DypB peroxidase for aflatoxin removal: New insights into the toxin degradation process.

Aflatoxin B1 (AFB1) is one of the most potent carcinogens and a widespread food and feed contaminant. As for other toxins, many efforts are devoted to find efficient and environmentally-friendly methods to degrade AFB1, such as enzymatic treatments, thus improving the safety of food and feed products. In this regard, the dye decolorizing peroxidase of type B (DypB) can efficiently degrade AFB1. The molecular mechanism, which is required to drive protein optimization in view of the usage of DypB as a mycotoxin reduction agent in large scale application, is unknown. Here, we focused on the role of four DypB residues in the degradation of AFB1 by alanine-scanning (residues 156, 215, 239 and 246), which were identified from biochemical assays to be kinetically relevant for the degradation. As a result of DypB degradation, AFB1 is converted into four products. Interestingly, the relative abundancy of these products depends on the replaced residues. Molecular dynamics simulations were used to investigate the role of these residues in the binding step between protein and manganese, a metal ion which is expected to be involved in the degradation process. We found that the size of the haem pocket as well as conformational changes in the protein structure could play a role in determining the kinetics of AFB1 removal and, consequently, guide the process towards specific degradation products.

Quasi-3D morphology and modulation of focal adhesions of human adult stem cells through combinatorial concave elastomeric surfaces with varied stiffness.

In vivo cell niches are complex architectures that provide a wide range of biochemical and mechanical stimuli to control cell behavior and fate. With the aim to provide in vitro microenvironments mimicking physiological niches, microstructured substrates have been exploited to support cell adhesion and to control cell shape as well as three dimensional morphology. At variance with previous methods, we propose a simple and rapid protein subtractive soft lithographic method to obtain microstructured polydimethylsiloxane substrates for studying stem cell adhesion and growth. The shape of adult renal stem cells and nuclei is found to depend predominantly on micropatterning of elastomeric surfaces and only weakly on the substrate mechanical properties. Differently, focal adhesions in their shape and density but not in their alignment mainly depend on the elastomer stiffness almost regardless of microscale topography. Local surface topography with concave microgeometry enhancing adhesion drives stem cells in a quasi-three dimensional configuration where stiffness might significantly steer mechanosensing as highlighted by focal adhesion properties.

High progesterone levels on the day of HCG administration do not affect the embryo quality and the reproductive outcomes of frozen embryo transfers.

OBJECTIVES: The aim of this study was to evaluate the impact of pre- mature progesterone rise on the day of human chorionic gonadotropin (hCG) administration on the outcome of in vitro fertilization (IVF) of frozen embryo transfer (FET) cycles using cleavage-stage embryos. METHODS: This was a retrospective, cohort study of 131 ovarian stimulation cycles followed by cleavage-stage frozen embryo transfers. The first group consisted of women undergoing FET due to premature luteinization during controlled ovarian stimulation (n = 56, P ≥1.2 ng/ml). The controls were represented by women undergoing FET not complicated by high progesterone levels at induction (n = 75, P < 1.2 ng/ml). For both groups, the progesterone was measured on the day of hCG administration and the fertilization rate, cleavage rate, implantation rate, clinical pregnancy rate, ongoing pregnancy rate and Top-Quality Embryos (TQE) rates were compared. RESULTS: The increase of progesterone in patients of the Group A had no significant effects on the number of oocytes retrieved or available for the insemination. The fertilization rate, cleavage rate and implantation rates, as well as the clinical pregnancy rate and ongoing pregnancy were very similar in both study groups. The analysis of TQE rates between the two groups indicated a roughly comparable result. CONCLUSIONS: The results of this study showed that progesterone elevation on the day of hCG administration did not affect the outcomes of IVF with frozen embryos at cleavage stage. This study therefore confirms that for patients with high progesterone levels the right way to obtain a healthy pregnancy should be to delay the embryo transfer at a successive FET cycle, not associated with the ovarian stimulation.

Acetyl-L-carnitine feeding to unloaded rats triggers in soleus muscle the coordinated expression of genes involved in mitochondrial biogenesis.

The expressional profile of mitochondrial transcripts and of genes involved in the mitochondrial biogenesis pathway induced by ALCAR daily supplementation in soleus muscle of control and unloaded 3-month-old rats has been analyzed. It has been found that ALCAR treatment is able to upregulate the expression level of mitochondrial transcripts (COX I, ATP6, ND6, 16 S rRNA) in both control and unloaded animals. Interestingly, ALCAR feeding to unloaded rats resulted in the increase of transcript level for master factors involved in mitochondrial biogenesis (PGC-1alpha, NRF-1, TFAM). It also prevented the unloading-induced downregulation of mRNA levels for kinases able to transduce metabolic (AMPK) and neuronal stimuli (CaMKIIbeta) into mitochondrial biogenesis. No significant effect on the expressional level of such genes was found in control ALCAR-treated rats. In addition, ALCAR feeding was able to prevent the loss of mitochondrial protein content due to unloading condition. Correlation analysis revealed a strong coordination in the expression of genes involved in mitochondrial biogenesis only in ALCAR-treated suspended animals, supporting a differentiated effect of ALCAR treatment in relation to the loading state of the soleus muscle. In conclusions, we demonstrated the ability of ALCAR supplementation to promote only in soleus muscle of hindlimb suspended rats an orchestrated expression of genes involved in mitochondrial biogenesis, which might counteract the unloading-induced metabolic changes, preventing the loss of mitochondrial proteins.

Tissue-specific effect of age and caloric restriction diet on mitochondrial DNA content.

The effect of age and caloric-restriction (CR) diet on mitochondrial DNA (mtDNA) content in different rat tissues was investigated. A decrease of the mtDNA content occurs with aging in liver and soleus muscle, whereas there is no age-related significant change of mtDNA content in brain. CR fully reverses the age-dependent loss of mtDNA in liver and soleus, whereas it results in a significant increase of mtDNA amount above the value of aged ad libitum fed rats in brain. These results further support the tissue-specific effect of CR, likely because of the different dependence of tissues on external nutrient uptake.