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OBJECTIVE Type 2 diabetes is associated with insulin resistance and skeletal muscle mitochondrial dysfunction. We have found that subjects with early-onset type 2 diabetes show incapacity to increase Vo(2max) in response to chronic exercise. This suggests a defect in muscle mitochondrial response to exercise. Here, we have explored the nature of the mechanisms involved. RESEARCH DESIGN AND METHODS Muscle biopsies were collected from young type 2 diabetic subjects and obese control subjects before and after acute or chronic exercise protocols, and the expression of genes and/or proteins relevant to mitochondrial function was measured. In particular, the regulatory pathway peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha/mitofusin-2 (Mfn2) was analyzed. RESULTS At baseline, subjects with diabetes showed reduced expression (by 26%) of the mitochondrial fusion protein Mfn2 and a 39% reduction of the alpha-subunit of ATP synthase. Porin expression was unchanged, consistent with normal mitochondrial mass. Chronic exercise led to a 2.8-fold increase in Mfn2, as well as increases in porin, and the alpha-subunit of ATP synthase in muscle from control subjects. However, Mfn2 was unchanged after chronic exercise in individuals with diabetes, whereas porin and alpha-subunit of ATP synthase were increased. Acute exercise caused a fourfold increase in PGC-1alpha expression in muscle from control subjects but not in subjects with diabetes. CONCLUSIONS Our results demonstrate alterations in the regulatory pathway that controls PGC-1alpha expression and induction of Mfn2 in muscle from patients with early-onset type 2 diabetes. Patients with early-onset type 2 diabetes display abnormalities in the exercise-dependent pathway that regulates the expression of PGC-1alpha and Mfn2. [ABSTRACT FROM AUTHOR]

Intestinal Spirochaetes of the genus Brachyspira cause important diseases in swine and poultry. An increasing problem in the presentation of resistant strains to the antimicrobial drugs usually used to treat the intestinal spirochaetosis in swine has been evidenced and this could be applicable to the isolations from poultry. There are very few reports of in vitro antimicrobial susceptibility of Brachyspira spp. isolated from birds. In this study the antimicrobial susceptibility of twelve Brachyspira pilosicoli isolates obtain from commercial layers was evaluated against tiamulin, tylosin and lincomycin establishing the Minimum Inhibitory Concentration (MIC) by agar dilution technique. All bacteria analyzed were sensitive to tiamulin (MIC ≤0,1μg/ml), and lincomycin (MIC 1μg/ml) and resistant to tylosin (MIC 5μg/ml).Las espiroquetas intestinales del género Brachyspira ocasionan enfermedades importantes en porcinos y aves. Se ha evidenciado un problema de incremento en la presentación de cepas resistentes a los antimicrobianos utilizados normalmente para tratar las espiroquetosis intestinales en porcinos, y esto podría ser aplicable a los aislamientos de aves. Hay muy pocos reportes de sensibilidad antimicrobiana in vitro de cepas de Brachyspira spp. Aisladas en aves. En este estudio se evaluó la sensibilidad de doce aislamientos de Brachyspira pilosicoli obtenidos de granjas de ponedoras comerciales a los agentes antimicrobianos tiamulina, tilosina y lincomicina, y se estableció la concentración mínima inhibitoria (CMI) mediante la técnica de dilución en agar. Todas las bacterias analizadas fueron sensibles a tiamulina (CMI ≤0,1 μg/ml) y lincomicina (CMI 1 μg/ml) y resistentes a tilosina (CMI 5 μg/ml).

C16H10F9N3O3S, triclinic, P1̅ (No. 2), a = 5.4635(2) Å, b = 10.266(1) Å, c = 17.645(2) Å, α = 105.731(6)°, β = 93.187(5)°, γ = 97.036(7)°, V = 941.3 Å3, Z = 2, Rgt(F) = 0.048, wRref(F2) = 0.134, T = 213 K.

C16H10F9N3O3S, monoclinic, P121/c1 (No. 14), a = 5.6549(3) Å, b = 12.2550(9) Å, c = 27.170(6) Å, β = 90.446(8)°, V = 1882.8 Å3, Z = 4, Rgt(F) = 0.088, wRref(F2) = 0.261, T = 213 K.

Abstract Background The tyramine producer Enterococcus durans IPLA655 contains all the necessary genes for tyramine biosynthesis, grouped in the TDC cluster. This cluster includes tyrS, an aminoacyl-tRNA synthetase like gene. Results This work shows that tyrS was maximally transcribed in absence of tyrosine at acidic pH, showing a greater than 10-fold induction in mRNA levels over levels occurring in presence of tyrosine. Mapping of the tyrS transcriptional start site revealed an unusually long untranslated leader region of 322 bp, which displays the typical features of the T box transcriptional attenuation mechanism. The tyrosine concentration regulation of tyrS was found to be mediated by a transcription antitermination system, whereas the specific induction at acidic pH was regulated at transcription initiation level. Conclusions The expression of the tyrS gene present in the TDC cluster of E. durans is transcriptionally regulated by tyrosine concentration and extracelular pH. The regulation is mediated by both an antitermination system and the promoter itself.

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) and its more severe form steatohepatitis (MASH) contribute to rising morbidity and mortality rates. The storage of fat in humans is closely associated with these diseases' progression. Thus, adipose tissue metabolic homeostasis could be key in both the onset and progression of MASH., Methods: We conducted a case-control observational research using a systems biology-based approach to analyse liver, abdominal subcutaneous adipose tissue (SAT), omental visceral adipose tissue (VAT), and blood of n = 100 patients undergoing bariatric surgery (NCT05554224). MASH was diagnosed through histologic assessment. Whole-slide image analysis, lipidomics, proteomics, and transcriptomics were performed on tissue samples. Lipidomics and proteomics profiles were determined on plasma samples., Findings: Liver transcriptomics, proteomics, and lipidomics revealed interconnected pathways associated with inflammation, mitochondrial dysfunction, and lipotoxicity in MASH. Paired adipose tissue biopsies had larger adipocyte areas in both fat depots in MASH. Enrichment analyses of proteomics and lipidomics data confirmed the association of liver lesions with mitochondrial dysfunction in VAT. Plasma lipidomics identified candidates with high diagnostic accuracy (AUC = 0.919, 95% CI 0.840-0.979) for screening MASH., Interpretation: Mitochondrial dysfunction is also present in VAT in patients with obesity-associated MASH. This may cause a disruption in the metabolic equilibrium of lipid processing and storage, which impacts the liver and accelerates detrimental adaptative responses., Funding: The project leading to these results has received funding from 'la Caixa' Foundation (HR21-00430), and from the Instituto de Salud Carlos III (ISCIII) (PI21/00510) and co-funded by the European Union., Competing Interests: Declaration of interests The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Obesity and type 2 diabetes (T2D) are widespread metabolic disorders that significantly impact global health today, affecting approximately 17% of adults worldwide with obesity and 9.3% with T2D. Both conditions are closely linked to disruptions in lipid metabolism, where peroxisomes play a pivotal role. Mitochondria and peroxisomes are vital organelles responsible for lipid and energy regulation, including the β-oxidation and oxidation of very long-chain fatty acids (VLCFAs), cholesterol biosynthesis, and bile acid metabolism. These processes are significantly influenced by estrogens, highlighting the interplay between these organelles' function and hormonal regulation in the development and progression of metabolic diseases, such as obesity, metabolic dysfunction-associated fatty liver disease (MAFLD), and T2D. Estrogens modulate lipid metabolism through interactions with nuclear receptors, like peroxisome proliferator-activated receptors (PPARs), which are crucial for maintaining metabolic balance. Estrogen deficiency, such as in postmenopausal women, impairs PPAR regulation, leading to lipid accumulation and increased risk of metabolic disorders. The disruption of peroxisomal-mitochondrial function and estrogen regulation exacerbates lipid imbalances, contributing to insulin resistance and ROS accumulation. This review emphasizes the critical role of these organelles and estrogens in lipid metabolism and their implications for metabolic health, suggesting that therapeutic strategies, including hormone replacement therapy, may offer potential benefits in treating and preventing metabolic diseases.

Trithorax-related H3K4 methyltransferases, KMT2C and KMT2D, are critical epigenetic modifiers. Haploinsufficiency of KMT2C was only recently recognized as a cause of neurodevelopmental disorder (NDD), so the clinical and molecular spectrums of the KMT2C-related NDD (now designated as Kleefstra syndrome 2) are largely unknown. We ascertained 98 individuals with rare KMT2C variants, including 75 with protein-truncating variants (PTVs). Notably, ∼15% of KMT2C PTVs were inherited. Although the most highly expressed KMT2C transcript consists of only the last four exons, pathogenic PTVs were found in almost all the exons of this large gene. KMT2C variant interpretation can be challenging due to segmental duplications and clonal hematopoesis-induced artifacts. Using samples from 27 affected individuals, divided into discovery and validation cohorts, we generated a moderate strength disorder-specific KMT2C DNA methylation (DNAm) signature and demonstrate its utility in classifying non-truncating variants. Based on 81 individuals with pathogenic/likely pathogenic variants, we demonstrate that the KMT2C-related NDD is characterized by developmental delay, intellectual disability, behavioral and psychiatric problems, hypotonia, seizures, short stature, and other comorbidities. The facial module of PhenoScore, applied to photographs of 34 affected individuals, reveals that the KMT2C-related facial gestalt is significantly different from the general NDD population. Finally, using PhenoScore and DNAm signatures, we demonstrate that the KMT2C-related NDD is clinically and epigenetically distinct from Kleefstra and Kabuki syndromes. Overall, we define the clinical features, molecular spectrum, and DNAm signature of the KMT2C-related NDD and demonstrate they are distinct from Kleefstra and Kabuki syndromes highlighting the need to rename this condition., Competing Interests: Declaration of interests R.W. is a consultant (equity) for Alamya Health., (Copyright © 2024 American Society of Human Genetics. All rights reserved.)

Background and Aim: The excessive accumulation of lipid droplets (LDs) is a defining characteristic of nonalcoholic fatty liver disease (NAFLD). The interaction between LDs and mitochondria is functionally important for lipid metabolism homeostasis. Exercise improves NAFLD, but it is not known if it has an effect on hepatic LD-mitochondria interactions. Here, we investigated the influence of exercise on LD-mitochondria interactions and its significance in the context of NAFLD., Approach and Results: Mice were fed high-fat diet (HFD) or HFD-0.1 % methionine and choline-deficient diet (MCD) to emulate simple hepatic steatosis or non-alcoholic steatohepatitis, respectively. In both models, aerobic exercise decreased the size of LDs bound to mitochondria and the number of LD-mitochondria contacts. Analysis showed that the effects of exercise on HOMA-IR and liver triglyceride levels were independent of changes in body weight, and a positive correlation was observed between the number of LD-mitochondria contacts and NAFLD severity and with the lipid droplet size bound to mitochondria. Cellular fractionation studies revealed that ATP-coupled respiration and fatty acid oxidation (FAO) were greater in hepatic peridroplet mitochondria (PDM) from HFD-fed exercised mice than from equivalent sedentary mice. Finally, exercise increased FAO and mitofusin-2 abundance exclusively in PDM through a mechanism involving the curvature of mitochondrial membranes and the abundance of saturated lipids. Accordingly, hepatic mitofusin-2 ablation prevented exercise-induced FAO in PDM., Conclusions: This study demonstrates that aerobic exercise has beneficial effects in murine NAFLD models by lessening the interactions between hepatic LDs and mitochondria, and by decreasing LD size, correlating with a reduced severity of NAFLD. Additionally, aerobic exercise increases FAO in PDM and this process is reliant on Mfn-2 enrichment, which modifies LD-mitochondria communication., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The relationship between lipid mediators and severe obesity remains unclear. Our study investigates the impact of severe obesity on plasma concentrations of oxylipins and fatty acids and explores the consequences of weight loss., Methods: In the clinical trial identifier NCT05554224 study, 116 patients with severe obesity and 63 overweight/obese healthy controls matched for age and sex (≈2:1) provided plasma. To assess the effect of surgically induced weight loss, we requested paired plasma samples from 44 patients undergoing laparoscopic sleeve gastrectomy one year after the procedure. Oxylipins were measured using ultra-high-pressure liquid chromatography coupled to a triple quadrupole mass spectrometer via semi-targeted lipidomics. Cytokines and markers of interorgan crosstalk were measured using enzyme-linked immunosorbent assays., Results: We observed significantly elevated levels of circulating fatty acids and oxylipins in patients with severe obesity compared to their metabolically healthier overweight/obese counterparts. Our findings indicated that sex and liver disease were not confounding factors, but we observed weak correlations in plasma with circulating adipokines, suggesting the influence of adipose tissue. Importantly, while weight loss restored the balance in circulating fatty acids, it did not fully normalize the oxylipin profile. Before surgery, oxylipins derived from lipoxygenase activity, such as 12-HETE, 11-HDoHE, 14-HDoHE, and 12-HEPE, were predominant. However, one year following laparoscopic sleeve gastrectomy, we observed a complex shift in the oxylipin profile, favoring species from the cyclooxygenase pathway, particularly proinflammatory prostanoids like TXB2, PGE2, PGD2, and 12-HHTrE. This transformation appears to be linked to a reduction in adiposity, underscoring the role of lipid turnover in the development of metabolic disorders associated with severe obesity., Conclusions: Despite the reduction in fatty acid levels associated with weight loss, the oxylipin profile shifts towards a predominance of more proinflammatory species. These observations underscore the significance of seeking mechanistic approaches to address severe obesity and emphasize the importance of closely monitoring the metabolic adaptations after weight loss., Competing Interests: Conflict of interest The authors declare that there are no competing interests in disclosing personal or financial interests, and there are no external factors potentially affecting the objectivity and quality of the information., (Copyright © 2023 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

The subfractionation of the endoplasmic reticulum (ER) is a widely used technique in cell biology. However, current protocols present limitations such as low yield, the use of large number of dishes, and contamination with other organelles. Here, we describe an improved method for ER subfractionation that solves other reported methods' main limitations of being time consuming and requiring less starting material. Our protocol involves a combination of different centrifugations and special buffer incubations as well as a fine-tuned method for homogenization followed by western blotting to confirm the purity of the fractions. This protocol contains a method to extract clean ER samples from cells using only five (150 mm) dishes instead of over 50 plates needed in other protocols. In addition, in this article we not only propose a new cell fractionation approach but also an optimized method to isolate pure ER fractions from one mouse liver instead of three, which are commonly used in other protocols. The protocols described here are optimized for time efficiency and designed for seamless execution in any laboratory, eliminating the need for special/patented reagents. Key features • Subcellular fractionation from cells and mouse liver. • Uses only five dishes (150 mm) or one mouse liver to extract highly enriched endoplasmic reticulum without mitochondrial-associated membrane contamination. • These protocols require the use of ultracentrifuges, dounce homogenizers, and/or Teflon Potter Elvehjem. As a result, highly enriched/clean samples are obtained. Graphical overview., Competing Interests: Competing interestsThe authors are not affected by any conflict of interest., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY license.)

In eukaryotic cells, different organelles interact at membrane contact sites stabilized by tethers. Mitochondrial mitofusin 2 (MFN2) acts as a membrane tether that interacts with an unknown partner on the endoplasmic reticulum (ER). In this work, we identified the MFN2 splice variant ERMIT2 as the ER tethering partner of MFN2. Splicing of MFN2 produced ERMIT2 and ERMIN2, two ER-specific variants. ERMIN2 regulated ER morphology, whereas ERMIT2 localized at the ER-mitochondria interface and interacted with mitochondrial mitofusins to tether ER and mitochondria. This tethering allowed efficient mitochondrial calcium ion uptake and phospholipid transfer. Expression of ERMIT2 ameliorated the ER stress, inflammation, and fibrosis typical of liver-specific Mfn2 knockout mice. Thus, ER-specific MFN2 variants display entirely extramitochondrial MFN2 functions involved in interorganellar tethering and liver metabolic activities.

Type 2 diabetes mellitus (T2D) affects millions of people worldwide and is one of the leading causes of morbidity and mortality. The skeletal muscle (SKM) is one of the most important tissues involved in maintaining glucose homeostasis and substrate oxidation, and it undergoes insulin resistance in T2D. In this study, we identify the existence of alterations in the expression of mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) in skeletal muscle from two different forms of T2D: early-onset type 2 diabetes (YT2) (onset of the disease before 30 years of age) and the classical form of the disease (OT2). GSEA analysis from microarray studies revealed the repression of mitochondrial mt-aaRSs independently of age, which was validated by real-time PCR assays. In agreement with this, a reduced expression of several encoding mt-aaRSs was also detected in skeletal muscle from diabetic (db/db) mice but not in obese ob/ob mice. In addition, the expression of the mt-aaRSs proteins most relevant in the synthesis of mitochondrial proteins, threonyl-tRNA, and leucyl-tRNA synthetases (TARS2 and LARS2) were also repressed in muscle from db/db mice. It is likely that these alterations participate in the reduced expression of proteins synthesized in the mitochondria detected in db/db mice. We also document an increased iNOS abundance in mitochondrial-enriched muscle fractions from diabetic mice that may inhibit aminoacylation of TARS2 and LARS2 by nitrosative stress. Our results indicate a reduced expression of mt-aaRSs in skeletal muscle from T2D patients, which may participate in the reduced expression of proteins synthesized in mitochondria. An enhanced mitochondrial iNOS could play a regulatory role in diabetes., Competing Interests: Declaration of competing interest The authors are not affected by any conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Phospholipids are the basic structure block of eukaryotic membranes, in both the outer and inner membranes, which delimit cell organelles. Phospholipids can also be damaged by oxidative stress produced by mitochondria, for instance, becoming oxidized phospholipids. These damaged phospholipids have been related to prevalent diseases such as atherosclerosis or non-alcoholic steatohepatitis (NASH) because they alter gene expression and induce cellular stress and apoptosis. One of the main sites of phospholipid synthesis is the endoplasmic reticulum (ER). ER association with other organelles through membrane contact sites (MCS) provides a close apposition for lipid transport. Additionally, an important advance in this small cytosolic gap are lipid transfer proteins (LTPs), which accelerate and modulate the distribution of phospholipids in other organelles. In this regard, LTPs can be established as an essential point within phospholipid circulation, as relevant data show impaired phospholipid transport when LTPs are defected. This review will focus on phospholipid function, metabolism, non-vesicular transport, and associated diseases.

Airline pilots are frequently exposed to numerous flights per week, changes in their circadian rhythms, and extended periods away from home. All these stressors make pilots susceptible to developing psychiatric disorders. Recently, emphasis has been placed on the need for molecular tests that help in the diagnosis of depression. The genes SLC6A4 and S100A10 encode serotonin transporter (SERT) and p11 protein, respectively. Their expression has been frequently associated with stress and depression. In this work, we quantified, by quantitative PCR, the expression of SERT and p11 in peripheral mononuclear cells of airline pilots compared to patients with depression and healthy volunteers. Moreover, by mass spectrometry, we quantified the serum serotonin levels in the same three groups. We found that SERT and p11 were overexpressed in the mononuclear cells of airline pilots and depressed patients compared to healthy volunteers. Although serum serotonin was not different between healthy volunteers and airline pilots, a decreasing trend was observed in the latter. As expected, serum serotonin in the patients was significantly lower. Alterations in SERT and p11 in airline pilots could be related to professional stress, a condition that could potentially affect their long-term mental health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Becerril-Villanueva, Olvera-Alvarez, Alvarez-Herrera, Maldonado-García, López-Torres, Ramírez-Marroquín, González-Ruiz, Nogueira-Fernández, Mendoza-Contreras, Sánchez-García, José-Alfallo, Valencia Baños, Torres-Serrano, Jiménez-Genchi, Mendieta-Cabrera, Pérez-Sánchez and Pavón.)

The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.

Importance: Truncating variants in the gene encoding filamin C (FLNCtv) are associated with arrhythmogenic and dilated cardiomyopathies with a reportedly high risk of ventricular arrhythmia., Objective: To determine the frequency of and risk factors associated with adverse events among FLNCtv carriers compared with individuals carrying TTN truncating variants (TTNtv)., Design, Setting, and Participants: This cohort study recruited 167 consecutive FLNCtv carriers and a control cohort of 244 patients with TTNtv matched for left ventricular ejection fraction (LVEF) from 19 European cardiomyopathy referral units between 1990 and 2018. Data analyses were conducted between June and October, 2020., Main Outcomes and Measures: The primary end point was a composite of malignant ventricular arrhythmia (MVA) (sudden cardiac death, aborted sudden cardiac death, appropriate implantable cardioverter-defibrillator shock, and sustained ventricular tachycardia) and end-stage heart failure (heart transplant or mortality associated with end-stage heart failure). The secondary end point comprised MVA events only., Results: In total, 167 patients with FLNCtv were studied (55 probands [33%]; 89 men [53%]; mean [SD] age at baseline evaluation, 43 [18] years). For a median follow-up of 20 months (interquartile range, 7-60 months), 29 patients (17.4%) reached the primary end point (19 patients with MVA and 10 patients with end-stage heart failure). Eight (44%) arrhythmic events occurred among individuals with baseline mild to moderate left ventricular systolic dysfunction (LVSD) (LVEF = 36%-49%). Univariable risk factors associated with the primary end point included proband status, LVEF decrement per 10%, ventricular ectopy (≥500 in 24 hours) and myocardial fibrosis detected on cardiac magnetic resonance imaging. The LVEF decrement (hazard ratio [HR] per 10%, 1.83 [95% CI, 1.30-2.57]; P < .001) and proband status (HR, 3.18 [95% CI, 1.12-9.04]; P = .03) remained independent risk factors on multivariable analysis (excluding myocardial fibrosis and ventricular ectopy owing to case censoring). There was no difference in freedom from MVA between FLNCtv carriers with mild to moderate or severe (LVEF ≤35%) LVSD (HR, 1.29 [95% CI, 0.45-3.72]; P = .64). Carriers of FLNCtv with impaired LVEF at baseline evaluation (n = 69) had reduced freedom from MVA compared with 244 TTNtv carriers with similar baseline LVEF (for mild to moderate LVSD: HR, 16.41 [95% CI, 3.45-78.11]; P < .001; for severe LVSD: HR, 2.47 [95% CI, 1.04-5.87]; P = .03)., Conclusions and Relevance: The high frequency of MVA among patients with FLNCtv with mild to moderate LVSD suggests that higher LVEF values than those currently recommended should be considered for prophylactic implantable cardioverter-defibrillator therapy in FLNCtv carriers.

Salmonella hijack host machinery in order to invade cells and establish infection. While considerable work has described the role of host proteins in invasion, much less is known regarding how natural variation in these invasion-associated host proteins affects Salmonella pathogenesis. Here we leveraged a candidate cellular GWAS screen to identify natural genetic variation in the ARHGEF26 (Rho Guanine Nucleotide Exchange Factor 26) gene that renders lymphoblastoid cells susceptible to Salmonella Typhi and Typhimurium invasion. Experimental follow-up redefined ARHGEF26's role in Salmonella epithelial cell infection. Specifically, we identified complex serovar-by-host interactions whereby ARHGEF26 stimulation of S. Typhi and S. Typhimurium invasion into host cells varied in magnitude and effector-dependence based on host cell type. While ARHGEF26 regulated SopB- and SopE-mediated S. Typhi (but not S. Typhimurium) infection of HeLa cells, the largest effect of ARHGEF26 was observed with S. Typhimurium in polarized MDCK cells through a SopB- and SopE2-independent mechanism. In both cell types, knockdown of the ARHGEF26-associated protein DLG1 resulted in a similar phenotype and serovar specificity. Importantly, we show that ARHGEF26 plays a critical role in S. Typhimurium pathogenesis by contributing to bacterial burden in the enteric fever murine model, as well as inflammation in the colitis infection model. In the enteric fever model, SopB and SopE2 are required for the effects of Arhgef26 deletion on bacterial burden, and the impact of sopB and sopE2 deletion in turn required ARHGEF26. In contrast, SopB and SopE2 were not required for the impacts of Arhgef26 deletion on colitis. A role for ARHGEF26 on inflammation was also seen in cells, as knockdown reduced IL-8 production in HeLa cells. Together, these data reveal pleiotropic roles for ARHGEF26 during infection and highlight that many of the interactions that occur during infection that are thought to be well understood likely have underappreciated complexity., Competing Interests: The authors have declared that no competing interests exist.

(1) Background: The transforming growth factor (TGF)-β plays a dual role in liver carcinogenesis. At early stages, it inhibits cell growth and induces apoptosis. However, TGF-β expression is high in advanced stages of hepatocellular carcinoma (HCC) and cells become resistant to TGF-β induced suppressor effects, responding to this cytokine undergoing epithelial-mesenchymal transition (EMT), which contributes to cell migration and invasion. Metabolic reprogramming has been established as a key hallmark of cancer. However, to consider metabolism as a therapeutic target in HCC, it is necessary to obtain a better understanding of how reprogramming occurs, which are the factors that regulate it, and how to identify the situation in a patient. Accordingly, in this work we aimed to analyze whether a process of full EMT induced by TGF-β in HCC cells induces metabolic reprogramming. (2) Methods: In vitro analysis in HCC cell lines, metabolomics and transcriptomics. (3) Results: Our findings indicate a differential metabolic switch in response to TGF-β when the HCC cells undergo a full EMT, which would favor lipolysis, increased transport and utilization of free fatty acids (FFA), decreased aerobic glycolysis and an increase in mitochondrial oxidative metabolism. (4) Conclusions: EMT induced by TGF-β in HCC cells reprograms lipid metabolism to facilitate the utilization of FFA and the entry of acetyl-CoA into the TCA cycle, to sustain the elevated requirements of energy linked to this process.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer. Due to its rising incidence and limited therapeutic options, HCC has become a leading cause of cancer-related death worldwide, accounting for 85% of all deaths due to primary liver cancers. Standard therapy for advanced-stage HCC is based on anti-angiogenic drugs such as sorafenib and, more recently, lenvatinib and regorafenib as a second line of treatment. The identification of novel therapeutic strategies is urgently required. Mitochondrial dynamics describes a group of processes that includes the movement of mitochondria along the cytoskeleton, the regulation of mitochondrial morphology and distribution, and connectivity mediated by tethering and fusion/fission events. In recent years, mitochondrial dynamic processes have emerged as key processes in the maintenance of liver mitochondrial homeostasis. In addition, some data are accumulating on the role played by mitochondrial dynamics during cancer development, and specifically on how such dynamics act directly on tumor cells or indirectly on cells responsible for tumor aggression and defense. Here, we review the data that suggest mitochondrial dynamics to be involved in the development of liver tumors.

Lipids are important molecules for human health. The quantity and quality of fats consumed in the diet have important effects on the modulation of both the natural biosynthesis and degradation of lipids. There is an important number of lipid-failed associated metabolic diseases and an increasing number of studies suggesting that certain types of lipids might be beneficial to the treatment of many metabolic diseases. The aim of the present work is to expose an overview of de novo biosynthesis, storage, and degradation of lipids in mammalian cells, as well as, to review the published data describing the beneficial effects of these processes and the potential of some dietary lipids to improve metabolic diseases.

Objectives: Dysregulation of CD59 may lead to increased complement-mediated end-organ injury in preeclampsia. We sought to determine if soluble CD59 concentrations are altered in preeclampsia with severe features., Study Design: Observational case-control study, which enrolled subjects prospectively from six centers in Colombia from 2015 to 2016. Cases had preeclampsia with severe features and controls were either healthy or had chronic hypertension, gestational hypertension, or preeclampsia without severe features. Trained coordinators collected clinical data, blood and urine. Analyses were by test of medians and Spearman's correlation., Main Outcome Measures: Soluble CD59 concentration in plasma and urine, using enzyme linked immunosorbent assays., Results: In total, 352 subjects were enrolled (104 cases; 248 controls). Compared to healthy women or those with other hypertensive disorders of pregnancy, women with preeclampsia with severe features had increased concentration of CD59 in plasma (P < 0.001) and decreased CD59 in urine (P = 0.01). In sub-group analyses, plasma CD59 concentrations were increased in preeclampsia with severe features compared to healthy controls (P < 0.001) or controls with either chronic hypertension (P = 0.002) or gestational hypertension (P = 0.02). Increased plasma CD59 concentrations correlated with decreased platelet count and increased lactate dehydrogenase, creatinine, aspartate transaminase, urine protein/creatinine ratio, systolic blood pressure and diastolic blood pressure (P < 0.01, all correlations)., Conclusion: In women with preeclampsia with severe features, soluble CD59 concentrations were increased in plasma and decreased in urine, and plasma levels correlated with increased blood pressure and end-organ injury. Soluble CD59 concentrations may help identify a subset of women with preeclampsia that have altered regulation of terminal complement proteins., (Copyright © 2020 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Background: The field of specific healthcare for transgender people has not been included in the official curriculum of professionals. This causes a lack of knowledge that can be presumed to become a barrier to healthcare. Currently, different methodologies are emerging to achieve meaningful learning for students and professionals. The objective of this study was to evaluate the increase in the level of knowledge of final-year nursing students, applying methodological strategies such as problem-based learning (PBL) and film-forum., Methods: 59 nursing students were randomly assigned to two intervention groups (G1 = 31 and G2 = 28), and another 57 were assigned to the control group (without specific classes or workshops on the subject of the study). The intervention consisted of a specific training course on transgender issues (TGSC&W, TransGender Specific Course and Workshop), where the type of meaningful learning strategy used depended on the group (G1 = film-forum and G2 = PBL). The study was carried out at the Faculty of Nursing Nuestra Señora de Candelaria of the Canary Islands Health Service. The randomization was done by blindly choosing a computer-generated code., Results: The main outcome was based on 116 participants, comparing their level of knowledge before and after the workshop. The comparison by pairs shows that there were statistically significant differences ( p = 0.000) between those undergoing the methodological interventions and the control group. Statistical significance between film-forum and PBL was not obtained ( p = 1.000): Both methodologies increased the level of knowledge, but there was no significant difference between them. The means for satisfaction with the learning methodology used did not show statistically significant differences., Conclusion: The workshop carried out was highly effective and significant in terms of increasing knowledge. No significant differences were observed in the level of knowledge, or in the degree of satisfaction, between the two methodologies used (PBL and film-forum).

Petunia × hybrida cv 'Mitchell Diploid' floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis ultimately produces floral volatiles derived sequentially from phenylalanine, cinnamic acid, and p-coumaric acid. In an attempt to better understand biochemical steps after p-coumaric acid production, we cloned and characterized three petunia transcripts with high similarity to p-coumarate 3-hydroxylase (C3H), hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT), and caffeoyl shikimate esterase (CSE). Transcript accumulation of PhC3H and PhHCT was highest in flower limb tissue during open flower stages. PhCSE transcript accumulation was also highest in flower limb tissue, but it was detected earlier at initial flower opening with a bell-shaped distribution pattern. Down regulation of endogenous PhC3H transcript resulted in altered transcript accumulation of many other FVBP network transcripts, a reduction in floral volatiles, and the emission of a novel floral volatile. Down regulation of PhHCT transcript did not have as large of an effect on floral volatiles as was observed for PhC3H down regulation, but eugenol and isoeugenol emissions were significantly reduced on the downstream floral volatiles. Together these results indicate that PhC3H is involved in FVBP biosynthesis and the reduction of PhC3H transcript influences FVBP metabolism at the network level. Additional research is required to illustrate PhHCT and PhCSE functions of petunia.

TP53INP2 positively regulates autophagy by binding to Atg8 proteins. Here, we uncover a novel role of TP53INP2 in death-receptor signaling. TP53INP2 sensitizes cells to apoptosis induced by death receptor ligands. In keeping with this, TP53INP2 deficiency in cultured cells or mouse livers protects against death receptor-induced apoptosis. TP53INP2 binds caspase-8 and the ubiquitin ligase TRAF6, thereby promoting the ubiquitination and activation of caspase-8 by TRAF6. We have defined a TRAF6-interacting motif (TIM) and a ubiquitin-interacting motif in TP53INP2, enabling it to function as a scaffold bridging already ubiquitinated caspase-8 to TRAF6 for further polyubiquitination of caspase-8. Mutations of key TIM residues in TP53INP2 abrogate its interaction with TRAF6 and caspase-8, and subsequently reduce levels of death receptor-induced apoptosis. A screen of cancer cell lines showed that those with higher protein levels of TP53INP2 are more prone to TRAIL-induced apoptosis, making TP53INP2 a potential predictive marker of cancer cell responsiveness to TRAIL treatment. These findings uncover a novel mechanism for the regulation of caspase-8 ubiquitination and reveal TP53INP2 as an important regulator of the death receptor pathway., (© 2019 The Authors.)

Non-alcoholic fatty liver is the most common liver disease worldwide. Here, we show that the mitochondrial protein mitofusin 2 (Mfn2) protects against liver disease. Reduced Mfn2 expression was detected in liver biopsies from patients with non-alcoholic steatohepatitis (NASH). Moreover, reduced Mfn2 levels were detected in mouse models of steatosis or NASH, and its re-expression in a NASH mouse model ameliorated the disease. Liver-specific ablation of Mfn2 in mice provoked inflammation, triglyceride accumulation, fibrosis, and liver cancer. We demonstrate that Mfn2 binds phosphatidylserine (PS) and can specifically extract PS into membrane domains, favoring PS transfer to mitochondria and mitochondrial phosphatidylethanolamine (PE) synthesis. Consequently, hepatic Mfn2 deficiency reduces PS transfer and phospholipid synthesis, leading to endoplasmic reticulum (ER) stress and the development of a NASH-like phenotype and liver cancer. Ablation of Mfn2 in liver reveals that disruption of ER-mitochondrial PS transfer is a new mechanism involved in the development of liver disease., (Copyright © 2019. Published by Elsevier Inc.)

Succinate is a signaling metabolite sensed extracellularly by succinate receptor 1 (SUNCR1). The accumulation of succinate in macrophages is known to activate a pro-inflammatory program; however, the contribution of SUCNR1 to macrophage phenotype and function has remained unclear. Here we found that activation of SUCNR1 had a critical role in the anti-inflammatory responses in macrophages. Myeloid-specific deficiency in SUCNR1 promoted a local pro-inflammatory phenotype, disrupted glucose homeostasis in mice fed a normal chow diet, exacerbated the metabolic consequences of diet-induced obesity and impaired adipose-tissue browning in response to cold exposure. Activation of SUCNR1 promoted an anti-inflammatory phenotype in macrophages and boosted the response of these cells to type 2 cytokines, including interleukin-4. Succinate decreased the expression of inflammatory markers in adipose tissue from lean human subjects but not that from obese subjects, who had lower expression of SUCNR1 in adipose-tissue-resident macrophages. Our findings highlight the importance of succinate-SUCNR1 signaling in determining macrophage polarization and assign a role to succinate in limiting inflammation.

Background : Transgender people have a gender identity different from the one allocated to them at birth. In many countries, transsexualism and transgenderism are considered mental illnesses under the diagnosis of gender dysphoria. This pathologization impacts on human rights. Main content : The United Nations (UN) has denounced violations against trans-people, including attacks, forced medical treatments, lack of legal gender recognition, and discrimination in the areas of education, employment, access to healthcare, and justice. The UN has linked these violations directly with discriminatory diagnostic classifications that pathologize gender diversity. Trans-people have been pathologized by psycho-medical classification and laws all around the world, with a different impact depending on countries. This paper argues that pathologization infringes infringes upon a wide range of human rights such as; civil, economic, social cultural and also the access to medical care. Conclusion s : The current situation for trans-people with respect to legal healthcare matters, depends on the country. Human rights are universal, not a question for cultural interpretation. They are the minimum that every human being must have assured only by the fact of being human. Countries must protect these rights by regulating trans-pathologization with special attention dedicated to intersex people and their specific needs., Competing Interests: The authors declare that they have no competing interests

Trans men are people who, based on their genitals, were assigned the status of female at birth. However, their identity and their way of living gender do not correspond to the socially established norms. In this paper, we discuss the different perspectives in relation to transgender people and their desire for parenthood. This review, and the basis of this paper, is inspired by the case of a trans man who desired gestation with his own genetic material. He began the cycle of assisted reproduction when he was a legally recognized woman, but that attempt ended with a miscarriage. From that assisted reproduction cycle, four embryos remained frozen. After the failed experience of gestation, the person completed his transition. Now legally a man, he attempted to gestate using his reproductive organs. This literature review aimed to identify relevant studies describing the relationship between transgender person and biological parenthood. This study comprehensively addresses important aspects one should know when considering a transgender pregnancy. These factors include biological, psychological, social, and legal issues. After reviewing the state-of-the-art information on trans parenthood, the main conclusion is that 'the desire to have a child is not a male or female desire but a human desire'., Competing Interests: The authors declare no conflict of interest.

Several fungal species, particularly some included in the Mucoromycotina, have been used to develop fermentation processes for the production of β-carotene. Oxygenated derivatives of β-carotene (xanthophylls) are desirable value-added products, and the preference by the market of carotenoids from biological sources has increased the research in different carotenoid-producing organisms. We currently use Mucor circinelloides f. lusitanicus as a model organism to develop strains with an increased content of new and more valuable carotenoids. The main carotenoid accumulated by M. circinelloides is β-carotene, although it has some hydroxylase activity and produces low amounts of zeaxanthin. On the other hand, in astaxanthin-producing organisms two enzymatic activities are required for the production of astaxanthin from β-carotene: a hydroxylase and a ketolase. In this chapter, we delineate part of our efforts to construct genetically modified strains that could advance in the improvement of carotenoid accumulation by this fungus and the diversification of its carotenoid content. Accordingly, we describe detailed and empirically tested protocols for the construction of functional expression vectors and gene fusions.

Acetaminophen (APAP) is the active component of many medications used to treat pain and fever worldwide. Its overuse provokes liver injury and it is the second most common cause of liver failure. Mitochondrial dysfunction contributes to APAP-induced liver injury but the mechanism by which APAP causes hepatocyte toxicity is not completely understood. Therefore, we lack efficient therapeutic strategies to treat this pathology. Here we show that APAP interferes with the formation of mitochondrial respiratory supercomplexes via the mitochondrial negative regulator MCJ, and leads to decreased production of ATP and increased generation of ROS. In vivo treatment with an inhibitor of MCJ expression protects liver from acetaminophen-induced liver injury at a time when N-acetylcysteine, the standard therapy, has no efficacy. We also show elevated levels of MCJ in the liver of patients with acetaminophen overdose. We suggest that MCJ may represent a therapeutic target to prevent and rescue liver injury caused by acetaminophen.

The molecular mechanisms responsible for the pathophysiological traits of type 2 diabetes are incompletely understood. Here we have performed transcriptomic analysis in skeletal muscle, and plasma metabolomics from subjects with classical and early-onset forms of type 2 diabetes (T2D). Focused studies were also performed in tissues from ob/ob and db/db mice. We document that T2D, both early and late onset, are characterized by reduced muscle expression of genes involved in branched-chain amino acids (BCAA) metabolism. Weighted Co-expression Networks Analysis provided support to idea that the BCAA genes are relevant in the pathophysiology of type 2 diabetes, and that mitochondrial BCAA management is impaired in skeletal muscle from T2D patients. In diabetic mice model we detected alterations in skeletal muscle proteins involved in BCAA metabolism but not in obese mice. Metabolomic analysis revealed increased levels of branched-chain keto acids (BCKA), and BCAA in plasma of T2D patients, which may result from the disruption of muscle BCAA management. Our data support the view that inhibition of genes involved in BCAA handling in skeletal muscle takes place as part of the pathophysiology of type 2 diabetes, and this occurs both in early-onset and in classical type 2 diabetes.

Transforming Growth Factor beta (TGF-β) induces tumor cell migration and invasion. However, its role in inducing metabolic reprogramming is poorly understood. Here we analyzed the metabolic profile of hepatocellular carcinoma (HCC) cells that show differences in TGF-β expression. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR), metabolomics and transcriptomics were performed. Results indicated that the switch from an epithelial to a mesenchymal/migratory phenotype in HCC cells is characterized by reduced mitochondrial respiration, without significant differences in glycolytic activity. Concomitantly, enhanced glutamine anaplerosis and biosynthetic use of TCA metabolites were proved through analysis of metabolite levels, as well as metabolic fluxes from U-13C6-Glucose and U-13C5-Glutamine. This correlated with increase in glutaminase 1 (GLS1) expression, whose inhibition reduced cell migration. Experiments where TGF-β function was activated with extracellular TGF-β1 or inhibited through TGF-β receptor I silencing showed that TGF-β induces a switch from oxidative metabolism, coincident with a decrease in OCR and the upregulation of glutamine transporter Solute Carrier Family 7 Member 5 (SLC7A5) and GLS1. TGF-β also regulated the expression of key genes involved in the flux of glycolytic intermediates and fatty acid metabolism. Together, these results indicate that autocrine activation of the TGF-β pathway regulates oxidative metabolism in HCC cells.

Risk, severity, and outcome of infection depend on the interplay of pathogen virulence and host susceptibility. Systematic identification of genetic susceptibility to infection is being undertaken through genome-wide association studies, but how to expeditiously move from genetic differences to functional mechanisms is unclear. Here, we use genetic association of molecular, cellular, and human disease traits and experimental validation to demonstrate that genetic variation affects expression of VAC14, a phosphoinositide-regulating protein, to influence susceptibility to Salmonella enterica serovar Typhi ( S Typhi) infection. Decreased VAC14 expression increased plasma membrane cholesterol, facilitating Salmonella docking and invasion. This increased susceptibility at the cellular level manifests as increased susceptibility to typhoid fever in a Vietnamese population. Furthermore, treating zebrafish with a cholesterol-lowering agent, ezetimibe, reduced susceptibility to S Typhi. Thus, coupling multiple genetic association studies with mechanistic dissection revealed how VAC14 regulates Salmonella invasion and typhoid fever susceptibility and may open doors to new prophylactic/therapeutic approaches., Competing Interests: The authors declare no conflict of interest.

Poly(3-hydroxybutyrate) (PHB) is an interesting biopolymer for replacing petroleum-based plastics, its biological production is performed in natural and engineered microorganisms. Current metabolic engineering approaches rely on high-throughput strain construction and screening. Analytical procedures have to be compatible with the small scale and speed of these approaches. Here, we present a method based on isotope dilution mass spectrometry (IDMS) and propanolysis extraction of poly(3-hydroxybutyrate) from an Escherichia coli strain engineered for PHB production. As internal standard (IS), we applied an uniformly labeled 13 C-cell suspension, of an E. coli PHB producing strain, grown on U- 13 C-glucose as C-source. This internal 13 C-PHB standard enables to quantify low concentrations of PHB (LOD of 0.01 µg/g CDW ) from several micrograms of biomass. With this method, a technical reproducibility of about 1.8% relative standard deviation is achieved. Furthermore, the internal standard is robust towards different sample backgrounds and dilutions. The early addition of the internal standard also enables higher reproducibility and increases sensitivity and throughput by simplified sample preparation steps., Competing Interests: The authors declare no conflict of interest.

Mitochondrial function can be influenced by mitochondrial shape and connectivity with other cellular organelles through fusion and fission processes. Disturbances in mitochondrial architecture and mitochondrial fusion-related genes are observed in situations of type 2 diabetes and obesity, leading to a highly fissioned mitochondrial network. To directly test the effect of reduced mitochondrial fusion on hepatic metabolism, we generated mice with a liver-specific deletion of the Mfn1 gene (Mfn1LKO) and monitored their energy homeostasis, mitochondrial function, and susceptibility to diet-induced insulin resistance. Livers from Mfn1LKO mice displayed a highly fragmented mitochondrial network. This was coupled to an enhanced mitochondrial respiration capacity and a preference for the use of lipids as the main energy source. Although Mfn1LKO mice are similar to control mice fed a low-fat diet, they are protected against insulin resistance induced by a high-fat diet. Importantly, Mfn1 deficiency increased complex I abundance and sensitized animals to the hypoglycemic effect of metformin. Our results suggest that targeting Mfn1 could provide novel avenues to ameliorate glucose homeostasis in obese patients and improve the effectiveness of metformin., (© 2016 by the American Diabetes Association.)