Establishing authenticity of hay milk: Detection of silage feeding through cyclopropane fatty acids analysis using 1H NMR spectroscopy.

Cyclopropane fatty acids (CPFAs) serve as indicators of silage feeding, verifying the authenticity of hay milk where silage feeding is forbidden. In this study, the authenticity of hay milk was determined by detecting CPFAs using proton nuclear magnetic resonance (1H NMR) spectroscopy. 245 milk samples were collected in South Tyrol (Italy), categorized as follows: 98 from grass silage-fed cows, 98 from maize silage-fed cows, and 49 authentic hay milk. The limit of detection of CPFAs was 12 µM, corresponding to 70 mg/kg of freeze-dried milk. The CPFAs were absent in all of the hay milk samples, verifying their authenticity. In contrast, 97 % of maize silage and 77 % of grass silage samples exhibited distinct CPFAs signals. These findings were further corroborated by gas chromatography-mass detector (GC-MS) analysis. The study highlights 1H NMR as a robust, and rapid technique for hay milk authentication, supporting alpine dairy production and increasing consumer trust in food authenticity.

Virtual Double-System Single-Box: A Nonequilibrium Alchemical Technique for Absolute Binding Free Energy Calculations: Application to Ligands of the SARS-CoV-2 Main Protease.

In the context of drug-receptor binding affinity calculations using molecular dynamics techniques, we implemented a combination of Hamiltonian replica exchange (HREM) and a novel nonequilibrium alchemical methodology, called virtual double-system single-box, with increased accuracy, precision, and efficiency with respect to the standard nonequilibrium approaches. The method has been applied for the determination of absolute binding free energies of 16 newly designed noncovalent ligands of the main protease (3CLpro) of SARS-CoV-2. The core structures of 3CLpro ligands were previously identified using a multimodal structure-based ligand design in combination with docking techniques. The calculated binding free energies for four additional ligands with known activity (either for SARS-CoV or SARS-CoV-2 main protease) are also reported. The nature of binding in the 3CLpro active site and the involved residues besides the CYS-HYS catalytic dyad have been thoroughly characterized by enhanced sampling simulations of the bound state. We have identified several noncongeneric compounds with predicted low micromolar activity for 3CLpro inhibition, which may constitute possible lead compounds for the development of antiviral agents in Covid-19 treatment.

Interaction of hydroxychloroquine with SARS-CoV2 functional proteins using all-atoms non-equilibrium alchemical simulations.

Using a combination of enhanced sampling molecular dynamics techniques and non-equilibrium alchemical transformations with full atomistic details, we have shown that hydroxychloroquine (HCQ) may act as a mild inhibitor of important functional proteins for SARS-CoV2 replication, with potency increasing in the series PLpro, 3CLpro, RdRp. By analyzing the bound state configurations, we were able to improve the potency for the 3CLpro target, designing a novel HCQ-inspired compound, named PMP329, with predicted nanomolar activity. If confirmed in vitro, our results provide a molecular rationale for the use of HCQ or of strictly related derivatives in the treatment of Covid-19.

Effect of surgery on pancreatic tumor-dependent lymphocyte asset: modulation of natural killer cell frequency and cytotoxic function.

OBJECTIVES: Tumor burden and invasiveness establish a microenvironment that surgery could alter. This study shows a comprehensive analysis of size, dynamics, and function of peripheral lymphocyte subsets in pancreatic cancer patients before and at different times after duodenopancreatectomy. METHODS: Lymphocyte frequency and natural cytotoxicity were evaluated by flow cytometry and in vitro assay on peripheral blood from initial and advanced-stage pancreatic cancer patients before (BS), at day 7 (PS7), and at day 30 (PS30) after surgery. RESULTS: An increase in natural killer (NK) cells and the diminution of B-cells occurred at PS30, whereas cytotoxicity decreased at PS7. The positive correlation between NK frequency and cytotoxicity at BS and PS7 revealed an altered NK behavior. The elevation of NK cell frequency at PS30, an initial defect in CD56bright NK, and the aberrant correlation between NK frequency and cytotoxicity remained significant in advanced-stage patients, whereas the diminution of NK cytotoxicity only affected initial stage patients. CONCLUSIONS: The NK cell functional ability is altered in presurgery patients; duodenopancreatectomy is associated with short-term impairment of NK function and with a long-term NK cell augmentation and reversion of the aberrant NK behavior, which may impact on immunosurveillance against residual cancer.