International Journal of Molecular Sciences

Brown adipose tissue (BAT) is a unique tissue with mitochondria specialized for thermogenesis via the BAT-specific uncoupling protein 1 (UCP1). Ucp1-/- mice cannot tolerate acute exposure to cold, illustrating the necessity of UCP1 for efficient mitochondrial thermogenesis. However, these mice adapt to low temperatures through a gradual acclimation process, suggesting a high degree of mitochondrial plasticity in brown and beige fat cells. This phenomenon, which remains to be fully elucidated, indicates the potential for these mitochondria to implement effective thermogenic mechanisms in the absence of uncoupling protein 1 (UCP1). Here, we investigated mitochondrial remodeling in beige and brown fat of Ucp1-/- mice to determine how they fulfill their thermogenic role. Upon gradual acclimation to a cold environment, Ucp1-/- mice exhibited body metabolic parameters and temperatures in the interscapular region similar to those of wild-type mice of BAT, highlighting effective thermogenesis. Interestingly, mitochondrial patch-clamp analysis and a mitochondrial Ca2+ swelling assay revealed a dramatic increase in Ca2+ uptake depending on the mitochondrial calcium uniporter (MCU) in BAT mitochondria from Ucp1-/- mice when robust thermogenesis was required. Mitochondrial remodeling was accompanied by markedly increased tethering between mitochondria and the endoplasmic reticulum (ER) in Ucp1-/- mice, confirming a significant restructuring of the contact sites between the ER and mitochondria, likely to adapt to a new Ca2+ homeostasis. Respiratory complexes also underwent significant reorganization, which partly led to a reduction in their assembly. Levels of ATP synthase and its F1 subcomplex increased, suggesting a major source of ATP consumption and energy expenditure. We propose a new role for MCU as a key regulator of mitochondrial plasticity, enabling efficient thermogenesis in beige and brown adipose tissues in the absence of UCP1.

Inflammatory bowel disease (IBD), comprising Ulcerative colitis (UC) and Crohns Disease, is a chronic relapsing immune-mediated inflammatory disorder of the gut. The intestinal mucus layer is a protective barrier that safeguards direct exposure of epithelium to luminal microbes and antigens. A prolonged disruption of the mucus layer may contribute to the development of IBD. Loss of mucin-producing goblet cells is a hallmark of UC. The underlying molecular mechanism controlling goblet regulation remains poorly understood. In the current work, we show a key role for NCoR1 (Nuclear corepressor 1) in goblet cell regulation. A specific downregulation of NCoR1 in intestinal crypts and goblet cells was observed in human UC and mice models. While NCoR1 was upregulated during goblet cell differentiation, inflammatory cues downregulated its expression. Experimental loss of NCoR1 resulted in exacerbated disease in a murine model of colitis, whereas its upregulation via Vitamin D led to a rescue. ChIP-seq led to the identification of KLF-16, a transcription factor, as a target of NCoR1. NCoR1 -KLF16 regulatory axis regulated key goblet cell proteins, including MUC2. Mechanistically, the regulation of MUC2 is modulated by the NCoR1-KLF16 axis, via mTOR signalling. In conclusion, this work shows a critical involvement of NCoR1-KLF16 in governing goblet cell function and intestinal homeostasis.

Caffeine, the most widely consumed stimulant worldwide and primarily sourced from coffee, is well known for its central nervous system effects. Emerging evidence indicates that caffeine also modulates key cellular processes, including DNA repair. It inhibits the kinase activity of ATM and ATR-essential DNA damage response proteins, and impairs homologous recombination (HR)-mediated repair through multiple mechanisms. However, its effects on nonhomologous end joining (NHEJ), a major double-strand break (DSB) repair pathway, have been underexplored. In a recent study, we reported that caffeine inhibits NHEJ primarily by interfering with Ligase IV/XRCC4 complex, using in vitro and ex vivo model systems. Given coffees role as a primary dietary caffeine source, this study investigates the impact of Coffea arabica decoction on NHEJ-mediated DSB repair. High-performance liquid chromatography (HPLC) quantified caffeine levels in the decoction, followed by in vitro and ex vivo assays to evaluate NHEJ efficiency. Results demonstrate that coffee decoction inhibits end joining of both compatible and noncompatible DNA ends in cell-free systems derived from normal and cancer cells. Extrachromosomal repair assays confirmed impaired intracellular NHEJ, leading to accumulation of unrepaired DSBs in human cells. Kinetic analysis of {gamma}-H2AX foci formation and resolution revealed persistent DNA breaks and reduced repair kinetics. Reconstitution experiments verified that the decoction specifically targets the Ligase IV/XRCC4 complex. These findings, building on our previous work, establish coffee decoction as a potent NHEJ inhibitor, mirroring purified caffeines effects. This underscores caffeines interference with endogenous DNA repair, with profound implications for cancer therapy by sensitizing tumors to genotoxic treatments.

Purpose: To evaluate the efficacy and safety of oral L-ergothioneine (EGT) in alleviating pain and associated symptoms in women with primary dysmenorrhea (PD). Methods: In this randomized, double-blind, placebo-controlled trial, 40 women with PD (aged 18-30 years) were randomized (1:1) to receive EGT capsules (120 mg/day) or a matching placebo for 3 consecutive menstrual cycles. Outcomes evaluated at baseline and post-cycle included peak pain (Visual Analog Scale, VAS), Dysmenorrhea Symptom Score, and the COX Menstrual Symptom Scale (CMSS). Results: EGT significantly improved PD symptoms over 3 cycles. Mean VAS for peak pain decreased from 4.80 {+/-} 1.12 to 2.32 {+/-} 1.59 in the EGT group (p < 0.001), compared to a non-significant reduction (4.10 {+/-} 1.30 to 3.45 {+/-} 1.69) in the placebo group. The between-group difference at cycle 3 was significant (p < 0.01). A linear mixed-model confirmed a significant Time x Group interaction (p < 0.001), showing an accelerated decline in symptom severity for EGT. Furthermore, 84% of EGT-treated patients achieved [&ge;]50% VAS reduction versus 35% in the placebo group (p = 0.003). Serum inflammatory biomarkers showed no significant between-group differences or correlation with VAS improvements, suggesting EGT's analgesic effects likely operate via cytoprotective pathways independent of classical inflammatory cascades. No adverse events were reported. Conclusion: Oral EGT supplementation (120 mg/day) effectively and progressively mitigates menstrual pain and systemic symptoms in PD, offering a well-tolerated, non-pharmacological intervention. Trial Registration: ChiCTR2500112557; Retrospectively registered on 2025-11-17.

This study aimed to investigate the dynamics of gene expression in pigs during heat stress (HS), focusing on both short-term (STHA) and long-term (LTHA) heat acclimation phases. A total of 12 castrated males were exposed to thermoneutral temperatures (24{degrees}C) for 14 days (TN) and then to a constant temperature of 30{degrees}C for 21 days. Rectal temperature measurements indicated a biphasic thermoregulatory response, with an initial peak followed by acclimation. Using whole blood transcriptome analysis at seven time points between day 5 before the initiation of HS challenge and day 13 post HS. A total of 525 genes were differentially expressed during the STHA (day 0-day 2) phase. A switch in the expression of most genes was observed around 20 hours after HS. Functional pathway enrichment analysis identified through shape-based clustering revealed the activation of the immune system, especially mediated through toll-like receptor signaling pathways. The LTHA phase (day 2-day 13) revealed 985 differentially expressed genes, with pathways associated with various metabolisms, including mitochondrial fatty acid beta-oxidation, and electron transport, ATP synthesis, and heat production by uncoupling proteins. Interestingly, oxidative phosphorylation was predicted to be activated during the LTHA, particularly in Complex V, whereas other complexes showed mixed regulation. Comparative pathway analysis indicated distinct metabolic adaptations between STHA and LTHA, with up-regulation of glucose and lipid metabolism in late STHA and down-regulation of lipid metabolism during LTHA. This study contributes to a better understanding of the time course of adaptation mechanisms in pigs to HS, underlying a coordinated regulation during STHA involving several stress-specific mechanisms (via the HSP) and metabolic variation to help pigs achieve homeothermy.

Synthetic 6-Br-Q0C10 has been shown to have a partial electron transfer activity of native coenzyme Q in the isolated mitochondria. It reduces energy coupling efficiency by 30 %, suggesting that it may be useful in the management of obesity. The effect of 6-Br-Q0C10 on cell growth has been confirmed by several cell lines. Whether or not it behaves in the same way in the animal, however, has not yet been tested. Recently, we investigated the effect of 6-Br-Q0C10 on growth of rats. When 6-Br-Q0C10 was dissolved in different media, such as carboxymethyl cellulose, ethanol, and mixture of oil and butter and then feed to rats It shows no toxicity and little negative effects on growth as measured body weight gains over a period of time. When higher concentration (0.5 mg) of 6-Br-Q0C10 was given to each rat in 0.3 mL of oil/butter (70%/30%) mixture via intragastric injection daily for a period, a significant reduction in body weight gains was observed. These results validate the earlier observation that 6-Br-Q0C10 reduces the growth (30-60%) of all cell lines tested, in a time- and concentration dependent manner. These results strengthen the idea of using 6-Br-Q0C10 to manage obesity. It is also implying that 6-BrQ0C10 may slow the growth rate of cancer cells and thus prolong life. (This study was partially funded by NIH grants AA030221 and DA046258 to S.L.Chang.)

Purified proteins are routinely flash frozen for use in functional and structural studies, providing a convenient way to reproduce results across complex experiments. Rho guanine-nucleotide exchange factors (RhoGEFs) are no exception to this practice, yet the effects of freezing on their activity and stability remain largely uncharacterized. This gap potentially affects the characterization of these important enzymes and how results are interpreted with respect to their prospective use as therapeutic targets. Here, we tested the isolated DH/PH tandems of P-Rex1, P-Rex2, and PRG under different cryoprotectant conditions and monitored activity and thermostability over time after flash freezing. Our results show a clear divergence between the activity of fresh and frozen purified RhoGEF protein samples in as little as one week for some conditions. Specifically, the variability in data collected on frozen samples was greatly increased. Despite these differences, thermostability seems to be preserved for much longer timepoints across RhoGEFs. Moreover, despite eventual changes in both activity and thermostability with respect to freezing, there are no obvious changes in global conformation between fresh and frozen samples of the isolated P-Rex2 DH/PH tandem. From our data, there are few generalizable trends between the different RhoGEFs and no single cryoprotective agent tested was a silver bullet to preserve both activity and thermostability across RhoGEFs. Overall, our findings emphasize the unpredictable effects of freezing RhoGEFs. As such, RhoGEF freezing should be carefully characterized for each protein and critically viewed when comparing analyses between different studies.

NLRP3 inflammasome is a cytosolic multi-protein complex that plays a crucial role in the immune system, responding to various exogenous and endogenous stimuli by triggering protective inflammatory responses. However, aberrant NLRP3 inflammasome activation is implicated in numerous inflammatory diseases. Therefore, the NLRP3 inflammasome is an important pharmacological target for the treatment of multiple diseases. In this context, we screened various US-FDA-approved drugs for NLRP3 inflammasome inhibition. We found that among various drugs, minoxidil hydrochloride (MXL) effectively inhibits NLRP3 inflammasome, evidenced by reduced secretion of IL-1{beta} and IL-18 in J774A.1 cells treated with MXL. The IC50 values of MXL for inhibition of IL-1{beta} and IL-18 were calculated to be 1.2 and 1.06 {micro}M, respectively. MXL was found to prevent ASC oligomerization, thereby inhibiting the NLRP3 inflammasome and leading to CASP1 cleavage. Further investigation revealed that MXL also utilizes AMPK-mediated autophagy to modulate NLRP3 inflammasome activity. Using siAMPK and bafilomycin A1, an end-stage autophagy inhibitor, we elucidated crosstalk between the NLRP3 inflammasome and autophagic pathways, which was modulated by MXL. Furthermore, we demonstrated the efficacy of MXL in two different mouse models of inflammation, involving the NLRP3 inflammasome. MXL at doses of 10 and 20 mg/kg effectively inhibited the activation of NLRP3 inflammasome by monosodium urate in the air pouch model and by ATP in the peritoneal inflammation model, as evidenced by reduced secretion of 1{beta} and IL-18 in the lavage. Our study identifies MXL as a potent NLRP3 inflammasome inhibitor, warranting further investigation as a potential therapeutic agent for inflammatory diseases.

Primary human bronchial epithelial cells (pHBECs) of the airways of smokers are chronically exposed to cigarette smoke, which may induce chronic obstructive pulmonary disease (COPD) ranked fourth among the most common global causes of death. Using an established protocol for differentiation of pHBECs to a pseudostratified epithelium at an air liquid interface (ALI), we analyzed functional expression of transient receptor potential vanilloid 4 (TRPV4) proteins after application of cigarette smoke extract (CSE), which upregulated seven smoke exposure regulated genes (SERGs). TRPV4 protein expression in the plasma membrane and localization next to the cilia of ciliated cells was reduced, while cell barrier function was not altered after chronic exposure to CSE for 28 days compared to untreated control cells. Accordingly, TRPV4-mediated Ca2+ influx was blocked in pHBECs after CSE exposure. Moreover, Os-9 protein, which after binding mediates protection from degradation of TRPV4 protein by polyubiquitination, was significantly less expressed in pHBECs upon CSE exposure. Most interestingly, overexpression of OS-9 in pHBECs rescued reduced TRPV4 protein levels induced by CSE. Our study identifies a novel molecular mechanism of toxicity by CSE interfering with TRPV4 and OS-9 expression in pHBECs, which may blaze the trail for new therapeutic options in COPD.

This report identifies a bidirectional signaling axis connecting lipid metabolism to nuclear mechanotransduction, with the potential to control fatty acid/triglyceride metabolism. The sterol regulatory element-binding (SREBP) family of transcription factors control fatty acid, triglyceride and cholesterol synthesis and metabolism. The family consists of three members: SREBP1a, SREBP1c, and SREBP2, that are regulated by intracellular cholesterol levels and insulin signaling. The SREBP2-dependent control of the LDL receptor gene is a well-established target for cholesterol-lowering therapeutics and the activity of SREBP1c is an attractive target in metabolic disease. In the current report, we identify SYNE4 (nesprin-4), a component of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, as a direct target of the SREBP family of transcription factors, and show that nesprin-4 in turn supports SREBP1c function. We identify functional SREBP binding sites in the human SYNE4 promoter and demonstrate that these are required for the sterol- and SREBP-dependent regulation of the promoter. Furthermore, we show that the endogenous SYNE4 gene is also regulated by SREBP1/2 and intracellular sterol levels. Interestingly, SREBP2 is responsible for the sterol regulation of the SYNE4 gene in HepG2 cells, while SREBP1 is the major regulator in MCF7 cells, demonstrating that diberent cell types use diberent SREBP paralogs to regulate the same promoter/gene. Importantly, we find that nesprin-4 is a positive regulator of SREBP1c expression and function in HepG2 cells and during the diberentiation of human adipose-derived stem cells. In summary, the current report identifies a novel regulatory interaction between lipid metabolism and the LINC complex. Importantly, we demonstrate that this signaling axis is bidirectional, forming a closed loop that has the potential to control SREBP1c activity and thereby fatty acid and triglyceride synthesis/metabolism. Based on our data, we propose that the nesprin-4-dependent regulation of SREBP1c could represent a novel therapeutic target in metabolic disease.

Equine asthma (EA) is a highly prevalent, chronic, inflammatory disease of the lower airways ranging from mild-to-moderate to severe clinical presentations. Diagnosis currently relies on bronchoalveolar lavage fluid (BALF) cytology, an invasive method associated with interobserver variability, which highlights the need for more reproducible approaches. MicroRNAs (miRNAs) are small noncoding RNAs involved in post-transcriptional gene regulation. They are stable and readily detectable in body fluids and have shown promising results as biomarkers in human asthma. The aim of this study was to characterize miRNA abundance profiles in BALF and serum from horses with distinct EA endotypes to evaluate their biomarker potential and explore their involvement in disease pathogenesis. A total of 43 horses were included and classified as either EA (n=32) or controls (n=11), based on clinical examination and BALF cytology. The EA horses were further divided into three endotypes based on BALF inflammatory cell composition: neutrophilic asthma (n=10), mastocytic asthma (n=15), and mixed asthma (n=7). RNA was isolated from both serum and BALF samples and analyzed by quantitative real-time PCR (qPCR) targeting 103 miRNAs linked to asthma and pulmonary inflammation in humans. Differential miRNA abundance was analyzed across EA endotypes. The most significantly differentially abundant miRNAs were used for in silico target prediction and pathway enrichment analyses. Horses with mixed EA had significantly lower levels of eca-miR-125a-3p and eca-miR-125b-5p in BALF compared to controls. Additionally, eca-miR-146a-5p abundance was significantly increased in BALF from horses with neutrophilic EA compared to mastocytic EA. Target and pathway enrichment analyses for eca-miR-146a-5p identified immune-relevant pathways, such as MAPK and T-cell receptor signaling, supporting its involvement in inflammatory processes associated with asthma. This study identified three promising candidates, eca-miR-125a-3p, eca-miR-125b-5p, and eca-miR-146a-5p, as potential biomarkers associated with different EA endotypes. These miRNAs are interesting candidates for further investigation in an independent cohort.

In our society, ageing, longevity, and neurodegenerative diseases are major concerns of public health. Recently, in Drosophila, we have identified a new cluster of three snoRNAs, including jouvence, and showed that each of them affect longevity and neurodegeneration. As these snoRNAs are required in the epithelium of the gut, these results point-out a causal relationship between the epithelium of the gut and the neurodegenerative lesions through the metabolic parameters, indicating a gut-brain axis. Here, we demonstrate that each snoRNA pseudouridylates a specific site on ribosomal-RNA, which consequently affects the amount of ribosomes as well as the translational efficacy. Moreover, using TRAP experiment assay, we also show that these lacks of pseudouridylations modify the translation of specific genes involved in lipid metabolism. Consequently, these lead to a chronic deregulation of trigycerides and sterols levels, whose correlate to an increase of neurogenerative lesions in old flies, as well as to a modification of longevity.

Eukaryotes use several distinct quality control pathways to resolve aberrant ribosomes and mRNAs. For example, the no-go decay mRNA pathway is stimulated after ribosome collisions caused by stalled ribosomes translating damaged or truncated mRNAs. Separate decay pathways for non-functional 40S and 60S subunits containing rRNA mutations affecting decoding and peptidyl transferase activity, respectively, have also been elucidated. To our knowledge, whether eukaryotes have evolved a quality control pathway to sense and process globally stalled ribosomes is unclear; however, such a pathway would be advantageous to eukaryotes during exposure to natural elongation inhibitors such as ricin and diphtheria toxin. Here, we test how prolonged robust inhibition of elongation using a high dose of cycloheximide (CHX) affects ribosome turnover. Despite no decrease in cell viability and that mammalian ribosomes have been classically characterized of having a half-life of 3-5 days, a single 24 hr high dose of CHX resulted in drastically shortened half-lives (<24 hr) of 28S and 18S rRNA in A549 cells. A [~]2-fold reduction in nearly all ribosome species was observed by polysome analysis in HeLa and A549 cells after prolonged CHX treatment. Depletion of ribosomes was also evident when assessing ribosomal proteins from both the 40S and 60S subunits by Western blot. Literature supports that ribosomes can be degraded by autophagy and the ubiquitin (Ub)-proteasome system. Upon testing inhibitors of both pathways, only proteasome inhibitors (i.e., MG132 and bortezomib) rescued both rRNA and ribosomal protein levels. Proteasome inhibitors also rescued ribosome levels in polysome profiling experiments. Remarkably, rRNA levels were not rescued during CHX treatment when co-treated with the Ub activating enzyme E1 inhibitor, TAK243. Polysome analysis also showed that the high prolonged dose of CHX did not cause robust accumulation of collided ribosomes compared to control treatments. Proteasome-dependent turnover of rRNA was also observed with high doses of other elongation inhibitors, namely anisomycin, homoharringtonine, and lactimidomycin. The recognition capabilities of the pathway were further expanded as we observed that 80S ribosomes not trapped on the mRNA were also targeted for degradation by the proteasome. Together, our findings define the framework of a regulatory pathway in mammalian cells that degrades both ribosomal subunits in response to prolonged periods of robust elongation inhibition.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays a crucial role in regulating various microglial functions, including phagocytosis, inflammation, chemotaxis, and proliferation. Recent studies have demonstrated that TREM2 cooperates with DAP12 to mediate intracellular signaling essential for these processes. Despite the importance of the TREM2-DAP12 complex in microglial physiology, the mechanisms controlling its expression and activity remain poorly understood. In this study, we report that the soluble ectodomain of TREM2 (sTREM2) regulates microglial phagocytic activity by attenuating the surface expression of DAP12. We found that stimulation of the microglial cell line BV2 with recombinant sTREM2 reduces the membrane expression of DAP12, but not that of TREM2. In addition, sTREM2 binds to full-length TREM2, leading to the uncoupling of TREM2 from DAP12. Furthermore, pre-treatment of BV2 cells with sTREM2 significantly inhibited amyloid-{beta} incorporation. These findings suggest that sTREM2 negatively regulates TREM2 signaling through the destabilization of the TREM2-DAP12 complex, and act as a novel bioactive molecule that modulates TREM2 signaling under physiological and pathological conditions.

Microproteins are proteins of <100 amino acids. They represent a major, and until recently, overlooked fraction of the human proteome. However, it has now been demonstrated that many of these proteins play key roles in cellular physiology. Our group reported the expression of a microprotein expressed from an ioORF within the 53BP1 CDS arising as a result of delayed translational reinitiation mediated by a small uORF within the 5 TL. We named this microprotein SEP53BP1. We have sought to expand these studies with the ultimate aim of establishing a function for this microprotein. Although this remains elusive, we report findings providing new insights into the elements regulating its translation and demonstrate that the SEP53BP1 sequence serves as a Golgi targeting tag. Lastly, despite the fact that subunits of the proteasome feature prominently on interactome studies we were unable to demonstrate an impact of microprotein over-expression on the activities of both the proteasome and immunoproteasome.

IP3R-Grp75-VDAC1 protein complex at the mitochondria-ER contact sites (MERCS) is involved in response to nutrients and control of glucose and energy metabolism, however, early alterations of the complex and MERCS in response to increased fat intake remain inconclusive. We investigated early effects of high-fat diet (HFD) on IP3R-Grp75-VDAC1 protein expression in correlation with ER-mitochondrial interaction in the liver of mice. Five-week-old mice were fed an HFD or a standard diet (SD) for 2 weeks (2W) or 8 weeks (8W). MERCS fractionation by a gradient ultracentrifugation, Western blot, transmission electron microscopy (TEM), Oroboros high-resolution respirometry were used to analyse liver tissues, while real-time PCR was used to profile genes responsive to HFD. No macroscopic morphological or functional alterations were observed in mice at 2W, while, expectedly, at 8W of HFD mice gained weight and glucose intolerance. Total IP3R protein was reduced at both 2W and 8W points by a post-transcriptional mechanism, while in MERCS, IP3R, VDAC1 and Grp75 were reduced at 8W time-point. TEM analysis revealed a significant reduction of mitochondrial coverage by MERCS, mitochondrial fragmentation and shortening of ER-mitochondria distance already at 2W time-point. Mitochondrial function and metabolism were largely spared. Markers of altered protein homeostasis such as Lmp2, Mecl-1 and Lmp7 showed an early upregulation. In conclusion, HFD induces early alterations in liver MERCS that precede gain of weight and glucose intolerance, suggesting their primary role in obesity and metabolic diseases and as potential therapeutic target.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

Bisphenol A (BPA) and Bisphenol S (BPS) exposure disrupt ovarian function and granulosa cell (GC) steroidogenesis. Extracellular vesicles (EVs) and their miRNA cargo, as mediators of cellular response to environmental stimuli, might be involved in fertility and folliculogenesis. This study explored modulation of microRNA expression after 48h BPA or BPS exposure (10 {micro}M) in ovine primary GC and EVs from corresponding conditioned medium (CM EVs). Small RNA sequencing of control (0h) and 48h treated GC, CM EVs as well as follicular fluid EVs allowed identification of 533 ovine miRNAs, including 129 new sequences. BPA did not alter miRNA expression in GC, while BPS decreased cellular oar-24b miR. In contrast, BPA modified expression of 4 miRNAs in CM-EVs, including 3 new sequences, and two miRNAs were modified by BPS. Both compounds reduced expression of sequence homologous to miR-1306. Further studies are required to decipher their roles in bisphenol toxicity in GC.

Metabolic dysfunction-associated steatohepatitis (MASH) is associated with increased expression of peroxisome proliferator-activated receptor gamma (PPAR{gamma}, Pparg) and reduced expression of genes involved in methionine metabolism in the liver. The nuclear receptor PPAR{gamma} is activated by fatty acids, and the knockout of Pparg in hepatocytes (Pparg{Delta}Hep) reduced the negative effects of MASH on methionine metabolism. Here, we sought to determine whether hepatocyte Pparg is required for the transcriptional regulation of genes involved in hepatic methionine metabolism in conditions with altered fatty acid flux to the liver: fasting, refeeding, and high-fat diet (HFD)-induced obesity/steatosis. Fasting induced liver steatosis and increased the expression of key genes involved in the methionine metabolism in the liver, while 6h-refeeding reversed these effects and reduced the expression of phosphatidylethanolamine N-methyltransferase (Pemt) and cystathionine beta synthase (Cbs). Overall, fasting and refeeding did not alter hepatocyte Pparg expression nor Pparg{Delta}Hep affected fasting and refeeding-mediated regulation of methionine metabolism gene expression. Diet-induced steatosis reduced hepatic Pemt expression in control (Pparg-intact) mice, and the thiazolidinedione (TZD)-mediated activation of PPAR{gamma} in diet-induced obese control (Pparg-intact) mice reduced the expression of betaine homocysteine S-methyltransferase (Bhmt) and Cbs. However, diet-induced steatosis increased hepatocyte Pparg expression, and Pparg{Delta}Hep blocked the negative effects of HFD and TZD on hepatic methionine metabolism. The PPAR{gamma}-dependent reduction of hepatic Bhmt and Cbs expression was confirmed in mouse primary hepatocytes. Taken together, hepatocyte Pparg may serve as a negative regulator of hepatic methionine metabolism in diet-induced obese mice and these actions could contribute to promoting the onset of MASH.

The importance of human aldehyde oxidase (hAOX1) has increased over the last decades due to its involvement in drug metabolism. Inhibition studies concerning hAOX1 are extensive and a common reducing agent, dithiothreitol (DTT), was recently found to inactivate the enzyme. However, in previous crystallographic studies of hAOX1, DTT was found to be essential for crystallization. To surpass this concern another reducing agent used in crystallization trials. Using tris(2-carboxyethyl)phosphine (TCEP), a sulphur-free reducing agent, it was possible to obtain well-ordered crystals from hAOX1 wild type and variant, hAOX1_6A, which diffracted beyond 2.3 [A]. Instead of the typical star-shaped crystals of hAOX1, at pH 4.7, plates are obtained in the orthorhombic space group (P22121) with two molecules in the asymmetric unit. Activity assays with the enzyme incubated with both reducing agents show that contrary to DTT, TCEP does not lead to irreversible inactivation of the enzyme. The replacement of DTT with TCEP in crystallization of hAOX1 provides a strategy to circumvent enzyme inactivation during crystallographic studies, allowing future applications of new assays, such as time-resolved crystallography.