Study on the intervention effect of Epimedium before and after suet-oil-processed on kidney yang deficiency rats based on intestinal flora and fecal metabolomics.

Epimedium is a Chinese herbal medicine commonly used in clinical practice to reinforce yang. Previous studies have shown that Epimedium fried with suet oil based has the best effect on warming kidney and promoting yang. Evidence suggests a relationship between kidney yang deficiency syndrome (KYDS) and metabolic disorders of the intestinal microflora. However, the specific interaction between KYDS and the intestinal microbiome, as well as the internal regulatory mechanism of the KYDS intestinal microbiome regulated by Epimedium fried with suet oil, remain unclear. The purpose of this study was to investigate the regulatory effects of different processed products of Epimedium on intestinal microflora and metabolites in rats with kidney yang deficiency, and to reveal the processing mechanism of Epimedium fried with suet oil warming kidney and helping yang. 16 S rRNA and LC-MS/MS technology were used to detect fecal samples. Combined with multivariate statistical analysis, differential intestinal flora and metabolites were screened. Then the content of differential bacteria was then quantified using quantitative real-time fluorescence PCR. Furthermore, the correlation between differential bacterial flora and metabolites was analyzed using Spearman's method. The study found that the composition of intestinal flora in rats with kidney yang deficiency changed compared to healthy rats. Epimedium fried with suet oil could increase the levels of beneficial bacteria, while significantly reducing the levels of harmful bacteria. Real-time quantitative PCR results were consistent with 16 S rRNA gene sequencing analysis. Fecal metabolomics revealed that KYDS was associated with 30 different metabolites, involving metabolic pathways steroid hormone biosynthesis etc. Moreover, differential bacteria were closely correlated with potential biomarkers. Epimedium could improve metabolic disorders associated with KYDS by acting on the intestinal flora, with Epimedium fried with suet oil demonstrating the most effective regulatory effect. Its potential mechanism may involve the regulation of abnormal metabolism and the impact on the diversity and structure of the intestinal flora.

Identification of Flavanone 3-Hydroxylase Gene Family in Strawberry and Expression Analysis of Fruit at Different Coloring Stages.

The color of strawberry fruit is an important appearance quality index that affects the marketability of fruit, and the content and type of anthocyanin are two of the main reasons for the formation of fruit color. At present, the research on anthocyanin synthesis mainly focuses on the phenylpropane metabolic pathway, and the F3H gene family is an important member of this metabolic pathway. Therefore, in order to clarify the role of flavanone 3-hydroxylase (F3H) in regulating anthocyanin accumulation in strawberry, we identified F3H gene family members in strawberry and analyzed their bioinformatics and expression at different fruit color stages. The results showed that the strawberry F3H family contains 126 members, which are distributed on seven chromosomes and can be divided into six subgroups. The promoter region of strawberry F3H gene family contains light response elements, abiotic stress response elements and hormone response elements. Intraspecic collinearity analysis showed that there were six pairs of collinearity of the F3H gene. Interspecific collinearity analysis showed that there were more collinearity relationships between strawberry and apple, grape and Arabidopsis, but less collinearity between strawberry and rice. Via tissue-specific expression analysis, we found that the expression levels of FvF3H48, FvF3H120 and FvF3H74 were higher in the stages of germination, growth, flowering and fruit setting. The expression levels of FvF3H42 and FvF3H16 were higher in seeds. The expression levels of FvF3H16 and FvF3H11 were higher in the ovary wall of stage 1, stage 2, stage 3 and stage 5. FvF3H15 and FvF3H48 were highly expressed in the pericardium, anther, receptacle and anther. Real-time fluorescence quantitative PCR showed the expression changes in F3H in the fruit coloring process. The results indicate that the expression levels of most members were higher during the S3 stage, such as FvF3H7, FvF3H16, FvF3H32, FvF3H82, FvF3H89, FvF3H92 and FvF3H112. FvF3H63 and FvF3H104 exhibited particularly high expression levels during the S1 stage, with some genes also showing elevated expression during the S4 stage, including FvF3H13, FvF3H27, FvF3H66 and FvF3H103. FvF3H58, FvF3H69, FvF3H79 and FvF3H80 showed higher expression levels during the S2 stage. These findings lay the groundwork for elucidating the biological functions of the strawberry F3H gene family and the selection of related genes.

[Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii].

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

Effect of Ivermectin on the Expression of P-Glycoprotein in Third-Stage Larvae of Haemonchus contortus Isolated from China.

Haemonchus contortus poses a severe hazard to the healthy development of the sheep industry and threatens the welfare of sheep. Ivermectin is the primary anthelmintic used for the prevention and treatment of H. contortus parasitism. However, the widespread and uncontrolled application of ivermectin has resulted in the development and spread of resistant strains of H. contortus. P-glycoprotein (P-gp) plays important roles in the pharmacology and toxicology of ivermectin, and changes in P-gp expression levels can be used to analyze the resistance of H. contortus to ivermectin. This study aimed to analyze the effects of ivermectin on P-gp expression in H. contortus L3 larvae isolated from China and to evaluate whether changes in P-gp expression levels can be used to analyze resistant H. contortus strains. In the absence of drug treatment, the ivermectin-resistant strains isolated in China showed increased expression of P-gp11 (p < 0.01) compared with sensitive strains from elsewhere, whereas the expressions of P-gp2 and P-gp9.1 were downregulated (p < 0.01). When the same strain was compared before and after drug treatment, obvious differences in expression were observed between the different strains. Ivermectin-induced P-gp expression was found to be very complex among the L3 larvae of different strains. In addition, it was confirmed that using P-gp to determine ivermectin resistance in H. contortus strains from different geographic environments can yield different results.

Development and validation of real-time fluorescence quantitative PCR method for murine virus / 中国生物制品学杂志

@#Objective To develop and validate the real-time fluorescent quantitative PCR(Q-PCR)method for the detection of 8 murine viruses. Methods The specificity,sensitivity and precision of the Q-PCR method were verified by four laboratories,and the virus simulated contamination test and blind sample detection were carried out simultaneously,of which the detection results were compared. The Q-PCR method was used to detect 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other products of murine origin. Results The Q-PCR method used for detecting 8 kinds of murine viruses had no cross reaction with the same family and genus or other murine viruses. Except the sensitivity of laboratory 2 to ectromelia virus(EctV/Mouse Pox,MPV)was 2 × 10~2copies/μL,the sensitivity of laboratory 2 to other 7 viruses and 3 other laboratories to 8 murine viruses was 2 × 10~1copies/μL. Except the inter-assay CV of the copy number of mouse adenovirus(MAdV)detected by laboratory 3 was 37. 58%,the intra-assay and inter-assay CVs of the Ct and copy number of other 7 viruses detected by laboratory 3 and those of 8 viruses detected by other 3 laboratories were all less than 25%.The sensitivity of virus simulated contamination test met the parameter requirements. The coincidence rate of blind sample detection results by 4 laboratories was 100%. All the 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other murine derived products were negative for 8 murine viruses. Conclusion Q-PCR method for murine virus has good specificity,sensitivity and precision,and can be used for the detection of murine derived biological products.

Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii / 中国中药杂志

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

Development of a low-cost multi-channel nucleic acid detection PCR instrument and clinical detection application of COVID-19.

Since the COVID-19 outbreak at the end of December 2019, a variety of novel Coronavirus nucleic acid detection methods have been proposed at home and abroad. Because of the disadvantages of most existing PCR instruments on the market such as long reaction time and high cost, this study developed a more timesaving and cheaper two-channel real-time quantitative PCR instrument. In this instrument, a PCR system combining a thermal cycle system and real-time fluorescence quantitative technology was designed. The software system and data processing, optical system, thermal cycle module, and hardware module of the PCR instrument were studied. The low-cost, portable real-time quantitative PCR system has been validated with consistent results compared to Bio-rad CFX Connect. At the same time, the same samples were used for the contract experiment with the hospital instrument, and the amplification result was better than the existing instrument in the hospital.

Development of Nested PCR and Duplex Real-Time Fluorescence Quantitative PCR Assay for the Simultaneous Detection of Theileria equi and Babesia caballi.

Equine piroplasmosis (EP) is a type of blood protozoan disease caused by tick-borne parasites, Theileria equi (T. equi), Babesia caballi (B. caballi) and Theileria haneyi. While many studies have been conducted on EP diagnosis, diagnostic methods exhibiting high sensitivity and specificity remain lacking. Therefore, nested PCR (nPCR) and duplex real-time fluorescence quantitative PCR (qPCR) that can simultaneously detect both T. equi and B. caballi causing agents were established and compared. The two techniques were used to analyze 36 horse blood samples for EP. This set of samples was also detected by a multinested PCR (mnPCR) targeting the EMA-1 gene of T. equi and the RAP-1 gene of B. caballi. By nPCR, duplex real-time fluorescence qPCR and mnPCR, infections with B. caballi were detected in 16.67% (6/36), 2.78% (1/36), 19.44% (7/36) of the horses, respectively. The T. equi prevalence was 58.33% (21/36) by the nPCR, 33.33% (12/36) by the duplex real-time fluorescence qPCR and 2.78% (1/36) by the mnPCR. The overall prevalence of infection with mixed parasites by nPCR was 5.56% (2/36), by duplex real-time fluorescence qPCR was 2.78% (1/36) and by mnPCR 0% (0/36). Results suggest that nPCR can detect T. equi and B. caballi positive samples with good specificity and sensitivity, although distinguishing between the two parasites requires an electrophoresis with 4% agarose gels. The duplex real-time fluorescence qPCR can readily distinguish between T. equi and B. caballi infection, but with low sensitivity.

Expressions of Toll Like Receptor (TLR) Genes in Paralichthys olivaceus After Induced by Different Extracts of Edwardsiella tarda.

Toll like receptors (TLRs) are the main innate immune 'pattern recognition receptors' of animals, which play a central role in host cell recognition and responses to invasive pathogens, particularly common structures of microbial pathogens. In this study, the gene expression profiles of TLRs in the spleen, head kidney, gill, small intestine, liver, muscle, and heart of healthy Paralichthys olivaceus were detected by real-time quantitative PCR (qPCR). The TLR family members were widely expressed in different tissues with different basic expression profiles. The highest expressions of TLR1, 5m, 7, 8, 9, 14, and 21 were found in the spleen; the highest expressions of TLR3 and TLR21 were found in the gill; the highest expressions of TLR2 and 5s were found in the small intestine. The second highest expressions of TLR3, 7, and 8 were found in small intestine. The gene expression profiles of TLRs stimulated with Edwardsiella tarda DNA, RNA, and lipopolysaccharide (LPS) were also detected in spleen, head kidney and gill. TLR9 and TLR21 were sensitive to E. tarda DNA; TLR 8 and TLR21 were sensitive to E. tarda RNA; and TLR1 and TLR14 were sensitive to E. tarda LPS. The expressions of the other TLR genes showed no significant changes. The results imply that the expressions of these TLR genes in P. olivaceus are differently regulated in the whole body and play important roles in the immune response against E. tarda infection.

Identification and antioxidant capacity of 4-hydroxyphenylpyruvate dioxygenase (HPPD), a new favored herbicide target, in Apis cerana cerana.

4-Hydroxyphenylpyruvate dioxygenase (HPPD), a nonheme oxygenase, catalyzes the second step of the tyrosine catabolic pathway, which is shared by almost all aerobic life forms. This demonstrates its importance in aerobic biology. We isolated an HPPD homolog from Apis cerana cerana and named it AccHPPD. AccHPPD has an open reading frame (ORF) length of 900 bp and encodes a 299 amino acid protein that has a predicted molecular weight of 34.67 kDa and an isoelectric point of 6.27. Amino acid analysis showed that AccHPPD contained three conserved metal ion active sites, H-101, H-184 and E-267. Real-time fluorescence quantitative PCR (RT-qPCR) analysis showed that AccHPPD mainly existed in specific tissue sites, mainly high in the legs and in the thorax and epidermis, and in specific developmental stages, mainly adults. Under temperature, pesticide, heavy metal and ultraviolet (UV) radiation treatments, the expression level was downregulated, but under H2O2 treatment, the expression level was upregulated. Exogenous expression of the recombinant AccHPPD plasmid in E. coli enhanced the resistance to HgCl2 and H2O2. Inhibition of AccHPPD activity was demonstrated by the upregulation of the tyrosine content after feeding with the inhibitor 2-(2-nitro-4-trifluoromethyl benzoyl)-1,3-cyclohexanedione (NTBC). After silencing of AccHPPD, the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) decreased, and the expression levels of AccBax- and AccCaspase8-related genes were upregulated. The antioxidant genes AccCAT, AccGSTZ1, AccGSTD, AccSOD2, AccTpx3, AccCYP4G11, AccGDTS4, AccGSTO2 and AccMSRA were all upregulated. These results suggest that AccHPPD may serve an integral function in the response of A. cerana cerana to oxidative stress.

Rapid and visual detection of Mycoplasma synoviae by recombinase-aided amplification assay combined with a lateral flow dipstick.

Mycoplasma synoviae (MS) is an important avian pathogen that has brought substantial economic losses to the global poultry industry. Fast and accurate diagnosis is one of the critical factors for the control of MS infection. This study established a simple, rapid and visual detection method for MS using a recombinase-aided amplification (RAA) combined with a lateral flow dipstick (LFD). The reaction temperature and time of the RAA-LFD assay were optimized after selecting the primers and probe, and the specificity and sensitivity rates were analyzed. The results showed that RAA could amplify the target gene in 20 min at a constant temperature of 38°C, and the amplification products could be visualized by LFD within 5 min. There was no cross-reaction with Mycoplasma gallisepticum (MG), Pasteurella multocida (P. multocida), Escherichia coli (E. coli), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and avian reovirus (ARV). Furthermore, the RAA-LFD assay exhibited high sensitivity with a detection limit of 10 copies/µL. A total of 128 clinical samples with suspected infection of MS were tested by RAA-LFD, PCR, and real-time fluorescence quantitative PCR (RFQ-PCR). The coincidence rate of the detection results was 95.3% between RAA-LFD and PCR, and 98.4% between RAA-LFD and RFQ-PCR. These results suggested that the RAA-LFD method established in the present study was easy to use and was associated with strong specificity and high sensitivity. This method was very suitable for the rapid detection of MS in clinical practice.

Hydrogen sulfide treatment retrieves the inhibition of growth and development characteristics in silkworm (Bombyx mori) via phosphoacetyl glucosamine mutase gene knock down.

Phosphoacetyl glucosamine mutase (PGM) is the key gene for glycolysis of important metabolic pathways in silkworm, and H2 S (7.5 µM) can promote the growth and development of silkworm. Herein, we used body cavity injection of small-interfering RNA (siRNA) to interfere with the PGM gene in H2 S-treated silkworms. After RNA interference (RNAi), we investigated the growth and development of the silkworm. H2 S treatment could significantly recover the inhibition of body weight, cocoon weight, cocoon shell weight, and cocoon shell ratio by knocking down PGM gene in silkworm, without significant effects on eggs laying and production, and then analyzed the mRNA expression of PGM gene. The interference of siRNA significantly decreased the expression of targeted PGM gene and was concentrated in 48 h followed by gradual recovery. Three interference fragments also showed different interference effects, and siRNA of PGM-3 exerted the highest interference effect to the target gene expression. Fat body had the highest mRNA expression of PGM gene, and the best interference effect was observed after siRNA injection. The results showed that the gene based on H2 S treatment may have an important impact on the growth and development of silkworm by affecting its metabolic pathway.

[Cloning and functional characterization of lignan glycosyltransferase gene IiUGT349 in Isatis indigotica].

Based on the transcriptome data of Isatis indigotica, a total of 110 putative glycosytransferases were identified. Through prokaryotic expression and enzymic activity assay in vitro, a novel lignan glycosyltransferase gene was screened out and named IiUGT349, which catalyzed lariciresinol into lariciresinol-4-O-ß-D-glucoside and lariciresinol-4'-O-ß-D-glucoside. Bioinformatics analysis suggested that IiUGT349 contained an open reading frame(ORF) of 1 401 bp encoding a protein of 467 amino acids. A protein analysis indicated that IiUGT349 have a predecited molecular weight of 52.77 kDa and pI of 5.96. Phylogenetic analysis showed that IiUGT349 belonging to UGT90 family shared low amino acid sequence identity with the reported lignan glycosyltransferases, which may represent a novel type of lignan glycosyltransferases. Quantitative real-time PCR(qRT-PCR) analysis showed that IiUGT349 was expressed in roots, stems, young leaves and leaves, with the highest expression level in stems. Further biochemical analysis showed that the optimal reaction time of IiUGT349 recombinant protein was 12 h and the optimal temperature was 45 ℃. Subcellular localization demonstrated that IiUGT349 was located in the cytoplasm and nucleus of plants. In this study, a new glucosyltransferase gene IiUGT349 from I. indigotica belonging to the UGT90 family was cloned, which laid a foundation to further investigate its' function and elucidate the lignan glycosides biosynthesis pathway and plays an important role for great significance for the synthetic biology of active lignan glycosides.

Significance of serum circRNA hsa_circ_0000437 in the auxiliary diagnosis and prognosis of gastric cancer / 中华检验医学杂志

Objective:To investigate the expression of hsa_circ_0000437 in the serum of patients with gastric cancer and its clinical value.Methods:The serum samples from 80 patients (57 males and 23 females) with pathologically confirmed gastric cancer (GC), 50 gastric benign disease (28 males and 22 females) and 80 healthy controls (46 males and 34 females) were collected from October 2018 to December 2020 in Affiliated Hospital of Nantong University.Serum samples from 35 of 80 gastric cancer patients after operation were collected. The expression of serum hsa_circ_0000437 was determined by real-time fluorescent quantitative PCR (RT-qPCR). Serum carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA 199) and carbohydrate antigen 724 (CA724) were determined by chemiluminescence method.Comparisons of serum hsa_circ_0000437 between groups were performed by Mann-Whitney U test.The correlation between serum expression of hsa_circ_0000437 in gastric cancer patients and its clinical pathological characteristics was performed by χ 2 test.Receiver operating characteristic (ROC) curve and the area under the curve of ROC (AUC) were used to evaluate their diagnosis efficiency. Kaplan-Meier survival curve analysis was used to analyze the relationship between the expression level of serum hsa_circ_0000437 and the prognosis of patients. Results:The relative expression of hsa_circ_0000437 in GC, gastric benign disease, healthy controls were 2.252 (1.235, 4.765), 1.598(1.139, 1.982) and 1.000 (0.818, 1.385) respectively.The relative expression of hsa_circ_0000437 in GC was significantly higher than that in gastric benign disease ( P<0.001) and healthy controls ( P<0.001). The difference between gastric benign disease and healthy controls was also statistically significant ( P<0.001).The differences of serum hsa_circ_0000437 expression in GC patients between T stage, N stage, and tumor differentiation were statistically significant. The AUC of hsa_circ_0000437, CEA, CA199 and CA724 in GC patients were 0.863, 0.619, 657 and 0.608 respectively compared with healthy controls. The AUC of above four-parameter panel was 0.892 and the sensitivity was up to 97.5% (78/80). Kaplan-Meier survival curve showed that the overall survival rate of patients with high serum hsa_circ_0000437 expression was significantly lower than that of patients with low expression ( P=0.008). Conclusion:Serum hsa_circ_0000437 could be a biomarker for the auxiliary diagnosis and prognosis of GC.

Establishment and application of detection method for interferon stimulated genes expression in children / 中华检验医学杂志

Objective:To establish the detection method for the interferon stimulated genes(ISGs), calculate the cut-off value and test it in clinical practice.Methods:Patients with type I interferonopathies who were admitted to Peking Union Medical College Hospital from November 2017 to September 2021 were chosen as the disease group, and healthy children were included as the control group. A total of 18 children were in the disease group, including 8 males and 10 females, with a median age of 8.5 years for the first test. From them 25 blood specimens were collected. A total of 28 healthy children, aged 1 to 18 years, with a median age of 10.5 years, including 15 males and 13 females, were included in the control group. Blood samples of 34 controls and 18 interferonopathies patients were collected, then total RNA extraction and cDNA synthesis were performed. Real-time quantitative polymerase chain reaction assays were run in duplicate to measure the expression of six ISGs: interferon induced protein with tetratricopeptide repeats 1 (IFIT1), interferon α inducible protein 27 (IFI27), interferon induced protein 44 like (IFI44L), interferon stimulated genes 15 (ISG15), sialic acid binding Ig like lectin 1 (SIGLEC1), and radical S-adenosyl methionine domain containing 2 (RSAD2). The relative abundances of each target transcript was normalized to the expression level of β-Actin and OAZ. The median fold change of the six ISGs was used to create an interferon score (IS) for each individual. Samples with abnormal expressions were removed and the cDNA mix of the remaining samples was used as a calibrator to calculate the IS. We define an abnormal IS as being greater than+2 standard deviations above the mean of controls. Differences in IS between groups were compared using t-test or Mann-Whitney U-test. Results:The mean IS of controls was 1.046, standard 0.755, and the cut-off value was 2.556. A total of 25 samples from 18 interferonopathies patients were tested. The mean value was 27.010 with a 15/18 abnormality rate. Compared with the control group, IS in patients was significantly higher, t=4.247( P=0.000 1). The accuracy, precision, sensitivity, and specificity were 91.30% (42/46), 7.47%(0.084/1.124), 15/18, and 96.43% (27/28), respectively. Conclusion:This study provides a new and reliable method for clinical screening and dynamic monitoring of type Ⅰ interferonopathies by detecting ISGs expression and creating an IS.

Establishment and preliminary application of a joint detection method for transplantation-associated infection pathogens / 中国输血杂志

【Objective】 To investigate the effectiveness of multilink real-time fluorescence quantitative PCR (qPCR) in the detection of common pathogens in transplantation. 【Methods】 The primers of the qPCR detection system were designed for 24 common infectious pathogens after clinical transplantation, and the standard plasmids of each pathogen were used to verify the qPCR reaction.After the primer probe effect and concentration of each pathogen reaction system in this experiment was optimized, the sensitivity, correlation coefficient (R2) and amplification efficiency (E) of qPCR method were analyzed and confirmed.Twenty-two samples from patients, who underwent liver and kidney transplantation in transplant ICU of Sichuan Provincial People′s Hospital, were used to verify the application of the detection system.The total nucleic acid of 100 μL was extracted from each individual and divided into two aliquots, which were detected by multi-link qPCR reaction system and analyzed by high-throughput sequencing method (NGS). At the same time, samples (2 mL each) were taken from the transplanted patients for microbial culture.The results of the three detection methods were compared, and the NGS method was taken as the gold standard to analyze the positive detection rate of the multi-link qPCR method and its difference with the culture method and NGS. 【Results】 The lower limit of qPCR detection for 24 pathogens in the established qPCR detection system was 101cp/μL(R2>0.99), with the positive rate of pathogens at 59.1% (13/22), showing significant difference versus microbial culture (18.2%, 4/22)(P<0.05), but not versus NGS (63.6%, 14/22)(P>0.05). Percentage of pathogens detected was as follows: human herpetic virus type 6 (HHV-6) 30.8% (4/13), cytomegalovirus (HCMV) 23.1% (3/13), Epstein-Barr virus (EBV) 23.1% (3/13), human parvovirus B19 15.4% (2/13), Haemophilus influenzae (Hin) 15.4% (2/13), Enterococcus faecium (EFM) 15.4% (2/13), Clostridium difficile 15.4% (2/13), Escherichia coli 7.7% (1/13), Stenotrophomonas maltophilia (Sma) 7.7% (1/13), Klebsiella pneumoniae (Kpn) 7.7% (1/13), Enterococcus faecalis (Efa) 7.7% (1/13) and Streptococcus pneumoniae (Spn) 7.7% (1/13). The consistency rate of pathogens detected by the three methods was 32% (7/22), among which the consistency rate of multi-link qPCR with NGS method was 59% (13/22), and multi-link qPCR with microbial culture was 41% (9/22). 【Conclusion】 Compared with the microbial culture, the multi-link qPCR method demonstrated high sensitivity, accurate quantification, short time and low cost for the detection of common pathogens in clinical transplantation.Multi-link qPCR combined with NGS and microbial culture is helpful to quickly predict the pathogen infection status of patients after transplantation.

Whole-Genome Sequencing and Potassium-Solubilizing Mechanism of Bacillus aryabhattai SK1-7.

To analyze the whole genome of Bacillus aryabhattai strain SK1-7 and explore its potassium solubilization characteristics and mechanism, thus providing a theoretical basis for analyzing the utilization and improvement of insoluble potassium resources in soil. Genome information for Bacillus aryabhattai SK1-7 was obtained by using Illumina NovaSeq second-generation sequencing and GridION Nanopore ONT third-generation sequencing technology. The contents of organic acids and polysaccharides in fermentation broth of Bacillus aryabhattai SK1-7 were determined by high-performance liquid chromatography and the anthrone sulfuric acid method, and the expression levels of the potassium solubilization-related genes ackA, epsB, gltA, mdh and ppc were compared by real-time fluorescence quantitative PCR under different potassium source culture conditions. The whole genome of the strain consisted of a complete chromosome sequence and four plasmid sequences. The sequence sizes of the chromosomes and plasmids P1, P2, P3 and P4 were 5,188,391 bp, 136,204 bp, 124,862 bp, 67,200 bp and 12,374 bp, respectively. The GC contents were 38.2, 34.4, 33.6, 32.8, and 33.7%. Strain SK1-7 mainly secreted malic, formic, acetic and citric acids under culture with an insoluble potassium source. The polysaccharide content produced with an insoluble potassium source was higher than that with a soluble potassium source. The expression levels of five potassium solubilization-related genes with the insoluble potassium source were higher than those with the soluble potassium source.

Diagnostic Value of Detection of Pregenomic RNA in Sera of Hepatitis B Virus-Infected Patients with Different Clinical Outcomes.

Pregenomic RNA (pgRNA) is a direct transcription product of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and it plays important roles in viral genome amplification and replication. This study was designed to investigate whether serum pgRNA is a strong alternative marker for reflecting HBV cccDNA levels and to analyze the correlation between serum pgRNA, serum HBV DNA, and hepatitis B surface antigen (HBsAg). A total of 400 HBV-infected patients who received nucleos(t)ide analog (NA) therapy with different clinical outcomes were involved in this research. Case groups included asymptomatic hepatitis B virus carrier (ASC), chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC) patients, with 100 patients in each group. The results showed that the levels of HBV pgRNA had significant differences between these 4 groups. Serum pgRNA levels correlated well with serum HBV DNA and HBsAg levels (HBV pgRNA levels versus HBV DNA levels, r = 0.58, P < 0.001; HBV pgRNA levels versus HBsAg levels, r = 0.47, P < 0.001). In addition, we focused on the 108 HBV-infected patients with HBV DNA levels of <500 IU/ml; it was surprising to find that in 17.57% (13/74) of cases, HBV pgRNA could be detected even when the HBV DNA level was below 20 IU/ml. In conclusion, HBV pgRNA levels in serum can be a surrogate marker for intrahepatic HBV cccDNA compared with serum HBV DNA and HBsAg. The detection of serum HBV pgRNA levels may provide a reference for clinical monitoring of cccDNA levels and the selection of appropriate timing for discontinuing antiviral therapy, especially when HBV DNA levels are below the detection limit.

[Study on Dynamic Changes of Human Papillomavirus 16 E5 Gene in the Occurrence of Cervical Cancer].

OBJECTIVE: To explore the dynamic changes of human papillomavirus (HPV) type 16 E5 gene in the development of cervical cancer and the significance of E5 mRNA in early screening of cervical cancer. METHODS: Paraffin specimens of cervical lesions were collected from 49 cases (HPV positive) during September 2015 to December 2017 According to the standard of FIGO, all cervical lesions were diagnosed as: 13 cases of cervicitis, cervical intraepithelial neoplasia disorders (CIN) Ⅰ in 5 cases, CIN Ⅱ in 18 cases, CIN Ⅲ in 5 cases, 8 cases of cervical cancer. Real-time fluorescence quantitative PCR was used to detect the integrity of E5 gene and the mRNA expression levels of E5, E6 and E 7in cervical tissues. RESULTS: All the 49 cases showed positive HPV16 infection. E5 genetic integrity in CINⅠwas higher than that in cervical inflammation, CIN Ⅱand cervical cancer (P < 0.05), which was also higher than that in CIN Ⅲ, but without statistically significance (P>0.05). The mRNA levels of E5, E6, E7 were the highest in CIN Ⅲ. Compared with E6 and E7, E5 presented superior expression in all types of cervical lesions (P < 0.05), while E 6and E7 mRNA expressions only increased in CIN Ⅲ and cervical cancer. CONCLUSION: In the patients with HPV16 infection, the integrity of E5 gene in cervical tissues may be related to the occurrence and development of cervical diseases. E5 gene is expected to be the target gene for early screening of cervical cancer.

Therapeutic effects of Erbin inhibitor on spinal cord contusion in mice.

Erbin has been shown to maintain the integrity of cell structure, regulate the proliferation and differentiation of cell and transconduct signals in the pathways. This study was conducted to assess the therapeutic effects of an Erbin inhibitor on spinal cord contusion in mice. Spinal contusion models of mouse were constructed and treated with an Erbin inhibitor. The experimental animals were divided into control (normal animal without any treatment), models with spinal cord injury (SIM), and models receiving Erbin inhibitor (Inhibitor). The contents of 5-hydroxytryptamine (5-HT) and reactive oxygen species (ROS) in the brain and spinal cord tissues were measured using ELISA. The expression of ERK1/2, MAPK, NF-kB and NRG1 was quantified using qRT-PCR, Western blot analysis and immunohistochemistry. Flow cytometry was used to determine the formation of macrophages. Erbin interference vector was constructed and its interference effect on the expression of these genes was characterized in cultured bone marrow cells. Spinal contusion models were successfully constructed. Administering Erbin inhibitor inhibited the expression of ERK1/2, MAPK and NF-kB and up-regulated the expression of NRG1. Flow cytometry showed that Erbin inhibitor induced the formation of a large number of macrophages, which are beneficial to the recovery of spinal cord injury. Experiments with Erbin interference vector showed similar impacts on the expression of genes at cellular level as the inhibitor did. Our work has demonstrated that the Erbin inhibitor is very effective to treat spinal cord contusion in mice. The possible mechanism of therapeutic effect is that the inhibitor suppresses the ERK1/2/MAPK and/or NF-kB/MAPK signal pathways and enhances the NRG1-ErbB signaling pathway by reducing the expression of Erbin, leading to the inhibition of apoptosis, promotion of proliferation and differentiation, and subsequent repair of the damaged spinal cord.