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The development of high-throughput sequencing techniques enabled a deeper and more comprehensive understanding of environmental microbiology. Specifically, the third-generation sequencing techniques represented by nanopore sequencing have greatly promoted the development of environmental microbiology research due to its advantages such as long sequencing reads, fast sequencing speed, real-time monitoring of sequencing data, and convenient machine carrying, as well as no GC bias and no PCR amplification requirement. This review briefly summarized the technical principle and characteristics of nanopore sequencing, followed by discussing the application of nanopore sequencing techniques in the amplicon sequencing, metagenome sequencing and whole genome sequencing of environmental microorganisms. The advantages and challenges of nanopore sequencing in the application of environmental microbiology research were also analyzed.
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Environmental Microbiology , High-Throughput Nucleotide Sequencing , Metagenome , Nanopore Sequencing , NanoporesABSTRACT
Objective:To study whether simvastatin could inhibit viral replication during human metapneumovirus (hMPV) infection.Methods:Human bronchial epithelial cells (16HBE) were infected with hMPV and then treated with or without simvastatin. Real-time quantitative PCR (qPCR) and Western blot were used to detect virus titers and the activation of autophagy and related pathways. BALB/c mice were infected with hMPV and then treated with simvastatin through intragastric administration. Pathological changes in lung tissues were observed. Changes in viral loads and the activation of autophagy and related pathways in proteins and RNA extracted from lung tissues were detected.Results:The in vitro experiment showed that the hMPV+ simvastatin group had decreased virus titer and enhanced autophagy than the hMPV group. The AKT/mTOR pathway in the hMPV+ simvastatin group was inhibited, which was verified by a further experiment using rapamycin, a specific inhibitor of AKT/mTOR pathway. The in vivo experiment showed that the virus titer in the hMPV+ simvastatin group was lower than that in the hMPV group, but there was no significant difference in the activation of autophagy. The AKT/mTOR pathway was down-regulated in the hMPV+ simvastatin group. HE staining revealed that obvious pathological changes were observed in the hMPV group, but the condition was improved after simvastatin intervention. Conclusions:Simvastatin can inhibit the replication of hMPV, which is associated with the activation of autophagy induced by AKT/mTOR pathway.
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Objective To investigate the efficacy and the course of desensitization treatment in bronchial asthma with positive specific IgE to dust mite in children. Methods A total of 105 children with bronchial asthma with positive specific IgE to dermatophagoides farinae allergens were randomized into the observation group and the control group. Children in the control group were treated to continue anti-asthma according to the routine of prevention and treatment children with asthma. Chinldren in the observation group were treated by dermatophagoides farinae drops in addition to the treatment of children in the control group. The recurrence of asthma was compared between two groups at 25 weeks post-treatment. At 25 weeks post-treatment , children in the observation group was randomly divided into the observation groupⅠand group Ⅱ. Children in the observation groupⅠreceived continuous treatment except for desensitization treatment. Children in the observation group II received the sublingual immunotherapy with dermatophagoides farinae drops (No.4) for 1 year in addition to the treatment in the observation groupⅠ. The recurrence of asthma was also compared between the two sub-groups. Results The rate and times of recurrence of asthma were lower in the observation group than those in the control group(P 0.05). Conclusion The recurrent rate and frequency of asthma could be reduced by the sublingual immunotherapy with dermatophagoides farinae drops in children with asthma of positive specific IgE to dust mite. The course of treatment may be half year long.
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To obtain human antibodies against the Gn protein of Severe fever with thrombocytopenia syndrome virus (SFTSV) with phage display technology, this study aimed to screen anti-Gn protein antibodies from an anti-SFTSV Fab human phage display library. Antibody genes were identified by sequence analysis and the specificity of antibodies was confirmed by ELISA. The Fab antibody genes were cloned into the HL51-14 vector and expressed in a mammalian cell expression system. IgG antibodies were then purified by protein A affinity chromatography,and the results were further confirmed by ELISA,IFA,western blotting assays and micro-neutralization tests. The results showed that, after three rounds of panning, there were 390 human Fab antibodies against SFTSV particles, of which 364 were specific for nucleoprotein. Coated with the Gn protein, eight different Fab antibodies specific for Gn protein were obtained after the determination of the subtype and subclass of antibodies by gene sequencing; five of these antibodies were from the Lambda library and three were from the Kappa library. The eight IgG antibodies could specifically bind to Gn protein according to the ELISA, IFA and Western blotting assays. The micro-neutralization test showed that these eight antibodies had no neutralizing activity,but they could still provide a reference for research in human monoclonal antibodies against SFTSV.
Subject(s)
Humans , Antibodies , Genetics , Allergy and Immunology , Bunyaviridae Infections , Genetics , Allergy and Immunology , Virology , Cell Line , Cloning, Molecular , Immunoglobulin Fab Fragments , Genetics , Allergy and Immunology , Immunoglobulin G , Genetics , Allergy and Immunology , Neutralization Tests , Phlebovirus , Genetics , Allergy and Immunology , Viral Proteins , Genetics , Allergy and ImmunologyABSTRACT
Objective To study the effect of Jingyebaidu granules on treating cytomegalovirus ( CMV) infection during mid-pregnancy. Methods The sexually mature guinea pigs with no CMV infection history served as the subjects. Put the male and female ones in the same cages. Then the female ones were randomly divided into three groups during mid-pregnancy. Model control group:15 guinea pigs which were inoculated 1 mL suspension of GPCMV intraperitoneally. Jingyebaidu Medicine group:15 guinea pigs which were treated with Jingyebaidu(3. 09 mL·kg-1 ) through stomach perfusion after inoculation for 14 days. Normal control group:15 normal mid-pregnant guinea pigs. Viremia rates were examined 7 days after infection. All animals were sacrificed 20 days after infection. The placenta infection rate, pup infection rate, still-born rate were examined. Results Compared with the normal controls, the still-born rate was increased in model control group(8. 33% vs 34. 55%, P<0. 05). In comparison to the model control group, the GPCMV maternal infection rate(86. 67% vs 33. 33%), placenta infection rate (91. 67% vs 61. 22%), pup infection rate(90. 91% vs 48. 28%), still-born rate(34. 55% vs 15. 52%) were significantly decreased in the Jinyebaidu group (all P<0. 05). Conclusion Jinyebaidu granules could reduce maternal infection,pup loss, and placenta infection caused by CMV inoculation during mid-pregnancy.
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Objective: This paper is aimed at to evaluate B7-H1 expression as induced by human cytomegalovirus [HCMV] in extravillous cytotrophoblast cell line HPT-8 and possible underlying mechanism
Method: Real time PCR and flow cytometry were used to determine B7-H1 mRNA and protein before and after HCMV infection in HPT-8 cells. Western blot analysis was used to determine the level of MAPK phosphorylation in HPT-8 cell lines infected with HCMV
Results: 100TCID50 was found to be the most effective dose, capable of stimulating B7-H1 mRNA and protein expression in HPT-8 cells. When empty control group was considered to have a B7-H1 mRNA value of 1, B7-H1 mRNA was 4.32 in 100TCID50 group. In flow cytometry study, mean fluorescence intensity [MFI] of 100TCID50 group was 16.14, while empty control group was 1.34. Both mRNA and protein expression were found to be significantly increased [P < 0.05] in 100TCID50 group compared to empty control group
The result of Western blot analysis showed increase in B7-H1 expression caused by the extracellular signaling that was related to ERK activation and the ERK inhibitor U0126 was found to reverse this increase
Conclusion: HCMV upregulates B7-H1 expression in human extravillous cytotrophoblast cell line HPT-8, which is related to MAPK activation. Our result would be helpful in finding better therapies against intrauterine HCMV infection
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This study examined the anti-viral effect of ursolic acid on guinea pig cytomegalovirus (GPCMV) and explored the steps of viral replication targeted by ursolic acid. Cytopathic effect assay and MTT method were employed to determine the 50% cellular cytotoxicity (CC(50)), 50% effective concentration (EC(50)) and therapeutic index (TI) with GPCMV. To investigate the specific anti-viral effect of ursolic acid at different temperatures and time points, two other medicines, ganciclovir and Jinyebaidu (JYBD), serving as controls, were studied for comparison. Our results showed that the CC50 of ganciclovir, JYBD and ursolic acid were 333.8, 3015.6, 86.7 μg/mL, respectively; EC(50) of ganciclovir, JYBD and ursolic acid was 48.1, 325.5 and 6.8 μg/mL, respectively; TI of ganciclovir, JYBD and ursolic acid was 7, 9, 13, respectively. Similar with ganciclovir, ursolic acid could inhibit the viral synthesis, but did not affect the viral adsorption onto and penetration into cells. We are led to conclude that the anti-cytomegalovirus effect of ursolic acid is significantly stronger than ganciclovir or JYBD, and the cytotoxic effect of ursolic acid lies in its ability to inhibit viral synthesis.
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This study examined the anti-viral effect of ursolic acid on guinea pig cytomegalovirus (GPCMV) and explored the steps of viral replication targeted by ursolic acid. Cytopathic effect assay and MTT method were employed to determine the 50% cellular cytotoxicity (CC(50)), 50% effective concentration (EC(50)) and therapeutic index (TI) with GPCMV. To investigate the specific anti-viral effect of ursolic acid at different temperatures and time points, two other medicines, ganciclovir and Jinyebaidu (JYBD), serving as controls, were studied for comparison. Our results showed that the CC50 of ganciclovir, JYBD and ursolic acid were 333.8, 3015.6, 86.7 μg/mL, respectively; EC(50) of ganciclovir, JYBD and ursolic acid was 48.1, 325.5 and 6.8 μg/mL, respectively; TI of ganciclovir, JYBD and ursolic acid was 7, 9, 13, respectively. Similar with ganciclovir, ursolic acid could inhibit the viral synthesis, but did not affect the viral adsorption onto and penetration into cells. We are led to conclude that the anti-cytomegalovirus effect of ursolic acid is significantly stronger than ganciclovir or JYBD, and the cytotoxic effect of ursolic acid lies in its ability to inhibit viral synthesis.
Subject(s)
Animals , Antiviral Agents , Pharmacology , Cells, Cultured , Guinea Pigs , Roseolovirus , Triterpenes , PharmacologyABSTRACT
The effect of human cytomegalovirus (HCMV) on invasive capability of early pregnant extravillous cytotrophoblasts (EVTs) was investigated in vitro. Primary EVTs were obtained by complex phosphoesterasum digestion and gradient centrifugation from villous tissue aseptically taken from healthy pregnant women. Cytokeratin7 (CK7), vimentin (Vim) and c-erbB-2 were immunocytochemically detected to identify source of cells, and HCMVpp65 antigen was assayed to determine the infection state of primary EVTs by immunocytochemical staining. The EVTs were divided into two groups: control group and HCMV group, and the expression of c-erbB-2, matrix metalloproteinase-2 (MMP-2) and MMP-9 proteins was detected in two groups by immunocytochemistry and Western blotting. Enzymic activity changes of MMP-2 and MMP-9 were tested by gelatin zymography in primary EVTs infected with HCMV. The invasion of primary EVTs was detected by cell invasion assay in vitro after they were infected by HCMV. The cell source identification showed that the cells obtained were highly-pure primary EVTs, and primary EVTs could be infected by HCMV. Primary EVTs could express c-erbB-2, MMP-2 and MMP-9 proteins, and as compared with control group, the protein expression was decreased significantly in HCMV groups (P<0.05). Primary EVTs could secrete active MMP-2 and MMP-9 in vitro, and the activity of two MMPs was decreased significantly in HCMV groups (P<0.05). The in vitro cell invasion assay showed that the number of primary EVTs permeating Matrigel in HCMV group was decreased (P<0.05). We are led to conclude that HCMV can infect primary EVTs and inhibit their invasion capability, suggesting that the impaired EVT's invasion capability might be related to the abnormal expression of c-erbB-2, MMP-2 and MMP-9 proteins.
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This study examined the impacts of intrauterine murine cytomegalovirus (MCMV) infection on the long-term learning and memory of offspring. Sexually matured male and female BALB/C mice without MCMV infection were identified by ELISA and then mated. Seventy pregnant mice were randomly divided into the virus group (n=40) and the control group (n=30), in which the pregnant mice were subjected to placenta inoculation of MCMV suspension (1 μL, 1×106 PFU) or the same amount of cell culture medium, respectively, at gestational age of 12.5 days. Some pregnant mice [virus group (n=20), control group (n=15)] were sacrificed by cervical dislocation at gestational age of 18.5 days, and the head circumference and brain weight of the mouse fetuses were measured, and the MCMV infection in their brain tissues was detected by PCR. The other pregnant mice [virus group (n=20), control group (n=15)] delivered naturally, and the learning and memory capability of the offspring at 70-day-old was analyzed by Morris water maze test. The results showed that 28.57% mouse fetuses in the virus group developed viral infection in the brain. Their head circumference and brain weight were significantly reduced as compared with those in the control group (P<0.01). The Morris water maze test revealed that the mouse offspring in the control group found the platform with straight-line trajectories after training. In contrast, the counterparts in the virus group intended to enter the central area, but looked for the platform with a circular trajectory. And the infected mice exhibited prolonged swimming distance and swimming latency (P<0.01). It was concluded that: (1) placenta inoculation of MCMV can cause fetal brain infection and intrauterine development retardation; (2) the offspring of MCMV placenta inoculation mice showed a long-term decline in learning and memory capability.
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The present study examined von Willebrand factor (vWF) levels and ADAMTS13 activity in pregnant and severe preeclamptic women in order to shed light on the prothrombotic state in severe preeclampsia. Thirty healthy women of childbearing age, 22 second trimester pregnant women, 30 third trimester pregnant women and 10 severe preeclamptic patients were recruited in this study. ADAMTS13 activity was determined by the FRETS-vWF73 assay and vWF antigen (vWF:Ag) levels by an enzyme-linked immunosorbent assay. The results showed that there were statistically significant differences in plasma vWF antigen levels between the severe preeclamptic and third trimester pregnant women, between third and second trimester pregnant women (P<0.05). The third trimester pregnant women had significantly lower plasma ADAMTS13 activity than second trimester pregnant women (P<0.05). Nevertheless, no significant differences in plasma ADAMTS13 activity were found between severe preeclamptic patients and the third trimester pregnant women (P>0.05). In conclusion, plasma ADAMTS13 activity is normal in severe preeclampsia despite the increased vWF:Ag levels. Prothrombotic state is involved in the pathogenesis of severe preeclampsia, as a result of endothelial injury.
Subject(s)
Adult , Female , Humans , Pregnancy , Young Adult , ADAM Proteins , Blood , Metabolism , ADAMTS13 Protein , Blood Coagulation , Physiology , Case-Control Studies , Pre-Eclampsia , Blood , Pregnancy Trimester, Third , von Willebrand Factor , MetabolismABSTRACT
The present study examined von Willebrand factor (vWF) levels and ADAMTS13 activity in pregnant and severe preeclamptic women in order to shed light on the prothrombotic state in severe preeclampsia. Thirty healthy women of childbearing age, 22 second trimester pregnant women, 30 third trimester pregnant women and 10 severe preeclamptic patients were recruited in this study. ADAMTS13 activity was determined by the FRETS-vWF73 assay and vWF antigen (vWF:Ag) levels by an enzyme-linked immunosorbent assay. The results showed that there were statistically significant differences in plasma vWF antigen levels between the severe preeclamptic and third trimester pregnant women, between third and second trimester pregnant women (P0.05). In conclusion, plasma ADAMTS13 activity is normal in severe preeclampsia despite the increased vWF:Ag levels. Prothrombotic state is involved in the pathogenesis of severe preeclampsia, as a result of endothelial injury.
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Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV. Trophoblast cells were obtained by compound enzymes digestion and discontinuous percoll gradient. Cells and purity were identified by using immunocytochemistry assay with anti-CK7, Vim and beta-hCG antibodies. HCMV AD169 strain replication in isolated trophoblast cells and cell apoptosis were detected at different time points post infection (p.i.). The results showed that highly purified trophoblast cells were obtained. Specific virus replication was increased dramatically at the 24th h p.i., and then increased slowly during 48 h and 72 h. Apoptosis rate of trophoblast cells infected with HCMV was (34.68+/-3.14)% at 24th h p.i., while that in control group was (15.32+/-2.34)% (P<0.05). It was suggested that highly purified trophoblast cells can be isolated by the simplified cell purification method. HCMV can infect human trophoblast cells, and be quickly replicated, resulting in the accelerated apoptosis of human trophoblast cells during early time.
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<p><b>OBJECTIVE</b>To study the in vitro anti-human cytomegalovirus effect and the cytotoxicity of Forsythia suspensa and its main active ingredient quercetin.</p><p><b>METHOD</b>The 0% toxic dose (TD0), minimum effective concentration (MEC) and therapeutic index (TI) of anti-human cytomegalovirus activity by F. suspensa and quercetin were detected with the cytopathic assay and MTT method. Ganciclovir was used as the control drug for comparison.</p><p><b>RESULT</b>The TD0 of ganciclovir, F. suspensa and quercetin were 10, 30, 30 mg L(-1), the MEC were 10, 30, 0.3 mg x L(-1), TI were 1, 1 and 100, respectively.</p><p><b>CONCLUSION</b>The anti-human cytomegalovirus effect of quercetin is much higher than ganciclovir and F. suspensa, and the cytotoxicity is equivalent to F. suspensa but lower than ganciclovir. Therefore, quercetin has the potential advantages of anti-human cytomegalovirus effect.</p>
Subject(s)
Humans , Antiviral Agents , Pharmacology , Toxicity , Cell Line , Cytomegalovirus , Drugs, Chinese Herbal , Pharmacology , Toxicity , Forsythia , Chemistry , Quercetin , Pharmacology , ToxicityABSTRACT
Objective To study the observation and nursing for exercise-induced fatigue by thread-burying in acupoint. Methods 41 athletes-in-training were treated by thread-burying in acu-point for 2 courses of treatment, choosing main acupointa such as Guan yuan, Shen yu, Pi yu, Ming men,Zu sanli, San yinjiao. Then the changes of athletes' training and hemoglobin were observed. Results After two courses of treatment, athletes' quality of sleep, mental condition in the morning, diet, desire to train, physical fitness, training quality and concentration improved siguificantly.Level of hemoglobin in-creased distinctly. Conclusions The treatment of thread-burying in acupoint could improve sport fa-tigue. Combination with Chinese and western integrative medicine, dialectical treatment, emotion and diet nurse, western integral appraisal procedure to observe and asepsis operation played a positive part in the recovery from sport fatigue.
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To study the effects of mouse cytomegalovirus (MCMV) on the in vitro maturation, fertilization, cleavage and blastula formation of mouse oocytes, the immature oocytes were infected in vitro by MCMVs of different dosages (100 TCID(50), 10 TCID(50) and 1 TCID(50)). The oocytes were then observed for in vitro maturation, fertilization, cleavage and blastula formation and the ultrastructural changes after the culture with the viruses. Our results showed that no significant differences were found in IVM, IVF, cleavage and blastula formation among the groups treated with of virus of various dosages. And ultrastructural abnormality was observed in the oocytes treated by 100 TCID(50) of viruses. It is concluded that MCMV did not have any conspicuous effects on IVM, IVF, cleavage and blastula formation of murine immature oocytes.
Subject(s)
Blastocyst , Cells, Cultured , Cleavage Stage, Ovum , Cytomegalovirus Infections , Fertilization , Muromegalovirus/pathogenicity , Oocytes/cytology , Oocytes/growth & development , Oocytes/virologyABSTRACT
To study the effects of mouse cytomegalovirus (MCMV) on the in vitro maturation, fertilization, cleavage and blastula formation of mouse oocytes, the immature oocytes were infected in vitro by MCMVs of different dosages (100 TCID50, 10 TCID50 and 1 TCID50). The oocytes were then observed for in vitro maturation, fertilization, cleavage and blastula formation and the ultrastructural changes after the culture with the viruses. Our results showed that no significant differences were found in IVM, IVF, cleavage and blastula formation among the groups treated with of virus of various dosages. And ultrastructural abnormality was observed in the oocytes treated by 100 TCID50 of viruses. It is concluded that MCMV did not have any conspicuous effects on IVM, IVF, cleavage and blastula formation of murine immature oocytes.
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The purpose is to study the prophylactic and therapeutic effect of the traditional Chinese Medicine (TCM)-Jinyebaidu (JYBD) to guinea pig cytomegalovirus (GPCMV) intrauterine infection. The virus-free female and male guinea pigs were screened with nest-polymerase chain reaction (N-PCR). After inbred, pregnant guinea pigs were selected and divided into 3 groups randomly: 5 guniea pigs of the blank control group were not given either GPCMV or JYBD. 31 guniea pigs of the positive control group were inoculated 1 mL (10(7) TCID50) suspension of GPCMV intraperitoneal. 10 guniea pigs of the experimental group were inoculated GPCMV firstly and then perfused stomach with JYBD for 14 days (Dosage in accordance with the modulus of the weight ratio of human to guniea pig). The effects of JYBD on the intrauterine infection of GPCMV were observed. The results showed that JYBD could decrease the maternal infection rate from 100% (31/31) to 50% (5/10) (P < 0.001), the intrauterine infection rate from 100% (72/72) to 75% (21/28) (P < 0.001), and the rate of abnormal outcome of pregnancy from 64.4% (29/45) to 25.0% (7/28) (P < 0.001), the infective symptoms being relieved. It can be concluded that traditional Chinese medicine- JYBD can prevent and treat (GPCMV intrauterine infection, and can be expected a prophylactic drug for HCMV intrauterine infection.
Subject(s)
Cytomegalovirus , Cytomegalovirus Infections/drug therapy , Drugs, Chinese Herbal/therapeutic use , Fetal Diseases/drug therapy , Fetal Diseases/prevention & control , Fetal Diseases/virology , Phytotherapy , Pregnancy Complications, Infectious/drug therapy , Random AllocationABSTRACT
The purpose is to study the prophylactic and therapeutic effect of the traditional Chinese Medicine (TCM)-Jinyebaidu (JYBD) to guinea pig cytomegalovirus (GPCMV) intrauterine infec tion. The virus-free female and male guinea pigs were screened with nest-polymerase chain reaction (N-PCR). After inbred, pregnant guinea pigs were selected and divided into 3 groups randomly: 5guniea pigs of the blank control group were not given either GPCMV or JYBD. 31 guniea pigs of the positive control group were inoculated 1 mL (107 TCID50 ) suspension of GPCMV intraperitoneal. 10 guniea pigs of the experimental group were inoculated GPCMV firstly and then perfused stomach with JYBD for 14 days (Dosage in accordance with the modulus of the weight ratio of human to guniea pig). The effects of JYBD on the intrauterine infection of GPCMV were observed.The results showed that JYBD could decrease the maternal infection rate from 100 % (31/31) to 50% (5/10) (P＜0. 001), the intrauterine infection rate from 100 % (72/72) to 75 % (21/28) (P＜0. 001), and the rate of abnormal outcome of pregnancy from 64.4 % (29/45) to 25.0 % (7/28)(P＜0. 001), the infective symptoms being relieved. It can be concluded that traditional Chinese medicine JYBD can prevent and treat GPCMV intrauterine infection, and can be expected a prophy lactic drug for HCMV intrauterine infection.
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The value of ELISA, N-PCR and RT-PCR in clinical practice for pregnant women with HCMV infection was investigated. 5581 pregnant women were screened by ELISA. Among them, 100 cases were positive for IgM (group 1), 69 for both IgM and serous DNA (group 2) and 69 for both IgM and mRNA (group 3). The infectious status, maternal-fetal transmission and pregnancy outcome were monitored. It was demonstrated that the accordance rate of group 3 and group 2 with group 1 was 56.25% and 43.75%, respectively. The maternal-fetal transmission rate in the group 1, 2 and 3 was 19.00%, 40.58% and 46.15%, respectively, with a significant difference found between group 2, 3 and group 1 (P < 0.01). Incidence of spontaneous abortion, fetal death, fetal abnormality and neonatal death in group 1, 2 and 3 was 10.00%, 15.94% and 30.77%, respectively, and that of group 3, 2 was 4 and 2 times as much as that of group 1, respectively (OR = 4.00, P < 0.001; OR = 2.343, P < 005, respectively). It was concluded that HCMV-IgM(+) can only be considered as an screening indicator for pregnant women with HCMV infection, while IgM(+) combined with serous DNA(+) or mRNA(+) indicates active infection and has a high incidence of maternal-fetal transmission and abnormal pregnancy outcome.