Genome-wide identification of FoxO-dependent gene networks in skeletal muscle during C26 cancer cachexia

BACKGROUND: Evidence from cachectic cancer patients and animal models of cancer cachexia supports the involvement of Forkhead box O (FoxO) transcription factors in driving cancer-induced skeletal muscle wasting. However, the genome-wide gene networks and associated biological processes regulated by FoxO during cancer cachexia are unknown. We hypothesize that FoxO is a central upstream regulator of diverse gene networks in skeletal muscle during cancer that may act coordinately to promote the wasting phenotype. METHODS: To inhibit endogenous FoxO DNA-binding, we transduced limb and diaphragm muscles of mice with AAV9 containing the cDNA for a dominant negative (d.n.) FoxO protein (or GFP control). The d.n.FoxO construct consists of only the FoxO3a DNA-binding domain that is highly homologous to that of FoxO1 and FoxO4, and which outcompetes and blocks endogenous FoxO DNA binding. Mice were subsequently inoculated with Colon-26 (C26) cells and muscles harvested 26 days later. RESULTS: Blocking FoxO prevented C26-induced muscle fiber atrophy of both locomotor muscles and the diaphragm and significantly spared force deficits. This sparing of muscle size and function was associated with the differential regulation of 543 transcripts (out of 2,093) which changed in response to C26. Bioinformatics analysis of upregulated gene transcripts that required FoxO revealed enrichment of the proteasome, AP-1 and IL-6 pathways, and included several atrophy-related transcription factors, including Stat3, Fos, and Cebpb. FoxO was also necessary for the cancer-induced downregulation of several gene transcripts that were enriched for extracellular matrix and sarcomere protein-encoding genes. We validated these findings in limb muscles and the diaphragm through qRT-PCR, and further demonstrate that FoxO1 and/or FoxO3a are sufficient to increase Stat3, Fos, Cebpb, and the C/EBPβ target gene, Ubr2. Analysis of the Cebpb proximal promoter revealed two bona fide FoxO binding elements, which we further establish are necessary for Cebpb promoter activation in response to IL-6, a predominant cytokine in the C26 cancer model. CONCLUSIONS: These findings provide new evidence that FoxO-dependent transcription is a central node controlling diverse gene networks in skeletal muscle during cancer cachexia, and identifies novel candidate genes and networks for further investigation as causative factors in cancer-induced wasting.R01 AR060217 - NIAMS NIH HHS; R01 AR060209 - NIAMS NIH HHS; T32 HD043730 - NICHD NIH HHS; R00 HL098453 - NHLBI NIH HHS; R00HL098453 - NHLBI NIH HHS; R01AR060209 - NIAMS NIH HHS; R01AR060217 - NIAMS NIH HH

Informing the design of a national screening and treatment programme for chronic viral hepatitis in primary care: qualitative study of at-risk immigrant communities and healthcare professionals

Background: Effective strategies are needed to provide screening and treatment for hepatitis B and C to immigrant groups in the UK at high risk of chronic infection. This study aimed to build an understanding of the knowledge, beliefs and attitudes towards these conditions and their management in a range of high-risk minority ethnic communities and health professionals, in order to inform the design of a screening and treatment programme in primary care. Methods: Qualitative data collection consisted of three sequential phases- (i) semi-structured interviews with key informants (n = 17), (ii) focus groups with people from Chinese, Pakistani, Roma, Somali, and French- and English-speaking African communities (n = 95), and (iii) semi-structured interviews with general practitioners (n = 6). Datasets from each phase were analysed using the Framework method. Results: Key informants and general practitioners perceived that there was limited knowledge and understanding about hepatitis B and C within high-risk immigrant communities, and that chronic viral hepatitis did not typically feature in community discourses about serious illness. Many focus group participants were confused about the differences between types of viral hepatitis, held misconceptions regarding transmission, and were unaware of the asymptomatic nature of chronic infection. Most welcomed the idea of a screening programme, but key informants and focus group participants also identified numerous practical barriers to engagement with primary care-based screening and treatment; including language and communication difficulties, limited time (due to long working hours), and (for some) low levels of trust and confidence in general practice-based care. General practitioners expressed concerns about the workload implications and sustainability of screening and treating immigrant patients for chronic viral hepatitis in primary care. Conclusions: Strategies to reduce the burden of chronic viral hepatitis in immigrant communities will need to consider how levels of understanding about hepatitis B and C within these communities, and barriers to accessing healthcare, may affect capacity to engage with screening and treatment. Services may need to work with community groups and language support services to provide information and wider encouragement for screening. Primary care services will need ongoing consultation regarding their support needs to deliver hepatitis screening and treatment programmes

Diaphragm Abnormalities in Patients with End-Stage Heart Failure: NADPH Oxidase Upregulation and Protein Oxidation

Patients with heart failure (HF) have diaphragm abnormalities that contribute to disease morbidity and mortality. Studies in animals suggest that reactive oxygen species (ROS) cause diaphragm abnormalities in HF. However, the effects of HF on ROS sources, antioxidant enzymes, and protein oxidation in the diaphragm of humans is unknown. NAD(P)H oxidase, especially the Nox2 isoform, is an important source of ROS in the diaphragm. Our main hypothesis was that diaphragm from patients with HF have heightened Nox2 expression and p47phox phosphorylation (marker of enzyme activation) that is associated with elevated protein oxidation. We collected diaphragm biopsies from patients with HF and brain-dead organ donors (controls). Diaphragm mRNA levels of Nox2 subunits were increased 2.5–4.6-fold over controls (p \u3c 0.05). Patients also had increased protein levels of Nox2 subunits (p47phox, p22phox, and p67phox) and total p47phox phosphorylation, while phospho-to-total p47phox levels were unchanged. The antioxidant enzyme catalase was increased in patients, whereas glutathione peroxidase and superoxide dismutases were unchanged. Among markers of protein oxidation, carbonyls were increased by ~40% (p \u3c 0.05) and 4-hydroxynonenal and 3-nitrotyrosines were unchanged in patients with HF. Overall, our findings suggest that Nox2 is an important source of ROS in the diaphragm of patients with HF and increases in levels of antioxidant enzymes are not sufficient to maintain normal redox homeostasis. The net outcome is elevated diaphragm protein oxidation that has been shown to cause weakness in animals

Predictors of student mask mandate policies in United States school districts during the COVID-19 pandemic

IntroductionAlthough factors such as urbanicity, population demographics, and political affiliation have been linked with COVID-19 masking behavior and policy in community settings, little work has investigated factors associated with school mask policies. We sought to characterize United States state and school district student COVID-19 masking policies during the 2021–22 school year and determine predictors of these mandates at four time points, including before and after federal guidance relaxed school mask recommendations in February 2022.MethodsStudent mask policies for US states and the District of Columbia, as well as a sample of 56 districts were categorized as prohibited, recommended, or required in September 2021, November 2021, January 2022, and March 2022 based on the Johns Hopkins eSchool+ Initiative School Reopening Tracker. Changes in policies over time were characterized. Generalized estimating equations and logistic regression were used to evaluate whether political affiliation of governor, urbanicity, economic disadvantage, and race/ethnic composition of district students, and county-level COVID-19 incidence predicted the presence of a district mask mandate at any time point and at all four time points.ResultsState and district policies changed over time. Districts that implemented student mandates at any point were more likely to be in states with Democratic governors (AOR: 5.52; 95% CI: 2.23, 13.64) or in non-rural areas (AOR: 8.20; 95% CI: 2.63, 25.51). Districts that retained mask mandates at all four time points were more likely to have Democratic governors (AOR: 5.39; 95% CI: 2.69, 10.82) and serve a smaller proportion of economically disadvantaged students (AOR: 0.97; 95% CI: 0.95, 0.99). Districts serving a larger proportion of students from minoritized racial/ethnic groups were more likely to have mask mandates at any or all timepoints. Notably, county-level COVID-19 prevalence was not related to the presence of a mask mandate at any or all time points. By March 2022, no factors were significantly associated with district mask policy.DiscussionPolitical, geographic, and demographic characteristics predicted the likelihood of student mask mandates in the 2021–22 school year. Public health promotion messages and policy must account for variation in these factors, potentially through centralized and consistent messaging and unbiased, trustworthy communication

Deducing the pathogenic contribution of recessive ABCA4 alleles in an outbred population

Accurate prediction of the pathogenic effects of specific genotypes is important for the design and execution of clinical trials as well as for meaningful counseling of individual patients. However, for many autosomal recessive diseases, it can be difficult to deduce the relative pathogenic contribution of individual alleles because relatively few affected individuals share the same two disease-causing variations. In this study, we used multiple regression analysis to estimate the pathogenicity of specific alleles of ABCA4 in patients with retinal phenotypes ranging from Stargardt disease to retinitis pigmentosa. This analysis revealed quantitative allelic effects on two aspects of the visual phenotype, visual acuity (P < 10−3) and visual field (P < 10−7). Discordance between visual acuity and visual field in individual patients suggests the existence of at least two non-ABCA4 modifying factors. The findings of this study will facilitate the discovery of factors that modify ABCA4 disease and will also aid in the optimal selection of subjects for clinical trials of new therapies

Informing the design of a national screening and treatment programme for chronic viral hepatitis in primary care: qualitative study of at-risk immigrant communities and healthcare professionals

n Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise statedThis paper presents independent research funded by the National Institute for Health Research (NIHR) under the Programme Grants for Applied Research programme (RP-PG-1209-10038).

Genome-wide identification of FoxO-dependent gene networks in skeletal muscle during C26 cancer cachexia

BACKGROUND: Evidence from cachectic cancer patients and animal models of cancer cachexia supports the involvement of Forkhead box O (FoxO) transcription factors in driving cancer-induced skeletal muscle wasting. However, the genome-wide gene networks and associated biological processes regulated by FoxO during cancer cachexia are unknown. We hypothesize that FoxO is a central upstream regulator of diverse gene networks in skeletal muscle during cancer that may act coordinately to promote the wasting phenotype. METHODS: To inhibit endogenous FoxO DNA-binding, we transduced limb and diaphragm muscles of mice with AAV9 containing the cDNA for a dominant negative (d.n.) FoxO protein (or GFP control). The d.n.FoxO construct consists of only the FoxO3a DNA-binding domain that is highly homologous to that of FoxO1 and FoxO4, and which outcompetes and blocks endogenous FoxO DNA binding. Mice were subsequently inoculated with Colon-26 (C26) cells and muscles harvested 26 days later. RESULTS: Blocking FoxO prevented C26-induced muscle fiber atrophy of both locomotor muscles and the diaphragm and significantly spared force deficits. This sparing of muscle size and function was associated with the differential regulation of 543 transcripts (out of 2,093) which changed in response to C26. Bioinformatics analysis of upregulated gene transcripts that required FoxO revealed enrichment of the proteasome, AP-1 and IL-6 pathways, and included several atrophy-related transcription factors, including Stat3, Fos, and Cebpb. FoxO was also necessary for the cancer-induced downregulation of several gene transcripts that were enriched for extracellular matrix and sarcomere protein-encoding genes. We validated these findings in limb muscles and the diaphragm through qRT-PCR, and further demonstrate that FoxO1 and/or FoxO3a are sufficient to increase Stat3, Fos, Cebpb, and the C/EBPβ target gene, Ubr2. Analysis of the Cebpb proximal promoter revealed two bona fide FoxO binding elements, which we further establish are necessary for Cebpb promoter activation in response to IL-6, a predominant cytokine in the C26 cancer model. CONCLUSIONS: These findings provide new evidence that FoxO-dependent transcription is a central node controlling diverse gene networks in skeletal muscle during cancer cachexia, and identifies novel candidate genes and networks for further investigation as causative factors in cancer-induced wasting.R01 AR060217 - NIAMS NIH HHS; R01 AR060209 - NIAMS NIH HHS; T32 HD043730 - NICHD NIH HHS; R00 HL098453 - NHLBI NIH HHS; R00HL098453 - NHLBI NIH HHS; R01AR060209 - NIAMS NIH HHS; R01AR060217 - NIAMS NIH HH

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Characterization of Azobenzene-based Photo-switches and their Evaluation for In Vivo Applications

Photoisomerization of azobenzene can be used to reversibly photo-control peptide and protein structure, thereby offering the potential to probe peptide and protein function directly in living systems. Most azobenzene photo-switches to date however require the use of UV light, which suffers from poor tissue penetration and can lead to cell damage. In addition, the unknown stability of azobenzene photo-switches within the reducing intracellular environment has limited these switches to extracellular applications. The ability to red-shift the photo-switching wavelength and tune the thermal cis-to-trans rate independently from one another has been a central challenge. Incorporating a piperazine-like moiety at the 4,4′-para positions red-shifts the π-π* band of an azobenzene-based cross-linker such that trans-to-cis photoisomerization can be triggered with blue light. Accompanying this red-shift was a faster cis-to-trans thermal rate, which allowed for fast secondary structural changes of the attached peptides (τ½ ~2s). Piperazine-like moieties at the 2,2′-ortho positions of an azobenzene-based cross-linker resulted in a similar red-shift in the π-π* band but a longer-lived cis isomer relative to its para-counterpart (τ½ ~minutes). These results suggest that the thermal rate could be tuned independently from the photo-switching wavelength by appropriate para or ortho substitution. The effect of 2,2′,6,6′-ortho-tetramethoxysubstitution provided an alternative approach to red-shifting the photo-switching wavelength. These groups caused an unconventional red-shift in the n-π* band of the trans isomer allowing for trans-to-cis and cis-to-trans photoisomerization to occur with green and blue light, respectively. In this case, the half-life of the cis isomer was not shortened, but rather extended relative to its parent compound (τ½ ~days versus minutes). These results provide progress in tuning the photo-switching wavelength independently from the thermal rate. A fluorescent reporter was developed to determine the stability of a commonly employed 4,4′-diamido derivative in vivo. Photoisomerization was found to cause time-dependent changes in fluorescein emission intensity. The reporter was microinjected in zebrafish embryos and photo-switching could be imaged for at least two days. This work provides the first direct evidence of azobenzene photo-switching in vivo, and indicates that it will be possible, in general, to photo-control peptide and protein function in living systems.Ph

Fluorogenic Real-Time Reporters of DNA Repair by MGMT, a Clinical Predictor of Antitumor Drug Response.

Common alkylating antitumor drugs, such as temozolomide, trigger their cytotoxicity by methylating the O6-position of guanosine in DNA. However, the therapeutic effect of these drugs is dampened by elevated levels of the DNA repair enzyme, O6-methylguanine DNA methyltransferase (MGMT), which directly reverses this alkylation. As a result, assessing MGMT levels in patient samples provides an important predictor of therapeutic response; however, current methods available to measure this protein are indirect, complex and slow. Here we describe the design and synthesis of fluorescent chemosensors that report directly on MGMT activity in a single step within minutes. The chemosensors incorporate a fluorophore and quencher pair, which become separated by the MGMT dealkylation reaction, yielding light-up responses of up to 55-fold, directly reflecting repair activity. Experiments show that the best-performing probe retains near-native activity at mid-nanomolar concentrations. A nuclease-protected probe, NR-1, was prepared and tested in tumor cell lysates, demonstrating an ability to evaluate relative levels of MGMT repair activity in twenty minutes. In addition, a probe was employed to evaluate inhibitors of MGMT, suggesting utility for discovering new inhibitors in a high-throughput manner. Probe designs such as that of NR-1 may prove valuable to clinicians in selection of patients for alkylating drug therapies and in assessing resistance that arises during treatment