Whole Genome Sequencing Reveals Antimicrobial Resistance and Virulence Genes of Both Pathogenic and Non-Pathogenic B. cereus Group Isolates from Foodstuffs in Thailand.

Members of the Bacillus cereus group are spore-forming Gram-positive bacilli that are commonly associated with diarrheal or emetic food poisoning. They are widespread in nature and frequently present in both raw and processed food products. Here, we genetically characterized 24 B. cereus group isolates from foodstuffs. Whole-genome sequencing (WGS) revealed that most of the isolates were closely related to B. cereus sensu stricto (12 isolates), followed by B. pacificus (5 isolates), B. paranthracis (5 isolates), B. tropicus (1 isolate), and "B. bingmayongensis" (1 isolate). The most detected virulence genes were BAS_RS06430, followed by bacillibactin biosynthesis genes (dhbA, dhbB, dhbC, dhbE, and dhbF), genes encoding the three-component non-hemolytic enterotoxin (nheA, nheB, and nheC), a gene encoding an iron-regulated leucine-rich surface protein (ilsA), and a gene encoding a metalloprotease (inhA). Various biofilm-associated genes were found, with high prevalences of tasA and sipW genes (matrix protein-encoding genes); purA, purC, and purL genes (eDNA synthesis genes); lytR and ugd genes (matrix polysaccharide synthesis genes); and abrB, codY, nprR, plcR, sinR, and spo0A genes (biofilm transcription regulator genes). Genes related to fosfomycin and beta-lactam resistance were identified in most of the isolates. We therefore demonstrated that WGS analysis represents a useful tool for rapidly identifying and characterizing B. cereus group strains. Determining the genetic epidemiology, the presence of virulence and antimicrobial resistance genes, and the pathogenic potential of each strain is crucial for improving the risk assessment of foodborne B. cereus group strains.

Production and immunological characterization of the novel single-chain variable fragment (scFv) antibodies against the epitopes on Opisthorchis viverrini cathepsin F (OvCatF).

BACKGROUND: Opisthorchis viverrini infection is a significant health problem in several countries, especially Southeast Asia. The infection causes acute gastro-hepatic symptoms and also long-term infection leading to carcinogenesis of an aggressive bile duct cancer (cholangiocarcinoma; CCA). Hence, the early diagnosis of O. viverrini infection could be the way out of this situation. Still, stool examination by microscopic-based methods, the current diagnostic procedure is restricted by low parasite egg numbers in the specimen and unprofessional laboratorians. The immunological procedure provides a better chance for diagnosis of the infection. Hence, this study aims to produce single-chain variable fragment (scFv) antibodies for use as a diagnostic tool for O. viverrini infection. METHODS: This study uses phage display technologies to develop the scFv antibodies against O. viverrini cathepsin F (OvCatF). The OvCatF-deduced amino acid sequence was analyzed and predicted for B-cell epitopes used for short peptide synthesis. The synthetic peptides were used to screen the phage library simultaneously with OvCatF recombinant protein (rOvCatF). The potentiated phages were collected, rescued, and reassembled in XL1-blue Escherichia coli (E. coli) as a propagative host. The positive clones of phagemids were isolated, and the single-chain variable (scFv) fragments were sequenced, computationally predicted, and molecular docked. The complete scFv fragments were digested from the phagemid, subcloned into the pOPE101 expression vector, and expressed in XL1-blue E. coli. Indirect ELISA and Western analysis were used to verify the detection efficiency. RESULTS: The scFv phages specific to OvCatF were successfully isolated, subcloned, and produced as a recombinant protein. The recombinant scFv antibodies were purified and refolded to make functional scFv. The evaluation of specific recognition of the particular epitopes and detection limit results by both computational and laboratory performances demonstrated that all three recombinant scFv antibodies against OvCatF could bind specifically to rOvCatF, and the lowest detection concentration in this study was only one hundred nanograms. CONCLUSION: Our produced scFv antibodies will be the potential candidates for developing a practical diagnostic procedure for O. viverrini infection in humans in the future.

Type I Cystatin Derived from Fasciola gigantica Suppresses Macrophage-Mediated Inflammatory Responses.

There is an inverse relationship between the high incidence of helminth infection and the low incidence of inflammatory disease. Hence, it may be that helminth molecules have anti-inflammatory effects. Helminth cystatins are being extensively studied for anti-inflammatory potential. Therefore, in this study, the recombinant type I cystatin (stefin-1) of Fasciola gigantica (rFgCyst) was verified to have LPS-activated anti-inflammatory potential, including in human THP-1-derived macrophages and RAW 264.7 murine macrophages. The results from the MTT assay suggest that rFgCyst did not alter cell viability; moreover, it exerted anti-inflammatory activity by decreasing the production of proinflammatory cytokines and mediators, including IL-1ß, IL-6, IL-8, TNF-α, iNOS, and COX-2 at the gene transcription and protein expression levels, as determined by qRT-PCR and Western blot analysis, respectively. Further, the secretion levels of IL-1ß, IL-6, and TNF-α determined by ELISA and the NO production level determined by the Griess test were decreased. Furthermore, in Western blot analysis, the anti-inflammatory effects involved the downregulation of pIKKα/ß, pIκBα, and pNF-κB in the NF-κB signaling pathway, hence reducing the translocation from the cytosol into the nucleus of pNF-κB, which subsequently turned on the gene of proinflammatory molecules. Therefore, cystatin type 1 of F. gigantica is a potential candidate for inflammatory disease treatment.

Molecular Cloning and Characterization of a Fasciola gigantica Nuclear Receptor Subfamily 1 (FgNR1).

Fasciola gigantica, a giant liver fluke, causes tremendous loss to the livestock economy in several regions throughout the world. The situation of drug resistance has been emerging increasingly; therefore, novel drugs and drug targets need to be discovered. The adult F. gigantica inhabits the major bile ducts where bile salts accumulate­these are steroid-like molecules that mediate several physiological processes in organisms through interacting with their specific nuclear receptors. However, the molecular mechanism of the interaction in the parasitic organisms have not been clearly understood. In this study, putative nuclear receptor subfamily 1 of F. gigantica (FgNR1) was identified. Nucleotide and amino acid sequences of the FgNR1 homolog were obtained from the transcriptome of F. gigantica and predicted for properties and functions using bioinformatics. The full-length cDNA was cloned and expressed in the bacterial expression system and then used for immunization. Western analysis and immunolocalization suggested that FgNR1 could be detected in the crude worm antigens and was highly expressed in the caeca and testes of the adult parasite. Moreover, the bile could significantly activate the expression of FgNR1 in cultured parasites. Our results indicated that FgNR1 has high potential for the development of a novel anthelminthic drug in the future.

Daily consumption of monosodium glutamate pronounced hypertension and altered renal excretory function in normotensive and hypertensive rats.

This study aimed to investigate the effects of monosodium glutamate (MSG) on the levels of arterial blood pressure (ABP) and renal excretory function. Male Wistar rats were divided into 2 groups (n = 24 each) namely sham operation (SO) and 2-kidneys-1-clip (2K1C) to develop the normotensive and hypertensive model, respectively. Four weeks after the operation, each group of rats were further divided into 4 subgroups (n = 6 each) which were orally administered of either distilled water or MSG at the doses of 80, 160, or 320 mg/kg BW/day once a day for 8 weeks. The body weight, the 24-hour water intake, and the 24-hour urine output were recorded weekly. Then, each rat was anesthetized and the ABP was measured via carotid artery. The renal excretory function was examined by using the clearance technique to measure the levels of the glomerular filtration rate and the renal blood flow. The levels of serum malondialdehyde (MDA) as a marker of oxidative stress were analyzed. The expression of tumor necrosis factor alpha (TNF-α) in the kidneys was also investigated using immunohistochemistry. It was found that all doses of MSG significantly increased the ABP in both SO and 2K1C groups. All doses of MSG significantly increased the serum MDA levels and the expression of TNF-α in the kidneys of the SO groups. Long-term intake of 320 mg/kg BW MSG significantly decreased the renal excretion of salt and water in both SO and 2K1C groups. As a whole, this study demonstrated that MSG consumption contributed to an increase in oxidative stress which could lead to alterations in the cardiovascular and renal function.

Camboginol and Morelloflavone from Garcinia dulcis (Roxb.) Kurz Flower Extract Promote Autophagic Cell Death against Human Glioblastoma Cells through Endoplasmic Reticulum Stress.

Garcinia dulcis is a tropical plant native to Southeast Asia that is traditionally used as a folk remedy to cure several pathological symptoms. Camboginol and morelloflavone have been revealed by previous studies as the principal bioactive compounds from the flower extract of G. dulcis. The disease-preventing properties of camboginol or morelloflavone, including anti-cancer, from various parts of G. dulcis have been revealed by recent studies. Glioblastoma is the aggressive malignant stage of brain cancer and suffers from chemotherapeutic resistance. This study aimed to test the anti-cancer effect of G. dulcis flower extract against the proliferation of A172 human glioblastoma cells. The extract had cytotoxic activity and promoted cell cycle arrest at the S and G2/M phases. Autophagic cell death was promoted by cytotoxic concentrations of the extract, as observed by enhancing autophagic flux and the expression of autophagic markers. Autophagic cell death induced by the extract might be associated with endoplasmic reticulum (ER) stress. Conclusively, it was indicated by this study that the extract from the flower of G. dulcis had a protective effect against the proliferation of A172 human glioblastoma cells through the induction of ER stress-mediated cytotoxic autophagy.