Candida albicans homologue of GGP1/GAS1 gene is functional in Saccharomyces cerevisiae and contains the determinants for glycosylphosphatidylinositol attachment

The GGP1/GAS1/CWH52 gene of Saccharomyces cerevisiae encodes a major exocellular 115 kDa glycoprotein (gp115) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI). The function of gp115 is still unknown but the analysis of null mutants suggests a possible role in the control of morphogenesis. PHR1 gene isolated from Candida alibicans is homologous to the GGP1 gene. In this report we have analysed the ability of PHR1 to complement a ggp1Δ mutation in S. cerevisiae. The expression of PHR1 controlled by its natural promoter or by the GGP1 promoter has been studied. In both cases we have observed a complete complementation of the mutant phenotype. Moreover, immunological analysis has revealed that PHR1 in budding yeast gives rise to a 75–80 kDa protein anchored to the membrane through a GPI, indicating that the signal for GPI attachment present in the C. albicans gene product is functional in S. cerevisiae.     

Beneficial and Sexually Dimorphic Response to Combined HDAC Inhibitor Valproate and AMPK/SIRT1 Pathway Activator Resveratrol in the Treatment of ALS Mice

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder. There is no cure and current treatments fail to slow the progression of the disease. Epigenetic modulation in the acetylation state of NF-kB RelA and the histone 3 (H3) protein, involved in the development of neurodegeneration, is a drugable target for the class-I histone deacetylases (HDAC) inhibitors, entinostat or valproate, and the AMP-activated kinase (AMPK)-sirtuin 1 pathway activator, resveratrol. In this study, we demonstrated that the combination of valproate and resveratrol can restore the normal acetylation state of RelA in the SOD1(G93A) murine model of ALS, in order to obtain the neuroprotective form of NF-kB. We also investigated the sexually dimorphic development of the disease, as well as the sex-sensibility to the treatment administered. We showed that the combined drugs, which rescued AMPK activation, RelA and the histone 3 acetylation state, reduced the motor deficit and the disease pathology associated with motor neuron loss and microglial reactivity, Brain-Derived Neurotrophic Factor (BDNF) and B-cell lymphoma-extra large (Bcl-xL) level decline. Specifically, vehicle-administered males showed earlier onset and slower progression of the disease when compared to females. The treatment, administered at 50 days of life, postponed the time of onset in the male by 22 days, but not in a significant way in females. Nevertheless, in females, the drugs significantly reduced symptom severity of the later phase of the disease and prolonged the mice’s survival. Only minor beneficial effects were produced in the latter stage in males. Overall, this study shows a beneficial and sexually dimorphic response to valproate and resveratrol treatment in ALS mice.     

Macrophage-Mediated Melanoma Reduction after HP-NAP Treatment in a Zebrafish Xenograft Model

The Helicobacter pylori Neutrophil Activating Protein (HP-NAP) is endowed with immunomodulatory properties that make it a potential candidate for anticancer therapeutic applications. By activating cytotoxic Th1 responses, HP-NAP inhibits the growth of bladder cancer and enhances the anti-tumor activity of oncolytic viruses in the treatment of metastatic breast cancer and neuroendocrine tumors. The possibility that HP-NAP exerts its anti-tumor effect also by modulating the activity of innate immune cells has not yet been explored. Taking advantage of the zebrafish model, we examined the therapeutic efficacy of HP-NAP against metastatic human melanoma, limiting the observational window to 9 days post-fertilization, well before the maturation of the adaptive immunity. Human melanoma cells were xenotransplanted into zebrafish embryos and tracked in the presence or absence of HP-NAP. The behavior and phenotype of macrophages and the impact of their drug-induced depletion were analyzed exploiting macrophage-expressed transgenes. HP-NAP administration efficiently inhibited tumor growth and metastasis and this was accompanied by strong recruitment of macrophages with a pro-inflammatory profile at the tumor site. The depletion of macrophages almost completely abrogated the ability of HP-NAP to counteract tumor growth. Our findings highlight the pivotal role of activated macrophages in counteracting melanoma growth and support the notion that HP-NAP might become a new biological therapeutic agent for the treatment of metastatic melanomas.     

The effect of cocoa fermentation and weak organic acids on growth and ochratoxin A production by Aspergillus species

The acidic characteristics of cocoa beans have influence on flavor development in chocolate. Cocoa cotyledons are not naturally acidic, the acidity comes from organic acids produced by the fermentative microorganisms which grow during the processing of cocoa. Different concentrations of these metabolites can be produced according to the fermentation practices adopted in the farms, which could affect the growth and ochratoxin A production by fungi. This work presents two independent experiments carried out to investigate the effect of some fermentation practices on ochratoxin A production by Aspergillus carbonarius in cocoa, and the effect of weak organic acids such as acetic, lactic and citric at different pH values on growth and ochratoxin A production by A. carbonarius and Aspergillus niger in culture media. A statistical difference (ρ<0.05) in the ochratoxin A level in the cured cocoa beans was observed in some fermentation practices adopted. The laboratorial studies demonstrate the influence of organic acids on fungal growth and ochratoxin A production, with differences according to the media pH and the organic acid present. Acetic acid was the most inhibitory acid against A. carbonarius and A. niger. From the point of view of food safety, considering the amount of ochratoxin A produced, fermentation practices should be conducted towards the enhancement of acetic acid, although lactic and citric acids also have an important role in lowering the pH to improve the toxicity of acetic acid.     

Cytoskeletal Protein 4.1G Is Essential for the Primary Ciliogenesis and Osteoblast Differentiation in Bone Formation

The primary cilium is a hair-like immotile organelle with specific membrane receptors, including the receptor of Hedgehog signaling, smoothened. The cilium organized in preosteoblasts promotes differentiation of the cells into osteoblasts (osteoblast differentiation) by mediating Hedgehog signaling to achieve bone formation. Notably, 4.1G is a plasma membrane-associated cytoskeletal protein that plays essential roles in various tissues, including the peripheral nervous system, testis, and retina. However, its function in the bone remains unexplored. In this study, we identified 4.1G expression in the bone. We found that, in the 4.1G-knockout mice, calcium deposits and primary cilium formation were suppressed in the trabecular bone, which is preosteoblast-rich region of the newborn tibia, indicating that 4.1G is a prerequisite for osteoblast differentiation by organizing the primary cilia in preosteoblasts. Next, we found that the primary cilium was elongated in the differentiating mouse preosteoblast cell line MC3T3-E1, whereas the knockdown of 4.1G suppressed its elongation. Moreover, 4.1G-knockdown suppressed the induction of the cilia-mediated Hedgehog signaling and subsequent osteoblast differentiation. These results demonstrate a new regulatory mechanism of 4.1G in bone formation that promotes the primary ciliogenesis in the differentiating preosteoblasts and induction of cilia-mediated osteoblast differentiation, resulting in bone formation at the newborn stage.     

Hybrid Nanoparticles and Composite Hydrogel Systems for Delivery of Peptide Antibiotics

The growing number of drug-resistant pathogenic bacteria poses a global threat to human health. For this reason, the search for ways to enhance the antibacterial activity of existing antibiotics is now an urgent medical task. The aim of this study was to develop novel delivery systems for polymyxins to improve their antimicrobial properties against various infections. For this, hybrid core–shell nanoparticles, consisting of silver core and a poly(glutamic acid) shell capable of polymyxin binding, were developed and carefully investigated. Characterization of the hybrid nanoparticles revealed a hydrodynamic diameter of approximately 100 nm and a negative electrokinetic potential. The nanoparticles demonstrated a lack of cytotoxicity, a low uptake by macrophages, and their own antimicrobial activity. Drug loading and loading efficacy were determined for both polymyxin B and E, and the maximal loaded value with an appropriate size of the delivery systems was 450 µg/mg of nanoparticles. Composite materials based on agarose hydrogel were prepared, containing both the loaded hybrid systems and free antibiotics. The features of polymyxin release from the hybrid nanoparticles and the composite materials were studied, and the mechanisms of release were analyzed using different theoretical models. The antibacterial activity against Pseudomonas aeruginosa was evaluated for both the polymyxin hybrid and the composite delivery systems. All tested samples inhibited bacterial growth. The minimal inhibitory concentrations of the polymyxin B hybrid delivery system demonstrated a synergistic effect when compared with either the antibiotic or the silver nanoparticles alone.     

Impact of Endocrine Disruptors upon Non-Genetic Inheritance

Similar to environmental factors, EDCs (endocrine-disrupting chemicals) can influence gene expression without modifying the DNA sequence. It is commonly accepted that the transgenerational inheritance of parentally acquired traits is conveyed by epigenetic alterations also known as “epimutations”. DNA methylation, acetylation, histone modification, RNA-mediated effects and extracellular vesicle effects are the mechanisms that have been described so far to be responsible for these epimutations. They may lead to the transgenerational inheritance of diverse phenotypes in the progeny when they occur in the germ cells of an affected individual. While EDC-induced health effects have dramatically increased over the past decade, limited effects on sperm epigenetics have been described. However, there has been a gain of interest in this issue in recent years. The gametes (sperm and oocyte) represent targets for EDCs and thus a route for environmentally induced changes over several generations. This review aims at providing an overview of the epigenetic mechanisms that might be implicated in this transgenerational inheritance.     

Different Contribution of Monocyte- and Platelet-Derived Microvesicles to Endothelial Behavior

Several contributions of circulating microvesicles (MVs) to the endothelial dysfunction have been reported in the past; a head-to-head comparison of platelet- and monocyte–derived MVs has however never been performed. To this aim, we assessed the involvement of these MVs in vessel damage related processes, i.e., oxidative stress, inflammation, and leukocyte-endothelial adhesion. Platelets and monocytes isolated from healthy subjects (HS, n = 15) were stimulated with TRAP-6 and LPS to release MVs that were added to human vascular endothelial cell (hECV) culture to evaluate superoxide anion production, inflammatory markers (IL-6, TNFα, NF-κB mRNA expression), and hECV adhesiveness. The effects of the MVs-induced from HS were compared to those induced by MVs spontaneously released from cells of patients with ST-segment elevation myocardial infarction (STEMI, n = 7). MVs released by HS-activated cells triggered a threefold increase in oxidative burst in a concentration-dependent manner. Only MVs released from monocytes doubled IL-6, TNFα, and NF-κB mRNA expression and monocyte-endothelial adhesion. Interestingly, the effects of the MVs isolated from STEMI-monocytes were not superimposable to previous ones except for adhesion to hECV. Conversely, MVs released from STEMI-platelets sustained both redox state and inflammatory phenotype. These data provide evidence that MVs released from activated and/or pathologic platelets and monocytes differently affect endothelial behavior, highlighting platelet-MVs as causative factors of impaired endothelial function in the acute phase of STEMI.     

The Ability of the Nitric Oxide Synthases Inhibitor T1023 to Selectively Protect the Non-Malignant Tissues

Previously, we showed that a nitric oxide synthase (NOS) inhibitor, compound T1023, induces transient hypoxia and prevents acute radiation syndrome (ARS) in mice. Significant efficacy (according to various tests, dose modifying factor (DMF)—1.6–1.9 against H-ARS/G-ARS) and safety in radioprotective doses (1/5–1/4 LD10) became the reason for testing its ability to prevent complications of tumor radiation therapy (RT). Research methods included studying T1023 effects on skin acute radiation reactions (RSR) in rats and mice without tumors and in tumor-bearing animals. The effects were evaluated using clinical, morphological and histological techniques as well as RTOG classification. T1023 administration prior to irradiation significantly limited the severity of acute RSR. This was due to a decrease in radiation alteration of the skin and underlying tissues, and the preservation of the functional activity of cell populations that are critical in the pathogenesis of radiation burn. The DMF values for T1023 for skin protection were 1.4–1.7. Moreover, its radioprotective effect was fully selective to normal tissues in RT models of solid tumors—T1023 reduced the severity of acute RSR and did not modify the antitumor effects of γ-radiation. The results indicate that T1023 can selectively protect the non-malignant tissues against γ-radiation due to hypoxic mechanism of action and potentiate opportunities of NOS inhibitors in RT complications prevention.     

Myelinosome Organelles in the Retina of R6/1 Huntington Disease (HD) Mice: Ubiquitous Distribution and Possible Role in Disease Spreading

Visual deficit is one of the complications of Huntington disease (HD), a fatal neurological disorder caused by CAG trinucleotide expansions in the Huntingtin gene, leading to the production of mutant Huntingtin (mHTT) protein. Transgenic HD R6/1 mice expressing human HTT exon1 with 115 CAG repeats recapitulate major features of the human pathology and exhibit a degeneration of the retina. Our aim was to gain insight into the ultrastructure of the pathological HD R6/1 retina by electron microscopy (EM). We show that the HD R6/1 retina is enriched with unusual organelles myelinosomes, produced by retinal neurons and glia. Myelinosomes are present in all nuclear and plexiform layers, in the synaptic terminals of photoreceptors, in the processes of retinal neurons and glial cells, and in the subretinal space. In vitro study shows that myelinosomes secreted by human retinal glial Müller MIO-M1 cells transfected with EGFP-mHTT-exon1 carry EGFP-mHTT-exon1 protein, as revealed by immuno-EM and Western-blotting. Myelinosomes loaded with mHTT-exon1 are incorporated by naive neuronal/neuroblastoma SH-SY5Y cells. This results in the emergence of mHTT-exon1 in recipient cells. This process is blocked by membrane fusion inhibitor MDL 28170. Conclusion: Incorporation of myelinosomes carrying mHTT-exon1 in recipient cells may contribute to HD spreading in the retina. Exploring ocular fluids for myelinosome presence could bring an additional biomarker for HD diagnostics.