Characterization of IncHI1B Plasmids Encoding Efflux Pump TmexCD2-ToprJ2 in Carbapenem-Resistant Klebsiella variicola, Klebsiella quasipneumoniae, and Klebsiella michiganensis Strains.

Tigecycline serves as one of the last-resort antibiotics to treat severe infections caused by carbapenem-resistant Enterobacterales. Recently, a novel plasmid-mediated resistance-nodulation-division (RND)-type efflux pump gene cluster, TmexCD1-ToprJ1, and its variants, TmexCD2-ToprJ2 and TmexCD3-ToprJ3, encoding tetracyclines and tigecycline resistance, were revealed. In this study, we reported three TmexCD2-ToprJ2-harboring Klebsiella species strains, collected from two teaching tertiary hospitals in China, including one K. quasipneumoniae, one K. variicola, and one K. michiganensis. The three strains were characterized by antimicrobial susceptibility testing (AST), conjugation assay, WGS, and bioinformatics analysis. AST showed that K. variicola and K. quasipneumoniae strains were resistant to tigecycline with MIC values of 4µg/ml, whereas the K. michiganensis was susceptible to tigecycline with an MIC value of 1µg/ml. The TmexCD2-ToprJ2 clusters were located on three similar IncHI1B plasmids, of which two co-harbored the metallo-ß-lactamase gene bla NDM-1. Conjugation experiments showed that all three plasmids were capable of self-transfer via conjugation. Our results showed, for the first time, that this novel plasmid-mediated tigecycline resistance mechanism TmexCD2-ToprJ2 has spread into different Klebsiella species, and clinical susceptibility testing may fail to detect. The co-occurrence of bla NDM-1 and TmexCD2-ToprJ2 in the same plasmid is of particular public health concern as the convergence of "mosaic" plasmids can confer both tigecycline and carbapenem resistance. Its further spread into other clinical high-risk Klebsiella clones will likely exacerbate the antimicrobial resistance crisis. A close monitoring of the dissemination of TmexCD-ToprJ encoding resistance should be considered.

Ultrasound-Mediated Co-Delivery of miR-34a and sPD-1 Complexed with Microbubbles for Synergistic Cancer Therapy.

BACKGROUND: miR-34a was downregulated and PD-L1 was upregulated in cervical cancer; however, the treatment of cervical cancer lacks precision and targeting. This study explored the ultrasound-mediated co-delivery of miR-34a and sPD-1 complexes with microbubbles for synergistic cancer therapy. METHODS: Cationic lipid microbubbles (CLMBs) were prepared by membrane hydration and mechanical oscillation. U14 subcutaneous xenograft mice were injected with CLMBs-loaded sPD-1 and miR-34a combined with ultrasound targeted destruction, and tumor volume and tumor weight of mice were measured. TUNEL apoptosis test and the mRNA expression of apoptosis-related gene Bcl-2 and Bax were analyzed by qRT-PCR. Antitumor immune-related cytokines IFN-γ were investigated by qRT-PCR, LDH Cytotoxicity Assay Kit were performed to test cytotoxic T lymphocytes (CTL). RESULTS: CLMBs were successfully prepared and the plasmid bound to its surface. The tumor volume and weight were specifically decreased by ultrasound-mediated co-delivery of miR-34a and sPD-1 complexes with microbubbles, apoptosis was induced and the apoptosis suppressor gene Bcl-2 was downregulated and proapoptotic gene Bax were upregulated. qRT-PCR analysis revealed that antitumor immunity-related IFN-γ was strongly upregulated in mice, which were treated with CLMBs-loaded sPD-1 and miR-34a combined with ultrasound targeted destruction, and the percentage of CTL was increased. CONCLUSION: These findings from the study demonstrated that CLMBs could deliver miR-34a and sPD-1, combined with ultrasound targeted destruction, could suppress the tumor tissue growing, induce apoptosis and enhance antitumor immunity in U14 subcutaneous xenograft mice.

The relation of microsatellite instability to expression of hTERT in human gastric carcinoma.

OBJECTIVE: To investigate the role of microsatellite instability (MSI) in the pathogenesis of gastric carcinoma and its relationship with the expression of hTERT gene. METHODS: 75 cases of gastric carcinoma and paired normal control tissues were included in this study. MSI of BAT-25, BAT-26, D5S346, D17S250 and D2S1235 were detected by PCR, native polyacrylamide gel electrophoresis, and silver staining while the expression of hTERT was localized by immunohistochemistry at the same time. RESULTS: MSI positive rates of BAT-25, BAT-26, D5S346, D17S250 and D2S123 were 14.7%, 12.00%, 26.67%, 16% and 21.3%. MSI was obviously related with lymph node metastasis and pathologic stages respectively (P<0.05), but not with age, gender, histologic type, or infiltration depth (P>0.05). hTERT was not expressed in normal gastric mucosa, but in intestinal metaplasia, dysplasia, and gastric carcinoma. The positive rate of hTERT was 76% (57/75) in 75 cases of gastric carcinoma tissues. The expression of hTERT was obviously related to histological type (P<0.05), but not to age, gender, lymph node metastasis, depth of invasion, or staging, respectively (P>0.05). The positive rate was higher in poorly differentiated cases than in moderately and well differentiated cases (P<0.05). MSI accounted for 28.1% of 57 hTERT positive cases while MSI accounted for 72.2% in 18 hTERT negative cases. Spearman rank correlation analysis showed that MSI was negatively related to hTERT expression (r=0.387, P=0.001). CONCLUSION: MSI may play an important role in the pathogenesis and progression of gastric carcinoma by affecting the expression of TERT gene.

Clinical value of the preoperative neutrophil-to-lymphocyte ratio and red blood cell distribution width in patients with colorectal carcinoma.

The aim of the present study was to investigate the clinical value of the preoperative neutrophil-to-lymphocyte ratio (NLR) and red blood cell distribution width (RDW) in the peripheral blood of colorectal carcinoma (CRC) patients. Clinical data obtained from 240 patients with CRC undergoing radical surgical resection in Shandong Provincial Hospital Affiliated to Shandong University (Jinan, Shandong, China) between January 2011 and April 2015 were retrospectively analyzed. Data were also collected from 110 patients with colon polyps and 48 healthy volunteers to serve as controls for comparative analysis. The clinicopathological characteristics of the patients in the low and high NLR and RDW groups were compared. The NLR and RDW values were compared prior to and following surgery. Kaplan-Meier analyses and Cox regression modeling were performed to predict overall survival (OS) and disease-free survival (DFS). The NLR and RDW levels in the CRC patients were markedly higher than those in the colon polyp patients and the healthy controls. The optimum NLR and RDW cutoff points for CRC were 2.06 and 13.45%, respectively. Significant differences were detected in tumor location, diameter, degree of differentiation, tumor depth, carcinoembryonic antigen and carbohydrate antigen 199 when comparing the high and low NLR groups (P<0.05). A high RDW was significantly associated with distant metastasis and older age in CRC patients. No significant difference was detected in the NLR and RDW levels of CRC patients prior to and following surgery (P>0.05). CRC patients with an increased RDW had significantly worse OS and DFS rates, particularly those with metastatic CRC (P<0.05). Patients with a high NLR exhibited a reduced DFS time in CRC (P=0.053), although this difference was not significant, and a significantly worse DFS time in metastatic CRC (P=0.047). In conclusion, it is convenient to use preoperative NLR and RDW to predict prognosis following surgery for patients with CRC.

Qa-2 mRNA in peripheral blood mononuclear cells: a potential marker of acute allograft rejection.

BACKGROUND: HLA-G displays an immunotolerogenic role and its expressions in grafts/sera are related to allograft acceptance. However, it is still unclear how its transcription level in peripheral blood mononuclear cells (PBMCs) changes during allograft rejection. The aim of the present study was to detect the expression changes of the murine homolog of HLA-G, Qa-2, serially during the whole process of graft rejection with mouse skin transplantation models and to investigate their relationship with the pathological grade of graft rejection and immunosuppressive therapy. MATERIAL/METHODS: Full-thickness skin derived from donor mice (C57BL/6j for syngeneic groups and Balb/c for allogeneic groups) was transplanted onto the dorsal thorax of recipient mice (C57BL/6j). Pathological allograft changes were classified into four grades. Qa-2 mRNA expression of PBMCs was detected serially by real-time RT-PCR. RESULTS: Qa-2 mRNA did not show any obvious change in the syngeneic recipients without immunosuppressive treatment, inferring that surgical stress might not influence Qa-2 expression. A significant increase in Qa-2 mRNA was observed in the immunosuppressant-treated recipients, suggesting that the increased Qa-2 mRNA level might be attributed to the immunosuppressive treatment. During allograft rejection, the Qa-2 mRNA level decreased significantly, especially with immunosuppressive treatment, and the decreased expression was detected when the allograft presented grade 2-3 pathological rejection, much earlier than when gross rejection was observed. CONCLUSIONS: These results suggest that decreased expression of Qa-2 mRNA in PBMCs may be a potential marker for predicting acute allograft rejection as well as a tool to monitor the effects of immunosuppressive treatment.

Expression of MHC-I mRNA in peripheral blood lymphocytes as an early marker of acute rejection following skin transplantation in mice.

Early prediction of acute rejection (AR) is important in clinical practice of organ and tissue transplantation. The aims of this study were to investigate the expression of major histocompatibility complex (MHC)-I and MHC-II genes in peripheral blood lymphocytes (PBLs) following skin grafting and whether their expression can be used as early markers for AR. Skin-grafted mice were selected as an animal model and PBL samples were collected daily for up to 2 weeks post-transplant. Full-thickness skin from the backs of C57BL/6 mice (H-2b) was transplanted onto that of BALB/c mice (H-2d) in allograft group (H-2b to H-2d) and in syngeneic graft group (H-2d to H-2d). The expression levels of MHC-I (H-2K, H-2D) and MHC-II (H-2Ia, H-2Ie) mRNAs were examined using real-time PCR. The histopathological changes of graft biopsies were also analyzed with hematoxylin-eosin staining. The real-time PCR analysis showed that MHC-I and MHC-II mRNA levels were increased in a bimodal distribution pattern during AR in allograft group, whereas no significant changes were detected in syngeneic graft group. The level of H-2K mRNA was significantly increased at day 5 post-transplants compared with those pre-transplant controls (p < 0.01). This increase was detected 5-6 days earlier before graft rejection observed macroscopically. H-2K mRNA level was increased significantly in 93.8% of mice (61/65) in allograft group. These results indicate that the expression of MHC-I and MHC-II mRNAs is up-regulated in PBLs during AR. Especially, the expression of H-2K mRNA can be used as an early marker for AR.