This study was aimed at creating an engineered strain of Bacillus subtilis for efficient expression of biologically active type 2 toxin B(TcdB2)derived from a highly virulent strain of Clostridium difficile.The TcdB2 gene was cloned from ST1/RT027 strain genome DNA,incorporated into the PHT01 vector,and then transformed into B.subtilis strain WB800N for prokaryotic expression.Cell toxicity assays revealed that the recombinant TcdB2 exhibited cytotoxic effects in various cells.The engineered B.subtilis strain effectively expressed biologically active TcdB2,thus providing a basis for further exploration of the pathogenic mechanisms of highly virulent strains of C.difficile and establishing a foundation for potential vaccine can-didate targets.

Objective To discuss the application value of CT scanning technology in cause of death determination of medical dispute cases. Methods From July 2017 to December 2018, postmortem CT imaging data of 12 medical dispute cases were collected. CT imaging diagnosis results and anatomy findings as well as differences between antemortem and postmortem CT diagnosis were compared. The advantages and disadvantages of CT routine tests of the cadavers in terms of the diagnosis of disease and damage were analyzed. Results The comparison between CT imaging diagnosis and anatomical findings showed that CT scans had advantages in the diagnosis of disease and damage with large differences in density changes, such as atelectasis, pneumonia, calcification, fracture and hemorrhage, etc. The comparison of CT diagnosis in antemortem and postmortem examination showed that the cadavers of medical dispute cases were well preserved and that postmortem CT scan was meaningful for the diagnosis of antemortem diseases. Conclusion Virtual anatomy technology has a relatively high application value in postmortem examination of medical dispute cases. It can provide effective information for the appraisers before the autopsy and can also provide a reference for cause of death analysis when the anatomy cannot be performed.
Autopsy ; Cadaver ; Dissent and Disputes ; Humans ; Postmortem Changes ; Tomography, X-Ray Computed

Objective To observe the clinical efficacy and safety of β-aescin sodium injection combined with bumetanide injection in the treatment of acute cerebral infarction with cerebral edema.Methods Ninety -eight patients of acute cerebral infarction with cerebral edema were randomly divided into control and treatment groups with 49 cases per group.Control group was given 20％ mannitol injection 250 mL per time,qd,intravenous infusion.Treatment group was treated with β-aescin sodium injection 20 mg per time,qd,intravenous infusion + bumetanide injection 2 mg per time,qd,intravenous injection,on the basis of control group.Two groups were treated for 7-10 days.The clinical efficacy,superoxide dismutase (SOD),interleukin-6 (IL-6),tumor necrosis factor-α (TNF-α) and adverse drug reactions were compared between two groups.Results After treatment,the total effective rates of treatment and control groups were 85.71％ (42 cases/49 cases) and 67.35 ％ (33 cases/49 cases) with significant difference (P ＜ 0.05).After treatment,the main indexes in treatment and control groups were compared:SOD were (64.71 ± 8.19) and (59.65 ± 8.52) kU · L-1,IL-6 were (54.03±7.53) and (65.49±6.93)ng· L-1,TNF-α were (8.59:±0.89) and (9.54 ±l.00)ng· L-1,the differences were statistically significant (all P ＜ 0.05).The adverse drug reactions in treatment group were hyperglycemia,electrolyte disorder and urinalysis abnormality,which in control group were electrolyte disorder and urinalysis abnormality.The total incidences of adverse drug reactions in treatment and control groups were 14.29％ and 12.24％ without significant difference (P ＞0.05).Conclusion β-aescin sodium injection combined with bumetanide injection has a definitive clinical efficacy in the treatment of acute cerebral infarction with cerebral edema,which can effectively improve the level of oxidative stress and inflammatory factors,without increasing the incidence of adverse drug reactions.

Objective To summarize clinical experience and explore application value of endoscopic clipping with histoacryl using in management of type 2 gastroesophageal varices. Methods Clinical data of 30 patients with type 2 gastroesophageal varices patients (including acute hemorrhage and primary prevention) from May 2015 to December 2016 were collected. Then evaluate therapeutic effect and safety of endoscopic clipping adjuvant therapy. Results Average glue dosage was (1.46 ± 0.70) ml, average using of clips were (5 ~ 6), and intraoperative needle pulling hemorrhage occurred in 2 cases. 14 patients (46.7%) underwent endoscopic re-examination, 3 patients (10.0%) achieved varicose vein elimination, 11 cases (36.7%) remained residual. Rebleeding occurred in 4 cases (13.3%), and 2 cases died (6.7%), one because of postoperative hematemesis and hemorrhagic shock, the other one died of spontaneous peritonitis and septic shock. For general curative effect, 2 cases (6.7%) were healed, 22 cases (73.3%) were improved, and 6 cases were unhealed (20.0%, 4 cases occurred rebleeding, 2 cases died); 17 cases underwent CT portal venograpy, abnormal embolization was not found in any patients, glue extrusion bleeding occurred in 1 case (3.3%), no patients had severe postoperative complications. Conclusion Endoscopic clipping with histoacryl can be used in the prevention and treatment of type 2 gastroesophageal varices to improve the treatment effect and reduce postoperative bleeding risk, may have good clinical practice value.

OBJECTIVETo explore possible mechanism of electroacupuncture (EA) for regulating immune function in anxiety disorder (AD) rats by observing the effect of acupuncture on the histology of thymus and expressions of atrial natriuretic peptide (ANP) and natriuretic peptide receptor type A (NPR- A) in thymus.

METHODSTotally 34 SD healthy rats were randomly divided into the blank control group (n = 10), the model group (n = 12), the EA group (n = 12). Anxiety model was established in rats of the model group and the EA group by using chronic unpredictable stress (CUS) stimulation. EA (15/25 Hz) at Neiguan (PC6) and Shenmen (HT7) was performed in the EA group, with 15-min needle retaining, once every other day, 15 days in total. Needle was fixed at same acupoints for 15 min without electric stimulus in the other two groups. Anxiety-like behavior was measured by elevated plus-maze (EPM) test. Pathological changes of thymus tissue were observed by optical microscope. Expressions of ANP and NPR-A in thymus were measured by immunohistochemical assay.

RESULTSThe thymus tissue in the model group was severely atrophied, with unclear structure of thymic lobules, unclear margin of thymic medulla, loosely arranged lymphocytes ,and obviously enlarged volume of thymic corpuscle. The thymus tissue in the EA group was mildly atrophied, with existent structure of thymic lobules, clear margin of thymic medulla, densely arranged lymphocytes in cortical region, and widened medullary area. Com- pared with the blank control group, the percentage of open-arms entries (OE%) in the total QE times ob- viously decreased in the model group (P < 0.05), ANP expression obviously increased (P < 0.05), and NPR-A expression obviously decreased (P < 0.01). Compared with the model group, OE% was obviously elevated (P < 0.05), ANP expression obviously decreased (P < 0.05), and NPR-A expression obviously increased (P < 0.01) in the EA group.

CONCLUSIONEA not only could reduce anxiety of rats, but also could improve chronic stress induced thymus injury through intervening synthesis and secretion of ANP, as well as the expression of NPR-A (a specific receptor of ANP).

Acupuncture Points ; Animals ; Anxiety Disorders ; therapy ; Atrial Natriuretic Factor ; metabolism ; Electroacupuncture ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Atrial Natriuretic Factor ; metabolism ; Thymus Gland ; pathology

Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.
Alkaloids ; biosynthesis ; genetics ; metabolism ; Atropa belladonna ; genetics ; metabolism ; Gene Expression Regulation, Plant ; genetics ; Hyoscyamine ; genetics ; metabolism ; Plants, Medicinal ; genetics ; metabolism ; Scopolamine Hydrobromide ; metabolism ; Tropanes ; metabolism

In 2013, the World Health Organization reported the first case of human infection with a new influenza A (H7N9) virus in China. This has caused damage and panic within certain areas in China. Therefore, analysis of this virus with bioinformatics technology is very necessary. Neuraminidase (NA) is one of the most important antigens of the influenza virus and an important target for anti-flu drugs. In this study, the nucleotide and protein sequences of NA gene of A/H7N9 influenza viruses were retrieved from the NCBI database, and MEGA 5.0 software was employed to construct a phylogenetic tree based on the nucleotide coding sequence; BioEdit software was used to align the nucleotide and protein sequences of NA and calculate the homologies of nucleotides and amino acids and then to analyze the important mutation sites of NA gene. The results demonstrated that the spread of influenza virus H7N9 showed certain geographical and temporal relations. The H7N9 virus isolated from China in 2013 belonged to Euroasiatic serotype, and its NA stalk region hadobvious variation, which may be one of the reasons that this virus infects human. These analyses may be very helpful for understanding the evolutionary relationship and mutation trend of A/H7N9 influenza viruses.
Databases, Genetic ; Evolution, Molecular ; Humans ; Influenza A Virus, H7N9 Subtype ; enzymology ; genetics ; Mutation ; Neuraminidase ; chemistry ; genetics ; Phylogeny ; Sequence Analysis

Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed.
Atropa belladonna ; genetics ; metabolism ; Atropine ; metabolism ; Gene Expression ; Mixed Function Oxygenases ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; Scopolamine Hydrobromide ; metabolism ; Solanaceae ; enzymology ; genetics

OBJECTIVETo establish the method of cytological examination and the normal reference values for hypertonic saline solution-induced sputum of healthy children (age range from 5 to 15 years) with physical examination in Guangzhou.

METHODA total of 352 children, 5 to 15 years old, were enrolled from primary school and middle school in Guangzhou from January to December, 2010. All subjects completed a standardized questionnaire on the presence of respiratory, allergic symptoms and family history, the medical history and the physical examination was performed by doctors, lung function (forced expiratory volume at 1 s in predicted normal, FEV(1)%) was determined. There were 266 healthy children (137 males, 129 females) who were selected and undergone hypertonic saline solution induction of sputum, and cytological examination was performed. Hypertonic saline (5%) was nebulized and inhaled for 15 - 30 min. No expectoration within 30 min was defined as failure, and the procedure was terminated. The part of opaque and higher density sputum samples was detected by cytology. The proportion of neutrophils, lymphocytes, eosinophils, macrophages and monocytes was calculated. This study was approved by the institutional Ethics Review Committee of First Affiliated Hospital of Guangzhou Medical College. Informed consent was obtained from the legal guardians of all participants following a detailed description of the purpose and potential benefits of the study.

RESULTThere were 175 subjects' induced sputum specimens (175/266, 65.8%), non-qualified sputum samples were obtained from 16 of the subjects. The proportions of median (IQR) of lymphocytes were 0.012 (0.020), 95%CI were ranged from 0.015 to 0.022; neutrophils 0.207 (0.330), 95%CI 0.266 - 0.356 macrophages 0.761 (0.327), 95%CI 0.607 - 0.699; eosinophils 0.004 (0.019), 95%CI 0.013 - 0.022. There were no significant differences in proportions of cytological findings of female or male, different age groups and second-hand smoking or not (all P > 0.05). The incidence of adverse event was 4.40% (7/159).

CONCLUSIONThe method and the preliminary data may be used for research, diagnosis and treatment of patients with chronic cough and airway inflammation.

Adolescent ; Child ; Child, Preschool ; China ; Cough ; diagnosis ; physiopathology ; Eosinophils ; cytology ; Female ; Forced Expiratory Volume ; Humans ; Leukocyte Count ; Lymphocyte Count ; Lymphocytes ; cytology ; Male ; Monocytes ; cytology ; Neutrophils ; cytology ; Reference Values ; Saline Solution, Hypertonic ; chemistry ; Sputum ; cytology ; metabolism

OBJECTIVETo assess the value of biochemical marker detection in risk stratification in hospitalized patients with acute coronary syndrome (ACS).

METHODSA total of 264 consecutive patients (180 male and 84 female patients) admitted for complaint of chest tightness or/and pain were evaluated for a decision of coronary angiography (CAG) within 24 h after admission. The patients were divided into two groups to receive emergency or elective CAG. The venous blood samples were taken from the patient immediately after admission for detection of amino-terminal pro-brain natriuretic peptide (NT-pro-BNP), high-sensitivity C-reactive protein (hs-CRP), myeloperoxidase (MPO), monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule (sICAM-1), soluble CD40 ligand (sCD40L), matrix metalloproteinase 9 (MMP-9), interleukin-6 (IL-6), interleukin 27 (IL-27) and creatine kinase isoenzyme (CK-MB) were detected.

RESULTSNo significant differences in NT-proBNP, hs-CRP, MPO, sCD40L, and MMP-9 were found between emergency CAG group and elective CAG group (P<0.05). Logistic regression identified significant differences in NT-proBNP, hs-CRP, MPO, IL-27 and CK-MB between the two groups, and a predictive model for risk stratification of ACS was established using these biomarkers. The ROC curves of this predictive model showed an area under the curve of 98.1, suggesting a high predictive value of this model in assessment of the changes or progression of ACS.

CONCLUSIONCombined detection of the biochemical markers can be helpful for risk stratification of the hospitalized patients with ACS early after admission.

Acute Coronary Syndrome ; blood ; Aged ; Biomarkers ; blood ; C-Reactive Protein ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Models, Statistical ; Natriuretic Peptide, Brain ; blood ; Peroxidase ; blood ; Predictive Value of Tests ; ROC Curve ; Risk Assessment ; Risk Factors