Metagenomic profiles of different types of Italian high-moisture Mozzarella cheese

The microbiota of different types of Italian high-moisture Mozzarella cheese produced using cow or buffalo milk, acidified with natural or selected cultures, and sampled at the dairy or at the mass market, was evaluated using a Next Generation Sequencing approach, in order to identify possible drivers of the bacterial diversity. Cow Mozzarella and buffalo Mozzarella acidified with commercial cultures were dominated by Streptococcus thermophilus, while buffalo samples acidified with natural whey cultures showed similar prevalence of L. delbrueckii subsp. bulgaricus, L. helveticus and S. thermophilus. Moreover, several species of non-starter lactic acid bacteria were frequently detected. The diversity in cow Mozzarella microbiota was much higher than that of water buffalo samples. Cluster analysis clearly separated cow's cheeses from buffalo's ones, the former having a higher prevalence of psychrophilic taxa, and the latter of Lactobacillus and Streptococcus. A higher prevalence of psychrophilic species and potential spoilers was observed in samples collected at the mass retail, suggesting that longer exposures to cooling temperatures and longer production-to-consumption times could significantly affect microbiota diversity. Our results could help in detecting some kind of thermal abuse during the production or storage of mozzarella cheese

Evaluating the SERCA2 and VEGF mRNAs as Potential Molecular Biomarkers of the Onset and Progression in Huntington's Disease

Abnormalities of intracellular Ca2+ homeostasis and signalling as well as the down-regulation of neurotrophic factors in several areas of the central nervous system and in peripheral tissues are hallmarks of Huntington\u2019s disease (HD). As there is no therapy for this hereditary, neurodegenerative fatal disease, further effort should be made to slow the progression of neurodegeneration in patients through the definition of early therapeutic interventions. For this purpose, molecular biomarker(s) for monitoring disease onset and/or progression and response to treatment need to be identified. In the attempt to contribute to the research of peripheral candidate biomarkers in HD, we adopted a multiplex real-time PCR approach to analyse the mRNA level of targeted genes involved in the control of cellular calcium homeostasis and in neuroprotection. For this purpose we recruited a total of 110 subjects possessing the HD mutation at different clinical stages of the disease and 54 sex- and agematched controls. This study provides evidence of reduced transcript levels of sarco-endoplasmic reticulum-associated ATP2A2 calcium pump (SERCA2) and vascular endothelial growth factor (VEGF) in peripheral blood mononuclear cells (PBMCs) of manifest and premanifest HD subjects. Our results provide a potentially new candidate molecular biomarker for monitoring the progression of this disease and contribute to understanding some early events that might have a role in triggering cellular dysfunctions in HD

NGS AND PERSONALIZED MEDICINE SMN2 target re-sequencing in spinal muscular atrophy patients

Genetic heterogeneity of individuals highlights the need to enhance personalized medicine to achieve effective treatments of human diseases. Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by degeneration of α-motorneurons in the spinal cord. The primary SMA-determining gene is SMN1, absent in about 95% of patients. The neighbouring nearly identical SMN2 gene fails to generate adequate levels of full-length SMN protein (FL-SMN). Still, SMN2 copy numbers can vary between 1 and 6, potentially modifying the severity of the disease. However, all SMN2 alleles are not functionally equivalent since they produce FL-transcripts with different efficiencies, most probably due to variations in their sequence. We established a new method to identify genetic polymorphisms in the complete genomic region of SMN2. Samples were sequenced by Illumina platform and a pooled indexing strategy. The estimated variant frequencies are usually indicative of the number of variant gene copies per subject when related to the individual’s SMN2 copy numbers. The method we described is ready to be used to identify variants/haplotypes associated with a particular SMA phenotype.openDottorato di ricerca in Scienze e tecnologie clinicheopenDUBSKY de WITTENAU, Giorgi

Primer and probe sequences used for the real-time detection of β-actin.

<p>Primer and probe sequences used for the real-time detection of β-actin.</p

mRNA quantification of VEGF relative to the age and clinical conditions of the patients.

<p>(A) Comparison of the level of VEGF mRNA in PBMCs from HD pre-manifest, manifest patients and age- and sex-matched healthy controls.Age 27–40: Controls = 15; pre-manifest = 12; manifest = 13; Age 43–58: Controls = 26; pre-manifest = 8; manifest = 42; Age 59–84: Controls = 13; manifest = 35; ** p<0.01; *** p<0.001. (B) Comparison of the level of VEGF mRNA in PBMCs of patients with different stages of the disease: pre-manifest = 20; early-manifest = 17; manifest = 55; late-manifest = 18;* p<0.05. The mean values ± SD of three independent assays are shown.</p

Primers used to evaluate the number of CAG repeats for the molecular diagnosis of HD.

<p>Primers used to evaluate the number of CAG repeats for the molecular diagnosis of HD.</p

RNA quantification of PMCA1, SERCA2, SERCA3 and VEGF in PBMCs from healthy controls and HD patients divided according to sex.

<p>Subjects analysed: 10 healthy male controls and 10 healthy female controls age-matched ± 3 years; 10 males HD, mean age of 44.5 ± 10.1; 10 females HD, mean age of 53.4 ± 9.6. *** p<0.005. Each column represents the relative mean value of three independent experiments ± SD.</p