The ibeA gene is located on a genomic island, GimA, which is involved in the pathogenesis of neonatal meningitis Escherichia coli (NMEC) and avian pathogenic E. coli (APEC). The prevalence of ibeA in the APEC collection in China was investigated, and 20 of 467 strains (4.3%) were positive. In addition, analysis of the association of the E. coli reference (ECOR) groups with positive strains revealed that ibeA was linked to group B2. The ibeA gene in DE205B was analyzed and compared to those of APEC and NMEC, which indicated that the specificity of ibeA was not consistent along pathotypes. The invasion of chicken embryo fibroblast DF-1 cells by APEC DE205B and RS218 was observed, which suggested that DF-1 cells could be a model to study the mechanism of APEC invasion. The inactivation of ibeA in APEC DE205B led to the reduced capacity to invade DF-1 cells, defective virulence in vivo, and decreased biofilm formation compared to the wild-type strain. In addition, strain AAEC189 expressing ibeA exhibited enhanced invasion capacity and biofilm formation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) analysis and animal system infection experiments indicated that the loss of ibeA decreased the colonization and proliferation capacities of APEC in the brain during system infection.Escherichia coli typically colonizes the mammalian and avian gastrointestinal tract and other mucosal surfaces. While many of these strains are commensal, certain pathogenic strains can cause severe diseases (33). Extraintestinal pathogenic E. coli (ExPEC) is a group of strains that have been implicated in a large range of infections in humans and animals, such as neonatal meningitis, urinary tract infections (UTIs), pneumonia, osteomyelitis, and septicemia (14,16,30,35). Among these, typical infections caused by ExPEC in humans are UTIs and neonatal meningitis (5). Similarly, systemic infections caused by avian pathogenic E. coli (APEC) are economically devastating to poultry industries (14, 16). APEC enters and colonizes the avian respiratory tract by inhalation of fecal dust, leading to localized infections, such as airsacculitis and pneumonia. In certain cases, they spread into various internal organs, typically causing pericarditis, perihepatitis, peritonitis, salpingitis, and other extraintestinal diseases. Systemic infection of poultry is characterized in its acute form by septicemia, commonly resulting in sudden death (14,16,52).Previous studies showed that certain subsets of ExPEC strains isolated from different host organisms are highly similar (17,31,44,51), thus increasing the need to study their zoonotic potential. Virulence determinants common to uropathogenic E. coli (UPEC), APEC, neonatal meningitis Escherichia coli (NMEC), or septicemia-associated E. coli (SEPEC), such as the aerobactin iron transport system, the K1 capsule, and type 1 and P fimbriae, have been identified (2,14,19,21,43,47,59). Furthermore, the function of the K1 capsule was similar in virulence to those of APEC and NMEC. The K1...
Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.
The ibeA gene, one of the important invasion-associated genes in neonatal meningitis Escherichia coli (NMEC), has been recently detected in avian pathogenic E. coli (APEC). Thus, it is necessary to close monitor the possible contamination of the poultry farms and its products to people. Here, a dot blot method for detecting the ibeA gene in E. coli was developed and validated. For the present study, probe sequence was designed and optimized for the specificity of dot blot. A 342-bp conserved fragment of ibeA gene was selected and labeled with digoxigenin (DIG)-dUTP according to the manufacturer's guidelines, which indicated that this probe hybridizes with ibeA. In our established method, the bacteria culture samples were directly spotted on the membrane, following simple lyses on the membrane. Hence, the extraction of genomic DNA is not required, which reduces the workload and shortens the time. Furthermore, this assay was very sensitive, which could detect as few as 2.5 × 10(3) CFU bacteria. The diagnostic reliability of this dot blot was evaluated on 467 APEC bacteria samples by using PCR analysis. Both methods showed that the result was in complete concordance. The dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify invasion-associated genes ibeA, which could be applied for initial screening of a large number of clinical samples or direct detection of bacteria culture.
Objectives: To measure the expression of matrix metalloproteinase (MMP)-2, tissue inhibitor of matrix metalloproteinase inhibitor (TIMP)-2, and CD147 in mice with chronic liver injury induced by carbon tetrachloride after treatment with the traditional Chinese medicine (TCM) “Compound T11”. Method: Sixty male ICR mice were divided randomly into 6 groups of 10: control (C), model (M), low-dose treatment (LT; 50 mg/mL of Compound T11), medium-dose treatment (MT, 100 mg/mL), high-dose treatment (HT, 150 mg/mL), and positive drug treatment (YT, 67.5 mg/mL). Each group was modeled for 7 weeks. Groups M, LT, MT, HT, and YT were injected (s.c.) with 20% carbon tetrachloride diluted with olive oil, and group C was given olive oil in the same way twice a week. After modeling, the treatment groups were administered Compound T11 at the concentrations shown above by oral gavage daily for 2 weeks, while group C was given 0.5% carboxymethyl cellulose sodium. After the final treatment, mice were killed and their liver tissues were excised. Immunohistochemical staining was performed to measure the protein expression of MMP-2, TIMP-2, and CD147, and western blotting was used to measure the protein expression of MMP-2, TIMP-2, CD147, and α-smooth muscle actin (SMA). MMP-2, TIMP-2, and CD147 mRNA expression was determined by quantitative fluorescence real-time PCR. Results: Compound T11 increased the protein expression of MMP-2 and CD147 and decreased the protein expression of TIMP-2 and α-SMA. Conclusions: Treatment of chronic liver injury by TCM Compound T11 may be associated with changes to the expression of MMP-2 and CD147, and the inhibition of TIMP-2 expression.
Dunaliella salina (D. salina) expression system shows a very attractive application prospect, but it currently has a technical bottleneck, namely the low or unstable expression of recombinant proteins. Given the characteristics of cell-penetrating peptides (CPPs) or/and nuclear localization signal (NLS) peptides, this study rstly attempted to improve the transformation rate of foreign gene with transactivating transcriptional (TAT) protein or/and NLS peptides. Using salt gradient method, exogenous plasmids were transferred into D. salina cells with TAT or TAT/NLS complexes simultaneously. The βglucuronidase (GUS) gene expression was identi ed by means of histochemical stain and RT-qPCR detection. Through observation with light microscope, TAT-mediating cells exhibit an apparent cytotoxicity even at ratios of 0.5, no signi cant toxicity was noted in the TAT/plasmid/NLS complex group. It is obvious that with the addition of peptides the toxicity decreases signi cantly. Histochemical staining showed that the transformants presented blue color. under light microscope, but the negative control and blank control are not. Furthermore, based on a TAT/plasmids ratio of 4 with 10 µg NLS peptides mediation, RT-qPCR results demonstrated that the transcripts of target gene were increased by 269 times than that of control group. Collectively, this study demonstrated that combination of TAT and NLS peptides can signi cantly improve the transformation rate and expression level of foreign gene in D. salina system. It offers a promising way for promoting the application and development of D. salina bioreactor.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.