This study evaluated the potential of antitumor activity of snake venom from Vipera ammodytes and L-amino acid oxidase from Crotalus adamanteus on different colorectal cancer cell lines through determination of cytotoxic activity by MTT assay, pro-apoptotic activity by acridine orange/ethidium bromide staining, and concentrations of redox status parameters (superoxide, reduced glutathione, lipid peroxidation) by colorimetric methods.
The expression of genes involved in the biotransformation process and metabolite efflux was determined by qPCR method, while protein expression of glutathione synthetase and P-glycoprotein were analysed by immunocytochemistry. The analysis of cell death shows that snake venom dominantly leads cells to necrosis. Induction of apoptosis by L-amino acid oxidase was in correlation with oxidative disbalance in cancer cells.
Gene expression profile of membrane transporters and CYP genes were different in each cell line and in correlation with their sensitivity of treatment. Our results show that L-amino acid oxidase from snake venom is a potent cytotoxic substance with pronounced pro-apoptotic activity. The inhibition of P-glycoprotein suggests that L-amino acid oxidase is a good substance for furter research of antitumor effect, with unexpressed potential for occurrence of drug resistance in vitro.

Selective NF-κB inducing kinase inhibitor attenuates the onset of chronic periodontitis

Chronic periodontitis is an inflammatory disease that represents a major public health issue nowadays. Here, we investigated the protective role of NF-κB inducing kinase (NIK)-inhibitor on chronic periodontitis and revealed the underlying molecular mechanism. NIK-inhibitor was synthesized, and its functions were examined in primary osteoclasts and wild-type (WT) and NIK-/- chronic periodontitis mouse model. Lipopolysaccharides (LPS) or activator of NF-κB was applied to stimulate inflammatory response of osteoclasts.
The qRT-PCR, ELISA and Western blot were used to measure the expression of pro-inflammatory and osteoclast-related genes, and the activation of NF-κB signaling. Osteoclastogenesis and bone damage were detected by TRAP staining and micro-CT. NIK knockdown mice had lower expression of osteoclast-related genes and improved CEJ-ABC damage.
Similarly, NIK-inhibitor administration inhibited inflammatory responses and CEJ-ABC damage in chronic periodontitis models. NIK-inhibitor suppressed osteoclastogenesis and osteoclast-related genes expression through inhibiting the non-canonical NF-κB signaling. NIK plays important role in bone destruction of chronic periodontitis and NIK-inhibitor represents a promising therapeutic strategy for this disease.

Proteomic analysis in diffuse large B-cell lymphoma identifies dysregulated tumor microenvironment proteins in non-GCB/ABC subtype patients

The complexity of the activated B-cell like (ABC) diffuse large B-cell lymphoma (DLBCL) subtype is probably not only explained by genetic alterations and methods to measure global protein expression could bring new knowledge regarding the pathophysiology. We used quantitative proteomics to analyze the global protein expression of formalin-fixed paraffin-embedded (FFPE) tumor tissues from 202 DLBCL patients.
We identified 6430 proteins and 498 were significantly regulated between the germinal center B-cell like (GCB) and non-GCB groups. A number of proteins previously not described to be upregulated in non-GCB or ABC DLBCL was found, e.g. CD64, CD85A, guanylate-binding protein 1 (GBP1), interferon-induced proteins with tetratricopeptide repeat (IFIT)2, and mixed lineage kinase domain-like protein (MLKL) and immunohistochemical staining showed higher expression of GBP1 and MLKL.
A cluster analysis revealed that the most prominent cluster contained proteins involved in the tumor microenvironment and regulation of the immune system. Our data suggest that the therapeutic focus should be expanded toward the tumor microenvironment in non-GCB/ABC subtype patients.

Role of the VirSR-VirAB system in biofilm formation of Listeria monocytogenes EGD-e

The ability of Listeria monocytogenes, an important foodborne pathogen, to form biofilms in food processing environments leads to increased opportunity for contamination of food products, which is a major concern for food safety. In this study, the role of a complex system composed of the VirSR two-component signal transduction system (TCS) and the ATP-binding cassette (ABC) transporter VirAB in biofilm formation of L. monocytogenes EGD-e was investigated.
Biofilm formation was measured using the microplate assay with crystal violet staining, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), and attachment and swarming motility were compared between strain EGD-e and its isogenic deletion mutants. Additionally, the relative expression levels of genes associated with the early steps of biofilm development in the wild-type and mutant strains were also determined by RT-qPCR. Results from microplate assay, CLSM and SEM showed that VirR is not required for biofilm formation in L. monocytogenes EGD-e.
A central finding of this study is that both VirAB and VirS are essential for biofilm formation and they could function as a whole in biofilm formation of L. monocytogenes EGD-e. The results also demonstrated that both VirAB and VirS are involved in attachment, but they are not associated with swarming motility. Results from RT-qPCR showed that flaA, motA and motB were downregulated in the mutant strains ΔvirAB and ΔvirS, which could be the possible reason for reduced attachment and biofilm formation in these mutants. This study provides a better understanding of the mechanisms involved in biofilm formation of L. monocytogenes, leading to improved processes to control this biofilm-forming foodborne pathogen.

Understanding the Interactions between Staphylococcus aureus and the Raw-Meat-Processing Environment Isolate Klebsiella oxytoca in Dual-Species Biofilms via Discovering an Altered Metabolic Profile

In a raw-meat-processing environment, members of the Enterobacteriaceae family can coexist with Staphylococcus aureus to form dual-species biofilms, leading to a higher risk of food contamination. However, very little is known about the effect of inter-species interactions on dual-species biofilm formation.
The aim of this study was to investigate the interactions between S. aureus and raw-meat-processing environment isolates of Klebsiella oxytoca in dual-species biofilms, by employing an untargeted metabolomics tool. Crystal violet staining assay showed that the biomass of the dual-species biofilm significantly increased and reached its maximum after incubation for 21 h, compared with that of single species grown alone.
L-amino acid oxidase from snake venom: Biotransformation and induction of apoptosis in human colon cancer cells
The number of K. oxytoca in the dual-species biofilm was significantly higher than that of S. aureus. Field emission scanning electron microscopy (FESEM) revealed that both species were evenly distributed, and were tightly wrapped by extracellular polymeric substances in the dual-species biofilms. Ultra-high-pressure liquid chromatography equipped with a quadrupole-time-of-flight mass spectrometer (UHPLC-Q-TOF MS) analysis exhibited a total of 8184 positive ions, and 6294 negative ions were obtained from all test samples.

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Multivariate data analysis further described altered metabolic profiling between mono- and dual-species biofilms. Further, 18 and 21 different metabolites in the dual-species biofilm were screened as biomarkers by comparing the mono-species biofilms of S. aureus and K. oxytoca, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were exclusively upregulated in the dual-species biofilm included ABC transporters, amino acid metabolism, and the two-component signal transduction system. Our results contribute to a better understanding of the interactive behavior of inter-species biofilm communities, by discovering altered metabolic profiling.

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