The Effect of Riboflavin/UVA Collagen Cross-linking Therapy on the Structure and Hydrodynamic Behaviour of the Ungulate and Rabbit Corneal Stroma

Purpose

To examine the effect of riboflavin/UVA corneal crosslinking on stromal ultrastructure and hydrodynamic behaviour.

Methods

One hundred and seventeen enucleated ungulate eyes (112 pig and 5 sheep) and 3 pairs of rabbit eyes, with corneal epithelium removed, were divided into four treatment groups: Group 1 (28 pig, 2 sheep and 3 rabbits) were untreated; Group 2 (24 pig) were exposed to UVA light (3.04 mW/cm²) for 30 minutes and Group 3 (29 pig) and Group 4 (31 pig, 3 sheep and 3 rabbits) had riboflavin eye drops applied to the corneal surface every 5 minutes for 35 minutes. Five minutes after the initial riboflavin instillation, the corneas in Group 4 experienced a 30 minute exposure to UVA light (3.04 mW/cm²). X-ray scattering was used to obtain measurements of collagen interfibrillar spacing, spatial order, fibril diameter, D-periodicity and intermolecular spacing throughout the whole tissue thickness and as a function of tissue depth in the treated and untreated corneas. The effect of each treatment on the hydrodynamic behaviour of the cornea (its ability to swell in saline solution) and its resistance to enzymatic digestion were assessed using in vitro laboratory techniques.

Results

Corneal thickness decreased significantly following riboflavin application (p<0.01) and also to a lesser extent after UVA exposure (p<0.05). With the exception of the spatial order factor, which was higher in Group 4 than Group 1 (p<0.01), all other measured collagen parameters were unaltered by cross-linking, even within the most anterior 300 microns of the cornea. The cross-linking treatment had no effect on the hydrodynamic behaviour of the cornea but did cause a significant increase in its resistance to enzymatic digestion.

Conclusions

It seems likely that cross-links formed during riboflavin/UVA therapy occur predominantly at the collagen fibril surface and in the protein network surrounding the collagen.     

First International Conference on “Biotechnology in Agriculture”

The 1st International Conference on “Biotechnology in Agriculture” was held at the Agricultural University of Tirana, Albania, from the 22nd to the 23rd of April 2014. The conference was organized as collaboration between the Agricultural University of Tirana (AUT) and the Friedrich Schiller University in Jena, Germany. About 200 scientists from different countries presented their work orally or as posters covering the three main topics of the conference: plant, animal and food biotechnology. The conference included a field trip as well to the Experimental Didactic Economy Station and a Fish Farm, both belonging to the Agricultural University of Tirana. In this report, we describe the main topics of the Conference and a summary of talks and posters presented in different sessions. The main conclusions provided at the end of the activity, the objectives for the future and a brief overview of the field trips and pre- and post-conference tours are displayed in this current report. Since the conference on “Biotechnology in Agriculture” was the first one organized in Albania and also an international one, it was of a great interest for the participants and also for the scientific community working in the biotechnology area. Therefore, one of the decisions made at the end of the conference was to hold similar conferences periodically, every two years, at AUT.     

Alteration in Endometrial Proteins during Early- and Mid-Secretory Phases of the Cycle in Women with Unexplained Infertility

Background

Compromised receptivity of the endometrium is a major cause of unexplained infertility, implantation failure and subclinical pregnancy loss. In order to investigate the changes in endometrial protein profile as a cause of unexplained infertility, the current study was undertaken to analyze the differentially expressed proteins of endometrium from early-secretory (LH+2) to mid-secretory phase (LH+7), in women with unexplained infertility.

Methods

2-D gel electrophoresis was performed to analyze the proteomic changes between early- (n = 8) and mid-secretory (n = 8) phase endometrium of women with unexplained infertility. The differentially expressed protein spots were identified by LC-MS analysis and validated by immunoblotting and immuno-histochemical analysis in early- (n = 4) and mid-secretory (n = 4) phase endometrium of infertile women. Validated proteins were also analyzed in early- (n = 4) and mid-secretory (n = 4) phase endometrium of fertile women.

Results

Nine proteins were found to be differentially expressed between early- and mid- secretory phases of endometrium of infertile women. The expression of Ras-related protein Rap-1b, Protein disulfide isomerase A3, Apolipoprotein-A1 (Apo-A1), Cofilin-1 and RAN GTP-binding nuclear protein (Ran) were found to be significantly increased, whereas, Tubulin polymerization promoting protein family member 3, Superoxide dismutase [Cu-Zn], Sorcin, and Proteasome subunit alpha type-5 were significantly decreased in mid- secretory phase endometrium of infertile women as compared to early-secretory phase endometrium of infertile women. Validation of 4 proteins viz. Sorcin, Cofilin-1, Apo-A1 and Ran were performed in separate endometrial biopsy samples from infertile women. The up-regulated expression of Sorcin and down-regulated expression of Cofilin-1 and Apolipoprotein-A1, were observed in mid-secretory phase as compared to early-secretory phase in case of fertile women.

Conclusions

De-regulation of the expression of Sorcin, Cofilin-1, Apo-A1 and Ran, during early- to mid-secretory phase may have physiological significance and it may be one of the causes for altered differentiation and/or maturation of endometrium, in women with unexplained infertility.     

Glucose-induced activation of H+-ATPase in Dunaliella salina and its role in hygromycin resistance

Dunaliella salina, a eukaryotic microalga, is known for its highly halophilic nature. The high level of salts in growth medium for this alga has made its genetic transformation a comparatively difficult procedure, particularly during the selection stage. The high salt content decreases the efficiency of most antibiotics which are being used as selection markers. Studies pertaining to the interrelationship between salt concentration and antibiotic sensitivity are scarce in Dunaliella. During our previous experiment at genetic transformation of Dunaliella, an inverse relationship between the amount of antibiotic hygromycin and sodium chloride in the medium was revealed. A possible link between plasma membrane activity and the hygromycin sensitivity was investigated in the present study by modulating plasma membrane H⁺-ATPase activity using glucose. Glucose-induced activation of H⁺-ATPase, reduced the tolerance of D. salina to the antibiotic hygromycin. Hygromycin concentration required for selection during genetic transformation of Dunaliella was lowered from 100 to 25 mg L^?1 in the presence of 10 mM glucose. Conversely, the inhibitors of the plasma membrane H⁺-ATPase, orthovanadate and diethylstilbestrol were found to inhibit the glucose activation at concentrations of 10 and 15 μM, respectively. The activation of H⁺-ATPase by glucose was further confirmed through H⁺-ATPase assay and medium acidification experiments. The results indicated that the sensitivity of Dunaliella to antibiotic is related to H⁺-ATPase and the possible involvement of pH gradient, created through H⁺-ATPase activation during drug transport.     

Design of inhibitors against guanase: synthesis and biochemical evaluation of analogues of azepinomycin

As part of a program to design rational, mechanism-based inhibitors of guanase, we report here the synthesis and biochemical screening of two analogues of azepinomycin (1 and 2), a naturally occurring inhibitor of guanase, known to mimic the transition-state of the enzyme-catalyzed reaction. Our biochemical results show that compounds 1 and 2 are competitive inhibitors with K(i) of 2.01+/-0.16 x 10(-5) and 5.36+/-0.14 x 10(-5) M, respectively.     

Imipramine blocks ethanol-induced ASMase activation, ceramide generation, and PP2A activation, and ameliorates hepatic steatosis in ethanol-fed mice

Our previous data showed the inhibitory effect of ethanol on AMP-activated protein kinase phosphorylation, which appears to be mediated, in part, through increased levels of hepatic ceramide and activation of protein phosphatase 2A (Liangpunsakul S, Sozio MS, Shin E, Zhao Z, Xu Y, Ross RA, Zeng Y, Crabb DW. Am J Physiol Gastrointest Liver Physiol 298: G1004-G1012, 2010). The effect of ethanol on AMP-activated protein kinase phosphorylation was reversed by imipramine, suggesting that the generation of ceramide via acid sphingomyelinase (ASMase) is stimulated by ethanol. In this study, we determined the effects of imipramine on the development of hepatic steatosis, the generation of ceramide, and downstream effects of ceramide on inflammatory, insulin, and apoptotic signaling pathways, in ethanol-fed mice. The effect of ethanol and imipramine (10 μg/g body wt ip) on ceramide levels, as well as inflammatory, insulin, and apoptotic signaling pathways, was studied in C57BL/6J mice fed the Lieber-DeCarli diet. Ethanol-fed mice developed the expected steatosis, and cotreatment with imipramine for the last 2 wk of ethanol feeding resulted in improvement in hepatic steatosis. Ethanol feeding for 4 wk induced impaired glucose tolerance compared with controls, and this was modestly improved with imipramine treatment. There was a significant decrease in total ceramide concentrations in response to imipramine in ethanol-fed mice treated with and without imipramine (287 ± 11 vs. 348 ± 12 pmol/mg tissue). The magnitude and specificity of inhibition on each ceramide species differed. A significant decrease was observed for C16 (28 ± 3 vs. 33 ± 2 pmol/mg tissue) and C24 (164 ± 9 vs. 201 ± 4 pmol/mg tissue) ceramide. Ethanol feeding increased the levels of the phosphorylated forms of ERK slightly and increased phospho-p38 and phospho-JNK substantially. The levels of phospho-p38 and phospho-JNK were reduced by treatment with imipramine. The activation of ASMase and generation of ceramide in response to ethanol feeding may underlie several effects of ethanol. ASMase inhibitors may be considered as a therapeutic target for alcohol-induced hepatic steatosis and activation of stress kinases.     

The Pronounced Th17 Profile in Systemic Sclerosis (SSc) Together with Intracellular Expression of TGFβ and IFNγ Distinguishes SSc Phenotypes

Background

Systemic sclerosis (SSc) is an autoimmune disease where controversy on Th1/Th2 balance dominates. We investigated whether the recently discovered Th17 pattern was present in SSc.

Methodology and Principal Findings

Patients were subdivided as having limited cutaneous SSc (lcSSc, n = 12) or diffuse cutaneous SSc (dcSSc, n = 24). A further arbitrary subdivision was made between early dcSSc (n = 11) and late dcSSc (n = 13) based upon the duration of disease. As a comparator group 14 healthy controls were studied. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, CD45Ro, CD45Ra, IL-23, GITR, CD69 and intracellular expression of IL-17, TGFβ and IFNγ using flow cytometry. Levels of IL-17, IL-6, IL-1α and IL-23 were measured using Bioplex assays. SSc patients had more and more activated CD4+ cells. In addition, CD4, CD45Ro and CD45Ra cells from all SSc patients highly expressed the IL23R, which was associated with a higher IL-17 expression as well. In contrast, IFNγ and TGFβ were selectively up regulated in SSc subsets. In line with these observation, circulating levels of IL-17 inducing cytokines IL-6, IL-23 and IL-1α were increased in all or subsets of SSc patients.

Conclusion and Significance

The combination of IL-17, IFNγ and TGFβ levels in CD45Ro and CD45Ra cells from SSc patients is useful to distinguish between lSSc, ldSSc or edSSc. Blocking Th17 inducing cytokines such as IL-6 and IL-23 may provide a useful tool to intervene in the progression of SSc.