Development and Evaluation of a Novel One-Step RT-qPCR Targeting the Vero Gene for the Identification of False-Positive Results Caused by Inactivated Virus Vaccine Contamination.

To identify false-positive SARS-CoV-2 test results caused by novel coronavirus inactivated vaccine contamination, a novel RT-qPCR targeting the ORF1ab and N genes of SARS-CoV-2 and Vero gene was developed. The amplification efficiency, precision, and lower limit of detection (LLOD) of the RT-qPCR assay were determined. A total of 346 clinical samples and 132 environmental samples were assessed, and the diagnostic performance was evaluated. The results showed that the amplification efficiency of the ORF1ab, N, and Vero genes was 95%, 97%, and 93%, respectively. The coefficients of variation of Ct values at a concentration of 3 × 104 copies/mL were lower than 5%. The LLOD for the ORF1ab, N, and Vero genes reached 8.0, 3.3, and 8.2 copies/reaction, respectively. For the 346 clinical samples, our RT-qPCR assay identified SARS-CoV-2-positive and SARS-CoV-2-negative samples with a sensitivity of 100.00% and a specificity of 99.30% and novel coronavirus inactivated vaccine-contaminated samples with a sensitivity of 100% and a specificity of 100%. For the environmental samples, our RT-qPCR assay identified novel coronavirus inactivated vaccine-contaminated samples with a sensitivity of 88.06% and a specificity of 95.38%. In conclusion, the RT-qPCR assay we established can be used to diagnose COVID-19 and, to a certain extent, false-positive results due to vaccine contamination.

Red Blood Cell Lysis Pretreatment Can Significantly Improve the Yield of Treponema pallidum DNA from Blood.

PCR can be a supplement to Treponema serological testing. However, its sensitivity is not satisfactory for blood sample testing. The aim of this study was to investigate whether pretreatment with red blood cell (RBC) lysis could enhance the yield of Treponema pallidum subsp. pallidum DNA extraction from blood. We developed and verified the efficacy of a quantitative PCR (qPCR) assay that utilizes TaqMan technology to specifically detect T. pallidum DNA by targeting the polA gene. Simulation media with 106 to 100 treponemes/mL were prepared in normal saline (NS), whole blood, plasma, and serum, and RBC lysis pretreatment was performed on a portion of whole blood. Then, blood samples drawn from 50 syphilitic rabbits were divided in parallel into five groups, labeled whole blood, whole blood/lysed RBCs, plasma, serum, and blood cells/lysed RBCs. DNA extraction and qPCR detection were performed. The detection rate and copy number were compared among different groups. The polA assay showed good linearity and an excellent amplification efficiency of 102%. In the simulated blood samples, the detection limit of the polA assay reached 1 × 102 treponemes/mL in whole blood/lysed RBCs, plasma, and serum. However, the detection limit was only 1 × 104 treponemes/mL in NS and whole blood. Among the blood samples from syphilitic rabbits, whole blood/lysed RBCs showed the best detection rate (82.0%), while the detection rate for whole blood was only 6%. The copy number of whole blood/lysed RBCs was higher than that of whole blood. RBC lysis pretreatment can significantly improve the yield of T. pallidum DNA from whole blood, and the yield is better than that from whole blood, plasma, serum, and blood cells/lysed RBCs. IMPORTANCE Syphilis is a sexually transmitted disease caused by T. pallidum that can spread into the blood. T. pallidum DNA can be detected in blood by PCR but with low sensitivity. Few studies have applied RBC lysis pretreatment to blood T. pallidum DNA extraction. This study shows that the detection limit, detection rate, and copy number of whole blood/lysed RBCs were better than those of whole blood, plasma, and serum. After RBC lysis pretreatment, the yield of low concentrations of T. pallidum DNA was improved, and the low sensitivity of blood-based T. pallidum PCR was improved. Therefore, whole blood/lysed RBCs are the ideal sample for acquiring blood T. pallidum DNA.

Identifying FBLN1 (Gene ID: 2192) as a Potential Melanoma Biomarker for Melanoma based on an Analysis of microRNA Expression Profiles in the GEO and TCGA Databases.

Aims: We analyzed and compared the gene expression profiles (GSE92763) from normal melanocytes with malignant melanoma cell lines to identify genes that were differentially expressed that could serve as potential biomarkers for melanoma diagnosis. Materials and Methods: Gene expression profiles from the GSE92763 dataset were downloaded from the Gene Expression Omnibus (GEO) database. By comparing normal human melanocytes with multiple melanoma cell lines we identified 127 differentially expressed genes whose expression was altered. These data were used to identify hub genes associated with protein-protein interaction networks using Cytoscape software. To explore the biological functions of the aforementioned hub genes, we utilized the clusterProfiler package in R studio to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. We then used the Gene Expression Profiling Interactive Analysis (GEPIA) website to determine the association of these hub genes with overall survival (OS). In addition, we utilized the Oncomine and Cancer Cell Line Encyclopedia (CCLE) databases to further analyze and compare the expression of these key genes associated with melanoma with other tumor types. Results: The hub genes included three upregulated and seven downregulated genes, which were linked with extracellular junctions, migration, paracrine and proliferation functions based on GO. In addition, we performed a confirmatory analysis of the hub genes using The Cancer Genome Atlas (TCGA) database. This analysis revealed that the expression of the Fibulin 1 (FBLN1; gene ID: 2192) gene was significantly downregulated in melanomas, and that its expression level in melanoma patients was significantly associated with OS with high expressors having better OS (log-rank p = 0.0034, hazard ratio = 1.5, p = 0.0036). We further analyzed the expression of FBLN1 in melanoma using the TCGA and Oncomine databases, and confirmed that FBLN1 is expressed at lower levels than in other cells (p = 2.03E-15, t = -15.586). FBLN1 has extremely high DNA copy number and low messenger RNA expression in melanoma cell lines according to the CCLE analysis. Conclusion: These results suggest that FBLN1 expression may be utilized as a biomarker and essential prognostic factor for melanoma; as well as provide an important theoretical basis for the development of melanoma treatments.

Protective effects of orally administered shark compound peptides from Chiloscyllium plagiosum against acute inflammation.

The anti-inflammatory effects of shark compound peptides (SCP) from Chiloscyllium plagiosum were investigated. Results showed that SCP enhanced the viability of RAW 264.7 macrophages in vitro in a dose-dependent manner. Orally administered SCP exhibited potent anti-inflammatory activity in lipopolysaccharide (LPS)-challenged mice by suppressing serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), as well as nitric oxide (NO). Moreover, SCP significantly inhibited the inflammatory rise of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and creatinine (CRE), while blocking the decline of cholinesterase (CHE), with an efficacy close to aspirin. This research showed that orally administered SCP from C. plagiosum notably downregulated uncontrolled inflammatory responses, and conferred substantial protection from endotoxin-induced acute hepatic damage and renal functional impairment. Therefore, oral supplementation of SCP can be used as a preventive approach to reduce the risk of inflammatory-related diseases.

The inhibitory kinetics and mechanism of glycolic acid on lipase.

Obesity is prone to cause a variety of chronic metabolic diseases, and it has aroused people's attention that the rapid increase in the global population of obese people in the past years. As a kind of weight-loss drug acting in the intestine, lipase inhibitor does not enter the bloodstream without producing central nervous side effects. Because they do not affect the metabolism system, lipase inhibitors and obesity have become one of the hot spots in recent years. Glycolic acid is a new substrate analog inhibitor with the value of the semi-inhibitory concentration of lipase is estimated to be 17.29 ± 0.14 mM. Using the plots of Lineweaver-Burk, the inhibition mechanism of lipase by glycolic acid was reversible and the inhibition type belongs to competitive inhibition with a KI value of 19.61 ± 0.26 mM. The inhibitory kinetics assay showed that the microscopic velocity constant k+0 of inhibition kinetics is 1.79 × 10-3 mM-1s-1, and k-0 is 0.73 × 10-3 s-1. The results of UV full-wavelength scanning on product cumulative, fluorescence quenching and molecular simulation also indicated that glycolic acid and substrate competitive with lipase by binding to Lys137. Thereby glycolic acid inhibiting the oxidation-catalyzed reaction and reducing the product of the enzyme and substrate. This adds a new direction for the search for lipase inhibitors and provides new ideas about the development of anti-obesity drugs.Communicated by Ramaswamy H. Sarma.

Bone-strengthening effects and safety of compound peptides from skin of Chiloscyllium plagiosum and Mustelus griseus.

Fish processing produces a lot of by-products highly containing large amount of proteins which mainly consist of collagen, implying great potential value for application as nutraceutical ingredients. In present study, two kinds of sharks, Chiloscyllium plagiosum and Mustelus griseus, were used as raw material to gain three kinds of "compound peptides" (CPs) by enzymolysis, FCP (CPs from the flesh of C. plagiosum), SCP (CPs from the skin of C. plagiosum), and SMG (CPs from the skin of M. griseus). According to a series of constituent analysis, the molecule weights of FCP, SCP, and SMG were under 800 Da; amino acids composition analysis of FCP, SCP, and SMG showed that there were high glycine, proline, and hydroxyproline and low cysteine contents in SCP and SMG, which is the characteristic of collagen peptides; their total protein contents were 87.500%, 91.875%, and 95.625%, respectively; and heavy metal contents of CPs were all beneath national standards. After three kinds of CPs were administrated intragastrically to C57BL/6 mice at a total dosage of 15 g/kg, bone-strengthening effects of SCP and SMG were manifested by osteoblasts activity promotion, bone mineral density (BMD) increase, and marrow adipocyte number decrease, yet nonsignificant effects were shown in FCP group. No index showed toxicity of SCP and SMG in subacute toxicology trial, indicating their safety as functional foods. Herein, industrial application foundation of the skins from these two sharks was explored but more efforts should subsequently be implemented for further exploitation.

Inhibitory effects of Cefazolin and Cefodizime on the activity of mushroom tyrosinase.

Tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones that form brown or black pigments. In the present paper, the effects of Cefazolin and Cefodizime on the activity of mushroom tyrosniase have been studied. The results showed that the Cephalosporin antibacterial drugs (Cefazolin and Cefodizime) could inhibit both monophenolase activity and diphenolase activity of the enzyme. For the monophenolase activity, Both Cefazolin and Cefodizime could lengthen the lag time and decrease the steady-state activities, and the IC(50) values were estimated as 7.0 mM and 0.13 mM for monophenolase activity, respectively. For the diphenolase activity, the inhibitory capacity of Cefodizime was obviously stronger than that of Cefazolin, and the IC(50) values were estimated as 0.02 mM and 0.21 mM, respectively. Kinetic analyses showed that inhibition by both compounds was reversible and their mechanisms were competitive and mixed-type, respectively. Their inhibition constants were also determined and compared. The research may offer a lead for designing and synthesizing novel and effective tyrosinase inhibitors and also under the application field of Cephalosporins.

Diosgenin Suppresses Cholangiocarcinoma Cells Via Inducing Cell Cycle Arrest And Mitochondria-Mediated Apoptosis.

PURPOSE: Diosgenin (DSG) is the precursor of steroid hormones and plays a crucial part in the proliferation of various carcinomas including human colorectal cancer and gastric carcinoma. Nevertheless, its specific features and mechanisms in human cholangiocarcinoma (CCA) remain unknown. METHODS: MTS assay, colony-forming assay, and EdU assay were performed to determine the role of DSG on the progression of human CCA cells. The distributions of cell cycle, the ratio of apoptosis, and the mitochondrial membrane potential (ΔΨm) were studied by flow cytometry (FCM). AO/EB and Hoechst 33258 staining were performed to observe the morphological features of cell apoptosis. TEM was performed to observe the ultrastructures of QBC939 and HuCCT1 cells. The mRNA and protein expression of mitochondrial apoptotic pathway and GSK3ß/ß-catenin pathway were further confirmed by qPCR and Western blotting. The xenograft tumor model of HuCCT1 cells was built. Immunohistochemistry of tumor tissues was performed. RESULTS: Our results indicated that DSG inhibited the progression of six CCA cell lines. In vivo tumor studies also indicated that DSG significantly inhibited tumor growth in xenografts in nude mice. The expression of mitosis-promoting factor cyclinB1 was decreased along with the elevating level of cell cycle inhibitor p21, resulting in arresting CCA cell cycles at G2/M phase. Furthermore, DSG induced apoptosis with the increased expressions of cytosol cytochrome C, cleaved-caspase-3, cleaved-PARP1 and the Bax/Bcl-2 ratio. Mechanistically, our study showed that GSK3ß/ß-catenin pathway was involved in the apoptosis of CCA cells. Thus, DSG might provide a new clue for the drug therapy of CCA. CONCLUSION: In our data, DSG was found to have efficient antitumor potential of human CCA cells in vitro and in vivo.

Inhibitory effects of cefotaxime on the activity of mushroom tyrosinase.

Tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones that form brown or black pigments. In the present paper, cefotaxime, a cephalosporin antibacterial drug, was tested as an inhibitor of tyrosinase. The results show that cefotaxime inhibits both the monophenolase and diphenolase activities of tyrosinase. For the monophenolase activity, cefotaxime increased the lag time and decreased the steady-state activity with an IC50 of 3.2 mM. For the diphenolase activity, the inhibition by cefotaxime is reversible and mix-I type with an IC50 of 0.14 mM. The inhibition constants (K(I) and K(IS)) were determined to be 0.14 and 0.36 mM, respectively. The molecular mechanism of inhibition of tyrosinase by cefotaxime was determined by fluorescence quenching and molecular docking. The results demonstrated that cefotaxime was a static quencher of tyrosinase and that cefotaxime could dock favorably in the active site of tyrosinase. This research may offer a lead for designing and synthesizing novel and effective tyrosinase inhibitors in the future.

Condensed tannins from Ficus virens as tyrosinase inhibitors: structure, inhibitory activity and molecular mechanism.

Condensed tannins from Ficus virens leaves, fruit, and stem bark were isolated and their structures characterized by 13C nuclear magnetic resonance spectrometry, high performance liquid chromatography electrospray ionization mass spectrometry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results showed that the leaves, fruit, and stem bark condensed tannins were complex mixtures of homo- and heteropolymers of B-type procyanidins and prodelphinidins with degrees of polymerization up to hexamer, dodecamer, and pentadecamer, respectively. Antityrosinase activities of the condensed tannins were studied. The results indicated that the condensed tannins were potent tyrosinase inhibitors. The concentrations for the leaves, fruit, and stem bark condensed tannins leading to 50% enzyme activity were determined to be 131.67, 99.89, and 106.22 µg/ml on monophenolase activity, and 128.42, 43.07, and 74.27 µg/ml on diphenolase activity. The inhibition mechanism, type, and constants of the condensed tannins on the diphenolase activity were further investigated. The results indicated that the condensed tannins were reversible and mixed type inhibitors. Fluorescence quenching, copper interacting, and molecular docking techniques were utilized to unravel the molecular mechanisms of the inhibition. The results showed that the hydroxyl group on the B ring of the condensed tannins could chelate the dicopper irons of the enzyme. Moreover, the condensed tannins could reduce the enzyme product o-quinones into colourless compounds. These results would contribute to the development and design of antityrosinase agents.

Synthesis and antityrosinase mechanism of benzaldehyde thiosemicarbazones: novel tyrosinase inhibitors.

p-Hydroxybenzaldehyde thiosemicarbazone (HBT) and p-methoxybenzaldehyde thiosemicarbazone (MBT) were synthesized and established by (1)H NMR and mass spectra. Both compounds were evaluated for their inhibition activities on mushroom tyrosinase and free-cell tyrosinase and melanoma production from B(16) mouse melanoma cells. Results showed that both compounds exhibited significant inhibitory effects on the enzyme activities. HBT and MBT decreased the steady state of the monophenolase activity sharply, and the IC(50) values were estimated as 0.76 and 7.0 µM, respectively. MBT lengthened the lag time, but HBT could not. HBT and MBT inhibited diphenolase activity dose-dependently, and their IC(50) values were estimated as 3.80 and 2.62 µM, respectively. Kinetic analyses showed that inhibition type by both compounds was reversible and their mechanisms were mixed-type. Their inhibition constants were also determined and compared. The research may supply the basis for the development of new food preservatives and cosmetic additives.

Irreversible competitive inhibitory kinetics of cardol triene on mushroom tyrosinase.

Cardol triene was first purified from cashew (Anacardium occidentale L.) nut shell liquid and identified by gas chromatography coupled to mass spectroscopy and nuclear magnetic resonance. The effects of this compound on the activity of mushroom tyrosinase were studied. The results of the kinetic study showed that cardol triene was a potent irreversible competitive inhibitor and the inactivation was of the complexing type. Two molecules of cardol triene could bind to one molecule of tyrosinase and lead to the complete loss of its catalytic activity. The microscopic rate constants were determined for the reaction of cardol triene with the enzyme. The anti-tyrosinase kinetic research of this study provides a comprehensive understanding of inhibitory mechanisms of resorcinolic lipids and is beneficial for the future design of novel tyrosinase inhibitors.

Inhibitory effects of methyl trans-cinnamate on mushroom tyrosinase and its antimicrobial activities.

The control of food browning and growth of disease-causing microorganisms is critical to maintaining the quality and safety of food. Tyrosinase is the key enzyme in food browning. The inhibitory effect of methyl trans-cinnamate on the activity of tyrosinase has been investigated. Methyl trans-cinnamate can strongly inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. When the concentration of methyl trans-cinnamate reached 2.5 mM, the lag time was lengthened from 32 to 160 s and the steady-state activity was lost about 65%. The IC(50) value was 1.25 mM. For the diphenolase activity, the inhibition of methyl trans-cinnamate displayed a reversible and noncompetitive mechanism. The IC(50) value was 1.62 mM, and the inhibition constant (K(I)) was determined to be 1.60 mM. Moreover, the antibacterial activity against Escherichia coli, Bacillus subtilis and Staphyloccocus aureus and antifungal activity against Candida albicans were tested. The results showed that methyl trans-cinnamate possessed an antimicrobial ability.