OBJECTIVETo investigate the diagnostic application of molecular detection of enterovirus type 71 (EV71) infection using post-mortem paraffin-embedded tissue.

METHODSTwo autopsy cases of EV71 infection were studied by histopathological and immunohistochemical methods. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the viral RNA in paraffin-embedded tissue samples.

RESULTSCharacteristic features of acute encephalitis were seen in the brain, with most prominent lesions found in the brain stem in both cases. Inflammatory cells were largely CD68-positive microglia with a few CD15-positive neutrophils in the areas of neuronal necrosis. The 5'-untranslated region of EV71 was detected in the medulla by RT-PCR using paraffin-embedded tissues of both cases. Sequencing analysis of the RT-PCR products showed 100% homology to the EV71 strain, recently submitted to the GenBank database from Fuyang, Anhui province.

CONCLUSIONSMolecular detection of EV71 can be performed on formalin-fixed, paraffin-embedded tissue samples from fatally infected patients. Timely and accurate diagnosis of the infection by such molecular approach is crucial for the proper clinical and public health intervention.

5' Untranslated Regions ; Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Autopsy ; Brain ; metabolism ; Encephalitis ; metabolism ; virology ; Enterovirus A, Human ; genetics ; isolation & purification ; Enterovirus Infections ; metabolism ; pathology ; virology ; Female ; Humans ; Infant ; Lewis X Antigen ; metabolism ; Male ; Paraffin Embedding ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, RNA

Cocktail probe substrates approach is a fast, sensitive and high through put method to determine cytochrome P450 enzymes activity. It has been widely used to screen early drug development, analyze drug metabolism types and confirm the metabolism pathways, study drug-drug interactions, optimize clinical regimen, evaluate post marketing drugs and help liver/kidney pathological studies. This article reviewed characteristics of Cocktail probe substrates, focused on the application to traditional Chinese medicine to CYP450 system as follows: the metabolic pathway research of Chinese herb active ingredients; processing way and compatibility of medical herbs affect CYP450; find out the metabolic characteristic of Chinese patent medicine, study in pharmacy of national minority; do research in liver protective effect of traditional Chinese medicine and evaluate traditional Chinese medicine syndromes in animal models. This article make a summary of existing research results and also make a comparison of cocktail probe substrates approach application to western medicine and Chinese medicine.

OBJECTIVETo obtain the recombinant rv1837c and rv3803c of Mycobacterium tuberculosis using gene engineering technology and explore their prokaryotic expression, purification, and immunogenicity.

METHODSThe Mycobacterium tuberculosis rv1837c and rv3803c genes were amplified by polymerase chain reaction, and then cloned into the vector pTA2, followed by the subclone into the expression vector pET30a (+). The resulting plasmids, named pET30a (+): rv1837c and pET30a (+): rv3803c, encode recombinant protein containing a hexa-histidine tag on its N-terminus. pET30a (+): rv1837c and pET30a (+): rv3803c were introduced into E. coli BL21 (DE3) by transformation respectively, and the recombinant gene was induced with 0.4 mmol/L isopropyl-D-thiogalactopyranoside. The expressed products were identified by Western blot with hexa-histidine tag antibody and serum from tuberculotic patients. The histidine tagged protein was purified by nickel nitrilotriacetic acid His-Bind resin. Rabbits were immunized with purified recombinant Rv1837c and Rv3803c proteins. Then the purified recombinant Rv1837c and Rv3803c proteins were used to detect antibody in rabbit serum, which had been immunized by Western blot.

RESULTSAfter transformation of the E. coli and induction with 0.4 mmol/L of isopropyl-D-thiogalactopyranoside, recombinant target proteins Rv1837c (relative molecular mass: 92000) and Rv3803c (relative molecular mass: 38 000) were expressed in pET30a (+): rv1837c and pET30a (+): rv3803c system. The expressed protein existed in cytoplasm in an unsoluble form and amounted to 30% and 50% of the total proteins of E. coli. The purity of the purified protein reached 90%. The immunogenicity of the recombinant proteins Rv1837c and Rv3803c was strong, as identified by Western blot.

CONCLUSIONThe prokaryotic expression recombinant plasmids pET30a (+): rv1837c and pET30a (+): rv3803c was successfully constructed and the recombinant proteins Rv1837c and Rv3803c were obtained, which laid a basis for the optimized diagnosis of active tuberculosis.

Antibodies ; metabolism ; Bacterial Proteins ; genetics ; immunology ; metabolism ; Blotting, Western ; Escherichia coli ; metabolism ; Genetic Vectors ; Mycobacterium tuberculosis ; genetics ; immunology ; metabolism ; Plasmids ; metabolism ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; metabolism

OBJECTIVE: To investigate the predictive value of cervical length（CL）changes in spontaneous preterm birth（SPTB）in twin pregnancies in the second and third trimesters of pregnancy.METHODS: A retrospective analysis was made of 166 cases of twin pregnant women who underwent transvaginal ultrasound to measure CL during the second trimester of pregnancy（20~25 weeks）and the third trimester of pregnancy（28~32 weeks）from January 2014 to December 2017 in the Third Hospital of Peking University and Tongzhou Maternal and Child Health Hospital of Beijing.Evaluate the predictive value of CL changes in SPTB before 32 and 34 weeks.The area under the receiver-operating characteristics（ROC）curve was compared by bootstrap method.Assessment of the value of CL in the third trimester of pregnancy and CL in the second trimester of pregnancy alone in predicting SPTB before 32 and 34 weeks.RESULTS: Of the 166 cases,90 were full-term delivery and 76 were premature delivery.The median CL of mid and late pregnancy was 34 mm and 29 mm respectively,and it was 35.5 mm and 31 mm,and in full-term delivery.32.5 mm and21 mm in premature delivery,respectively.There were significant differences among the three groups（All P<0.001）.According to whether the shortening of CL was more than 20%,they are divided into shortening group（CL shortening more than 20%,78 cases）and stabilizing group（no shortening of CL or shortening less than 20%,88 cases）.The preterm birth rate of shortening group was 2.22 times and 1.85 times higher than that of stabilizing group before 32 weeks and 34 weeks,but there was no significant difference.Of 40 cases with CL≤ 25 mm,there were 34 cases in shortening group,6 cases in stabilizing group.Excluding these twin pregnant women,the preterm birth rate in the shortening group increased by 7.58 times and 2.09 times than that in the stabilizing group before 32 weeks and 34 weeks.There was a significant difference in the preterm birth rate between the shortening group and the stabilizing group before 32 weeks（P=0.049）.The area under ROC curve of preterm birth predicted by CL in late pregnancy was increased compared with that in mid-pregnancy before 32 weeks and 34 weeks,but the difference was not statistically significant.CONCLUSION: In twin pregnancies,the CL shortening ≥ 20% at 28~32 weeks of gestation increases the risk of preterm birth before 32 weeks;with CL>25 mm and CL shortening≥ 20%,it can better predict preterm birth before 32 weeks.

ObjectiveTo explore the role of transient receptor potential vanilloid 1 （TRPV1） channel in reducing cardiomyocyte toxicity of Aconiti Kusnezoffii Radix processed with Chebulae Fructus. MethodH9c2 cardiomyocytes cultured in vitro were used as a model to assess cell viability by methyl thiazolyl tetrazolium （MTT） assay， the expression of TRPV1 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the leakage rate of lactate dehydrogenase （LDH）， the changes of nucleus， reactive oxygen species （ROS）， mitochondrial membrane potential and Ca²⁺ contents were detected by enzyme linked immunosorbent assay （ELISA）. ResultCompared with the blank group， when the concentration was ≥0.5 g·L^-1， the cell viability was significantly decreased （P<0.01）， the leakage rate of LDH， the release of ROS and Ca²⁺ were increased， the mitochondrial membrane potential was decreased， and the nucleus was pyknosis or even broken in raw Aconiti Kusnezoffii Radix and Aconiti Kusnezoffii Radix processed with Chebulae Fructus groups. When the concentration was ≥0.5 g·L^-1， compared with the same mass concentration of raw Aconiti Kusnezoffii Radix group， the cell viability increased significantly （P<0.01）， the leakage rate of LDH， the release of ROS and Ca²⁺ decreased， the mitochondrial membrane potential increased， and the nuclear morphology improved in Aconiti Kusnezoffii Radix processed with Chebulae Fructus group. Application of the same mass concentration of raw Aconiti Kusnezoffii Radix to H9c2 cardiomyocytes pretreated with the TRPV1 inhibitor BCTC significantly increased cell viability， decreased leakage rate of LDH， ROS and Ca²⁺ release， increased mitochondrial membrane potential and improved nuclear pyknosis compared with untreated H9c2 cardiomyocytes. Application of the same mass concentration of Aconiti Kusnezoffii Radix processed with Chebulae Fructus to H9c2 cardiomyocytes pretreated with BCTC decreased cell viability， increased LDH leakage rate， ROS and Ca²⁺ release， reduced mitochondrial membrane potential compared with untreated H9c2 cardiomyocytes. Real-time PCR results showed that both raw Aconiti Kusnezoffii Radix and Chebulae Fructus decoction could increase the expression of TRPV1 mRNA in cardiomyocytes in a concentration dependent manner. ConclusionRaw Aconiti Kusnezoffii Radix can induce cardiomyocyte apoptosis and cardiotoxicity by activating TRPV1 channel， while Aconiti Kusnezoffii Radix processed with Chebulae Fructus can attenuate the toxicity through TRPV1 channel， which may be related to the synergistic effect of acid components in Chebulae Fructus and alkaloids in Aconiti Kusnezoffii Radix on TRPV1 channel.

Lower respiratory tract infection (LRTI) induced by respiratory syncytial virus (RSV) is an important cause of hospitalization for infants. Compared with adults, infants are more likely to cause serious respiratory diseases after RSV infection due to the specific immature airway structure and immune system. The balance of immune resistance and immune tolerance of the host is critical to effective virus clearance and disease control. This paper reviews the relationship between RSV infection and respiratory diseases in infancy, the influence factors of the high pathogenicity of RSV infection in early life, as well as the research progress of anti-RSV therapy, and expands the specific molecular events regulating immune resistance and immune tolerance. We expect to present new ideas for the prevention and treatment of RSV-related respiratory diseases in clinical practice.
Humans ; Infant ; Respiration Disorders ; Respiratory Syncytial Virus Infections ; Respiratory Syncytial Viruses ; Respiratory Tract Infections

Objective: To explore the clinical efficacy and safety of modified Huqianwan in treatment of rheumatoid arthritis (RA) liver-kidney Yin deficiency syndrome, and investigate its possible mechanism. Method: A total of 184 patients with RA liver-kidney Yin deficiency syndrome were randomly divided into Chinese medicine group (62 cases), western medicine group (57 cases) and integrated Chinese and western medicine group (65 cases) according to the digital table method. The patients in Chinese medicine group were treated with Huqianwan; the patients in western medicine group were treated with methotrexate tablets and leflunomide tablets; and the patients in integrated Chinese and western medicine group received Huqianwan+methotrexate tablets and leflunomide tablets,with a treatment course of 12 weeks in all groups. The pain visual analog scale (VAS), swelling and tenderness scores of 28 joints (DAS28), average hands grip strength, morning stiffness time and liver-kidney Yin deficiency syndrome differentiation of traditional Chinese medicine (TCM) syndrome score were compared between groups before and after treatment. The changes of erythrocyte sedimentation rate (ESR), C reactive protein (CRP), immunoglobulin (Ig) G, tumor necrosis factor-alpha (TNF-α) and rheumatoid factor (RF) were detected in all groups after treatment. Clinical efficacy, and incidence of adverse reactions such as gastrointestinal response, liver injury, leukopenia, serum glutamate oxaloacetic aminotransferase (GOT) and platelet (PLT) level changes were compared between the groups, so as to investigate the efficiency and safety of the different medicines. Result: After 12 weeks of treatment, the total clinical effective rate was 79.0%, 80.7%, and 92.3% respectively in Chinese medicine group, western medicine group, and integrated Chinese and western medicine group; the integrated Chinese and western medicine group was significantly better than the Chinese medicine group and western medicine group (P<0.01), but there was no difference between the Chinese medicine group and western medicine group. Every treatment group can effectively improve liver and kidney Yin deficiency syndrome in RA patients (P<0.05), and the effect in integrated Chinese and western medicine group was superior to that in Chinese medicine group (P<0.05); the effect in Chinese medicine group was superior to that in western medicine group (P<0.05). The incidence of adverse reactions was Chinese medicine group (1.61%, 1/62)Conclusion: The efficacy in treating RA liver and kidney Yin deficiency syndrome shows no significant difference between modified Huqianwan and methotrexate tablets+leflunomide tablets. In the treatment of RA liver and kidney Yin deficiency syndrome, Huqianwan has fewer adverse reactions. Huqianwan combined with methotrexate tablets+leflunomide tablets is superior to that in methotrexate tablets+leflunomide tablets in treatment of RA liver-kidney Yin deficiency syndrome.

Objective: To investigate the clinicopathological features as well as BRAF V600E and MYD88 L265P mutation status of nodal marginal zone B cell lymphoma (NMZL). Methods: Thirty-two cases of NMZL were diagnosed from September 2009 to February 2021 at the Henan Provincial People's Hospital and Peking University School of Basic Medical Sciences. The clinicopathologic characteristics were obtained and analyzed. BRAF V600E and MYD88 L265P mutation status were identified using PCR and Sanger sequencing, respectively. Results: There were 20 males and 12 females patients with a median age of 69 years (ranging 36-82 years). The most prevalent clinical manifestation was multiple lymph nodes enlargement in head and neck (22/32, 68.8%), followed by inguinal (12/32, 37.5%), axillary (11/32, 34.4%), mediastinum (5/32, 15.6%) and retroperitoneal lymph nodes (4/32, 12.5%). Most of the patients were in Ann Arbor stage Ⅰ/Ⅱ (21 cases). The morphologic features included diffuse (24/32, 75.0%), nodular (5/32, 15.6%), interfollicular (2/32,6.3%) and perifollicular (1/32,3.1%) types. The tumor cells showed monocyte-like, centrocyte-like, small lymphocyte-like and plasma cell-like differentiation. Immunophenotyping revealed diffuse expression of CD20 in all tumor cells, whereas CD43 (11/32, 34.4%), bcl-2 (20/32, 62.5%), MNDA (13/32, 40.6%) and CD5 (2/32, 6.3%) were partially expressed. Ki-67 proliferation index varied from 10% to 40%. BRAF V600E mutation was found in two cases (2/32, 6.3%), but MYD88 L265P mutation was not detected. Eighteen patients survived and three died at the end of follow-up period which ranged 6 to 110 months. Conclusions: The morphologic features of NMZL varies across individuals, it should be differentiated from various B-cell lymphomas; however immunological biomarkers with high specificity for NMZL are still lacking. No MYD88 L265P mutation is found in NMZL. Some cases may harbor BRAF V600E mutation and yet the prevalence remains indeterminate; further researches are warranted.
Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Lymphoma, B-Cell, Marginal Zone/pathology* ; Male ; Middle Aged ; Mutation ; Myeloid Differentiation Factor 88/genetics* ; Polymerase Chain Reaction ; Proto-Oncogene Proteins B-raf/genetics*

Objective: To evaluate the efficacy within the first 24 h post extracorporeal membrane pulmonary oxygenation (ECMO) and the impact of early efficacy on the prognosis of adult patients with fulminant myocarditis (FM). Methods: This retrospective case analysis study included hospitalized patients (age≥18 years) who were diagnosed with fulminant myocarditis from November 2016 to May 2021 in the First Affiliated Hospital of Zhengzhou University. Patients were divided into survival or non-survival groups according to treatment outcomes. The age, sex, treatments, drug use, ECMO use, clinical and laboratory data (before and 24 h after the use of ECMO) were analyzed. The change rate of clinical and laboratory data after 24 h use of ECMO was calculated to find differences between two groups. Multivariate logistic regression was used to analyze the related factors with in-hospital death and complication between the two groups. Results: A total of 38 FM patients treated with ECMO were included. There were 23 cases (60.5%) in the survival group, aged (39.6±13.7) years, and 17 (73.9%) cases were female. The total ECMO time was (134.4±71.3)h. There were 15 cases (39.5%) in non-survival group, aged (40.0±15.8) years, and there were 12(80.0%) female, the ECMO time was (120.1±72.4) h in this group. The proportion of tracheal intubation and continuous renal replacement therapy in the survivor group and dosage of norepinephrine within 24 h after ECMO implantation were significantly less than in non-survival group (all P<0.05). There was no significant difference in all efficacy related biochemical indexes between two groups before ECMO use. The levels of lactic acid, procalcitonin, creatinine, alanine aminotransferase, aspartate aminotransferase, creatine kinase-MB, cardiac troponin I and N-terminal B-type natriuretic peptide prosoma were significantly less in survival group than in non-survival group at 24 h after the use of ECMO (all P<0.05). Results of multivariate logistic regression analysis showed that the higher 24 h change rate of creatinine (OR=0.587, 95%CI 0.349-0.986, P=0.044) and creatine kinase-MB (OR=0.177, 95%CI 0.037-0.841, P=0.029) were positively correlated with reduced risk of in-hospital mortality. The central hemorrhage and acute kidney injury in survival group were less than in non-survivor group (P<0.05). Conclusions: After 24 h early use of ECMO in FM patients, the improvement of various efficacy related biochemical test indexes in the survival group was better than that in the non-survival group. Faster reduction of creatine kinase-MB and creatinine values within 24 h ECMO use is positively correlated with reduced risk of in-hospital mortality in adult patients with FM.
Adolescent ; Adult ; Extracorporeal Membrane Oxygenation/methods* ; Female ; Hospital Mortality ; Humans ; Middle Aged ; Myocarditis/therapy* ; Retrospective Studies ; Treatment Outcome ; Young Adult

Some Chinese herbal medicine needs to be processed before it can be used as medicine, especially toxic Chinese medicine. Highly toxic Aconti Kusnezoffii Radix(Caowu in Chinese) is widely used in traditional Chinese medicine and Mongolian medicine. In traditional Chinese medicine, Caowu is usually processed by boiling with water(CW) until no white part inside and being tasted without tongue-numbing. In Mongolian medicine, it is usually soaked in Chebulae Fructus(Hezi in Chinese) decoction for several days(CH). Both methods could reduce toxicity according to reports. The biggest difference between CW and CH is that CW needs to be heated for 4-6 h, while CH needs Hezi as processing adjuvants. To explore the toxicity reduction mechanism of CW and CH, we studied the contents of various compounds in Caowu processed by two methods by UPLC-Orbitrap-MS. The results indicated that CW had 14 new ingredients, such as 14-O-anisoylneoline and dehydro-mesaconitine, while N-demethyl-mesaconitine and aconitine disappeared. At the same time, it could significantly decrease the content of diester diterpenoid alkaloids and increase the contents of monoester diterpenoid alkaloids and amine-diterpenoid alkaloids. CH had 9 new ingredients from Hezi, like gallic acid, chebulic acid and shikimic acid. Neither the kinds nor the contents of compositions from Caowu in CH changed little. This suggested that the processing mechanism of CW reduced highly toxic components(diester diterpenoid alkaloids) and increased the content of lowly toxic components(monoester diterpenoid alkaloids and amine-diterpenoid alkaloids). Attenuated principle of CH may be related to the components of Hezi. In this experiment, the conclusion shows that the chemical constituents of CW and CH are essentially different, and the two methods have different toxicity reduction principles.
Aconitine ; Aconitum/chemistry* ; Alkaloids/analysis* ; Chemistry, Pharmaceutical/methods* ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal/analysis* ; Mass Spectrometry ; Medicine, Chinese Traditional