Mitochondrial Sirtuins in Chronic Degenerative Diseases: New Metabolic Targets in Colorectal Cancer

Sirtuins (SIRTs) are a family of class III histone deacetylases (HDACs) consisting of seven members, widely expressed in mammals. SIRTs mainly participate in metabolic homeostasis, DNA damage repair, cell survival, and differentiation, as well as other cancer-related biological processes. Growing evidence shows that SIRTs have pivotal roles in chronic degenerative diseases, including colorectal cancer (CRC), the third most frequent malignant disease worldwide. Metabolic alterations are gaining attention in the context of CRC development and progression, with mitochondrion representing a crucial point of complex and intricate molecular mechanisms. Mitochondrial SIRTs, SIRT2, SIRT3, SIRT4 and SIRT5, control mitochondrial homeostasis and dynamics. Here, we provide a comprehensive review on the latest advances on the role of mitochondrial SIRTs in the initiation, promotion and progression of CRC. A deeper understanding of the pathways by which mitochondrial SIRTs control CRC metabolism may provide new molecular targets for future innovative strategies for CRC prevention and therapy.     

Plastid Transformation: New Challenges in the Circular Economy Era

In a circular economy era the transition towards renewable and sustainable materials is very urgent. The development of bio-based solutions, that can ensure technological circularity in many priority areas (e.g., agriculture, biotechnology, ecology, green industry, etc.), is very strategic. The agricultural and fishing industry wastes represent important feedstocks that require the development of sustainable and environmentally-friendly industrial processes to produce and recover biofuels, chemicals and bioactive molecules. In this context, the replacement, in industrial processes, of chemicals with enzyme-based catalysts assures great benefits to humans and the environment. In this review, we describe the potentiality of the plastid transformation technology as a sustainable and cheap platform for the production of recombinant industrial enzymes, summarize the current knowledge on the technology, and display examples of cellulolytic enzymes already produced. Further, we illustrate several types of bacterial auxiliary and chitinases/chitin deacetylases enzymes with high biotechnological value that could be manufactured by plastid transformation.     

Tobacco Plastid Transformation as Production Platform of Lytic Polysaccharide MonoOxygenase Auxiliary Enzymes

Plant biomass is the most abundant renewable resource in nature. In a circular economy perspective, the implementation of its bioconversion into fermentable sugars is of great relevance. Lytic Polysaccharide MonoOxygenases (LPMOs) are accessory enzymes able to break recalcitrant polysaccharides, boosting biomass conversion and subsequently reducing costs. Among them, auxiliary activity of family 9 (AA9) acts on cellulose in synergism with traditional cellulolytic enzymes. Here, we report for the first time, the production of the AA9 LPMOs from the mesophilic Trichoderma reesei (TrAA9B) and the thermophilic Thermoascus aurantiacus (TaAA9B) microorganisms in tobacco by plastid transformation with the aim to test this technology as cheap and sustainable manufacture platform. In order to optimize recombinant protein accumulation, two different N-terminal regulatory sequences were used: 5′ untranslated region (5′-UTR) from T7g10 gene (DC41 and DC51 plants), and 5′ translation control region (5′-TCR), containing the 5′-UTR and the first 14 amino acids (Downstream Box, DB) of the plastid atpB gene (DC40 and DC50 plants). Protein yields ranged between 0.5 and 5% of total soluble proteins (TSP). The phenotype was unaltered in all transplastomic plants, except for the DC50 line accumulating AA9 LPMO at the highest level, that showed retarded growth and a mild pale green phenotype. Oxidase activity was spectrophotometrically assayed and resulted higher for the recombinant proteins without the N-terminal fusion (DC41 and DC51), with a 3.9- and 3.4-fold increase compared to the fused proteins.     

Selective Inhibition of Genomic and Non-Genomic Effects of Thyroid Hormone Regulates Muscle Cell Differentiation and Metabolic Behavior

Thyroid hormones (THs) are key regulators of different biological processes. Their action involves genomic and non-genomic mechanisms, which together mediate the final effects of TH in target tissues. However, the proportion of the two processes and their contribution to the TH-mediated effects are still poorly understood. Skeletal muscle is a classical target tissue for TH, which regulates muscle strength and contraction, as well as energetic metabolism of myofibers. Here we address the different contribution of genomic and non-genomic action of TH in skeletal muscle cells by specifically silencing the deiodinase Dio2 or the β3-Integrin expression via CRISPR/Cas9 technology. We found that myoblast proliferation is inversely regulated by integrin signal and the D2-dependent TH activation. Similarly, inhibition of the nuclear receptor action reduced myoblast proliferation, confirming that genomic action of TH attenuates proliferative rates. Contrarily, genomic and non-genomic signals promote muscle differentiation and the regulation of the redox state. Taken together, our data reveal that integration of genomic and non-genomic signal pathways finely regulates skeletal muscle physiology. These findings not only contribute to the understanding of the mechanisms involved in TH modulation of muscle physiology but also add insight into the interplay between different mechanisms of action of TH in muscle cells.     

Semi-solid casting (SSC) of zinc alloy nanocomposites

An innovative casting method that combines semi-solid casting (SSC) and metal matrix nanocomposite (MMNC) technology was successfully demonstrated. The method uses the grain refining properties of the nanoparticles in the MMNC to produce slurry with the appropriate globular structure for semi-solid casting. In this way no additional material processing or mechanical agitation is necessary to achieve the desirable slurry microstructure. The grain refining attributes of the nanoparticles were shown to stem from the promotion of nucleation in the solidifying matrix alloy. Differential scanning calorimetry (DSC) showed a reduced undercooling necessary for nucleation owing to the nucleation catalytic potency of the nanoparticles. Using this method zinc alloy AC43A nanocomposite with 0.5 wt.% SiC β nanoparticle additions were cast at a 30% solid fraction. The resulting castings showed increased ductility, reduced shrinkage, and increased strength compared to their monolithic liquid cast counterparts.     

The Genoa 2005 outbreak. Determination of putative palytoxin in Mediterranean Ostreopsis ovata by a new liquid chromatography tandem mass spectrometry method

A new method for sensitive, specific, and direct determination of palytoxin is proposed herein. It is based on combination of reversed-phase liquid chromatography with mass spectrometry (LC-MS). The new method was set up on a turbo ion spray-triple quadrupole MS instrument operating in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) acquisition modes (positive ions). The minimum detection levels for matrix-free toxin on column were thus estimated from the data to be 200 and 125 pg in SIM and MRM modes, respectively. Spiking experiments before and after extraction allowed us to assess limits of detection and quantitation for palytoxin in matrix, accuracy, and intraday and interday reproducibility of the method. The developed method was decisive for the analysis of a plankton sample collected along Genoa coasts in July 2005 when respiratory illness in people exposed to marine aerosols occurred. It is suggested that putative palytoxin was the causative agent responsible for patients' symptoms and demonstrated for the first time the presence of such a toxin in Italian waters.     

Buffalo Milk Whey Activates Necroptosis and Apoptosis in a Xenograft Model of Colorectal Cancer

Recent pharmacological research on milk whey, a byproduct of the dairy industry, has identified several therapeutic properties that could be exploited in modern medicine. In the present study, we investigated the anticancer effects of whey from Mediterranean buffalo (Bubalus bubalis) milk. The antitumour effect of delactosed milk whey (DMW) was evaluated using the HCT116 xenograft mouse model of colorectal cancer (CRC). There were no discernible differences in tumour growth between treated and untreated groups. Nevertheless, haematoxylin and eosin staining of the xenograft tissues showed clearer signs of different cell death in DMW-treated mice compared to vehicle-treated mice. Detailed biochemical and molecular biological analyses revealed that DMW was able to downregulate the protein expression levels of c-myc, phospho-Histone H3 (ser 10) and p-ERK. Moreover, DMW also activated RIPK1, RIPK3, and MLKL axis in tumour tissues from xenograft mice, thus, suggesting a necroptotic effect. The necroptotic pathway was accompanied by activation of the apoptotic pathway as revealed by increased expression of both cleaved caspase-3 and PARP-1. At the molecular level, DMW-induced cell death was also associated with (i) upregulation of SIRT3, SIRT6, and PPAR-γ and (ii) downregulation of LDHA and PPAR-α. Overall, our results unveil the potential of whey as a source of biomolecules of food origin in the clinical setting of novel strategies for the treatment of CRC.     

Determination of Homoarginine,Arginine, NMMA,ADMA, and SDMA in Biological Samples by HPLC-ESI-Mass Spectrometry

N^G,N^G-dimethyl-l-arginine (ADMA) and N^G-methyl-l-arginine (NMMA) are endogenous inhibitors of nitric oxide synthase (NOS). In contrast, N^G,N′^G-dimethyl-Larginine (SDMA) possesses only a weak inhibitory potency towards neuronal NOS and it is known to limit nitric oxide (NO) production by competing with l-arginine for cellular uptake. The inhibition of NOS is associated with endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. l-Homoarginine (HArg), a structural analog of l-arginine (Arg), is an alternative but less efficient substrate for NOS. Besides, it inhibits arginase, leading to an increased availability of l-arginine for NOS to produce NO. However, its relation with cardiovascular disease remains unclear. To date, several analytical methods for the quantitative determination of Arg, HArg, NMMA, AMDA, and SDMA in biological samples have been described. Here, we present a simple, fast, and accurate HPLC-ESI-MS/MS method which allows both the simultaneous determination and quantification of these compounds without needing derivatization, and the possibility to easily modulate the chromatographic separation between HArg and NMMA (or between SDMA and ADMA). Data on biological samples revealed the feasibility of the method, the minimal sample preparation, and the fast run time which make this method very suitable and accurate for analysis in the basic and clinical settings.     

Transcutaneous oximetry in smokers with moderate hypertension and peripheral arterial disease treated with amlodipine and defibrotide, also with total smoking cessation

To evaluate the hemorheological influence on oxygen release after a period of 4 months of suspension from smoking and of antihypertensive treatment with amlodipine 10 mg o.d. and defibrotide 400 mg o.d. we have studied 14 smokers with II moderate hypertension (according to the World Health Organization) with hypertensive retinopathy II and slight left ventricular hypertrophy and II stage type a peripheral arterial disease according to Leriche-Fontaine classification. The total suspension for a period of 4 months from smoking associated with a Ca-antagonist such as amlodipine and an hemorheological, antithrombotic drug such as defibrotide together could bring about an improvement on the treatment of hypertension and a notable reduction in the risks linked to the complications found in hypertensives with PAOD II type a.