👤 Sébastien Bonnet

A study of the lipidome and proteome was performed on milk fat globule membranes (MFGM) originating from milk samples from high (HL) and low (LL) lipolysis groups of cows. Combined univariate and multivariate statistical analyses proposed a set of variables highly associated to contrasted samples with regard to milk lipolysis. Milk from HL group were related to 4 phosphatidylinositols, 8 phosphatidylcholines, 1 sphingomyelin and 27 proteins, among them the phosphatidylcholine/phosphatidylethanolamine ratio and ORM1 may contribute to the membrane remodeling of the MFGM. The abundance of CP, CHI3L1, NECTIN2, A2M were strongly positively correlated with HL. Conversely, 3 phosphatidylinositols, 1 phosphatidylcholine and 2 phosphatidylethanolamines were assigned to the LL group. The HL group in cows is associated with a specific MFGM phospholipids and proteins profile, suggesting an impact on membrane fluidity and lipid rafts composition intervening in LPL anchoring and activation, as well as on pro-inflammatory lipids and proteins. Show less

Limbal stem cell deficiency (LSCD) is a sight-threatening condition caused by the loss and/or dysfunction of limbal stem cells (LSCs), which are essential for corneal epithelial regeneration and homeostasis and are critical for maintaining corneal transparency. We have previously shown that specific inactivation of the endothelial mineralocorticoid receptor (MR) inhibits corneal neovascularization (CN) and that MR antagonists (MRA) improve corneal epithelial wound healing. This study investigated the therapeutic potential of MRA in LSCD and their mechanisms of action. Using a rat model of LSCD, systemic administration of spironolactone (SPL) or a more specific MRA, eplerenone, similarly reduced CN and corneal oedema, demonstrating MR-specific effects. SPL further limited inflammation, enhanced the corneal epithelial barrier, reduced corneal conjunctivalization and promoted nerve regeneration, highlighting its potential to improve corneal integrity. Transcriptomic analysis revealed that SPL upregulated genes associated with LSC maintenance (Tp63, Wnt6), corneal epithelial differentiation (Vdr, Fermt1, Ehf) and nerve regeneration (Sprr1a, Anxa1), while downregulating genes associated with angiogenesis (Kdr, Scube2), inflammation (Ccl2, Cxcl1) and fibrosis (Fbln1, Snai1). Conversely, transgenic rats overexpressing human NR3C2 encoding MR showed corneal epithelial irregularities and dysregulation of genes related to extracellular matrix remodeling and fibrosis (Matn3, Serpine2, Fmod, Bgn, Ddr2), angiogenesis (Nrp2, Scube1) and limbal cell function (Ifitm3). These findings demonstrate that activation of the MR pathway disrupts limbal and corneal homeostasis and that SPL effectively modulates critical mechanisms in LSCD, offering promising therapeutic potential to reduce CN and improve corneal epithelial barrier integrity. Show less

Transcription factor EB (TFEB) is a key transcription factor that orchestrates the cellular response to stress. Dysregulation of TFEB is associated with a range of human diseases, and understanding the regulatory mechanisms of TFEB is crucial for identifying potential drug targets. In this study, we used Show less

Pulmonary hypertension (PH)-induced right ventricular (RV) failure (PH-RVF) is a significant prognostic determinant of mortality and is characterized by RV hypertrophy, endothelial-to-mesenchymal transition (EndMT), fibroblast-to-myofibroblast transition (FMT), fibrosis, and extracellular matrix (ECM)-remodeling. Despite the importance of RV function in PH, the mechanistic details of PH-RVF, especially the regulatory control of RV EndMT, FMT, and fibrosis, remain unclear. The action of transcription factor Snai1 is shown to be mediated through LOXL2 recruitment, and their co-translocation to the nucleus, during EndMT progression. We hypothesize that RV EndMT and fibrosis in PH-RVF are governed by the TGFβ1-Snai1-LOXL2 axis. Furthermore, targeting Snai1 could serve as a novel therapeutic strategy for PH-RVF. Adult male Sprague Dawley rats (250-300g) received either a single subcutaneous injection of Monocrotaline (MCT, 60mg/kg, n=9; followed for 30-days) or Sugen (SU5416 20mg/kg, n=9; 10% O PH-RVF had increased RVSP and Fulton index and decreased RV fractional area change (RVFAC %). RV RNASeq demonstrated EndMT as the common top-upregulated pathway between rat (MCT, SuHx, and PAB) and human PH-RVF. Immunofluorescence using EndMT- and FMT-specific markers demonstrated increased EndMT and FMT in RV of MCT and SuHx rats and PH-RVF patients. Further, RV expression of TGFβ1, Snai1, and LOXL2 was increased in MCT and SuHx. Nuclear co-localization and increased immunoreactivity, transcript, and protein levels of Snai1 and LOXL2 were observed in MCT and SuHx rats and human RVs. MCT rats treated with Snai1-siRNA demonstrated decreased Snai1 expression, RVSP, Fulton index, and increased RVFAC. Snai1-KD resulted in decreased RV-EndMT, FMT, and fibrosis RV-specific targeting of Snai1 rescues PH-RVF by inhibiting EndMT and Fibrosis Show less

To examine the changes in diagnostic practices and clinical management of patients with 5α-reductase type 2 (SRD5A2) or 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) deficiency since molecular diagnoses became available. Clinical, laboratory, and therapeutic data were retrieved from the medical records of 52 patients with a molecular diagnosis of SRD5A2 (n = 31) or HSD17B3 (n = 21) deficiency. Temporal trends regarding age at assessment and initial sex assignment over 1994-2020 were qualitatively analyzed. Age at molecular diagnosis was compared between two subgroups of patients according to their year of birth. Fifty-eight percent (n = 30) patients were diagnosed during the perinatal period, 33% (n = 17) during infancy, and 9% (n = 5) during adolescence or adulthood. Over the studied period, the patients' age at initial assessment and diagnosis frankly decreased. The median (range) age at diagnostic confirmation was 10.5 (0-53.2) years for patients born before 2007 and 0.4 (0-9.3) years for those born in 2007 or later (P = 0.029). Genetic testing identified 27 different variants for the SRD5A2 gene (30% novel, n = 8) and 18 for the HSD17B3 gene (44% novel, n = 8). Before 2002, most patients were initially assigned as females (95%, n = 19), but this proportion dropped for those born later (44%, n = 14; P < 0.001). The influence of initial genital appearance on these decisions seemingly decreased in the most recent years. Therapeutic interventions differed according to the sex of rearing. Ten percent (n = 2) patients requested female-to-male reassignment during adulthood. This study showed, over the past two decades, a clear trend toward earlier diagnosis and assignment of affected newborns as males. Show less

This study aimed to investigate the genetic cause of food-dependent Cushing syndrome (FDCS) observed in patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) and adrenal ectopic expression of the glucose-dependent insulinotropic polypeptide receptor. Germline ARMC5 alterations have been reported in about 25% of PBMAH index cases but are absent in patients with FDCS. A multiomics analysis of PBMAH tissues from 36 patients treated by adrenalectomy was performed (RNA sequencing, single-nucleotide variant array, methylome, miRNome, exome sequencing). The integrative analysis revealed 3 molecular groups with different clinical features, namely G1, comprising 16 patients with ARMC5 inactivating variants; G2, comprising 6 patients with FDCS with glucose-dependent insulinotropic polypeptide receptor ectopic expression; and G3, comprising 14 patients with a less severe phenotype. Exome sequencing revealed germline truncating variants of KDM1A in 5 G2 patients, constantly associated with a somatic loss of the KDM1A wild-type allele on 1p, leading to a loss of KDM1A expression both at messenger RNA and protein levels (P = 1.2 × 10 KDM1A inactivation explains about 90% of FDCS PBMAH. Genetic screening for ARMC5 and KDM1A can now be offered for most PBMAH operated patients and their families, opening the way to earlier diagnosis and improved management. Show less

Essential fatty acids (EFA) and conjugated linoleic acids (CLA) are unsaturated fatty acids with immune-modulatory effects, yet their synergistic effect is poorly understood in dairy cows. This study aimed at identifying differentially abundant proteins (DAP) and their associated pathways in dairy cows supplied with a combination of EFA and CLA during the transition from antepartum (AP) to early postpartum (PP). Sixteen Holstein cows were abomasally infused with coconut oil as a control (CTRL) or a mixture of EFA (linseed + safflower oil) and CLA (Lutalin, BASF) (EFA + CLA) from - 63 to + 63 days relative to parturition. Label-free quantitative proteomics was performed on plasma samples collected at days - 21, + 1, + 28, and + 63. During the transition time, DAP, consisting of a cluster of apolipoproteins (APO), including APOE, APOH, and APOB, along with a cluster of immune-related proteins, were related to complement and coagulation cascades, inflammatory response, and cholesterol metabolism. In response to EFA + CLA, specific APO comprising APOC3, APOA1, APOA4, and APOC4 were increased in a time-dependent manner; they were linked to triglyceride-enriched lipoprotein metabolisms and immune function. Altogether, these results provide new insights into metabolic and immune adaptation and crosstalk between them in transition dairy cows divergent in EFA + CLA status. Show less

Bardet-Biedl syndrome (BBS) is a multisystemic disorder characterized by rod-cone dystrophy, truncal obesity, postaxial polydactyly, cognitive impairment, male hypogonadotropic hypogonadism, complex female genitourinary malformations, and renal abnormalities. There is a large clinical and also genetic heterogeneity in BBS. Here, we report a patient with polydactyly, hyperechogenic kidneys increased in size with normal corticomedullary differentiation, anal imperforation, and malformation of genitals with presence of a genital tubercle with ventral urethral meatus associated with two unfused lateral genital swelling and absent urethral folds, in the context of 46, XY karyotype. Karyotype and solo exome sequencing were performed to look for a genetic etiology for the features described in our patient. We identified a homozygous in-frame deletion of exons 4 to 6 in the BBS4 gene (NM-033028 (BBS4-i001): c.[(157-?)₍₄₀₅ +?)del] p.(Ala53-Trp135del), which is classified as pathogenic variant. This analysis allowed the molecular diagnosis of BBS type 4 in this patient. Complex genital malformations are only reported in female BBS6 patients yet, and genital abnormalities and anal imperforation are not reported in male BBS4 patients to date. We discuss the possible hypotheses for this phenotype, including the phenotypic overlap between ciliopathies. Show less

Optimal molecular diagnosis of primary dyslipidemia is challenging to confirm the diagnosis, test and identify at risk relatives. The aim of this study was to test the application of a single targeted next-generation sequencing (NGS) panel for hypercholesterolemia, hypocholesterolemia, and hypertriglyceridemia molecular diagnosis. NGS workflow based on a custom AmpliSeq panel was designed for sequencing the most prevalent dyslipidemia-causing genes (ANGPTL3, APOA5, APOC2, APOB, GPIHBP1, LDLR, LMF1, LPL, PCSK9) on the Ion PGM Sequencer. One hundred and forty patients without molecular diagnosis were studied. In silico analyses were performed using the NextGENe software and homemade tools for detection of copy number variations (CNV). All mutations were confirmed using appropriate tools. Eighty seven variations and 4 CNV were identified, allowing a molecular diagnosis for 40/116 hypercholesterolemic patients, 5/13 hypocholesterolemic patients, and 2/11, hypertriglyceridemic patients respectively. This workflow allowed the detection of CNV contrary to our previous strategy. Some variations were found in previously unexplored regions providing an added value for genotype-phenotype correlation and familial screening. In conclusion, this new NGS process is an effective mutation detection method and allows better understanding of phenotype. Consequently this assay meets the medical need for individualized diagnosis of dyslipidemia. Show less

We evaluated whether the association of insulin sensitivity with HDL cholesterol (HDL) and triglycerides is influenced by major plasma apolipoproteins, as suggested by recent experimental evidence. This study included a cross-sectional analysis of the RISC Study, a multicenter European clinical investigation in 1,017 healthy volunteers balanced in sex (women 54%) and age strata (range 30-60 years). Insulin sensitivity (M/I in µmol ⋅ min(-1) ⋅ kgFFM(-1) ⋅ nM(-1)) was measured by the clamp technique and apolipoproteins (ApoB, -C3, -A1, and -E) by Multiplex Technology. The center-, sex-, and age-adjusted standardized regression coefficients (STDβ) with M/I were similar for HDL and triglycerides (+19.9 ± 1.9 vs. -20.0 ± 2.0, P < 0.0001). Further adjustment for triglycerides (or HDL), BMI, and adiponectin (or nonesterified fatty acid) attenuated the strength of the association of M/I with both HDL (STDβ +6.4 ± 2.3, P < 0.01) and triglycerides (-9.5 ± 2.1, P < 0.001). Neither ApoA1 nor ApoE and ApoB showed any association with M/I independent from plasma HDL cholesterol and triglycerides. ApoC3, in contrast, in both men and women, was positively associated with M/I independently of plasma lipids. A relative enrichment of plasma lipids with ApoC3 is associated with lower body fat percentage and lower plasma alanine amino transferase. Our results suggest that HDL cholesterol modulates insulin sensitivity through a mechanism that is partially mediated by BMI and adiponectin but not by ApoA1. Similarly, the influence of triglycerides on insulin sensitivity is in part mediated by BMI and is unrelated to ApoE or ApoB, but it is significantly modulated by ApoC3, which appears to protect from the negative effect of plasma lipids. Show less

The reliability of reverse transcription quantitative real-time PCR (RT-qPCR) depends on normalising the mRNA abundance using carefully selected, stable reference genes. Our aim was to propose sets of reference genes for normalisation in bovine or caprine adipose tissue (AT), mammary gland, liver and muscle. All of these tissues contribute to nutrient partitioning and metabolism and, thus, to the profitability of ruminant productions (i.e. carcasses, meat and milk). In this study, eight commonly used reference genes that belong to different functional classes (CLN3, EIF3K, MRPL39, PPIA, RPLP0, TBP, TOP2B and UXT) were analysed using the geNorm procedure to determine the most stable reference genes in bovine and/or caprine tissues. Abundances and rankings of reference genes varied between tissues, species and the combination of tissues and/or species. Therefore, we proposed 29 sets of reference genes that differed depending on the tissue and/or species. As examples of the 29 sets, EIF3K, TOP2B and UXT were proposed as the most stable reference genes in bovine AT; UXT, EIF3K and RPLP0 were the most stable reference genes in bovine and caprine AT. The optimal number of reference genes for data normalisation was 3 for 27 of the proposed 29 sets. In two of the 29 sets, four to five reference genes were necessary for data normalisation when the number of studied tissues was increased. For example, UXT, EIF3K, TBP, TOP2B and CLN3 were required for data normalisation in bovine mammary gland, AT, muscle and liver. We have evaluated some of our proposed sets of reference genes for the normalisation of CD36 gene expression. Normalisation using the three most stable reference genes has revealed downregulation of CD36 gene expression in bovine mammary gland by a concentrate-based diet that is supplemented with sunflower oil and upregulation of CD36 gene expression in caprine liver by including a rapidly degradable starch in the diet. The dietary regulation of the gene expression of CD36 has been erased by normalisation with the least stable reference genes, which may result in misinterpretation of CD36 gene regulation. To conclude, our results provide valuable reference gene sets for other studies that aim to measure tissue and/or species-specific mRNA abundance in ruminants. Show less

The incidence and the magnitude of lipodystrophy and dyslipidemia in HIV-treated people reported in previous studies are very variable. Several predisposing factors have been identified, but there are few data on genetic factors. To search for a correlation between APOC3 polymorphisms and lipid disorders and lipodystrophy in HIV patients under d4T and protease inhibitors (PI)-containing HAART, we designed a monocenter, cross-sectional study in a University Hospital in Southern France during the period 2001-2004. Forty patients under HAART were included, with d4T for > or = 2 years and PI for > or = 1 year. We determined body mass composition by DXA, lipoprotein markers, and the -455/-482 apo C3 genotypes. Carriers of APOC3 variant alleles (-455 1/-482 1) displayed higher levels of triglycerides (3.72 vs. 2.57 mmol/liter), apo C3 (45.3 vs. 34.5 mg/liter), and apo E (130.2 vs. 66.5 mg/liter) and a lower fat mass (13.9 vs. 19.7%) than patients with nonvariant alleles (-455 0/-482 0). APOC3 polymorphisms might be associated with both dyslipidemia and lipoatrophy in HAART-treated patients. Show less

Active anti-HIV therapy can induce hypertriglyceridemia, low high-density lipoprotein (HDL) and insulin resistance, eventually accompanied by clinical lipodystrophy, associated loss of subcutaneous adipose tissue and an increase in abdominal adiposity. The frequency of these metabolic disorders is approximately 50% and host genetic factors might confer particular susceptibility. Variants of apolipoproteins (apo) A5 and C3, interacting with APOE genotypes, have been associated with the severity of antiretroviral therapy-induced dyslipidemia and with occurrence of lipodystrophy, and for APOC3, with objective criteria of fat redistribution. Genetic polymorphisms of the nuclear transcription-factor sterol response element-binding proteins (SREBP1c) and of tumor necrosis factor-alpha (TNFalpha) have yielded contrasting results. Other candidate genes will be explored to define a pharmacogenomic strategy to identify patients at high risk of metabolic disorders upon antiretroviral therapy. Show less