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Introduction: The primary analysis (median follow-up 34.9 mo across all arms) of the phase 3 POSEIDON study revealed a statistically significant overall survival (OS) improvement with first-line tremelimumab plus durvalumab and chemotherapy (T+D+CT) versus CT in patients with EGFR and ALK wild-type metastatic NSCLC (mNSCLC). D+CT had a trend for OS improvement versus CT that did not reach statistical significance. This article reports prespecified OS analyses after long-term follow-up (median >5 y)., Methods: A total of 1013 patients were randomized (1:1:1) to T+D+CT, D+CT, or CT, stratified by tumor cell programmed cell death ligand-1 (PD-L1) expression (≥50% versus <50%), disease stage (IVA versus IVB), and tumor histologic type (squamous versus nonsquamous). Serious adverse events were collected during follow-up., Results: After a median follow-up of 63.4 months across all arms, T+D+CT had sustained OS benefit versus CT (hazard ratio [HR] = 0.76, 95% confidence interval [CI]: 0.64-0.89; 5-y OS: 15.7% versus 6.8%). OS improvement with D+CT versus CT (HR = 0.84, 95% CI: 0.72-1.00; 5-y OS: 13.0%) was consistent with the primary analysis. OS benefit with T+D+CT versus CT remained more pronounced in nonsquamous (HR = 0.69, 95% CI: 0.56-0.85) versus squamous (HR = 0.85, 95% CI: 0.65-1.10) mNSCLC. OS benefit with T+D+CT versus CT was still evident regardless of PD-L1 expression, including patients with PD-L1 tumor cell less than 1%, and remained evident in STK11-mutant (nonsquamous), KEAP1-mutant, and KRAS-mutant (nonsquamous) mNSCLC. No new safety signals were identified., Conclusions: After a median follow-up of more than 5 years, T+D+CT had durable long-term OS benefit versus CT, supporting its use as first-line treatment in mNSCLC, including in patient subgroups with harder-to-treat disease., Competing Interests: Disclosure Peters has received grants (paid to institution) from Amgen, Arcus, AstraZeneca, BeiGene, Boehringer Ingelheim, Bristol-Myers Squibb, F. Hoffmann-La Roche/Genentech, GlaxoSmithKline, iTeos, Merck Sharp & Dohme, Merck Serono, Mirati, PharmaMar, Promontory Therapeutics, and Seattle Genetics; has received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events (paid to institution) from AiCME, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, ecancer, Eli Lilly, F. Hoffmann-La Roche/Genentech, Foundation Medicine, GlaxoSmithKline, Illumina, Imedex, Ipsen, Medscape, Merck Sharp & Dohme, Mirati, MJH Life Sciences, Novartis, Peerview, Pfizer, RTP, and Takeda; and has participation on a data safety monitoring board or advisory board (payment to institution) for AbbVie, AiCME, Amgen, Arcus, AstraZeneca, Bayer, BeiGene, BerGenBio, Biocartis, Bioinvent, Blueprint Medicines, Boehringer Ingelheim, Bristol-Myers Squibb, Clovis, Daiichi Sankyo, Debiopharm, ecancer, Eli Lilly, Elsevier, F. Hoffmann-La Roche/Genentech, Fishawack, Foundation Medicine, F-Star, Genzyme, Gilead, GlaxoSmithKline, Hutchmed, Illumina, Imedex, Incyte, Ipsen, IQVIA, iTeos, Janssen, Medscape, Medtoday, Merck Sharp & Dohme, Merck Serono, Merrimack, Mirati, MJH Life Sciences, Novartis, Novocure, Nykode Therapeutics, OncologyEducation, Peerview, Pharma Mar, Pfizer, Promontory Therapeutics, Regeneron, RMEI, RTP, Sanofi, Seattle Genetics, and Takeda. Cho reports receiving research funding from MOGAM Institute, LG Chem, Oscotec, Interpark Bio Convergence Corp., GIInnovation, GI-Cell, Abion, AbbVie, AstraZeneca, Bayer, Blueprint Medicines, Boehringer Ingelheim, Champions Oncology, CJ Bioscience, CJ Blossom Park, Cyrus, Dizal Pharma, Genexine, Janssen, Lilly, Merck Sharp & Dohme, Novartis, Nuvalent, Oncternal, Ono, Regeneron, Dong-A ST, Bridgebio Therapeutics, Yuhan, ImmuneOncia, Illumina, Kanaph Therapeutics, Therapex, JINTSbio, Hanmi, CHA Bundang Medical Center, and Vertical Bio AG; receiving royalties from Champions Oncology, Crown Bioscience, Imagen, and PearlRiver Bio GmbH; receiving consulting fees from Abion, BeiGene, Novartis, AstraZeneca, Boehringer Ingelheim, Roche, Bristol-Myers Squibb, CJ, CureLogen, Cyrus Therapeutics, Ono, Onegene Biotechnology, Yuhan, Pfizer, Eli Lilly, GI-Cell, Guardant, HK Inno-N, Imnewrun Biosciences Inc., Janssen, Takeda, Merck Sharp & Dohme, Janssen, Medpacto, Blueprint Medicines, RandBio, and Hanmi; receiving payment or honoraria for presentations from ASCO, AstraZeneca, Guardant, Roche, ESMO, International Association for the Study of Lung Cancer, Korean Cancer Association, Korean Society of Medical Oncology, Korean Society of Thyroid-Head and Neck Surgery, Korean Cancer Study Group, Novartis, Merck Sharp & Dohme, The Chinese Thoracic Oncology Society, and Pfizer; having scientific advisory board participation for Kanaph Therapeutics Inc., Bridgebio Therapeutics, Cyrus Therapeutics, Guardant Health, Oscotec Inc., J INTS Bio, Therapex Co., Ltd., Gilead, and Amgen; having membership of the board of directors for J INTS BIO; having stock ownership in TheraCanVac Inc., Gencurix Inc., Bridgebio Therapeutics, Kanaph Therapeutics Inc., Cyrus Therapeutics, Interpark Bio Convergence Corp., and J INTS BIO; having employment with Yonsei University Health System; and being a founder of DAAN Biotherapeutics. Alatorre-Alexander has received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Amgen, AstraZeneca, Bristol-Myers Squibb, Janssen, Merck Sharp & Dohme, and Roche; received support for attending meetings and/or travel from AstraZeneca, Merck Sharp & Dohme, and Roche; and participated on a data safety monitoring board or advisory board for Amgen, AstraZeneca, Bristol-Myers Squibb, Janssen, Merck Sharp & Dohme, and Roche. Geater has received research funding (paid to institution) from AstraZeneca, Boehringer Ingelheim, Merck Sharp & Dohme, Novartis, and Roche; received honoraria from AstraZeneca and Boehringer Ingelheim; and performed an advisory role for Merck Sharp & Dohme and Pfizer. Laktionov has received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events for AstraZeneca, Biocad, Bristol-Myers Squibb, Merck Sharp & Dohme, and Roche AG, and has participated on a data safety monitoring board or advisory board for AstraZeneca, Biocad, Bristol-Myers Squibb, Merck Sharp & Dohme, Pfizer, and Roche AG. Kim has received grants (paid to institution) from Yuhan and support for attending an investigator meeting from AstraZeneca. Hussein has received consulting fees from IntegraConnect, Coherus Biosciences, Athenex, Karyopharm Therapeutics, Bristol-Myers Squibb, AstraZeneca, Mirati Therapeutics, Exelixis, Biopharma, Oncocyte, Aptitude Health, IntrinsiQ, GIntrinsiQ, National Community Oncology Dispensing Association, Integra PrescisionQ, AbbVie, and CTI BioPharma Corp. Araujo has received grants from Lilly, Boehringer, Merck Sharp & Dohme, Bristol-Myers Squibb, Roche, Pfizer, AstraZeneca, Novartis, and Merck; consulting fees from Merck Sharp & Dohme, Roche, AstraZeneca, Bristol-Myers Squibb, Lilly, Illumina, and Sanofi; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Merck Sharp & Dohme, Bristol-Myers Squibb, AstraZeneca, Boehringer, Merck, Roche, Pfizer, and Lilly; and support for attending meetings and/or travel from Daiichi Sankyo, Bristol-Myers Squibb, and AstraZeneca. Saito has received grants from AstraZeneca, Bristol-Myers Squibb, Chugai Pharmaceutical, and ONO Pharmaceutical, and payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Chugai Pharmaceutical, ONO Pharmaceutical, and Pfizer. Reinmuth has received consulting fees from Amgen, AstraZeneca, Bristol-Myers Squibb, Daiichi Sankyo, GlaxoSmithKline, Hoffmann-La Roche, Janssen, Merck Sharp & Dohme, and Pfizer; received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Amgen, AstraZeneca, Bristol-Myers Squibb, Boehringer Ingelheim, Daiichi Sankyo, Eli Lilly, GlaxoSmithKline, Hoffmann-La Roche, Janssen, Merck Sharp & Dohme, Pfizer, Sanofi, and Takeda; received support for attending meetings and/or travel from Janssen, Sanofi, and Takeda; and participated on a data safety monitoring board or advisory board for Symphogen. Lowery, Mann, Stewart, and Jiang are employees of, and own stocks in, AstraZeneca. Garon has received grants paid to their institution from ABL-Bio, AstraZeneca, Bristol-Myers Squibb, Daiichi Sanko, Dynavax Technologies, Eli Lilly, EMD Serono, Genentech, Gilead, Iovance Biotherapeutics, Merck, Mirati Therapeutics, Neon, and Novartis; consulting fees paid to their institution from AbbVie, ABL-Bio, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Dracen Pharmaceuticals, EMD Serono, Eisai, Eli Lilly, Gilead, GlaxoSmithKline, Merck, Natera, Novartis, Personalis, Regeneron, Sanofi, Shionogi, Xilio, and Zymeworks; and support for attending meetings and/or travel from A2 Bio and Novartis. Mok reports receiving grants paid to their institution from AstraZeneca, Bristol-Myers Squibb, G1 Therapeutics, Merck Sharp & Dohme, Merck Serono, Novartis, Pfizer, Roche, SFJ, Takeda, and Xcovery; consulting fees from AbbVie Inc., ACEA Pharma, Adagene, Alpha Biopharma Co., Ltd., Amgen, Amoy Diagnostics Co., AVEO Pharmaceuticals, Inc., Bayer Healthcare Pharmaceuticals Ltd., BeiGene, Berry Oncology, Boehringer Ingelheim, Blueprint Medicines Corporation, Bristol-Myers Squibb, Bowtie Life Insurance Company Limited, Bridge Biotherapeutics Inc., Covidien LP, C4 Therapeutics Inc., Cirina Ltd., CStone Pharmaceuticals, Curio Science, D3 Bio Ltd., Da Volterra, Daiichi Sankyo, Eisai, Elevation Oncology, F. Hoffmann-La Roche Ltd./Genentech, Fishawack Facilitate Ltd., G1 Therapeutics Inc., geneDecode Co., Ltd., Gilead Sciences, Inc., GLG’s Healthcare, Gritstone Oncology, Inc., Guardant Health, Hengrui Therapeutics Inc., HutchMed, Ignyta, Inc., Illumina Inc., Incyte Corporation, Inivata, IQVIA, Janssen, Lakeshore Biotech Ltd., Lilly, Lunit USA, Inc., Loxo-Oncology, Lucence Health Inc., Medscape LLC/WebMD, Medtronic, Merck Serono, Merck Sharp & Dohme, Mirati Therapeutics Inc., MiRXES, MoreHealth, Novartis, Novocure GmbH, Omega Therapeutics Inc., OrigiMed, OSE Immunotherapeutics, PeerVoice, Pfizer, PrIME Oncology, Prenetics, Puma Biotechnology Inc., Qiming Development (HK) Ltd., Regeneron Pharmaceuticals Inc., Roche Pharmaceuticals/Diagnostics/Foundation One, Sanofi-Aventis, SFJ Pharmaceutical Ltd., Simcere of America Inc., Synergy Research, Summit Therapeutics Sub, Inc., Takeda Pharmaceuticals HK Ltd., Tigermed, Vertex Pharmaceuticals, Virtus Medical Group, XENCOR, Inc., and Yuhan Corporation; receiving payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from ACEA Pharma, Alpha Biopharma Co. Ltd., Amgen, Amoy Diagnostics Co. Ltd., AstraZeneca (before January 1, 2019), BeiGene, Boehringer Ingelheim, Bristol-Myers Squibb, Daiichi Sankyo, Daz Group, Fishawack Facilitate Ltd., InMed Medical Communications, Janssen Pharmaceutica NV, Jiahui Holdings Co. Ltd., LiangYiHui Healthcare, Lilly, Lucence Health Inc., MD Health Brazil, Medscape LLC, Merck Pharmaceuticals HK Ltd., Merck Sharp & Dohme, MiRXES, Novartis, OrigiMed Co. Ltd., P. Permanyer SL, PeerVoice, Physicians’ Education Resource, Pfizer, PrIME Oncology, Research to Practice, Roche Pharmaceuticals/Diagnostics/Foundation One, Sanofi-Aventis, Shanghai BeBirds Translation & Consulting Co. Ltd., Taiho Pharmaceutical Co. Ltd., Takeda Oncology, and Touch Independent Medical Education Ltd.; receiving support for attending meetings and/or travel from Boehringer Ingelheim, Pfizer, Bristol-Myers Squibb, Merck Sharp & Dohme (paid to institution), Novartis, Roche (paid to self and institution), AstraZeneca, Daiichi Sankyo, MiRXES, AbbVie, Zai Lab, and Liangyihui (paid to self); having participation on a data safety monitoring board or advisory board for AbbVie Inc., ACEA Pharma, Amgen, AstraZeneca, Berry Oncology, Blueprint Medicines Corporation, Boehringer Ingelheim, Bowtie Life Insurance Co., Ltd., Bristol-Myers Squibb, C4 Therapeutics Inc., Covidien LP, CStone Pharmaceuticals, Curio Science, D3 Bio Ltd., Daiichi Sankyo Inc., Eisai, Fishawack Facilitate Ltd., G1 Therapeutics Inc., Gilead Sciences Inc., Gritstone Oncology Inc., Guardant Health, geneDecode Co. Ltd. (uncompensated), Hengrui Therapeutics Inc., HutchMed, Ignyta Inc., Incyte Corporation, Imagene AI Ltd., Inivata, IQVIA, Janssen, Lakeshore Biotech, Lilly, Loxo-Oncology Inc., Lunit Inc., Merck Serono, Merck Sharp & Dohme, Mirati Therapeutics Inc., MiRXES Group, Novartis, OrigiMed, Pfizer, Puma Biotechnology Inc., Roche/Genentech, Regeneron Pharmaceuticals Inc., Sanofi-Aventis R&D, SFJ Pharmaceutical, Simcere of America Inc., Simcere Zaiming, Inc., Takeda, Vertex Pharmaceuticals, Virtus Medical Group, XENCOR, Inc., and Yuhan Corporation; having leadership or fiduciary role in other board, society, committee, or advocacy group, paid or unpaid, with AstraZeneca PLC, HutchMed, and Aurora; and having stock or stock options in AstraZeneca, Aurora Tele-Oncology Ltd., Biolidics Ltd., HutchMed, Prenetics, D3 Bio, Lunit, Bowtie Life Insurance, Lakeshore Biotech Ltd., Loxo-oncology, Virtus Medical Group, and Phanes Therapeutics, Inc. Johnson has received research funding paid to their institution from AbbVie, Acerta, Adaptimmune, Amgen, Apexigen, Arcus Biosciences, Array BioPharma, Artios Pharma, AstraZeneca, Atreca, BeiGene, BerGenBio, BioAtla, Black Diamond, Boehringer Ingelheim, Calithera Biosciences, Carisma Therapeutics, Corvus Pharmaceutical, Curis, CytomX, Daiichi Sankyo, Dracen Pharmaceuticals, Dynavax, Lilly, Elicio Therapeutics, EMD Serono, EQRx, Erasca, Exelixis, Fate Therapeutics, Genentech/Roche, Genmab, Genocea Biosciences, GlaxoSmithKline, Gritstone Oncology, Guardant Health, Harpoon, Helsinn Healthcare SA, Hengrui Therapeutics, Hutchison MediPharma, IDEAYA Biosciences, IGM Biosciences, Immunitas Therapeutics, Immunocore, Incyte, Janssen, Kadmon Pharmaceuticals, Kartos Therapeutics, Loxo-Oncology, Lycera, Memorial Sloan-Kettering, Merck, Merus, Mirati Therapeutics, NeoImmune Tech, Neovia Oncology, Novartis, Numab Therapeutics, Nuvalent, OncoMed Pharmaceuticals, Palleon Pharmaceuticals, Pfizer, PMV Pharmaceuticals, Rain Therapeutics, Regeneron Pharmaceuticals, Relay Therapeutics, Revolution Medicines, Ribon Therapeutics, Rubius Therapeutics, Sanofi, Seven and Eight Biopharmaceuticals/Birdie Biopharmaceuticals, Shattuck Labs, Silicon Therapeutics, Stem CentRx, Syndax Pharmaceuticals, Takeda Pharmaceuticals, Tarveda, TCR2 Therapeutics, Tempest Therapeutics, Tizona Therapeutics, TMUNITY Therapeutics, Turning Point Therapeutics, University of Michigan, Vyriad, Y-mAbs Therapeutics, Bristol-Myers Squibb, Checkpoint Therapeutics, City of Hope National Medical Center, Jounce Therapeutics, Mythic Therapeutics, RasCal Therapeutics, WindMIL Therapeutics, ArriVent BioPharma, Bayer, LockBody Therapeutics, and Taiho Oncology; and receiving consulting fees paid to their institution from AbbVie, Amgen, Arrivent, Alentis Therapeutics, AstraZeneca, Bristol-Myers Squibb, D3 Bio Limited, Daiichi Sankyo, Fate Therapeutics, Genentech/Roche, Genmab, Genocea Biosciences, GlaxoSmithKline, Gritstone Oncology, Hookipa Biotech, Janssen, Merck, Mirati Therapeutics, Molecular Axiom, Novartis, Pyramid Biosciences, Revolution Medicines, Sanofi-Aventis, SeaGen, Takeda Pharmaceuticals, VBL Therapeutics, Arcus Biosciences, Immunocore, Jazz Pharmaceuticals, Synthekine, Boehringer Ingelheim, Gilead Sciences, Normunity, Lilly, Novocure, and Pfizer. The remaining authors delcare no conflict of interest., (Copyright © 2024 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Rising global temperatures can lead to heat waves, which in turn can pose health risks to the community. However, a notable gap remains in highlighting the primary contributing factors that amplify heat-health risk among vulnerable populations. This study aims to evaluate the precedence of heat stress contributing factors in urban and rural vulnerable populations living in hot and humid tropical regions. A comparative cross-sectional study was conducted, involving 108 respondents from urban and rural areas in Klang Valley, Malaysia, using a face-to-face interview and a validated questionnaire. Data was analyzed using the principal component analysis, categorizing factors into exposure, sensitivity, and adaptive capacity indicators. In urban areas, five principal components (PCs) explained 64.3% of variability, with primary factors being sensitivity (health morbidity, medicine intake, increased age), adaptive capacity (outdoor occupation type, lack of ceiling, longer residency duration), and exposure (lower ceiling height, increased building age). In rural, five PCs explained 71.5% of variability, with primary factors being exposure (lack of ceiling, high thermal conductivity roof material, increased building age, shorter residency duration), sensitivity (health morbidity, medicine intake, increased age), and adaptive capacity (female, non-smoking, higher BMI). The order of heat-health vulnerability indicators was sensitivity > adaptive capacity > exposure for urban areas, and exposure > sensitivity > adaptive capacity for rural areas. This study demonstrated a different pattern of leading contributors to heat stress between urban and rural vulnerable populations., (© 2024. The Author(s).)

Continued heat exposure can cause physiological and cellular responses. This study investigated the association between physiological responses and heat shock protein 70 (HSP70) expressions in Kuala Lumpur's urban vulnerable population. We conducted a cross-sectional study involving 54 participants from four areas classified as experiencing moderate to strong heat stress. Physiological measurements included core body temperature, heart rate, and diastolic and systolic blood pressure. RT-qPCR and ELISA were also performed on blood samples to assess HSP70 gene and protein expressions. Despite indoor heat stress, participants maintained normal physiological parameters while there were significant indications of HSP70 expression at both the gene and protein levels. However, our study found no significant correlation ( p  > 0.05) between physiological responses and HSP70 expressions. This study shows no interaction between physiological responses and HSP70 expressions in the study population, revealing the complex mechanisms of indoor heat stress in vulnerable individuals.

Introduction: We evaluated efficacy and safety of cabozantinib plus atezolizumab or cabozantinib alone in advanced NSCLC previously treated with an immune checkpoint inhibitor (ICI)., Methods: COSMIC-021 (NCT03170960) is a phase 1b, multicenter study in advanced solid tumors. This analysis included patients with stage IV non-squamous NSCLC without actionable genomic aberrations in EGFR, ALK, ROS1 , or BRAF - V600E who progressed on one prior ICI and less than or equal to two prior lines of systemic anticancer therapy. Patients received cabozantinib 40 mg orally/day plus atezolizumab 1200 mg intravenously every three weeks (combination cohort) or cabozantinib 60 mg orally/day (single-agent cabozantinib cohort). Primary end point of the combination cohort was objective response rate per Response Evaluation Criteria in Solid Tumors v1.1 by investigator. Outcomes in the single-agent cabozantinib cohort were exploratory., Results: Eighty-one patients assigned to combination therapy and 31 assigned to single-agent cabozantinib received greater than or equal to one dose of study treatment. Median (range) follow-up was 26.1 months (12.1-44.2) and 22.4 months (1.5-29.0), respectively. Objective response rate was 20% (95% confidence interval: 11.7%-30.1%) in combination cohort and 6% (95% confidence interval: 0.8%-21.4%) in single-agent cabozantinib cohort. Treatment-related adverse events (TRAEs) occurred in 86% of patients in the combination cohort and 90% in the single-agent cabozantinib cohort; grade 3/4 TRAEs were 44% and 48%, respectively. There were two grade 5 TRAEs: pneumonitis (n = 1, combination) and gastric ulcer hemorrhage (n = 1, single-agent). Neither PD-L1 expression in tumor cells nor tumor mutation burden correlated with outcomes., Conclusions: Cabozantinib plus atezolizumab demonstrated modest clinical activity and manageable toxicity in advanced NSCLC after progression on prior ICI., Competing Interests: Dr. Neal reported receiving honoraria from Biomedical Learning Institute CME, Clinical Care Options, CME Matter, Medscape CME, MJH Life Sciences CME, MLI Peerview CME, Prime Oncology CME, Projects in Knowledge CME, Research to Practice CME, Rockpointe CME, Medical Educator Consortium, HMP Education, a consulting or advisory role with AstraZeneca, Blueprint Pharmaceuticals, Calithera Biosciences, D2G Oncology, Exelixis, Genentech, Roche, Jounce Therapeutics, Eli Lilly and Company, Natera, Regeneron, Sanofi, Surface Oncology, Takeda Pharmaceuticals, Turning Point Therapeutics, and receiving institutional research funding from 10.13039/100006483AbbVie, Adaptimmune, Boehringer Ingelheim, 10.13039/100010544Exelixis, 10.13039/100004328Genentech, 10.13039/100004337Roche, GSK, 10.13039/100005565Janssen, 10.13039/100004334Merck, Nektar Therapeutics, 10.13039/100004336Novartis, Takeda Pharmaceuticals. Dr. Armando Santoro reported having a consulting or advisory role with Sanofi, Incyte, receiving honoraria from AbbVie, Amgen, ArQule, AstraZeneca, Bayer, BMS, Celgene, Eisai, Gilead Sciences, Lilly, MSD, Novartis, Pfizer, Roche, Sandoz, Servier, Takeda, and participating in a data safety or advisory board with BMS, Servier, Gilead, Pfizer, Eisai, Bayer, MSD. Dr. Maria Gonzalez-Cao reported receiving grants or contracts from Roche, AstraZeneca, and Novartis, honoraria from Bristol Myers Squibb, MSD, AstraZeneca, Novartis, and Pierre Fabre, support for travel, accommodations, and expenses from MSD, AstraZeneca, and Pierre Fabre, and having a leadership or fiduciary role with the Spanish Melanoma Group. Dr. Ryan D. Gentzler reported receiving honoraria from Targeted Oncology, OncLive, Clinical Care Options, Society for Immunotherapy of Cancer, American Society of Clinical Oncology, MedStar Health, Aptitude Health, support for attending meetings from International Association for the Study of Lung Cancer, American Society of Clinical Oncology, Dava Oncology, Tempus, having a consulting or advisory role with AstraZeneca, Daiichi Sankyo, Gilead, Janssen, Jazz Pharmaceuticals, Mirati Therapeutics, OncoCyte, Sanofi, Takeda, Merus, Regeneron, receiving institutional research funding from 10.13039/100002429Amgen, Alliance Foundation, 10.13039/100004325AstraZeneca, Big Ten Research Consortium, 10.13039/100002491Bristol Myers Squibb, 10.13039/100010795Chugai, 10.13039/501100022274Daiichi Sankyo, 10.13039/100008130Helsinn Therapeutics, Hoosier Cancer Research Network, 10.13039/100005565Janssen, 10.13039/100016765Jounce Therapeutics, Merck, 10.13039/100016957Mirati Therapeutics, 10.13039/100004319Pfizer, 10.13039/100008199RTI International, Takeda, Dizal, Puma, and having leadership or committee roles with Hoosier Cancer Research Network, ASCO, Journal of Clinical Oncology, NCI Investigational Drug Steering Committee, and International Association for the Study of Lung Cancer. Dr. Jerome Goldschmidt reported receiving honoraria from G1 Therapeutics, and support for travel, accommodations, and expenses from Sara Cannon Research Institute. Dr. Claudia Proto reports receiving honoraria from AstraZeneca, Bristol Myers Squibb, MSD, Roche, Sanofi, having a consulting or advisory role with AstraZeneca, MSD and Roche, institutional research funding from MSD, Lilly, Pfizer, support for travel, accommodations, expenses from AstraZeneca, MSD, and Roche, and is the principal investigator in clinical trials for Janssen, Pfizer, Lilly, Spectrum Pharmaceuticals, Roche, MSD, BMS, and AstraZeneca. Dr. Shiven Patel reported receiving a consulting or advisory role with AstraZeneca, Blueprint Medicines, Boehringer Ingelheim, Regeneron, Sanofi, TerSera Therapeutics LLC, Total Health Conferencing, Natera, Takeda, Merck, BMS, and receiving institutional research funding from AstraZeneca, Janssen, Merck, and Takeda. Dr. Sonam Puri reported having an institutional consulting or advisory role with Jazz Pharma, receiving consulting fees from G1 Therapeutics, Pfizer, Bristol-Myers Squibb, receiving honoraria from Aptitude Health, and support for travel, accommodations, and expenses from Dava Oncology. Dr. Stephen V. Liu reported having a consulting or advisory role with AbbVie, Amgen, AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Catalyst, Daiichi Sankyo, Eisai, Elevation Oncology, Genentech, Roche, Gilead Sciences, Guardant Health, Janssen Oncology, Jazz Pharmaceuticals, Merck, Merus, Mirati, Novartis, Regeneron, Sanofi, Takeda, and Turning Point Therapeutics, receiving institutional research funding from AbbVie, 10.13039/100018529Alkermes, Elevation Oncology, Ellipses, Genentech, Roche, 10.13039/100005564Gilead Sciences, Merck, Merus, Nuvalent, Inc., RAPT Therapeutics, and Turning Point Therapeutics, and participation in a data safety monitoring board or advisory board with Candel Therapeutics. Dr. Erminia Massarelli reported receiving honoraria from AstraZeneca, Merck, Eli Lilly and Company, Takeda Pharmaceuticals, Mirati, and having a consulting or advisory role with Bristol Myers Squibb, Daiichi Sankyo Co., Fusion Therapeutics, Janssen, Eli Lilly and Company, Sanofi, AbbVie, Iovance Therapeutics, and Mirati. Ms. Denise Williamson, Drs. Martin Schwickart, Christian Scheffold, and Svetlana Adrianova are employees of and reported having stock with Exelixis. In addition, Dr. Christian Scheffold reported a patent: Combinations of Cabozantinib and Atezolizumab to Treat Cancer; patent number 11198731. Dr. Enriqueta Felip reported having a consulting or advisory role with AbbVie, Amgen, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, Eli Lilly, F. Hoffmann-La Roche, Gilead, GlaxoSmithKline, Janssen, Merck Serono, MSD, Novartis, Peptomyc, Pfizer, Regeneron, Sanofi, Takeda, Turning Point, Daiichi Sankyo, honoraria from Amgen, AstraZeneca, Bristol Myers Squibb, Daiichi Sankyo, Eli Lilly, F. Hoffmann-La Roche, Genentech, Janssen, Medical Trends, Medscape, Merck Serono, MSD, Peervoice, Pfizer, Sanofi, Takeda, Touch Oncology, and receiving support for travel, accommodations, and expenses from AstraZeneca, Janssen, Roche. The remaining authors declare no conflict of interest., (© 2024 The Authors.)

Objectives: In the phase 3 POSEIDON study, first-line tremelimumab plus durvalumab and chemotherapy significantly improved overall survival and progression-free survival versus chemotherapy in metastatic non-small-cell lung cancer (NSCLC). We present patient-reported outcomes (PROs)., Patients and Methods: Treatment-naïve patients were randomized 1:1:1 to tremelimumab plus durvalumab and chemotherapy, durvalumab plus chemotherapy, or chemotherapy. PROs (prespecified secondary endpoints) were assessed using the European Organisation for Research and Treatment of Cancer 30-item core quality of life questionnaire version 3 (QLQ-C30) and its 13-item lung cancer module (QLQ-LC13). We analyzed time to deterioration (TTD) of symptoms, functioning, and global health status/quality of life (QoL) from randomization by log-rank test and improvement rates by logistic regression., Results: 972/1013 (96 %) patients randomized completed baseline QLQ-C30 and QLQ-LC13 questionnaires, with scores comparable between treatment arms. Patients receiving tremelimumab plus durvalumab and chemotherapy versus chemotherapy had longer median TTD for all PRO items. Hazard ratios for TTD favored tremelimumab plus durvalumab and chemotherapy for all items except diarrhea; 95 % confidence intervals did not cross 1.0 for global health status/QoL, physical functioning, cognitive functioning, pain, nausea/vomiting, insomnia, constipation, hemoptysis, dyspnea, and pain in other parts. For durvalumab plus chemotherapy, median TTD was longer versus chemotherapy for all items except nausea/vomiting and diarrhea. Hazard ratios favored durvalumab plus chemotherapy for all items except appetite loss; 95 % confidence intervals did not cross 1.0 for global health status/QoL, physical functioning, role functioning, dyspnea, and pain in other parts. For both immunotherapy plus chemotherapy arms, improvement rates in all PRO items were numerically higher versus chemotherapy, with odds ratios > 1., Conclusions: Tremelimumab plus durvalumab and chemotherapy delayed deterioration in symptoms, functioning, and global health status/QoL compared with chemotherapy. Together with significant improvements in survival, these results support tremelimumab plus durvalumab and chemotherapy as a first-line treatment option in metastatic NSCLC., Competing Interests: Declaration of Competing Interest Edward B. Garon reports grants or contracts from ABL Bio, AstraZeneca, Bristol-Myers Squibb, Daiichi-Sankyo, Dynavax Technologies, Eli Lilly, EMD Serono, Genentech, Gilead, Iovance Biotherapeutics, Merck, Mirati Therapeutics, Neon, and Novartis; consulting fees from AbbVie, ABL Bio, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Dracen Pharmaceuticals, EMD Serono, Eisai, Eli Lilly, Gilead, GlaxoSmithKline, Merck, Natera, Novartis, Personalis, Regeneron, Sanofi, Shionogi, Xilio, and Zymeworks; and travel support from A2 Bio and Novartis. Byoung Chul Cho reports consulting fees from Abion, AstraZeneca, BeiGene, Blueprint Medicines, Boehringer Ingelheim, Bridgebio Therapeutics, Bristol-Myers Squibb, CJ, CureLogen, Cyrus Therapeutics, Eli Lilly, GI-Cell, Hanmi, HK Inno-N, Imnewrun Biosciences, Janssen, KANAPH Therapeutic, Medpacto, MSD, Novartis, Ono Pharmaceutical, Onegene Biotechnology, Oscotec, Pfizer, RandBio, Roche, Takeda, and Yuhan; advisory board participation for Bridgebio Therapeutics, Cyrus Therapeutics, Guardant Health, Kanaph Therapeutics, and Oscotec; honoraria from ASCO, AstraZeneca, ESMO, Guardant Health, IASLC, Korean Cancer Association, Korean Cancer Study Group, Korean Society of Medical Oncology, Korean Society of Thyroid-Head and Neck Surgery, MSD, Novartis, Pfizer, Roche, and The Chinese Thoracic Oncology Society; research funding from AbbVie, Abion, AstraZeneca, Bayer, Blueprint Medicines, Boehringer Ingelheim, Bridgebio Therapeutics, CHA Bundang Medical Center, Champions Oncology, CJ Bioscience, CJ Blossom Park, Cyrus Therapeutics, Dizal Pharma, Dong-A ST, Eli Lilly, Genexine, GI-Cell, GIInnovation, Hanmi, Illumina, ImmuneOncia, Interpark Bio, Janssen, J Ints Bio, Kanaph Therapeutics, LG Chem, Medpacto, MOGAM Institute, MSD, Novartis, Nuvalent, Oncternal, Ono Pharmaceutical, Oscotec, Regeneron, Therapex, and Yuhan; royalties from Champions Oncology, Crown Bioscience, and Imagen; board membership for Interpark Bio and J Ints Bio; stock ownership in Bridgebio Therapeutics, Cyrus Therapeutics, Gencurix, Interpark Bio, Kanaph Therapeutics, J Ints Bio, and TheraCanVac; and other relationships for DAAN Biotherapeutics (Founder) and Korean University Health System (Employment). Alexander Luft has nothing to disclose. Jorge Alatorre-Alexander reports advisory board participation for Amgen, AstraZeneca, Bristol-Myers Squibb, Janssen, MSD, and Roche; travel support from AstraZeneca, MSD, and Roche; and honoraria from Amgen, AstraZeneca, Bristol-Myers Squibb, Janssen, MSD, and Roche. Sarayut Lucien Geater reports research funding (to institution) from AstraZeneca, Boehringer Ingelheim, MSD, Novartis and Roche; honoraria from AstraZeneca, and Boehringer Ingelheim; and advisory board participation for Pfizer. Sang-We Kim reports research funding (to institution) from Yuhan; and travel support from AstraZeneca. Grygorii Ursol has nothing to disclose. Maen Hussein reports consulting fees from AbbVie, Aptitude Health, AstraZeneca, Athenex, Biopharma, Bristol-Myers Squibb, Coherus Biosciences, CTI BioPharma, Exelixis, GIntrinsiQ, IntegraConnect, Integra PrecisionQ, IntrinsiQ, Karyopharm Therapeutics, Mirati Therapeutics, National Community Oncology Dispensing Association, and Oncocyte. Farah Louise Lim has nothing to disclose. Cheng-Ta Yang has nothing to disclose. Luiz Henrique Araujo reports consulting fees from AstraZeneca, Bristol-Myers Squibb, Illumina, Lilly, MSD, Roche, and Sanofi; honoraria from AstraZeneca, Bristol-Myers Squibb, Boehringer Ingelheim, Lilly, Merck, MSD, Pfizer, and Roche; travel support from AstraZeneca, Bristol-Myers Squibb, and Daiichi-Sankyo; and grants or contracts from AstraZeneca, Bristol-Myers Squibb, Boehringer Ingelheim, Lilly, Merck, MSD, Novartis, Pfizer, and Roche. Haruhiro Saito reports honoraria from AstraZeneca and ONO Pharmaceutical; and grants or contracts from AstraZeneca, Bristol-Myers Squibb, Chugai Pharmaceutical, and ONO Pharmaceutical. Niels Reinmuth reports honoraria from AstraZeneca, Bristol-Myers Squibb, Lilly, MSD, and Roche; and consulting fees from AstraZeneca, Bristol-Myers Squibb, MSD, and Roche. Nenad Medic, Helen Mann and Xiaojin Shi are full time employees of and own stock in AstraZeneca. Solange Peters reports honoraria (to institution) from AstraZeneca, Bristol-Myers Squibb, Boehringer Ingelheim, ecancer, Eli Lilly, Fishawack, Imedex, IQVIA, Medscape, MSD, Novartis, Oncology Education, PER, Pfizer, Prime Oncology, RMEI Medical Education, Research to Practice, Roche/Genentech, and Takeda; advisory board participation for AbbVie, Amgen, AstraZeneca, Bayer, BeiGene, Boehringer Ingelheim, Biocartis, Bioinvent, Bristol-Myers Squibb, Clovis Oncology, Daiichi-Sankyo, Debiopharm Group, Eli Lilly, Foundation Medicine, Illumina, Incyte, Janssen, Merck Serono, MSD, Merrimack, Novartis, Pfizer, PharmaMar, Phosplatin Therapeutics, Regeneron, Roche/Genentech, Sanofi, Seattle Genetics, and Takeda; and research funding (to institution) from Amgen, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Clovis, Illumina, Iovance Biotherapeutics, Lilly, Merck Serono, MSD, Novartis, Pharma Mar, Pfizer, Phosplatin Therapeutics, Takeda, Sanofi, Seattle Genetics, and Roche/Genentech. Tony Mok reports honoraria from ACEA Pharma, Alpha Biopharma, Amgen, Amoy Diagnostics, AstraZeneca (before 1/1/19), BeiGene, Boehringer Ingelheim, Bristol-Myers Squibb, Daiichi-Sankyo, Daz, Fishawack Facilitate, InMed Medical Communication, Janssen, Jiahui Holdings, LiangYiHui Healthcare, Lilly, Lucence Health, MD Health Brazil, Medscape, Merck, MiRXES, MSD, Novartis, OrigiMed, P. Permanyer SL, PeerVoice, PER, Pfizer, Prime Oncology, Research to Practice, Roche Pharmaceuticals/Diagnostics/Foundation One, Sanofi-Aventis, Shanghai BeBirds Translation & Consulting, Taiho Pharmaceutical, Takeda, and Touch Independent Medical Education; consulting fees from for AbbVie, ACEA Pharma, Adagene, Alpha Biopharma, Amgen, Amoy Diagnostics, Bayer, BeiGene, Berry Oncology, Boehringer Ingelheim, Blueprint Medicines, Bristol-Myers Squibb, Covidien, C4 Therapeutics, Cirina, CStone Pharmaceuticals, Curio Science, D3 Bio, Da Volterra, Daiichi-Sankyo, Eisai, Elevation Oncology, G1 Therapeutics, geneDecode, Gilead, Gritstone Oncology, Guardant Health, Hengrui Therapeutics, HutchMed, Ignyta, Inivata, IQVIA, Janssen, Lilly, Lunit USA, Loxo Oncology, Lucence Health, Medscape/WebMD, Merck Serono, MSD, Mirati Therapeutics, MiRXES, MoreHealth, Novartis, Omega Therapeutics, OrigiMed, OSE Immunotherapeutics, PeerVoice, Pfizer, Prime Oncology, Puma Biotechnology, Qiming Development, Roche/Genetech, Roche Pharmaceuticals/Diagnostics/Foundation One, Sanofi-Aventis, SFJ Pharmaceutical, Simcere of America, Synergy Research, Takeda, Tigermed, Vertex Pharmaceuticals, Virtus Medical Group, and Yuhan; advisory board participation for AbbVie, ACEA Pharma, Amgen, AstraZeneca, Berry Oncology, Blueprint Medicines, Boehringer Ingelheim, Bowtie Life Insurance, Bristol-Myers Squibb, C4 Therapeutics, Covidien, CStone Pharmaceuticals, Curio Science, D3 Bio, Daiichi-Sankyo, Eisai, Fishawack Facilitate, G1 Therapeutics, Gilead, Gritstone Oncology, Guardant Health, geneDecode (uncompensated), Hengrui Therapeutics, HutchMed, Ignyta, Incyte, Inivata, IQVIA, Janssen, Lakeshore Biotech, Lilly, Loxo Oncology, Lunit, Merck Serono, Mirati Therapeutics, MiRXES, MSD, Novartis, OrigiMed, Pfizer, Puma Biotechnology, Roche/Genentech, Sanofi-Aventis, SFJ Pharmaceutical, Simcere of America, Takeda, Vertex Pharmaceuticals, Virtus Medical Group, Yuhan; leadership roles for ACT Genomics-Sanomics, AstraZeneca, Aurora Tele-Oncology, HutchMed, and Lunit USA; stock/stock options in Act Genomics-Sanomics, AstraZeneca, Aurora Tele-Oncology, Biolidics, and HutchMed; research funding (to institution) from AstraZeneca, Bristol-Myers Squibb, G1 Therapeutics, MSD, Merck Serono, Novartis, Pfizer, Roche, SFJ, Takeda, and XCovery; and travel support (some to institution) from AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Daiichi-Sankyo, MiRXES, MSD, Novartis, Pfizer, Roche. Melissa Johnson reports research funding (paid to institution) from AbbVie, Acerta, Adaptimmune, Amgen, Apexigen, Arcus Biosciences, Array BioPharma, Artios Pharma, AstraZeneca, Atreca, BeiGene, BerGenBio, BioAtla, Black Diamond, Boehringer Ingelheim, Bristol-Myers Squibb, Calithera Biosciences, Carisma Therapeutics, Checkpoint Therapeutics, City of Hope National Medical Center, Corvus Pharmaceuticals, Curis, CytomX, Daiichi-Sankyo, Dracen Pharmaceuticals, Dynavax, Lilly, Elicio Therapeutics, EMD Serono, Erasca, EQRx, Exelixis, Fate Therapeutics, Genentech/Roche, Genmab, Genocea Biosciences, GlaxoSmithKline, Gritstone Oncology, Guardant Health, Harpoon, Helsinn Healthcare, Hengrui Therapeutics, Hutchison MediPharma, IDEAYA Biosciences, IGM Biosciences, Immunitas Therapeutics, Immunocore, Incyte, Janssen, Jounce Therapeutics, Kadmon Pharmaceuticals, Kartos Therapeutics, Loxo Oncology, Lycera, Memorial-Sloan Kettering, Merck, Merus, Mirati Therapeutics, Mythic Therapeutics, NeoImmune Tech, Neovia Oncology, Novartis, Numab Therapeutics, OncoMed Pharmaceuticals, Palleon Pharmaceuticals, Pfizer, PMV Pharmaceuticals, Rain Therapeutics, RasCal Therapeutics, Regeneron Pharmaceuticals, Relay Therapeutics, Revolution Medicines, Ribon Therapeutics, Rubius Therapeutics, Sanofi, Seven and Eight Biopharmaceuticals/Birdie Biopharmaceuticals, Shattuck Labs, Silicon Therapeutics, StemCentRx, Syndax Pharmaceuticals, Takeda, Tarveda, TCR2 Therapeutics, Tempest, Therapeutics, Tizona Therapeutics, TMUNITY Therapeutics, Turning Point Therapeutics, University of Michigan, Vyriad, WindMIL, and Y-mAbs Therapeutics; and consulting fees (paid to institution) from AbbVie, Arcus Biosciences, Amgen, Arrivent, Astellas, AstraZeneca, Axelia Oncology, Black Diamond, Calithera, Daiichi-Sankyo, EcoR1, Genentech/Roche, Genmab, Genocea Biosciences, GlaxoSmithKline, Gritstone Oncology, Ideaya Biosciences, Immunocore, iTeos, Janssen, Jazz Pharmaceuticals, Merck, Mirati Therapeutics, Molecular Axiom, Novartis, Oncorus, Pyramid Biosciences, Regeneron Pharmaceuticals, Revolution Medicines, SeaGen, Sanofi-Aventis, Takeda, Turning Point Therapeutics, Synethekine, and VBL Therapeutics., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Approximately 10-15% of pediatric B-cell acute lymphoblastic leukemia (B-ALL) are high risk at diagnosis or relapsed/ refractory. Prior to the availability of chimeric antigen receptor T-cell (CAR-T) in Singapore and the region, the treatment options for these paediatric and young adults are conventional salvage chemotherapy or chemo-immunotherapy regimens as a bridge to allogeneic total body irradiation-based hematopoietic stem cell transplantation (allo-HSCT). This results in significant acute and long-term toxicities, with suboptimal survival outcomes. Finding a curative salvage therapy with fewer long-term toxicities would translate to improved quality-adjusted life years in these children and young adults. In this review, we focus on the burden of relapsed/refractory pediatric B-ALL, the limitations of current strategies, the emerging paradigms for the role of CAR-T in r/r B-ALL, our local perspectives on the health economics and future direction of CAR-T therapies in pediatric patients., Competing Interests: Professor William YK Hwang reports Honorarium for talk from Novartis, outside the submitted work. The authors report no other conflicts of interest in this work., (© 2023 Seng et al.)

Background: Acute cutaneous inflammation causes microbiome alterations as well as ultrastructural changes in epidermis stratification. However, the interactions between keratinocyte proliferation and differentiation status and the skin microbiome have not been fully explored., Objectives: Hypothesizing that the skin microbiome contributes to regulation of keratinocyte differentiation and can modify antimicrobial responses, we examined the effect of exposure to commensal (Staphylococcus epidermidis, SE) or pathogenic (Staphylococcus aureus, SA) challenge on epidermal models., Methods: Explant biopsies were taken to investigate species-specific antimicrobial effects of host factors. Further investigations were performed in reconstituted epidermal models by bulk transcriptomic analysis alongside secreted protein profiling. Single-cell RNA sequencing analysis was performed to explore the keratinocyte populations responsible for SA inflammation. A dataset of 6391 keratinocytes from control (2044 cells), SE challenge (2028 cells) and SA challenge (2319 cells) was generated from reconstituted epidermal models., Results: Bacterial lawns of SA, not SE, were inhibited by human skin explant samples, and microarray analysis of three-dimensional epidermis models showed that host antimicrobial peptide expression was induced by SE but not SA. Protein analysis of bacterial cocultured models showed that SA exposure induced inflammatory mediator expression, indicating keratinocyte activation of other epidermal immune populations. Single-cell DropSeq analysis of unchallenged naive, SE-challenged and SA-challenged epidermis models was undertaken to distinguish cells from basal, spinous and granular layers, and to interrogate them in relation to model exposure. In contrast to SE, SA specifically induced a subpopulation of spinous cells that highly expressed transcripts related to epidermal inflammation and antimicrobial response. Furthermore, SA, but not SE, specifically induced a basal population that highly expressed interleukin-1 alarmins., Conclusions: These findings suggest that SA-associated remodelling of the epidermis is compartmentalized to different keratinocyte populations. Elucidating the mechanisms regulating bacterial sensing-triggered inflammatory responses within tissues will enable further understanding of microbiome dysbiosis and inflammatory skin diseases, such as atopic eczema., Competing Interests: Conflicts of interest The authors declare they have no conflicts of interest., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Association of Dermatologists.)

Purpose: The open-label, phase III POSEIDON study evaluated tremelimumab plus durvalumab and chemotherapy (T + D + CT) and durvalumab plus chemotherapy (D + CT) versus chemotherapy alone (CT) in first-line metastatic non-small-cell lung cancer (mNSCLC)., Methods: Patients (n = 1,013) with EGFR / ALK wild-type mNSCLC were randomly assigned (1:1:1) to tremelimumab 75 mg plus durvalumab 1,500 mg and platinum-based chemotherapy for up to four 21-day cycles, followed by durvalumab once every 4 weeks until progression and one additional tremelimumab dose; durvalumab plus chemotherapy for up to four 21-day cycles, followed by durvalumab once every 4 weeks until progression; or chemotherapy for up to six 21-day cycles (with or without maintenance pemetrexed; all arms). Primary end points were progression-free survival (PFS) and overall survival (OS) for D + CT versus CT. Key alpha-controlled secondary end points were PFS and OS for T + D + CT versus CT., Results: PFS was significantly improved with D + CT versus CT (hazard ratio [HR], 0.74; 95% CI, 0.62 to 0.89; P = .0009; median, 5.5 v 4.8 months); a trend for improved OS did not reach statistical significance (HR, 0.86; 95% CI, 0.72 to 1.02; P = .0758; median, 13.3 v 11.7 months; 24-month OS, 29.6% v 22.1%). PFS (HR, 0.72; 95% CI, 0.60 to 0.86; P = .0003; median, 6.2 v 4.8 months) and OS (HR, 0.77; 95% CI, 0.65 to 0.92; P = .0030; median, 14.0 v 11.7 months; 24-month OS, 32.9% v 22.1%) were significantly improved with T + D + CT versus CT. Treatment-related adverse events were maximum grade 3/4 in 51.8%, 44.6%, and 44.4% of patients receiving T + D + CT, D + CT, and CT, respectively; 15.5%, 14.1%, and 9.9%, respectively, discontinued treatment because of treatment-related adverse events., Conclusion: D + CT significantly improved PFS versus CT. A limited course of tremelimumab added to durvalumab and chemotherapy significantly improved OS and PFS versus CT, without meaningful additional tolerability burden, representing a potential new option in first-line mNSCLC.

Novel therapeutic options for cancer offer hope for patients and their families, particularly when the cancer has not responded to established treatment regimens. The CAR-T cell therapeutic approach has changed the treatment paradigm for relapsed or refractory lymphoma, extending the capacity of the patient's own T cells to detect and eliminate cancer cells through genetic modification of T-cell surface receptors. The process of establishing treatment centers and developing clinical expertize in this novel treatment strategy is complex. Time, resources, and a commitment to focusing health budgets on a new area are required. Currently, Singapore is the only country in southeast and south Asia with market authorization of the CAR-T product, tisagenlecleucel. Availability of CAR-T treatment across international borders provides patients in neighboring countries with choice in therapeutic options. This paper describes the unique hub-and-spoke cross-border collaboration developed between Singapore and its neighbors to provide access to CAR-T cell therapy for patients with relapsed or refractory lymphoma. To date in 2022, four patients have been included in the CAR-T treatment cross-border collaboration. Their stay in Singapore has been at least 2 months' duration, including the pre-treatment evaluation, apheresis, CAR-T cell infusion and post-treatment monitoring. Patient support from referring and treating physicians, critical to the success of the undertaking, is characterized by early communication, patient selection, multi-disciplinary care, post-treatment monitoring, and attention to detail. The patient journey and the development and implementation of this unique collaboration are discussed., (© 2022 John Wiley & Sons Ltd.)

Keratinocytes produce lipids that are critical for the skin barrier, however, little is known about the impact of age on fatty acid (FA) biosynthesis in these cells. We have examined the relationship between keratinocyte FA composition, lipid biosynthetic gene expression, gene promoter methylation and age. Expression of elongase (ELOVL6 and 7) and desaturase (FADS1 and 2) genes was lower in adult versus neonatal keratinocytes, and was associated with lower concentrations of n-7, n-9 and n-10 polyunsaturated FA in adult cells. Consistent with these findings, transient FADS2 knockdown in neonatal keratinocytes mimicked the adult keratinocyte FA profile in neonatal cells. Interrogation of methylation levels across the FADS2 locus (53 genomic sites) revealed differential methylation of 15 sites in neonatal versus adult keratinocytes, of which three hypermethylated sites in adult keratinocytes overlapped with a SMARCA4 protein binding site in the FADS2 promoter., Competing Interests: Declaration of competing interest Messenger D.J.(2), Barrett K.E.(2), Hyliands D.(2), Talbot D.(2), Fowler M.I.(2), Kawatra T.(2), Gunn D.A.(2), Lim FL.(2), Wainwright L.J.(2), Jenkins G.(2), are employed by Unilever R&D. Griffiths H.R.(1,3) received part funding from Unilever R&D and BBSRC to undertake this study., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The olfactory system combines input from multiple receptor types to represent odor information, but there are few explicit examples relating olfactory receptor (OR) activity patterns to odor perception. To uncover these relationships, we performed genome-wide scans on odor-perception phenotypes for ten odors in 1000 Han Chinese and validated results for six of these odors in an ethnically diverse population (n = 364). In both populations, consistent with previous studies, we replicated three previously reported associations (β-ionone/OR5A1, androstenone/OR7D4, cis-3-hexen-1-ol/OR2J3 LD-band), but not for odors containing aldehydes, suggesting that olfactory phenotype/genotype studies are robust across populations. Two novel associations between an OR and odor perception contribute to our understanding of olfactory coding. First, we found a SNP in OR51B2 that associated with trans-3-methyl-2-hexenoic acid, a key component of human underarm odor. Second, we found two linked SNPs associated with the musk Galaxolide in a novel musk receptor, OR4D6, which is also the first human OR shown to drive specific anosmia to a musk compound. We noticed that SNPs detected for odor intensity were enriched with amino acid substitutions, implying functional changes of odor receptors. Furthermore, we also found that the derived alleles of the SNPs tend to be associated with reduced odor intensity, supporting the hypothesis that the primate olfactory gene repertoire has degenerated over time. This study provides information about coding for human body odor, and gives us insight into broader mechanisms of olfactory coding, such as how differential OR activation can converge on a similar percept., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: The study was funded in part by Unilever R&D (the Netherlands). M.A.M.S. and F.-L.L. are employees of Unilever. The other authors declare that they have no competing interests.

Objective: Chemoradiation (CRT) may induce a change in systemic inflammatory state which could affect clinical outcomes in oesophageal cancer. We aimed to evaluate the changes and prognostic significance of systemic inflammatory markers following definitive CRT in oesophageal squamous cell carcinoma., Methods: A total of 53 patients treated with concurrent CRT were included in this retrospective analysis. We compared neutrophils, lymphocytes, platelets, neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) before and after CRT using Wilcoxon signed-rank test. Overall survival (OS) and progression-free survival (PFS) were calculated. Univariable and multivariable survival analysis were performed using Cox regression analysis. Clinical univariable survival prognostic factors with p < 0.1 were included in a multivariable cox regression analysis for backward stepwise model selection., Results: Both NLR (median ∆+2.8 [IQR -0.11, 8.62], p < 001) and PLR (median ∆+227 [81.3-523.5], p < 0.001) increased significantly after CRT. Higher levels of pre-CRT, post-CRT and change (∆) in NLR and PLR were associated with inferior OS and PFS. Post-CRT NLR (HR 1.04, 95% CI 1.02-1.07, p < 0.001), post-CRT platelets (HR 1.03, 95% CI 1.01-1.05, p = 0.005), cT-stage (HR 3.83, 95% CI 1.39-10.60, p = 0.01) and RT dose (HR 0.41, 95% CI 0.21-0.81, p = 0.01) were independent prognostic factors for OS in multivariable analysis. Change in NLR (HR 1.04, 95% CI 1.01-1.06, p = 0.001), post-CRT platelets (HR 1.03, 95% CI 1.01-1.05, p = 0.002), cT-stage (HR 3.98, 95% CI 1.55-10.25, p = 0.004) and RT dose (HR 0.41, 95% CI 0.21-0.80, p = 0.009) were independent prognostic factors for PFS., Conclusion: Both NLR and PLR increased following definitive CRT. Post-CRT NLR and ∆NLR were associated with adverse survival in oesophageal SCC., Advances in Knowledge: We showed that CRT increased PLR and NLR, possibly reflecting a systemic inflammatory state which were associated with poor clinical outcomes in oesophageal SCC.

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders., Competing Interests: KR, ES, RC, EL, SAn, RB, MD, PB, DM, and The University of Texas MD Anderson Cancer Center (MDACC) have an institutional financial conflict of interest with Takeda Pharmaceutical for the licensing of the technology related to CAR-NK cells. MD Anderson has implemented an Institutional Conflict of Interest Management and Monitoring Plan to manage and monitor the conflict of interest with respect to MDACC's conduct of any other ongoing or future research related to this relationship. KR, ES, RB, EL, SAn, DM and The University of Texas MD Anderson Cancer Center has an institutional financial conflict of interest with Affimed GmbH. Because MD Anderson is committed to the protection of human subjects and the effective management of its financial conflicts of interest in relation to its research activities, MD Anderson is implementing an Institutional Conflict of Interest Management and Monitoring Plan to manage and monitor the conflict of interest with respect to MD Anderson's conduct of any other ongoing or future research related to this relationship. ES participates on Scientific Advisory Board for Bayer, Novartis, Magenta, Adaptimmune, Mesoblast and Axio. KR participates on Scientific Advisory Board for GemoAb, AvengeBio, Kiadis, GSK and Bayer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mendt, Daher, Basar, Shanley, Kumar, Wei Inng, Acharya, Shaim, Fowlkes, Tran, Gokdemir, Uprety, Nunez-Cortes, Ensley, Mai, Kerbauy, Melo-Garcia, Lin, Shen, Mohanty, Lu, Li, Nandivada, Wang, Banerjee, Reyes-Silva, Liu, Ang, Gilbert, Li, Wan, Gu, Zhao, Baran, Muniz-Feliciano, Wilson, Kaur, Gagea, Konopleva, Marin, Tang, Chen, Champlin, Rezvani and Shpall.)

OBJECTIVE: To examine the associations between endotoxin and (1,3)-β-glucan concentrations in office dust and respiratory symptoms and airway inflammation among 695 office workers in Malaysia. METHODS: Health data were collected using a questionnaire, sensitisation testing and measurement of fractional exhaled nitric oxide (FeNO). Indoor temperature, relative air humidity (RH) and carbon dioxide (CO₂) were measured in the offices and settled dust was vacuumed and analysed for endotoxin and (1,3)-β-glucan concentrations. Associations were analysed by two level multiple logistic regression. RESULTS: Overall, 9.6% of the workers had doctor-diagnosed asthma, 15.5% had wheeze, 18.4% had daytime attacks of breathlessness and 25.8% had elevated FeNO (≥25 ppb). The median levels in office dust were 11.3 EU/mg endotoxin and 62.9 ng/g (1,3)-β-glucan. After adjusting for personal and home environment factors, endotoxin concentration in dust was associated with wheeze ( P = 0.02) and rhinoconjunctivitis ( P = 0.007). The amount of surface dust ( P = 0.04) and (1,3)-β-glucan concentration dust ( P = 0.03) were associated with elevated FeNO. CONCLUSION: Endotoxin in office dust could be a risk factor for wheeze and rhinoconjunctivitis among office workers in mechanically ventilated offices in a tropical country. The amount of dust and (1,3)-β-glucan (a marker of indoor mould exposure) were associated with Th2 driven airway inflammation.

Background: Hemolysis at the time of graft infusion is one of the immediate complications in major ABO-incompatible allogeneic hematopoietic stem cell transplants (HSCTs). We conducted a retrospective analysis to evaluate the efficacy of donor-type fresh frozen plasma (FFP) in reducing isohemagglutinin titer and preventing hemolysis, as well as its effect on delayed red cell engraftment., Materials and Methods: This is a single-center study on a series of 380 allogeneic HSCT between 2005 and 2015; of which 99 were either major (n = 74) or bidirectional (n = 25) ABO mismatched. Pre-transplant infusion of FFP, post-transplant complications and transfusion requirements were determined by retrospective review of individual medical records. Laboratory results were also reviewed for evidence of hemolysis and pure red cell aplasia (PRCA)., Results: Clinical manifestation of hemolysis attributable to ABO mismatch was present in one recipient of major ABO-incompatible peripheral blood stem cell (PBSC) with a titer of 64. Another recipient of major ABO-incompatible PBSC with a titer of 64 showed biochemical evidence of hemolysis. Both patients recovered with supportive treatment. Hemolysis did not occur in any patients with titer of 32 or less at the time of stem cell infusion. We were unable to demonstrate the influence of any variables on the incidence of PRCA., Conclusion: Our experience demonstrated that donor-type FFP is safe and effective in preventing acute hemolysis in major ABO-mismatched HSCT. We have also established the titer of 64 as the threshold that may cause hemolysis and therefore efforts should be made to reduce titer to below this level., (© 2018 AABB.)

The in situ control of redox insult in human organs is of major clinical relevance, yet remains incompletely understood. Activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the "master regulator" of genes controlling cellular redox homeostasis, is advocated as a therapeutic strategy for diseases with severely impaired redox balance. It remains to be shown whether this strategy is effective in human organs, rather than only in isolated human cell types. We have therefore explored the role of Nrf2 in a uniquely accessible human (mini-) organ: scalp hair follicles. Microarray and qRT-PCR analysis of human hair follicles after Nrf2 activation using sulforaphane identified the modulation of phase II metabolism, reactive oxygen species clearance, the pentose phosphate pathway, and glutathione homeostasis. Nrf2 knockdown (small interfering RNA) in cultured human hair follicles confirmed the regulation of key Nrf2 target genes (i.e., heme oxygenase-1, NAD(P)H dehydrogenase, quinone 1, glutathione reductase, glutamate-cysteine ligase catalytic subunit, ABCC1, peroxiredoxin 1). Importantly, Nrf2 activation significantly reduced reactive oxygen species levels and associated lipid peroxidation. Nrf2 preactivation reduced premature catagen and hair growth inhibition induced by oxidative stress (H 2 O 2 or menadione), significantly ameliorated the H 2 O 2 -dependent increase in matrix keratinocyte apoptosis and reversed the reactive oxygen species-induced reduction in hair matrix proliferation. This study thus provides direct evidence for the crucial role of Nrf2 in protecting human organ function (i.e., scalp hair follicles) against redox insult., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Recent evidence suggests a role of fibrogenesis in intervertebral disc (IVD) degeneration. We aim to explore if fibrotic genes may serve as IVD degeneration indicators, and if their expression is associated with myofibroblast activity., Design: Transcriptional expression of fibrosis markers (COL1A1, COL3A1, FN1, HSP47, MMP12, RASAL1) were analyzed in degenerated (D) and non-degenerated (ND) human nucleus pulposus (NP) and annulus fibrosus (AF) cells, along with traditional (SOX9, ACAN) and newly established degeneration markers (CDH2, KRT19, KRT18, FBLN1, MGP, and COMP). Protein expression was investigated by immunohistochemistry in human IVDs, and in rodent IVDs undergoing natural ageing or puncture-induced degeneration. Co-expression with myofibroblast markers was examined by double staining on human and rat specimens. Disc degeneration severity and extent of fibrosis were determined by histological scoring and picrosirius red staining respectively., Results: Human D-NP showed more intensive staining for picrosirius red than ND-NP. Among the genes examined, D-NP showed significantly higher MMP12 expression along with lower KRT19 expression. Protein expression analysis revealed increased MMP12(+) cells in human D-IVD. Histological scoring indicated mild degeneration in the punctured rat discs and discs of ageing mouse. Higher MMP12 positivity was found in peripheral NP and AF of the degenerative rat discs and in NP of the aged mice. In addition, human D-NP and D-AF showed increased α-SMA(+) cells, indicating enhanced myofibroblast activity. MMP12 was found co-expressed with α-SMA, FSP1 and FAP-α in human and rat degenerative IVDs., Conclusions: Our study suggests that in addition to a reduced KRT19 expression, an increased expression of MMP12, a profibrotic mediator, is characteristic of disc degenerative changes. Co-expression study indicates an association of the increased MMP12 positivity with myofibroblast activity in degenerated IVDs. Overall, our findings implicate an impact of MMP12 in disc cell homeostasis. The precise role of MMP12 in IVD degeneration warrants further investigation., (Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Nucleus pulposus (NP) cells of the intervertebral disc are essential for synthesizing extracellular matrix that contributes to disc health and mechanical function. NP cells have a unique morphology and molecular expression pattern derived from their notochordal origin, and reside in N-cadherin (CDH2) positive cell clusters in vivo. With disc degeneration, NP cells undergo morphologic and phenotypic changes including loss of CDH2 expression and ability to form cell clusters. Here, we investigate the role of CDH2 positive cell clusters in preserving healthy, biosynthetically active NP cells. Using a laminin-functionalized hydrogel system designed to mimic features of the native NP microenvironment, we demonstrate NP cell phenotype and morphology is preserved only when NP cells form CDH2 positive cell clusters. Knockdown (CRISPRi) or blocking CDH2 expression in vitro and in vivo results in loss of a healthy NP cell. Findings also reveal that degenerate human NP cells that are CDH2 negative can be promoted to re-express CDH2 and healthy, juvenile NP matrix synthesis patterns by promoting cell clustering for controlled microenvironment conditions. This work also identifies CDH2 interactions with β-catenin-regulated signaling as one mechanism by which CDH2-mediated cell interactions can control NP cell phenotype and biosynthesis towards maintenance of healthy intervertebral disc tissues.

Objective: There are few studies on fractional exhaled nitric oxide (FeNO) and respiratory symptoms among adults in tropical areas. The aim was to study associations between FeNO and selected personal factors, respiratory symptoms, allergies, office characteristics and indoor office exposures among office workers (n = 460) from a university in Malaysia., Methods: Information on health was collected by a questionnaire, skin prick test and FeNO measurement. Temperature, relative air humidity, carbon monoxide and carbon dioxide were measured in the offices. Settled dust was vacuumed in the offices and analyzed for endotoxin, (1,3)-β-glucan and house dust mites allergens, namely Dermatophagoides pteronyssinus (Der p 1) and Dermatophagoides farinae (Der f 1). Two-level linear mixed models and multiple logistic regression were used to analyze the associations., Results: One-fourth (25.9%) of the office workers had elevated FeNO level (≥ 25 ppb) and 61.5% had HDM, cat, seafood or pollen allergy. Male gender (p < 0.001), current smoking (p = 0.037), height (p < 0.001) and atopy (p < 0.001) were associated with FeNO. The amount of vacuumed dust was associated with FeNO among atopic subjects (p = 0.009). Asthma and rhinitis symptoms were associated with FeNO (p < 0.05), especially among atopic subjects. In particular, a combination of atopy and elevated FeNO were associated with doctor-diagnosed asthma (p < 0.001), rhinitis (p < 0.001) and airway symptoms last 12 months (p < 0.001)., Conclusion: Gender, smoking, height and atopy are important risk factors for elevated FeNO levels. A combination of allergy testing and FeNO measurement could be useful in respiratory illness epidemiology studies and patient investigations in tropical areas.

Sorghum bicolor is an important cereal crop grown on the arid and semi-arid regions of >98 different countries. These regions are such that this crop is often subjected to low water conditions, which can compromise yields. Stay-green sorghum plants are able to retain green leaf area for longer under drought conditions and as such have higher yields than their senescent counterparts. However, the molecular and physiological basis of this drought tolerance is yet to be fully understood. Here, a transcriptomic approach was used to compare gene expression between stay-green (B35) and senescent (R16) sorghum varieties. Ontological analysis of the differentially expressed transcripts identified an enrichment of genes involved with the 'response to osmotic stress' Gene Ontology (GO) category. In particular, delta1-pyrroline-5-carboxylate synthase 2 (P5CS2) was highly expressed in the stay-green line compared with the senescent line, and this high expression was correlated with higher proline levels. Comparisons of the differentially expressed genes with those that lie in known stay-green qualitative trait loci (QTLs) revealed that P5CS2 lies within the Stg1 QTL. Polymorphisms in known cis-elements were identified in the putative promoter region of P5CS2 and these could be responsible for the differences in the expression of this gene. This study provides greater insight into the stay-green trait in sorghum. This will be greatly beneficial not only to improve our understanding of drought tolerance mechanisms in sorghum, but also to facilitate the improvement of future sorghum cultivars by marker-assisted selection (MAS)., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

There are few studies on sick building syndrome (SBS) including clinical measurements for atopy and fractional exhaled nitric oxide (FeNO). Our aim was to study associations between SBS symptoms, selected personal factors, office characteristics and indoor office exposures among office workers from a university in Malaysia. Health data were collected by a questionnaire (n=695), skin prick test (SPT) (n=463) and FeNO test (n=460). Office settled dust was vacuumed and analyzed for endotoxin, (1,3)-β-glucan and house dust mites (HDM) allergens group 1 namely Dermatophagoides pteronyssinus (Der p 1) and Dermatophagoides farinae (Der f 1). Office indoor temperature, relative air humidity (RH), carbon monoxide (CO) and carbon dioxide (CO2) were measured by a direct reading instrument. Associations were studied by two-levels multiple logistic regression with mutual adjustment and stratified analysis. The prevalence of weekly dermal, mucosal and general symptoms was 11.9%, 16.0% and 23.0% respectively. A combination of SPT positivity (allergy to HDM or cat) and high FeNO level (≥25 ppb) was associated with dermal (p=0.002), mucosal (p<0.001) and general symptoms (p=0.05). Der f1 level in dust was associated with dermal (p<0.001), mucosal (p<0.001) and general (p=0.02) symptoms. Among those with allergy to D. farinae, associations were found between Der f 1 levels in dust and dermal (p=0.003), mucosal (p=0.001) and general symptoms (p=0.007). Office-related symptoms were associated with Der f 1 levels in dust (p=0.02), low relative air humidity (p=0.04) and high office temperature (p=0.05). In conclusion, a combination of allergy to cat or HDM and high FeNO is a risk factor for SBS symptoms. Der f 1 allergen in dust can be a risk factor for SBS in the office environment, particularly among those sensitized to Der f 1 allergen., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A prevalence study was conducted among office workers in Malaysia (N= 695). The aim of this study was to examine associations between asthma, airway symptoms, rhinitis and house dust mites (HDM) and cat allergy and HDM levels in office dust. Medical data was collected by a questionnaire. Skin prick tests were performed for HDM allergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae) and cat allergen Felis domesticus. Indoor temperature and relative air humidity (RH) were measured in the offices and vacuumed dust samples were analyzed for HDM allergens. The prevalence of D. pteronyssinus, D. farinae and cat allergy were 50.3%, 49.0% and 25.5% respectively. Totally 9.6% had doctor-diagnosed asthma, 15.5% had current wheeze and 53.0% had current rhinitis. The Der p 1 (from D. pteronyssinus) and Der f 1 (from D. farinae) allergens levels in dust were 556 ng/g and 658 ng/g respectively. Statistical analysis was conducted by multilevel logistic regression, adjusting for age, gender, current smoking, HDM or cat allergy, home dampness and recent indoor painting at home. Office workers with HDM allergy had more wheeze (p= 0.035), any airway symptoms (p= 0.032), doctor-diagnosed asthma (p= 0.005), current asthma (p= 0.007), current rhinitis (p= 0.021) and rhinoconjuctivitis (p< 0.001). Cat allergy was associated with wheeze (p= 0.021), wheeze when not having a cold (p= 0.033), any airway symptoms (p= 0.034), doctor-diagnosed asthma (p= 0.010), current asthma (p= 0.020) and nasal allergy medication (p= 0.042). Der f 1 level in dust was associated with daytime breathlessness (p= 0.033) especially among those with HDM allergy. Der f 1 levels were correlated with indoor temperature (p< 0.001) and inversely correlated with RH (p< 0.001). In conclusion, HDM and cat allergies were common and independently associated with asthma, airway symptoms and rhinitis. Der f 1 allergen can be a risk factor for daytime breathlessness.

Aims: At our centre, ductal carcinoma in situ (DCIS) was commonly treated with breast-conservation therapy (BCT). Local recurrence after BCT is a major concern. The aims of our study were to review the outcomes of DCIS treatment in our patients and to evaluate a nomogram from Memorial Sloan Kettering Cancer Centre (MSKCC) for predicting ipsilateral breast tumour recurrence (IBTR) in our Asian population., Materials and Methods: Chart reviews of 716 patients with pure DCIS treated from 1992 to 2011 were carried out. Univariable Cox regression analyses were used to evaluate the effects of the 10 prognostic factors of the MSKCC nomogram on IBTR. We constructed a separate National Cancer Centre Singapore (NCCS) nomogram based on multivariable Cox regression via reduced model selection by applying the stopping rule of Akaike's information criterion to predict IBTR-free survival. The abilities of the NCCS nomogram and the MSKCC nomogram to predict IBTR of individual patients were evaluated with bootstrapping of 200 sets of resamples and the NCCS dataset, respectively. Harrell's c-index was calculated for each nomogram to evaluate the concordance between predicted and observed responses of individual subjects., Results: Study patients were followed up for a median of 70 months. Over 95% of patients received adjuvant radiotherapy. The 5 and 10 year actuarial IBTR-free survival rates for the cohort were 95.5 and 92.6%, respectively. In the multivariate analysis, independent prognostic factors for IBTR included use of adjuvant endocrine therapy, presence of comedonecrosis and younger age at diagnosis. These factors formed the basis of the NCCS nomogram, which had a similar c-index (NCCS: 0.696; MSKCC: 0.673) compared with the MSKCC nomogram., Conclusion: The MSKCC nomogram was validated in an Asian population. A simpler NCCS nomogram using a different combination of fewer prognostic factors may be sufficient for the prediction of IBTR in Asians, but requires external validation to compare for relative performance., (Copyright © 2014 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.)

Objective: Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti-dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays., Methods: Microarray analysis was performed to profile gene expression changes in climbazole-treated primary human keratinocyte cells. Results were independently validated using qPCR and analysis of protein expression using ELISA and immunocytochemistry., Results: Microarray analysis of climbazole-treated keratinocytes showed statistically significant expression changes in genes associated with the gene ontology groups encompassing epidermal differentiation, keratinization, cholesterol biosynthesis and immune response. Upregulated genes included a number encoding cornified envelope proteins such as group 3 late-cornified envelope proteins, LCE3 and group 2 small-proline-rich proteins, SPRR2. Protein analysis studies of climbazole-treated primary keratinocytes using ELISA and immunocytochemistry were able to demonstrate that the increase in gene transcripts translated into increased protein expression of these cornified envelope markers., Conclusion: Climbazole treatment of primary keratinocytes results in an upregulation in expression of a number of genes including those encoding proteins involved in cornified envelope formation with further studies demonstrating this did translate into increased protein expression. A climbazole-driven increase in cornified envelope proteins may improve the scalp skin barrier, which is known to be weaker in dandruff. These studies suggest climbazole, besides its antifungal activity, is delivering positive skin benefits helping to relive dandruff symptoms effectively., (© 2014 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

Background: Abiotic stresses which include drought and heat are amongst the main limiting factors for plant growth and crop productivity. In the field, these stress types are rarely presented individually and plants are often subjected to a combination of stress types. Sorghum bicolor is a cereal crop which is grown in arid and semi-arid regions and is particularly well adapted to the hot and dry conditions in which it originates and is now grown as a crop. In order to better understand the mechanisms underlying combined stress tolerance in this important crop, we have used microarrays to investigate the transcriptional response of Sorghum subjected to heat and drought stresses imposed both individually and in combination., Results: Microarrays consisting of 28585 gene probes identified gene expression changes equating to ~4% and 18% of genes on the chip following drought and heat stresses respectively. In response to combined stress ~20% of probes were differentially expressed. Whilst many of these transcript changes were in common with those changed in response to heat or drought alone, the levels of 2043 specific transcripts (representing 7% of all gene probes) were found to only be changed following the combined stress treatment. Ontological analysis of these 'unique' transcripts identified a potential role for specific transcription factors including MYB78 and ATAF1, chaperones including unique heat shock proteins (HSPs) and metabolic pathways including polyamine biosynthesis in the Sorghum combined stress response., Conclusions: These results show evidence for both cross-talk and specificity in the Sorghum response to combined heat and drought stress. It is clear that some aspects of the combined stress response are unique compared to those of individual stresses. A functional characterization of the genes and pathways identified here could lead to new targets for the enhancement of plant stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of these abiotic stress types.

Natural human skin colour is determined both by environmental exposure to ultraviolet light and through inherited genetic variation in a very limited number of genes. Variation of a non-synonymous single-nucleotide polymorphism (nsSNP; rs1426654) in the gene (SLC24A5) encoding the NCKX5 protein is associated with differences in constitutive skin colour in South Asians. The nsSNP encodes the substitution of alanine for threonine at residue 111 (A111T) near a transmembrane region required for exchanger activity, a region which is highly conserved across different species and between NCKX family members. We have shown that NCKX5 is located at the trans-Golgi network of melanocytes and functions as a potassium-dependent sodium-calcium exchanger. When heterologously expressed, the 111T variant of NCKX5 shows significantly lower exchanger activity than the A111 variant. We have postulated that lower exchanger activity causes the reduced melanogenesis and lighter skin in Thr111-positive individuals. We used gene expression microarrays with qPCR replication and validation to assess the impact of siRNA-mediated knockdown of SLC24A5 on the transcriptome of cultured normal human melanocytes (NHM). Very few genes associated with melanogenesis were altered at the transcript level except for MC1R, suggesting that SLC24A5 interacts with at least one well-characterized melanogenic signalling pathway. More surprisingly, the expression of a number of cholesterol homeostatic genes was altered after SLC24A5 knockdown, and the total cholesterol content of NHM was increased. Cholesterol has previously been identified as a potential melanogenic regulator, and our data imply that NCKX5 exchanger function influences natural variation in skin pigmentation via a novel, unknown mechanism affecting cellular sterol levels.

The effect of hypoxia on global gene expression in human adipocytes has been examined using DNA microarrays. Adipocytes (Zen-Bio, day 12 post-differentiation) were exposed to hypoxia (1% O(2)) or 'normoxia' (21% O(2)) for 24 h and extracted RNA probed with Agilent arrays containing 41,152 probes. A total of 1346 probes were differentially expressed (>2.0-fold change, P < 0.01) in response to hypoxia; 650 genes were up-regulated (including LEP, IL6, VEGF, ANGPTL4) and 650 down-regulated (including ADIPOQ, UCP2). Major genes not previously identified as hypoxia-sensitive in adipocytes include AQP3, FABP3, FABP5 and PPARGC1A. Ingenuity analysis indicated that several pathways and functions were modulated by hypoxia, including glucose utilization, lipid oxidation and cell death. Network analysis indicated a down-regulation of p38/MAPK and PGC-1α signalling in the adipocytes. It is concluded that hypoxia has extensive effects on human adipocyte gene expression, consistent with low O(2) tension underlying adipose tissue dysfunction in obesity.

Senescence is thought to play an important role in the progressive age-related decline in tissue integrity and concomitant diseases, but not much is known about the complex interplay between upstream regulators and downstream effectors. We profiled whole genome gene expression of non-stressed and rotenone-stressed human fibroblast strains from young and oldest old subjects, and measured senescence associated β-gal activity. Microarray results identified gene sets involved in carbohydrate metabolism, Wnt/β-catenin signaling, the cell cycle, glutamate signaling, RNA-processing and mitochondrial function as being differentially regulated with chronological age. The most significantly differentially regulated mRNA corresponded to the p16 gene. p16 was then investigated using qPCR, Western blotting and immunocytochemistry. In conclusion, we have identified cellular pathways that are differentially expressed between fibroblast strains from young and old subjects., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The aim of this study was to examine the effects of macrophage secretions on global gene expression in human preadipocytes using microarrays. Preadipocytes were cultured with unconditioned or conditioned medium from U937 macrophages, and gene expression examined with Agilent arrays (43,000 probes). 472 transcripts were differentially regulated (>2-fold difference; P<0.05) between preadipocytes in the conditioned medium compared to the unconditioned; 401 were upregulated and 71 downregulated. The upregulated transcripts were particularly linked to inflammation, including IL-1β, IL-6, and CCL20 (16.8-, 10.0-, and 8.9-fold increases, respectively) together with matrix metalloproteinases (MMP3, MMP9 and MMP12). Major pathways regulated by the conditioned medium were linked to inflammation, macrophage infiltration and lipid accumulation. Network analysis identified NFkB and IL-1β as central nodes in the upregulation of multiple inflammation-related genes. Treatment with an IL-1β neutralising antibody abolished the stimulation of IL-6 secretion by conditioned medium, indicating that IL-1β is a key regulator of preadipocyte IL-6 production. Macrophages evoke extensive changes in preadipocyte gene expression., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

White adipose tissue exhibits inflammation as tissue mass expands in obesity, involving macrophage infiltration and a direct inflammatory response by adipocytes. DNA microarrays and conditioned medium have been used to examine the effects of macrophages on global gene expression in human adipocytes. SGBS adipocytes, differentiated in culture, were treated with macrophage-conditioned medium (U937 cells) for 4 or 24 h; control cells received unconditioned medium. Agilent arrays comprising 44,000 probes were used to analyse gene expression. Microarray analysis identified 1,088 genes differentially expressed in response to the conditioned medium at both 4 and 24 h (754 up-regulated, 334 down-regulated at 24 h); these included genes associated with inflammation and macrophage infiltration. A cluster of matrix metalloproteinase genes were highly up-regulated at both time-points, including MMP1, MMP3, MMP9, MMP10, MMP12 and MMP19. At 4 and 24 h, MMP1 was the most highly up-regulated gene (>2,400-fold increase in mRNA at 24 h). ELISA measurements indicated that substantial quantities of MMP1 and MMP3 were released from adipocytes incubated with conditioned medium, with little release by control adipocytes. Treatment with TNFalpha induced substantial increases in MMP1 (>100-fold) and MMP3 (27-fold) mRNA level and MMP1 and MMP3 release in adipocytes, suggesting that this cytokine could contribute to the stimulation of MMP expression by macrophages. In conclusion, macrophage-secreted factors induce a major inflammatory response in human adipocytes, with expression of MMP family members being strongly up-regulated. The induction of MMP1 and other MMPs suggests that macrophages stimulate tissue remodelling during adipose tissue expansion in obesity.

One of the most conserved methods to significantly increase lifespan in animals is through dietary restriction (DR). The mechanisms by which DR increases survival are controversial but are thought to include improvements in mitochondrial function concomitant with reductions in reactive oxygen species production and alterations in the insulin signalling pathway, resulting in global metabolic adaptation. In order to identify novel genes that may be important for lifespan extension of Brown Norway rats, we compared gene expression profiles from skeletal muscle of 28-month-old animals fed ad libitum or DR diets using whole-genome arrays. Following DR, 426 transcripts were significantly down-regulated whilst only 52 were up-regulated. Included in the up-regulated transcripts were three functionally related previously unidentified DR-regulated genes: Nr4a1, Nr4a2, and Nr4a3. Up-regulation of all three Nr4a receptors was also observed in liver - but not brain - of DR-fed animals. Furthermore, RT-PCR revealed up-regulation of several NR4A transcriptional targets (Ucp-3, Ampk-gamma3, Pgc-1alpha and Pgc-1beta) in skeletal muscle of DR animals. Due to the proposed roles of the NR4A nuclear receptors in sensing and responding to changes in the nutritional environment and in regulating glucose and lipid metabolism and insulin sensitivity, we hypothesise that these proteins may contribute to DR-induced metabolic adaptation.

A number of physiological changes follow prolonged skeletal muscle unloading as occurs in spaceflight, bed rest, and hindlimb suspension (HLS) and also in aging. These include muscle atrophy, fiber type switching, and loss of the ability to switch between lipid and glucose usage, or metabolic inflexibility. The signaling and genomic events that precede these physiological manifestations have not been investigated in detail, particularly in regard to loss of metabolic flexibility. Here we used gene arrays to determine the effects of 24-h HLS on metabolic remodeling in mouse muscle. Acute unloading resulted in differential expression of a number of transcripts in soleus and gastrocnemius muscle, including many involved in lipid and glucose metabolism. These include the peroxisome proliferator-activated receptors (PPARs). In contrast to Ppar-alpha and Ppar-gamma, which were downregulated by acute HLS, Ppar-delta was upregulated concomitant with increased expression of its downstream target, uncoupling protein-3 (Ucp-3). However, differential expression of Ppar-delta was both acute and transient in nature, suggesting that regulation of PPARdelta may represent an adaptive, compensatory response aimed at regulating fuel utilization and maintaining metabolic flexibility.

Ferroportin disease is an autosomal dominant form of hemochromatosis associated with siderosis in cells of the mononuclear phagocyte system and, to varying degrees, in hepatocytes. Ferroportin was investigated as a candidate gene in two pedigrees with hyperferritinaemia and siderosis in mononuclear phagocytes. The entire ferroportin coding region was sequenced and hepatic iron concentration, histology and response to treatment were determined. The results were compared with previously reported cases. The A77D mutation was detected in patient 1, his father (patient 2) and his brother (patient 3), who had portal fibrosis. The V162del mutation was detected in patient 4, who developed anemia after the third weekly venesection. While the disease is rare, A77D and V162del are the most common ferroportin mutations in Caucasians. The spectrum of clinical expression of these two mutations was reviewed in all cases described to date. These mutations were associated with fibrosis in about a third of cases. For A77D and V162del, this analysis confirms that the threshold hepatic iron concentration for development of fibrosis may be higher than for classical hemochromatosis. These two mutations, which both decreased iron export in cell culture studies, give rise to similar patterns of clinical expression and morbidity, although the highest hepatic iron concentrations have been observed with A77D. It is important for clinicians to consider ferroportin disease in cases where there are features of iron overload unrelated to HFE, autosomal dominant inheritance and/or iron deposition in mononuclear phagocytes.

DNA microarrays can be used to measure environmental stress responses. If they are to be predictive of environmental impact, we need to determine if altered gene expression translates into negative impacts on individuals and populations. A large cDNA microarray (14000 spots) was created to measure molecular stress responses to cadmium in Daphnia magna,the mostwidely used aquatic indicator species, and relate responses to population growth rate (pgr). We used the array to detect differences in the transcription of genes in juvenile D. magna (24 h old) after 24 h exposure to a control and three cadmium concentrations (6, 20, and 37 microg Cd2+ L(-1)). Stress responses at the population level were estimated following a further 8 days exposure. Pgr was approximately linear negative with increasing cadmium concentration over this range. The microarray profile of gene expression in response to acute cadmium exposure begins to provide an overview of the molecular responses of D. magna, especially in relation to growth and development. Of the responding genes, 29% were involved with metabolism including carbohydrate, fat and peptide metabolism, and energy production, 31% were involved with transcription/translation, while 40% of responding genes were associated with cellular processes like growth and moulting, ion transport, and general stress responses (which included oxidative stress). Our production and application of a large Daphnia magna microarray has shown that measured gene responses can be logically linked to the impact of a toxicant such as cadmium on somatic growth and development, and consequently pgr.

Current attitudes toward organ donation among university students in mainland China and the differences in attitudes between Chinese students in mainland China versus overseas are unknown. To address these issues, we conducted a cross-sectional survey using questionnaires among 922 Chinese undergraduates from mainland China and overseas regions of the world. Data were analyzed by descriptive statistics, Student t tests, chi-square tests, and a logistic regression analysis. We found that blood donors showed significantly better awareness of heart, liver, lung, skin, and tendon donation among commonly transplanted organs/tissues. As to the willingness for cadaveric organ donation, 61.3% of respondents consented, 8.5% objected, and 30.3% answered "not sure." The percentage holding an organ donor card was 15.7% among students from Hong Kong; 3.0%, mainland China; 2.8%, Macau; 2.6%, Taiwan, and 4.0%, other regions of the world. In a logistic regression analysis, female students (odds ratio [OR], 2.24; 95% confidence interval [CI], 1.35 to 3.72) and blood donors (OR, 1.91; 95% CI, 1.10 to 3.32) did, but age and study specialty (medical vs nonmedical) did not show significantly more positive attitudes toward cadaveric organ donation. Compared with students from mainland China, overseas Chinese students from various regions did not show significantly different attitudes toward cadaveric organ donation. In summary, blood donors among university students have a greater knowledge of transplantation and a more positive attitude toward organ donation. Since university students are an important source of blood donors in China, they will be a potential pool of organ donors in the future.

The circadian clock controls many aspects of mammalian physiology and behaviour with a periodicity of approximately 24 h. These include the anticipation of, and adaptation to, daily environmental changes such as the light-dark cycle, temperature fluctuations and the availability of food. The toxicity of many drugs is dependent on the circadian phase at which they are administered, and recent work has begun to unravel the molecular basis for circadian variations in sensitivity to xenobiotic exposure. Between 2 and 10% of the transcriptome is expressed in a circadian manner, including many key genes associated with the metabolism and transport of xenobiotics. Furthermore, a number of xenobiotics may directly alter the expression of genes that control circadian rhythms. This review discusses the emerging evidence for the regulation of circadian rhythm genes having an important impact on molecular response to xenobiotics.

We have used global gene expression profiling, combined with pathway analysis tools, to identify in rats the molecular events associated with paraquat toxicity in the lung. Early (2, 8 and 18h) gene expression changes induced following intraperitoneal (i.p.) exposure to paraquat were measured in the caudal lobe of lungs using Affymetrix rat genome GeneChips (31,042 probe sets). A single high dose of paraquat dichloride (20mg/kg) was used that has been shown previously to cause in rats extensive lung fibrosis after 10 days. Hierarchical clustering of 543 paraquat-responsive genes (false discovery rate<0.05) revealed that under these conditions of exposure paraquat induces a staged transcriptional response in the rat lung that precedes the appearance of lung damage. We report here that many of the transcriptional responses to paraquat were rapid (being maximal at 2h post-dose), and that the predominant molecular functions and biological processes associated with these genes include membrane transport, oxidative stress, lung development, epithelial cell differentiation and transforming growth factor beta (TGF-beta) signalling. These data provide novel insights into the molecular pathways that lead to toxicity after exposure of the rat lung to paraquat.

We have previously used genome-wide transcript profiling to investigate the relationships between changes in gene expression and physiological alterations during the response of the immature mouse uterus to estrogens. Here we describe the identification of a functionally inter-related group of estrogen-responsive genes associated with iron homeostasis, including the iron-binding protein lactotransferrin, the ferroxidase ceruloplasmin, the iron delivery protein lipocalin 2 and the iron-exporter ferroportin. Quantitative real-time PCR revealed that the expression of these genes increases with time during the uterotrophic response, reaching maximal levels in the post-proliferative phase (between 48 and 72 h). In contrast, the heme biosynthesis genes aminolevulinic acid synthase 1 and 2 were maximally induced by estrogen at 2 and 4 h, respectively, prior to increased cell proliferation. Together, these data reveal that estrogen induces the temporally coordinated expression of iron homeostasis genes in the mouse uterus, and suggest an important role for iron metabolism during sex steroid hormone-induced uterine cell growth and differentiation.

Toxicogenomics has the potential to reveal the molecular pathways and cellular processes that mediate the adverse responses to a toxicant. However, the initial output of a toxicogenomic experiment often consists of large lists of genes whose expression is altered after toxicant exposure. To interpret gene expression changes in the context of underlying biological pathways and processes, new bioinformatics methods must be developed. We have used global gene expression profiling combined with an evaluation of Gene Ontology (GO) and pathway mapping tools as unbiased methods for identifying the molecular pathways and processes affected upon toxicant exposure. We chose to use the acute effects caused by the non-genotoxic carcinogen and peroxisome proliferator (PP) diethylhexylphthalate (DEHP) in the mouse liver as a model system. Consistent with what is known about the mode of action of DEHP, our GO analysis of transcript profiling data revealed a striking overrepresentation of genes associated with the peroxisomal cellular component, together with genes involved in carboxylic acid and lipid metabolism. Furthermore we reveal gene expression changes associated with additional biological functions, including complement activation, hemostasis, the endoplasmic reticulum overload response, and circadian rhythm. Together, these data reveal potential new pathways of PP action and shed new light on the mechanisms by which non-genotoxic carcinogens control hepatocyte hypertrophy and proliferation. We demonstrate that GO mapping can identify, in an unbiased manner, both known and novel DEHP-induced molecular changes in the mouse liver and is therefore a powerful approach for elucidating modes of toxicity based on toxicogenomic data.

Estrogen receptor (ER)-negative breast carcinomas do not respond to hormone therapy, making their effective treatment very difficult. The re-expression of ERalpha in ER-negative MDA-MB-231 breast cancer cells has been used as a model system, in which hormone-dependent responses can be restored. Paradoxically, in contrast to the mitogenic activity of 17beta-estradiol (E2) in ER-positive breast cancer cells, E2 suppresses proliferation in ER-negative breast cancer cells in which ERalpha has been re-expressed. We have used global gene expression profiling to investigate the mechanism by which E2 suppresses proliferation in MDA-MB-231 cells that express ERalpha through adenoviral infection. We show that a number of genes known to promote cell proliferation and survival are repressed by E2 in these cells. These include genes encoding the anti-apoptosis factor SURVIVIN, positive cell cycle regulators (CDC2, CYCLIN B1, CYCLIN B2, CYCLIN G1, CHK1, BUB3, STK6, SKB1, CSE1 L) and chromosome replication proteins (MCM2, MCM3, FEN1, RRM2, TOP2A, RFC1). In parallel, E2-induced the expression of the negative cell cycle regulators KIP2 and QUIESCIN Q6, and the tumour-suppressor genes E-CADHERIN and NBL1. Strikingly, the expression of several of these genes is regulated in the opposite direction by E2 compared with their regulation in ER-positive MCF-7 cells. Together, these data suggest a mechanism for the E2-dependent suppression of proliferation in ER-negative breast cancer cells into which ERalpha has been reintroduced.

A major challenge in the emerging field of toxicogenomics is to define the relationships between chemically induced changes in gene expression and alterations in conventional toxicologic parameters such as clinical chemistry and histopathology. We have explored these relationships in detail using the rodent uterotrophic assay as a model system. Gene expression levels, uterine weights, and histologic parameters were analyzed 1, 2, 4, 8, 24, 48, and 72 hr after exposure to the reference physiologic estrogen 17 beta-estradiol (E2). A multistep analysis method, involving unsupervised hierarchical clustering followed by supervised gene ontology-driven clustering, was used to define the transcriptional program associated with E2-induced uterine growth and to identify groups of genes that may drive specific histologic changes in the uterus. This revealed that uterine growth and maturation are preceded and accompanied by a complex, multistage molecular program. The program begins with the induction of genes involved in transcriptional regulation and signal transduction and is followed, sequentially, by the regulation of genes involved in protein biosynthesis, cell proliferation, and epithelial cell differentiation. Furthermore, we have identified genes with common molecular functions that may drive fluid uptake, coordinated cell division, and remodeling of luminal epithelial cells. These data define the mechanism by which an estrogen induces organ growth and tissue maturation, and demonstrate that comparison of temporal changes in gene expression and conventional toxicology end points can facilitate the phenotypic anchoring of toxicogenomic data.

We used gene expression profiling to investigate whether the molecular effects induced by estrogens of different provenance are intrinsically similar. In this article we show that the physiologic estrogen 17-beta-estradiol, the phytoestrogen genistein, and the synthetic estrogen diethylstilbestrol alter the expression of the same 179 genes in the intact immature mouse uterus under conditions where each chemical has produced an equivalent gravimetric and histologic uterotrophic effect, using the standard 3-day assay protocol. Data are also presented indicating the limitations associated with comparison of gene expression profiles for different chemicals at times before the uterotrophic effects are fully realized. We conclude that the case has yet to be made for regarding synthetic estrogens as presenting a unique human hazard compared with phytoestrogens and physiologic estrogens. Key words: diethylstilbestrol, estrogen, gene expression, genistein, microarray, phytoestrogen, toxicogenomics, uterus.

The MADS-box transcription factor Mcm1p and forkhead (FKH) transcription factor Fkh2p act in a DNA-bound complex to regulate cell-cycle dependent expression of the CLB2 cluster in Saccharomyces cerevisiae. Binding of Fkh2p requires prior binding by Mcm1p. Here we have investigated the molecular determinants governing the formation of the Mcm1p- Fkh2p complex. Fkh2p exhibits cooperativity in complex formation with Mcm1p and we have mapped a small region of Fkh2p located immediately upstream of the FKH DNA binding domain that is required for this cooperativity. This region is lacking in the related protein Fkh1p that cannot form ternary complexes with Mcm1p. A second region is identified that inhibits Mcm1p-independent DNA binding by Fkh2p. The spacing between the Mcm1p and Fkh2p binding sites is also a critical determinant for complex formation. We also show that Fkh2p can form ternary complexes with the human counterpart of Mcm1p, serum response factor (SRF). Mutations at analogous positions in Mcm1p, which are known to affect SRF interaction with its partner protein Elk-1, abrogate complex formation with Fkh2p, demonstrating evolutionary conservation of coregulatory protein binding surfaces. Our data therefore provide molecular insights into the mechanisms of Mcm1p- Fkh2p complex formation and more generally aid our understanding of MADS-box protein function.

The yeast MADS-box transcription factor Mcm1p plays an important regulatory role in several diverse cellular processes. In common with a subset of other MADS-box transcription factors, Mcm1p elicits substantial DNA bending. However, the role of protein-induced bending by MADS-box proteins in eukaryotic gene regulation is not understood. Here, we demonstrate an important role for Mcm1p-mediated DNA bending in determining local promoter architecture and permitting the formation of ternary transcription factor complexes. We constructed mutant mcm1 alleles that are defective in protein-induced bending. Defects in nuclear division, cell growth or viability, transcription, and gene expression were observed in these mutants. We identified one likely cause of the cell growth defects as the aberrant formation of the cell cycle-regulatory Fkh2p-Mcm1p complex. Microarray analysis confirmed the importance of Mcm1p-mediated DNA bending in maintaining correct gene expression profiles and revealed defects in Mcm1p-mediated repression of Ty elements and in the expression of the cell cycle-regulated YFR and CHS1 genes. Thus, we discovered an important role for DNA bending by MADS-box proteins in the formation and function of eukaryotic transcription factor complexes.

Hybridization array technology is increasingly being used for the analysis of gene expression in the yeast Saccharomyces cerevisiae. It is a powerful technique in which the relative abundance of all the mRNA molecules transcribed under a particular condition may be simultaneously measured. However, most studies performed using this technique are carried out in batch culture where the growth rate and environment are continuously changing. Often, the experimental condition being studied also impacts on the growth rate of the cells. Changes in growth rate affect the pattern of gene expression. Consequently, the analysis and interpretation of experimental results obtained in this way are inherently problematic due to the difficulty in discriminating between effects due to the experimental condition per se and concomitant growth rate-related effects. Here, we present a method that addresses this problem by exploiting chemostat culture, in which the cells can be grown at a fixed growth rate, in combination with hybridization array technology. We use two experimental examples to illustrate the advantages of using this approach and then describe a specific application of this approach to investigate the effect of carbon and nitrogen limitation at the transcriptome level.

In the yeast Saccharomyces cerevisiae, the MADS-box protein Mcm1, which is highly related to mammalian SRF (serum response factor), forms a ternary complex with SFF (Swi five factor) to regulate the cell cycle expression of genes such as SWI5, CLB2 and ACE2. Here we show that the forkhead protein Fkh2 is a component of SFF and is essential for ternary complex formation on the SWI5 and ACE2 promoters. Fkh2 is essential for the correct cell cycle periodicity of SWI5 and CLB2 gene expression and is phosphorylated with a timing that is consistent with a role in this expression. Furthermore, investigation of the relationship between Fkh2 and a related forkhead protein Fkh1 demonstrates that these proteins act in overlapping pathways to regulate cell morphology and cell separation. This is the first example of a eukaryotic transcription factor complex containing both a MADS-box and a forkhead protein, and it has important implications for the regulation of mammalian gene expression.