Protein Requirements for Maximal Muscle Mass and Athletic Performance Are Achieved with Completely Plant-Based Diets Scaled to Meet Energy Needs: A Modeling Study in Professional American Football Players.

American football players consume large quantities of animal-sourced protein in adherence with traditional recommendations to maximize muscle development and athletic performance. This contrasts with dietary guidelines, which recommend reducing meat intake and increasing consumption of plant-based foods to promote health and reduce the risk of chronic disease. The capacity of completely plant-based diets to meet the nutritional needs of American football players has not been studied. This modeling study scaled dietary data from a large cohort following completely plant-based diets to meet the energy requirements of professional American football players to determine whether protein, leucine, and micronutrient needs for physical performance and health were met. The Cunningham equation was used to estimate calorie requirements. Nutrient intakes from the Adventist Health Study 2 were then scaled to this calorie level. Protein values ranged from 1.6-2.2 g/kg/day and leucine values ranged from 3.8-4.1 g/meal at each of four daily meals, therefore meeting and exceeding levels theorized to maximize muscle mass, muscle strength, and muscle protein synthesis, respectively. Plant-based diets scaled to meet the energy needs of professional American football players satisfied protein, leucine, and micronutrient requirements for muscle development and athletic performance. These findings suggest that completely plant-based diets could bridge the gap between dietary recommendations for chronic disease prevention and athletic performance in American football players.

Dissection of molecular mechanisms of liver injury induced by microcystin-leucine arginine via single-cell RNA-sequencing.

The occurrence of poisoning incidents caused by cyanobacterial blooms has aroused wide public concern. Microcystin-leucine arginine (MC-LR) is a well-established toxin produced by cyanobacterial blooms, which is widely distributed in eutrophic waters. MC-LR is not only hazardous to the water environment but also exerts multiple toxic effects including liver toxicity in both humans and animals. However, the underlying mechanisms of MC-LR-induced liver toxicity are unclear. Herein, we used advanced single-cell RNA sequencing technology to characterize MC-LR-induced liver injury in mice. We established the first single-cell atlas of mouse livers in response to MC-LR. Our results showed that the differentially expressed genes and pathways in diverse cell types of liver tissues of mice treated with MC-LR are highly heterogeneous. Deep analysis showed that MC-LR induced an increase in a subpopulation of hepatocytes that highly express Gstm3, which potentially contributed to hepatocyte apoptosis in response to MC-LR. Moreover, MC-LR increased the proportion and multiple subtypes of Kupffer cells with M1 phenotypes and highly expressed proinflammatory genes. Furthermore, the MC-LR increased several subtypes of CD8+ T cells with highly expressed multiple cytokines and chemokines. Overall, apart from directly inducing hepatocytes apoptosis, MC-LR activated proinflammatory Kupffer cell and CD8+ T cells, and their interaction may constitute a hostile microenvironment that contributes to liver injury. Our findings not only present novel insight into underlying molecular mechanisms but also provide a valuable resource and foundation for additional discovery of MC-LR-induced liver toxicity.

Molnupiravir's real-world effectiveness in COVID-19 outpatients at high risk of severe disease: a single-center study.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic started in March 2020. Since then, there has been an urgent need for effective therapeutic methods to manage the disease. We aimed to assess the effectiveness of molnupiravir in reducing the need for hospitalization in at-risk, non-hospitalized COVID-19 patients. METHODOLOGY: This was a single-center, non-randomized, observational retrospective study of non-hospitalized patients with confirmed COVID-19, treated at the Clinic for Infectious and Tropical Diseases, University Clinical Center in Belgrade, Serbia. RESULTS: The study was conducted between 15 December 2021 and 15 February 2022 and included 320 patients. Of these, 165 (51.6%) received treatment with molnupiravir. The study and control groups were similar in gender and age distribution. The study group had a higher proportion of vaccination (75.2% vs. 51%, p < 0.001). There was no statistically significant difference in presence of comorbidity within the groups. Majority of the patients who received molnupiravir did not require hospitalization; and this was statistically significant in comparison to control group (92.7 vs. 24.5%, p < 0.001). Oxygen supplementation was less frequently required in the study group compared to the control group (0.6% vs. 31%, p < 0.001). During the follow-up period of 12.12 ± 3.5 days, significantly less patients from the study group were admitted to the intensive care unit (p < 0.001). Molnupiravir significantly reduced the risk of hospitalization by 97.9% (HR 0.021; 95% CI 0.005-0.089; p < 0.001). CONCLUSIONS: Molnupiravir is an effective therapy in preventing the development of severe forms of COVID-19 and hospitalization.

Vesicle-like Nanocapsules Formed by Self-Assembly of Peptides with Oligoproline and -Leucine.

Since drug carriers are envisaged to be used in a wide variety of situations and environments, nanocarriers with diverse properties, such as biocompatibility, biodegradability, nonimmunogenicity, adequate particle size, robustness, and cell permeability, are required. Here, we report the construction of novel nanocapsules with the above-mentioned features by the self-assembly of peptides composed of oligoproline and oligoleucine (i.e., H-Pro10Leu4-NH2 and H-Pro10Leu6-NH2). The peptides self-organized via hydrogen bonds and hydrophobic interactions between oligoleucine moieties to form vesicle-like nanocapsules with cationic oligoproline exposed on the surface. The guest encapsulation experiments revealed that the nanocapsules were capable of uptake of both water-soluble and insoluble compounds. Furthermore, positively charged and/or oligoproline-based peptides are known to improve cell permeability and cellular uptake, suggesting that the peptide nanocapsules are good candidates for nanocarriers to complement liposomes and polymer micelles.

Nirmatrelvir/ritonavir or Molnupiravir for treatment of non-hospitalized patients with COVID-19 at risk of disease progression.

BACKGROUND: In randomized controlled trials, Nirmatrelvir/ritonavir (NMV/r) and Molnupiravir (MPV) reduced the risk of severe/fatal COVID-19 disease. Real-world data are limited, particularly studies directly comparing the two agents. METHODS: Using the VA National COVID-19 database, we identified previously uninfected, non-hospitalized individuals with COVID-19 with ≥1 risk factor for disease progression who were prescribed either NMV/r or MPV within 3 days of a positive test. We used inverse probability of treatment weights (IPTW) to account for providers' preferences for a specific treatment. Absolute risk difference (ARD) with 95% confidence intervals were determined for those treated with NMV/r vs. MPV. The primary outcome was hospitalization or death within 30 days of treatment prescription using the IPTW approach. Analyses were repeated using propensity-score matched groups. RESULTS: Between January 1 and November 30, 2022, 9,180 individuals were eligible for inclusion (6,592 prescribed NMV/r; 2,454 prescribed MPV). The ARD for hospitalization/death for NMV/r vs MPV was -0.25 (95% CI -0.79 to 0.28). There was no statistically significant difference in ARD among strata by age, race, comorbidities, or symptoms at baseline. Kaplan-Meier curves did not demonstrate a difference between the two groups (p-value = 0.6). Analysis of the propensity-score matched cohort yielded similar results (ARD for NMV/r vs. MPV -0.9, 95% CI -2.02 to 0.23). Additional analyses showed no difference for development of severe/critical/fatal disease by treatment group. CONCLUSION: We found no significant difference in short term risk of hospitalization or death among at-risk individuals with COVID-19 treated with either NMV/r or MPV.

Development of a Biosafety Level 1 Cellular Assay for Identifying Small-Molecule Antivirals Targeting the Main Protease of SARS-CoV-2: Evaluation of Cellular Activity of GC376, Boceprevir, Carmofur, Ebselen, and Selenoneine.

While research has identified several inhibitors of the main protease (Mpro) of SARS-CoV-2, a significant portion of these compounds exhibit reduced activity in the presence of reducing agents, raising concerns about their effectiveness in vivo. Furthermore, the conventional biosafety level 3 (BSL-3) for cellular assays using viral particles poses a limitation for the widespread evaluation of Mpro inhibitor efficacy in a cell-based assay. Here, we established a BSL-1 compatible cellular assay to evaluate the in vivo potential of Mpro inhibitors. This assay utilizes mammalian cells expressing a tagged Mpro construct containing N-terminal glutathione S-transferase (GST) and C-terminal hemagglutinin (HA) tags and monitors Mpro autodigestion. Using this method, GC376 and boceprevir effectively inhibited Mpro autodigestion, suggesting their potential in vivo activity. Conversely, carmofur and ebselen did not exhibit significant inhibitory effects in this assay. We further investigated the inhibitory potential of selenoneine on Mpro using this approach. Computational analyses of binding energies suggest that noncovalent interactions play a critical role in facilitating the covalent modification of the C145 residue, leading to Mpro inhibition. Our method is straightforward, cost-effective, and readily applicable in standard laboratories, making it accessible to researchers with varying levels of expertise in infectious diseases.

SARS-CoV-2 Spike Protein Enhances Carboxypeptidase Activity of Angiotensin-Converting Enzyme 2.

In this study, we investigated whether severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein may modify angiotensin-converting enzyme 2 (ACE2) activity in the plasma, heart, kidney, liver, lung, and six brain regions (amygdala, brain stem, cortex, hippocampus, hypothalamus, and striatum) of diabetic and hypertensive rats. We determine ACE2 activity in the plasma and lysates of heart, kidney, liver, lung, and six brain regions. MLN-4760 inhibits ACE2 activity in the plasma and all organs. On the other hand, soluble ACE2 (sACE2) activity increased in the plasma of diabetic rats, and there was no change in the plasma of hypertensive rats. ACE2 activity was augmented in the liver, brain stem, and striatum, while it decreased in the kidney, amygdala, cortex, and hippocampus of diabetic rats. ACE2 activity increased in the kidney, liver, and lung, while it decreased in the heart, amygdala, cortex, and hypothalamus of hypertensive rats. We measured the ACE2 content via enzyme-linked immunosorbent assay and found that ACE2 protein levels increased in the heart, while it decreased in the plasma, kidney, brain stem, cortex, hippocampus, hypothalamus, and striatum of diabetic rats. ACE2 protein levels decreased in the brain stem, cortex, hippocampus, and hypothalamus of hypertensive rats. Our data showed that the spike protein enhanced ACE2 activity in the liver and lungs of diabetic rats, as well as in the heart and three of the brain regions (cortex, hypothalamus, and striatum) of hypertensive rats.

Comparison of the precursor, amino acid oxidation, and end-product methods for the evaluation of protein turnover in senior dogs.

Stable isotope methods have been used to study protein metabolism in humans; however, there application in dogs has not been frequently explored. The present study compared the methods of precursor (13C-Leucine), end-products (15N-Glycine), and amino acid oxidation (13C-Phenylalanine) to determine the whole-body protein turnover rate in senior dogs. Six dogs (12.7 ± 2.6 years age, 13.6 ± 0.6 kg bodyweight) received a dry food diet for maintenance and were subjected to all the above-mentioned methods in succession. To establish 13C and 15N kinetics, according to different methodologies blood plasma, urine, and expired air were collected using a specifically designed mask. The volume of CO2 was determined using respirometry. The study included four methods viz. 13C-Leucine, 13C-Phenylalanine evaluated with expired air, 13C-Phenylalanine evaluated with urine, and 15N-Glycine, with six dogs (repetitions) per method. Data was subjected to variance analysis and means were compared using the Tukey test (P<0.05). In addition, the agreement between the methods was evaluated using Pearson correlation and Bland-Altman statistics. Protein synthesis (3.39 ± 0.33 g.kg-0,75. d-1), breakdown (3.26 ± 0.18 g.kg-0.75.d-1), and flux estimations were similar among the four methods of study (P>0.05). However, only 13C-Leucine and 13C-Phenylalanine (expired air) presented an elevated Pearson correlation and concordance. This suggested that caution should be applied while comparing the results with the other methodologies.

Real-life effectiveness of sofosbuvir/velpatasvir/voxilaprevir in hepatitis C patients previously treated with sofosbuvir/velpatasvir or glecaprevir/pibrentasvir.

BACKGROUND: Sofosbuvir, velpatasvir and voxilaprevir (SOF/VEL/VOX) is the recommended rescue therapy for patients with chronic hepatitis C infection who fail direct-acting antivirals (DAAs). Data are limited on the effectiveness of this treatment after the current first-line therapies. Our aim was to analyse the effectiveness and safety of SOF/VEL/VOX among patients failing sofosbuvir/velpatasvir (SOF/VEL) or glecaprevir/pibrentasvir (GLE/PIB). METHODS: Retrospective multicentre study (26 Spanish hospitals), including chronic hepatitis C patients unsuccessfully treated with SOF/VEL or GLE/PIB, and retreated with SOF/VEL/VOX ± ribavirin for 12 weeks between December 2017 and December 2022. RESULTS: In total, 142 patients included: 100 (70.4%) had failed SOF/VEL and 42 (29.6%) GLE/PIB. Patients were mainly men (84.5%), White (93.9%), with hepatitis C virus genotype (GT) 3 (49.6%) and 47.2% had liver cirrhosis. Sustained virological response (SVR) was evaluated in 132 patients who completed SOF/VEL/VOX and were followed 12 weeks after end of treatment; 117 (88.6%) achieved SVR. There were no significant differences in SVR rates according to initial DAA treatment (SOF/VEL 87.9% vs. GLE/PIB 90.2%, p = 0.8), cirrhosis (no cirrhosis 90% vs. cirrhosis 87.1%, p = 0.6) or GT3 infection (non-GT3 91.9% vs. GT3 85.5%, p = 0.3). However, when considering the concurrent presence of SOF/VEL treatment, cirrhosis and GT3 infection, SVR rates dropped to 82.8%. Ribavirin was added in 8 (6%) patients, all achieved SVR. CONCLUSION: SOF/VEL/VOX is an effective rescue therapy for failures to SOF/VEL or GLE/PIB, with an SVR of 88.6%. Factors previously linked to lower SVR rates, such as GT3 infection, cirrhosis and first-line therapy with SOF/VEL were not associated with lower SVRs.

Two remarkable serine/leucine polymorphisms in Helicobacter pylori: functional importance for serine protease HtrA and adhesin BabA.

Single nucleotide polymorphisms (SNPs) account for significant genomic variability in microbes, including the highly diverse gastric pathogen Helicobacter pylori. However, data on the effects of specific SNPs in pathogen-host interactions are scarce. Recent functional studies unravelled how a serine/leucine polymorphism in serine protease HtrA affects the formation of proteolytically active trimers and modulates cleavage of host cell-to-cell junction proteins during infection. A similar serine/leucine mutation in the carbohydrate binding domain of the adhesin BabA controls binding of ABO blood group antigens, enabling binding of either only the short Lewis b/H antigens of blood group O or also the larger antigens of blood groups A and B. Here we summarize the functional importance of these two remarkable bacterial SNPs and their effect on the outcome of pathogen-host interactions.

Antiviral activity of Vigna radiata extract against feline coronavirus in vitro.

Feline infectious peritonitis (FIP) is a fatal illness caused by a mutated feline coronavirus (FCoV). This disease is characterized by its complexity, resulting from systemic infection, antibody-dependent enhancement (ADE), and challenges in accessing effective therapeutics. Extract derived from Vigna radiata (L.) R. Wilczek (VRE) exhibits various pharmacological effects, including antiviral activity. This study aimed to investigate the antiviral potential of VRE against FCoV, addressing the urgent need to advance the treatment of FIP. We explored the anti-FCoV activity, antiviral mechanism, and combinational application of VRE by means of in vitro antiviral assays. Our findings reveal that VRE effectively inhibited the cytopathic effect induced by FCoV, reduced viral proliferation, and downregulated spike protein expression. Moreover, VRE blocked FCoV in the early and late infection stages and was effective under in vitro ADE infection. Notably, when combined with VRE, the polymerase inhibitor GS-441524 or protease inhibitor GC376 suppressed FCoV more effectively than monotherapy. In conclusion, this study characterizes the antiviral property of VRE against FCoV in vitro, and VRE possesses therapeutic potential for FCoV treatment.

DNA damage induces p53-independent apoptosis through ribosome stalling.

In response to excessive DNA damage, human cells can activate p53 to induce apoptosis. Cells lacking p53 can still undergo apoptosis upon DNA damage, yet the responsible pathways are unknown. We observed that p53-independent apoptosis in response to DNA damage coincided with translation inhibition, which was characterized by ribosome stalling on rare leucine-encoding UUA codons and globally curtailed translation initiation. A genetic screen identified the transfer RNAse SLFN11 and the kinase GCN2 as factors required for UUA stalling and global translation inhibition, respectively. Stalled ribosomes activated a ribotoxic stress signal conveyed by the ribosome sensor ZAKα to the apoptosis machinery. These results provide an explanation for the frequent inactivation of SLFN11 in chemotherapy-unresponsive tumors and highlight ribosome stalling as a signaling event affecting cell fate in response to DNA damage.

Conformational Preferences of the Non-Canonical Amino Acids (2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5'-Difluoroleucine in a Protein.

Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.

(2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5'-Difluoroleucine in Escherichia coli PpiB: Protein Production, 19F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect.

Global substitution of leucine for analogues containing CH2F instead of methyl groups delivers proteins with multiple sites for monitoring by 19F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. 19F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The 19F chemical shifts correlate with the three-bond 1H-19F couplings (3JHF), providing the first experimental verification of the γ-gauche effect predicted by [Feeney, J. J. Am. Chem. Soc. 1996, 118, 8700-8706] and establishing the effect as the predominant determinant of the 19F chemical shifts of CH2F groups. Individual CH2F groups can be confined to single rotameric states by the protein environment, but most CH2F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5'-difluoroleucine bias the CH2F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the 19F chemical shift to the rotameric state of the CH2F groups potentially renders them particularly sensitive for detecting allosteric effects.

Unravelling caramelization and Maillard reactions in glucose and glucose + leucine model cakes: Formation and degradation kinetics of volatile markers extracted during baking.

A large number of volatile compounds are formed during the baking of foods by reactions such as caramelization and Maillard reactions. Elucidating the reaction mechanisms may be useful to predict and control food quality. Ten reaction volatile markers were extracted during baking of solid model cakes implemented with known amounts of precursors (glucose with or without leucine) and then quantified by Thermal desorption-Gas chromatography-Mass spectrometry. The kinetic data showed that the level of air convection in the oven had no significant influence on the reaction rates. In contrast, increasing baking temperatures had a nonlinear accelerating impact on the generation of newly formed volatile compounds with a bell-shaped kinetic curve found for most of the markers at 200 °C. The presence of leucine triggered the activation of the Maillard and Strecker routes with a specific and very rapid formation of 3-Methylbutanal and pyrazines. A dynamic model was developed, combining evaporation flow rate and kinetic formation and consumption of reaction markers. It can be used to describe, for two furanic compounds of different volatilities, the vapor concentrations in the oven from the concentrations measured in the model cakes.

Synthesis, taste characteristics and taste mechanism of N-lactoyl leucine from soy sauce using sensory analysis and UPLC-MS/MS.

Recently, amino acid derivatives gradually gained attention, but studies on N-lactoyl-leucine (Lac-Leu) and N-lactoyl-isoleucine (Lac-Ile) are limited. This study aims to explore the contributions of Lac-Leu and Lac-Ile to soy sauce. Lac-Leu and Lac-Ile were synthesized via enzymatic synthesis method catalyzed by Tgase. The mixed solutions containing Lac-Leu were found to have greater taste improvement than those containing Lac-Ile. Sensory evaluation indicated the sour, bitter, and astringent taste of Lac-Leu in water as well as its kokumi, astringent, and umami-enhancing taste in MSG solution. The taste threshold and umami-enhancing threshold of Lac-Leu measured by TDA and cTDA, respectively, were 0.08 mg/mL and 0.16 mg/mL. Molecular docking of Lac-Leu and Lac-Ile with the kokumi receptor CaSR and the umami receptors T1R1 and T1R3 indicated that Lac-Leu had higher affinities with receptors than Lac-Ile. These findings demonstrated the underlying contribution Lac-Leu made to soy sauce, indicating its potential to improve the flavor quality of soy sauce.

Glycyrrhizic acid conjugates with amino acid methyl esters target the main protease, exhibiting antiviral activity against wild-type and nirmatrelvir-resistant SARS-CoV-2 variants.

COVID-19 pandemic is predominantly caused by SARS-CoV-2, with its main protease, Mpro, playing a pivotal role in viral replication and serving as a potential target for inhibiting different variants. In this study, potent Mpro inhibitors were identified from glycyrrhizic acid (GL) derivatives with amino acid methyl/ethyl esters. Out of the 17 derivatives semisynthesized, Compounds 2, 6, 9, and 15, with methionine methyl esters, D-tyrosine methyl esters, glutamic acid methyl esters, and methionines in the carbohydrate moiety, respectively, significantly inhibited wild-type SARS-CoV-2 Mpro-mediated proteolysis, with IC50 values ranging from 0.06 µM to 0.84 µM. They also demonstrated efficacy in inhibiting trans-cleavage by mutant Mpro variants (Mpro_P132H, Mpro_E166V, Mpro_P168A, Mpro_Q189I), with IC50 values ranging from 0.05 to 0.92 µM, surpassing nirmatrelvir (IC50: 1.17-152.9 µM). Molecular modeling revealed stronger interactions with Valine166 in the structural complex of Mpro_E166V with the compounds compared to nirmatrelvir. Moreover, these compounds efficiently inhibited the post-entry viral processes of wild-type SARS-CoV-2 single-round infectious particles (SRIPs), mitigating viral cytopathic effects and reducing replicon-driven GFP reporter signals, as well as in vitro infectivity of wild-type, Mpro_E166V, and Mpro_Q189I SRIPs, with EC50 values ranging from 0.02 to 0.53 µM. However, nirmatrelvir showed a significant decrease in inhibiting the replication of mutant SARS-CoV-2 SRIPs carrying Mpro_E166V (EC50: >20 µM) and Mpro_Q189I (EC50: 13.2 µM) compared to wild-type SRIPs (EC50: 0.06 µM). Overall, this study identifies four GL derivatives as promising lead compounds for developing treatments against various SARS-CoV-2 strains, including Omicron, and nirmatrelvir-resistant variants.

The efficacy and safety of leucine-enriched essential amino acids in knee osteoarthritis patients: A randomized controlled trial.

BACKGROUND: Supplementation with leucine-enriched essential amino acids (LEAAs) has shown efficacy in the recovery of muscle injury and activation of muscle synthesis. Muscle function in knee osteoarthritis is a crucial factor for managing pain and preserving ambulatory function. However, the efficacy and safety of LEAAs supplementation in patients with knee osteoarthritis have not been evaluated. METHODS: In this prospective analysis, we evaluated the efficacy and safety of supplementation with 12 g of LEAAs daily for 8 weeks in knee-symptomatic osteoarthritis patients. For assessing the efficacy, clinical pain, calf circumference, and disability were assessed using questionnaires (visual analog scale, Knee Society Score, and 36-item short form survey [SF-36]), laboratory analyses (total protein and albumin), and radiologic study (dual-energy X-ray absorptiometry [DEXA]) for muscle and bone density. To evaluate safety, generalized or localized protein allergic reactions, complete blood count, liver and kidney function, and serum glucose were measured. RESULTS: Sixty-five participants, categorized into the experimental (n = 32) and control (n = 33) groups, were included in this 8-week trial from March 2022 to July 2022. A significantly higher efficacy was observed in the experimental group than in the control group, as indicated by muscle density in the DEXA scan (P = .001) and SF-36 (P < .001). The safety evaluation revealed no related generalized or local protein allergy. Hematological findings, serum glucose, and kidney and liver toxicity were not significantly different between the groups. CONCLUSION: Supplementation with leucine-enriched proteins is safe and efficacious in the improvement of muscle density and quality of life.

Leucine promotes energy metabolism and stimulates slow-twitch muscle fibers expression through AMPK/mTOR signaling in equine skeletal muscle satellite cells.

Previous research has shown that leucine (Leu) can stimulate and enhance the proliferation of equine skeletal muscle satellite cells (SCs). The gene expression profile associated with Leu-induced proliferation of equine SCs has also been documented. However, the specific role of Leu in regulating the expression of slow-twitch muscle fibers (slow-MyHC) and mitochondrial function in equine SCs, as well as the underlying mechanism, remains unclear. During this investigation, equine SCs underwent culturing in differentiation medium and were subjected to varying concentrations of Leu (0 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM) over a span of 3 days. AMP-activated protein kinase (AMPK) inhibitor Compound C and mammalian target of rapamycin complex (mTOR) inhibitor Rapamycin were utilized to explore its underlying mechanism. Here we showed that the expression of slow-MyHC at 2 mM Leu level was significantly higher than the concentration levels of 0 mM,0.5 mM and 10 mM (P <0.01), and there was no significant difference compared to other groups (P > 0.05); the basal respiration, maximum respiration, standby respiration and the expression of slow-MyHC, PGC-1α, Cytc, ND1, TFAM, and COX1 were significantly increased with Leu supplementation (P < 0.01). We also found that Leu up-regulated the expression of key proteins on AMPK and mTOR signaling pathways, including LKB1, p-LKB1, AMPK, p-AMPK, S6, p-S6, 4EBP1, p-4EBP1, mTOR and p-mTOR (P < 0.05 or P < 0.01). Notably, when we treated the equine SCs with the AMPK inhibitor Compound C and the mTOR inhibitor Rapamycin, we observed a reduction in the beneficial effects of Leu on the expression of genes related to slow-MyHC and signaling pathway-related gene expressions. This study provides novel evidence that Leu promotes slow-MyHC expression and enhances mitochondrial function in equine SCs through the AMPK/mTOR signaling pathways, shedding light on the underlying mechanisms involved in these processes for the first time.

SRX3177, a CDK4/6-PI3K-BET inhibitor, in combination with an RdRp inhibitor, Molnupiravir, or an entry inhibitor MU-UNMC-2, has potent antiviral activity against the Omicron variant of SARS-CoV-2.

Despite considerable progress in developing vaccines and antivirals to combat COVID-19, the rapid mutations of the SARS-CoV-2 genome have limited the durability and efficacy of the current vaccines and therapeutic interventions. Hence, it necessitates the development of novel therapeutic approaches or repurposing existing drugs that target either viral life cycle, host factors, or both. Here, we report that SRX3177, a potent triple-activity CDK4/6-PI3K-BET inhibitor, blocks replication of the SARS-CoV-2 Omicron variant with IC50 values at sub-micromolar concentrations without any impact on the cell proliferation of Calu-3 cells at and below its IC50 concentration. When SRX3177 is combined with EIDD-1931 (active moiety of a small-molecule prodrug Molnupiravir) or MU-UNMC-2 (a SARS-CoV-2 entry inhibitor) at a fixed doses matrix, a synergistic effect was observed, leading to the significant reduction in the dose of the individual compounds to achieve similar inhibition of SARS-CoV-2 replication. Herein, we report that the combination of SRX3177/MPV or SRX3177/UM-UNMC-2 has the potential for further development as a combinational therapy against SARS-CoV-2 and in any future outbreak of beta coronavirus.