Preoperative prediction of microvascular invasion and perineural invasion in pancreatic ductal adenocarcinoma with 18F-FDG PET/CT radiomics analysis.

AIM: To develop and validate a predictive model based on 2-[18F]-fluoro-2-deoxy-d-glucose (18F-FDG) positron-emission tomography (PET)/computed tomography (CT) radiomics features and clinicopathological parameters to preoperatively identify microvascular invasion (MVI) and perineural invasion (PNI), which are important predictors of poor prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). MATERIALS AND METHODS: Preoperative 18F-FDG PET/CT images and clinicopathological parameters of 170 patients in PDAC were collected retrospectively. The whole tumour and its peritumoural variants (tumour dilated with 3, 5, and 10 mm pixels) were applied to add tumour periphery information. A feature-selection algorithm was employed to mine mono-modality and fused feature subsets, then conducted binary classification using gradient boosted decision trees. RESULTS: For MVI prediction, the model performed best on a fused subset of 18F-FDG PET/CT radiomics features and two clinicopathological parameters, with an area under the receiver operating characteristic curve (AUC) of 83.08%, accuracy of 78.82%, recall of 75.08%, precision of 75.5%, and F1-score of 74.59%. For PNI prediction, the model achieved best prediction results only on the subset of PET/CT radiomics features, with AUC of 94%, accuracy of 89.33%, recall of 90%, precision of 87.81%, and F1 score of 88.35%. In both models, 3 mm dilation on the tumour volume produced the best results. CONCLUSIONS: The radiomics predictors from preoperative 18F-FDG PET/CT imaging exhibited instructive predictive efficacy in the identification of MVI and PNI status preoperatively in PDAC. Peritumoural information was shown to assist in MVI and PNI predictions.

[A retrospective analysis of clinical characteristics and prognostic factors for 152 cases of Staphylococcus aureus bloodstream infection].

To understand the clinical characteristics of Staphylococcus aureus bloodstream infection and the main risk factors affecting clinical prognosis, providing a reference for clinical prevention and control of Staphylococcus aureus bloodstream infection. In this study, the clinical data of 152 patients with Staphylococcus aureus bloodstream infection admitted to Guangdong Provincial People's Hospital from January 2019 to December 2021 were retrospectively analyzed by reviewing the electronic medical record system, including underlying diseases, clinical characteristics, risk factors, and bacterial resistance. Statistical methods such as Chi-Squared Test and t Test were used to analyze the related risk factors that may affect the clinical characteristics and prognosis of patients with Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection, then the variables with P<0.05 in univariate analysis were included in the multivariate logistic regression model to analyze the independent risk factors of poor prognosis. The results showed among 152 patients with Staphylococcus aureus bloodstream infection, 50 patients (32.89%) were infected with MRSA. In comparison, 102 patients (67.11%) were infected with methicillin-sensitive Staphylococcus aureus (MSSA). Except for rifampicin, the resistance rate of MRSA to commonly used antibiotics was all higher than that of MSSA, and the difference was statistically significant (Chi-square values were 8.272, 11.972, 4.998, 4.776, respectively;all P-values are less than 0.05). Strains resistant to vancomycin, linezolid, and quinupristin/dalfopristin were not found. In the MRSA group, indwelling catheter and drainage tube, carbapenems, and ß-lactamase inhibitor treatment were significantly higher than the MSSA group. The difference was statistically significant (P<0.05). The incidence of poor prognosis of bloodstream infection in the MRSA group was higher than that in the MSSA group (34.00% vs 13.73%), and the difference was statistically significant (χ2=8.495, P<0.05). No independent risk factors associated with poor prognosis were found in the included patients with MRSA bloodstream infection.Multivariate Logistic regression model analysis showed that solid malignant tumors (OR=13.576, 95%CI: 3.352-54.977, P<0.05), mechanical ventilation (OR=7.468, 95%CI: 1.398-39.884, P<0.05) were the most important independent risk factors for poor prognosis in patients with Staphylococcus aureus bloodstream infection. In summary, the poor prognosis rate of MRSA bloodstream infection is higher than that of MSSA. The clinical evaluation of related risk factors should be strengthened, targeted prevention and control interventions should be taken to improve the prognosis of patients with Staphylococcus aureus bloodstream infection, and the use of antibiotics should be rational and standardized, to control bacterial infection and drug resistance effectively.

[Establishment of a rapid method for detection of influenza A/B virus' antigens].

This study aims to develop a rapid and convenient test card for simultaneous detection of influenza A and influenza B viruses using quantum dot-based immunochromatographic assay. The test card consists of a test strip and a plastic casing. The test strip is composed of absorbent paper, a buffer pad, nitrocellulose membrane (NC membrane), sample pad, quantum dot-labeled antibody pad, and polyvinyl chloride (PVC) board. The NC membrane is coated with mouse monoclonal antibodies against influenza A and influenza B viruses for the T lines (test lines), and reference proteins A and B for the C line (control line). The quantum dot-labeled antibody pad contains mouse monoclonal antibody-quantum dot conjugates against influenza A and influenza B viruses. The results showed that the detection limit of the test card for both viruses ranged from 1.51 ×102 to 2.71×103 TCID50/ml, indicating its sensitivity for accurate detection of influenza A and influenza B viruses without being affected by various variants. The test card exhibited specific reactions with different subtypes of influenza A and influenza B virus culture fluids and showed no cross-reactivity with adenovirus, novel coronavirus, Mycoplasma pneumoniae, respiratory syncytial virus, Staphylococcus aureus, and other pathogens. Overall, the sensitivity and specificity of the test card for simultaneous detection of influenza A and influenza B viruses meet the requirements for clinical use. It offers the advantages of simplicity, rapidity, and no requirement for special equipment, enabling quick auxiliary diagnosis to prevent disease transmission.

[Research progress of CRISPR/Cas biosensors based on different signal amplification strategies].

CRISPR/Cas(the clustered regularly interspaced short palindromic repeats-CRISPR associated)system exists in most bacteria and all archaea. It is an important strategy for bacteria and archaea to resist foreign nucleic acid invasion and use for self-defense. The CRISPR/Cas system is a simple, fast, and specific diagnostic tool, which is widely used in agriculture, industry, animal husbandry, and medicine. This article mainly introduces and discusses recently advantages and limitations of biosensors combining CRISPR/Cas system with fluorescence, visualization and surface enhanced raman related technologies, as well as future research directions.

[Constructions of the scale of difficulty in the extraction of impacted mandibular third molars by using Delphi method].

OBJECTIVE: To evaluate the relevant indicators affecting difficulty in the extraction of impacted mandibular third molars and score difficulty of different operation and risk indicators, so as to build an intuitive and accurate scale to help operators make more accurate analysis and prediction of difficulty before the operation. METHODS: Based on literature and the clinical review, the difficulty indicators of tooth extraction were summarized. Firstly, 10 doctors from Peking University School and Hospital of Stomatology who had been engaged in alveolar surgery for a long time established an expert nominal group, and then rated whether the summarized indicators needed to be retained in the form of face-to-face questionnaires. A level 1 and 2 item frame for evaluating difficulty in the tooth extraction was formed after discussion; Then Delphi method was used to send a questionnaire to 30 experts by e-mail. After two rounds of scoring and modification, the scale of difficulty in the extraction of impacted mandibular third molars was formed. RESULTS: The recycling rate of two rounds of questionnaires was 100.0%, which showed that the experts were very enthusiastic about the study; The authority coefficients (Cr) of the two rounds of Delphi expert consultation were both 0.92, which showed that the results were representative and authoritative. After two rounds of grading and revision, the variable coefficient (CV) decreased and the Kendall's concordance coefficient (W) increased, which were statistically significant: In the first round, the CV was 0.24 and W was 0.56 (P < 0.001), and in the second, the CV was 0.19 and W was 0.72 (P < 0.001), which indicated that there was a good convergence among the expert opinions. Finally, a scale of difficulty in the tooth extraction containing 12 items at level A and 37 items at level B was formed, including operation difficulty indicators, risk difficulty indicators and common difficulty indicators. CONCLUSION: Based on comprehensive literature retrieval, the study has put forward the concept that difficulty in the extraction of impacted mandibular third molars is composed of operation difficulty and risk difficulty. Using Delphi method, the long-term clinical experience and professional knowledge of experts are transformed into quantitative indicators as a scoring scale. The scale has certain representativeness and authority.

[Advances in the relationship between lung cancer and microbiota].

Interaction exists in lung cancer and microbiota. Lung microecological homeostasis can improve the immune tolerance, enhance immune suppression, and inhibit inflammatory responses, to reduce the lung cancer; while lung cancer can lead to pulmonary microecological imbalance, change the lung environment, and promote tumor cell proliferation. Therefore, modulating microbial flora and microecological immunotherapy may be a potential and preventive treatment for lung cancer, to restore tumor immunosuppression and improve patient survival. However, the individual differences in the lung microecology, because of different genetics, ethnic characteristics, and dietary habits, increasing the difficulty of precise diagnosis and treatment, which is also the current bottleneck in the application of microecological immunotherapy. Otherwise, the effectiveness of regulatory measures such as probiotics, prebiotics or antimicrobials is questionable. The research on microbial flora is still in its infancy, and further exploration is needed to form a standardized, effective, and precise treatment plan. So, standardized, effective, and precise microbial flora treatment strategies need to be further explored.

Evaluation of the probiotic and functional potential of Lactobacillus agilis 32 isolated from pig manure.

Escherichia coli is a symbiotic bacterium in humans and animals and an important pathogen of humans and animals. Prevention and suppression of E. coli infection is of great concern. In this study, we isolated a strain of Lactobacillus agilis 32 from pig manure and evaluated its biological characteristics, and found that its bacterial survival rate was 25% after 4 h of treatment at pH 2, and under the condition of 0·5% bile concentration, its survival rate exceeds 30%. In addition, L. agilis 32 has a cell surface hydrophobicity of 77·8%, and exhibits 67·1% auto-aggregation and 63·2% aggregation with Enterotoxigenic E. coli 10 (ETEC 10). FITC fluorescence labelling showed that the fluorescence intensity of cecum was significantly higher than that of duodenum, jejunum or colon (P < 0·05), but no significant difference from ileum. Lactobacillus agilis 32 bacterial culture and CFS showed average inhibition zone diameters of 14·2 and 15·4 mm respectively. Lactobacillus agilis 32 CFS treatment can significantly reduce the pathogenicity of ETEC 10. These results show that L. agilis 32 is an active and potential probiotic, and it has a good antibacterial effect on ETEC10, which provides basic research for probiotics to prevent and treat intestinal diarrhoea pathogen infection.

[The prognostic value of myoglobin difference in sepsis related chronic critical illness].

Objective: To investigate the predictive value of myoglobin (Mb) for the prognosis of sepsis related chronic critical illness (CCI). Methods: Retrospective study was conducted on septic patients with the length of ICU stay equal or greater than 14 days, and sepsis-related organ failure assessment (SOFA) score equal or greater than 2 on the 14th day in ICU in the First Department of Critical Care Medicine at the First Affiliated Hospital of Sun Yat-sen University from January 2017 to March 2020. Patients' clinical and laboratory data were collected on the 1st and 14th day in ICU. The survival on day 28 in ICU was recorded. According to the myoglobin levels on day 1 and day 14, all subjects were divided into myoglobin elevation group and decline group. Kaplan-Meier survival curve was used to compare the cumulative survival rate at day 28. Cox regression analysis was used to analyze the independent risk factors of mortality. Receiver operating characteristic (ROC) curve was used to analyze the prognostic value of myoglobin. Results: A total of 131 patients with sepsis related CCI were recruited, including 58 patients in the elevation group and 73 in the decline group. The Mb level in elevation group on day 1 was significantly lower than that in decline group [172.40(59.99, 430.53) µg/L vs. 413.60(184.40, 1 328.50) µg/L, Z=3.749, P=0.000], and the Mb level on day 14 was the opposite change in two groups [483.65(230.38, 1 471.75)µg/L in elevation group vs. 132.20(76.86, 274.35)µg/L in decline group, Z=5.595, P=0.000]. Kaplan-Meier survival curve analysis showed that the 28-day cumulative survival rate of the elevation group was significantly lower than that of decline group (χ²=7.051, P=0.008). Cox ratio regression analysis suggested that elevated myoglobin was an independent risk factor for 28-day mortality in septic patients with CCI (OR=2.534, 95%CI 1.212-5.295, P=0.013). ROC curve analysis suggested that the sensitivity of myoglobin elevation in predicting mortality related to CCI within 28 days was 64.5%, and the specificity was 32.0% with area under the curve(AUC) 0.661(95%CI 0.550-0.773,P=0.007) and Jorden Index was 0.325. Conclusion: Elevated myoglobin, an independent risk factor for mortality within 28 days in ICU, can predict the prognosis of sepsis related chronic critical illness.

An evaluation of direct PCR assays for the detection and quantification of Porphyromonas gingivalis.

Porphyromonas gingivalis has been linked to the development and progression of oesophageal squamous cell carcinoma (ESCC), and is considered to be a high-risk factor for ESCC. Currently, the commonly used methods for P. gingivalis detection are culture or DNA extraction-based, which are either time and labour intensive especially for high-throughput applications. We aimed to establish and evaluate a rapid and sensitive direct quantitative polymerase chain reaction (qPCR) protocol for the detection of P. gingivalis without DNA extraction which is suitable for large-scale epidemiological studies. Paired gingival swab samples from 192 subjects undergoing general medical examinations were analysed using two direct and one extraction-based qPCR assays for P. gingivalis. Tris-EDTA buffer-based direct qPCR (TE-direct qPCR), lysis-based direct qPCR (lysis-direct qPCR) and DNA extraction-based qPCR (kit-qPCR) were used, respectively, in 192, 132 and 60 of these samples for quantification of P. gingivalis. The sensitivity and specificity of TE-direct qPCR was 95.24% and 100% compared with lysis-direct qPCR, which was 100% and 97.30% when compared with kit-qPCR; TE-direct qPCR had an almost perfect agreement with lysis-direct qPCR (κ = 0.954) and kit-qPCR (κ = 0.965). Moreover, the assay time used for TE-direct qPCR was 1.5 h. In conclusion, the TE-direct qPCR assay is a simple and efficient method for the quantification of oral P. gingivalis and showed high sensitivity and specificity compared with routine qPCR.

Environmental impacts of nitrogen emissions in China and the role of policies in emission reduction.

Atmospheric reactive nitrogen (Nr) has been a cause of serious environmental pollution in China. Historically, China used too little Nr in its agriculture to feed its population. However, with the rapid increase in N fertilizer use for food production and fossil fuel consumption for energy supply over the last four decades, increasing gaseous Nr species (e.g. NH3 and NOx) have been emitted to the atmosphere and then deposited as wet and dry deposition, with adverse impacts on air, water and soil quality as well as plant biodiversity and human health. This paper reviews the issues associated with this in a holistic way. The emissions, deposition, impacts, actions and regulations for the mitigation of atmospheric Nr are discussed systematically. Both NH3 and NOx make major contributions to environmental pollution but especially to the formation of secondary fine particulate matter (PM2.5), which impacts human health and light scattering (haze). In addition, atmospheric deposition of NH3 and NOx causes adverse impacts on terrestrial and aquatic ecosystems due to acidification and eutrophication. Regulations and practices introduced by China that meet the urgent need to reduce Nr emissions are explained and resulting effects on emissions are discussed. Recommendations for improving future N management for achieving 'win-win' outcomes for Chinese agricultural production and food supply, and human and environmental health, are described. This article is part of a discussion meeting issue 'Air quality, past present and future'.

Macrophage populations show an M1-to-M2 transition in an experimental model of coronal pulp tissue engineering with mesenchymal stem cells.

AIM: To assess M1/M2 macrophage phenotypes in a coronal pulp regeneration model in rats, under the hypothesis that there are dynamic M1/M2 phenotype changes during the different stages of the pulp regeneration. METHODOLOGY: The maxillary first molars of Wistar rats were pulpotomized, and biodegradable hydrogel-made scaffolds carrying rat bone marrow mesenchymal stem cells were implanted in the pulp chamber. After 3, 7 and 14 days, samples were processed for (i) histological analysis and double immunoperoxidase staining for CD68 (a general macrophage marker) and one of either CCR7 (an M1 marker), CD163 (an M2 marker) or CD206 (an M2 marker); (ii) real-time PCR for AIF1 (an M1 marker), CD163, CD206, IL-10 and TNF-α mRNA expression; and (iii) Western blotting for the detection of CD68, CCR7 and CD206 proteins. RESULTS: Histological analysis of the implanted region revealed sparse cellular distribution at 3 days, pulp-like tissue with a thin dentine bridge-like structure at 7 days, and dentine bridge-like mineralized tissue formation and resorption of most scaffolds at 14 days. CCR7+ macrophages had the highest density at 3 days, and then significantly decreased until 14 days (P < 0.05). In contrast, M2 marker (CD163 or CD206) expressing macrophages had the lowest density at 3 days and significantly increased until 14 days (P < 0.05). AIF1 and TNF-α mRNA levels, and CD68 and CCR7 protein levels were highest at 3 days. CD163 and CD206 mRNA levels, and CD206 protein levels increased with time and showed the highest at 14 days. IL-10 mRNA was highest at 3 days, decreased at 7 days and increased at 14 days. CONCLUSIONS: Macrophages in the regenerating pulp tissue underwent a distinct transition from M1-dominant to M2-dominant, suggesting that the M1-to-M2 transition of macrophages plays an important role in creating a favourable microenvironment necessary for pulp tissue regeneration.

[Gastrointestinal leiomyoma with interstitial cells of Cajal: mimicker of gastrointestinal stromal tumor].

Objective: To study clinical and pathologic characteristics of leiomyomas of the gastrointestinal tract, and to investigate the distribution characteristics of interstitial cells of Cajal ( ICCs ) in gastrointestinal leiomyomas. Methods: One hundred and forty-seven cases of leiomyomas of gastrointestinal tract were collected at the Second Affiliated Hospital of Zhengzhou University from June 2012 to June 2017. Clinical and pathologic findings were analyzed, combined with immunohistochemistry, Alcian blue-osafranin staining and molecular study. Results: The age of patients ranged from 13-82 years with mean age of 52 years. Male to female ratio was about 1∶2. Histologically, all tumors were composed of ovoid to spindle cells arranged in short intersecting fascicles. All tumors were diffusely and strongly positive for smooth muscle antibodies, desmin and h-caldesmon by immunohistochemical staining. A prominent interspersed subpopulation of elongated/dendritic-like cells with CD117 and DOG1 positivity (accounting for 1% to 30% of all tumor cells) and negative for Alcian blue-osafranin staining was identified in all esophageal leiomyomas, 16 of 20 (80%) gastric leiomyomas and 3 of 12 small bowel leiomyomas, but none in colonic/rectal leiomyomas. Mutational analysis in 16 cases showed absence of mutation in exons 9, 11, 13 or 17 of C-KIT and exons 12 or 18 of PDGFRA. Conclusions: ICCs are identified in esophageal and gastric leiomyomas, as well as in small percentage of intestinal leiomyomas. Such findings may bring significant diagnostic pitfalls for misdiagnosis as gastrointestinal stromal tumor. Careful attention to the distribution of CD117 and DOG1 positive cells and molecular mutation analysis of C-KIT and PDGFRA may be necessary to establish the correct diagnosis.

Close association between intestinal microbiota and irritable bowel syndrome.

Trillions of microbes inhabiting the intestine form a complex ecological community, affecting the normal physiology and pathological susceptibility through their collective metabolic activities and interactions with the host. Increased numbers of diseases have been found to be associated with disturbances in this ecosystem. There is evidence that intestinal microflora undergoes alterations in patients with irritable bowel syndrome (IBS). IBS is a frequent functional gut disease with negative impact on the patient's quality of life. Although the etiology and pathology of IBS remain largely unknown, it is generally accepted that the interaction between the microbiota and the host is associated with IBS. However, there are no specific or effective therapies for the treatment of IBS at present. In recent years, researchers have shown a growing interest in seeking safer and more effective alternatives for the treatment of IBS by focusing their attention on the potential role of probiotics and prebiotics. In this review, we will discuss alterations in intestinal microbiota and how these alterations may exacerbate IBS, and introduce several new IBS treatment options aiming at re-establishing a healthy and beneficial ecosystem.

Performance of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry system for rapid identification of streptococci: a review.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been developed as a new type of soft ionization mass spectrometry in recent years. An increasing number of clinical microbiological laboratories consider it as an innovative approach for bacterial identification. This study was undertaken in order to evaluate the use of MALDI-TOF MS for rapid identification of the clinical streptococci. A systematic review was conducted based on a literature search of the Medline and Embase databases. Fixed-effects models based on the P-value and the I-square were used for meta-analysis while considering the possibility of heterogeneity between studies. Statistical analyses were performed by using STATA 11.0. Twenty-seven studies covering 3,540 streptococci were included in our meta-analysis. The MALDI-TOF MS correctly identified the species of 96% (I2 = 92.8, P < 0.1) of the streptococci. The MALDI-TOF MS correctly identified the species of 99% of the Streptococcus pneumoniae (I2 = 85.2%, P < 0.1), 100% of the Streptococcus pyogenes (I2 = 32.8%, P > 0.1), and 100% of Streptococcus agalactiae (I2 = 20.7%, P > 0.2). What's more, it also had high confidence in other Streptococcus. But the accuracy of bovis needs to be improved. The overall performance of both MALDI-MS systems was different. Notably, the identifying accuracy rate of streptococci by VITEK MS was 98%, compared to 94% by the MALDI biotyper system. Interestingly, when analyzing the incorrect identification of MALDI-TOF MS, 36 out of the 38 strains of Streptococcus mitis/oralis were inaccurately identified as Streptococcus pneumoniae by the MALDI biotyper system. In conclusion, the results of this review indicated that MALDI-TOF MS could be a reliable and rapid method for identification of the streptococci.

First report of the qnrA determinant in Shigella sonnei isolated from China.

We investigated the first presence of qnrA among Shigella sonnei clinical isolates in Jiangsu Province, China. The qnrA-positive isolates coexisted with the mutation in gyrA at codon 83, these isolates were resistant to nalidixic acid and 22·2% (2 of 9) of them were resistant to norfloxacin.

[Specific cytotoxicity of a novel HER2-based chimeric antigen receptor modified T lymphocytes against HER2-positive tumor cells].

Objective: To construct the third generation chimeric antigen receptor based on a novel humanized anti-HER2 H1-2 scFv, and to investigate the specific cytotoxicity of H1-2 CAR modified T lymphocytes(CAR-T) against HER2(+) tumor cells. Method: The expression cassette of the third generation CAR gene and anti-HER2 H1-2 scFv were constructed and cloned into lentivirus transfer plasmid, and then the third generation H1-2 CAR was transduced into human T lymphocytes using lentivirus.Enzyme linked immunosorbent assay was used to detect the expression of cytokines IL2, and LDH release assay was used to detect the cytotoxic effect of the H1-2 CAR-T.Finally, NOD/SCID mice and HER2(+) breast cancer cell line SKBR3 were used to detect the anti-tumor effect of H1-2 CAR-T in vivo. Results: The third generation H1-2 CAR was successfully constructed.H1-2 CAR-T secreted high dose of IL2 after confrontation with HER2(+) breast cancer cells.In vitro, the cytolytic rate of H1-2 CAR-T on high expression HER2(+) tumor cells was significantly higher than that in low expression HER2 or non-expression HER2 tumor cells. At the efficacy to target ratio of 20, the cytolytic rate of H1-2 CAR-T against breast cancer cell SK-BR-3 could reach (90.1±2.8)%, while the cytolytic rate of H1-2 CAR-T against HER2(-) breast cancer cell MDA-MB-231 was only (13.5±4.7)%. In the mouse xenograft tumor model, H1-2 CAR-T cells inhibited breast cancer growth in vivo.At the end of the experiments, the average tumor weight in the H1-2 CAR-T cell treatment group was (0.7±0.1) g, the non-transfected T cell therapeutic group was (1.2±0.2) g, and the PBS group was (1.2±0.2) g. There was significant difference between the H1-2 CAR-T therapeutic group and the non-transfected T cell therapeutic group (P<0.05). However, there was no significant difference between the non-transfected T cell therapeutic group and the PBS treatment group (P>0.05). Conclusion: The HER2-sepcific H1-2 CAR-T cells specifically kill HER2 positive cells, and further studies on CAR-T cells for the treatment of HER2(+) cancers are useful.

[Establishment and validation of human cancer cell lines with stable Cas9 expression].

Objective: To establish human cancer cell strains with stable Cas9 expression, and to validate the gene editing activity of Cas9 for simple gene editing in future study. Methods: Fifteen cancer cell lines of different tissue origins were infected with pLv-EF1α-Cas9-Flag-Neo or pLv-EF1α-Cas9-Flag-Puro by lentivirus and clone selection was employed to screen Cas9 stably expressed cancer cell lines. Afterward designed guide RNA vectors targeting TSC22 gene were transiently transfected into 3 of cell lines, and subsequently the gene editing activity of Cas9 was evaluated by genomic PCR, sequencing and Western blot. Results: Sixty-nine human cancer cell strains with stable Cas9 expression from different cancers were established, and by transient transfection with designed guide RNA, long fragment deletion was detected in TSC22 gene. Conclusions: Sixty-nine human cancer cell strains are successfully established with stable expression of Cas9 protein and gene editing activity. These cell strains may be employed in large-scale drug screening, screening of new drug targets and gene function investigation.

Novel mutations in quinolone resistance-determining regions of gyrA, gyrB, parC and parE in Shigella flexneri clinical isolates from eastern Chinese populations between 2001 and 2011.

The aim of this study was to evaluate the prevalence of fluoroquinolone resistance and mechanisms of selected fluoroquinolone resistance in Shigella flexneri isolates. A total of 624 S. flexneri strains isolated between 2001 and 2011 in Jiangsu Province of China were analysed for their fluoroquinolone susceptibility. The quinolone resistance-determining region of gyrA, gyrB, parC and parE were amplified and sequenced. In general, 90.5 % of S. flexneri exhibited resistance to nalidixic acid. The mean norfloxacin resistance rate was 22.4 % during the 11 years from 2001 to 2011 (6.4 % from 2001 to 2005 and 36.8 % from 2006 to 2011). Sequencing of gyrA, gyrB, parC and parE genes of all S. flexneri isolates showed that the mutation rate was as high as 93.9 %. In addition, 91.8 % and 92.3 % of S. flexneri harboured mutations in gyrA and parC, respectively. About 35.2 % of S. flexneri isolates susceptible to nalidixic acid contained mutations. Meanwhile, mutations were detected in 91.2 % of norfloxacin-susceptible strains, and almost all S. flexneri isolates resistant to fluoroquinolone contained mutations. To the best of our knowledge, this is the first study reporting the occurrence of point mutations Asn57Lys and His80Pro in gyrA and Ala85Thr, Asp111His and Ser129Pro in parC. Emerging fluoroquinolone resistance with a significantly high mutation rate of the gyrA and parC genes in S. flexneri in Jiangsu Province deserves attention, and monitoring antibiotic susceptibility is important for the effective management of S. flexneri infections.

Prevalence, resistance patterns, and characterization of integrons of Shigella flexneri isolated from Jiangsu Province in China, 2001-2011.

OBJECTIVE: To provide the epidemiology, resistance pattern, and characterization of integrons in Shigella flexneri isolated between 2001 and 2011 in Jiangsu Province. METHOD: A total of 624 strains of S. flexneri were collected from both outpatients and inpatients in hospitals in Jiangsu Province from January 2001 to December 2011. The Kirby-Bauer disk diffusion method was used to perform the antimicrobial susceptibility test. Polymerase chain reaction (PCR) was used in the detection of integrons. Pulsed-field gel electrophoresis (PFGE) was applied in the homology studies. RESULT: Serotype 2a accounted for the largest proportion in S. flexneri, namely 26.4 %. Notably, an increasing trend was detected in the resistance to common antimicrobial agents during the period 2001-2011. In recent years, more than 80.0 % isolates of S. flexneri have proved to be resistant to ampicillin, nalidixic acid, and tetracycline. The positive rates of class 1, class 2, and the atypical class 1 integrons in S. flexneri are 69.3 %, 87.8 %, and 89.2 % respectively. Most integrons detected in our research carry genes encoding resistance to trimethoprim and streptomycin. CONCLUSION: Antimicrobial resistance in S. flexneri has demonstrated a continuous rising trend in Jiangsu Province. A high prevalence of integrons and gene cassettes play an important role in the transmission of drug resistance in S. flexneri. Effective measures are urgently needed to control the spread of multi-drug-resistant S. flexneri, and more continuing active surveillance of antimicrobial resistance should be established worldwide, especially in developing countries.