Evaluation of the use of phase-specific gene promoters for the expression of enological enzymes in an industrial wine yeast strain

Summary Genes as POT1, HSP104 and SSA3, which are late expressed in laboratory culture conditions are expressed only during the first few days in microvinifications in wine yeast cells. This effect is probably due to the different growth conditions and leads to useless levels of enzyme activity for a reporter gene. However the ACT1 promoter, which is constitutively expressed in laboratory conditions, produces sufficient amounts of enzyme activity in late fermentation phases.     

Effect of tyrosine modification on the biological and immunological properties of equine chorionic gonadotropin

The tyrosine residues of equine chorionic gonadotropin have been nitrated with tetranitromethane and the resulting effects on the biological and immunological activities of the hormone studied. All of the tyrosine residues in equine chorionic gonadotropin were found to react with tetranitromethane when a 100-fold molar excess of reagent was used or with an 8.6 molar excess in the presence of 5 M guanidine hydrochloride. Complete nitration abolished the biological activities and decreased the immunological activity of the hormone. The nitration of one tyrosine residue resulted in the loss of 70% of the LH activity of equine chorionic gonadotropin; the FSH activity declined in a similar fashion. Maximal nitration resulted in the loss of about 50% of the immunological activity of the native hormone. Nitrated derivatives of equine chorionic gonadotropin were unable to compete with the native hormone in the rat Leydig cell assay for LH. The results indicate that the tyrosine residues of equine chorionic gonadotropin play an important role in the manifestation of both the FSH and LH activity of the hormone.     

Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development

Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in <0.1 μl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes.     

Exploring macrophage cell therapy on Diabetic Kidney Disease

Alternatively activated macrophages (M2) have regenerative properties and shown promise as cell therapy in chronic kidney disease. However, M2 plasticity is one of the major hurdles to overcome. Our previous studies showed that genetically modified macrophages stabilized by neutrophil gelatinase‐associated lipocalin (NGAL) were able to preserve their M2 phenotype. Nowadays, little is known about M2 macrophage effects in diabetic kidney disease (DKD). The aim of the study was to investigate the therapeutic effect of both bone marrow‐derived M2 (BM‐фM2) and ф‐NGAL macrophages in the db/db mice. Seventeen‐week‐old mice with established DKD were divided into five treatment groups with their controls: D+BM‐фM2; D+ф‐BM; D+ф‐NGAL; D+ф‐RAW; D+SHAM and non‐diabetic (ND) (db/‐ and C57bl/6J) animals. We infused 1 × 10⁶ macrophages twice, at baseline and 2 weeks thereafter. BM‐фM2 did not show any therapeutic effect whereas ф‐NGAL significantly reduced albuminuria and renal fibrosis. The ф‐NGAL therapy increased the anti‐inflammatory IL‐10 and reduced some pro‐inflammatory cytokines, reduced the proportion of M1 glomerular macrophages and podocyte loss and was associated with a significant decrease of renal TGF‐β1. Overall, our study provides evidence that ф‐NGAL macrophage cell therapy has a therapeutic effect on DKD probably by modulation of the renal inflammatory response caused by the diabetic milieu.     

Presence of Blastocystis in gut microbiota is associated with cognitive traits and decreased executive function

Growing evidence implicates the gut microbiome in cognition. Blastocystis is a common gut single-cell eukaryote parasite frequently detected in humans but its potential involvement in human pathophysiology has been poorly characterized. Here we describe how the presence of Blastocystis in the gut microbiome was associated with deficits in executive function and altered gut bacterial composition in a discovery (n = 114) and replication cohorts (n = 942). We also found that Blastocystis was linked to bacterial functions related to aromatic amino acids metabolism and folate-mediated pyrimidine and one-carbon metabolism. Blastocystis-associated shifts in bacterial functionality translated into the circulating metabolome. Finally, we evaluated the effects of microbiota transplantation. Donor’s Blastocystis subtypes led to altered recipient’s mice cognitive function and prefrontal cortex gene expression. In summary, Blastocystis warrant further consideration as a novel actor in the gut microbiome-brain axis.Subject terms: Biomarkers, Pathogenesis, Diagnosis     

Effects of roscovitine on the nuclear and cytoskeletal components of calf oocytes and their subsequent development

Roscovitine, a potent inhibitor of M-phase promoting factor kinase activity, was used to maintain calf oocytes at the germinal vesicle stage for a 24h culture period. Cumulus-oocyte complexes were first prematured for 24h in the presence of different levels of roscovitine (12.5, 25, 50 and 100 microM). Roscovitine was shown to block germinal vesicle breakdown in calf oocytes in a concentration dependent manner. Significantly greater inhibitory effect was observed at 50 and 100 microM with 64.6% and 63.2% oocytes being blocked in the germinal vesicle stage when compared to the control (0.0%) and the 12.5 microM (2.9%) and 25 microM (18.8%) groups. However, this inhibitory effect of roscovitine was fully reversible since a substantial number of the oocytes resumed meiosis and reached the metaphase II stage after a further 24h of culture in a permissive medium. Cleavage rates and blastocyst yields were not significantly different for oocytes cultured under 50 microM roscovitine inhibition compared to oocytes not subjected to prematuration culture (rates of 76.7% cleavage and 8.7% blastocysts for control oocytes compared to 69.8% and 6.3%, respectively, for oocytes pretreated with 50 microM roscovitine). The morphology of the meiotic spindle was typical of metaphase II in 75.8% and 82.1% of the oocytes reaching the metaphase II stage after pretreatment with 50 microM roscovitine compared to control, respectively. A normal distribution of actin filaments was observed in 97.0% and 98.2% of oocytes exposed to 50 microM roscovitine compared to control, respectively. These results demonstrate the feasibility of maintaining calf oocytes in artificial meiotic arrest without compromising their subsequent developmental competence.