O-GlcNAc regulates anti-fibrotic genes in lung fibroblasts through EZH2.

Epigenetic modifications are involved in fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and contribute to the silencing of anti-fibrotic genes. H3K27me3, a key repressive histone mark, is catalysed by the methyltransferase enhancer of Zeste homologue 2 (EZH2), which is regulated by the post-translational modification, O-linked N-Acetylglucosamine (O-GlcNAc). In this study, we explored the effects of O-GlcNAc and EZH2 on the expression of antifibrotic genes, cyclooxygenase-2 (Cox2) and Heme Oxygenase (Homx1). The expression of Cox2 and Hmox1 was examined in primary IPF or non-IPF lung fibroblasts with or without EZH2 inhibitor EZP6438, O-GlcNAc transferase (OGT) inhibitor (OSMI-1) or O-GlcNAcase (OGA) inhibitor (thiamet G). Non-IPF cells were also subjected to TGF-ß1 with or without OGT inhibition. The reduced expression of Cox2 and Hmox1 in IPF lung fibroblasts is restored by OGT inhibition. In non-IPF fibroblasts, TGF-ß1 treatment reduces Cox2 and Hmox1 expression, which was restored by OGT inhibition. ChIP assays demonstrated that the association of H3K27me3 is reduced at the Cox2 and Hmox1 promoter regions following OGT or EZH2 inhibition. EZH2 levels and stability were decreased by reducing O-GlcNAc. Our study provided a novel mechanism of O-GlcNAc modification in regulating anti-fibrotic genes in lung fibroblasts and in the pathogenesis of IPF.

Impact of socioeconomic status on presentation, care quality and outcomes of patients attended by emergency medical services for dyspnoea: a population-based cohort study.

BACKGROUND: Low socioeconomic status (SES) has been linked to poor outcomes in many conditions. It is unknown whether these disparities extend to individuals presenting with dyspnoea. We aimed to evaluate the relationship between SES and incidence, care quality and outcomes among patients attended by emergency medical services (EMS) for dyspnoea. METHODS: This population-based cohort study included consecutive patients attended by EMS for dyspnoea between 1 January 2015 and 30 June 2019 in Victoria, Australia. Data were obtained from individually linked ambulance, hospital and mortality datasets. Patients were stratified into SES quintiles using a composite census-derived index. RESULTS: A total of 262 412 patients were included. There was a stepwise increase in the age-adjusted incidence of EMS attendance for dyspnoea with increasing socioeconomic disadvantage (lowest SES quintile 2269 versus highest quintile 889 per 100 000 person years, ptrend<0.001). Patients of lower SES were younger and more comorbid, more likely to be from regional Victoria or of Aboriginal or Torres Strait Islander heritage and had higher rates of respiratory distress. Despite this, lower SES groups were less frequently assigned a high acuity EMS transport or emergency department (ED) triage category and less frequently transported to tertiary centres or hospitals with intensive care unit facilities. In multivariable models, lower SES was independently associated with lower acuity EMS and ED triage, ED length of stay>4 hours and increased 30-day EMS reattendance and mortality. CONCLUSION: Lower SES was associated with a higher incidence of EMS attendances for dyspnoea and disparities in several metrics of care and clinical outcomes.

Chest Pain Management Using Prehospital Point-of-Care Troponin and Paramedic Risk Assessment.

Importance: Prehospital point-of-care troponin testing and paramedic risk stratification might improve the efficiency of chest pain care pathways compared with existing processes with equivalent health outcomes, but the association with health care costs is unclear. Objective: To analyze whether prehospital point-of-care troponin testing and paramedic risk stratification could result in cost savings compared with existing chest pain care pathways. Design, Setting, and Participants: In this economic evaluation of adults with acute chest pain without ST-segment elevation, cost-minimization analysis was used to assess linked ambulance, emergency, and hospital attendance in the state of Victoria, Australia, between January 1, 2015, and June 30, 2019. Interventions: Paramedic risk stratification and point-of-care troponin testing. Main Outcomes and Measures: The outcome was estimated mean annualized statewide costs for acute chest pain. Between May 17 and June 25, 2022, decision tree models were developed to estimate costs under 3 pathways: (1) existing care, (2) paramedic risk stratification and point-of-care troponin testing without prehospital discharge, or (3) prehospital discharge and referral to a virtual emergency department (ED) for low-risk patients. Probabilities for the prehospital pathways were derived from a review of the literature. Multivariable probabilistic sensitivity analysis with 50 000 Monte Carlo iterations was used to estimate mean costs and cost differences among pathways. Results: A total of 188 551 patients attended by ambulance for chest pain (mean [SD] age, 61.9 [18.3] years; 50.5% female; 49.5% male; Indigenous Australian, 2.0%) were included in the model. Estimated annualized infrastructure and staffing costs for the point-of-care troponin pathways, assuming a 5-year device life span, was $2.27 million for the pathway without prehospital discharge and $4.60 million for the pathway with prehospital discharge (incorporating virtual ED costs). In the decision tree model, total annual cost using prehospital point-of-care troponin and paramedic risk stratification was lower compared with existing care both without prehospital discharge (cost savings, $6.45 million; 95% uncertainty interval [UI], $0.59-$16.52 million; lower in 94.1% of iterations) and with prehospital discharge (cost savings, $42.84 million; 95% UI, $19.35-$72.26 million; lower in 100% of iterations). Conclusions and Relevance: Prehospital point-of-care troponin and paramedic risk stratification for patients with acute chest pain could result in substantial cost savings. These findings should be considered by policy makers in decisions surrounding the potential utility of prehospital chest pain risk stratification and point-of-care troponin models provided that safety is confirmed in prospective studies.

Epigenetic regulation of IPF fibroblast phenotype by glutaminolysis.

OBJECTIVE: Excessive extra-cellular-matrix production and uncontrolled proliferation of the fibroblasts are characteristics of many fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). The fibroblasts have enhanced glutaminolysis with up-regulated glutaminase, GLS1, which converts glutamine to glutamate. Here, we investigated the role of glutaminolysis and glutaminolysis-derived metabolite α-ketoglutarate (α-KG) on IPF fibroblast phenotype and gene expression. METHODS: Reduced glutamine conditions were carried out either using glutamine-free culture medium or silencing the expression of GLS1 with siRNA, with or without α-KG compensation. Cell phenotype has been characterized under these different conditions, and gene expression profile was examined by RNA-Seq. Specific profibrotic genes (Col3A1 and PLK1) expression were examined by real-time PCR and western blots. The levels of repressive histone H3K27me3, which demethylase activity is affected by glutaminolysis, were examined and H3K27me3 association with promoter region of Col3A1 and PLK1 were checked by ChIP assays. Effects of reduced glutaminolysis on fibrosis markers were checked in an animal model of lung fibrosis. RESULTS: The lack of glutamine in the culture medium alters the profibrotic phenotype of activated fibroblasts. The addition of exogenous and glutaminolysis-derived metabolite α-KG to glutamine-free media barely restores the pro-fibrotic phenotype of activated fibroblasts. Many genes are down-regulated in glutamine-free medium, α-KG supplementation only rescues a limited number of genes. As α-KG is a cofactor for histone demethylases of H3K27me3, the reduced glutaminolysis alters H3K27me3 levels, and enriches H3K27me3 association with Col3A1 and PLK1 promoter region. Adding α-KG in glutamine-free medium depleted H3K27me3 association with Col3A1 promoter region but not that of PLK1. In a murine model of lung fibrosis, mice with reduced glutaminolysis showed markedly reduced fibrotic markers. CONCLUSIONS: This study indicates that glutamine is critical for supporting pro-fibrotic fibroblast phenotype in lung fibrosis, partially through α-KG-dependent and -independent mechanisms, and supports targeting fibroblast metabolism as a therapeutic method for fibrotic diseases.

Epidemiology, outcomes and predictors of mortality in patients transported by ambulance for dyspnoea: A population-based cohort study.

OBJECTIVES: There are currently limited data to inform the management of patients transported by emergency medical services (EMS) with dyspnoea. We aimed to describe the incidence, aetiology and outcomes of patients transported by EMS for dyspnoea using a large population-based sample and to identify factors associated with 30-day mortality. METHODS: Consecutive EMS attendances for dyspnoea in Victoria, Australia from January 2015 to June 2019 were included. Data were individually linked to hospital and mortality records to determine incidence, diagnoses, and outcomes. Factors associated with 30-day mortality were assessed using multivariable logistic regression. RESULTS: During the study period, there were 2 505 324 cases attended by EMS, of whom 346 228 (14%) met inclusion criteria for dyspnoea. The incidence of EMS attendances for dyspnoea was 1566 per 100 000 person-years, and was higher in females, older patients and socially disadvantaged areas. Of the 271 204 successfully linked cases (median age 76 years; 51% women), 79% required hospital admission with a 30-day mortality of 9%. The most common final diagnoses (and 30-day mortality rates) were lower respiratory tract infection (13%, mortality 11%), chronic obstructive pulmonary disease (13%, mortality 6.4%), heart failure (9.1%, mortality 9.8%), arrhythmias (3.9%, mortality 4.4%), acute coronary syndromes (3.9%, mortality 9.5%) and asthma (3.2%, mortality 0.5%). Predictors of mortality included older age, male sex, pre-existing chronic kidney disease, heart failure or cancer, abnormal respiratory status or vital signs and pre-hospital intubation. CONCLUSION: Dyspnoea is a common presentation with a broad range of causes and is associated with high rates of hospitalisation and death.

Cost-Effectiveness of Empagliflozin in Patients With Heart Failure and Preserved Ejection Fraction.

BACKGROUND: Empagliflozin is the first medication to demonstrate clinical benefit in patients with heart failure with preserved ejection fraction, but its cost-effectiveness is unknown. We aimed to evaluate the cost-effectiveness of adding empagliflozin to standard therapy in patients with heart failure with preserved ejection fraction. METHODS: A Markov model from the perspective of the Australian health care system was constructed to compare empagliflozin plus standard care to standard care alone among a hypothetical cohort of patients with heart failure with preserved ejection fraction. Clinical probabilities were derived from The EMPEROR-Preserved (Empagliflozin Outcome Trial in Patients with Chronic Heart Failure with Preserved Ejection Fraction). Costs and utilities were derived from published sources. The main outcome was the incremental cost-effectiveness ratio per quality-adjusted life-year gained. Deterministic and probabilistic sensitivity analyses were performed to assess model uncertainty. Costs and benefits were discounted at 5% annually. RESULTS: Over a lifetime, the addition of empagliflozin to standard care prevented 167 heart failure hospitalizations and 155 heart failure-related urgent care visits for every 1000 patients treated and increased mean quality-adjusted survival by 0.16 quality adjusted life-years per patient. Mean lifetime costs in the empagliflozin and standard care groups were AUD$63 218 and AUD$58 478 per patient, respectively. This resulted in an incremental cost-effectiveness ratio of AUD$29 202 per quality adjusted life-year gained. In probabilistic sensitivity analyses, empagliflozin was cost-effective in 85% of 10 000 Monte Carlo simulations at a willingness-to-pay threshold of AUD$50 000 per quality adjusted life-year gained. CONCLUSIONS: In patients with heart failure with preserved ejection fraction, adding empagliflozin to standard care is likely to be cost-effective when compared with standard care alone in the Australian health care setting.

Modulation of H4K16Ac levels reduces pro-fibrotic gene expression and mitigates lung fibrosis in aged mice.

Histone H4 lysine16 acetylation (H4K16Ac) modulates chromatin structure by serving as a switch from a repressive to a transcriptionally active state. This euchromatin mark is associated with active transcription. In this study, we investigated the effects of H4K16Ac on the expression of pro-fibrotic genes in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and in an aging murine model of lung fibrosis. Methods: The lung tissues and fibroblasts from human IPF/non-IPF donors and from aged mice with/without bleomycin induced lung fibrosis were used in this study. The H4K16Ac levels were examined by immunohistochemistry or western blots. RNA silencing of H4K16Ac acetyltransferase Mof was used to reduce H4K16Ac levels in IPF fibroblasts. The effects of reduced H4K16Ac on pro-fibrotic gene expression were examined by western blots and real-time PCR. The association of H4K16Ac with these genes' promoter region were evaluated by ChIP assays. The gene expression profile in siRNA Mof transfected IPF cells were determined by RNA-Seq. The impact of H4K16Ac levels on lung fibrosis was evaluated in an aging murine model. Results: Aged mice with bleomycin induced lung fibrosis showed increased H4K16Ac levels. Human lung fibroblasts with siRNA Mof silencing demonstrated reduced H4K16Ac, and significantly down-regulated profibrotic genes, such as α-smooth muscle actin (α-SMA), collagen I, Nox4, and survivin. ChIP assays confirmed the associations of these pro-fibrotic genes' promoter region with H4K16Ac, while in siRNA Mof transfected cells the promoter/H4K16Ac associations were depleted. RNA-seq data demonstrated that Mof knockdown altered gene expression and cellular pathways, including cell damage and repair. In the aging mice model of persistent lung fibrosis, 18-month old mice given intra-nasal siRNA Mof from week 3 to 6 following bleomycin injury showed improved lung architecture, decreased total hydroxyproline content and lower levels of H4K16Ac. Conclusions: These results indicate a critical epigenetic regulatory role for histone H4K16Ac in the pathogenesis of pulmonary fibrosis, which will aid in the development of novel therapeutic strategies for age-related diseases such as IPF.

Sex-Specific Outcomes Following Percutaneous Coronary Intervention Versus Coronary Artery Bypass Grafting for Left Main Disease: A Systematic Review and Meta-Analysis.

OBJECTIVE: To assess whether outcomes following percutaneous coronary intervention (PCI) versus coronary artery bypass grafting (CABG) for left main coronary artery (LMCA) disease differ between men and women. BACKGROUND: Current guidelines recommend either PCI or CABG for patients with unprotected LMCA disease and low-to-intermediate anatomical complexity. However, it is unclear whether these guidelines apply to women, who are underrepresented in clinical trials. METHODS: An electronic search was performed to identify studies reporting sex-specific outcomes after PCI versus CABG in patients with LMCA disease. Trial level hazard ratios (HRs) and 95% confidence intervals (CIs) were pooled by random-effects modelling. RESULTS: Eight (8) studies met inclusion criteria, comprising 13,066 patients (24.3% women). In both sexes, there was no difference between PCI and CABG with respect to the primary composite endpoint of death, myocardial infarction or stroke (HR in women: 1.03, 95% CI 0.76-1.40; HR in men: 1.04, 95% CI 0.92-1.17). However, both sexes were more likely to require repeat revascularisation after PCI. There was no interaction between sex and treatment effect for the primary composite endpoint nor for the individual outcomes of death, stroke and repeat revascularisation. However, in women the risk of myocardial infarction was higher after PCI compared with CABG (HR 1.84, 95% CI 1.06-3.18), with a trend toward the opposite in men (HR 0.78, 95% CI 0.54-1.13; p-interaction=0.01). CONCLUSION: Percutaneous coronary intervention and CABG have a comparable risk of the composite outcome of death, stroke or myocardial infarction in patients undergoing revascularisation for LMCA disease, with no significant interaction between sex and treatment effect.

Intravascular Ultrasound Versus Angiography-Guided Drug-Eluting Stent Implantation: A Health Economic Analysis.

BACKGROUND: There is increasing evidence that use of intravascular ultrasound (IVUS) guidance during percutaneous coronary intervention (PCI) is associated with improved clinical outcomes compared with angiography guidance alone. However, concern regarding the cost-effectiveness of IVUS has limited use of this technology worldwide. In this study, we aimed to evaluate the cost-effectiveness of IVUS-guided PCI compared with angiography-guided PCI in patients undergoing drug-eluting stent implantation. METHODS: A decision-analytic Markov model was constructed to compare the cost-effectiveness of IVUS to angiography guidance from the Australian healthcare system perspective. Procedure-related morbidity and mortality were estimated from the literature. Costs were obtained from Australian sources. The population of interest was all-comers undergoing PCI with drug-eluting stent. Outcomes of interest included costs, life-expectancy, and quality-adjusted life years (QALYs) for both treatment groups. RESULTS: In the base case, IVUS guidance was cost-effective compared with angiography guidance alone. With 5% annual discounting, IVUS was associated with increased lifetime costs of Australian dollars (AUD) $823 (USD $597) per person and benefits of 0.04 life years and 0.05 QALYs compared with angiography, yielding an incremental cost-effectiveness ratio of AUD $17 539 (USD $12 730) per QALY gained. Results were robust to sensitivity analyses, with IVUS being cost-effective in 99% of 10 000 Monte Carlo iterations assuming a willingness-to-pay threshold of AUD $50 000 per QALY gained. In a worst-case scenario analysis, IVUS remained the cost-effective option, with an ICER of AUD $36 651 (USD $26 601) per QALY gained. Exploratory subgroup analysis revealed that cost-effectiveness may be greatest among patients with left main and complex coronary lesions. CONCLUSIONS: Use of IVUS guidance during PCI is likely to be cost-effective compared with angiography guidance alone among patients undergoing drug-eluting stent implantation.

Thirst interneurons that promote water seeking and limit feeding behavior in Drosophila.

Thirst is a motivational state that drives behaviors to obtain water for fluid homeostasis. We identified two types of central brain interneurons that regulate thirsty water seeking in Drosophila, that we term the Janu neurons. Janu-GABA, a local interneuron in the subesophageal zone, is activated by water deprivation and is specific to thirsty seeking. Janu-AstA projects from the subesophageal zone to the superior medial protocerebrum, a higher order processing area. Janu-AstA signals with the neuropeptide Allatostatin A to promote water seeking and to inhibit feeding behavior. NPF (Drosophila NPY) neurons are postsynaptic to Janu-AstA for water seeking and feeding through the AstA-R2 galanin-like receptor. NPF neurons use NPF to regulate thirst and hunger behaviors. Flies choose Janu neuron activation, suggesting that thirsty seeking up a humidity gradient is rewarding. These findings identify novel central brain circuit elements that coordinate internal state drives to selectively control motivated seeking behavior.

The effects of a simple optical clearing protocol on the mechanics of collagenous soft tissue.

Optical clearing of biological tissues improves imaging depth for light transmission imaging modalities such as two-photon microscopy. In studies that investigate the interplay between microstructure and tissue-level mechanics, mechanical testing of cleared tissue may be useful. However, the effects of optical clearing on soft tissue mechanics have not been investigated. Thus, we set out to quantify the effects of a simple and effective optical clearing protocol on the mechanics of soft collagenous tissues using ovine mitral valve anterior leaflets as a model system. First, we demonstrate the effectiveness of an isotonic glycerol-DMSO optical clearing protocol in two-photon microscopy. Second, we evaluate the mechanical effects of optical clearing on leaflets under equibiaxial tension in a dependent study design. Lastly, we quantify the shrinkage strain while traction-free and the contractile forces while constrained during clearing. We found the optical clearing protocol to improve two-photon imaging depth from ~100 µm to ~500-800 µm, enabling full-thickness visualization of second-harmonic generation, autofluorescent, and fluorophore-tagged structures. Under equibiaxial tension, cleared tissues exhibited reduced circumferential (p < 0.001) and radial (p = 0.009) transition stretches (i.e. stretch where collagen is recruited), and reduced radial stiffness (p = 0.031). Finally, during clearing we observed ~10-15% circumferential and radial compressive strains, and when constrained, ~2mN of circumferential and radial traction forces. In summary, we suggest the use of this optical clearing agent with mechanical testing be done with care, as it appears to alter the tissue's stress-free configuration and stiffness, likely due to tissue dehydration.

Cost-Effectiveness of Transcatheter Versus Surgical Aortic Valve Replacement in Low-Risk Patients With Severe Aortic Stenosis.

BACKGROUND: Recent studies have shown that transcatheter aortic valve implantation (TAVI) is non-inferior to surgical aortic valve replacement (SAVR) for treatment of low-risk patients with severe aortic stenosis (AS). However, the cost-effectiveness of TAVI in this population is unknown. We sought to evaluate the cost-effectiveness of both balloon-expandable and self-expanding TAVI compared to SAVR in patients with severe AS at low operative risk. METHODS: We developed a Markov model comparing TAVI to SAVR over a lifetime horizon. Key data inputs were drawn from the Placement of AoRTic TraNscathetER Valve (PARTNER) 3 trial for balloon-expandable TAVI and the Evolut Low-Risk trial for self-expanding TAVI. Costs were obtained from Australian sources. The perspective was that of the Australian health care system. RESULTS: Although procedural costs were higher for TAVI compared to SAVR, these were offset by a shorter length of hospitalisation and lower acute complication costs in the TAVI group. Over a lifetime horizon, balloon-expandable TAVI was associated with increased costs of A$702 compared to SAVR, and increased quality-adjusted survival by 0.20 years, yielding an incremental cost-effectiveness ratio of A$3,521 per quality-adjusted life year (QALY) saved. Self-expanding TAVI was associated with lower lifetime costs compared to SAVR, and increased quality-adjusted survival by 0.08 years, and was therefore economically dominant. In probabilistic sensitivity analyses, balloon-expandable TAVI was cost-effective in 78% of iterations (at a cost-effectiveness threshold of A$50,000 per QALY gained) and self-expanding TAVI was cost-effective in 70% of iterations. CONCLUSIONS: Among low-risk AS patients, both balloon-expandable and self-expanding TAVI are likely to be cost-effective relative to SAVR.

SUMOylation Protects FASN Against Proteasomal Degradation in Breast Cancer Cells Treated with Grape Leaf Extract.

Existing therapeutic strategies for breast cancer are limited by tumor recurrence and drug-resistance. Antioxidant plant-derived compounds such as flavonoids reduce adverse outcomes and have been identified as a potential source of antineoplastic agent with less undesirable side effects. Here, we describe the novel regulation of fatty-acid synthase (FASN), the key enzyme in de novo fatty-acid synthesis, whereby Vitis vinifera L. cv Vermentino leaf hydroalcoholic extract lowers its protein stability that is regulated by small ubiquitin-like modifier (SUMO)ylation. The phenolic compounds characterization was performed by liquid chromatography-mass spectrometry (LC-MS), whereas mass spectrometry (LC-MS/MS), Western blotting/co-immunoprecipitation (Co-IP) and RT-PCR, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenicity assays, and FACS analysis were used to measure the expression of targets and tumorigenicity. Vermentino extract exhibits antitumorigenic effects, and we went on to determine that FASN and ubiquitin-conjugating enzyme 9 (UBC9), the sole E2 enzyme required for SUMOylation, were significantly reduced. Moreover, FASN was found SUMOylated in human breast cancer tissues and cell lines, and lack of SUMOylation caused by SUMO2 silencing reduced FASN protein stability. These results suggest that SUMOylation protects FASN against proteasomal degradation and may exert oncogenic activity through alteration of lipid metabolism, whereas Vermentino extract inhibits these effects which supports the additional validation of the therapeutic value of this compound in breast cancer.

Cost-effectiveness of transcatheter aortic valve implantation compared to surgical aortic valve replacement in the intermediate surgical risk population.

BACKGROUND: The recent PARTNER S3i trial compared transcatheter aortic valve implantation (TAVI) using the third-generation SAPIEN 3 device to surgical aortic valve replacement (SAVR) in intermediate-risk patients with severe symptomatic aortic stenosis. Using data from PARTNER S3i, we performed a contemporary cost-effectiveness analysis of current-generation TAVI versus SAVR from the Australian healthcare system perspective. METHODS: A Markov model with monthly cycles and a ten-year horizon was constructed to estimate costs, life-years and quality adjusted life-years (QALYs) associated with TAVI and SAVR. Efficacy inputs were derived from the PARTNER S3i study. Costs were estimated from published sources. Deterministic and probabilistic sensitivity analyses were performed to assess model uncertainty. RESULTS: TAVI was found to have higher immediate procedural costs than SAVR, driven primarily by the cost of the transcatheter valve. This was offset by a shorter length of hospitalisation following TAVI, such that the combined cost of initial procedure and hospitalisation was lower in TAVI compared to SAVR. With 5% annual discounting, total costs over ten-years were $50,515 AUD in TAVI and $60,144 AUD in SAVR, and TAVI was found to produce 0.33 more life years and 0.31 more QALYs than SAVR. Thus, from a health economic perspective, TAVI was dominant compared to SAVR. Results were robust to sensitivity analyses, with TAVI being dominant in 68% of 10,000 Monte Carlo iterations and cost-effective in 92% of iterations at a willingness-to-pay threshold of $50,000/QALY gained. CONCLUSIONS: TAVI is likely to be highly cost-effective compared to SAVR in intermediate-risk patients with severe aortic stenosis.

A Click Chemistry Approach Reveals the Chromatin-Dependent Histone H3K36 Deacylase Nature of SIRT7.

Using an engineered pyrrolysyl-tRNA synthetase mutant together with tRNACUAPyl, we have genetically encoded Nε-(7-azidoheptanoyl)-l-lysine (AzHeK) by amber codon in Escherichia coli for recombinant expression of a number of AzHeK-containing histone H3 proteins. We assembled in vitro acyl-nucleosomes from these recombinant acyl-H3 histones. All these acyl-nucleosomes contained an azide functionality that allowed quick click labeling with a strained alkyne dye for in-gel fluorescence analysis. Using these acyl-nucleosomes as substrates and click labeling as a detection method, we systematically investigated chromatin deacylation activities of SIRT7, a class III NAD+-dependent histone deacylase with roles in aging and cancer biology. Besides confirming the previously reported histone H3K18 deacylation activity, our results revealed that SIRT7 has an astonishingly high activity to catalyze deacylation of H3K36 and is also catalytically active to deacylate H3K37. We further demonstrated that this H3K36 deacylation activity is nucleosome dependent and can be significantly enhanced when appending the acyl-nucleosome substrate with a short double-stranded DNA that mimics the bridging DNA between nucleosomes in native chromatin. By overexpressing SIRT7 in human cells, we verified that SIRT7 natively removes acetylation from histone H3K36. Moreover, SIRT7-deficient cells exhibited H3K36 hyperacetylation in whole cell extracts, at rDNA sequences in nucleoli, and at select SIRT7 target loci, demonstrating the physiologic importance of SIRT7 in determining endogenous H3K36 acetylation levels. H3K36 acetylation has been detected at active gene promoters, but little is understood about its regulation and functions. Our findings establish H3K36 as a physiologic substrate of SIRT7 and implicate this modification in potential SIRT7 pathways in heterochromatin silencing and genomic stability.

Star-related lipid transfer protein 10 (STARD10): a novel key player in alcohol-induced breast cancer progression.

BACKGROUND: Ethanol abuse promotes breast cancer development, metastasis and recurrence stimulating mammary tumorigenesis by mechanisms that remain unclear. Normally, 35% of breast cancer is Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2)-positive that predisposes to poor prognosis and relapse, while ethanol drinking leads to invasion of their ERBB2 positive cells triggering the phosphorylation status of mitogen-activated protein kinase. StAR-related lipid transfer protein 10 (STARD10) is a lipid transporter of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); changes on membrane composition of PC and PE occur before the morphological tumorigenic events. Interestingly, STARD10 has been described to be highly expressed in 35-40% of ERBB2-positive breast cancers. In this study, we demonstrate that ethanol administration promotes STARD10 and ERBB2 expression that is significantly associated with increased cell malignancy and aggressiveness. MATERIAL AND METHODS: We investigated the effect of ethanol on STARD10-ERBB2 cross-talk in breast cancer cells, MMTV-neu transgenic mice and in clinical ERBB2-positive breast cancer specimens with Western Blotting and Real-time PCR. We also examined the effects of their knockdown and overexpression on transient transfected breast cancer cells using promoter activity, MTT, cell migration, calcium and membrane fluidity assays in vitro. RESULTS: Ethanol administration induces STARD10 and ERBB2 expression in vitro and in vivo. ERBB2 overexpression causes an increase in STARD10 expression, while overexpression of ERBB2's downstream targets, p65, c-MYC, c-FOS or c-JUN induces STARD10 promoter activity, correlative of enhanced ERBB2 function. Ethanol and STARD10-mediated cellular membrane fluidity and intracellular calcium concentration impact ERBB2 signaling pathway as evaluated by enhanced p65 nuclear translocation and binding to both ERBB2 and STARD10 promoters. CONCLUSION: Our finding proved that STARD10 and ERBB2 positively regulate each other's expression and function. Taken together, our data demonstrate that ethanol can modulate ERBB2's function in breast cancer via a novel interplay with STARD10.

Sequential activation of the MEK-extracellular signal-regulated kinase and MKK3/6-p38 mitogen-activated protein kinase pathways mediates oncogenic ras-induced premature senescence.

In primary mammalian cells, oncogenic ras induces premature senescence, depending on an active MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. It has been unclear how activation of the mitogenic MEK-ERK pathway by ras can confer growth inhibition. In this study, we have found that the stress-activated MAPK, p38, is also activated during the onset of ras-induced senescence in primary human fibroblasts. Constitutive activation of p38 by active MKK3 or MKK6 induces senescence. Oncogenic ras fails to provoke senescence when p38 activity is inhibited, suggesting that p38 activation is essential for ras-induced senescence. Furthermore, we have demonstrated that p38 activity is stimulated by ras as a result of an activated MEK-ERK pathway. Following activation of MEK and ERK, expression of oncogenic ras leads to the accumulation of active MKK3/6 and p38 activation in a MEK-dependent fashion and subsequently induces senescence. Active MEK1 induces the same set of changes and provokes senescence relying on active p38. Therefore, oncogenic ras provokes premature senescence by sequentially activating the MEK-ERK and MKK3/6-p38 pathways in normal, primary cells. These studies have defined the molecular events within the ras signaling cascade that lead to premature senescence and, thus, have provided new insights into how ras confers oncogenic transformation in primary cells.