Valeria Mezzolla

: Mitochondrial dysfunction and oxidative stress are prominent features of a plethora of human disorders. Dysregulation of mitochondrial functions represents a common pathogenic mechanism of diseases such as neurodegenerative disorders and cancer. The maintenance of the Nicotinamide adenine dinucleotide (NAD+) pool, and a positive NAD+/NADH ratio, are essential for mitochondrial and cell functions. The synthesis and degradation of NAD+ and transport of its key intermediates among cell compartments play an important role in maintaining optimal NAD levels, for the regulation of NAD+-utilizing enzymes, such as sirtuins (Sirt), poly-ADP-ribose polymerases, and CD38/157 enzymes, either intracellularly as well as extracellularly. In this review, we present and discuss the links between NAD+, NAD+-consuming enzymes, mitochondria functions, and diseases. Attempts to treat various diseases with supplementation of NAD+ cycling intermediates and inhibitors of sirtuins and ADP-ribosyl transferases may highlight a possible therapeutic approach for therapy of cancer and neurodegenerative diseases.

PHA synthases (PhaC) are grouped into four classes based on the kinetics and mechanisms of reaction. The grouping of PhaC enzymes into four classes is dependent on substrate specificity, according to the preference in forming short-chain-length (scl) or medium-chain-length (mcl) polymers: Class I, Class III and Class IV produce scl-PHAs depending on propionate, butyrate, valerate and hexanoate precursors, while Class II PhaC synthesize mcl-PHAs based on the alkane (C6 to C14) precursors. PHA synthases of Class I, in particular PhaC_Cs from Chromobacterium USM2 and PhaC_Cn/RePhaC1 from Cupriavidus necator/Ralstonia eutropha, have been analysed and the crystal structures of the C-domains have been determined. PhaC_Cn/RePhaC1 was also studied by X-ray absorption fine-structure (XAFS) analysis. Models have been proposed for dimerization, catalysis mechanism, substrate recognition and affinity, product formation, and product egress route. The assays based on amino acid substitution by mutagenesis have been useful to validate the hypothesis on the role of amino acids in catalysis and in accommodation of bulky substrates, and for the synthesis of PHB copolymers and medium-chain-length PHA polymers with optimized chemical properties.

PHA synthases (PhaC) are grouped into four classes based on the kinetics and mechanisms of reaction. The grouping of PhaC enzymes into four classes is dependent on substrate specificity, according to the preference in forming short chain length (scl) or medium chain length (mcl) polymers: class I, class III, and class IV produce scl-PHAs depending on propionate, butyrate, valerate and hexanoate precursors, while class II phaC synthesize mcl-PHAs based on the alkane (C6 to C14) precursors. PHA synthases of class I, in particular PhaCCs from Chromobacterium USM2 and PhaCCn/RePhaC1 from Cupriavidus necator/R. eutropha, have been analysed and the crystal structures of the C-domains have been determined. PhaCCn/RePhaC1 was also studied by small angle X-ray scattering (SAXS) analysis. Models have been proposed for dimerization, catalysis mechanism, substrate recognition and affinity, product formation and product egress route. The assays based on amino acid substitution by mutagenesis have been useful to validate the hypothesis on the role of amino acids in catalysis and in accommodation of bulky substrates, for the synthesis of PHB co-polymers and medium chain length-PHA polymers with optimized chemical properties.

Polyhydroxyalcanoates (PHAs) are biodegradable polymers synthesized in cytoplasmic granules in bacteria, such as Cupriavidus necator (Ralstonia eutropha), Alcaligenes latus, Pseudomonas spp., Comamonas spp. and other species. PHAs accumulation occurs in response to stress conditions, i.e. under high carbon and low nitrogen (24:1 ratio). PHA can be synthesized using recombinant microorganisms (provided with the operon phbA/phbB/phbC), escaping the constrains of nutrient request, except addition of high amount of sugar (glucose, lactose, fructose). In this study; E. coli was genetically modified for PHB production in biofermentors. The production of PHA at industrial scale requires a continuous supplementation of fermentable sugars to support the availability of nutrients and to assess the level of the exponential growth phase; since sugars are required either for bacterial growth either for PHA synthesis and energy storage. The biofermentors need to be run in automated system. Sensors are used at many points in fermentators; in the evaluation of parameters: consumption of sugars; cell density; quantification of PHB synthesis. The need of operational control during the fermentation has prompted us to application of three measurements; one unit linked to a Nanodrop to evaluate OD; one linked to a reaction chamber to measure sugars consumed by enzyme based fluorescence detection; and one for bacteria Nile Blue staining and fluorescence intensity reads. The growth of bacteria on three different plant by-products was monitored and PHB production in four days using a banana by-product feed was optimised. These detectors will make possible to exploit the full potential of bioreactors optimizing the time of use and maximizing the number of bacteria synthesizing PHA.

Poly-hydroxy-alcanoates (PHAs) are biodegradable and biocompatible polymers synthesized and accumulated in intracellular compartments in several bacterial species. Polyhydroxyalcanoates (PHAs) are synthesized by numerous prokaryotes, such as Cupriavidus necator (Ralstonia eutropha), Pseudomonas spp., Comamonas spp., in response to stress conditions, i.e., under high carbon and low nitrogen (24:1 ratio). PHA can be synthesized using recombinant microorganisms (provided with the operon phbA/phbB/phbC), escaping the constrains of nutrient request, except addition of high amount of sugar (glucose, lactose, fructose). Recombinant E. coli systems were studied to produce PHB using metabolic engineering. In biofermentors, the critical points are the excess of fermentable sugars and the ratio of nutrients versus cell optical density. In order to allow production in biofermentors in automated system, sensors are envisaged to evaluate critical parameters such as sugar consumption, bacteria concentration and level of synthesis of PHA. The need of fermentors and operation control has compelled for application of three biosensing units, one linked to a Nanodrop to evaluate OD, one linked to an enzymatic reaction chamber to measure sugars consumed by enzyme linked sugar biosensing tools, and one for sampling the bacteria, Nile Blue staining, and fluorescence intensity reads. These detectors will make possible to exploit the full potential of bioreactors optimizing the time of use and maximizing the number of bacteria synthesizing PHA.

Food pathogens are the cause of foodborne epidemics, therefore there is a need to detect the pathogens in food productions rapidly. A pre-enrichment culture followed by selective agar plating are standard detection methods. Molecular methods such as qPCR have provided a first rapid protocol for detection of pathogens within 24 h of enrichment culture. Biosensors also may provide a rapid tool to individuate a source of Salmonella contamination at early times of pre-enrichment culture. Forty mL of Salmonella spp. enrichment culture were processed by immunoseparation using the Pathatrix, as in AFNOR validated qPCR protocols. The Salmonella biosensor combined with immunoseparation showed a limit of detection of 100 bacteria/40 mL, with a 400 fold increase to previous results. qPCR analysis requires processing of bead-bound bacteria with lysis buffer and DNA clean up, with a limit of detection of 2 cfu/50 μL. Finally, a protein chip was developed and tested in screening and identification of 5 common pathogen species, Salmonella spp., E. coli, S. aureus, Campylobacter spp. and Listeria spp. The protein chip, with high specificity in species identification, is proposed to be integrated into a Lab-on-Chip system, for rapid and reproducible screening of Salmonella spp. and other pathogen species contaminating food productions.

Renewable energies is an important sector that needs to be sustained and increased by the action of policies and economic support.

Malignant gliomas are lethal primary intracranial tumors. To date, little information on the role of deregulated genes in gliomas have been identified. As the involvement of miRNAs in the carcinogenesis is well known, we carried out a pilot study to identify, as potential biomarkers, differentially expressed microRNAs in blood samples of patients affected by glioma. We studied the miRNAs’ expression, by means of microarray and Real-Time PCR, in 30 blood samples from glioma patients and in 82 blood samples of patients suffering from: (a) various neurological disorders (n=30), (b) primary B-lymphoma of the Central Nervous System (PCNSL, n=36) and (c) secondary brain metastases (n=16). By quantitative real time reverse-transcriptase polymerase chain reaction (qRT-PCR), we identified significantly increased levels of two candidate biomarkers, miR-15b and miR-21, in blood of patients affected by gliomas. ROC analysis of miR-15b biomarker levels allowed to differentiate patients with tumour from patients without glioma. Furthermore, combined expression analyses of miR15b and miR-21 distinguished between patients with and without glioma (90% sensitivity and 100% specificity). In addition, a decrement in the expression levels of miR-16 characterized glioblastomas compared to low grade and anaplastic gliomas. In conclusion, this pilot study suggest that it’s possible to identify the disease state by meaning miR-15b and miR-21 markers in blood, while miR-16 can be used to distinguish glioblastoma from other grade gliomas. They can potentially be used as biomarkers for non-invasive diagnosis of gliomas; further studies are mandatory to confirm our preliminary findings.

Food pathogens frequently cause foodborne diseases. There is a need to rapidly identify the source of the bacteria in order to contain their spread and epidemics. A pre-enrichment culture or a direct culture on agar plate are standard microbiological methods. In this review, we present an update on alternative molecular methods to nucleic acid-based detection for species identification. Biosensor-based methods rely on the recognition of antigen targets or receptors by antibodies, aptamers or high-affinity ligands. The captured antigens may be then directly or indirectly detected through an antibody or high-affinity and high-specificity recognition molecule. Various different detection methods are discussed, from label-free sensors and immunosensors to fluorescence-based ones. Each method shows advantages and disadvantages in terms of equipment, sensitivity, simplicity and cost-effectiveness. Finally, lab-on-a-chip (LOC) devices are introduced briefly, with the potential to be fast, sensitive and useful for on-site bacteria detection in food processing laboratories to check potential contamination by sample monitoring combined with a rapid pre-enrichment step.

MicroRNAs are aberrantly expressed in many cancers and can exert tumour‐suppressive or oncogenic functions. As oncomirs promote growth of cancer cells and support survival during chemotherapy, thus microRNA‐silencing therapies could be a valuable approach to be associated with anticancer drugs and chemotherapy treatments. miR‐155 microRNA was found overexpressed in different types of cancer, such as leukaemias (PML, B‐cell lymphomas), lung cancer and glioblastoma. GABA‐A receptor downregulation was found correlated with glioma grading, with decreasing levels associated with higher grade of malignancies. A relationship between knock‐down of miR‐155 and re‐expression of GABRA 1 protein in vivo was recently individuated. This finding has implication on the effectiveness of RNA‐silencing approaches against miR‐155 with the scope to control proliferation and signalling pathways regulated by GABA‐A receptor. Applying microRNAs for treatment of brain tumours poses several problems, and fields to be solved are mainly the passage of the brain–blood barrier and the targeted delivery to specific cell types. Glioblastoma multiforme cells bud off microvesicles that deliver cytoplasmic contents to nearby cells. Thus, the exploitation of these mechanisms to deliver antagomir therapeutics targeting microvescicles in the brain could take the lead in the near future in the treatment for brain cancers in substitution of invasive surgical intervention.