Sperm glyceraldehyde 3-phosphate dehydrogenase gene expression in asthenozoospermic spermatozoa

It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have investigated the chemical and kinetic properties of the enzyme glyceraldehyde 3‐phosphate dehydrogenase to identify any changes in the regulation of glycolysis and sperm motility. In contrast, there are few studies analyzing the genetic basis of hypokinesis. For this reason, we investigated the glyceraldehyde 3‐phosphate dehydrogenase gene in human sperm to evaluate whether asthenozoospermia was correlated with any changes in its expression. Semen examination and glyceraldehyde 3‐phosphate dehydrogenase gene expression studies were carried out on 116 semen samples divided into two groups – Group A consisted of 58 normokinetic samples and Group B of 58 hypokinetic samples. Total RNA was extracted from spermatozoa, and real‐time PCR quantification of mRNA was carried out using specific primers and probes. The expression profiles for the Groups A and B were very similar. The mean delta Ct was as follows – Group A, 5.79 ± 1.04; Group B, 5.47 ± 1.27. Our study shows that in human sperm, there is no difference in glyceraldehyde 3‐phosphate dehydrogenase gene expression between samples with impaired motility and samples with normal kinetics. We believe that this study could help in the understanding of the molecular mechanisms of sperm kinetics, suggesting that hypomotility may be due to a possible posttranscriptional impairment of the control mechanism, such as mRNA splicing, or to posttranslational changes

PPARG: Gene Expression Regulation and Next-Generation Sequencing for Unsolved Issues

Peroxisome proliferator-activated receptor gamma (PPARγ) is one of the most extensively studied ligand-inducible transcription factors (TFs), able to modulate its transcriptional activity through conformational changes. It is of particular interest because of its pleiotropic functions: it plays a crucial role in the expression of key genes involved in adipogenesis, lipid and glucid metabolism, atherosclerosis, inflammation, and cancer. Its protein isoforms, the wide number of PPARγ target genes, ligands, and coregulators contribute to determine the complexity of its function. In addition, the presence of genetic variants is likely to affect expression levels of target genes although the impact of PPARG gene variations on the expression of target genes is not fully understood. The introduction of massively parallel sequencing platforms—in the Next Generation Sequencing (NGS) era—has revolutionized the way of investigating the genetic causes of inherited diseases. In this context, DNA-Seq for identifying—within both coding and regulatory regions of PPARG gene—novel nucleotide variations and haplotypes associated to human diseases, ChIP-Seq for defining a PPARγ binding map, and RNA-Seq for unraveling the wide and intricate gene pathways regulated by PPARG, represent incredible steps toward the understanding of PPARγ in health and disease

A novel splicing mutation in the ABCA1 gene, causing Tangier disease and familial HDL deficiency in a large family.

International audience; Tangier disease is a rare disorder of lipoprotein metabolism that presents with extremely low levels of HDL cholesterol and apoprotein A-I. It is caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene. Clinical heterogeneity and mutational pattern of Tangier disease are poorly characterized. Moreover, also familial HDL deficiency may be caused by mutations in ABCA1 gene. ATP-binding cassette transporter A1 (ABCA1) gene mutations in a patient with Tangier disease, who presented an uncommon clinical history, and in his family were found and characterized. He was found to be compound heterozygous for two intronic mutations of ABCA1 gene, causing abnormal pre-mRNAs splicing. The novel c.1510-1G > A mutation was located in intron 12 and caused the activation of a cryptic splice site in exon 13, which determined the loss of 22 amino acids of exon 13 with the introduction of a premature stop codon. Five heterozygous carriers of this mutation were also found in proband's family, all presenting reduced HDL cholesterol and ApoAI (0.86 ± 0.16 mmol/L and 92.2 ± 10.9 mg/dL respectively), but not the typical features of Tangier disease, a phenotype compatible with the diagnosis of familial HDL deficiency. The other known mutation c.1195-27G > A was confirmed to cause aberrant retention of 25 nucleotides of intron 10 leading to the insertion of a stop codon after 20 amino acids of exon 11. Heterozygous carriers of this mutation also showed the clinical phenotype of familial HDL deficiency. Our study extends the catalog of pathogenic intronic mutations affecting ABCA1 pre-mRNA splicing. In a large family, a clear demonstration that the same mutations may cause Tangier disease (if in compound heterozygosis) or familial HDL deficiency (if in heterozygosis) is provided

Lactic Fermentation of Cereal Flour: Feasibility Tests on Rice, Oat and Wheat

Background and objective: Consumers show increasing interests in probiotic foods and lactic acid fermentations. Cereal flour, can be a good fermentable substrate due to its prebiotic nature; from which, synbiotic products can be prepared. The aim of the current study was to investigate if three various cereal flours rice, oat and wheat would be good potentially functional foods.Material and methods: Fermentation tests were carried out on rice, oat and wheat flours, using Lactobacillus paracasei CBA L74 and 1.5-L fermenter with 1-L working volume. After 24 h, microbial growth, pH value, lactic acid production and starch consumption were assessed.Results and conclusion: In all three flours, pH reduction was seen; particularly in rice flour. The highest Lactobacillus growth and lactic acid production were achieved at the end of rice fermentation. The greatest starch consumption was reported at the end of rice fermentation. In conclusion, lactic fermentation of cereals as potentially functional foods was possible for the three flours. However, the best result belonged to rice flour.Conflict of interest: The authors declare no conflict of interest.

Iron (II) citrate complex as a food supplement: Synthesis, characterization and complex stability

Iron deficiency represents a widespread problem for a large part of the population, especially for women, and has received increasing attention in food/supplement research. The contraindications of the iron supplements commercially available (e.g., imbalances in the levels of other essential nutrients, low bioavailability, etc.) led us to search for a possible alternative. In the present work, a rapid and easy method to synthetize a solid iron (II) citrate complex from iron filings and citric acid was developed to serve, eventually, as a food supplement or additive. In order to state its atomic composition and purity, an assortment of analytical techniques was employed (e.g., combustion analysis, thermogravimetry, X-ray diffractometry, UV/Vis spectrophotometry, etc.). Results demonstrate that the synthesized crystalline solid corresponds to the formula FeC6H6O7∙H2O and, by consequence, contains exclusively iron (II), which is an advantage with respect to existing commercial products, because iron (II) is better absorbed than iron (III) (high bioavailability of iron)

Effects of the Glucose Addition during Lactic Fermentation of Rice, Oat and Wheat Flours

 Background and objective: Consumer interests in probiotic foods have increased in recent decades. Food industries respond to these growing interests by developing innovative products and guaranteeing high production efficiency. Cereals, due to their prebiotic nature, are good fermentable substrates; from which, potentially functional foods could be achieved. The aim of this study was to verify effects of D-glucose addition on fermentation of rice, oat and wheat flours.Material and methods: Suspensions of 15% of cereals flours (rice, oat and wheat) in distilled water added with increasing glucose concentrations (2, 5, 7 and 10% w v-1) were fermented by Lactobacillus paracasei CBA L74 for 24 h. Then, pH, microbial growth and lactic acid production were assessed.Results and conclusion: Rice fermentation was not affected by glucose addition. For oat and wheat, addition of D-glucose increased bacterial concentration, as well as lactic acid production. In particular, the best growth was achieved by the addition of 2 and 5% of glucose. Furthermore, lactic acid concentration increased with increased glucose concentration. In conclusion, D-glucose addition seems to be unnecessary for the improvement of rice fermentation. On the contrary, oat and wheat fermentations need further available carbon sources for a better Lactobacillus growth and a higher lactic acid production.Conflict of interest: The authors declare no conflict of interest

Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages

BackgroundA bidirectional crosstalk between tumor cells and the surrounding microenvironment contributes to tumor progression and response to therapy. Our previous studies have demonstrated that bcl-2 affects melanoma progression and regulates the tumor microenvironment. The aim of this study was to evaluate whether bcl-2 expression in melanoma cells could influence tumor-promoting functions of tumor-associated macrophages, a major constituent of the tumor microenvironment that affects anticancer immunity favoring tumor progression.MethodsTHP-1 monocytic cells, monocyte-derived macrophages and melanoma cells expressing different levels of bcl-2 protein were used. ELISA, qRT-PCR and Western blot analyses were used to evaluate macrophage polarization markers and protein expression levels. Chromatin immunoprecipitation assay was performed to evaluate transcription factor recruitment at specific promoters. Boyden chamber was used for migration experiments. Cytofluorimetric and immunohistochemical analyses were carried out to evaluate infiltrating macrophages and T cells in melanoma specimens from patients or mice.ResultsHigher production of tumor-promoting and chemotactic factors, and M2-polarized activation was observed when macrophages were exposed to culture media from melanoma cells overexpressing bcl-2, while bcl-2 silencing in melanoma cells inhibited the M2 macrophage polarization. In agreement, the number of melanoma-infiltrating macrophages in vivo was increased, in parallel with a greater expression of bcl-2 in tumor cells. Tumor-derived interleukin-1β has been identified as the effector cytokine of bcl-2-dependent macrophage reprogramming, according to reduced tumor growth, decreased number of M2-polarized tumor-associated macrophages and increased number of infiltrating CD4+IFNγ+and CD8+IFNγ+effector T lymphocytes, which we observed in response to in vivo treatment with the IL-1 receptor antagonist kineret. Finally, in tumor specimens from patients with melanoma, high bcl-2 expression correlated with increased infiltration of M2-polarized CD163+macrophages, hence supporting the clinical relevance of the crosstalk between tumor cells and microenvironment.ConclusionsTaken together, our results show that melanoma-specific bcl-2 controls an IL-1β-driven axis of macrophage diversion that establishes tumor microenvironmental conditions favoring melanoma development. Interfering with this pathway might provide novel therapeutic strategies