Ncx3-Induced Mitochondrial Dysfunction in Midbrain Leads to Neuroinflammation in Striatum of A53t-α-Synuclein Transgenic Old Mice

The exact mechanism underlying selective dopaminergic neurodegeneration is not completely understood. The complex interplay among toxic alpha-synuclein aggregates, oxidative stress, altered intracellular Ca²⁺-homeostasis, mitochondrial dysfunction and disruption of mitochondrial integrity is considered among the pathogenic mechanisms leading to dopaminergic neuronal loss. We herein investigated the molecular mechanisms leading to mitochondrial dysfunction and its relationship with activation of the neuroinflammatory process occurring in Parkinson’s disease. To address these issues, experiments were performed in vitro and in vivo in mice carrying the human mutation of α-synuclein A53T under the prion murine promoter. In these models, the expression and activity of NCX isoforms, a family of important transporters regulating ionic homeostasis in mammalian cells working in a bidirectional way, were evaluated in neurons and glial cells. Mitochondrial function was monitored with confocal microscopy and fluorescent dyes to measure mitochondrial calcium content and mitochondrial membrane potential. Parallel experiments were performed in 4 and 16-month-old A53T-α-synuclein Tg mice to correlate the functional data obtained in vitro with mitochondrial dysfunction and neuroinflammation through biochemical analysis. The results obtained demonstrated: 1. in A53T mice mitochondrial dysfunction occurs early in midbrain and later in striatum; 2. mitochondrial dysfunction occurring in the midbrain is mediated by the impairment of NCX3 protein expression in neurons and astrocytes; 3. mitochondrial dysfunction occurring early in midbrain triggers neuroinflammation later into the striatum, thus contributing to PD progression during mice aging.     

Development and characterization of an implantable biosensor for telemetric monitoring of ethanol in the brain of freely moving rats

Ethanol is one of the most widespread psychotropic agents in western society. While its psychoactive effects are mainly associated with GABAergic and glutamatergic systems, the positive reinforcing properties of ethanol are related to activation of mesolimbic dopaminergic pathways resulting in a release of dopamine in the nucleus accumbens. Given these neurobiological implications, the detection of ethanol in brain extracellular fluid (ECF) is of great importance. In this study, we describe the development and characterization of an implantable biosensor for the amperometric detection of brain ethanol in real time. Ten different designs were characterized in vitro in terms of Michaelis-Menten kinetics (V(MAX) and K(M)), sensitivity (linear region slope, limit of detection (LOD), and limit of quantification (LOQ)), and electroactive interference blocking. The same parameters were monitored in selected designs up to 28 days after fabrication in order to quantify their stability. Finally, the best performing biosensor design was selected for implantation in the nucleus accumbens and coupled with a previously developed telemetric device for the real-time monitoring of ethanol in freely moving, untethered rats. Ethanol was then administered systemically to animals, either alone or in combination with ranitidine (an alcohol dehydrogenase inhibitor) while the biosensor signal was continuously recorded. The implanted biosensor, integrated in the low-cost telemetry system, was demonstrated to be a reliable device for the short-time monitoring of exogenous ethanol in brain ECF and represents a new generation of analytical tools for studying ethanol toxicokinetics and the effect of drugs on brain ethanol levels.     

Occurrence of methicillin-resistant Staphylococcus aureus in dairy cattle herds,related swine farms,and humans in contact with herds

In this study we investigated the circulation of methicillin-resistant Staphylococcus aureus (MRSA) in 2 dairy cattle farms (farm A and B), previously identified as MRSA-positive in bulk tank milk samples, and epidemiologically related to swine farms. Collected specimens included quarter milk samples and nasal swabs from dairy cows, pig nasal swabs collected at both the farm and slaughterhouse level, environmental dust samples, and human nasal swabs from the farms’ owners and workers. The prevalence of MRSA was estimated at the herd level by testing quarter milk samples. The prevalence of MRSA was 4.8% (3/63; 95% confidence interval = 0–10.2%) and 60% (33/55; 95% confidence interval = 47.05–72.95) in farm A and B, respectively. In farm A, MRSA was also isolated from humans, pigs sampled at both farm and slaughterhouse level, and from environmental samples collected at the pig facilities. The dairy cattle facilities of farm A tested negative for MRSA. In farm B, MRSA was isolated from environmental dust samples in both the cattle and pig facilities, whereas nasal swabs collected from cows and from humans tested negative. Sixty-three selected MRSA isolates obtained from different sources in farm A and B were genetically characterized by multilocus sequence typing, spa-typing, ribosomal spacer-PCR, and also tested for the presence of specific virulence genes and for their phenotypical antimicrobial susceptibility by broth microdilution method. Different clonal complex (CC) and spa-types were identified, including CC398, CC97, and CC1, CC already reported in livestock animals in Italy. The MRSA isolates from quarter milk of farm A and B mostly belonged to CC97 and CC398, respectively. Both lineages were also identified in humans in farm A. The CC97 and CC398 quarter milk isolates were also identified as genotype GTBE and GTAF by ribosomal spacer-PCR respectively, belonging to distinct clusters with specific virulence and resistance patterns. The GTBE and GTAF clusters also included swine, environmental, and human isolates from both farms. A high heterogeneity in the genetic and phenotypic profiles was observed in environmental isolates, in particular from farm B. These results demonstrate the possibility of a dynamic sharing and exchange of MRSA lineages or genotypes between different species and farm compartments in mixed-species farms. The risk of transmission between swine and related dairy cattle herds should be considered. Our findings also confirm the zoonotic potential of livestock-associated MRSA and underline the importance of applying biosecurity measures and good hygiene practices to prevent MRSA spread at the farm level and throughout the food production chain.     

Neoantigen-Specific T-Cell Immune Responses: The Paradigm of NPM1-Mutated Acute Myeloid Leukemia

The C-terminal aminoacidic sequence from NPM1-mutated protein, absent in normal human tissues, may serve as a leukemia-specific antigen and can be considered an ideal target for NPM1-mutated acute myeloid leukemia (AML) immunotherapy. Different in silico instruments and in vitro/ex vivo immunological platforms have identified the most immunogenic epitopes from NPM1-mutated protein. Spontaneous development of endogenous NPM1-mutated-specific cytotoxic T cells has been observed in patients, potentially contributing to remission maintenance and prolonged survival. Genetically engineered T cells, namely CAR-T or TCR-transduced T cells, directed against NPM1-mutated peptides bound to HLA could prospectively represent a promising therapeutic approach. Although either adoptive or vaccine-based immunotherapies are unlikely to be highly effective in patients with full-blown leukemia, these strategies, potentially in combination with immune-checkpoint inhibitors, could be promising in maintaining remission or preemptively eradicating persistent measurable residual disease, mainly in patients ineligible for allogeneic hematopoietic stem cell transplant (HSCT). Alternatively, neoantigen-specific donor lymphocyte infusion derived from healthy donors and targeting NPM1-mutated protein to selectively elicit graft-versus-leukemia effect may represent an attractive option in subjects experiencing post-HSCT relapse. Future studies are warranted to further investigate dynamics of NPM1-mutated-specific immunity and explore whether novel individualized immunotherapies may have potential clinical utility in NPM1-mutated AML patients.     

Yogurt-like beverages made of a mixture of cereals, soy and grape must: microbiology, texture, nutritional and sensory properties

Cereal (rice, barley, emmer and oat) and soy flours and concentrated red grape must were used for making vegetable yogurt-like beverages (VYLB). Two selected strains of Lactobacillus plantarum were used for lactic acid fermentation, according to a process which included the flour gelatinization. All VLYB had values of pH lower than 4.0 and both selected starters remained viable at ca. 8.4 log cfu/g throughout storage. All VLYB showed high values of apparent viscosity and water holding capacity. During fermentation, lactic acid bacteria consumed glucose, fructose, and malic acid, which was supplied with grape must. Compared to control vegetable yogurt-like beverages (CVYLB), without bacterial inoculum, an increase of total free amino acids (FAA) was found during fermentation and storage. Also the concentration of polyphenolic compounds and ascorbic acid (ASC) was higher in VLYB compared to CVYLB. This was reflected on the antioxidant activity. As determined by Solid Phase Micro-Extraction/Gas-Chromatography/Mass-Spectrometry analysis, several volatile compounds were identified. Beverages made with the mixture of rice and barley or emmer flours seemed to possess the best combination textural, nutritional and sensory properties.     

Comparison of personal, indoor, and outdoor exposures to hazardous air pollutants in three urban communities

Two-day average concentrations of 15 individual volatile organic compounds (VOCs) were measured concurrently in (a) ambient air in three urban neighborhoods, (b) air inside residences of participants, and (c) personal air near the breathing zone of 71 healthy, nonsmoking adults. The outdoor (O), indoor (I), and personal (P) samples were collected in the Minneapolis/St. Paul metropolitan area over three seasons (spring, summer, and fall) in 1999 using charcoal-based passive air samplers (3M model 3500 organic vapor monitors). A hierarchical, mixed-effects statistical model was used to estimate the mutually adjusted effects of monitor location, community, and season while accounting for within-subject and within-time-index (monitoring period) correlation. Outdoor VOC concentrations were relatively low compared to many other urban areas, and only minor seasonal differences were observed. A consistent pattern of P > I > O was observed across both communities and seasons for 13 of 15 individual VOCs (exceptions were carbon tetrachloride and chloroform). Results indicate that ambient VOC measurements at central monitoring sites can seriously underestimate actual exposures for urban residents, even when the outdoor measurements are taken in their own neighborhoods.     

Utilization of African grains for sourdough bread making

Abstract: Acha and Iburu flours were singly subjected to sourdough fermentation with previously selected autochthonous starters. Sourdoughs were used (30%, wt/wt) as aroma carriers and acidifiers during short time fermentation with the addition of baker's yeast. Acha and Iburu sourdough breads were compared to wheat sourdough bread started with the same strains and to breads made with the same formula but using baker's yeast alone. During Acha and Iburu sourdough fermentations, starter lactic acid bacteria reached almost the same cell density found in wheat sourdoughs. Acidification was more intense. Iburu sourdough bread had the highest total titratable acidity, the lowest pH, and contained the highest levels of free amino acids and phytase activity. The values of in vitro protein digestibility did not differ between Acha sourdough and wheat sourdough breads, while Iburu sourdough bread showed a slightly lower value. Acha and Iburu sourdough breads showed lower specific volume and higher density with respect to wheat sourdough breads. Nevertheless, Acha and Iburu sourdough breads were preferred for hardness and resilience. As shown by sensory analysis, Acha and especially Iburu sourdough breads were appreciated for color, acid taste and flavor, and overall acceptability. Practical Application: This study was aimed at evaluating the technological and nutritional properties of the African cereals Acha and Iburu. Sourdough fermentation and the use of selected starters increased the nutritional and sensory qualities and the potential application for bakery industry.     

Blue-on-yellow perimetry with a scanning laser ophthalmoscope: small alterations in the central macula with aging.

The sensitivity of short-wavelength-sensitive (SWS) cone pathways was measured in the central fields of 74 normal subjects, aged 17-86 yr, with healthy maculas. The new fundus perimetry technique used a research scanning laser ophthalmoscope with a small entrance pupil to present blue static perimetry targets on a bright yellow background. Simultaneous infrared imaging aided target positioning and rapid assessment of potential pathology in elderly subjects. Targets were positioned peripheral to fixation, avoiding both the SWS-cone-free area and the peak macular pigment, determined in 11 subjects. Sensitivity declined 0.019 log unit per decade, while intraindividual variability across loci increased. The nasal-temporal asymmetry remained constant. Sensitivity of older subjects was relatively less for the most central targets but was unrelated to transmission through macular pigment. Retinal changes with age occur to differing extents or at differing rates and are readily detectable in the central macula.     

The P2X7R-NLRP3 and AIM2 Inflammasome Platforms Mark the Complexity/Severity of Viral or Metabolic Liver Damage

P2X7R-NLRP3 and AIM2 inflammasomes activate caspase-1 and the release of cytokines involved in viral-related liver disease. Little is known about their role in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steato-hepatitis (NASH). We characterized the role of inflammasomes in NAFLD, NASH, and HCV. Gene expression and subcellular localization of P2X7R/P2X4R-NLRP3 and AIM2 inflammasome components were examined in histopathological preparations of 46 patients with biopsy-proven viral and metabolic liver disease using real-time PCR and immunofluorescence. P2X7R, P2X4R, and Caspase-1 are two- to five-fold more expressed in patients with NAFLD/NASH associated with chronic HCV infection than those with metabolic damage only (p ≤ 0.01 for all comparisons). The AIM2 inflammasome is 4.4 times more expressed in patients with chronic HCV infection, regardless of coexistent metabolic abnormalities (p = 0.0006). IL-2, a cytokine playing a pivotal role during chronic HCV infection, showed a similar expression in HCV and NASH patients (p = 0.77) but was virtually absent in NAFLD. The P2X7R-NLRP3 complex prevailed in infiltrating macrophages, while AIM2 was localized in Kupffer cells. Caspase-1 expression correlated with elastography-based liver fibrosis (r = 0.35, p = 0.02), whereas P2X7R, P2X4R, NRLP3, Caspase-1, and IL-2 expression correlated with circulating markers of disease severity. P2X7R and P2X4R play a major role in liver inflammation accompanying chronic HCV infection, especially when combined with metabolic damage, while AIM2 is specifically expressed in chronic viral hepatitis. We describe for the first time the hepatic expression of IL-2 in NASH, so far considered a peculiarity of HCV-related liver damage.