Loss of testosterone induces postprandial insulin resistance and increases the expression of the hepatic antioxidant flavin-containing monooxygenases in mice exposed to intermittent hypoxia.

AIM: We tested the hypothesis that low testosterone alters the effects of intermittent hypoxia (IH) on glucose homeostasis, hepatic oxidative stress, and transcriptomic profile in male mice. METHODS: We used sham-operated or orchiectomized (ORX) mice exposed to normoxia (Nx) or IH for 2 weeks. We performed fasting insulin and glucose tolerance tests and assessed fasting and postprandial insulin resistance with the HOMA-IR. The activity of hepatic prooxidant (NADPH oxidase-NOX), antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase-SOD, Cat, GPx), lipid peroxidation (MDA concentration), and the total concentration of glutathione (GSH) were measured under postprandial conditions. mRNA sequencing and pathway enrichment analyses were used to identify hepatic genes underlying the interactions between IH and testosterone. RESULTS: In Sham mice, IH improves fasting insulin sensitivity and glucose tolerance, while there are no effects of IH in ORX mice. In ORX mice, IH induces postprandial hyperinsulinemia, insulin resistance, and a prooxidant profile of enzyme activity (low SOD activity) without altering hepatic MDA and GSH content. ORX and IH altered the expression of genes involved in oxidoreductase activities, cytochromes-dependent pathways, and glutathione metabolism. Among the genes upregulated in ORX-IH mice, the flavin-containing monooxygenases (FMO) are particularly relevant since these are potent hepatic antioxidants that could help prevent overt oxidative stress in ORX-IH mice. CONCLUSION: Low levels of testosterone in male mice exposed to IH induce post-prandial hyperinsulinemia and insulin resistance and determine the mechanisms by which the liver handles IH-induced oxidative stress.

Hedgehog signaling regulates Wolffian duct development through the primary cilium†.

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). Although mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; IFT88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix, mesenchymal-epithelial transition, canonical Hh and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

HCK and ABAA: A Newly Designed Pipeline to Improve Fungi Metabarcoding Analysis.

INTRODUCTION: The fungi ITS sequence length dissimilarity, non-specific amplicons, including chimaera formed during Polymerase Chain Reaction (PCR), added to sequencing errors, create bias during similarity clustering and abundance estimation in the downstream analysis. To overcome these challenges, we present a novel approach, Hierarchical Clustering with Kraken (HCK), to classify ITS1 amplicons and Abundance-Base Alternative Approach (ABAA) pipeline to detect and filter non-specific amplicons in fungi metabarcoding sequencing datasets. MATERIALS AND METHODS: We compared the performances of both pipelines against QIIME, KRAKEN, and DADA2 using publicly available fungi ITS mock community datasets and using BLASTn as a reference. We calculated the Precision, Recall, F-score using the True-Positive, False-positive, and False-negative estimation. Alpha diversity (Chao1 and Shannon metrics) was also used to evaluate the diversity estimation of our method. RESULTS: The analysis shows that ABAA reduced the number of false-positive with all metabarcoding methods tested, and HCK increases precision and recall. HCK, coupled with ABAA, improves the F-score and bring alpha diversity metric value close to that of the BLASTn alpha diversity values when compared to QIIME, KRAKEN, and DADA2. CONCLUSION: The developed HCK-ABAA approach allows better identification of the fungi community structures while avoiding use of a reference database for non-specific amplicons filtration. It results in a more robust and stable methodology over time. The software can be downloaded on the following link: https://bitbucket.org/GottySG36/hck/src/master/.

Glutamine supplementation improves the efficacy of miltefosine treatment for visceral leishmaniasis.

BACKGROUND: The disturbance of host metabolic pathways by Leishmania parasites has crucial consequences for the activation status of immune cells and the outcome of infection. Glutamine has been described as an immunomodulatory amino acid, yet its role during Leishmania infection is still unknown. METHODS: We performed transcriptomics in uninfected and L. donovani-infected macrophages 6 hours post-infection. Glutamine quantification by HPLC was assessed in the supernatant of macrophages throughout the infection course. For experimental L. donovani infections, mice were infected with 1.0 x 108 stationary L. donovani promastigotes. Glutaminase (GLS) chemical inhibition was performed using BPTES and glutamine was administered throughout infection. For combined therapy experiment, a daily administration of miltefosine and glutamine was performed by oral gavage. Parasite burden was determined using a Taqman-based assay. Immune cell phenotyping and cytotoxicity were performed in splenic cells using flow cytometry. FINDINGS: We show that glutamine is essential for the control of L. donovani infection. Transcriptomic analysis of L. donovani-infected macrophages demonstrated an upregulation of genes involved in glutamine metabolism. Pharmacological inhibition of glutaminolysis significantly increased the susceptibility to infection, accompanied by an increased recruitment of anti-inflammatory myeloid cells and impaired T cell responses. Remarkably, the supplementation of glutamine to mice infected with L. donovani during miltefosine treatment potentiates parasite clearance through the development of a more effective anti-Leishmania adaptive immune response. CONCLUSIONS: Our data indicates that dietary glutamine supplementation may act as a promising adjuvant for the treatment of visceral leishmaniasis.

The Absence of HIF-1α Increases Susceptibility to Leishmania donovani Infection via Activation of BNIP3/mTOR/SREBP-1c Axis.

Hypoxia-inducible factor-1 alpha (HIF-1α) is considered a global regulator of cellular metabolism and innate immune cell functions. Intracellular pathogens such as Leishmania have been reported to manipulate host cell metabolism. Herein, we demonstrate that myeloid cells from myeloid-restricted HIF-1α-deficient mice and individuals with loss-of-function HIF1A gene polymorphisms are more susceptible to L. donovani infection through increased lipogenesis. Absence of HIF-1α leads to a defect in BNIP3 expression, resulting in the activation of mTOR and nuclear translocation of SREBP-1c. We observed the induction of lipogenic gene transcripts, such as FASN, and lipid accumulation in infected HIF-1α-/- macrophages. L. donovani-infected HIF-1α-deficient mice develop hypertriglyceridemia and lipid accumulation in splenic and hepatic myeloid cells. Most importantly, our data demonstrate that manipulating FASN or SREBP-1c using pharmacological inhibitors significantly reduced parasite burden. As such, genetic deficiency of HIF-1α is associated with increased lipid accumulation, which results in impaired host-protective anti-leishmanial functions of myeloid cells.

Complexity of the microRNA transcriptome of cow milk and milk-derived extracellular vesicles isolated via differential ultracentrifugation.

MicroRNAs (miRNAs) are small gene-regulatory noncoding RNA that are highly enriched in cow milk. They are encapsulated in different extracellular vesicle (EV) subsets that protect them from the extracellular milieu and the harsh conditions of the gastrointestinal tract during digestion. Here, we isolated pellets enriched in 4 different EV subsets, via differential ultracentrifugation of commercial cow milk: 12,000 × g (P12K), 35,000 × g (P35K), 70,000 × g (P70K), and 100,000 × g (P100K). Small RNA sequencing (sRNA-Seq) analyses revealed an unprecedented level of diversity in the complete miRNA repertoire and features of unfractionated cow milk and derived EV subsets. Although 5 miRNA sequences represented more than 50% of all miRNAs, milk EV exhibited heterogeneous content of miRNAs and isomeric variants (termed isomiR): P100K EV were enriched in reference miRNA sequences, and P12K and P35K EV in related isomiR. Incubation of milk EV with human cultured HeLa cells led to cellular enrichment in miRNA miR-223, which was concomitant with decreased expression of a reporter gene placed under the control of miR-223, thereby demonstrating the functionality of miR-223. These results suggest that cow milk EV may transfer their miRNAs to human cells and regulate recipient cell gene expression programming in a manner as complex as that of their miRNA transcriptome. The biological activity and relevance of the different milk EV subsets and bioactive mediators, including small noncoding RNA, in health and disease, warrants further investigation.

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer.

Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM -/- patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors.

RNA sequencing data of human prostate cancer cells treated with androgens.

Prostate cancer (PCa) is the most frequent cancer in North American men and PCa cells rely on the androgen receptor (AR) for growth and survival. To understand the effect of AR in cancer cells, we have treated LNCaP and LAPC4 cells, two immortalized human PCa cells in vitro, with the synthetic androgen R1881 and then performed RNA-seq analyses. High quality sequencing data have been analyzed using our bioinformatic pipeline which consists of FastQC for quality controls, Trimmomatic for trimming, and Kallisto for pseudoalignment to the transcriptome. Differentially expressed genes were identified using DESeq2 after adjustment for false-discovery rate (FDR q values < 0.05) and Relative Log Expression (RLE) normalization. Gene Set Enrichment Analysis (GSEA) was also performed to identify biological pathways significantly modulated by androgens. GSEA analyses identified the androgen signaling pathway, as well as several metabolic pathways, as significantly enriched following androgen stimulation. These analyses highlight the most significant metabolic pathways up-regulated following AR activation. Raw and processed RNA-seq data were deposited and made publicly available on the Gene Expression Omnibus (GEO; GSE128749). These data have been incorporated in a recent article describing the functions of AR as a master regulator of PCa cell metabolism. For more details about interpretation of these results, please refer to "Functional genomics studies reveal the androgen receptor as a master regulator of cellular energy metabolism in prostate cancer" by Gonthier et al. (doi: 10.1016/j.jsbmb.2019.04.016).

The Ovulatory Signal Precipitates LRH-1 Transcriptional Switching Mediated by Differential Chromatin Accessibility.

In the ovary, follicular growth and maturation are complicated processes that involve a series of morphological and physiological changes in oocytes and somatic cells leading to ovulation and luteinization, essential processes for fertility. Given the complexity of ovulation, characterization of genome-wide regulatory elements is essential to understand the mechanisms governing the expression of specific genes in the rapidly differentiating follicle. We therefore employed a systems biology approach to determine global transcriptional mechanisms during the early stages of the ovulatory process. We demonstrate that, following the hormonal signal that initiates ovulation, granulosa cells undergo major modification of distal regulatory elements, which coincides with cistrome reprogramming of the indispensable orphan nuclear receptor liver receptor homolog-1 (LRH-1). This cistromic reorganization correlates with the extensive changes in gene expression in granulosa cells leading to ovulation. Together, our study yields a highly detailed transcriptional map delineating ovarian cell differentiation during the initiation of ovulation.

Genetic sequence variations of BRCA1-interacting genes AURKA, BAP1, BARD1 and DHX9 in French Canadian families with high risk of breast cancer.

Breast cancer is a heterogeneous disease displaying some degree of familial clustering. Highly penetrant breast cancer susceptibility genes represent approximately 20-25% of the familial aggregation of breast cancer. A significant proportion of this familial aggregation of breast cancer is thus yet to be explained by other breast cancer susceptibility genes. Given the high susceptibility conferred by the two major breast cancer predisposition genes, BRCA1 and BRCA2 and the implication of these genes in many key cellular processes, assessment of genes encoding BRCA1-interacting proteins as plausible breast cancer candidate genes is thus attractive. In this study, four genes encoding BRCA1-interacting proteins were analyzed in a cohort of 96 breast cancer individuals from high-risk non-BRCA1/BRCA2 French Canadian families. Although no deleterious truncating germline mutations or aberrant spliced mRNA species were identified, a total of 10, 4, 11 and 6 variants were found in the AURKA, BAP1, BARD1 and DHX9 genes, respectively. The allele frequency of each variant was further ascertained in a cohort of 98 healthy French Canadian unrelated women and a difference in allele frequency was observed for one BARD1 variant based on single-marker analysis. Haplotype estimation, haplotype blocks and tagging SNPs identification were then performed for each gene, providing a valuable tool for further searches of common disease-associated variants in these genes and therefore further analyses on these genes in larger cohorts is warranted in the search of low-to-moderate penetrance breast cancer susceptibility alleles.

Germline mutations in the breast cancer susceptibility gene PTEN are rare in high-risk non-BRCA1/2 French Canadian breast cancer families.

Cowden syndrome is a disease associated with an increase in breast cancer susceptibility. Alleles in PTEN and other breast cancer susceptibility genes would be responsible for approximately 25% of the familial component of breast cancer risk, BRCA1 and BRCA2 being the two major genes responsible for this inherited risk. In order to evaluate the proportion of high-risk French Canadian non-BRCA1/BRCA2 breast/ovarian cancer families potentially harboring a PTEN germline mutation, the whole coding and flanking intronic sequences were analyzed in a series of 98 breast cancer cases. Although no germline mutation has been identified in the coding region, our study led to the identification of four intronic variants. Further investigations were performed to analyze the effect of these variants, alone and/or in combination, on splicing and PTEN protein levels. Despite suggestive evidence emerging from in silico analyses, the presence of these intronic variants do not seem to alter RNA splicing or PTEN protein levels. In addition, as loss of PTEN or part of it has been reported, Western blot analysis has also been performed. No major deletion could be identified in our cohort. Therefore, assuming a Poisson distribution for the frequency of deleterious mutation in our cohort, if the frequency of such deleterious mutation was 2%, we would have had a 90% or greater chance of observing at least one such mutation. These results suggest that PTEN germline mutations are rare and are unlikely to account for a significant proportion of familial breast cancer cases in the French Canadian population.