Anti-VIM monoclonal antibody, clone 2B0

Epithelial to mesenchymal transition (EMT) contributes to fibrosis associated pathologies including scarring of different ocular tissues. Recently targeting EMT is seen as an appropriate therapeutic approach for different fibrosis related eye diseases such as macular degeneration or glaucoma surgery related fibrosis. Nevertheless, for ocular surface diseases, target genes specific for particular cell type or condition are still undefined. This study aimed to expose the complex regulatory mechanisms that trigger EMT in human conjunctival epithelial (HCjE) cells. EMT was induced by prolonged treatment with two TGF-beta isoforms, TGF-beta 1 and TGF-beta 2, and their combination. TGF-beta 1 showed the strongest potential for initiating EMT in HCjE cells, reflected on morphological changes, cell migration and the levels of mRNA expression of different epithelial (CDH1, OCLN, DSP) and mesenchymal (CDH2, FN1, VIM, SNAI1, ZEB2, TWIST1) marker genes. Co-treatment with the DNA demethylating agent 5-Azacytidine (5-AzaC) was capable of stopping the transition of HCjE cells towards a mesenchymal phenotype, based on morphological features, reduced cell mobility and mRNA and protein expression levels of epithelial and mesenchymal marker genes. An EMT qRT-PCR-based array revealed that EMT induced considerable alterations in gene expression, with downregulation of the majority of epithelial marker genes and upregulation of genes specific for the mesenchymal state. The major effect of 5-AzaC treatment was observed as a suppression of mesenchymal marker genes, suggesting the involvement of upstream negative regulator(s) whose promoter demethylation and subsequent expression will in turn promote EMT switch off. The expression level of miRNAs potentially important for EMT induction was determined using qRT-PCR-based array which pointed at members of miR-200 family as main regulators of EMT process in HCjE cells. 5-AzaC treatment induced increased expression of miR-200a, -200b, -200c and miR-141 towards the control level, indicating important role of DNA methylation in their regulation. The DNA methylation status of both miR-200 family clusters, analyzed with high-resolution melting (HRM) and bisulfite sequencing (Bis-Seq), revealed that TGF-01-induced EMT was accompanied by increase in promoter CpG methylation of both miR-200 loci, which was reverted after 5-AzaC treatment. In conclusion, our results indicate that DNA demethylation of promoters of miR-200 loci is critically important for stopping and reverting the EMT in human conjunctival epithelial cells, suggesting the potential for the development of novel epigenetic-based therapeutic strategies for treating conjunctival conditions associated with EMT.

The presence of extended-spectrum beta-lactamase (ESBL)-producing bacteria in environmental sources has been reported worldwide and constitutes a serious risk of community-acquired infections with limited treatment options. The current study aimed to explore the presence of these worrisome bacteria in a pond located at the Norwegian University of Life Sciences in angstrom s, Norway. A total of 98 bacterial isolates survived growth on selective chromogenic media and were identified by 16S rRNA Sanger sequencing. All strains were evaluated for the presence of the most commonly found beta-lactamases and ESBLs in clinical settings (bla(CTX-M) groups 1, 2, and 9, bla(CMY), bla(SHV) and bla(TEM)) and carbapenemases (bla(IMP), bla(KPC), bla(NDM), bla(OXA), bla(SFC1), bla(VIM)) through multiplex PCR. A total of eight strains were determined to contain one or more genes of interest. Phenotypic resistance to 18 antimicrobial agents was assessed and isolates were subjected to whole genome sequencing through a combination of Oxford Nanopore's MinION and lllumina's MiSeq. Results revealed the presence of beta-lactamase and ESBL-producing Escherichia coli, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and a Paraburkholderia spp. Identified beta-lactamases and ESBLs include bla(CTX-M), bla(TEM), bla(CMY), bla(SHV) and a possible bla(KPC)-like gene, with both documented and novel sequences established. In addition, two inducible beta-lactamases were found, a class A beta-lactamase (L1) and a cephalosporinase (L2). All strains were determined to be multidrug resistant and numerous resistance genes to non-beta-lactams were observed. In conclusion, this study demonstrates that environmental sources are a potential reservoir of clinically relevant ESBL-producing bacteria that may pose a health risk to humans upon exposure.