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Background: Data on the safety profiles and clinical outcomes of patients with solid tumors and cardiac metastasis treated with immune checkpoint inhibitors (ICIs) are limited., Methods: This is an international multicenter retrospective study of patients with cancer and cardiac metastasis at baseline. Patients who had received ≥1 dose of ICI were included. Treatment-related adverse events (trAEs) were graded per Common Terminology Criteria for Adverse Event V.5.0. Objective response rates (ORR) were evaluated by Response Evaluation Criteria in Solid Tumors V.1.1 when available. Overall survival (OS) and progression-free survival (PFS) were estimated by the Kaplan-Meier method., Results: Among 110 pts, median age at ICI initiation was 65 (IQR: 59-75). Median follow-up time since ICI initiation was 36 (95% CI: 26 to 51) months. Melanoma (38%, n=42) and non-small cell lung cancer (24%, n=26) were the most common. 68 (62%) patients received ICIs as first-line, and 29 (26%) patients were treated with combination anti-programmed death-1 and anti-cytotoxic T-lymphocyte antigen 4. The most common location of cardiac metastasis was in the atria (37%, n=41) and ventricles (35%, n=39). 15 patients (13.6%) had bilateral cardiac/pericardial metastasis, 44 (40%) had left-sided, and 43 (39.8%) had right-sided. At ICI initiation, 21% (n=23) had a cardiac thrombus. Cardiology referrals and cardiac MRIs at the time of cancer diagnosis were completed on 58 (53%) and 52 (47%) patients, respectively. Cardiac events occurred in 40 (36%) patients, including arrhythmias (n=14, 13%), arterial/venous emboli (n=4, 3.6%), and cardiac tamponade (n=3, 2.7%). 53 (47%) patients developed trAEs; most common were colitis/diarrhea (n=16, 15%), dermatitis (n=13, 12%), and hepatitis (n=9, 8.2%). ICI-related major cardiac trAEs occurred in 2 (1.8%) patients. 22 patients (20%) developed grade ≥3 trAE. Patients with multiple cardiac metastases had significantly lower responses to ICI-based regimens compared with patients with single cardiac metastasis (11% vs 63%, p=0.02). For melanoma, ORR, median PFS, and median OS were 38%, 9.0 months, and 28.9 months, respectively. 83% of patients with melanoma had concordant responses in overall disease burden and cardiac disease. 91 patients discontinued ICIs, and the main reason was progression or death in 55 (49%) patients., Conclusions: Among patients with pre-existing cardiac metastasis, ICIs demonstrated meaningful clinical efficacy with no increase in safety signals. Most patients had concordant responses in the overall disease burden and cardiac mass. Multidisciplinary teams are crucial for the appropriate management of patients with cardiac metastasis., Competing Interests: Competing interests: AHN receives honoraria from OncLive, TEMPUS, and Korean Society for Medical Oncology. Consulting fees: Guidepoint Global. EHY: Research funding/grants from CSL Behring, Boehringer Ingelheim and Eli and Lilly, Bristol Myers Squibb, and Amgen. Consulting fees from Pfizer, Xencor. RRM: consultant/advisor: AstraZeneca, Ambrx, Aveo, Bayer, Blue Earth Diagnostics, Bristol-Myers Squibb, Calithera, Caris, Dendreon, Exelixis, Eisai, Johnson & Johnson, Lilly, Merck, Myovant, Novartis, Pfizer, Sanofi, Seagen, Sorrento, Telix, Tempus. AD: Ad Board/Consulting for: SeaGen, Astra Zeneca, Merck, EMD Serono, Exelixis, Eli Lilly, Infinity, Roche.DJP: Lecture fees: Bayer Healthcare, Astra Zeneca, EISAI, Bristol Myers-Squibb, Roche, Ipsen; Travel expenses: Bristol Myers-Squibb, Roche, Bayer Healthcare; Consulting fees: Mina Therapeutics, Boehringer Ingelheim, Ewopharma, EISAI, Ipsen, Roche, H3B, Astra Zeneca, DaVolterra, Mursla, Avammune Therapeutics, LiFT Biosciences, Exact Sciences; Research funding (to institution): MSD, BMS, GSK.Alessio Cortellini received grants for consultancies/advisory boards: BMS, MSD, OncoC4, IQVIA, Roche, GSK, AstraZeneca, Access Infinity, Ardelis Health and REGENERON. He also received speaker fees from AstraZeneca, EISAI, MSD, SANOFI/REGENERON and Pierre-Fabre. KN receives honorarium from Pfizer, Eisai, AstraZeneca, Eli Lilly, Takeda, Chugai, Fuji Film Pharma, PDR pharma, MSD, Boehringer Ingelheim, Ono, Daiichi-Sankyo, Bayer, Jansen, and Sanofi. Advisory board: Eisai, AstraZeneca, Sanofi, Genmab, Gliad, OncXerna, Takeda, Novartis, MSD, Henlius. Research support (to institution): MSD, Daiichi-Sankyo, Merck Biopharma, AstraZeneca, Taiho, Pfizer, Novartis, Takeda, Chugai, Ono, Sanofi, Seattle Genetics, Eisai, Eli Lilly, Genmab, Boehringer Ingelheim, Kyowa Hakko Kirin, Nihon Kayaku, Haihe. Principal investigator: MSD, Daiichi-Sankyo, AstraZeneca, Taiho, Merck Biopharma Pfizer, Novartis, Takeda, Chugai, Ono, Sanofi, Seattle Genetics, Eisai, Eli Lilly, Genmab, Boehringer Ingelheim, Kyowa Hakko Kirin, Nihon Kayaku, Haihe. SG received honoraria from BMS, MSD, Sanofi, Novartis, Medison, Merck Serono. JE receives honoraria from Roche, AstraZeneca, and BMS. Consulting: Roche. Expert testimony: Roche. Travel expenses: MSD. SD: consultancy role with AstraZeneca, Gilead Sciences, Pfizer, Novartis, Myocardial Solutions, Eli Lilly none relevant to this manuscript. Funding: None. TKC reports: Institutional and/or personal, paid and/or unpaid support for research, advisory boards, consultancy, and/or honoraria past 5 years, ongoing or not, from: Alkermes, AstraZeneca, Aravive, Aveo, Bayer, Bristol Myers-Squibb, Calithera, Circle Pharma, Deciphera Pharmaceuticals, Eisai, EMD Serono, Exelixis, GlaxoSmithKline, Gilead, HiberCell, IQVA, Infinity, Ipsen, Jansen, Kanaph, Lilly, Merck, Nikang, Neomorph, Nuscan/PrecedeBio, Novartis, Oncohost, Pfizer, Roche, Sanofi/Aventis, Scholar Rock, Surface Oncology, Takeda, Tempest, Up-To-Date, CME events (Peerview, OncLive, MJH, CCO and others), outside the submitted work; Institutional patents filed on molecular alterations and immunotherapy response/toxicity, and ctDNA; Equity: Tempest, Pionyr, Osel, Precede Bio, CureResponse, InnDura Therapeutics, Primium; Committees: NCCN, GU Steering Committee, ASCO/ESMO, ACCRU, KidneyCan; Medical writing and editorial assistance support may have been funded by Communications companies in part; No speaker’s bureau; Mentored several non-US citizens on research projects with potential funding (in part) from non-US sources/Foreign Components; The institution (Dana-Farber Cancer Institute) may have received additional independent funding from drug companies or/and royalties potentially involved in research around the subject matter; TKC is supported in part by the Dana-Farber/Harvard Cancer Center Kidney SPORE (2P50CA101942-16) and Program 5P30CA006516-56, the Kohlberg Chair at Harvard Medical School and the Trust Family, Michael Brigham, Pan Mass Challenge, Hinda and Arthur Marcus Fund and Loker Pinard Funds for Kidney Cancer Research at DFCI. ARN reports Funding to Institution for Trials he is PI on: Loxo@Lilly, Surface Oncology, ADC Therapeutics, IGM Biosciences, EMD Serono, Aravive, Nikang Therapeutics, Inspirna, Exelexis, Revolution Medicine, Jacobio, Pionyr, Jazz Pharmaceuticals, NGM Biopharmaceuticals. ARN receives Consultant Editor Compensation: JCO Precision Oncology. Consulting/Advisory Board: Foundation Med. ARN reports Travel Compensation from: SITC/ AACR/ Conquer Cancer Foundation, Jazz Pharmaceuticals, Binay Tara Foundation, Foundation Med Funding: None., (© Author(s) (or their employer(s)) 2025. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Study Question: How does two-consecutive single embryo transfer (2xSET) affect reproductive outcomes of IVF and ICSI compared to double embryo transfer (DET)?, Summary Answer: Two-consecutive SET may provide greater or comparable live birth rate (LBR); with lower multiple birth, preterm birth, and pregnancy loss or neonatal death rates compared to DET., What Is Known Already: Elective SET in IVF/ICSI is widely encouraged over DET to minimize the risk of multiple births and associated morbidities. Despite this, multiple birth rates following IVF remain higher than the 10% target across Europe and the USA. Currently, the majority of evidence regarding SET and DET is based on various studies assessing outcomes such as LBR per treatment cycle, as opposed to per oocyte retrieval. As such, the representation of SET is mostly unfavourable. Analysis of cumulative LBR following the transfer of two embryos over consecutive cycles, rather than in one transfer event (DET) is more effective at distinguishing the two methods and will therefore provide more valuable information relevant to clinical practice., Study Design, Size, Duration: This retrospective cohort study was conducted using Human Fertilisation and Embryology Authority (HFEA) register data, which encompasses national data from all IVF clinics in the UK. All women who underwent their first oocyte retrieval and IVF or ICSI treatment cycle with subsequent SET, DET, or 2xSET between 2010 and 2019 using blastocyst embryos were included (N = 71 807)., Participants/materials, Setting, Methods: The rate of live birth, liveborn baby rate, multiple birth, preterm birth, and pregnancy loss or neonatal death was compared between SET, DET, and 2xSET IVF/ICSI pregnancies using blastocyst-stage embryos, where data were stratified by maternal age. Data analysis was conducted in RStudio v4.2, alpha equals 0.05., Main Results and the Role of Chance: Blastocyst-stage 2xSET achieved a greater median LBR of 0.47 (interquartile range [IQR] 0.13) than SET, 0.41 (IQR 0.13), and DET, 0.38 (IQR 0.13) (P < 0.05). Using SET as the reference standard, 2xSET was associated with a significantly lower odds of multiple births compared to DET ((odds ratio [OR] 6.87, 95% CI 6.14-7.68) vs 28.20, 95% CI 25.20-31.57). The odds of preterm birth were also lower following 2xSET (OR 1.11, 95% CI 1.06-1.15) compared to DET (OR 2.80, 95% CI 2.67-2.94). Similarly, the odds of pregnancy loss or neonatal death were lower following 2xSET (OR 1.14, 95% CI 1.08-1.21) compared to DET (OR 2.11, 95% CI 1.98-2.24). LBR was consistently higher following 2xSET than DET and SET in women aged 39 years and under (P < 0.05). However, results were comparable in women over 39 years (P > 0.05). Across all age groups, DET pregnancies had the highest multiple birth rate (P < 0.05). In women aged 39 years and under, DET was associated with the highest preterm birth rate (P < 0.05), whereas the rate was comparable across cohorts in women over 39 (P > 0.05). Moreover, pregnancy loss and neonatal death rates were highest following DET in women aged 37 years and under (P < 0.05), and comparable to SET and 2xSET in women over 37 years (P > 0.05)., Limitations, Reasons for Caution: Certain confounders are not recorded within HFEA registry data, including patient BMI, evaluation of embryo quality, and endometrial thickness at embryo transfer. Consequently, while our analysis identifies broad trends in embryo transfer success and morbidity, results may differ within certain patient populations., Wider Implications of the Findings: Blastocyst-stage 2xSET may provide greater LBR in women aged 39 years and under, and comparable LBR in women over 39 years old, with overall lower multiple birth and morbidity than DET. 2xSET should be considered first-line among certain patient cohorts, including women with advanced maternal age to improve reproductive outcomes and reduce the risk of morbidity following ART., Study Funding/competing Interest(s): No external funding was used for this study. None of the authors has any conflicts of interest., Trial Registration Number: This cohort study did not require registration. Following consultation with the Institutional Review Board at Imperial College London, ethical approval was not deemed necessary., (© The Author(s) 2025. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

The incidence of Intracranial Metastatic Disease (IMD) continues to increase in part due to improvements in systemic therapy resulting in durable control of extra-cranial disease (ECD). Contrast-enhanced Magnetic Resonance Imaging (CE-MRI) is the preferred method for imaging IMD, but has limitations particularly in follow-up surveillance scans to optimise patient care. We investigate a new diagnostic approach of hybrid ([ 18 ]F]fluoropivalate (FPIA) Positron Emission Tomography-multiparametric MRI (PET-mpMRI), in 12 treatment-naïve and 10 stereotactic radiosurgery (SRS)-treated patients (± combination therapy within 4-8 weeks). High FPIA uptake was seen in all IMD compared to contralateral white matter, regardless of ECD tumour-of-origin (p = 0.0001) and FPIA-PET volumes extended beyond CE-MRI volumes in treatment-naïve but not SRS-treated tumours. Patients with maximum PET Standardised Uptake Value, (SUVmax) ≥ 2.0 showed particularly short overall-survival (median 4 v 15 months, p = 0.0136), while CE-MRI was uninformative regarding outcome; a PET-mpMRI grade-measure also provided non-invasive prediction of overall-survival, warranting larger studies of PET-mpMRI. Independent metabolomics analyses were consistent with shared adaptation of IMD to utilise or accumulate monocarboxylates and acylcarnitines, respectively, providing a common phenotypic basis to FPIA-PET. Reprogrammed monocarboxylate metabolism-related FPIA-PET provides new insights into annotating IMD, to be expounded in future opportunities for therapy decisions for the growing number of cancer patients with IMD [Trial registration reference: Clinicaltrials.gov NCT04807582; 3rd November 2021, retrospectively registered]., Competing Interests: Declarations. Ethics approval and consent to participate: Study approval for the PET-mpMRI imaging study was obtained from the South Central - Berkshire Research Ethics Committee and United Kingdom Health Regulator Authority (Ethics reference 20/SC/0134). Approval for administration of radioactive substances was obtained from the United Kingdom Administration of Radioactive Substances Advisory Committee. All procedures involving human participants were done in accordance with the ethical standards of the institutional and/or national research committee and with the principles of the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards and written-informed consent was obtained from all individual participants included in the study. Furthermore, the authors affirm that human research participants provided informed consent for publication of the images displayed in the figures. Institutional ethical approval for retrospective analysis of human tissue was obtained, under R18019. The clinical study was retrospectively registered under Clinicaltrials.gov NCT04807582. The date of Registration– (Initial Release Date) was 03-11-2021 and date of enrolment of the first participant was 19-10-2020. URL of Registry: https://clinicaltrials.gov/study/NCT04807582 . Conflict of interest: EOA is on the Scientific Advisory Board of Radiopharm Theranostics; EOA is inventor of carboxylate imaging agents including FPIA. EA has been a Consultant for Radiopharm Theranostics. SI acknowledges unrestricted educational grant (PhD fellowship) support from Bayer Healthcare., (© 2025. The Author(s).)

Pancreatic ductal adenocarcinoma has a dismal prognosis. A comprehensive analysis of single-cell multi-omic data from matched tumour-infiltrated CD45+ cells and peripheral blood in 12 patients, and two published datasets, reveals a complex immune infiltrate. Patients have either a myeloid-enriched or adaptive-enriched tumour microenvironment. Adaptive immune cell-enriched is intrinsically linked with highly distinct B and T cell clonal selection, diversification, and differentiation. Using TCR data, we see the largest clonal expansions in CD8 effector memory, senescent cells, and highly activated regulatory T cells which are induced within the tumour from naïve cells. We identify pathways that potentially lead to a suppressive microenvironment, including investigational targets TIGIT/PVR and SIRPA/CD47. Analysis of patients from the APACT clinical trial shows that myeloid enrichment had a shorter overall survival compared to those with adaptive cell enrichment. Strategies for rationale therapeutic development in this disease include boosting of B cell responses, targeting immunosuppressive macrophages, and specific Treg cell depletion approaches., Competing Interests: Competing interests: S.S. held a personal fellowship from BMS during this study with a grant provided to conduct experiments and also has research funding from Alchemab, has received speaker fees and travel grants from Astrazeneca, Novartis and Servier; and also conducts trials with Astrazeneca, Novartis, Roche, Genentech and BioNTech. E.A.B. is a contributor to intellectual property licensed by Oxford University Innovation to AstraZeneca and held a research award from Guts UK/Dr Falk during this project. D.J.R., M.W., S.C., T.L., P.V.S., and A.V.R. are current or former employees and shareholders of BMS. M.R.M. reports grants from GRAIL, Roche, Astrazeneca, BMS, Infinitopes, Immunocore, and study fees from BMS, Pfizer, MSD, Regeneron, BiolineRx, Replimune and Novartis outside of the submitted work. M.L.D. is on the SAB for Adaptimmune and Singula Bio, consults for Molecular Partners, Enara Bio, Labgenius and Astra Zeneca, and undertakes research supported by BMS, Cue Biopharma, Boehringer Ingelheim, Regeneron and Evolveimmune outside the submitted work. The remaining authors declare no competing interests. R.B.-R. is a co-founder of Alchemab Therapeutics Ltd and consultant for Alchemab Therapeutics Ltd, Roche, Enara Bio, UCB and GSK., (© 2025. The Author(s).)

Childhood obesity poses a significant public health challenge, yet the molecular intricacies underlying its pathobiology remain elusive. Leveraging extensive multi-omics profiling (methylome, miRNome, transcriptome, proteins and metabolites) and a rich phenotypic characterization across two parts of Europe within the population-based Human Early Life Exposome project, we unravel the molecular landscape of childhood obesity and associated metabolic dysfunction. Our integrative analysis uncovers three clusters of children defined by specific multi-omics profiles, one of which characterized not only by higher adiposity but also by a high degree of metabolic complications. This high-risk cluster exhibits a complex interplay across many biological pathways, predominantly underscored by inflammation-related cascades. Further, by incorporating comprehensive information from the environmental risk-scape of the critical pregnancy period, we identify pre-pregnancy body mass index and environmental pollutants like perfluorooctanoate and mercury as important determinants of the high-risk cluster. Overall, our work helps to identify potential risk factors for prevention and intervention strategies early in the life course aimed at mitigating obesity and its long-term health consequences., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Two APOBEC DNA cytosine deaminase enzymes, APOBEC3A and APOBEC3B, generate somatic mutations in cancer, thereby driving tumour development and drug resistance. Here, we used single-cell RNA sequencing to study APOBEC3A and APOBEC3B expression in healthy and malignant mucosal epithelia, validating key observations with immunohistochemistry, spatial transcriptomics and functional experiments. Whereas APOBEC3B is expressed in keratinocytes entering mitosis, we show that APOBEC3A expression is confined largely to terminally differentiating cells and requires grainyhead-like transcription factor 3 (GRHL3). Thus, in normal tissue, neither deaminase appears to be expressed at high levels during DNA replication, the cell-cycle stage associated with APOBEC-mediated mutagenesis. In contrast, in squamous cell carcinoma we find that, there is expansion of GRHL3expression and activity to a subset of cells undergoing DNA replication and concomitant extension of APOBEC3A expression to proliferating cells. These findings suggest that APOBEC3A may play a functional role during keratinocyte differentiation, and offer a mechanism for acquisition of APOBEC3A mutagenic activity in tumours., Competing Interests: Disclosure and competing interests statement. TRF is an advisory board member of and holds stock options in APOBEC Discovery Ltd., (© 2024. The Author(s).)

Introduction: Gastroesophageal reflux disease (GORD) and aspiration are risk factors in the development of bronchiolitis obliterans syndrome (BOS) in the lung transplant population. The aim of this study was to investigate if allograft function and survival improved after anti-reflux surgery (ARS) in lung transplant recipients., Methods: In accordance with PRISMA guidelines, we conducted a systematic search of MEDLINE, Embase, and the Cochrane library databases from inception until 13/01/2024. Articles reporting outcomes of ARS following lung transplantation were included. A random effects model was used for meta-analysis., Results: The search identified 20 which were used for quantitative analysis. Overall, FEV1 and rate of change of FEV1 had improved following ARS by 0.141 L/s (95% CI; -02.82, -0.001) and -1.153 mL/d (95% CI; -12.117, -0.188), respectively. Survival hazard ratio post-ARS was 0.39 (95% CI; 0.19, 0.60). Nissen fundoplication was the most effective anti-reflux procedure with the greatest effect on reduction in the rate of change of FEV1, with an improvement of -2.353 mL/d (95% CI; -3.058, -1.649)., Conclusion: ARS in lung transplant recipients improves allograft function and survival. Given the increased incidence of GORD in lung transplant recipients, there should be a low threshold for investigation of GORD and subsequent ARS., Competing Interests: Declarations. Disclosures: Oliver Krahelski, Iihan Ali, Christopher Namgoong, Kavita Dave, Anna Reed, Hutan Ashrafian, Marcus Reddy, Omar Khan, Bibek Das, and Matyas Fehervari have no conflicts of interest or financial ties to disclose., (© 2024. The Author(s).)

Introduction: Patients with advanced ALK-positive NSCLC typically have poor response to immunotherapy; the benefit of consolidation durvalumab in patients with unresectable stage III ALK-positive NSCLC remains unclear. Herein, we compare the efficacy and safety of consolidation ALK tyrosine kinase inhibitor (TKI) versus durvalumab or observation after concurrent chemoradiation., Methods: We conducted a retrospective study using a multicenter study of 17 institutions globally. Patients with unresectable stage III ALK-positive NSCLC treated between 2015 and 2022 were included. Patients received ALK TKI, durvalumab, or observation after concurrent chemoradiation. Real-world progression-free survival (rwPFS) and overall survival (OS) were estimated using Kaplan-Meier method. Treatment-related adverse events (trAEs) were classified by Common Terminology Criteria for Adverse Events version 5.0. Outcomes were assessed by multivariable Cox regression analysis., Results: A total of 67 patients were included, of whom 39 (58%) were female. Median age was 57 (interquartile range: 49-67) years. Furthermore, 15 received consolidation ALK TKI, 30 received durvalumab, and 22 underwent observation. Baseline characteristics were similar across the three groups other than differences in race. After adjusting for stage, age, and nodal status, median rwPFS was significantly longer for ALK TKI (rwPFS not reached, 95% confidence interval [CI]: 22.7- not reached) versus durvalumab (11.3 mo, 95% CI: 8.9-18.5, hazard ratio [HR] = 0.12, 95% CI: 0.026-0.5, p-adjusted [p-adj] = 0.006) or observation (7.2 mo, 95% CI: 3.4-10.6, HR = 0.04, 95% CI: 0.009-0.2, p-adj < 0.0001). Durvalumab significantly improved median rwPFS compared with observation (HR = 0.37, 95% CI: 0.19-0.71, p-adj = 0.002). Median OS in the ALK TKI and durvalumab cohorts was significantly improved compared with patients on observation (ALK TKI-observation: p = 0.04; durvalumab-observation: p = 0.03). TrAE of any grade occurred in eight (53%) and 11 (37%) patients treated with ALK TKI and durvalumab, respectively. Grade greater than or equal to three trAEs occurred in 27% (n = 4) of patients treated with ALK TKI and 6.7% of patients treated with durvalumab., Conclusions: Patients with ALK-positive NSCLC experience significantly improved rwPFS when treated with consolidation ALK TKI therapy, surpassing outcomes found with either durvalumab or observation. Although both ALK TKI therapy and durvalumab offer an extension in OS compared with observation alone, it seems that ALK TKI therapy is the superior choice, underscoring its pivotal role in enhancing patient survival., Competing Interests: Disclosure Dr. Nassar receives honoraria from OncLive, TEMPUS, and Korean Society for Medical Oncology and consulting fees from Guidepoint Global and Putnam Associates. Dr. Goldberg receives research funding from AstraZeneca, Boehringer Ingelheim, and Mirati; is an advisory board member for AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Amgen, Blueprint Medicine, Sanofi Genzyme, Daiichi Sankyo, Takeda, Janssen, Summit Therapeutics, Merck, Regeneron, and Eli Lilly; and receives honoraria for lectures for Amgen and AstraZeneca. Dr. Naqash receives funding to institution for trials he is PI on Loxo@Lilly, Surface Oncology, ADC Therapeutics, IGM Biosciences, EMD Serono, Aravive, Nikang Therapeutics, Inspirna, Exelexis, Revolution Medicine, Jacobio, Pionyr, Jazz Pharmaceuticals, NGM Biopharmaceuticals, Immunocore, Phanes Therapeutics, Deka, Kymera, IDEAYA, IGM Biosciences, Selexine, and Chipscreen Biosciences; receives consultant editor compensation from JCO Precision Oncology; receives travel compensation from SITC, American Association for Cancer Reasearch, Conquer Cancer Foundation, BinayTara Foundation, Foundation Med, Caris Life Sciences, Sarah Cannon Research Institute, Jazz Pharmaceuticals, ASCO, and ASTRO; serves on the advisory board of Foundation Med and NGM Biosciences; receives current research grant support from Swog Hope Foundation, FDA Broad Agency Award, and OSCTR; and has genomics research agreements from Tempus, Caris, and Foundation One. Dr. Kim receives institutional funding from Loxo@Lilly, AstraZeneca, Boehringer Ingelheim, Genentech, Bristol-Myers Squibb, DynamiCure, Seagen, Genmab, and Nykode Therapeutics. Dr. Reuss reports serving on the advisory board/as consultant of Sanofi/Genzyme, Personalis, Guardant, AstraZeneca, Bristol-Myers Squibb, Arcus, AbbVie, Daiichi Sankyo, Catalym, Seagen, Gilead, Janssen, Novocure, Regeneron, and Summit Therapeutics; receiving research funding (to institution) from Genentech/Roche, Verastem, Nuvalent, and Exelixis; and receiving honoraria from Merck and AstraZeneca. Dr. Cortellini reports receiving grants for consultancies/advisory boards from Merck Sharp & Dohme, Bristol-Myers Squibb, OncoC4, IQVIA, AstraZeneca, Regeneron, Access Infinity, Ardelis Health, Alpha Sight, Guidepoint, and Roche; receiving speaker fees from AstraZeneca, Pierre-Fabre, Merck Sharp & Dohme, and Sanofi/Regeneron; having writing/editorial activity for Bristol-Myers Squibb and Merck Sharp & Dohme; and receiving travel support from Sanofi/Regeneron and Merck Sharp & Dohme. Dr. Marron currently or has previously served on advisory and/or data safety monitoring boards for Rockefeller University, Regeneron, AbbVie, Merck, Bristol-Myers Squibb, Boehringer Ingelheim, Atara, AstraZeneca, Genentech, Celldex, Chimeric, DrenBio, Glenmark, Simcere, Surface, G1 Therapeutics, NGMbio, DBV Technologies, Arcus, Fate, Ono, Larkspur, Avammune, and Astellas; and has received research grants from Regeneron, Genentech, Bristol-Myers Squibb, Merck, and Boehringer Ingelheim. Dr. Fitzgerald receives institutional research funding from Genentech/Roche, PPD, and bioatla and travel funding from IASLC. Dr. Dilling has served on the advisory boards for AstraZeneca and is a member of the NCCN Non–Small Cell Lung Cancer Panel. Dr. Bar is an advisor of AbbVie, Amgen, AstraZeneca, Bayer, Merck Sharp & Dohme, Merck-Serono, Roche, and Takeda; has writing/speaker engagement for Bristol-Myers Squibb, Medison, and Pfizer, and has received research funding from Immunai, OncoHost, Merck Sharp & Dohme, AstraZeneca, Roche, and AbbVie. Dr. Aredo reports receiving consulting fees from AstraZeneca. Dr. Lin has served as a compensated consultant for Genentech, C4 Therapeutics, Blueprint Medicines, Nuvalent, Bayer, Elevation Oncology, Novartis, Mirati Therapeutics, AnHeart Therapeutics, CLaiM Therapeutics, Ellipses, AstraZeneca, Bristol-Myers Squibb, Daiichi Sankyo, Yuhan, Merus, Regeneron, Pfizer, and Turning Point Therapeutics; received institutional research funds from Hengrui Therapeutics, Turning Point Therapeutics, Neon Therapeutics, Relay Therapeutics, Bayer, Elevation Oncology, Roche, Linnaeus Therapeutics, Nuvalent, and Novartis; and has received travel support from Pfizer and Merus. Dr. Kwiatkowski receives research funding from AADI and Genentech; and consults for Genentech, AADI, Expertconnect, Guidepoint, Bridgebio, Slingshot Insights, William Blair, MEDACorp, and Radyus Research. Dr. Yu has done consulting for AstraZeneca, Daiichi, Janssen, Amgen, Black Diamond, Blueprint, Cullinan, Takeda, and Taiho. Dr. Feng reports receiving speaker honoraria from AstraZeneca Canada. Dr. Velazquez reports receiving consulting fees from AstraZeneca, Merus, Novocure, and Regeneron., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Lactobacillus species dominance of the vaginal microbiome is a hallmark of vaginal health. Pathogen displacement of vaginal lactobacilli drives innate immune activation and mucosal barrier disruption, increasing the risks of STI acquisition and, in pregnancy, of preterm birth. We describe differential TLR mediated activation of the proinflammatory transcription factor NF-κB by vaginal pathogens and commensals. Vaginal Lactobacillus strains associated with optimal health selectively interact with anti-inflammatory innate immune receptors whereas species associated with suboptimal health including L. iners and Gardnerella vaginalis interact with both pro- and anti-inflammatory receptors. Anti-inflammatory action of L. crispatus is regulated by surface layer protein (SLPs)-mediated shielding of TLR ligands and selective interaction with the anti-inflammatory receptor DC-SIGN. Detection of SLPs within cervicovaginal fluid samples is associated with decreased concentrations of pro-inflammatory cytokines in Lactobacillus crispatus-dominated samples. These data offer mechanistic insights into how vaginal microbiota modulate host immune response and thus reproductive health and disease states., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Mutations in the FUT2 gene that result in a lack of expression of histo-blood group antigens on secreted glycoproteins may shape the vaginal microbiota with consequences for birth outcome. To test this, we analysed the relationship between secretor status, vaginal microbiota and gestational length in an ethnically diverse cohort of 302 pregnant women, including 82 who delivered preterm. Lactobacillus gasseri and L. jensenii were found to have distinct co-occurrence patterns with other microbial taxa in non-secretors. Moreover, non-secretors with Lactobacillus spp. depleted high diversity vaginal microbiota in early pregnancy had significantly shorter gestational length than Lactobacillus spp. dominated non-secretors (mean of 241.54 days (sd=47.14) versus 266.21 (23.61); P -value=0.0251). Similar gestational length differences were observed between non-secretors with high vaginal diversity and secretors with Lactobacillus spp. dominance (mean of 262.52 days (SD=27.73); p - value =0.0439) or depletion (mean of 266.05 days (SD=20.81); p - value =0.0312). Our data highlight secretor status and blood-group antigen expression as being important mediators of vaginal microbiota-host interactions in the context of preterm birth risk.

Background and Purpose: Neuronal ceroid lipofuscinoses are a group of neurodegenerative disorders. Recently, enzyme replacement therapy (ERT) was approved for neuronal ceroid lipofuscinosis type 2 (CLN2), a subtype of neuronal ceroid lipofuscinoses. The aim of this study was to quantify brain volume loss in CLN2 disease in patients on ERT in comparison with a natural history cohort using MRI., Materials and Methods: Nineteen (14 female, 5 male) patients with CLN2 disease at 1 UK center were studied using serial 3D T1-weighted MRI (follow-up time, 1-9 years). Brain segmentation was performed using FreeSurfer. Volume measurements for supratentorial gray and white matter, deep gray matter (basal ganglia/thalami), the lateral ventricles, and cerebellar gray and white matter were recorded. The volume change with time was analyzed using a linear mixed-effects model excluding scans before treatment onset. Comparison was made with a published natural history cohort of 12 patients (8 female, 4 male), which was re-analyzed using the same method., Results: Brain volume loss of all segmented brain regions was much slower in treated patients compared with the natural history cohort. For example, supratentorial gray matter volume in treated patients decreased by a mean of 3% (SD, 0.74%) ( P < .001) annually compared with an annual volume loss of a mean of 16.8% (SD, 1.5%) ( P < .001) in the natural history cohort., Conclusions: Our treatment cohort showed a significantly slower rate of brain parenchymal volume loss compared with a natural history cohort in several anatomic regions. Our results complement prior clinical data that found a positive response to ERT. We demonstrate that automated MRI volumetry is a sensitive tool to monitor treatment response in children with CLN2 disease., (© 2024 by American Journal of Neuroradiology.)

Intrathecal synthesis of immunoglobulins (Igs) is a key hallmark of multiple sclerosis (MS). B cells are known to accumulate in the leptomeninges of MS patients and associate with pathology in the underlying cortex and a more severe disease course. However, the role of locally produced antibodies in MS brain pathology is poorly understood. Here, we quantified the protein levels of IgA, IgM, IgG and albumin in serum and cerebrospinal fluid (CSF) samples of 80 MS patients and 28 neurological controls to calculate Ig indices. In addition, we quantified presence of meningeal IgA + , IgM + and IgG + B cells in post-mortem brain tissue of 20 MS patients and 6 controls using immunostainings. IgM and IgG, but not IgA, indices were increased in CSF of MS patients compared to controls, with no observed differences between MS disease types. Both IgM and IgG indices correlated significantly with neurofilament light (NfL) levels in CSF, but not with clinical or radiological parameters of disease. Similarly, IgG + and IgM + B cells were increased in MS meninges compared to controls, whereas IgA + B cells were not. Neuronal loss did not differ between sections with low or high IgA + , IgM + and IgG + B cells, but was increased in sections with high numbers of all CD19 + meningeal B cells. Similarly, high presence of CD19 + meningeal B cells and IgG + meningeal B cells associated with increased microglial density in the underlying cortex. Taken together, intrathecal synthesis of IgG and IgM is elevated in MS, which corresponds to an increased number of IgG + and IgM + B cells in MS meninges. The significant correlation between intrathecal IgG and IgM production and NfL levels, and increased microglial activation in cortical areas adjacent to meningeal infiltrates with high levels of IgG + B cells indicate a role for intrathecal IgM- and IgG-producing B cells in neuroinflammatory and degenerative processes in MS., (© 2024. The Author(s).)

Transcription factor (TF) binding to DNA is critical to transcription regulation. Although the binding properties of numerous individual TFs are well-documented, a more detailed comprehension of how TFs interact cooperatively with DNA is required. We present COBIND, a novel method based on non-negative matrix factorization (NMF) to identify TF co-binding patterns automatically. COBIND applies NMF to one-hot encoded regions flanking known TF binding sites (TFBSs) to pinpoint enriched DNA patterns at fixed distances. We applied COBIND to 5699 TFBS datasets from UniBind for 401 TFs in seven species. The method uncovered already established co-binding patterns and new co-binding configurations not yet reported in the literature and inferred through motif similarity and protein-protein interaction knowledge. Our extensive analyses across species revealed that 67% of the TFs shared a co-binding motif with other TFs from the same structural family. The co-binding patterns captured by COBIND are likely functionally relevant as they harbor higher evolutionarily conservation than isolated TFBSs. Open chromatin data from matching human cell lines further supported the co-binding predictions. Finally, we used single-molecule footprinting data from mouse embryonic stem cells to confirm that the COBIND-predicted co-binding events associated with some TFs likely occurred on the same DNA molecules., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Post-COVID- 19 syndrome (PCS) significantly impacts the quality of life of survivors. There is, however, a lack of a standardized approach to PCS diagnosis and management. Our bidirectional cohort study aimed to estimate PCS incidence, identify risk factors and biomarkers, and classify clinical phenotypes for enhanced management to improve patient outcomes., Methods: A bidirectional prospective cohort study was conducted at five medical sites in Hatyai district in Songkhla Province, Thailand. Participants were randomly selected from among the survivors of COVID-19 aged≥18 years between May 15, 2022, and January 31, 2023. The selected participants underwent a scheduled outpatient visit for symptom and health assessments 12 to 16 weeks after the acute onset of infection, during which PCS was diagnosed and blood samples were collected for hematological, inflammatory, and serological tests. PCS was defined according to the World Health Organization criteria. Univariate and multiple logistic regression analyses were used to identify biomarkers associated with PCS. Moreover, three clustering methods (agglomerative hierarchical, divisive hierarchical, and K-means clustering) were applied, and internal validation metrics were used to determine clustering and similarities in phenotypes., Findings: A total of 300 survivors were enrolled in the study, 47% of whom developed PCS according to the World Health Organization (WHO) definition. In the sampled cohort, 66.3% were females, and 79.4% of them developed PCS (as compared to 54.7% of males, p-value <0.001). Comorbidities were present in 19% (57/300) of all patients, with 11% (18/159) in the group without PCS and 27.7% (39/141) in the group with PCS. The incidence of PCS varied depending on the criteria used and reached 13% when a quality of life indicator was added to the WHO definition. Common PCS symptoms were hair loss (22%) and fatigue (21%), while mental health symptoms were less frequent (insomnia 3%, depression 3%, anxiety 2%). According to our univariate analysis, we found significantly lower hematocrit and IgG levels and greater ALP levels in PCS patients than in patients who did not develop PCS (p-value < 0.05). According to our multivariable analysis, adjusted ALP levels remained a significant predictor of PCS (OR 1.02, p-value= 0.005). Clustering analysis revealed four groups characterized by severe clinical symptoms and mental health concerns (Cluster 1, 4%), moderate physical symptoms with predominant mental health issues (Cluster 2, 9%), moderate mental health issues with predominant physical symptoms (Cluster 3, 14%), and mild to no PCS (Cluster 4, 77%). The quality of life and ALP levels varied across the clusters., Interpretation: This study challenges the prevailing diagnostic criteria for PCS, emphasizing the need for a holistic approach that considers quality of life. The identification of ALP as a biomarker associated with PCS suggests that its monitoring could be used for early detection of the onset of PCS. Cluster analysis revealed four distinct clinical phenotypes characterized by different clinical symptoms and mental health concerns that 'exhibited varying impacts on quality of life. This finding suggested that accounting for the reduced quality of life in the definition of PCS could enhance its diagnosis and management and that moving toward personalized interventions could both improve patient outcomes and help reduce medicalization and optimally target the available resources., Funding: The research publication received funding support from Medical Council of Thailand (Police General Dr. Jongjate Aojanepong Foundation), Hatyai Hospital Charity and Wellcome Trust., (© 2024. The Author(s).)

Finding effective therapies to manage age-related conditions is an emerging public health challenge. Although disease-targeted treatments are important, a preventive approach focused on aging can be more efficient. Pharmacological targeting of aging-related processes can extend lifespan and improve health in animal models. However, drug development and translation are particularly challenging in geroscience. Preclinical studies have survival as a major endpoint for drug screening, which requires years of research in mammalian models. Shorter-lived invertebrates can be exploited to accelerate this process. In particular, the fruit fly Drosophila melanogaster allows the validation of new drug targets using precise genetic tools and proof-of-concept experiments on drugs impacting conserved aging processes. Screening for clinically approved drugs that act on aging-related targets may further accelerate translation and create new tools for aging research. To date, 31 drugs used in clinical practice have been shown to extend the lifespan of flies. Here, we describe recent advances in the pharmacology of aging, focusing on Drosophila as a tool to repurpose these drugs and study age-related processes., Competing Interests: Conflict of Interest The authors declare that they have no conflict of interest.

Abstract: Blood endothelial cells control the hemostatic and inflammatory response by secreting von Willebrand factor (VWF) and P-selectin from storage organelles called Weibel-Palade bodies (WPBs). Actin-associated motor proteins regulate this secretory pathway at multiple points. Before fusion, myosin Va forms a complex that anchors WPBs to peripheral actin structures, allowing for the maturation of content. After fusion, an actomyosin ring/coat is recruited and compresses the WPB to forcibly expel the largest VWF multimers. Here, we provide, to our knowledge, the first evidence for the involvement of class I myosins during regulated VWF secretion. We show that the unconventional myosin-1C (Myo1c) is recruited after fusion via its pleckstrin homology domain in an actin-independent process. This provides a link between the actin ring and phosphatidylinositol 4,5-bisphosphate (PIP2) at the membrane of the fused organelle and is necessary to ensure maximal VWF secretion. This is an active process requiring Myo1c ATPase activity because inhibition of class I myosins using the inhibitor pentachloropseudilin or expression of an ATPase-deficient Myo1c rigor mutant perturbs the expulsion of VWF and alters the kinetics of the exocytic actin ring. These data offer a novel insight into the control of an essential physiological process and provide a new way in which it can be regulated., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

During pregnancy the immune system needs to maintain immune tolerance of the foetus while also responding to infection, which can cause premature activation of the inflammatory pathways leading to the onset of labour and preterm birth. The vaginal microbiome is an important modifier of preterm birth risk, with Lactobacillus dominance during pregnancy associated with term delivery while high microbial diversity is associated with an increased risk of preterm birth. Glycans on glycoproteins along the lower female reproductive tract are fundamental to microbiota-host interactions and the mediation of inflammatory responses. However, the specific glycan epitopes involved in these processes are not well understood. To address this, we conducted glycomic analyses of cervicovaginal fluid (CVF) from 36 pregnant women at high risk of preterm birth and 4 non-pregnant women. Our analysis of N- and O-glycans revealed a rich CVF glycome. While O-glycans were shown to be the main carriers of ABO blood group epitopes, the main features of N-glycans were the presence of abundant paucimannose and high mannose glycans, and a remarkable diversity of complex bi-, tri-, and tetra-antennary glycans decorated with fucose and sialic acid. We identified immuno-regulatory epitopes, such as Lewis antigens, and found that fucosylation was negatively correlated to pro-inflammatory factors, such as IL-1β, MMP-8, C3a and C5a, while glycans with only sialylated antennae were mainly positively correlated to those. Similarly, paucimannose glycans showed a positive correlation to pro-inflammatory factors. We revealed a high abundance of glycans which have previously been identified as hallmarks of cancer and viral glycosylation, such as Man8 and Man9 high mannose glycans. Although each pregnant woman had a unique glycomic profile, longitudinal studies showed that the main glycosylation features were consistent throughout pregnancy in women who delivered at term, whereas women who experienced extreme preterm birth exhibited sharp changes in the CVF glycome shortly before delivery. These findings shed light on the processes underlying the role of glycosylation in maintaining a healthy vaginal microbiome and associated host immune responses. In addition, these discoveries facilitate our understanding of the lower female reproductive tract which has broad implications for women's health., (© 2024. The Author(s).)

We introduce XGR-model (or XGRm), a web server made accessible at http://www.xgrm.pro, with the aim of meeting the increasing demand for effectively interpreting summary-level genomic data in model organisms. Currently, it hosts two enrichment analysers and two subnetwork analysers to support enrichment and subnetwork analyses for user-input mouse genomic data, whether gene-centric or genomic region-centric. The enrichment analysers identify ontology term enrichments for input genes (GElyser) or for genes linked from input genomic regions (RElyser). The subnetwork analysers rely on our previously established network algorithm to identify gene subnetworks from input gene-centric summary data (GSlyser) or from input region-centric summary data (RSlyser), leveraging network information about either functional interactions or pathway-derived interactions. Collectively, XGRm offers an all-in-one solution for gaining systems biology insights into summary-level genomic data in mice, underpinned by our commitment to regular updates as well as natural extensions to other model organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Environmental deprivation can have deleterious effects on adaptive myelination and oligodendroglia function. Early stage Huntington disease (HD) is characterised by white-matter myelin abnormalities in both humans and animal models. However, whether deprived environments exacerbate myelin-related pathological features of HD is not clearly understood. Here, we investigated the impact of deprivation and social isolation on ultrastructural features of myelin in the corpus callosum of the YAC128 mouse model of HD and wildtype (WT) mice using transmission electron microscopy. HD pathology on its own leads to increased representation of altered myelin features, such as thinner sheaths and compromised morphology. Interestingly, deprivation mirrors these effects in WT mice but does not greatly exacerbate the already aberrant myelin in HD mice, indicating a disease-related floor effect in the latter animals. These novel findings indicate that environmental deprivation causes abnormalities in myelin ultrastructure in the otherwise healthy corpus callosum of wild-type mice but has distinct effects on HD mice, where compromised myelin integrity is manifest from early stages of the disease., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The human ileum contains a high density of enteroendocrine L-cells, which release the appetite-suppressing hormones glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) in response to food intake. Recent evidence highlighted the potential role of food structures in PYY release, but the link between food structures, ileal metabolites, and appetite hormone release remains unclear owing to limited access to intact human ileum. In a randomized crossover trial (ISRCTN11327221; isrctn.com), we investigated the role of human ileum in GLP-1 and PYY release by giving healthy volunteers diets differing in fiber and food structure: high-fiber (intact or disrupted food structures) or low-fiber disrupted food structures. We used nasoenteric tubes to sample chyme from the intact distal ileum lumina of humans in the fasted state and every 60 min for 480 min postprandially. We demonstrate the highly dynamic, wide-ranging molecular environment of the ileum over time, with a substantial decrease in ileum bacterial numbers and bacterial metabolites after food intake. We also show that high-fiber diets, independent of food structure, increased PYY release compared with a low-fiber diet during 0 to 240 min postprandially. High-fiber diets also increased ileal stachyose, and a disrupted high-fiber diet increased certain ileal amino acids. Treatment of human ileal organoids with ileal fluids or an amino acid and stachyose mixture stimulated PYY expression in a similar profile to blood PYY concentrations, confirming the role of ileal metabolites in PYY release. Our study demonstrates the diet-induced changes over time in the metabolite environment of intact human ileum, which play a role in PYY release.

The identification of genes that confer either extension of life span or accelerate age-related decline was a step forward in understanding the mechanisms of aging and revealed that it is partially controlled by genetics and transcriptional programs. Here, we discovered that the human DNA sequence C16ORF70 encodes a protein, named MYTHO (macroautophagy and youth optimizer), which controls life span and health span. MYTHO protein is conserved from Caenorhabditis elegans to humans and its mRNA was upregulated in aged mice and elderly people. Deletion of the orthologous myt-1 gene in C. elegans dramatically shortened life span and decreased animal survival upon exposure to oxidative stress. Mechanistically, MYTHO is required for autophagy likely because it acts as a scaffold that binds WIPI2 and BCAS3 to recruit and assemble the conjugation system at the phagophore, the nascent autophagosome. We conclude that MYTHO is a transcriptionally regulated initiator of autophagy that is central in promoting stress resistance and healthy aging.

The canonical model of glucose-induced increase in insulin secretion involves the metabolism of glucose via glycolysis and the citrate cycle, resulting in increased ATP synthesis by the respiratory chain and the closure of ATP-sensitive K+ (KATP) channels. The resulting plasma membrane depolarization, followed by Ca2+ influx through L-type Ca2+ channels, then induces insulin granule fusion. Merrins and colleagues have recently proposed an alternative model whereby KATP channels are controlled by pyruvate kinase, using glycolytic and mitochondrial phosphoenolpyruvate (PEP) to generate microdomains of high ATP/ADP immediately adjacent to KATP channels. This model presents several challenges. First, how mitochondrially generated PEP, but not ATP produced abundantly by the mitochondrial F1F0-ATP synthase, can gain access to the proposed microdomains is unclear. Second, ATP/ADP fluctuations imaged immediately beneath the plasma membrane closely resemble those in the bulk cytosol. Third, ADP privation of the respiratory chain at high glucose, suggested to drive alternating, phased-locked generation by mitochondria of ATP or PEP, has yet to be directly demonstrated. Finally, the approaches used to explore these questions may be complicated by off-target effects. We suggest instead that Ca2+ changes, well known to affect both ATP generation and consumption, likely drive cytosolic ATP/ADP oscillations that in turn regulate KATP channels and membrane potential. Thus, it remains to be demonstrated that a new model is required to replace the existing, mitochondrial bioenergetics-based model., (© 2024 by the American Diabetes Association.)

Importance: Prenatal exposure to ubiquitous endocrine-disrupting chemicals (EDCs) may increase the risk of metabolic syndrome (MetS) in children, but few studies have studied chemical mixtures or explored underlying protein and metabolic signatures., Objective: To investigate associations of prenatal exposure to EDC mixtures with MetS risk score in children and identify associated proteins and metabolites., Design, Setting, and Participants: This population-based, birth cohort study used data collected between April 1, 2003, and February 26, 2016, from the Human Early Life Exposome cohort based in France, Greece, Lithuania, Norway, Spain, and the UK. Eligible participants included mother-child pairs with measured prenatal EDC exposures and complete data on childhood MetS risk factors, proteins, and metabolites. Data were analyzed between October 2022 and July 2023., Exposures: Nine metals, 3 organochlorine pesticides, 5 polychlorinated biphenyls, 2 polybrominated diphenyl ethers (PBDEs), 5 perfluoroalkyl substances (PFAS), 10 phthalate metabolites, 3 phenols, 4 parabens, and 4 organophosphate pesticide metabolites measured in urine and blood samples collected during pregnancy., Main Outcomes and Measures: At 6 to 11 years of age, a composite MetS risk score was constructed using z scores of waist circumference, systolic and diastolic blood pressures, triglycerides, high-density lipoprotein cholesterol, and insulin levels. Childhood levels of 44 urinary metabolites, 177 serum metabolites, and 35 plasma proteins were quantified using targeted methods. Associations were assessed using bayesian weighted quantile sum regressions applied to mixtures for each chemical group., Results: The study included 1134 mothers (mean [SD] age at birth, 30.7 [4.9] years) and their children (mean [SD] age, 7.8 [1.5] years; 617 male children [54.4%] and 517 female children [45.6%]; mean [SD] MetS risk score, -0.1 [2.3]). MetS score increased per 1-quartile increase of the mixture for metals (β = 0.44; 95% credible interval [CrI], 0.30 to 0.59), organochlorine pesticides (β = 0.22; 95% CrI, 0.15 to 0.29), PBDEs (β = 0.17; 95% CrI, 0.06 to 0.27), and PFAS (β = 0.19; 95% CrI, 0.14 to 0.24). High-molecular weight phthalate mixtures (β = -0.07; 95% CrI, -0.10 to -0.04) and low-molecular weight phthalate mixtures (β = -0.13; 95% CrI, -0.18 to -0.08) were associated with a decreased MetS score. Most EDC mixtures were associated with elevated proinflammatory proteins, amino acids, and altered glycerophospholipids, which in turn were associated with increased MetS score., Conclusions and Relevance: This cohort study suggests that prenatal exposure to EDC mixtures may be associated with adverse metabolic health in children. Given the pervasive nature of EDCs and the increase in MetS, these findings hold substantial public health implications.

Variants at the SLC30A8 locus are associated with type 2 diabetes (T2D) risk. The lead variant, rs13266634, encodes an amino acid change, Arg325Trp (R325W), at the C-terminus of the secretory granule-enriched zinc transporter, ZnT8. Although this protein-coding variant was previously thought to be the sole driver of T2D risk at this locus, recent studies have provided evidence for lowered expression of SLC30A8 mRNA in protective allele carriers. In the present study, we examined multiple variants that influence SLC30A8 allele-specific expression. Epigenomic mapping has previously identified an islet-selective enhancer cluster at the SLC30A8 locus, hosting multiple T2D risk and cASE associations, which is spatially associated with the SLC30A8 promoter and additional neighboring genes. Here, we show that deletion of variant-bearing enhancer regions using CRISPR-Cas9 in human-derived EndoC-βH3 cells lowers the expression of SLC30A8 and several neighboring genes and improves glucose-stimulated insulin secretion. While downregulation of SLC30A8 had no effect on beta cell survival, loss of UTP23, RAD21, or MED30 markedly reduced cell viability. Although eQTL or cASE analyses in human islets did not support the association between these additional genes and diabetes risk, the transcriptional regulator JQ1 lowered the expression of multiple genes at the SLC30A8 locus and enhanced stimulated insulin secretion., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

We tracked the consequences of in utero protein restriction in mice throughout their development and life course using a luciferase-based allelic reporter of imprinted Cdkn1c. Exposure to gestational low-protein diet (LPD) results in the inappropriate expression of paternally inherited Cdkn1c in the brains of embryonic and juvenile mice. These animals were characterised by a developmental delay in motor skills, and by behavioural alterations indicative of reduced anxiety. Exposure to LPD in utero resulted in significantly more tyrosine hydroxylase positive (dopaminergic) neurons in the midbrain of adult offspring as compared to age-matched, control-diet equivalents. Positron emission tomography (PET) imaging revealed an increase in striatal dopamine synthesis capacity in LPD-exposed offspring, where elevated levels of dopamine correlated with an enhanced sensitivity to cocaine. These data highlight a profound sensitivity of the developing epigenome to gestational protein restriction. Our data also suggest that loss of Cdkn1c imprinting and p57 KIP2 upregulation alters the cellular composition of the developing midbrain, compromises dopamine circuitry, and thereby provokes behavioural abnormalities in early postnatal life. Molecular analyses showed that despite this phenotype, exposure to LPD solely during pregnancy did not significantly change the expression of key neuronal- or dopamine-associated marker genes in adult offspring., (© 2024. The Author(s).)

Aryl hydrocarbon receptor (AHR) signalling integrates biological processes that sense and respond to environmental, dietary, and metabolic challenges to ensure tissue homeostasis. AHR is a transcription factor that is inactive in the cytosol but upon encounter with ligand translocates to the nucleus and drives the expression of AHR targets, including genes of the cytochrome P4501 family of enzymes such as Cyp1a1. To dynamically visualise AHR activity in vivo, we generated reporter mice in which firefly luciferase (Fluc) was non-disruptively targeted into the endogenous Cyp1a1 locus. Exposure of these animals to FICZ, 3-MC or to dietary I3C induced strong bioluminescence signal and Cyp1a1 expression in many organs including liver, lung and intestine. Longitudinal studies revealed that AHR activity was surprisingly long-lived in the lung, with sustained Cyp1a1 expression evident in discrete populations of cells including columnar epithelia around bronchioles. Our data link diet to lung physiology and also reveal the power of bespoke Cyp1a1-Fluc reporters to longitudinally monitor AHR activity in vivo., (© 2024. The Author(s).)

Background: The Coronavirus 2019 (COVID-19) pandemic has favored the growth of telemedicine systems and in this context the idea of Metaverse was born and developed. A 3D reality in which people can interact with each other through digital reproductions of themselves. Metaverse has already been tested in numerous medical fields due to its ability to combine visual and auditory information with tactile sensations. The purpose of this study is to highlight its potential also in its ability to be used as a telementoring place where the skills and knowledge of surgeons from all over the world can be combined., Material and Methods: The first HPB Surgery Workshop was held at the "Metaverse Surgical Hospital, USA". During the workshop, surgeons located in various parts of the world reported on hepatic, pancreatic and biliary tract surgery and remotely supported the execution of a robotic liver resection., Results: The Metaverse gave the opportunity for surgeons to meet and discuss HPB pathologies and its surgical strategies and for surgeons in training to interface with experts by participating in a moment of advanced training., Conclusion: In the Metaverse, telementoring can be used at very low cost to improve clinical and surgical practice., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objective: Analysis of postmortem multiple sclerosis (MS) tissues combined with in vivo disease milestones suggests that whereas perivascular white matter infiltrates are associated with demyelinating activity in the initial stages, leptomeningeal immune cell infiltration, enriched in B cells, and associated cortical lesions contribute to disease progression. We systematically examine the association of inflammatory features and white matter demyelination at postmortem with clinical milestones., Methods: In 269 MS brains, 20 sites were examined using immunohistochemistry for active lesions (ALs) and perivenular inflammation (PVI). In a subset of 22, a detailed count of CD20+ B cells and CD3+ T cells in PVIs was performed., Results: ALs were detected in 22%, whereas high levels of PVI were detected in 52% of cases. ALs were present in 35% of cases with high levels of PVI. Shorter time from onset of progression to death was associated with increased prevalence and higher levels of PVI (both p < 0.0001). Shorter time from onset of progression to wheelchair use was associated with higher prevalence of ALs (odds ratio [OR] = 0.921, 95% confidence interval [CI] = 0.858-0.989, p = 0.0230) and higher level of PVI (OR = 0.932, 95% CI = 0.886-0.981, p = 0.0071). High levels of PVI were associated with meningeal inflammation and increased cortical demyelination and significantly higher levels of B lymphocytes within the PVI., Interpretation: ALs, a feature of early disease stage, persist up to death in a subgroup with high levels of PVI. These features link to a rapid progressive phase and higher levels of meningeal inflammation and B-cell infiltrates, supporting the hypothesis that chronic inflammation drives progression in MS. ANN NEUROL 2024;95:706-719., (© 2023 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Three quarters of all breast cancers express the estrogen receptor (ER, ESR1 gene), which promotes tumor growth and constitutes a direct target for endocrine therapies. ESR1 mutations have been implicated in therapy resistance in metastatic breast cancer, in particular to aromatase inhibitors. ESR1 mutations promote constitutive ER activity and affect other signaling pathways, allowing cancer cells to proliferate by employing mechanisms within and without direct regulation by the ER. Although subjected to extensive genetic and transcriptomic analyses, understanding of protein alterations remains poorly investigated. Towards this, we employed an integrated mass spectrometry based proteomic approach to profile the protein and phosphoprotein differences in breast cancer cell lines expressing the frequent Y537N and Y537S ER mutations. Global proteome analysis revealed enrichment of mitotic and immune signaling pathways in ER mutant cells, while phosphoprotein analysis evidenced enriched activity of proliferation associated kinases, in particular CDKs and mTOR. Integration of protein expression and phosphorylation data revealed pathway-dependent discrepancies (motility vs proliferation) that were observed at varying degrees across mutant and wt ER cells. Additionally, protein expression and phosphorylation patterns, while under different regulation, still recapitulated the estrogen-independent phenotype of ER mutant cells. Our study is the first proteome-centric characterization of ESR1 mutant models, out of which we confirm estrogen independence of ER mutants and reveal the enrichment of immune signaling pathways at the proteomic level., (© 2024. The Author(s).)

Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallization has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 15 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7 and provide insights into the mechanisms contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. These results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design., (© 2024. The Author(s).)

Background: Ovarian cancer is the most lethal and endometrial cancer the most common gynaecological cancer in the UK, yet neither have a screening program in place to facilitate early disease detection. The aim is to evaluate whether online search data can be used to differentiate between individuals with malignant and benign gynaecological diagnoses., Methods: This is a prospective cohort study evaluating online search data in symptomatic individuals (Google user) referred from primary care (GP) with a suspected cancer to a London Hospital (UK) between December 2020 and June 2022. Informed written consent was obtained and online search data was extracted via Google takeout and anonymised. A health filter was applied to extract health-related terms for 24 months prior to GP referral. A predictive model (outcome: malignancy) was developed using (1) search queries (terms model) and (2) categorised search queries (categories model). Area under the ROC curve (AUC) was used to evaluate model performance. 844 women were approached, 652 were eligible to participate and 392 were recruited. Of those recruited, 108 did not complete enrollment, 12 withdrew and 37 were excluded as they did not track Google searches or had an empty search history, leaving a cohort of 235., Results: The cohort had a median age of 53 years old (range 20-81) and a malignancy rate of 26.0%. There was a difference in online search data between those with a benign and malignant diagnosis, noted as early as 360 days in advance of GP referral, when search queries were used directly, but only 60 days in advance, when queries were divided into health categories. A model using online search data from patients (n = 153) who performed health-related search and corrected for sample size, achieved its highest sample-corrected AUC of 0.82, 60 days prior to GP referral., Conclusions: Online search data appears to be different between individuals with malignant and benign gynaecological conditions, with a signal observed in advance of GP referral date. Online search data needs to be evaluated in a larger dataset to determine its value as an early disease detection tool and whether its use leads to improved clinical outcomes., (© 2024. The Author(s).)

Two APOBEC (apolipoprotein-B mRNA editing enzyme catalytic polypeptide-like) DNA cytosine deaminase enzymes (APOBEC3A and APOBEC3B) generate somatic mutations in cancer, driving tumour development and drug resistance. Here we used single cell RNA sequencing to study APOBEC3A and APOBEC3B expression in healthy and malignant mucosal epithelia, validating key observations with immunohistochemistry, spatial transcriptomics and functional experiments. Whereas APOBEC3B is expressed in keratinocytes entering mitosis, we show that APOBEC3A expression is confined largely to terminally differentiating cells and requires Grainyhead-like transcription factor 3 (GRHL3). Thus, in normal tissue, neither deaminase appears to be expressed at high levels during DNA replication, the cell cycle stage associated with APOBEC-mediated mutagenesis. In contrast, we show that in squamous cell carcinoma tissues, there is expansion of GRHL3 expression and activity to a subset of cells undergoing DNA replication and concomitant extension of APOBEC3A expression to proliferating cells. These findings indicate a mechanism for acquisition of APOBEC3A mutagenic activity in tumours., Competing Interests: Competing Interests T.R.F. is an advisory board member of and holds stock options in APOBEC Discovery Ltd,

Tilt table testing (TTT) has been used for decades to study short-term blood pressure (BP) and heart rate regulation during orthostatic challenges. TTT provokes vasovagal reflex in many syncope patients as a background of widespread use. Despite the availability of evidence-based practice syncope guidelines, proper application and interpretation of TTT in the day-to-day care of syncope patients remain challenging. In this review, we offer practical information on what is needed to perform TTT, how results should be interpreted including the Vasovagal Syncope International Study classification, why syncope induction on TTT is necessary in patients with unexplained syncope and on indications for TTT in syncope patient care. The minimum requirements to perform TTT are a tilt table with an appropriate tilt-down time, a continuous beat-to-beat BP monitor with at least three electrocardiogram leads and trained staff. We emphasize that TTT remains a valuable asset that adds to history building but cannot replace it, and highlight the importance of recognition when TTT is abnormal even without syncope. Acknowledgement by the patient/eyewitness of the reproducibility of the induced attack is mandatory in concluding a diagnosis. TTT may be indicated when the initial syncope evaluation does not yield a certain, highly likely, or possible diagnosis, but raises clinical suspicion of (1) reflex syncope, (2) orthostatic hypotension (OH), (3) postural orthostatic tachycardia syndrome or (4) psychogenic pseudosyncope. A therapeutic indication for TTT in the patient with a certain, highly likely or possible diagnosis of reflex syncope, may be to educate patients on prodromes. In patients with reflex syncope with OH TTT can be therapeutic to recognize hypotensive symptoms causing near-syncope to perform physical countermanoeuvres for syncope prevention (biofeedback). Detection of hypotensive susceptibility requiring therapy is of special value., (© 2023 The Authors. Clinical Physiology and Functional Imaging published by John Wiley & Sons Ltd on behalf of Scandinavian Society of Clinical Physiology and Nuclear Medicine.)

Exploring the molecular basis of disease severity in rare disease scenarios is a challenging task provided the limitations on data availability. Causative genes have been described for Congenital Myasthenic Syndromes (CMS), a group of diverse minority neuromuscular junction (NMJ) disorders; yet a molecular explanation for the phenotypic severity differences remains unclear. Here, we present a workflow to explore the functional relationships between CMS causal genes and altered genes from each patient, based on multilayer network community detection analysis of complementary biomedical information provided by relevant data sources, namely protein-protein interactions, pathways and metabolomics. Our results show that CMS severity can be ascribed to the personalized impairment of extracellular matrix components and postsynaptic modulators of acetylcholine receptor (AChR) clustering. This work showcases how coupling multilayer network analysis with personalized -omics information provides molecular explanations to the varying severity of rare diseases; paving the way for sorting out similar cases in other rare diseases., (© 2024. The Author(s).)

Comprehensive proteomic analysis is essential to elucidate molecular pathways and protein functions. Despite tremendous progress in proteomics, current studies still suffer from limited proteomic coverage and dynamic range. Here, we utilize micropillar array columns (µPACs) together with wide-window acquisition and the AI-based CHIMERYS search engine to achieve excellent proteomic comprehensiveness for bulk proteomics, affinity purification mass spectrometry and single cell proteomics. Our data show that µPACs identify ≤50% more peptides and ≤24% more proteins, while offering improved throughput, which is critical for large (clinical) proteomics studies. Combining wide precursor isolation widths of m/z 4-12 with the CHIMERYS search engine identified +51-74% and +59-150% more proteins and peptides, respectively, for single cell, co-immunoprecipitation, and multi-species samples over a conventional workflow at well-controlled false discovery rates. The workflow further offers excellent precision, with CVs <7% for low input bulk samples, and accuracy, with deviations <10% from expected fold changes for regular abundance two-proteome mixes. Compared to a conventional workflow, our entire optimized platform discovered 92% more potential interactors in a protein-protein interaction study on the chromatin remodeler Smarca5/Snf2h. These include previously described Smarca5 binding partners and undescribed ones including Arid1a, another chromatin remodeler with key roles in neurodevelopmental and malignant disorders., (© 2024. The Author(s).)

Background: The serotonergic neurotransmitter system is involved in many ethanol-induced changes, including many behavioural alterations, as well as contributing to alcohol dependence and its withdrawal., Aims: This review has evaluated microdialysis studies where alterations in the serotonin system, that is, serotonin, 5-HT, or its metabolite 5-hydroxyindoleacetic acid, 5-HIAA, have been reported during different ethanol intoxication states, as well as in animals showing alcohol preference or not. Changes in 5-HT receptors and the 5-HT transporter are briefly reviewed to comprehend the significance of changes in microdialysate 5-HT concentrations., Materials and Methods: Changes in 5-HT content following acute, chronic and during ethanol withdrawal states are evaluated. In addition, the serotoninergic system was assessed in animals that have been genetically selected for alcohol preference to ascertain whether changes in this monoamine microdialysate content may contribute to alcohol preference., Results and Discussion: Changes occurred in 5-HT signalling in the limbic brain regions, increasing after acute ethanol administration in specific brain regions, particularly at higher doses, while chronic alcohol exposure essentially decreased serotonergic transmission. Such changes may play a pivotal role in emotion-driven craving and relapse. Depending on the dosage, mode of administration and consumption rate, ethanol affects specific brain regions in different ways, enhancing or reducing 5-HT microdialysate content, thereby inducing behavioural and cognitive functions and enhancing ethanol consumption., Conclusion: Microdialysis studies demonstrated that ethanol induces several alterations in 5-HT content as well as its metabolites, 5-HIAA and 5-HTOL, not only in its release from a specific brain region but also in the modifications of its different receptor subtypes and its transporter., (© 2023 Société Française de Pharmacologie et de Thérapeutique. Published by John Wiley & Sons Ltd.)

During development, the rate of tissue growth is determined by the relative balance of cell division and cell death. Cell competition is a fitness quality-control mechanism that contributes to this balance by eliminating viable cells that are less fit than their neighbours. The mutations that confer cells with a competitive advantage and the dynamics of the interactions between winner and loser cells are not well understood. Here, we show that embryonic cells lacking the tumour suppressor p53 are 'super-competitors' that eliminate their wild-type neighbours through the direct induction of apoptosis. This elimination is context dependent, as it does not occur when cells are pluripotent and it is triggered by the onset of differentiation. Furthermore, by combining mathematical modelling and cell-based assays we show that the elimination of wild-type cells is not through competition for space or nutrients, but instead is mediated by short-range interactions that are dependent on the local cell neighbourhood. This highlights the importance of the local cell neighbourhood and the competitive interactions within this neighbourhood for the regulation of proliferation during early embryonic development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: People with severe COVID anxiety have poor mental health and impaired functioning, but the course of severe COVID anxiety is unknown and the quality of evidence on the acceptability and impact of psychological interventions is low., Methods: A quantitative cohort study with a nested feasibility trial. Potential participants aged 18 and over, living in the UK with severe COVID anxiety, were recruited online and from primary care services. We examined levels of COVID anxiety in the six months after recruitment, and factors that influenced this, using linear regression. Those scoring above 20 on the short Health Anxiety Inventory were invited to participate in a feasibility trial of remotely delivered Cognitive Behavioural Therapy for Health Anxiety (CBT-HA). Exclusion criteria were recent COVID-19, current self-isolation, or current receipt of psychological treatment. Key outcomes for the feasibility trial were the level of uptake of CBT-HA and the rate of follow-up., Results: 204 (70.2%) of 285 people who took part in the cohort study completed the six month follow-up, for whom levels of COVID anxiety fell from 12.4 at baseline to 6.8 at six months (difference = -5.5, 95% CI = -6.0 to -4.9). Reductions in COVID anxiety were lower among older people, those living with a vulnerable person, those with lower baseline COVID anxiety, and those with higher levels of generalised anxiety and health anxiety at baseline. 36 (90%) of 40 participants enrolled in the nested feasibility trial were followed up at six months. 17 (80.9%) of 21 people in the active arm of the trial received four or more sessions of CBT-HA. We found improved mental health and social functioning among those in the active, but not the control arm of the trial (Mean difference in total score on the Work and Social Adjustment Scale between baseline and follow up, was 9.7 (95% CI = 5.8-13.6) among those in the active, and 1.0 (95% C.I. = -4.6 to 6.6) among those in the control arm of the trial., Conclusions: While the mental health of people with severe COVID anxiety appears to improve over time, many continue to experience high levels of anxiety and poor social functioning. Health anxiety is highly prevalent among people with severe COVID anxiety and may provide a target for psychological treatment., Trial Registration: Retrospectively registered at ISRCTN14973494 on 09/09/2021., (© 2023. The Author(s).)

JASPAR (https://jaspar.elixir.no/) is a widely-used open-access database presenting manually curated high-quality and non-redundant DNA-binding profiles for transcription factors (TFs) across taxa. In this 10th release and 20th-anniversary update, the CORE collection has expanded with 329 new profiles. We updated three existing profiles and provided orthogonal support for 72 profiles from the previous release's UNVALIDATED collection. Altogether, the JASPAR 2024 update provides a 20% increase in CORE profiles from the previous release. A trimming algorithm enhanced profiles by removing low information content flanking base pairs, which were likely uninformative (within the capacity of the PFM models) for TFBS predictions and modelling TF-DNA interactions. This release includes enhanced metadata, featuring a refined classification for plant TFs' structural DNA-binding domains. The new JASPAR collections prompt updates to the genomic tracks of predicted TF binding sites (TFBSs) in 8 organisms, with human and mouse tracks available as native tracks in the UCSC Genome browser. All data are available through the JASPAR web interface and programmatically through its API and the updated Bioconductor and pyJASPAR packages. Finally, a new TFBS extraction tool enables users to retrieve predicted JASPAR TFBSs intersecting their genomic regions of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Programmed death ligand-1 (PD-L1) expression on circulating tumor cells (CTCs) has been suggested to provide prognostic information in non-small cell lung cancer (NSCLC), but consensus relative to treatment outcomes is lacking. We conducted the first comprehensive meta-analysis exploring its potential as a prognostic and predictive marker, and assessed the concordance between PD-L1 + CTCs and paired tumor tissue in NSCLC patients., Method: A comprehensive search was applied to PubMed and EMBASE to identify 26 studies that evaluated PD-L1 + CTCs and their association with survival outcomes in 1236 NSCLC patients., Results: The meta-analysis estimated a mean PD-L1 + CTCs detection rate of 61% (95% CI, 49-72). Subgroup analysis based on treatment showed that PD-L1 + CTCs was not significantly associated with better overall survival (OS) in NSCLC patients treated with immune checkpoint inhibitors (ICIs) (Hazard Ratio (HR) = 0.96, 95% CI, 0.35-2.65, P = 0.944), but was predictive of worse OS in those treated with other therapies (HR = 2.11, 95% CI, 1.32-3.36, P = 0.002). Similarly, PD-L1 + CTCs was not significantly associated with superior progressing free survival (PFS) in NSCLCs treated with ICIs (HR = 0.67, 95% CI, 0.41-1.09, P = 0.121), but was significantly associated with shorter PFS in patients treated with other therapies (HR = 1.91, 95% CI, 1.24-2.94, P = 0.001). The overall estimate for the concordance between PD-L1 expression on CTCs and tumor cells was 63% (95% CI, 44-80)., Conclusion: The average detection rate of PD-L1 + CTCs was comparable to the rate of PD-L1 expression in NSCLC tumors. There was a trend towards better PFS in ICI-treated NSCLC patients with PD-L1 + CTCs. Larger longitudinal studies on the association of PD-L1 + CTCs with clinical outcomes in NSCLC patients treated with ICIs are warranted., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Malignant transformation is characterised by aberrant phospholipid metabolism of cancers, associated with the upregulation of choline kinase alpha (CHKα). Due to the metabolic instability of choline radiotracers and the increasing use of late-imaging protocols, we developed a more stable choline radiotracer, [ 18 F]fluoromethyl-[1,2- 2 H 4 ]choline ([ 18 F]D4-FCH). [ 18 F]D4-FCH has improved protection against choline oxidase, the key choline catabolic enzyme, via a 1 H/ 2 D isotope effect, together with fluorine substitution. Due to the promising mechanistic and safety profiles of [ 18 F]D4-FCH in vitro and preclinically, the radiotracer has transitioned to clinical development. [ 18 F]D4-FCH is a safe positron emission tomography (PET) tracer, with a favourable radiation dosimetry profile for clinical imaging. [ 18 F]D4-FCH PET/CT in lung and prostate cancers has shown highly heterogeneous intratumoral distribution and large lesion variability. Treatment with abiraterone or enzalutamide in metastatic castrate-resistant prostate cancer patients elicited mixed responses on PET at 12-16 weeks despite predominantly stable radiological appearances. The sum of the weighted tumour-to-background ratios (TBRs-wsum) was associated with the duration of survival.

Precise localization and dissection of gene promoters are key to understanding transcriptional gene regulation and to successful bioengineering applications. The core RNA polymerase II initiation machinery is highly conserved among eukaryotes, leading to a general expectation of equivalent underlying mechanisms. Still, less is known about promoters in the plant kingdom. In this study, we employed cap analysis of gene expression (CAGE) at three embryonic developmental stages in barley to accurately map, annotate, and quantify transcription initiation events. Unsupervised discovery of de novo sequence clusters grouped promoters based on characteristic initiator and position-specific core-promoter motifs. This grouping was complemented by the annotation of transcription factor binding site (TFBS) motifs. Integration with genome-wide epigenomic data sets and gene ontology (GO) enrichment analysis further delineated the chromatin environments and functional roles of genes associated with distinct promoter categories. The TATA-box presence governs all features explored, supporting the general model of two separate genomic regulatory environments. We describe the extent and implications of alternative transcription initiation events, including those that are specific to developmental stages, which can affect the protein sequence or the presence of regions that regulate translation. The generated promoterome dataset provides a valuable genomic resource for enhancing the functional annotation of the barley genome. It also offers insights into the transcriptional regulation of individual genes and presents opportunities for the informed manipulation of promoter architecture, with the aim of enhancing traits of agronomic importance., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

The liver plays a key role in sensing nutritional and hormonal inputs to maintain metabolic homeostasis. Recent studies into pre-mRNA splicing and alternative splicing (AS) and their effects on gene expression have revealed considerable transcriptional complexity in the liver, both in health and disease. While the contribution of these mechanisms to cell and tissue identity is widely accepted, their role in physiological and pathological contexts within tissues is just beginning to be appreciated. In this review, we showcase recent studies on the splicing and AS of key genes in metabolic pathways in the liver, the effect of metabolic signals on the spliceosome, and therapeutic intervention points based on RNA splicing., Competing Interests: Declaration of interests A.M.J., S.Y., H.A.B.P., and S.V. are inventors on a patent covering the use of SSOs to modify liver metabolism. I.C.’s partner has stock options in Ochre Bio. The other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Neuronal homeostasis prevents hyperactivity and hypoactivity. Age-related hyperactivity suggests homeostasis may be dysregulated in later life. However, plasticity mechanisms preventing age-related hyperactivity and their efficacy in later life are unclear. We identify the adult cortical plasticity response to elevated activity driven by sensory overstimulation, then test how plasticity changes with age. We use in vivo two-photon imaging of calcium-mediated cellular/synaptic activity, electrophysiology and c-Fos-activity tagging to show control of neuronal activity is dysregulated in the visual cortex in late adulthood. Specifically, in young adult cortex, mGluR5-dependent population-wide excitatory synaptic weakening and inhibitory synaptogenesis reduce cortical activity following overstimulation. In later life, these mechanisms are downregulated, so that overstimulation results in synaptic strengthening and elevated activity. We also find overstimulation disrupts cognition in older but not younger animals. We propose that specific plasticity mechanisms fail in later life dysregulating neuronal microcircuit homeostasis and that the age-related response to overstimulation can impact cognitive performance., (© 2023. The Author(s).)

Pancreatic ductal adenocarcinoma (PDAC) has a very poor survival. The intra-tumoural microbiome can influence pancreatic tumourigenesis and chemoresistance and, therefore, patient survival. The role played by bile microbiota in PDAC is unknown. We aimed to define bile microbiome signatures that can effectively distinguish malignant from benign tumours in patients presenting with obstructive jaundice caused by benign and malignant pancreaticobiliary disease. Prospective bile samples were obtained from 31 patients who underwent either Endoscopic Retrograde Cholangiopancreatography (ERCP) or Percutaneous Transhepatic Cholangiogram (PTC). Variable regions (V3-V4) of the 16S rRNA genes of microorganisms present in the samples were amplified by Polymerase Chain Reaction (PCR) and sequenced. The cohort consisted of 12 PDAC, 10 choledocholithiasis, seven gallstone pancreatitis and two primary sclerosing cholangitis patients. Using the 16S rRNA method, we identified a total of 135 genera from 29 individuals (12 PDAC and 17 benign). The bile microbial beta diversity significantly differed between patients with PDAC vs. benign disease (Permanova p = 0.0173). The separation of PDAC from benign samples is clearly seen through unsupervised clustering of Aitchison distance. We found three genera to be of significantly lower abundance among PDAC samples vs. benign, adjusting for false discovery rate (FDR). These were Escherichia (FDR = 0.002) and two unclassified genera, one from Proteobacteria (FDR = 0.002) and one from Enterobacteriaceae (FDR = 0.011). In the same samples, the genus Streptococcus (FDR = 0.033) was found to be of increased abundance in the PDAC group. We show that patients with obstructive jaundice caused by PDAC have an altered microbiome composition in the bile compared to those with benign disease. These bile-based microbes could be developed into potential diagnostic and prognostic biomarkers for PDAC and warrant further investigation.

Core promoters are stretches of DNA at the beginning of genes that contain information that facilitates the binding of transcription initiation complexes. Different functional subsets of genes have core promoters with distinct architectures and characteristic motifs. Some of these motifs inform the selection of transcription start sites (TSS). By discovering motifs with fixed distances from known TSS positions, we could in principle classify promoters into different functional groups. Due to the variability and overlap of architectures, promoter classification is a difficult task that requires new approaches. In this study, we present a new method based on non-negative matrix factorisation (NMF) and the associated software called seqArchR that clusters promoter sequences based on their motifs at near-fixed distances from a reference point, such as TSS. When combined with experimental data from CAGE, seqArchR can efficiently identify TSS-directing motifs, including known ones like TATA, DPE, and nucleosome positioning signal, as well as novel lineage-specific motifs and the function of genes associated with them. By using seqArchR on developmental time courses, we reveal how relative use of promoter architectures changes over time with stage-specific expression. seqArchR is a powerful tool for initial genome-wide classification and functional characterisation of promoters. Its use cases are more general: it can also be used to discover any motifs at near-fixed distances from a reference point, even if they are present in only a small subset of sequences., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Nikumbh, Lenhard. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: We investigated intestinal permeability and fecal, plasma, and urine metabolomic profiles in methotrexate-treated active psoriatic arthritis (PsA) and how this related to clinical response following one sham or fecal microbiota transplantation (FMT)., Methods: This exploratory study is based on the FLORA trial cohort, in which 31 patients with moderate-to-high peripheral PsA disease activity, despite at least 3 months of methotrexate-treatment, were included in a 26-week, double-blind, 1:1 randomized, sham-controlled trial. Participants were randomly allocated to receive either one healthy donor FMT (n = 15) or sham (n = 16) via gastroscopy. The primary trial end point was the proportion of treatment failures through 26 weeks. We performed a lactulose-to-mannitol ratio (LMR) test at baseline (n = 31) and at week 26 (n = 26) to assess small intestinal permeability. Metabolomic profiles in fecal, plasma, and urine samples collected at baseline, weeks 4, 12, and 26 were measured using 1 H Nuclear Magnetic Resonance., Results: Trial failures (n = 7) had significantly higher LMR compared with responders (n = 19) at week 26 (0.027 [0.017-0.33]) vs. 0.012 [0-0.064], P = 0.013), indicating increased intestinal permeability. Multivariate analysis revealed a significant model for responders (n = 19) versus failures (n = 12) at all time points based on their fecal (P < 0.0001) and plasma (P = 0.005) metabolomic profiles, whereas urine metabolomic profiles did not differ between groups (P = 1). Fecal N-acetyl glycoprotein GlycA correlated with Health Assessment Questionnaire Disability Index (coefficient = 0.50; P = 0.03) and fecal propionate correlated with American College of Rheumatology 20 response at week 26 (coefficient = 27, P = 0.02)., Conclusion: Intestinal permeability and fecal and plasma metabolomic profiles of patients with PsA were associated with the primary clinical trial end point, failure versus responder., (© 2023 The Authors. ACR Open Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)