Background: Urinary tract infection (UTI) is one of the most common bacterial infections in the world, and uropathogenic Escherichia Coli (UPEC) is the principal pathogen of UTI. Escherichia coli (E. coli) outer membrane protease T (OmpT), a member of the omptin family with high substrate specificity, is supposed to be involved in the pathogenesis of E.coli. Methods: In order to explore the role of OmpT in UPEC-induced UTI, an ompT gene deletion mutant of UPEC strain CFT073 was constructed by using the λ RED recombineering, then its biological properties were analyzed with in vitro model of human unthelial cell strain 5637 and in vivo model of C57B/L6 mouse. Results: The in vitro adhesion showed that the adhesion rate for the mutant (COTD) was about 60% of the parent strain CFT073 (P < 0.05). For the in vitro invasion assay, the invasion rate of the CFT073 was as 2.5 times as that of the mutant (P < 0.01). For the in vivo invasion assay, the E. coli strains wild-type and mutant (COTD) was inoculated into the bladder of the C57B/L6 mice, respectively. After 12 h of inoculation, the bladder tissues were collected and used for colony counting. The results showed that the colonization number of the mutant (COTD) was significantly fewer than that of the CFT073. The colonization number of the mutants was about 4.0 × 106 cfu, which was also obviously fewer than that of the CFT073 (1.9 × 107 cfu) (P < 0.01). By using the method of β-galactosidase in situ staining, we found that the mutant was defective in the ability of intracellular bacterial community (IBC) formation, which is an important indicator of pathogenicity for UPEC. Conclusion: Taken together, this study has suggested that ompT plays an important role of the pathogenesis of uropathogenic Escherichia Coli, however, the detailed regulatory mechanisms between the genes still need further study.
To test the effect of the c-Met inhibitor cabozantinib in inhibiting infections by Listeria monocytogenes (LM) in mice.C57BL/6 mice at 6 weeks of age were subjected to intraperitoneal injection of LM and randomized into 4 groups for treatment with intraperitoneal injection of PBS, intragastric administration of cabozantinib (20 µg/g), intraperitoneal injection of ampicillin (Amp, 20 µg/g), or cabozantinib plus Amp. The survival curves were drawn for each group, and the number of bacteria in the blood and brain tissues was determined; serum IL-10 level and NF-κB p65 level in the cerebrospinal fluid (CSF) were assayed, and Evans Blue (EB) content and pathological changes in brain were examined.Compared with PBS-treated mice, the mice treated with cabozantinib showed a significantly higher survival rate, lower bacterial counts in the blood and brain (P<0.05 or 0.001), lower IL-10 (P<0.05) and NF-κB p65 levels (P<0.01), lower brain EB content (P<0.001), and milder pathological changes in the brain. The blood and brain bacterial counts (P<0.001), IL-10 (P<0.01) and NF-κB p65 levels (P<0.001), and brain EB content (P<0.001) were all significantly lower in mice treated with the combination of drugs than in mice treated with cabozantinib alone.Cabozantinib can inhibit LM infection in mice and has important values in developing new anti-intracellular infection drug.
Abstract Cancer mutations play essential roles in tumor development by promote cell proliferation or immune evasion. However, some recent researches indicate that cancer cells with these mutation burden also expose their vulnerability to immune-based therapies such as checkpoint blockade. Here we performed comprehensive CRISPR knockout library screens in syngeneic mouse models on cancer driver mutation genes. We identified a set of genes including Kmt2d, Arid2, Rhob, Eloc, Ptpn11 and Xpo1, which have high frequency mutation in several human cancer types and their deficiency made tumor cells better respond to anti-PD-1 treatment. Furthermore, some of these deletions were validated to sensitize the tumor cells to cytotoxic T cells by in vitro co-culture with CD8+ T cells. The findings of Kmt2d was in accordance with a recent report for its role as a sensitizing modulator of immune checkpoint blockade from mouse model and patient prediction. These findings provide a new insight for understanding the interaction between the tumor heterogeneity and microenvironment. These results revealed CRISPR in vivo screen as a robust tool for both context and target discovery for tumor immunotherapy. Citation Format: Wenrong Zhou, Zhengang Peng, Dawei Huang, Min Long, Tianyu Song, Siyuan Ni, Yong Cang. Cancer driver screens identifyregulators of immune checkpoint blockade therapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1600.
Background: Cryptococcus neoformans infection has significantly increased recently, particularly in AIDS patients and immunocompromised individuals. At present, it is unclear how C. neoformans traversesthe alveolar-capillary interface and causes infection. Previous study has show that C. neoformans capsular hyaluronic acid (HA) was a virulent factor and CD44 is the major HA receptor on HBMEC during C. neoformans infection. Methods & Materials: Our study examined and characterized the interaction of C. neoformans with human pulmonary microvascular endothelial cells (HPMEC), which constitute the alveolar-capillary barrier. Cryptococcus neoformans wild-type and CPS1 gene mutant (HA-deficient strains) were used to infect HPMEC. Immune suppressed mice model was also used. Then, the ability to associate with HPMEC was done by fungi counting. CD44 expression and actin rearrangement of HPMEC were observed by the immunofluorescent techniques. Balb/c mice were immunosuppressed with cyclophosphamide (CTX), and inhaled Cryptococcus neoformans using ultrasonic atomized inhalation. The number of CFU of Cryptococci was quantized in blood and brain. CD44 expression in lung tissue was examined by the immunofluorescent method. Results: We found that C. neoformans association with HPMEC was reduced apparently using CPS1 mutant strains. Expression of CD44 signal was increased in membrane of HPMEC infected by C. neoformans but no change infected by CPS1 mutant strain. C. neoformans CPS1 mutant strain was less virulent in inducing actin cytoskeleton change of HPMEC. And C. neoformans infection increased the expression of CD44 signal in lung tissue of the immunosuppressive Balb/c mouse and caused the fungal invasion across the alveolar-capillary interface into blood. Conclusion: Our results suggest that the capsular hyaluronic acid (HA) contributes to Cryptococcus neoformans penetrating across the alveolar-capillary barrier.
Non-small-cell lung carcinomas (NSCLCs) exhibit poor prognosis and are usually resistant to conventional chemotherapy. Absence of p21WAF1/CIP1, a cyclin-dependent kinase (cdk) inhibitor, has been linked to drug resistance in many in vitro cellular models. RNA activation (RNAa) is a transcriptional activation phenomena guided by double-strand RNA (dsRNA) targeting promoter region of target gene.In this study, we explored the effect of up-regulation of p21 gene expression on drug-resistance in A549 non-small-cell lung carcinoma cells by transfecting the dsRNA targeting the promoter region of p21 into A549 cells.Enhanced p21 expression was observed in A549 cells after transfection of dsRNA, which was correlated with a significant growth inhibition and enhancement of chemosensitivity to cisplatin in A549 cells in vitro. Moreover, in vivo experiment showed that saRNA targeting the promoter region of p21 could significantly inhibit A549 xenograft tumor growth.These results indicate that p21 plays a role in lung cancer drug-resistance process. In addition, this study also provides evidence for the usage of saRNA as a therapeutic option for up-regulating lower-expression genes in lung cancer.
To construct the recombinant plasmid containing catalase (KatA) of Helicobacter pylori (Hp), analyze its nucleic acid sequence, express it in E. coli and study its antigenicity.KatA fragments were amplified from Hp chromosomal DNA by PCR. Its T-A was cloned, sequenced and compared with other HP strains on the GenBank. Then the gene cloned into pGEX-4T-1 fusion expression vector was expressed in E. coli and purified by GST-affinity chromatography. The purified product was used to identify 29 stains of mouse anti Hp monoclonal antibodies and analyze antigenicity with serum of Hp-infected patients by Western blot.KatA fragments were composed of 1,515 bp (GenBank No. DQ333889) and the nucleotide homology with other Hp strains on the GenBank was 96%-97%. 85 kDa of the recombinant KatA-pGEX-4T-1 was expressed in E. coli. 4 of 29 anti-Hp mouse monoclonal antibodies were against KatA. Western blot analysis proved that KatA was specifically recognized in the serum of Hp-infected patients.The recombinant KatA has original antigenicity. It is of great value to clinical sero-diagnosis and vaccine study of Hp.
BACKGROUND Serum retinol-binding protein (RBP) is the primary transport protein of circulating vitamin A. RBP has a crucial role in maintaining nutrient metabolism and physiologic homeostasis. Several studies have indicated that serum RBP participates in the progression of diabetes and diabetes-related complications. However, the impact of serum RBP on lower limb atherosclerosis has not been determined in individuals with type 2 diabetes mellitus (T2DM). AIM To determine the association between serum RBP and lower limb atherosclerosis in individuals with T2DM. METHODS This retrospective study enrolled 4428 eligible T2DM patients and divided the patients into non-lower limb atherosclerosis (n = 1913) and lower limb atherosclerosis groups (n = 2515) based on lower limb arterial ultrasonography results. At hospital admission, baseline serum RBP levels were assessed, and all subjects were categorized into three groups (Q1-Q3) based on RBP tertiles. Logistic regression, restricted cubic spline regression, subgroup analysis, and machine learning were used to assess the association between RBP levels and lower limb atherosclerosis risk. RESULTS Among 4428 individuals with T2DM, 2515 (56.80%) had lower limb atherosclerosis. Logistic analysis showed that lower limb atherosclerosis risk increased by 1% for every 1 unit rise in serum RBP level (odds ratio = 1.01, 95% confidence interval: 1.00-1.02, P = 0.004). Patients in the highest tertile group (Q3) had a higher lower limb atherosclerosis risk compared to the lowest tertile group (Q1) (odds ratio = 1.36, 95% confidence interval: 1.12-1.67, P = 0.002). The lower limb atherosclerosis risk gradually increased with an increase in RBP tertile (P for trend = 0.005). Restricted cubic spline analysis indicated a linear correlation between serum RBP levels and lower limb atherosclerosis risk (non-linear P < 0.05). Machine learning demonstrated the significance and diagnostic value of serum RBP in predicting lower limb atherosclerosis risk. CONCLUSION Elevated serum RBP levels correlate with an increased lower limb atherosclerosis risk in individuals with T2DM.
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