Sirt3 and metabolic reprogramming mediate the antiproliferative effects of whey in human colon cancer cells

Emerging strategies to improve healthy aging include dietary interventions as a tool to promote health benefits and reduce the incidence of aging-related comorbidities. The health benefits of milk are also linked to its richness in betaines and short-chain acylcarnitines, which act synergisti-cally in conferring anticancer, anti-inflammatory, and antioxidant properties. Whey, despite being a dairy by-product, still has a considerable content of bioactive betaines and acylcarnitines. Here, we investigated the anticancer properties of whey from Mediterranean water buffalo (Bubalus bubalis) milk by testing its antiproliferative effects in colorectal cancer (CRC) cells HT-29, HCT 116, LoVo and SW480. Results indicated that treatment with whey for 72 h inhibited cell proliferation (p < 0.001), induced cell cycle arrest, and apoptosis via caspase-3 activation, and modulated cell metabolism by limiting glucose uptake and interfering with mitochondrial energy metabolism with the highest effects observed in HT-29 and HCT 116 cells. At molecular level, these effects were accompanied by upregulation of sirtuin 3 (SIRT3) (p < 0.01) and peroxisome proliferator-activated receptor (PPAR)-γ expression (p < 0.001), and downregulation of lactate dehydrogenase A (LDHA) (p < 0.01), sterol regulatory-element binding protein 1 (SREBP1) (p < 0.05), and PPAR-α (p < 0.01). Transient SIRT3 gene silencing blocked the effects of whey on the LDHA, PPAR-γ, and PPAR-α protein expressions (p < 0.01) suggesting that the whey capacity of perturbating the metabolic homeostasis in CRC cell lines is mediated by SIRT3

Detection of Brucella abortus DNA and RNA in different stages of development of the sucking louse Haematopinus tuberculatus

Background: Brucellosis is considered the world’s most widespread zoonotic infection. It causes abortion and sterility in livestock leading to serious economic losses and has even more serious medical impact in humans, since it can be a trigger to more than 500,000 infections per year worldwide. The aim of this study was to evaluate the role of Haematopinus tuberculatus, a louse that can parasitize several ruminants, as a new host of brucellosis. Louse specimens were collected from seropositive and seronegative water buffaloes and divided in 3 developmental stages: adults, nymphs and nits. All samples were separately screened for Brucella spp. DNA and RNA detection by Real Time PCR. In particular, primers and probes potentially targeting the 16S rRNA and the Brucella Cell Surface 31 kDalton Protein (bcsp31) genes were used for Real Time PCR and buffalo β actin was used as a housekeeping gene to quantify host DNA in the sample. A known amount of B. abortus purified DNA was utilized for standard curve preparation and the target DNA amount was divided by the housekeeping gene amount to obtain a normalized target value. A further molecular characterization was performed for Brucella strain typing and genotyping by the Bruce-ladder, AMOS-PCR and MLVA assays. Data were statistically analysed by ANOVA. Results: Brucella abortus DNA and RNA were detected in all developmental stages of the louse, suggesting the presence of viable bacteria. Data obtained by MLVA characterization support this finding, since the strains present in animals and the relative parasites were not always identical, suggesting bacterial replication. Furthermore, the detection of Brucella DNA and RNA in nits samples demonstrate, for the first time, a trans-ovarial transmission of the bacterium into the louse. Conclusions: These findings identified H. tuberculatus as a new host of brucellosis. Further studies are needed to establish the role of this louse in the epidemiology of the disease, such as vector or reservoir

Synergistic effect of dietary betaines on sirt1-mediated apoptosis in human oral squamous cell carcinoma cal 27

Betaines are food components widely distributed in plants, animals, microorganisms, and dietary sources. Among betaines, δ-valerobetaine (N,N,N-trimethyl-5-aminovaleric acid, δVB) shares a metabolic pathway common to γ-butyrobetaine (γBB). The biological properties of δVB are particularly attractive, as it possesses antioxidant, anti-inflammatory and anticancer activities. Here, we investigated the possible synergism between δVB and the structurally related γBB, to date unexplored, by testing the in vitro anticancer activity in head and neck squamous cell carcinoma cell lines, FaDu, UM-SCC-17A and Cal 27. Among cell lines tested, results indicated that betaines showed the highest effect in reducing Cal 27 cell proliferation up to 72 h (p &lt; 0.01). This effect was enhanced when betaines were administered in combination (δVB plus γBB) (p &lt; 0.001). Inhibition of cell growth by δVB plus γBB involved reactive oxygen species (ROS) accumulation, upregulation of sirtuin 1 (SIRT1), and apoptosis (p &lt; 0.001). SIRT1 gene silencing by small interfering RNA decreased the apoptotic effect of δVB plus γBB by modulating downstream procaspase-3 and cyclin B1 (p &lt; 0.05). These findings might have important implications for novel prevention strategies for tongue squamous cell carcinoma by targeting SIRT1 with naturally occurring betaines

SIRT3 Modulates Endothelial Mitochondrial Redox State during Insulin Resistance

Emerging evidence indicates that defects in sirtuin signaling contribute to impaired glucose and lipid metabolism, resulting in insulin resistance (IR) and endothelial dysfunction. Here, we examined the effects of palmitic acid (PA) treatment on mitochondrial sirtuins (SIRT2, SIRT3, SIRT4, and SIRT5) and oxidative homeostasis in human endothelial cells (TeloHAEC). Results showed that treatment for 48 h with PA (0.5 mM) impaired cell viability, induced loss of insulin signaling, imbalanced the oxidative status (p &lt; 0.001), and caused negative modulation of sirtuin protein and mRNA expression, with a predominant effect on SIRT3 (p &lt; 0.001). Restoration of SIRT3 levels by mimic transfection (SIRT3+) suppressed the PA-induced autophagy (mimic NC+PA) (p &lt; 0.01), inflammation, and pyroptosis (p &lt; 0.01) mediated by the NLRP3/caspase-1 axis. Moreover, the unbalanced endothelial redox state induced by PA was counteracted by the antioxidant δ-valerobetaine (δVB), which was able to upregulate protein and mRNA expression of sirtuins, reduce reactive oxygen species (ROS) accumulation, and decrease cell death. Overall, results support the central role of SIRT3 in maintaining the endothelial redox homeostasis under IR and unveil the potential of the antioxidant δVB in enhancing the defense against IR-related injuries

Different non-structural carbohydrates/crude proteins (NCS/CP) ratios in diet shape the gastrointestinal microbiota of water buffalo

The microbiota of the gastrointestinal tract (GIT) are crucial for host health and production efficiency in ruminants. Its microbial composition can be influenced by several endogenous and exogenous factors. In the beef and dairy industry, the possibility to manipulate gut microbiota by diet and management can have important health and economic implications. The aims of this study were to characterize the different GIT site microbiota in water buffalo and evaluate the influence of diet on GIT microbiota in this animal species. We characterized and compared the microbiota of the rumen, large intestine and feces of water buffaloes fed two different diets with different non-structural carbohydrates/crude proteins (NSC/CP) ratios. Our results indicated that Bacteroidetes, Firmicutes and Proteobacteria were the most abundant phyla in all the GIT sites, with significant differences in microbiota composition between body sites both within and between groups. This result was particularly evident in the large intestine, where beta diversity analysis displayed clear clustering of samples depending on the diet. Moreover, we found a difference in diet digestibility linked to microbiota modification at the GIT level conditioned by NSC/CP levels. Diet strongly influences GIT microbiota and can therefore modulate specific GIT microorganisms able to affect the health status and performance efficiency of adult animals

Enhanced GRK2 expression and desensitization of betaAR vasodilatation in hypertensive patients.

Increased levels of G protein coupled receptor kinase GRK2 appear to participate in hypertension presumably through the desensitization of beta adrenergic receptors (betaARs) that mediate vasodilatation. There are contrasting data on the occurrence of betaAR desensitization in the vasculature, we therefore investigated betaAR vasodilatation and desensitization in normotensives and in hypertensive humans. In blood lymphocytes, we assessed betaAR signaling and GRK2 expression and found betaAR signaling alterations and, consistent with desensitization, ncreased GRK2 levels in hypertensives. We studied in vivo vasodilatation to the betaAR agonist isoproterenol (ISO) injected in the brachia artery in control conditions and during the concomitant infusion of heparin, a known in vitro nonspecific GRK inhibitor. ISO induced a dose-dependent vasorelaxation that was attenuated in hypertensives indicating a loss of betaAR signaling. Intra-arterial infusion of heparin nhibited lymphocyte GRK2 activity and prevented desensitization of betaAR vasodilatation in normotensives. In hypertensives, heparin restored vasodilatation to ISO, to levels observed in normotensives. Our results suggest that betaAR desensitization does indeed occur at the vascular levels in vivo, and that heparin by acting as a GRK inhibitor prevents this in normotensives and restores impaired betaAR vasodilation in hypertensives. We conclude that desensitization participates to impaired betaAR vasodilation in hypertension

ROS-Mediated Apoptotic Cell Death of Human Colon Cancer LoVo Cells by Milk δ-Valerobetaine

δ-Valerobetaine (δVB) is a constitutive milk metabolite with antioxidant and anti-inflammatory activities. Here, we tested the antineoplastic properties of milk δVB on human colorectal cancer cells. CCD 841 CoN (non-tumorigenic), HT-29 (p53 mutant adenocarcinoma) and LoVo (APC/RAS mutant adenocarcinoma) cells were exposed to 3 kDa milk extract, δVB (2 mM) or milk+δVB up to 72 h. Results showed a time- and dose-dependent capability of δVB to inhibit cancer cell viability, with higher potency in LoVo cells. Treatment with milk+δVB arrested cell cycle in G2/M and SubG1 phases by upregulating p21, cyclin A, cyclin B1 and p53 protein expressions. Noteworthy, δVB also increased necrosis (P &lt; 0.01) and when used in combination with milk it improved its activity on live cell reduction (P &lt; 0.05) and necrosis (P &lt; 0.05). δVB-enriched milk activated caspase 3, caspase 9, Bax/Bcl-2 apoptotic pathway and reactive oxygen species (ROS) production, whereas no effects on ROS generation were observed in CCD 841 CoN cells. The altered redox homeostasis induced by milk+δVB was accompanied by upregulation of sirtuin 6 (SIRT6). SIRT6 silencing by small interfering RNA blocked autophagy and apoptosis activated by milk+δVB, unveiling the role of this sirtuin in the ROS-mediated apoptotic LoVo cell death

Multifunctional Core@Satellite Magnetic Particles for Magnetoresistive Biosensors

Magnetoresistive (MR) biosensors combine distinctive features such as small size, low cost, good sensitivity, and propensity to be arrayed to perform multiplexed analysis. Magnetic nanoparticles (MNPs) are the ideal target for this platform, especially if modified not only to overcome their intrinsic tendency to aggregate and lack of stability but also to realize an interacting surface suitable for biofunctionalization without strongly losing their magnetic response. Here, we describe an MR biosensor in which commercial MNP clusters were coated with gold nanoparticles (AuNPs) and used to detect human IgG in water using an MR biochip that comprises six sensing regions, each one containing five U-shaped spin valve sensors. The isolated AuNPs (satellites) were stuck onto an aggregate of individual iron oxide crystals (core) so that the resulting core@satellite magnetic particles (CSMPs) could be functionalized by the photochemical immobilization technique an easy procedure that leads to oriented antibodies immobilized upright onto gold. The morphological, optical, hydrodynamic, magnetic, and surface charge properties of CSMPs were compared with those exhibited by the commercial MNP clusters showing that the proposed coating procedure endows the MNP clusters with stability and ductility without being detrimental to magnetic properties. Eventually, the high-performance MR biosensor allowed us to detect human IgG in water with a detection limit of 13 pM (2 ng mL-1). Given its portability, the biosensor described in this paper lends itself to a point-of-care device; moreover, the features of the MR biochip also make it suitable for multiplexed analysis