to misfolding and aggregation. On the other hand, the formation of amyloid fibrils from antibody light chains is related to amyloidosis. Although aggregation of antibody light chain is an important issue, atomic-level structural examinations of antibody light chain aggregates are sparse. In this study, we present an antibody light chain that maintains an equilibrium between its monomeric and tetrameric states. According to data from X-ray crystallography, thermodynamic and kinetic measurements, as well as theoretical studies, this antibody light chain engages in 3D domain swapping within its variable region. Here, a pair of domain-swapped dimers creates a tetramer through hydrophobic interactions, facilitating the revelation of the domain-swapped structure. The negative cotton effect linked to the β-sheet structure, observed around 215 nm in the circular dichroism (CD) spectrum of the tetrameric variable region, is more pronounced than that of the monomer. This suggests that the monomer contains less β-sheet structures and exhibits greater flexibility than the tetramer in solution. These findings not only clarify the domain-swapped structure of the antibody light chain but also contribute to controlling antibody quality and advancing the development of future molecular recognition agents and drugs.

Background: Bullous pemphigoid is an autoimmune bullous disease that frequently affects a large skin surface area, but it can also present in localized areas. It has been hypothesized that bullous pemphigoid affects the systemic functioning of different organs because inflammatory cells and cytokines circulate throughout numerous organs. Results: Recent clinical and experimental studies have revealed an association between bullous pemphigoid and systemic organ disorders. To avoid the emergence of systemic organ diseases, the significance of systemic treatment in cases of severe bullous pemphigoid should be emphasized. Conclusions: Here, we discuss the specific molecular processes underlying typical systemic organ inflammatory diseases associated with bullous pemphigoids.

BACKGROUND: Evolutionary pressure has led to the emergence of SARS-CoV-2 variants, with the most recent Omicron variant containing an unparalleled 30 mutations in the spike protein. Many of these mutations are expected to increase immune evasion, thus making breakthrough cases and re-infection more common. METHODS: From June 2020 to December 2021 serial blood samples (initial post recovery, 6 months, 12 months) were collected from a COVID-19 convalescent cohort in Boston, MA. Plasma was isolated for use in Mesoscale Discovery based antibody binding assays. Unvaccinated donors or those vaccinated prior to the primary blood draw were excluded from this analysis, as were those who did not have at least two blood draws. Wilcoxon signed rank tests were used to compare pre- and post-vaccination titers and antibody response against different variants, while McNemar tests were used to compare the proportions of achieving ≥ 4 fold increases against different variants. FINDINGS: Forty-eight COVID convalescent donors with post-infection vaccination (hybrid immunity) were studied to evaluate the levels of cross-reactive antibodies pre- and post- vaccination against various SARS-CoV-2 Spike and receptor binding domain (RBD) proteins. Vaccination with BNT162b2, mRNA-1273 or Ad26.COV2.S led to a 6·3 to 7·8 fold increase in anti-Spike antibody titers and a 7·0 to 7·4 fold increase in anti-WT, Alpha and Delta RBD antibody. However, a lower response was observed for Beta and Omicron RBDs with only 7/48 (15%) and 15/48 (31%) donors having a ≥4 fold increase in post-vaccination titers against Beta and Omicron RBDs. Structural analysis of the Beta and Omicron RBDs reveal a shared immune escape strategy involving residues K417-E484-N501 that is exploited by these variants of concern. INTERPRETATION: Through mutations of the K417-E484-N501 triad, SARS-CoV-2 has evolved to evade neutralization by the class I/II anti-RBD antibody fraction of hybrid immunity plasma as the polyclonal antibody response post-vaccination shows limitations in the ability to solve the structural requirements to bind the mutant RBDs. FUNDING: Massachusetts Consortium on Pathogen Readiness (280870.5116709.0016) and the National Institute of Allergy and Infectious Diseases (1R01AI161152-01A1).

Objectives This study documents the time elapsed from the diagnosis of osteonecrosis of the femoral head (ONFH) to surgery, exploring the factors that influence ONFH severity.Design Retrospective observational study of a nationwide database.Setting The Kaplan-Meier method with log-rank tests was applied to examine the period from definitive diagnosis of ONFH to surgery using any surgery as the end point. For bilateral cases, the date of the first surgery was the endpoint.Participants This study included 2074 ONFH cases registered in 34 university hospitals and highly specialised hospitals of the multicentre sentinel monitoring system of the Japanese Investigation Committee between 1997 and 2018.Main outcome measure The primary outcome was the time from diagnosis to surgery. The secondary outcome was the proportion of subjects remaining without surgery at 3, 6 and 9 months, and at 1, 2 and 5 years after diagnosis.Results The median time to surgery was 9 months (IQR 4–22 months) after diagnosis of ONFH. The time to surgery was significantly shorter in the alcohol alone group and the combined corticosteroid and alcohol group than in the corticosteroid alone group (p=0.018 and p

Abstract Duchenne muscular dystrophy (DMD) is an inherited disorder with mutations in the dystrophin gene characterized by progressive muscle degeneration and weakness. Therapy such as administration of glucocorticoids, exon skipping of mutant genes and introduction of dystrophin mini-genes have been tried, but there is no radical therapy for DMD. In this study, we used C. elegans carrying mutations in the dys-1 gene as a model of DMD to examine the effects of febuxostat (FBX). We applied FBX to dys-1 mutant animals harboring a marker for muscle nuclei and mitochondria, and found that FBX ameliorates the muscle loss. We next used a severer model dys-1; unc-22 double mutant and found the dys-1 mutation causes a weakened muscle contraction. We applied FBX and other compounds to the double mutant animals and assayed the movement. We found that the administration of FBX in combination of uric acid has the best effects on the DMD model.

Abstract Serine/threonine kinase, cell division cycle 7 (CDC7) is critical for initiating DNA replication. TAK-931 is a specific CDC7 inhibitor, which is a next-generation replication stress (RS) inducer. This study preclinically investigates TAK-931 antitumor efficacy and immunity regulation. TAK-931 induce RS, generating senescence-like aneuploid cells, which highly expressed inflammatory cytokines and chemokines (senescence-associated secretory phenotype, SASP). In vivo multilayer-omics analyses in gene expression panel, immune panel, immunohistochemistry, RNA sequencing, and single-cell RNA sequencing reveal that the RS-mediated aneuploid cells generated by TAK-931 intensively activate inflammatory-related and senescence-associated pathways, resulting in accumulation of tumor-infiltrating immune cells and potent antitumor immunity and efficacy. Finally, the combination of TAK-931 and immune checkpoint inhibitors profoundly enhance antiproliferative activities. These findings suggest that TAK-931 has therapeutic antitumor properties and improved clinical benefits in combination with conventional immunotherapy.