Rapid detection of a novel B1-β-lactamase gene, blaAFM-1 using a loop-mediated isothermal amplification (LAMP) assay

Background BlaAFM-1 (GenBank Accession No. 143105.1) is a new B1 subclass metallo-β-lactamase gene discovered by our group, and isolated from an Alcaligenes faecalis plasmid that renders carbapenem antibiotics ineffective. In this study, we generated a fast and reliable assay for blaAFM-1 detection. Methods We designed optimum loop-mediated isothermal amplification (LAMP) primers and constructed a recombinant plasmid AFM-1 to specifically detect blaAFM-1. Optimal LAMP primers were used to assess sensitivity of the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (simulated sputum and simulated feces). Fifty two samples, without blaAFM-1, were used to assess LAMP real-time assay specificity; these samples were verified by conventional PCR and sequencing for the absence of blaAFM-1. Three hundred clinical Gram-negative carbapenem-resistant strains were tested by LAMP assay for strains carrying blaAFM-1, which were confirmed by conventional PCR and Sanger sequencing. We calculated the sensitivity and its 95% confidence interval (95% CI), specificity and its 95% CI, and predictive values of the LAMP assay and conventional PCR/sequencing by investigating positive and negative clinical strains. Results The lowest limit of detection for the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (in both simulated sputum and simulated feces) was 101 copies/reaction. All amplification curves of the 52 blaAFM-1-free bacteria strains were negative, suggesting the LAMP assay had excellent specificity for detecting blaAFM-1. Among the 300 clinical strains, eight were positive for blaAFM-1 using LAMP. These LAMP results were consistent with conventional PCR and Sanger sequencing data. As with conventional PCR/sequencing, the LAMP method exhibits 100% sensitivity (95% CI 59.8–100%) and 100% specificity (95% CI 98.4–100%) for blaAFM-1 detection. The LAMP assay is also time-efficient (1 h) for blaAFM-1 detection. Conclusions We established a new LAMP assay with high sensitivity and specificity to detect the novel B1-β-lactamase gene, blaAFM-1.

Expression comparison between two genes encoding CSF3 recombinant proteins having different codon composition at N-terminal in Escherichia coli

Colony-stimulating factor 3 (CSF3) is a glycoprotein with many therapeutic applications. In the Escherichia coli expression system, mRNA folding and stability near the translation initiation region (TIR) are known to influence protein expression significantly. We have successfully constructed the recombinant plasmid carrying genes encoding CSF3.1 and CSF3.2, which have different synonymous codon usage at N-terminal. In this study, we compared both expressions of CSF3.1 and CSF3.2 recombinant proteins in E. coli host. Recombinant plasmid pJ414-CSF3.1 and pJ414-CSF3.2 were transformed individually into E. coli NiCo21(DE3) competent cells by a heat-shock method, then spread on solid Lysogeny Broth (LB) medium containing ampicillin. Eight transformant colonies were selected and then expressed in 2xYT medium with the addition of IPTG inducer. Expression analysis was carried out using 15% SDS-PAGE gel. No significantly different band was observed in CSF3.1 protein expression compared to the negative control. In contrast, CSF3.2 protein can be expressed with a good amount at its expected size of 18 kDa. This result was strengthened by bioinformatics analysis which demonstrated the more open TIR of CSF3.2 than that of CSF3.1 Our study highlighted that AU-rich mRNA at the N-terminal is essential for efficient recognition of the ribosome binding site.

ALKBH5 Is Lowly Expressed in Esophageal Squamous Cell Carcinoma and Inhibits the Malignant Proliferation and Invasion of Tumor Cells

Background. Modification of N6-methyladenosine (m6A) and RNA m6A regulatory factors is required in cancer advancement. The contribution of m6A and its alteration in esophageal squamous cell carcinoma (ESCC) is still unclear. Results. ALKBH5 was lowly expressed in ESCC tissues, which the total m6A level was increased in ESCC tissue than the presentation in normal healthy tissue. The pcDNA3.1-ALKBH5 recombinant plasmid was transfected into KYSE-150 and Eca-109 cells. The overexpression of ALKBH5 is responsible for a significant reduction of the total m6A levels in Eca-109 and KYSE150 cells, inhibiting the proliferation capability, migration, and cell invasion. Conclusions. ALKBH5 as a demethylase was lowly expressed in cancer progression of ESCC and acts as a crucial component in ESCC progression.

In silico recombinant plasmid design of pHA171 with phdABCD insertion for ethidium bromide degradation

Background: Ethidium bromide is a common reagent that is used in nucleic acid staining. However, ethidium bromide has toxic and carcinogenic properties that are harmful to the environment. Phenanthrene dioxygenase (encoded by phdA, phdB, phdC, and phdD genes) in Nocardioides sp. KP7 can oxidize the phenanthridine structure aim to eliminate carcinogenic properties. Objective: This study aims to visualize and predict the structure, active site, and characteristics of the phenanthrene dioxygenase using bioinformatics tools. Methods: Plasmid design were prepared by inserting genes of interest phdA, phdB, phdC, and phdD from the NCBI database. Furthermore, several protein analysis tools were used for structure visualization, active site enzyme improvement, and protein characteristic of phenanthrene dioxygenase. Results: The prediction results found that phenanthrene dioxygenase reacts with the ethidium bromide substrate through the interaction of Fe3+ ions with water. The solubility level of phenanthrene dioxygenase protein is 0.404, suggesting that the protein has low solubility. The protein isoelectric point (pI) is between 5.17 to 5.36, and the protein molecular weight is 121.143 kDa. Conclusion: In silico analysis has supported that recombinant plasmid met characteristics for the construct which consists of gene interest and protein library.

Identification of African Elephant Polyomavirus in wild elephants and the creation of a vector expressing its viral tumor antigens to transform elephant primary cells

Wild elephant populations are declining rapidly due to rampant killing for ivory and body parts, range fragmentation, and human-elephant conflict. Wild and captive elephants are further impacted by viruses, including highly pathogenic elephant endotheliotropic herpesviruses. Moreover, while the rich genetic diversity of the ancient elephant lineage is disappearing, elephants, with their low incidence of cancer, have emerged as a surprising resource in human cancer research for understanding the intrinsic cellular response to DNA damage. However, studies on cellular resistance to transformation and herpesvirus reproduction have been severely limited, in part due to the lack of established elephant cell lines to enable in vitro experiments. This report describes creation of a recombinant plasmid, pAelPyV-1-Tag, derived from a wild isolate of African Elephant Polyomavirus (AelPyV-1), that can be used to create immortalized lines of elephant cells. This isolate was extracted from a trunk nodule biopsy isolated from a wild African elephant, Loxodonta africana, in Botswana. The AelPyV-1 genome contains open-reading frames encoding the canonical large (LTag) and small (STag) tumor antigens. We cloned the entire early region spanning the LTag and overlapping STag genes from this isolate into a high-copy vector to construct a recombinant plasmid, pAelPyV-1-Tag, which effectively transformed primary elephant endothelial cells. We expect that the potential of this reagent to transform elephant primary cells will, at a minimum, facilitate study of elephant-specific herpesviruses.

Inhibition Human Nasopharyngeal Carcinoma Cells TNF-α Gene Transfected Cytokine-Induced Killer Cells Based on Nanomaterial Polyphthalamide Monoamine Dendrimer Nanomaterial

The present study aims to investigate the possibility of TNF-α gene transfection into CIK (cytokine-induced killer) cells using the nanomaterial PAMAM and the inhibitory effects of these cells on the growth of the human nasopharyngeal carcinoma cell line CNE-2. The pEGFP-N1-TNF-α recombinant plasmid was constructed and used to transfect the CIK cells using the nanomaterial PAMAM. Subsequently, the transfection efficiency was measured. The ELISA method was used to analyze the CIK cell culture supernatant. TNF-α concentration in fluid, and CIK cell phenotype was analyzed by the flow cytometry. The MTT assay was used to detect the inhibitory activity of CIK cells on the growth of nasopharyngeal carcinoma cell line CNE-2 after transfection. The CIK cells were transfected with the nanomaterial PAMAM using the successfully constructed recombinant plasmid pEGFP-N1-TNF-α. The growth characteristics and phenotypic characteristics of the transfected CIK cells were not changed, and an increase in the TNF-α secretion was observed, indicating that the CIK cells can significantly inhibit CNE-2 cell growth (P < 0005).

Cloning of a Recombinant Plasmid Encoding PpSP42 Protein Fragment of Phlebotomus papatasi and expressing it in HEK-293T Eukaryotic Cell

The article's abstract is no available.