Understanding the basis of corneal shape and transparency

Corneal transparency is a feature that can be explained by the regular arrangement of collagen fibrils and proteoglycans across the whole tissue. This special assembly promotes destructive interference of scattered light and allows only the light going forward to pass through the corneal stromal layers. There are four proteoglycans (PGs) occurring in corneal stroma: decorin, lumican, mimecan, and keratocan. Thanks to their negative charges they make a hydrated interfibrillar gel around collagen fibrils so that the fibrils do not touch each other. However, the way the proteoglycans interact with collagen to promote this spacial arrangement is not known. The first model described in this thesis was achieved from 3-D reconstruction of the tissue. We compared our results with bovine and mouse cornea and reached the conclusion that they are not arranged in any special order around the collagen fibrils. However, they are found in sufficient number in the corneal stroma to be responsible for corneal transparency. We decided to compare our results from the human to the fish cornea to get more general view of the corneal structure. The fish is known to have smaller and more closely spaced fibrils than human. The curvature of the cornea is different as the function of these two tissues is different. Fish is more flat, while the human is more spherical. The lamellar arrangement also differs between these animals. The fish cornea is more circular, while the human is orthogonal. The fibril diameter and collagen spacing is increasing towards the end of these both corneas and following on from this the transparency is reduced as we go to the periphery. We also had a chance to examine guinea pig corneas as a model of Climatic Droplet Keratopathy – a disease which is related to the loss of corneal transparency. They are similar to the human cornea in terms of the demand for ascorbic acid which is responsible for protection against UVB. The results confirmed our assumption: the animals fed on low ascorbic acid diet demonstrated some structural changes connected with UVB radiation

Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may lead to tumoral transformation due to synthesis of impaired splice variants with oncogenic potential. In this paper we hypothesized that disturbed alternative splicing in ccRCC may result from improper expression of splicing factors, mediators of splicing reactions. METHODOLOGY/PRINCIPAL FINDINGS: Using real-time PCR and Western-blot analysis we analyzed expression of seven splicing factors belonging to SR proteins family (SF2/ASF, SC35, SRp20, SRp75, SRp40, SRp55 and 9G8), and one non-SR factor, hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) in 38 pairs of tumor-control ccRCC samples. Moreover, we analyzed splicing patterns of five genes involved in carcinogenesis and partially regulated by analyzed splicing factors: RON, CEACAM1, Rac1, Caspase-9, and GLI1. CONCLUSIONS/SIGNIFICANCE: We found that the mRNA expression of splicing factors was disturbed in tumors when compared to paired controls, similarly as levels of SF2/ASF and hnRNP A1 proteins. The correlation coefficients between expression levels of specific splicing factors were increased in tumor samples. Moreover, alternative splicing of five analyzed genes was also disturbed in ccRCC samples and splicing pattern of two of them, Caspase-9 and CEACAM1 correlated with expression of SF2/ASF in tumors. We conclude that disturbed expression of splicing factors in ccRCC may possibly lead to impaired alternative splicing of genes regulating tumor growth and this way contribute to the process of carcinogenesis

TGF-β and microRNA Interplay in Genitourinary Cancers

Genitourinary cancers (GCs) include a large group of different types of tumors localizing to the kidney, bladder, prostate, testis, and penis. Despite highly divergent molecular patterns, most GCs share commonly disturbed signaling pathways that involve the activity of TGF-β (transforming growth factor beta). TGF-β is a pleiotropic cytokine that regulates key cancer-related molecular and cellular processes, including proliferation, migration, invasion, apoptosis, and chemoresistance. The understanding of the mechanisms of TGF-β actions in cancer is hindered by the “TGF-β paradox” in which early stages of cancerogenic process are suppressed by TGF-β while advanced stages are stimulated by its activity. A growing body of evidence suggests that these paradoxical TGF-β actions could result from the interplay with microRNAs: Short, non-coding RNAs that regulate gene expression by binding to target transcripts and inducing mRNA degradation or inhibition of translation. Here, we discuss the current knowledge of TGF-β signaling in GCs. Importantly, TGF-β signaling and microRNA-mediated regulation of gene expression often act in complicated feedback circuits that involve other crucial regulators of cancer progression (e.g., androgen receptor). Furthermore, recently published in vitro and in vivo studies clearly indicate that the interplay between microRNAs and the TGF-β signaling pathway offers new potential treatment options for GC patients

Identification and characterization of tetracycline resistance in Lactococcus lactis isolated from Polish raw milk and fermented artisanal products

To assess the occurrence of antibiotic-resistant Lactic Acid Bacteria (LAB) in Polish raw milk and fermented artisanal products, a collection comprising 500 isolates from these products was screened. Among these isolates, six strains (IBB28, IBB160, IBB161, IBB224, IBB477 and IBB487) resistant to tetracycline were identified. The strains showing atypical tetracycline resistance were classified as Lactococcus lactis: three of them were identified as L. lactis subsp. cremoris (IBB224, IBB477 and IBB487) and the other three (IBB28, IBB160, IBB161) were identified as L. lactis subsp. lactis. The mechanism involving Ribosomal Protection Proteins (RPP) was identified as responsible for tetracycline resistance. Three of the tested strains (IBB28, IBB160 and IBB224) had genes encoding the TetS protein, whereas the remaining three (IBB161, IBB477 and IBB487) expressed TetM. The results also demonstrated that the genes encoding these proteins were located on genetic mobile elements. The tet(S) gene was found to be located on plasmids, whereas tet(M) was found within the Tn916 transposon

Intra- and Interspecies Conjugal Transfer of Tn916-Like Elements from Lactococcus lactis In Vitro and In Vivo▿

Tetracycline-resistant Lactococcus lactis strains originally isolated from Polish raw milk were analyzed for the ability to transfer their antibiotic resistance genes in vitro, using filter mating experiments, and in vivo, using germfree rats. Four of six analyzed L. lactis isolates were able to transfer tetracycline resistance determinants in vitro to L. lactis Bu2-60, at frequencies ranging from 10−5 to 10−7 transconjugants per recipient. Three of these four strains could also transfer resistance in vitro to Enterococcus faecalis JH2-2, whereas no transfer to Bacillus subtilis YBE01, Pseudomonas putida KT2442, Agrobacterium tumefaciens UBAPF2, or Escherichia coli JE2571 was observed. Rats were initially inoculated with the recipient E. faecalis strain JH2-2, and after a week, the L. lactis IBB477 and IBB487 donor strains were introduced. The first transconjugants were detected in fecal samples 3 days after introduction of the donors. A subtherapeutic concentration of tetracycline did not have any significant effect on the number of transconjugants, but transconjugants were observed earlier in animals dosed with this antibiotic. Molecular analysis of in vivo transconjugants containing the tet(M) gene showed that this gene was identical to tet(M) localized on the conjugative transposon Tn916. Primer-specific PCR confirmed that the Tn916 transposon was complete in all analyzed transconjugants and donors. This is the first study showing in vivo transfer of a Tn916-like antibiotic resistance transposon from L. lactis to E. faecalis. These data suggest that in certain cases food lactococci might be involved in the spread of antibiotic resistance genes to other lactic acid bacteria

Epigenetic Regulation of Thyroid Hormone Receptor Beta in Renal Cancer

<div><p>Abstract</p><p>Thyroid hormone receptor beta (<i>THRB</i>) gene is commonly deregulated in cancers and, as strengthened by animal models, postulated to play a tumor-suppressive role. Our previous studies revealed downregulation of <i>THRB</i> in clear cell renal cell carcinoma (ccRCC), but the culpable mechanisms have not been fully elucidated. Since epigenetic regulation is a common mechanism influencing the expression of tumor suppressors, we hypothesized that downregulation of <i>THRB</i> in renal cancer results from epigenetic aberrances, including CpG methylation and microRNA-dependent silencing. Our study revealed that ccRCC tumors exhibited a 56% decrease in <i>THRB</i> and a 37% increase in DNA methyltransferase 1 (DNMT1) expression when compared with paired non-neoplastic control samples. However, <i>THRB</i> CpG methylation analysis performed using BSP, SNaPshot and MSP-PCR consistently revealed no changes in methylation patterns between matched tumor and control samples. <i>In silico</i> analysis resulted in identification of four microRNAs (miR-155, miR-425, miR-592, and miR-599) as potentially targeting <i>THRB</i> transcript. Luciferase assay showed direct binding of miR-155 and miR-425 to 3′UTR of <i>THRB,</i> and subsequent in vivo analyses revealed that transfection of UOK171 cell line with synthetic miR-155 or miR-425 resulted in decreased expression of endogenous <i>TRHB</i> by 22% and 64%, respectively. Finally, real-time PCR analysis showed significant upregulation of miR-155 (354%) and miR-425 (162%) in ccRCC when compared with matched controls. Moreover, microRNA levels were negatively correlated with the amount of <i>THRB</i> transcript in tissue samples. We conclude that CpG methylation is not the major mechanism contributing to decreased <i>THRB</i> expression in ccRCC. In contrast, <i>THRB</i> is targeted by microRNAs miR-155 and miR-425, whose increased expression may be responsible for downregulation of <i>THRB</i> in ccRCC tumors.</p></div

Analysis of <i>THRB</i> CpG methylation.

<p><b>A.</b> The effect of 5-aza-2′ deoxycytidine (5-aza-dC) on <i>THRB</i> mRNA expression in UOK171 cell line. The results are shown as percent of control (the cells cultured without 5-aza-dC supplementation). The plot shows results of three independent biological experiments, measured in triplicates. Statistical analysis was performed using t-test. *p<0.05. <b>B.</b> The sequence of <i>THRB</i> gene with the promoter region (GenBank Acc.no. KF669869). CpG island, encompassing the promoter and 1^st exon (region −873 to +355) is shadowed blue. TATA box is bolded. CpG dinucleotides are shadowed gray. Lower case letters indicate 1^st exon of the TRβ transcript. <b>C.</b> Representative electrophoretic analysis of MSP-PCR. U: PCR products obtained with primers specific to unmethylated sequence; M: PCR products obtained with primers specific to methylated sequence. C: control samples, T: tumor samples.</p