Rectifying METTL4-Mediated N6-Methyladenine Excess in Mitochondrial DNA Alleviates Heart Failure.

BACKGROUND: Myocardial mitochondrial dysfunction underpins the pathogenesis of heart failure (HF), yet therapeutic options to restore myocardial mitochondrial function are scarce. Epigenetic modifications of mitochondrial DNA (mtDNA), such as methylation, play a pivotal role in modulating mitochondrial homeostasis. However, their involvement in HF remains unclear. METHODS: Experimental HF models were established through continuous angiotensin II and phenylephrine (AngII/PE) infusion or prolonged myocardial ischemia/reperfusion injury. The landscape of N6-methyladenine (6mA) methylation within failing cardiomyocyte mtDNA was characterized using high-resolution mass spectrometry and methylated DNA immunoprecipitation sequencing. A tamoxifen-inducible cardiomyocyte-specific Mettl4 knockout mouse model and adeno-associated virus vectors designed for cardiomyocyte-targeted manipulation of METTL4 (methyltransferase-like protein 4) expression were used to ascertain the role of mtDNA 6mA and its methyltransferase METTL4 in HF. RESULTS: METTL4 was predominantly localized within adult cardiomyocyte mitochondria. 6mA modifications were significantly more abundant in mtDNA than in nuclear DNA. Postnatal cardiomyocyte maturation presented with a reduction in 6mA levels within mtDNA, coinciding with a decrease in METTL4 expression. However, an increase in both mtDNA 6mA level and METTL4 expression was observed in failing adult cardiomyocytes, suggesting a shift toward a neonatal-like state. METTL4 preferentially targeted mtDNA promoter regions, which resulted in interference with transcription initiation complex assembly, mtDNA transcriptional stalling, and ultimately mitochondrial dysfunction. Amplifying cardiomyocyte mtDNA 6mA through METTL4 overexpression led to spontaneous mitochondrial dysfunction and HF phenotypes. The transcription factor p53 was identified as a direct regulator of METTL4 transcription in response to HF-provoking stress, thereby revealing a stress-responsive mechanism that controls METTL4 expression and mtDNA 6mA. Cardiomyocyte-specific deletion of the Mettl4 gene eliminated mtDNA 6mA excess, preserved mitochondrial function, and mitigated the development of HF upon continuous infusion of AngII/PE. In addition, specific silencing of METTL4 in cardiomyocytes restored mitochondrial function and offered therapeutic relief in mice with preexisting HF, irrespective of whether the condition was induced by AngII/PE infusion or myocardial ischemia/reperfusion injury. CONCLUSIONS: Our findings identify a pivotal role of cardiomyocyte mtDNA 6mA and the corresponding methyltransferase, METTL4, in the pathogenesis of mitochondrial dysfunction and HF. Targeted suppression of METTL4 to rectify mtDNA 6mA excess emerges as a promising strategy for developing mitochondria-focused HF interventions.

Mechanisms of Resistance to Noncovalent Bruton's Tyrosine Kinase Inhibitors.

BACKGROUND: Covalent (irreversible) Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood. METHODS: We performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors. RESULTS: Among 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors. CONCLUSIONS: Resistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.).

Exosomal miR-155-5p drives ibrutinib resistance in B-cell lymphoma.

Ibrutinib, a Bruton Tyrosine Kinase (BTK) inhibitor, has shown effectiveness against various B-cell lymphoid malignancies. However, prolonged usage can induce resistance, affecting treatment outcomes. The oncogenic microRNA, miR-155-5p, is associated with poor prognosis in B-cell lymphomas, prompting our investigation into the mechanism of acquired ibrutinib resistance in these cells. We generated ibrutinib-resistant OCI-Ly1 cells (OCI-Ly1-IbtR) through continuous exposure to 1 µM and 2 µM of ibrutinib. We conducted microRNA profiling of OCI-Ly1-IbtR and isolated exosomes via ultracentrifugation. Comparative studies of microRNA levels in cells and exosomes, as well as exploration of targets of up-regulated microRNAs in OCI-Ly1-IbtR, were performed. Target validation involved transfection of candidate microRNAs, and co-culture experiments utilized OCI-Ly1 cells with exosomes from OCI-Ly1-IbtR. Elevated levels of miR-155-5p were observed in OCI-Ly1-IbtR and its exosomes, correlating with AKT and NF-κB activation. Transfection of miR-155-5p induced AKT/NF-κB pathway activation in OCI-Ly1, resulting in ibrutinib resistance, enhanced colony formation, and sustained BTK activity. Primary cell lines from ibrutinib-refractory B-cell lymphoma patients exhibited similar signaling protein activation. Target evaluation identified KDM5B and DEPTOR as miR-155-5p targets, confirmed by downregulation after transfection. We observed KDM5B and DEPTOR enrichment in Ago2 during ibrutinib resistance and miR-155-5p transfection. Co-culture experiments demonstrated exosome-mediated transfer of miR-155-5p, inducing ibrutinib resistance and KDM5B/DEPTOR downregulation in OCI-Ly1. Our findings suggest that miR-155-5p overexpression is associated with AKT and NF-κB pathway activation in ibrutinib-resistant cells, proposing a potential role for acquired miR-155-5p upregulation in B-cell lymphoma ibrutinib resistance.

Extended Postexposure Protection Against Vaginal Simian/Human Immunodeficiency Virus Infection With Tenofovir Alafenamide Fumarate/Elvitegravir Inserts in Macaques.

Vaginal inserts that can be used on demand before or after sex may be a desirable human immunodeficiency virus (HIV) prevention option for women. We recently showed that inserts containing tenofovir alafenamide fumarate (TAF, 20 mg) and elvitegravir (EVG, 16 mg) were highly protective against repeated simian/human immunodeficiency virus (SHIV) vaginal exposures when administered to macaques 4 hours before or after virus exposure (93% and 100%, respectively). Here, we show in the same macaque model that insert application 8 hours or 24 hours after exposure maintains high efficacy (94.4% and 77.2%, respectively). These data extend the protective window by TAF/EVG inserts and inform their clinical development for on-demand prophylaxis in women.

Bruton's tyrosine kinase (BTK) inhibitors alter blood glucose and insulin in obese mice but reduce inflammation independent of BTK.

Obesity is associated with metabolic inflammation, which can contribute to insulin resistance, higher blood glucose, and higher insulin indicative of prediabetes progression. The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a metabolic danger sensor implicated in metabolic inflammation. Many features of metabolic disease can activate the NLRP3 inflammasome; however, it is not yet clear which upstream triggers to target, and there are no clinically approved NLRP3 inflammasome inhibitors for metabolic disease. Bruton's tyrosine kinase (BTK) mediates activation of the NLRP3 inflammasome. Ibrutinib is the most-studied pharmacological inhibitor of BTK, and it can improve blood glucose control in obese mice. However, inhibitors of tyrosine kinases are permissive, and it is unknown if BTK inhibitors require BTK to alter endocrine control of metabolism or metabolic inflammation. We tested whether ibrutinib and acalabrutinib, a new generation BTK inhibitor with higher selectivity, require BTK to inhibit the NLRP3 inflammasome, metabolic inflammation, and blood glucose in obese mice. Chronic ibrutinib administration lowered fasting blood glucose and improved glycemia, whereas acalabrutinib increased fasting insulin levels and increased markers of insulin resistance in high-fat diet-fed CBA/J mice with intact Btk. These metabolic effects of BTK inhibitors were absent in CBA/CaHN-Btkxid/J mice with mutant Btk. However, ibrutinib and acalabrutinib reduced NF-κB activity, proinflammatory gene expression, and NLRP3 inflammasome activation in macrophages with and without functional BTK. These data highlight that the BTK inhibitors can have divergent effects on metabolism and separate effects on metabolic inflammation that can occur independently of actions on BTK.NEW & NOTEWORTHY Bruton's tyrosine kinase (BTK) is involved in immune function. It was thought that BTK inhibitors improve characteristics of obesity-related metabolic disease by lowering metabolic inflammation. However, tyrosine kinase inhibitors are permissive, and it was not known if different BTK inhibitors alter host metabolism or immunity through actions on BTK. We found that two BTK inhibitors had divergent effects on blood glucose and insulin via BTK, but inhibition of metabolic inflammation occurred independently of BTK in obese mice.

Autophagy defects at weaning impair complement-dependent synaptic pruning and induce behavior deficits.

Autophagy is crucial for synaptic plasticity and the architecture of dendritic spines. However, the role of autophagy in schizophrenia (SCZ) and the mechanisms through which it affects synaptic function remain unclear. In this study, we identified 995 single nucleotide polymorphisms (SNPs) across 19 autophagy-related genes that are associated with SCZ. Gene Set Enrichment Analysis (GSEA) of data from the Gene Expression Omnibus public database revealed defective autophagy in patients with SCZ. Using a maternal immune activation (MIA) rat model, we observed that autophagy was downregulated during the weaning period, and early-life activation of autophagy with rapamycin restored abnormal behaviors and electrophysiological deficits in adult rats. Additionally, inhibition of autophagy with 3-Methyladenine (3-MA) during the weaning period resulted in aberrant behaviors, abnormal electrophysiology, increased spine density, and reduced microglia-mediated synaptic pruning. Furthermore, 3-MA treatment significantly decreased the expression and synaptosomal content of complement, impaired the recognition of C3b and CR3, indicating that autophagy deficiency disrupts complement-mediated synaptic pruning. Our findings provide evidence for a significant association between SCZ and defective autophagy, highlighting a previously underappreciated role of autophagy in regulating the synaptic and behavioral deficits induced by MIA.

Group2 innate lymphoid cells ameliorate renal fibrosis and dysfunction associated with adenine-induced CKD.

Renal fibrosis is a common pathway of chronic kidney disease (CKD) progression involving primary kidney injury and kidney diseases. Group 2 innate lymphoid cells (ILC2s) mediate type 2 immune responses irrespective of antigen presentation and play a reno-protective role in kidney injury and disease. In the present study, we observed a decrease in kidney-resident ILC2s in CKD and found that enrichment of ILC2s in the kidney ameliorates renal fibrosis. In CKD kidney, ILC2s preferentially produced IL-13 over IL-5 in response to IL-33 stimulation, regardless of ST2L expression. Moreover, GATA3 expression was decreased in ILC2s, and T-bet+ ILC1s and RORγt+ ILC3s were increased in CKD kidney. Adoptive transfer of kidney ILC2s into adenine-induced CKD model mouse improved renal function and fibrosis. Renal fibroblasts cultured with IL33-activated kidney ILC2s suppressed myofibroblast trans-differentiation through Acta2 and Fn-1 regulation. These results suggest that kidney ILC2s prevent CKD progression via improvement of renal fibrosis. Our findings also suggest that ILC2s may contribute to the development of new therapeutic agents and strategies for tissue fibroses.

High efficacy of bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) in Black adults in the United States, including those with pre-existing HIV resistance and suboptimal adherence.

BRAAVE (NCT03631732), a Phase 3b, multicenter, open-label US study, demonstrated the efficacy of switching to bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) among Black individuals with suppressed HIV through 48 weeks. Here, 72-week resistance, adherence, and virologic outcomes are presented. Enrollment criteria permitted nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistance (R), protease inhibitor (PI)-R, and certain nucleos(t)ide reverse transcriptase inhibitor (NRTI)-R (M184V/I allowed; ≥3 thymidine analog mutations [TAMs] excluded); but excluded primary integrase strand transfer inhibitor (INSTI)-R. Pre-existing resistance was determined using historical genotypes and retrospective baseline proviral DNA genotyping. Adherence, virologic outcomes, and viral blips were assessed. Of 489 participants receiving B/F/TAF with ≥1 post-switch HIV-1 RNA measurement: pre-existing NRTI-R (15% of participants), M184V/I (11%), ≥1 TAMs (8%), NNRTI-R (22%), and PI-R (13%) were observed; pre-existing INSTI-R substitutions (2%) were detected post-randomization; mean viral blip frequency was 0.9% across all timepoints (unassociated with virologic failure); 24% of participants had <95% adherence (98% of whom had HIV-1 RNA <50 copies/mL at last visit); none had treatment-emergent study-drug resistance. Overall, 99% of participants, including all with baseline NRTI-R/INSTI-R, had HIV-1 RNA <50 copies/mL at the last visit, demonstrating that B/F/TAF maintained virologic suppression through 72 weeks regardless of pre-existing resistance, viral blips, and suboptimal adherence.

Two Weeks of Continuous Opioid Treatment in an Adenine-Induced Mouse Model of Chronic Kidney Disease Exacerbates the Bone Inflammatory State and Increases Osteoclasts.

Patients with chronic kidney disease (CKD) report high pain levels, but reduced renal clearance eliminates many analgesic options; therefore, 30-50% of CKD patients have chronic opioid prescriptions. Opioid use in CKD is associated with higher fracture rates. Opioids may directly alter bone turnover directly through effects on bone cells and indirectly via increasing inflammation. We hypothesized that continuous opioid exposure would exacerbate the high bone turnover state of CKD and be associated with elevated measures of inflammation. Male C57Bl/6J mice after 8 weeks of adenine-induced CKD (AD) and non-AD controls (CON) had 14-day osmotic pumps (0.25-µL/hr release) containing either saline or 50-mg/mL oxycodone (OXY) surgically implanted in the subscapular region. After 2 weeks, all AD mice had elevated blood urea nitrogen, parathyroid hormone, and serum markers of bone turnover compared to controls with no effect of OXY. Immunohistochemical staining of the distal femur showed increased numbers of osteocytes positive for the mu opioid and for toll-like receptor 4 (TLR4) due to OXY. Osteocyte protein expression of tumor necrosis factor-α (TNF-α) and RANKL were higher due to both AD and OXY so that AD + OXY mice had the highest values. Trabecular osteoclast-covered surfaces were also significantly higher due to both AD and OXY, resulting in AD + OXY mice having 4.5-fold higher osteoclast-covered surfaces than untreated CON. These data demonstrate that opioids are associated with a pro-inflammatory state in osteocytes which increases the pro-resorptive state of CKD.

The nonreceptor tyrosine kinase SRMS inhibits autophagy and promotes tumor growth by phosphorylating the scaffolding protein FKBP51.

Nutrient-responsive protein kinases control the balance between anabolic growth and catabolic processes such as autophagy. Aberrant regulation of these kinases is a major cause of human disease. We report here that the vertebrate nonreceptor tyrosine kinase Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites (SRMS) inhibits autophagy and promotes growth in a nutrient-responsive manner. Under nutrient-replete conditions, SRMS phosphorylates the PHLPP scaffold FK506-binding protein 51 (FKBP51), disrupts the FKBP51-PHLPP complex, and promotes FKBP51 degradation through the ubiquitin-proteasome pathway. This prevents PHLPP-mediated dephosphorylation of AKT, causing sustained AKT activation that promotes growth and inhibits autophagy. SRMS is amplified and overexpressed in human cancers where it drives unrestrained AKT signaling in a kinase-dependent manner. SRMS kinase inhibition activates autophagy, inhibits cancer growth, and can be accomplished using the FDA-approved tyrosine kinase inhibitor ibrutinib. This illuminates SRMS as a targetable vulnerability in human cancers and as a new target for pharmacological induction of autophagy in vertebrates.

Computational Investigation of the Covalent Inhibition Mechanism of Bruton's Tyrosine Kinase by Ibrutinib.

Covalent inhibitors represent a promising class of therapeutic compounds. Nonetheless, rationally designing covalent inhibitors to achieve a right balance between selectivity and reactivity remains extremely challenging. To better understand the covalent binding mechanism, a computational study is carried out using the irreversible covalent inhibitor of Bruton tyrosine kinase (BTK) ibrutinib as an example. A multi-µs classical molecular dynamics trajectory of the unlinked inhibitor is generated to explore the fluctuations of the compound associated with the kinase binding pocket. Then, the reaction pathway leading to the formation of the covalent bond with the cysteine residue at position 481 via a Michael addition is determined using the string method in collective variables on the basis of hybrid quantum mechanical-molecular mechanical (QM/MM) simulations. The reaction pathway shows a strong correlation between the covalent bond formation and the protonation/deprotonation events taking place sequentially in the covalent inhibition reaction, consistent with a 3-step reaction with transient thiolate and enolates intermediate states. Two possible atomistic mechanisms affecting deprotonation/protonation events from the thiolate to the enolate intermediate were observed: a highly correlated direct pathway involving proton transfer to the Cα of the acrylamide warhead from the cysteine involving one or a few water molecules and a more indirect pathway involving a long-lived enolate intermediate state following the escape of the proton to the bulk solution. The results are compared with experiments by simulating the long-time kinetics of the reaction using kinetic modeling.

Autophagy-mediated ferroptosis is involved in development of severe acute pancreatitis.

BACKGROUND: Ferroptosis is a newly recognized form of regulatory cell death characterized by severe lipid peroxidation triggered by iron overload and the production of reactive oxygen species (ROS). However, the role of ferroptosis in severe acute pancreatitis(SAP) has not been fully elucidated. METHODS: We established four severe acute pancreatitis models of rats including the sham control group, the SAP group, the Fer -1-treated SAP (SAP + Fer-1) group, the 3-MA-treated SAP (SAP + 3-MA) group. The SAP group was induced by retrograde injection of sodium taurocholate into the pancreatic duct. The other two groups were intraperitoneally injected with ferroptosis inhibitor (Fer-1) and autophagy inhibitor (3-MA), respectively. The model of severe acute pancreatitis with amylase crest-related inflammatory factors was successfully established. Then we detected ferroptosis (GPX4, SLC7A1 etc.) and autophagy-related factors (LC3II, p62 ect.) to further clarify the relationship between ferroptosis and autophagy. RESULTS: Our study found that ferroptosis occurs during the development of SAP, such as iron and lipid peroxidation in pancreatic tissues, decreased levels of reduced glutathione peroxidase 4 (GPX 4) and glutathione (GSH), and increased malondialdehyde(MDA) and significant mitochondrial damage. In addition, ferroptosis related proteins such as GPX4, solute carrier family 7 member 11(SLC7A11) and ferritin heavy chain 1(FTH1) were significantly decreased. Next, the pathogenesis of ferroptosis in SAP was studied. First, treatment with the ferroptosis inhibitor ferrostatin-1(Fer-1) significantly alleviated ferroptosis in SAP. Interestingly, autophagy occurs during the pathogenesis of SAP, and autophagy promotes the occurrence of ferroptosis in SAP. Moreover, 3-methyladenine (3-MA) inhibition of autophagy can significantly reduce iron overload and ferroptosis in SAP. CONCLUSIONS: Our results suggest that ferroptosis is a novel pathogenesis of SAP and is dependent on autophagy. This study provides a new theoretical basis for the study of SAP.

Development of tenofovir monobenzyl ester phosphonoamidate prodrugs with improved anti-hepatitis B virus activity and intrahepatic tenofovir enrichment.

Various tenofovir (TFV) prodrugs have been developed by introducing masking groups to the hydroxyls of the monophosphonate group to enhance intestinal absorption efficiency and therapeutic effects. However, the reported TFV prodrugs have drawbacks such as low bioavailability, systemic toxicity caused by their breakdown in non-targeted tissues, and potential low intracellular conversion efficiency. In the present study, we developed a class of TFV monobenzyl ester phosphonoamidate prodrugs without substitutions on the benzene ring. Compared with previous TFV prodrugs, compounds 3a and 3b developed in the present study showed higher anti-hepatitis B virus activity, stronger stability and higher levels of intrahepatic enrichment of the metabolic product (TFV), indicating the potential of these compounds as novel prodrugs with high efficiency and low systemic toxicity for the treatment of hepatitis B.

Acroamine A, a 2-Amino Adenine Alkaloid from the Marine Soft Coral Acrozoanthus australiae and Its Semisynthetic Derivatives That Inhibit cAMP-Dependent Protein Kinase A Catalytic Subunit Alpha.

A high throughput screen performed to identify catalytic inhibitors of the oncogenic fusion form of cAMP-dependent protein kinase A catalytic subunit alpha (J-PKAcα) found an individual fraction from an organic extract of the marine soft coral Acrozoanthus australiae as active. Bioassay-guided isolation led to the identification of a 2-amino adenine alkaloid acroamine A (1), the first secondary metabolite discovered from this genus and previously reported as a synthetic product. As a naturally occurring protein kinase inhibitor, to unambiguously assign its chemical structure using modern spectroscopic and spectrometric techniques, five N-methylated derivatives acroamines A1-A5 (2-6) were semisynthesized. Three additional brominated congeners A6-A8 (7-9) were also semisynthesized to investigate the structure-activity relationship of the nine compounds as J-PKAcα inhibitors. Compounds 1-9 were tested for J-PKAcα and wild-type PKA inhibitory activities, which were observed exclusively in acroamine A (1) and its brominated analogs (7-9) achieving moderate potency (IC50 2-50 µM) while none of the N-methylated analogs exhibited kinase inhibition.

Fisetin ameliorates fibrotic kidney disease in mice via inhibiting ACSL4-mediated tubular ferroptosis.

Kidney fibrosis is the hallmark of chronic kidney disease (CKD) progression, whereas no effective anti-fibrotic therapies exist. Recent evidence has shown that tubular ferroptosis contributes to the pathogenesis of CKD with persistent proinflammatory and profibrotic responses. We previously reported that natural flavonol fisetin alleviated septic acute kidney injury and protected against hyperuricemic nephropathy in mice. In this study, we investigated the therapeutic effects of fisetin against fibrotic kidney disease and the underlying mechanisms. We established adenine diet-induced and unilateral ureteral obstruction (UUO)-induced CKD models in adult male mice. The two types of mice were administered fisetin (50 or 100 mg·kg-1·d-1, i.g.) for 3 weeks or 7 days, respectively. At the end of the experiments, the mice were euthanized, and blood and kidneys were gathered for analyzes. We showed that fisetin administration significantly ameliorated tubular injury, inflammation, and tubulointerstitial fibrosis in the two types of CKD mice. In mouse renal tubular epithelial (TCMK-1) cells, treatment with fisetin (20 µM) significantly suppressed adenine- or TGF-ß1-induced inflammatory responses and fibrogenesis, and improved cell viability. By quantitative real-time PCR analysis of ferroptosis-related genes, we demonstrated that fisetin treatment inhibited ferroptosis in the kidneys of CKD mice as well as in injured TCMK-1 cells, as evidenced by decreased ACSL4, COX2, and HMGB1, and increased GPX4. Fisetin treatment effectively restored ultrastructural abnormalities of mitochondrial morphology and restored the elevated iron, the reduced GSH and GSH/GSSG as well as the increased lipid peroxide MDA in the kidneys of CKD mice. Notably, abnormally high expression of the ferroptosis key marker ACSL4 was verified in the renal tubules of CKD patients (IgAN, MN, FSGS, LN, and DN) as well as adenine- or UUO-induced CKD mice, and in injured TCMK-1 cells. In adenine- and TGF-ß1-treated TCMK-1 cells, ACSL4 knockdown inhibited tubular ferroptosis, while ACSL4 overexpression blocked the anti-ferroptotic effect of fisetin and reversed the cytoprotective, anti-inflammatory, and anti-fibrotic effects of fisetin. In summary, we reveal a novel aspect of the nephroprotective effect of fisetin, i.e. inhibiting ACSL4-mediated tubular ferroptosis against fibrotic kidney diseases.

Autophagy regulates bone loss via the RANKL/RANK/OPG axis in an experimental rat apical periodontitis model.

AIM: Autophagy is involved in human apical periodontitis (AP). However, it is not clear whether autophagy is protective or destructive in bone loss via the receptor activator of nuclear factor-κB ligand (RANKL)/RANK/osteoprotegerin (OPG) axis. This study aimed to investigate the involvement of autophagy via the RANKL/RANK/OPG axis during the development of AP in an experimental rat model. METHODOLOGY: Twenty-four female Sprague-Dawley rats were divided into control, experimental AP (EAP) + saline, and EAP + 3-methyladenine (An autophagy inhibitor, 3-MA) groups. The control group did not receive any treatment. The EAP + saline group and the EAP + 3-MA group received intraperitoneal injections of saline and 3-MA, respectively, starting 1 week after the pulp was exposed. Specimens were collected for microcomputed tomography (micro-CT) scanning, histological processing, and immunostaining to examine the expression of light chain 3 beta (LC3B), RANK, RANKL, and OPG. Data were analysed using one-way analysis of variance (p < .05). RESULTS: Micro-CT showed greater bone loss in the EAP + 3-MA group than in the EAP + saline group, indicated by an elevated trabecular space (Tb.Sp) (p < .05). Inflammatory cell infiltration was observed in the EAP + saline and EAP + 3-MA groups. Compared with EAP + saline group, the EAP + 3-MA group showed weaker expression of LC3B (p < .01) and OPG (p < .05), more intense expression of RANK (p < .01) and RANKL (p < .01), and a higher RANKL/OPG ratio (p < .05). CONCLUSION: Autophagy may exert a protective effect against AP by regulating the RANKL/RANK/OPG axis, thereby inhibiting excessive bone loss.

Mitigation of benzyl butyl phthalate toxicity in male germ cells with combined treatment of parthenolide, N-acetylcysteine, and 3-methyladenine.

Benzyl butyl phthalate (BBP) is a widely used plasticizer that poses various potential health hazards. Although BBP has been extensively studied, the direct mechanism underlying its toxicity in male germ cells remains unclear. Therefore, we investigated BBP-mediated male germ cell toxicity in GC-1 spermatogonia (spg), a differentiated mouse male germ cell line. This study investigated the impact of BBP on reactive oxygen species (ROS) generation, apoptosis, and autophagy regulation, as well as potential protective measures against BBP-induced toxicity. A marked dose-dependent decrease in GC-1 spg cell proliferation was observed following treatment with BBP at 12.5 µM. Exposure to 50 µM BBP, approximating the IC50 of 53.9 µM, markedly increased cellular ROS generation and instigated apoptosis, as evidenced by augmented protein levels of both intrinsic and extrinsic apoptosis-related markers. An amount of 50 µM BBP induced marked upregulation of autophagy regulator proteins, p38 MAPK, and extracellular signal-regulated kinase and substantially downregulated the phosphorylation of key kinases involved in regulating cell proliferation, including phosphoinositide 3-kinase, protein kinase B, mammalian target of rapamycin (mTOR), c-Jun N-terminal kinase. The triple combination of N-acetylcysteine, parthenolide, and 3-methyladenine markedly restored cell proliferation, decreased BBP-induced apoptosis and autophagy, and restored mTOR phosphorylation. This study provides new insights into BBP-induced male germ cell toxicity and highlights the therapeutic potential of the triple inhibitors in mitigating BBP toxicity.

Is resveratrol really effective in kidney disease?: A different perspective than ever before.

BACKGROUND: Chronic kidney disease (CKD) is a global health problem and it is stated that the use of resveratrol supplement contributes to the protection of kidney health. In this study, it was aimed to evaluate the effect of resveratrol supplementation on kidney function, inflammation and histopathological findings in rats with experimental adenine-induced kidney damage. METHODS: Three different groups of 10 randomly selected rats were formed. The first group was the negative control group, the second group was the uremic control group (KDG), and the third group was the group in which uremia was created and resveratrol was applied (RG). Kidney damage was induced by administration of 200 mg/kg adenine. Resveratrol supplementation was administered at 20 mg/kg after kidney damage. Serum urea, creatinine, indoxyl sulfate (IS), p-cresol, glomerular filtration rate, C-reactive protein (CRP); interleukin (IL)-6 and tumor necrosis factor (TNF)-α gene expression levels and histopathological findings were evaluated. RESULTS: It was determined that resveratrol supplement applied after the formation of connective tissue in renal failure didn't have an improvement effect on the urine amount, kidney function and inflammatory parameters and histopathological changes (p > 0.05). Just, the increase in the CRP value of KDG (p < 0.05) was not observed in RG. CONCLUSION: The findings suggest that resveratrol administered after kidney damage with adenine has no effect on kidney disease.

Inhibiting Bruton's Tyrosine Kinase to Counteract Chemoresistance and Stem Cell-Like Properties in Osteosarcoma.

Osteosarcoma, a highly aggressive bone cancer, often develops resistance to conventional chemotherapeutics, leading to poor prognosis and survival rates. The malignancy and chemoresistance of osteosarcoma pose significant challenges in its treatment, highlighting the critical need for novel therapeutic approaches. Bruton's tyrosine kinase (BTK) plays a pivotal role in B-cell development and has been linked to various cancers, including breast, lung, and oral cancers, where it contributes to tumor growth and chemoresistance. Despite its established importance in these malignancies, the impact of BTK on osteosarcoma remains unexplored. Our study delves into the expression levels of BTK in osteosarcoma tissues by data from the GEO and TCGA database, revealing a marked increase in BTK expression compared with primary osteoblasts and a potential correlation with primary site progression. Through our investigations, we identified a subset of osteosarcoma cells, named cis-HOS, which exhibited resistance to cisplatin. These cells displayed characteristics of cancer stem cells (CSCs), demonstrated a higher angiogenesis effect, and had an increased migration ability. Notably, an upregulation of BTK was observed in these cisplatin-resistant cells. The application of ibrutinib, a BTK inhibitor, significantly mitigated these aggressive traits. Our study demonstrates that BTK plays a crucial role in conferring chemoresistance in osteosarcoma. The upregulation of BTK in cisplatin-resistant cells was effectively countered by ibrutinib. These findings underscore the potential of targeting BTK as an effective strategy to overcome chemoresistance in osteosarcoma treatment.

Enhancing Fat Graft Survival via Upregulating Autophagy of Adipocytes.

BACKGROUND: Autophagy is a cellular self-protection mechanism. The upregulation of adipose-derived stem cells' (ADSCs) autophagy can promote fat graft survival. However, the effect of interfering with adipocyte autophagy on graft survival is still unknown. In addition, autophagy is involved in adipocyte dedifferentiation. We investigated the effect of autophagy on adipocyte dedifferentiation and fat graft survival. METHODS: The classic autophagy regulatory drugs rapamycin (100 nM) and 3-methyladenine (3-MA; 10 mM) were used to treat adipocytes, adipocyte dedifferentiation was observed, and their effects on ADSCs were detected. In our experiments, 100 nM rapamycin, 10 mM 3-MA and saline were mixed with human adipose tissue and transplanted into nude mice. At 2, 4, 8 and 12 weeks postoperatively, the grafts were harvested for histological and immunohistochemical analysis. RESULTS: Rapamycin and 3-MA can promote and inhibit adipocyte dedifferentiation by regulating autophagy. Both drugs can inhibit ADSC proliferation, and 10 mM 3-MA can inhibit ADSC adipogenesis. At weeks 8 and 12, the volume retention rate of the rapamycin group (8 weeks, 64.77% ± 6.36%; 12 weeks, 56.13% ± 4.73%) was higher than the control group (8 weeks, 52.62% ± 4.04%; P < 0.05; 12 weeks, 43.17% ± 6.02%; P < 0.05) and the rapamycin group had more viable adipocytes and better vascularization. Compared with the control group, the volume retention rate, viable adipocytes and vascularization of the 3-MA group decreased. CONCLUSIONS: Rapamycin can promote adipocyte dedifferentiation by upregulating autophagy to promote fat graft survival. 3-MA can inhibit graft survival, but its mechanism includes the inhibition of adipocyte dedifferentiation and ADSC proliferation and adipogenesis. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .