ObjectiveTo explore the feasibility of constructing a programmed death receptor-1（PD-1） molecular probe for non-invasive micro-positron emission tomography/computed tomography （Micro-PET/CT） imaging of PD-1 protein in mouse S180 sarcoma. MethodsA transgenic PD-1 C57 S180 sarcoma mouse model was established using the S180 sarcoma cell injection. Furthermore， ¹²⁴I-anti-PD-1 monoclonal antibody probe was synthesized. 18.5 MBq of the ¹²⁴I-anti-PD-1 probe was injected into the tail vein of transgenic PD-1 C57 mice. Subsequently， S180 sarcoma was imaged using Micro-PET/CT. ResultsStudy successfully established a transgenic PD-1 C57 S180 sarcoma mouse model. Immunohistochemical （IHC） results showed PD-1 protein expression in S180 sarcoma. Micro-PET/CT imaging successfully visualized the PD-1 protein receptor in S180 sarcoma at different time points （20， 48， 72， and 120 h） after probe injection. ConclusionThe ¹²⁴I-anti-PD-1 monoclonal antibody molecular probe successfully targets the PD-1 receptor in S180 sarcoma of transgenic PD-1 C57 mice， and presents clear Micro-PET/CT immunoassay results， thus it potentially enables the non-invasive screening of patients with PD-1 positive malignant tumors.

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In order to understand the genetic variation and whole genome characteristics of goose as-trovirus(GoAstV)in Jiangxi Province,a strain of GoAstV was successfully isolated from a typical gosling gout case sample and the whole genome sequencing and genetic characteristics of the isola-ted strain were analyzed.The results showed that the JXNC1 strain could be stably passaged on LMH cells and could cause mild cytopathic effects in LMH cells.Sequencing and analysis showed that the full-length genome of the strain was 7 173 bp,and its genetic relationship was the closest to the GZ2301(PP966939)reference strain,belonging to the GoAstV-Ⅱ genotype.The complete genome of JXNC1 strain shared 98.1％-98.8％nucleotide similarity with 22 GoAstV-Ⅱ reference strains,and the amino acid similarity of ORF2 gene was 97.6％-99.6％.At the same time,the analysis results showed that the mutation of the strain mainly occurred in the ORF2 gene,and there were 13 amino acid site mutations,of which T630I was a unique mutation.Animal regression experiments showed that the inoculation of JXNC1 strain could cause urate deposition in the or-gans of goslings,congestion and dilatation of hepatic sinusoids,and small focal necrosis of some hepatocytes.Renal tissue tubular dilatation,renal interstitial connective tissue hyperplasia.The re-sults of this study laid a foundation for accurate prevention and control of the disease.

In order to understand the genetic variation and whole genome characteristics of goose as-trovirus(GoAstV)in Jiangxi Province,a strain of GoAstV was successfully isolated from a typical gosling gout case sample and the whole genome sequencing and genetic characteristics of the isola-ted strain were analyzed.The results showed that the JXNC1 strain could be stably passaged on LMH cells and could cause mild cytopathic effects in LMH cells.Sequencing and analysis showed that the full-length genome of the strain was 7 173 bp,and its genetic relationship was the closest to the GZ2301(PP966939)reference strain,belonging to the GoAstV-Ⅱ genotype.The complete genome of JXNC1 strain shared 98.1％-98.8％nucleotide similarity with 22 GoAstV-Ⅱ reference strains,and the amino acid similarity of ORF2 gene was 97.6％-99.6％.At the same time,the analysis results showed that the mutation of the strain mainly occurred in the ORF2 gene,and there were 13 amino acid site mutations,of which T630I was a unique mutation.Animal regression experiments showed that the inoculation of JXNC1 strain could cause urate deposition in the or-gans of goslings,congestion and dilatation of hepatic sinusoids,and small focal necrosis of some hepatocytes.Renal tissue tubular dilatation,renal interstitial connective tissue hyperplasia.The re-sults of this study laid a foundation for accurate prevention and control of the disease.

Parabacteroides distasonis is a gram-negative bacterium initially isolated from a clinical specimen in the 1930s. The strain was re-classified to form the new genus Parabacteroides in 2006. P. distasonis can regulate intestinal barrier function and plays a key role in immune response and metabolic regulation of bodies. Traditional Chinese medicine(TCM) is closely related to the intestinal microbiota. Polysaccharides, saponins, and other ingredients of TCM can treat diseases by interacting with P. distasonis, but the specific mechanisms underlying these processes are still unclear, requiring further exploration. This study reviewed the roles and related mechanisms of P. distasonis in inflammatory-immune diseases, metabolic diseases, cardiovascular disease, neuropsychiatric diseases, cancer, and other diseases and summarized the relevant research results of TCM to prevent and treat diseases by regulating P. distasonis. This study provides a reference for subsequent exploration of P. distasonis and research on the interaction between TCM and intestinal microbiota.
Humans ; Gastrointestinal Microbiome/drug effects* ; Medicine, Chinese Traditional ; Animals ; Bacteroidetes ; Drugs, Chinese Herbal/pharmacology*

OBJECTIVE: To investigate the value of coronary computed tomographic angiography (CCTA)-based fractional flow reserve (CT-FFR) and plaque quantitative analysis in predicting adverse outcomes in patients with non-obstructive coronary heart disease (CAD). METHODS: Clinical data of patients with non-obstructive CAD who underwent CCTA at the Affiliated Hospital of Jiangnan University from March 2014 to March 2018 were retrospectively analyzed and followed up, and the occurrence of major adverse cardiovascular event (MACE) was recorded. The patients were divided into MACE and non-MACE groups according to the occurrence of MACE. The clinical data, CCTA plaque characteristics including plaque length, stenosis degree, minimum lumen area, total plaque volume, non-calcified plaque volume, calcified plaque volume, plaque burden (PB) and remodelling index (RI), and CT-FFR were compared between the two groups. Multivaritate Cox proportional risk model was used to evaluate the relationship between clinical factors, CCTA parameters and MACE. The receiver operator characteristic curve (ROC curve) was used to assess the predictive power of outcome prediction model based on different CCTA parameters. RESULTS: Finally 217 patients were included, of which 43 (19.8%) had MACE and 174 (80.2%) did not. The median follow-up interval was 24 (16, 30) months. The CCTA showed that patients in the MACE group had more severe stenosis than that in the non-MACE group [(44.3±3.8)% vs. (39.5±2.5)%], larger total plaque volume and non-calcified plaque volume [total plaque volume (mm³): 275.1 (197.1, 376.9), non-calcified plaque volume (mm³): 161.5 (114.5, 307.8) vs. 117.9 (77.7, 185.5)], PB and RI were larger [PB: 50.2% (42.1%, 54.8%) vs. 45.1% (38.2%, 51.7%), RI: 1.19 (0.93, 1.29) vs. 1.03 (0.90, 1.22)], CT-FFR value was lower [0.85 (0.80, 0.88) vs. 0.92 (0.87, 0.97)], and the differences were statistically significant (all P < 0.05). Cox regression analysis showed that non-calcified plaques volume [hazard ratio (HR) = 1.005. 95% confidence interval (95%CI) was 1.025-4.866], PB ≥ 50% (HR = 3.146, 95%CI was 1.443-6.906), RI ≥ 1.10 (HR = 2.223, 95%CI was 1.002-1.009) and CT-FFR ≤ 0.87 (HR = 2.615, 95%CI was 1.016-6.732) were independent predictors of MACE (all P < 0.05). The model based on CCTA stenosis degree+CT-FFR+quantitative plaque characteristics (including non-calcified plaque volume, RI, PB) [area under the ROC curve (AUC) = 0.91, 95%CI was 0.87-0.95] had significantly better predictive efficacy for adverse outcomes than the model based on CCTA stenosis degree (AUC = 0.63, 95%CI was 0.54-0.71) and the model based on CCTA stenosis degree+CT-FFR (AUC = 0.71, 95%CI was 0.63-0.79; both P < 0.01). CONCLUSIONS CT-FFR and plaque quantitative analysis based on CCTA are helpful in predicting adverse outcomes in patients with non-obstructive CAD. Non-calcified plaque volume, RI, PB and CT-FFR are important predictors of MACE. Compared with the prediction model based on stenosis degree and CT-FFR, the combined plaque quantitative index can significantly improve the prediction efficiency of adverse outcomes in patients with non-obstructive CAD.
Humans ; Fractional Flow Reserve, Myocardial ; Coronary Angiography/methods* ; Constriction, Pathologic ; Retrospective Studies ; ROC Curve ; Predictive Value of Tests ; Plaque, Atherosclerotic/diagnostic imaging* ; Coronary Stenosis/diagnostic imaging* ; Tomography, X-Ray Computed ; Coronary Artery Disease/diagnostic imaging*

OBJECTIVE: To investigate the anti-coronavirus potential and the corresponding mechanisms of the two ingredients of Reduning Injection: quercetin and luteolin. METHODS: A pseudovirus system was designed to test the efficacy of quercetin and luteolin to inhibit SARS-CoV-2 infection and the corresponding cellular toxicity. Luteolin was tested for its activities against the pseudoviruses of SARS-CoV-2 and its variants. Virtual screening was performed to predict the binding sites by Autodock Vina 1.1.230 and PyMol. To validate docking results, surface plasmon resonance (SPR) was used to measure the binding affinity of the compounds with various proteins of the coronaviruses. Quercetin and luteolin were further tested for their inhibitory effects on other coronaviruses by indirect immunofluorescence assay on rhabdomyosarcoma cells infected with HCoV-OC43. RESULTS: The inhibition of SARS-CoV-2 pseudovirus by luteolin and quercetin were strongly dose-dependent, with concentration for 50% of maximal effect (EC₅₀) of 8.817 and 52.98 µmol/L, respectively. Their cytotoxicity to BHK21-hACE2 were 177.6 and 405.1 µmol/L, respectively. In addition, luetolin significantly blocked the entry of 4 pseudoviruses of SARS-CoV-2 variants, with EC₅₀ lower than 7 µmol/L. Virtual screening and SPR confirmed that luteolin binds to the S-proteins and quercetin binds to the active center of the 3CLpro, PLpro, and helicase proteins. Quercetin and luteolin showed over 99% inhibition against HCoV-OC43. CONCLUSIONS The mechanisms were revealed of quercetin and luteolin inhibiting the infection of SARS-CoV-2 and its variants. Reduning Injection is a promising drug for COVID-19.
Humans ; SARS-CoV-2 ; COVID-19 ; Luteolin ; Quercetin

L-homophenylalanine (L-HPA) is an important non-natural amino acid that has been used as a key intermediate for the synthesis of Puli drugs for the treatment of hypertension. At present, L-HPA is synthesized using chemical methods, which has the disadvantages of expensive raw materials, tedious steps and serious pollution. Therefore, researchers have conducted in-depth research on the enzymatic production of L-HPA. This review summarizes the research progress on the enzymatic synthesis of L-HPA, including the dehydrogenase process, the transaminase process, the hydantoinase process, and the decarboxylase process, with the hope to facilitate the industrial production of L-HPA.
Amino Acids ; Environmental Pollution ; Industry ; Protein Biosynthesis

Limonene and its derivative perillic acid are widely used in food, cosmetics, health products, medicine and other industries as important bioactive natural products. However, inefficient plant extraction and high energy-consuming chemical synthesis hamper the industrial production of limonene and perillic acid. In this study, limonene synthase from Mentha spicata was expressed in Saccharomyces cerevisiae by peroxisome compartmentalization, and the yield of limonene was 0.038 mg/L. The genes involved in limonene synthesis, ERG10, ERG13, tHMGR, ERG12, ERG8, IDI1, MVD1, ERG20ww and tLS, were step-wise expressed via modular engineering to study their effects on limonene yield. The yield of limonene increased to 1.14 mg/L by increasing the precursor module. Using the plasmid with high copy number to express the above key genes, the yield of limonene significantly increased up to 86.74 mg/L, which was 4 337 times higher than that of the original strain. Using the limonene-producing strain as the starting strain, the production of perillic acid was successfully achieved by expressing cytochrome P450 enzyme gene from Salvia miltiorrhiza, and the yield reached 4.42 mg/L. The results may facilitate the construction of cell factory with high yield of monoterpene products by S. cerevisiae.
Saccharomyces cerevisiae/metabolism* ; Limonene/metabolism* ; Metabolic Engineering ; Monoterpenes/metabolism*

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGAD^{S24R/D88R/Y309K} was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and Lpgad^{S24R/D88R/Y309K} genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD₆₀₀) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.
Glutamate Decarboxylase/genetics* ; Lactobacillus plantarum/genetics* ; Catalysis ; gamma-Aminobutyric Acid ; Hydrogen-Ion Concentration ; Glutamic Acid