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 evaluate the safety of preoxygenation with high-flow nasal oxygenation in elderly patients during induction of general anesthesia with endotracheal intubation. METHODS: Fifty-six elderly patients without difficult airway were randomized equally into high-flow nasal oxygen group (HF group) and conventional mask oxygen group (M group). Preoxygenation was performed for 5 min before induction of general anesthesia and endotracheal intubation. Oxygenation was maintained during laryngoscopy in HF group, and ventilation lasted until laryngoscopy in M group. For all the patients, the general data, cross-sectional area (CSA) of the gastric antrum measured by ultrasonography, arterial partial pressure of oxygen (PaO₂), arterial partial pressure of carbon dioxide (PaCO₂) and arterial oxygen saturation (cSO₂) were recorded before preoxygenation (T1), at 5 min of preoxygenation (T2) and immediately after intubation (T3). The safety time of asphyxia, intubation time, times of mask ventilation and postoperative complications were compared between the two groups. RESULTS: The general data were comparable between the two groups. After 5 min of preoxygenation, PaO₂ and cSO₂ were significantly increased in both groups, and PaO₂ was significantly higher in HF group than in M group (F=118.108 vs 9.511, P < 0.05). Both PaO₂ and cSO₂ decreased after intubation, but PaO₂ decreased more slowly in HF group and still remained higher than that at T1; cSO₂ decreased significantly in M group to a lower level than that at T1. Compared with those in M group, the patients in HF group showed a significantly longer safety time of asphyxia (t=5.305, P < 0.05) with fewer times of mask ventilation (χ²= 6.720, P < 0.05). PaCO₂ increased after intubation in both groups but was comparable between the two groups (F=3.138, P > 0.05). CONCLUSION High-flow nasal oxygen is safe, simple and effective for pre-oxygenation, which, as compared with the conventional oxygen mask, improves arterial oxygen partial pressure and prolongs the safety time of asphyxia to ensure the safety of airway management during induction of general anesthesia in elderly patients with endotracheal intubation.
Aged ; Anesthesia, General ; Asphyxia ; Humans ; Intubation, Intratracheal ; Oxygen ; Partial Pressure

Solitary kidney, renal duplication and malrotation are rare congenital renal malformations in urology department, and probably contributed to some complications such as obstruction, hydronephrosis, infection, stones. In this case report, we firstly presented a male patient with rarely multiple renal malformations, including solitary kidney, renal duplication, misaligned malrotation of upper and lower moieties, and accompanied by complete staghorn stones and hydronephrosis, who was treated with open pyelolithotomy under general anesthesia. After the operation, obstruction, hydronephrosis, and infection were relieved.

OBJECTIVE: To study the value of anti-neutrophil cytoplasmic antibody (ANCA) in assessing the severity of bronchiolitis obliterans (BO) in children. METHODS: A prospective analysis was performed on 59 children who were diagnosed with BO from June 2009 to October 2014. ELISA was used to measure the concentrations of myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA in serum. According to the results of ELISA, the children were divided into three groups: double-negative ANCA (n=22), single-positive ANCA (n=17), and double-positive ANCA (n=20). The three groups were compared in terms of the scores of BO risk factors, clinical symptoms, chest high-resolution computed tomography (HRCT), and lung pathology on admission, as well as the changes in the expression level of ANCA and the scores of clinical symptoms and chest HRCT over time. RESULTS: Compared with the double-negative ANCA group, the double-positive ANCA group had a significantly higher score of BO risk factors (P<0.05), and the single-positive ANCA group and the double-positive ANCA group had significantly higher scores of clinical symptoms, chest HRCT, and lung pathology (P<0.05). The children were followed up for 6 months after discharge, and there were significant reductions in MPO-ANCA and PR3-ANCA titers from admission and discharge to the end of follow-up (P<0.05), as well as a significant reduction in the score of clinical symptoms from admission to the end of follow-up (P<0.05), while there was no significant change in the score of chest HRCT from admission to the end of follow-up (P>0.05). The single-positive ANCA and double-positive ANCA groups still had a significantly higher score of clinical symptoms than the double-negative ANCA group (P<0.05). CONCLUSIONS The expression level of ANCA is correlated with the severity of BO in children and thus has certain clinical significance in disease evaluation.
Antibodies, Antineutrophil Cytoplasmic ; Bronchiolitis Obliterans ; Child ; Humans ; Myeloblastin ; Peroxidase ; Prospective Studies

Objective A/H1N1(pdm09) viruses were the dominant strains in Shanghai during 2018-2019 influenza surveillance year.This study is to provide a scientific reference for clinical drug use by investigating the susceptibility of A/H1N1(pdm09) viruses to neuraminidase inhibitors(NAIs). Methods Sixty strains of A/H1N1(pdm09) viruses were randomly selected for testing the susceptibility and drug resistance to Oseltamivir and Zanamivir by means of neutaminidase inhibition and neuraminidase (NA) gene sequencing. Results The 60 epidemic strains all proved to be susceptible to Oseltamivir and Zanamivir and the susceptibility was not observed to be decreased or remarkably decreased.In genetic sequencing, NA was not observed to present amino acid mutation at the key sites and auxiliary sites in catalytic activity, which confirmed the results of the phototypic detection of neuraminidase inhibition. Conclusion The subtype influenza viruses A/H1N1(pdm09) circulating in Shanghai during 2018-2019 surveillance year are still sensitive to NAIs, which provides a scientific reference for clinical use of drugs.However, we monitored only a number of strains and think that the work monitoring antiviral susceptibility should be continued with the wide use of the drugs.

In order to determine the quality evaluation method for standard decoction of Coptidis Rhizoma,15 batches of standard decoction of Coptidis Rhizoma were prepared by using standardized process. Parameters such as traits,p H value,indicative component content,fingerprint similarity,composition transfer rate and dry extract rate were selected as the indexes for quality evaluation. Similarity evaluation and cluster analysis were performed for HPLC fingerprint of standard decoction,and mathematical model was used to study the correlation between dry extract rate,berberine content,berberine transfer rate in standard decoction and berberine content in decoction pieces. The results showed that the similarity of fingerprints was greater than 0. 99 for these 15 batches of standard decoctions of Coptidis Rhizoma. In cluster analysis,the standard decoctions of Coptidis Rhizoma from 4 producing areas were classified into 3 categories,consistent with the content determination results,indicating that there were quality differences among different producing areas.R2 in three linear regression mathematical models established was all greater than 0. 9,with significant difference. The validation of three batches of data showed that the models had good accuracy. Therefore,this model can be used to predict the quality of standard decoction prepared from different Coptidis Rhizoma pieces. In the standard decoction process established in this study,the integrity of the traditional process was greatly preserved,and the established quality evaluation method could be used to comprehensively examine the quality of the standard decoction,which can provide a demonstration for the related research of water extraction preparation containing Coptidis Rhizoma pieces.
Berberine/analysis* ; Chromatography, High Pressure Liquid ; Coptis/chemistry* ; Coptis chinensis ; Drugs, Chinese Herbal/analysis* ; Linear Models ; Quality Control ; Rhizome/chemistry*

ObjectiveTo investigate the relationship of oxidative stress with DNA integrity and semen parameters in infertile men with varicocele (VC).

METHODSThis prospective study included 98 infertile males with VC. According to the levels of reactive oxygen species (ROS) in the semen, we divided the patients into a high ROS group (n=44) and a low ROS group (n=54), determined the sperm DNA fragmentation index (DFI), motility and morphology, and analyzed their correlation with ROS in the two groups of patients.

RESULTSCompared with the patients of the low ROS group, those of the high ROS group showed a significantly higher DFI (27.38±8.10 vs 34.49±6.05, P=0.039) and a higher concentration of seminal leukocytes (［0.65±0.15］×10⁶/ml vs ［0.86±0.41］×10⁶/ml, P=0.022), but lower sperm motility (［36.16±22.83］% vs ［18.22±25.21］%, P=0.017), percentage of progressively motile sperm (［23.34±11.53］% vs ［16.34±9.22］%, P=0.041), sperm curvilinear velocity (［27.03±6.21］ vs ［20.62±4.38］ μm/s, P=0.013), and sperm linearity (［29.75±8.24］% vs ［18.30±7.93］%, P=0.024). Spearman correlation analysis indicated that the ROS level was correlated positively with the concentration of seminal leukocytes (r=0.41, P<0.01) and DFI (r=0.21, P=0.006), but negatively with sperm curvilinear velocity (r=－0.24, P=0.017), linearity (r=－0.24, P=0.021), motility (r=－0.31, P=0.002), and the percentage of progressively motile sperm (r=－0.41, P=0.012). Additionally, the sperm DFI manifested a significant negative correlation with sperm motility (r=－0.29, P<0.01) and the percentage of progressively motile sperm (r=－0.34, P<0.01).

CONCLUSIONSThe level of seminal ROS is positively correlated with the sperm DFI in infertile men with varicocele, and both the ROS level and DNA integrity are associated with semen parameters.

DNA Fragmentation ; Humans ; Infertility, Male ; complications ; Male ; Oxidative Stress ; Prospective Studies ; Reactive Oxygen Species ; metabolism ; Semen ; Sperm Motility ; Spermatozoa ; pathology ; Varicocele ; complications

Objective To investigate elastic modulus of double-stranded DNA (dsDNA) biofilm adsorbed on microcantilever substrate. Methods Parsegian’s empirical potentials based on mesoscopic continuum liquid crystal theory was employed to describe the interaction energy among coarse-grained DNA cylinders; Monte Carlo method was used to simulate the distribution pattern of DNA chains before and after loading. The thought experiment method combined with the compression bar model in the sense of macroscopic continuum mechanics was adopted to predict the elastic modulus of DNA biofilm. Results The elastic modulus of dsDNA biofilm ranged from 0.1 MPa to 80 MPa. Conclusions It was found out that the classic hypothesis with uniform hexagonal pattern may underestimate the elastic modulus of DNA biofilm when compared with that in random pattern. Moreover, either the increase of packing density or the decrease of buffer salt concentration will help to enhance elastic modulus of DNA biofilm. These results have great significances in further understanding the mechanical properties and regulation rules of DNA biofilm related with clinical work.

OBJECTIVETo investigate the functional relations between the putative proteins YpCD1.08, YpCD1.09, YpCD1.16 encoded in pCD1 plasmid of Yersinia pestis and its type III secretion system (T3SS).

METHODSMutants of YpCD1.08, YpCD1.09, YpCD1.16 were constructed using λ-Red recombinant system. The growth curves of the mutant strains cultivated in TMH medium with or without calcium at 26 °C and 37 °C were determined to analyze the low calcium response phenotype. The transcription levels of ΔYpCD1.08, ΔYpCD1.09, ΔYpCD1.16 in Yersinia pestis and the dependence to temperature were determined using real time RT-PCR after cultivation at 26 °C and 37 °C and extraction of RNA. A β-lactamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS.

RESULTSWhen grown in TMH medium without calcium at 26 °C and 37 °C, the growth curve of the YpCD1.08, YpCD1.09, YpCD1.16 mutants were similar to that of the wild-type strain, indicating that the low calcium response of all the mutants were normal. The ratios of YpCD1.08, YpCD1.09, YpCD1.16 gene transcriptional level at 37 °C and 26 °C were 2.3 ± 0.3, 2.3 ± 0.5 and 3.2 ± 0.7, respectively, indicating that these genes were transcribed in Yersinia pestis and their transcription regulations showed a temperature-dependence that was consistent with the well established temperature-dependent expression of Yersinia T3SS genes. The β-lactamases reporter assays demonstrated that ΔYpCD1.08 could translocate much higher level of YopE into HeLa cells, since that the light intensity ratio of 477/520 nm at 140 min was 2.5, whereas it was 1.8 for the wild-type strain, and the values in ΔYpCD1.09 and ΔYpCD1.16 were similar to the wild-type strain.

CONCLUSIONYpCD1.08, YpCD1.09, YpCD1.16 gene are likely to be the new members of T3SS, and the putative protein YpCD1.08 could play some roles in YopE secretion and translocation.

Bacterial Outer Membrane Proteins ; secretion ; Bacterial Secretion Systems ; genetics ; Genes, Bacterial ; Plasmids ; Protein Interaction Mapping ; Yersinia pestis ; genetics ; metabolism ; pathogenicity

Amniotic Fluid ; metabolism ; Biomarkers ; metabolism ; Diagnosis, Differential ; Embolism, Amniotic Fluid ; metabolism ; pathology ; Female ; Humans ; Infant, Newborn ; Keratin-13 ; metabolism ; Pregnancy ; Respiratory Aspiration ; metabolism ; pathology