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

1-N6-Etheno-ADP-ribosylation of elongation factor-2 by diphtheria toxin

AbstractDiphtheria toxin fragment A is able to inhibit protein synthesis in the eukaryotic cell by ADP-ribosylating the diphthamide residue of elongation factor-2 (EF-2) [(1980) J. Biol. Chem. 255, 10710-10720]. The reaction requires NAD as ADP-ribose donor. This work reports on the capacity of an NAD analog, the nicotinamide 1-N6-ethenoadenine dinucleotide (ϵNAD), to be a substrate of diphtheria toxin fragment A in the transferring reaction of the fluorescent moiety, the ϵADP-ribose, to the EF-2. As a consequence of the transfer of the ϵADP-ribosyl moiety to the EF-2, there is an increase in the emission intensity of the fluorophore and a blue shift in its emission maximum. The ϵADP-ribosylated EF-2, like ADP-ribosylated EF-2, retains the capacity to bind GTP and ribosome. The utility of introducing a fluorescent probe in a well defined point of the EF-2 molecule for conformational or binding studies is discussed

Combining electrostatic, hindrance and diffusive effects for predicting particle transport and separation efficiency in deterministic lateral displacement microfluidic devices

Microfluidic separators based on Deterministic Lateral Displacement (DLD) constitute a promising technique for the label-free detection and separation of mesoscopic objects of biological interest, ranging from cells to exosomes. Owing to the simultaneous presence of different forces contributing to particle motion, a feasible theoretical approach for interpreting and anticipating the performance of DLD devices is yet to be developed. By combining the results of a recent study on electrostatic effects in DLD devices with an advection-diffusion model previously developed by our group, we here propose a fully predictive approach (i.e., ideally devoid of adjustable parameters) that includes the main physically relevant effects governing particle transport on the one hand, and that is amenable to numerical treatment at affordable computational expenses on the other. The approach proposed, based on ensemble statistics of stochastic particle trajectories, is validated by comparing/contrasting model predictions to available experimental data encompassing different particle dimensions. The comparison suggests that at low/moderate values of the flowrate the approach can yield an accurate prediction of the separation performance, thus making it a promising tool for designing device geometries and operating conditions in nanoscale applications of the DLD technique

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

Influence of days after calving and thermal stress on the efficacy of a progesterone-based treatment in acyclic italian mediterranean buffalo

The aim of this study was to evaluate the efficacy of a progesterone-based treatment on anoestrus in buffaloes. Primiparous acyclic buffaloes (n = 276), were divided into three classes according to their days in milk (DIM): from 50 to 90 (Class I; n = 86), from 91 to 150 (Class II; n = 102) and from 150 to 200 (Class III; n = 88). Animals were synchronized using P4 vaginal implants, followed by timed artificial insemination (TAI). They were then allowed to enter into a larger group of buffaloes for natural mating 15 days after AI was performed, and pregnancy status was monitored from then on at 15-day intervals. Finally, the temperature–humidity index (THI) was calculated. Statistical analysis was performed by ANOVA by means and both multiple and linear regression. The total pregnancy rate (PR) was 87.7%, with no differences among DIM classes (88.0, 92.4, and 80.0% in Classes I, II, and III, respectively). However, the PR at TAI tended to be higher (p = 0.07) in buffaloes in Class II. The follicle (FL) area in Class II buffaloes was larger (p < 0.01) than that of the other classes. No influence of the THI on the total PR was recorded. The pregnancy outcome at TAI was affected by the FL area (odds ratio = 2.237; p < 0.05) and body condition score (BCS) (odds ratio = 1.256; p < 0.05). In conclusion, treatment with vaginal P4 optimizes pregnancy rates in anoestrus buffaloes, particularly when the animals are in mid-lactation and show an optimal BCS. Furthermore, the THI does not seem to affect the efficiency of the progesterone treatment