The microRNA Expression Profile in Donation after Cardiac Death (DCD) Livers and Its Ability to Identify Primary Non Function

Donation after cardiac death (DCD) livers are marginal organs for transplant and their use is associated with a higher risk of primary non function (PNF) or early graft dysfunction (EGD). The aim was to determine if microRNA (miRNA) was able to discriminate between DCD livers of varying clinical outcome. DCD groups were categorized as PNF retransplanted within a week (n=7), good functional outcome (n=7) peak aspartate transaminase (AST) ≤ 1000 IU/L and EGD (n=9) peak AST ≥ 2500 IU/L. miRNA was extracted from archival formalin fixed post-perfusion tru-cut liver biopsies. High throughput expression analysis was performed using miRNA arrays. Bioinformatics for expression data analysis was performed and validated with real time quantitative PCR (RT-qPCR). The function of miRNA of interest was investigated using computational biology prediction algorithms. From the array analysis 16 miRNAs were identified as significantly different (p<0.05). On RT-qPCR miR-155 and miR-940 had the highest expression across all three DCD clinical groups. Only one miRNA, miR-22, was validated with marginal significance, to have differential expression between the three groups (p=0.049). From computational biology miR-22 was predicted to affect signalling pathways that impact protein turnover, metabolism and apoptosis/cell cycle. In conclusion, microRNA expression patterns have a low diagnostic potential clinically in discriminating DCD liver quality and outcome.     

Toward immunomodulation of witches broom disease of lime (WBDL) by targeting immunodominant membrane protein (IMP) of candidatus Phytoplasma aurantifolia

The witches' broom disease of lime (WBDL) caused by Candidatus Phytoplasma aurantifolia is the most devastating disease of acidian lime in southern part of Iran as it destroy thousands of trees yearly throughout these regions. Traditional methods such as eradication of infected trees and insect vector control have shown limited effect on this case. Therefore, alternative approaches such as plantibody-mediated resistance, have been considered. Throughout present study we prepared sufficient amount of antigen that is required for generation of specific monoclonal recombinant antibodies against Immunodominant membrane protein (IMP) which will be exploited for plantibody-mediated resistance approach. The gene encoding IMP protein was obtained by PCR amplification using specific primers and DNA extracted from the infected plants. Amplified fragment was then inserted into T/A cloning vector. Intact clones containing the right sequence was selected after digestion, PCR amplification and subsequent sequencing analysis. IMP encoding region having the right sequence was sub-cloned into pET28a bacterial expression vector. Large scale expression of His tagged recombinant protein was performed in the BL21-de3 strain of E. coli and purification under native conditions was carried out through immobilized metal ion affinity chromatography (IMAC) in a column containing Ni-NTA agarose beads. Successful expression and purification steps were confirmed by SDS-PAGE and western blotting analyses. The results obtained indicated the successful production of about 18 mg purified recombinant IMP protein with a low level of contamination in one liter cultured medium. Finally the purified protein was dialyzed in phosphate saline buffer and applied for immunization of mice.     

Expression of Acyl-lipid △12-desaturase Gene in Prokaryotic and Eukaryotic Cells and Its Effect on Cold Stress Tolerance of Potato

We report the expression profile of acyl-lipid Δ12-desaturase (desA) gene from Synechocystis sp. PCC6803 and its effect on cell membrane lipid composition and cold tolerance in prokaryotic (Escherichia coli) and eukaryotic (Solanum tuberosum) cells. For this purpose, a hybrid of desA and reporter gene encoding thermostable lichenase (licBM3) was constructed and used to transform these cells. The expression of this hybrid gene was measured using qualitative (Petri dish test, electrophoregram and zymogram) and quantitative methods (spectrometry and gas liquid chromatography assays). The maximum level of linoleic acid in the bacterial cells containing hybrid gene was 1.9% of total fatty acids. Cold stress tolerance assays using plant damage index and growth parameters showed that cold tolerance was enhanced in primary transgenic lines because of increased unsaturated fatty acid concentration in their lipids. The greatest content of 18:2 and 18:3 fatty acids in primary transgenic plants was observed for lines 2 (73%) and 3 (41%). Finally, our results showed that desaturase could enhance tolerance to cold stress in potato, and desaturase and lichenase retain their functionality in the structure of the hybrid protein where the enzymatic activity of target gene product was higher than in the case of reporter lichenase gene absence in the construction.     

Suppression of Sulfonylurea- and Glucose-Induced Insulin Secretion In Vitro and In Vivo in Mice Lacking the Chloride Transport Protein ClC-3

Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl⁻-fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic β cells. In ClC-3^−/− mice, insulin secretion evoked by membrane depolarization (high extracellular K⁺, sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a 80% reduction in depolarization-evoked β cell exocytosis (monitored as increases in cell capacitance) in single ClC-3^−/− β cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl⁻ fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process.     

Differences in islet-enriched miRNAs in healthy and glucose intolerant human subjects

Many microRNAs (miRNAs) are known to be cell-type specific and are implicated in development of diseases. We investigated the global expression pattern of miRNAs in human pancreatic islets compared to liver and skeletal muscle, using bead-based technology and quantitative RT-PCR. In addition to the known islet-specific miR-375, we also found enrichment of miR-127-3p, miR-184, miR-195 and miR-493∗ in the pancreatic islets. The expression of miR-375, miR-127-3p, miR-184 and the liver-enriched miR-122 is positively correlated to insulin biosynthesis, while the expression of miR-127-3p and miR-184 is negatively correlated to glucose-stimulated insulin secretion (GSIS). These correlations were absent in islets of glucose intolerant donors (HbA1c ? 6.1). We suggest that the presence of an islet-specific miRNA network, which consists of at least miR-375, miR-127-3p and miR-184, potentially involved in insulin secretion. Our results provide new insight into miRNA-mediated regulation of insulin secretion in healthy and glucose intolerant subjects.     

The potential impact of trigonelline loaded micelles on Nrf2 suppression to overcome oxaliplatin resistance in colon cancer cells

Molecular Biology Reports - Nuclear factor erythroid 2-related factor 2 (Nrf2) has a pivotal role in promoting chemoresistance by regulation of antioxidants and detoxification enzymes. Trigonelline...     

Improving the provision of ecosystem services from urban forest by integrating the species’ potential environmental functions in tree selecting process

Due to the fact that urban environments and population demands are evolving rapidly and species selection is inevitable, it is possible to gain substantial environmental benefit by implementing more effective urban tree planting programs, especially with the aim of increasing the upcoming provision of multiple ecosystem services (ES) through proper species selection. In this paper, we used a new approach to improve the potential of urban trees in optimizing more desirable environmental benefits. This was done by selecting the most appropriate tree species among a vast range of species based on their potential environmental function (both services and disservices) in Tabriz city, Iran. Also, three main planting scenarios (divided to six sub-scenarios) were developed so as to understand the long-term effectiveness of introducing the selected tree species in improving the environmental benefits (both urban forest structure and ES) in comparison with planting the existing tree species. The results indicate that regardless of the quantity of planting, the benefits of introducing the selected trees will be more than planting the existing species. Moreover, as the amount of the annual planting of the recommended species increases, so does the improvement in the projected tree characteristics and ES. This approach creates more opportunities which enable urban forest managers and policymakers to understand the importance of selecting the proper urban tree species when looking for a nature-based solution to promote the wellbeing of the urban population, to create more livable and ecologically sustainable cities and to mitigate urban environmental problems.

     

Candida galli Strain PGO6: A Novel Isolated Yeast Strain Capable of Transformation of Isoeugenol into Vanillin and Vanillic Acid

Candida galli strain PGO6 isolated from oil-contaminated water is the first isolated yeast strain which is capable to form vanillin and vanillic acid during isoeugenol biotransformation. The products were confirmed by thin-layer chromatography (TLC), changes in the UV absorption pattern and high-performance liquid chromatography (HPLC). The phenotypic and physiochemical characteristics as well as molecular phylogenetic analysis based on amplification the ITS1-5.8S-ITS2 rDNA regions indicated the isolated strain PGO6 was identified as C. galli (GenBank accession number HM641231). Resting cells of C. galli PGO6 from the late-exponential of growth phase were used as biocatalysts for the biotransformation of isoeugenol. The optimal molar conversion of vanillin (48%) and vanillic acid (19%) was obtained after a 30 h incubation using 0.1% (v/v) of isoeugenol and 6 mg of dry weight of cells per ml without further optimization. Under these conditions, the total amount of vanillin and vanillic acid was 583 mg l(-1). Further biotransformation was carried out using 0.5% (v/v) of isoeugenol under the resting cells conditions, yielding a vanillin concentration of 1.12 g l(-1) (molar yield 25.7%) after 60 h incubation. This study brings the first evidence for biotransformation of isoeugenol to vanillin and vanillic acid by a yeast strain.