Evaluation of the Inhibition Potency of Nirmatrelvir against Main Protease Mutants of SARS-CoV-2 Variants.

SARS-CoV-2 continues to pose a threat to public health. Main protease (Mpro) is one of the most lucrative drug targets for developing specific antivirals against SARS-CoV-2 infection. By targeting Mpro, peptidomimetic nirmatrelvir is able to inhibit viral replication of SARS-CoV-2 and reduce the risk for progression to severe COVID-19. However, multiple mutations in the gene encoding Mpro of emerging SARS-CoV-2 variants raise a concern of drug resistance. In the present study, we expressed 16 previously reported SARS-CoV-2 Mpro mutants (G15S, T25I, T45I, S46F, S46P, D48N, M49I, L50F, L89F, K90R, P132H, N142S, V186F, R188K, T190I, and A191V). We evaluated the inhibition potency of nirmatrelvir against these Mpro mutants and solved the crystal structures of representative Mpro mutants of SARS-CoV-2 bound to nirmatrelvir. Enzymatic inhibition assays revealed that these Mpro variants remain susceptible to nirmatrelvir as the wildtype. Detailed analysis and structural comparison provided the inhibition mechanism of Mpro mutants by nirmatrelvir. These results informed the ongoing genomic surveillance of drug resistance of emerging SARS-CoV-2 variants to nirmatrelvir and facilitate the development of next-generation anticoronavirus drugs.

Effects of Complex Antioxidants Added to Chicken Diet on Growth Performance, Serum Biochemical Indices, Meat Quality, and Antioxidant Capacity.

This study aimed to evaluate the effects of diets supplemented with various levels of complex antioxidants (CA) containing tertiary butylhydroquinone (TBHQ) and tea polyphenols (TP) on growth performance, meat quality of breast and leg muscles, serum biochemistry, and antioxidant capacity of serum, liver, breast meat, jejunum, and ileum in broilers. A total of 600 one-day-old Arbor Acres male broilers with similar body weights were randomly divided into three groups (10 replicates/group, 20 broilers/replicate). Birds in the three experimental groups were fed a basal diet with CA at 0, 300, and 500 mg/kg. The results showed that supplementing with 300 mg/kg CA significantly increased (p < 0.05) 42 d BW and 22-42 d ADG, and markedly decreased (p < 0.05) 22-42 d F: G ratio in comparison to the control group. Birds fed a diet with 300 mg/kg CA had a higher (p < 0.05) pH of chicken meat at 24 h and 48 h post mortem and lower (p < 0.05) yellowness values (b*) of chicken meat at 45 min and 24 h post mortem, along with a lower (p < 0.05) cooking loss. Supplementing with 300 mg/kg CA significantly increased (p < 0.05) serum and liver T-SOD activity, serum T-AOC level, as well as jejunual GST activity, and significantly decreased (p < 0.05) liver MDA content when compared with the control group. These results indicate that diet supplementation with 300 mg/kg CA containing TBHQ and TP could improve growth performance and meat quality by increasing the antioxidant capacity of broilers.

Structural Basis for Coronaviral Main Proteases Inhibition by the 3CLpro Inhibitor GC376.

The main protease (Mpro) of coronaviruses participates in viral replication, serving as a hot target for drug design. GC376 is able to effectively inhibit the activity of Mpro, which is due to nucleophilic addition of GC376 by binding covalently with Cys145 in Mpro active site. Here, we used fluorescence resonance energy transfer (FRET) assay to analyze the IC50 values of GC376 against Mpros from six different coronaviruses (SARS-CoV-2, HCoV-229E, HCoV-HUK1, MERS-CoV, SARS-CoV, HCoV-NL63) and five Mpro mutants (G15S, M49I, K90R, P132H, S46F) from SARS-CoV-2 variants. The results showed that GC376 displays effective inhibition to various coronaviral Mpros and SARS-CoV-2 Mpro mutants. In addition, the crystal structures of SARS-CoV-2 Mpro (wide type)-GC376, SARS-CoV Mpro-GC376, MERS-CoV Mpro-GC376, and SARS-CoV-2 Mpro mutants (G15S, M49I, S46F, K90R, and P132H)-GC376 complexes were solved. We found that GC376 is able to fit into the active site of Mpros from different coronaviruses and different SARS-CoV-2 variants properly. Detailed structural analysis revealed key molecular determinants necessary for inhibition and illustrated the binding patterns of GC376 to these different Mpros. In conclusion, we not only proved the inhibitory activity of GC376 against different Mpros including SARS-CoV-2 Mpro mutants, but also revealed the molecular mechanism of inhibition by GC376, which will provide scientific guidance for the development of broad-spectrum drugs against SARS-CoV-2 as well as other coronaviruses.

Metabolomics analysis to interpret changes in physiological and metabolic responses to chronic heat stress in Pekin ducks.

Heat stress (HS) is a major environmental threat that affects duck production in subtropical and tropical regions, especially in summer. This study aimed to evaluate the physiological and metabolic responses of Pekin ducks to chronic HS conditions via liquid chromatography-mass spectrometry (LC-MS) using a paired-fed (PF) experimental design. On the basis of equivalent feed intake (HS vs. PF), HS significantly reduced growth performance and the percentage of leg and breast muscles, however, markedly increased the percentage of abdominal fat and breast skin fat. Serum metabolomics results revealed that heat-stressed ducks showed enhanced glycolysis and pentose phosphate pathways, as demonstrated by higher glucose 6-phosphate and 6-phogluconic acid levels in the PF vs. HS comparison. HS decreased hepatic mRNA levels of mitochondrial fatty acid ß-oxidation-related genes (MCAD and SCAD) compared to the PF group, resulting in acetylcarnitine accumulation in serum. Moreover, HS elevated the concentrations of serum amino acids and mRNA levels of ubiquitination-related genes (MuRF1 and MAFbx) in the skeletal muscle and amino acid transporter-related genes (SLC1A1 and SLC7A1) and gluconeogenesis-related genes (PCK1 and PCase) in the liver compared to the PF group. When compared to the normal control group (NC), HS further decreased growth performance, but it elevated the abdominal fat rate. However, increased mRNA levels of ubiquitination-related genes and serum amino acid accumulation were not observed in the HS group compared to the NC group, implying that reduced feed intake masked the effect of HS on skeletal muscle breakdown and is a form of protection for the organism. These results suggest that chronic HS induces protein degradation in the skeletal muscle to provide amino acids for hepatic gluconeogenesis to provide sufficient energy, as Pekin ducks under HS conditions failed to efficiently oxidise fatty acids and ketones in the mitochondria, leading to poor growth performance and slaughter characteristics.

Crystal structures of main protease (Mpro) mutants of SARS-CoV-2 variants bound to PF-07304814.

There is an urgent need to develop effective antiviral drugs to prevent the viral infection caused by constantly circulating SARS-CoV-2 as well as its variants. The main protease (Mpro) of SARS-CoV-2 is a salient enzyme that plays a vital role in viral replication and serves as a fascinating therapeutic target. PF-07304814 is a covalent inhibitor targeting SARS-CoV-2 Mpro with favorable inhibition potency and drug-like properties, thus making it a promising drug candidate for the treatment of COVID-19. We previously solved the structure of PF-07304814 in complex with SARS-CoV-2 Mpro. However, the binding modes of PF-07304814 with Mpros from evolving SARS-CoV-2 variants is under-determined. In the current study, we expressed six Mpro mutants (G15S, K90R, M49I, S46F, V186F, and Y54C) that have been identified in Omicron variants including the recently emerged XBB.1.16 subvariant and solved the crystal structures of PF-07304814 bound to Mpro mutants. Structural analysis provided insight into the key molecular determinants responsible for the interaction between PF-07304814 and these mutant Mpros. Patterns for PF-07304814 to bind with these investigated Mpro mutants and the wild-type Mpro are generally similar but with some differences as revealed by detailed structural comparison. Structural insights presented in this study will inform the development of novel drugs against SARS-CoV-2 and the possible conformation changes of Mpro mutants when bound to an inhibitor.

Sirtuin 1 participates in intervertebral disc degeneration via the nicotinamide phosphoribosyl transferase/nicotinamide adenine dinucleotide/sirtuin 1 pathway responsible for regulating autophagy of nucleus pulposus cells.

Disc degeneration is the main cause of discogenic low back pain, disc herniation, degenerative stenosis of spinal canal, lumbar spondylolisthesis and other diseases. In the process of intervertebral disc degeneration, water and extracellular matrix of nucleus pulposus tissues are lost, so the normal tension in the intervertebral disc cannot be maintained, which worsens the living environment of nucleus pulposus cells. Low back pain (LBP), with a high incidence rate of disability, has become an increasing health concern and a social and economic problem. The present study aimed to analyze the action mechanisms of nicotinamide phosphoribosyl transferase (Nampt) and sirtuin 1 (SIRT1) in intervertebral disc degeneration (IVDD). In total 26 patients with lumbar disc herniation who had surgical resection at The Third Affiliated Hospital of Jinzhou Medical University were recruited as the experimental group and their degenerative nucleus pulposus (DNP) tissues of intervertebral disc were collected. In addition, nucleus pulposus tissues of intervertebral disc were collected from 20 patients with burst fracture of lumbar spine at the same hospital (control). Nucleus pulposus cells from primary culturing were separated for subsequent experimentation. LC3 II/I, beclin-1, SIRT1 and NAMPT mRNA and protein expression levels were determined using reverse transcription-quantitative PCR and western blotting, respectively. Nicotinamide adenine dinucleotide (NAD) contents in nucleus pulposus cells was determined by NAD assay kit. The mRNA and protein expression levels of SIRT1 in DNP tissues were reduced compared with the control tissues and decreased with increasing disease severity. The expression of autophagy-associated LC3 II/I and beclin-1 in DNP tissues was reduced compared with control tissues. SIRT1 regulated the LC3 II/I and beclin-1 expression levels in nucleus pulposus cells. Treatment with resveratrol and inhibitor of SIRT1 showed that Nampt/NAD+/SIRT1 pathway participated in the process of IVDD by regulating autophagy of nucleus pulposus cells. SIRT1 serves a role in the process of IVDD through Nampt/NAD+/SIRT1 pathway that regulates autophagy of nucleus pulposus cells. SIRT1 may become a biological target for the treatment of IVDD.

Evaluation of CD98 light chain-LAT1 as a potential marker of cancer stem-like cells in glioblastoma.

OBJECTIVE: Glioma stem cells (GSCs) are a minority population of glioma cells that regarded as the cause of tumor formation and recurrence. Identifying new molecular strategies targeting GSCs must be urgently developed to treat glioblastoma. In this study, one of CD98 light chain-L type amino acid transporter 1 (LAT1) was found as a potential GSC marker. LAT1 served as EAA transporter has been shown to be closely related with tumor invasion, metastasis, angiogenesis, and radiosensitivity. METHODS: LAT1+ and LAT1- glioma cells were sorted by flow cytometry. Cellular immunofluorescence, sphere-formation arrays, and in vitro limiting dilution experiments were used to identify cell stemness. Differentiated glioma stem cells were cultured, and the expressions of ß-tubulinIII, GFAP, and LAT1 were detected by Western blot. Nude mouse models were constructed to observe tumor formation and metastasis in nude mice. RESULTS: LAT1+ glioma cells were testified a small percentage of all cells and selected as the subsequent sorting marker. LAT1+ cells were separated from U87 and U251 cells could express high level of stem cell markers, and possessed GSC properties including self-renewal ability and multi-directional differentiation potential. But LAT1- cells did not have these characteristics. In addition, LAT1+ cells were able to generate tumors in vivo, tumor size of LAT1+ cells formed were much bigger than that of LAT1- cells. CONCLUSION: Our study, including molecular, cell, vitro and vivo experiments, has shown that LAT1+ cells possess GSC properties, and present for the first time that LAT1 can be used as a new marker for GSCs screening.

Effects of Fat Pre-Emulsification on the Growth Performance, Serum Biochemical Index, Digestive Enzyme Activities, Nutrient Utilization, and Standardized Ileal Digestibility of Amino Acids in Pekin Ducks Fed Diets with Different Fat Sources.

This experiment was conducted to evaluate the effects of fat pre-emulsification on growth performance, the serum biochemical index, intestinal digestive enzyme activities, nutrient utilization, and the standardized ileal digestibility of amino acids (SIDAA) in Pekin ducks fed diets containing different fat sources. Three hundred and twenty healthy ten-day-old Pekin male ducks (409 ± 27 g) were assigned to a 2 × 2 factorial design and given one of two types of poultry fat (duck fat or a mixed type of fat composed of chicken fat and duck fat in a 1:1 ratio) that had been pre-emulsified or not. This resulted in four treatments of eight replicates, with each replicate having ten ducks. The results showed that fat pre-emulsification (preE) significantly increased (p < 0.05) body weight and body weight gain and decreased (p < 0.05) the feed-to-gain ratio, the liver index, the activity of aspartate aminotransferase (AST) and the concentration of total cholesterol (TC) in the serum. Fat preE also tended to decrease the activity of lipase (p = 0.07) and significantly reduced (p < 0.05) the activity of trypsin in the duodenum. The utilization of dietary dry matter, ether extract (EE), energy, and total phosphorus, as well as apparent metabolizable energy (AME) and the SID of serine (p = 0.090), were improved by fat preE. Duck fat markedly increased (p < 0.05) the serum TC concentration and the utilization of dietary EE; however, it also tended to decrease the serum triglyceride (TG) concentration (p = 0.09) and markedly decreased (p < 0.05) the activity of trypsin in the jejunum and duodenum. These results indicated that fat preE contributed to the utilization of dietary nutrients, serum lipid metabolism, intestinal digestive enzyme activities, and liver health, thereby improving the growth performance of ducks. Duck fat has higher bioavailability for ducks based on dietary EE utilization.

Insulin negatively regulates dedifferentiation of mouse adipocytes in vitro.

Insulin plays an important role during adipogenic differentiation of animal preadipocytes and the maintenance of mature phenotypes. However, its role and mechanism in dedifferentiation of adipocyte remains unclear. This study investigated the effects of insulin on dedifferentiation of mice adipocytes, and the potential mechanisms. The preadipocytes were isolated from the subcutaneous white adipose tissue of wild type (WT), TNFα gene mutant (TNFα-/-), leptin gene spontaneous point mutant (db/db) and TNFα-/-/db/db mice and were then induced for differentiation. Interestingly, dedifferentiation of these adipocytes occurred once removing exogenous insulin from the adipogenic medium. As characteristics of dedifferentiation of the adipocytes, downregulation of adipogenic markers, upregulation of stemness markers and loss of intracellular lipids were observed from the four genotypes. Notably, dedifferentiation was occurring earlier if the insulin signal was blocked. These dedifferentiated cells regained the potentials of the stem cell-like characteristics. There is no significant difference in the characteristics of the dedifferentiation between the adipocytes. Overall, the study provided evidence that insulin plays a negative regulatory role in the dedifferentiation of adipocytes. We also confirmed that both dedifferentiation of mouse adipocytes, and effect of the insulin on this process were independent of the cell genotypes, while it is a widespread phenomenon in the adipocytes.

[Theophylline in the treatment of chronic obstructive pulmonary disease: a randomized, double-blind, placebo-controlled study].

OBJECTIVE: To observe the benefits and safety of low-dose, slow-release oral theophylline for long-term treatment of stable chronic obstructive pulmonary disease (COPD). METHODS: This was a randomized, parallel-group, double-blind, placebo-controlled trial. Slow-release theophylline (200 mg/d) twice daily or placebo (matching theophylline) was randomly given to 110 patients with stable COPD in the rural area of Shaoguan, Guangdong Province, for one year. Efficacy measures were spirometry and exacerbations, quality of life, dyspnea scores, satisfaction with treatments and adverse effects. Comparison of benefits was performed using superiority test. RESULTS: Of 110 patients, 85 (42 subjects in theophylline group and 43 subjects in placebo group) completed the study. An analysis for intention-to-treat (ITT) individuals showed that individuals with the treatment of theophylline experienced statistically fewer numbers [(0.8 +/- 1.2) times/year, (1.7 +/- 2.6) times/year, Z = -1.674, P = 0.047] and days of exacerbations [(4.6 +/- 7.9) d, (12.5 +/- 22.8) d, Z = -1.699, P = 0.045] in comparison to subjects receiving placebo, that patients receiving theophylline were less likely than the placebo group to experience moderate exacerbations [(0.4 +/- 1.0) times/year, (1.0 +/- 1.8) times/year, Z = -2.136, P = 0.017], and that more individuals satisfied with treatments in the theophylline group than the placebo group (n = 16, 3, Z = -2.198, P = 0.014), and that statistically greater improvement in pre-bronchodilators FEV(1) [(0.006 +/- 0.180) L, (-0.053 +/- 0.169) L, t = 1.789, P = 0.038] were found in the theophylline group in comparison to the placebo group. The similar results were observed in an analysis for per-protocol (PP) subjects. Statistical improvement on quality of life was observed in the PP subjects of theophylline group than in placebo group (-28 +/- 20, -20 +/- 23, F = 2.893, P = 0.047). Time to the first exacerbation in patients receiving theophylline was also delayed in comparison to placebo (365 d, 276 d, chi(2) = 3.880, P = 0.049). But no statistical difference was found between the two groups in post-bronchodilators FEV(1) in both ITT and PP subjects (t = -0.012, P = 0.495 and t = 0.040, P = 0.484 respectively). Drug-related adverse events (8.8%) such as insomnia, palpitation, stomach discomforts or stomachache, and headache were observed in the theophylline group. CONCLUSION: Slow-released oral theophylline (200 mg/d) may be beneficial and safe in long-term treatment of stable COPD in rural area.

Recovery from rat sciatic nerve injury in vivo through the use of differentiated MDSCs in vitro.

In this study, muscle-derived stem cells (MDSCs) whose differentiation into neuron-like cells was induced by ciliary neurotrophic factor (CNTF) and Salvia (Salvia miltiorrhiza) in vitro were used to repair rat sciatic nerve injuries in vivo, in order to investigate their multifunctional characteristics as pluripotent stem cells. The sciatic nerve in the right side of the lower limb was exposed under the anesthetized condition of 10% chloral hydrate (0.3 ml/100 g) injection into the abdominal cavity. The tissue which was 0.5 cm above the sciatic nerve bifurcation was broken using a hemostat. After induction, MDSCs were transferred in sodium hyaluronate gel and were placed into the damaged area. An untreated control group was also included in this study. The surgical area was sutured after washing with gentamycin sulfate solution. Sciatic nerve function index (SFI) was calculated, electrophysiological tests were performed and the recovery rate of gastrocnemius muscle wet weight was also calculated. Four weeks post-surgery, the SFI and the recovery rate of gastrocnemius muscle wet weight in the MDSC group were significantly higher than those in the control group (P<0.05). MDSCs whose differentiation is induced by CNTF and Salvia play an active role in the repair of peripheral nerve injury.

Positive benefits of theophylline in a randomized, double-blind, parallel-group, placebo-controlled study of low-dose, slow-release theophylline in the treatment of COPD for 1 year.

OBJECTIVE AND BACKGROUND: Increasing evidence suggests that low-dose theophylline has anti-inflammatory benefits and is safe in the treatment of COPD. This study aims to evaluate the efficacy and safety of low-dose, slow-release oral theophylline administered over a 1-year period in patients with COPD. METHODS: A randomized, double-blind, parallel-group, placebo-controlled trial was carried out. In total, 110 participants with COPD were randomly assigned to receive slow-release theophylline (100 mg b.i.d.) or placebo for 1 year. Use of medicine and symptoms recorded by diary cards; pulmonary function, exacerbations of COPD, quality of life and the use of rescue medicine were evaluated. Superiority test was used to estimate the efficacy. RESULTS: Of 110 participants, 85 (77.3%) complied with the protocol, with 42 subjects in theophylline and 43 subjects on placebo. In both intention-to-treat and per-protocol population analysis, greater improvement in pre-bronchodilator FEV(1) (P = 0.038 and P = 0.070, respectively), lower frequency of COPD exacerbations (P = 0.047 and P = 0.035, respectively), fewer days of COPD exacerbations (P = 0.045 and P = 0.046, respectively), lower frequency of clinical visits (P = 0.017 and P = 0.039, respectively), greater improvement in satisfaction with treatment (P = 0.014 and P = 0.004, respectively) were found in the theophylline group than in the placebo group. In per-protocol population, greater improvements in quality of life (P = 0.047) were also observed in the theophylline group and the mean time to the first exacerbation was delayed in theophylline group in comparison with placebo group (P = 0.047). Drug-related adverse events such as stomach discomfort (3.51%), headache (3.51%), insomnia (1.75%) and palpitation (1.75%) were found in the theophylline group. CONCLUSIONS: Low-dose, slow-release oral theophylline is effective and well-tolerated in the long term treatment of stable COPD, although it does not improve post-bronchodilator lung function.

[Study on the prevalence rate of chronic obstructive pulmonary disease in northern part of Guangdong province].

OBJECTIVE: To investigate the prevalence of chronic obstructive pulmonary disease (COPD) and its risk factors in population over 40 years old in northern part of Guangdong province. METHODS: Using uniform scheme, procedures and questionnaire, a cluster-randomized-sampling survey for the population aged over 40 years in a rural area of Shaoguan in the northern part of Guangdong province was performed. Spirometry was performed for every participant, followed by a bronchodilatation test when bronchial obstruction was present. RESULTS: There were 1468 cases with complete data from 1498 people aged >or= 40 years including 640 males, 828 females with an average age of 54.3 years old. The total prevalence of COPD was 12.0%. The prevalence of COPD in males was significantly higher than that in females (18.3% vs. 7.1%, P < 0.01). Only 80.7% of the patients with COPD presented one or more symptoms as cough, phlegm, or dyspnoea. Underdiagnosis of COPD would be quite serious. Only 26.1% of the cases was previously diagnosed to have chronic bronchitis, emphysema, or COPD. Smoking was an important risk factor to COPD and 78.4% of the patients with COPD were smokers. However, relation of biomass and COPD called for further investigation. CONCLUSION: Prevalence of COPD was much higher than expected in the northern part of Guangdong while smoking was an most important risk factor of COPD. Lung function test seemed to be of great importance to COPD diagnosis, especially in the earlier period of COPD.