miR-122-5p as a plasma biomarker of liver injury in fish exposed to microcystin-LR.

Recent studies have shown the presence of large amounts of microRNAs (miRNAs; miRs) from damaged cells in the peripheral blood. In this study, we investigated the levels of miRNAs circulating in the blood plasma of whitefish (Coregonus lavaretus) after exposure to microcystin-LR. We used real-time PCR to examine the relative expression of plasma levels of 4 miRNAs (miR-122-5p and let-7c-5p, the liver-enriched microRNAs, miR-148a-3p which promotes the hapatospecific phenotype in mammals, and miR-92a-3p, a cell proliferation and angiogenesis promoter, potentially hepatocarcinogenic) during the first 48 h after exposure to MC-LR. We observed a rapid increase of miR-122-5p levels 8 h after exposure (P < 0.05), which continued to the end of the experiment. Our results demonstrated that the plasma miR-122-5p was indicative of MC-LR-induced liver injury, exhibiting areas under the curve close to 1 in ROC analysis (AUC = 0.976, P < 0.001). Although plasma levels of miR-148a-3p and miR-92a-3p were significantly elevated by the end of the experiment, their discriminative power was lower than reported for the miR-122-5p. Based on these results and reports on miRNA-based diagnosis of liver injuries in mammals, plasma miR-122-5p could be considered as a robust, new generation diagnostic biomarker in fish, helpful for the non-invasive diagnosis of liver damage.

MicroRNA expression profiles in liver and colon of sexually immature gilts after exposure to Fusarium mycotoxins.

To improve our knowledge of the role of microRNAs (miRs) in responses of the porcine digestive system to two Fusarium mycotoxins, zearalenone (ZEN) and deoxynivalenol (DON), we examined the expression of 7 miRs (miR-9, miR-15a, miR-21, miR-34a, miR-122, miR-125b, and miR-192), previously found to be deregulated in diseased liver and colon cells. In this study, immature gilts were exposed to NOEL doses of ZEN (40 µg/kg/d), DON (12 µg/kg/d), ZEN + DON (40 + 12 µg/kg/d), andplacebo (negative control group) for 7, 14, 21, 28, 35, and 42 days. Before the treatment, expression levels of the selected miRs were measured in the liver, the duodenum, the jejunum, and the ascending and the descending colon of the gilts. Hierarchical clustering of the tissues by their miR expression profiles was consistent with what would be expected based on the anatomical locations and the physiological functions of the organs, suggesting that functions of the miRs are related to the specificities of the tissues in which they are expressed. A subset of 2 pairs of miRs (miR-21+miR-192 and miR-15a+miR-34a), which were assigned to two distinct clusters based on their tissue abundance, was then evaluated in the liver and the ascending and the descending colon during the treatment. The most meaningful results were obtained from the ascending colon, where a significant effect of the treatment was observed, suggesting that during the exposure to mycotoxins, the pathways involved in cell proliferation and survival were disordered. Changes in miR expression in the liver and the descending colon of the treated gilts were smaller, and were associated more with treatment duration than the exposure to ZEN, DON, or ZEN + DON. Further research should focus on identification of genes whose expression is regulated by these aberrantly expressed miRs. This should facili- tate understanding of the miRNA-regulated biological effects of mycotoxins.

c-myc in whitefish (Coregonus lavaretus): structure, expression, and insights into possible posttranscriptional regulatory mechanism.

c-myc has a crucial function in growth control, differentiation, and apoptosis of vertebrate cells. Despite the important role of c-myc in mediating the biological effects, studies of c-myc gene expression and factors that control it in organisms other than mammals, such as fish, have been rare. In the current study, we asked whether c-myc mRNA of whitefish, a feasible organism for pollution monitoring in aquatic systems and a model in toxicological research, contains activity sites for regulatory motifs in its 5'- and 3'-UTRs, similar to those found in mammals. We were particularly interested in whether miRNA-34, a known negative regulator of c-myc's in mammals, is able to regulate c-myc in fish. To answer these questions, we determined the mRNA sequence of whitefish c-myc and inferred the structure of the protein that it codes for. We found that the active sites of mRNA and structures of the inferred c-myc protein are similar to those found in mammals and other fish. Remarkably, levels of c-myc mRNA expression were very high in ovaries compared to other tissues of whitefish, thus corroborating previous data in fish. Using bioinformatic searches on c-myc 3'-UTR, we confirmed the presence of two miRNA-34a (miR-34a) response elements. Luciferase reporter assay showed that activity of reporters containing either the miR response elements or entire c-myc 3'-UTR was significantly reduced (p < 0.001) by ectopic expression of miR-34a. Therefore, we further investigated possible involvement of miR-34a in c-myc gene silencing by profiling the expression of both genes in livers of whitefish treated for 8, 24, 48 h with MC-LR, a potent c-myc inducer in mammals. Although the difference was only significant at p = 0.08, the expression of c-myc mRNA in challenged whitefish after 24 h of the treatment was notably higher than that in livers of control fish. Concurrently, we noticed slight but significant up-regulation of miR-34a after 24 and 48 h of the challenge (p < 0.05); however, we found no significant correlation of the c-myc mRNA levels and miR-34a expression. Together, these results suggest that miR-34a might regulate c-myc gene expression in whitefish liver; however, their involvement in MC-LR hepatotoxicity should be clarified in future studies.

SSP2, a sporulation-specific gene necessary for outer spore wall assembly in the yeast Saccharomyces cerevisiae.

Sporulation in yeast consists of two highly coordinated processes. First, a diploid cell that is heterozygous at the mating-type locus undergoes meiosis, in which one round of DNA replication is followed by two rounds of nuclear division. Second, the meiotic products are packaged into spore cells that remain within the mother cell. A large number of genes are induced specifically during sporulation, and their products carry out different sporulation-specific events. Expression of these sporulation-specific genes is controlled by several regulators which function at different stages of the sporulation program, resulting in a cascade of gene expression following induction of meiosis. Here we describe one sporulation-specific gene, SSP2, which is induced midway through meiosis. Ssp2 shows significant homology to the predicted product of a hypothetical ORF in Candida albicans. Homozygous mutant ssp2 diploid cells fail to sporulate. In the mutant background, meiotic recombination and nuclear divisions remain normal; however, viability declines rapidly. Following meiosis, ssp2 cells form the prospore membrane, but fail to form the outer layer of the spore wall. The Ssp2 protein localizes to the spore wall after meiosis II. In addition, the ssp2 defect is also associated with delayed and reduced expression of late sporulation-specific genes. Our results suggest that SSP2 function is required after meiosis II and during spore wall formation.

Identification and characterization of mycobacterial proteins differentially expressed under standing and shaking culture conditions, including Rv2623 from a novel class of putative ATP-binding proteins.

The environmental signals that affect gene regulation in Mycobacterium tuberculosis remain largely unknown despite their importance to tuberculosis pathogenesis. Other work has shown that several promoters, including acr (also known as hspX) (alpha-crystallin homolog), are upregulated in shallow standing cultures compared with constantly shaking cultures. Each of these promoters is also induced to a similar extent within macrophages. The present study used two-dimensional gel electrophoresis and mass spectrometry to further characterize differences in mycobacterial protein expression during growth under standing and shaking culture conditions. Metabolic labeling of M. bovis BCG showed that at least 45 proteins were differentially expressed under standing and shaking culture conditions. Rv2623, CysA2-CysA3, Gap, and Acr were identified from each of four spots or gel bands that were specifically increased in bacteria from standing cultures. An additional standing-induced spot contained two comigrating proteins, GlcB and KatG. The greatest induction was observed with Rv2623, a 32-kDa protein of unknown function that was strongly expressed under standing conditions and absent in shaking cultures. Analysis using PROBE, a multiple sequence alignment and database mining tool, classified M. tuberculosis Rv2623 as a member of a novel class of ATP-binding proteins that may be involved in M. tuberculosis's response to environmental signals. These studies demonstrate the power of combined proteomic and computational approaches and demonstrate that subtle differences in bacterial culture conditions may have important implications for the study of gene expression in mycobacteria.

Intracellular passage within macrophages affects the trafficking of virulent tubercle bacilli upon reinfection of other macrophages in a serum-dependent manner.

SETTING: The interaction of tubercle bacilli with macrophages is central to understanding of tuberculosis disease. OBJECTIVE: The objective was to determine whether prior passage within macrophages affects the behavior of Mycobacterium tuberculosis (Mtb) upon re-entry into other macrophages. DESIGN: Transmission electron microscopy was used to monitor fusion of bacterial phagosomes with late endosomal/lysosomal compartments using thoria as a fluid phase marker. Two-dimensional polyacrylamide gel electrophoresis was used to study bacterial protein expression within macrophages. RESULTS: H37Rv and BCG expressed novel proteins within macrophages. H37Rv also underwent less fusion after intracellular (IC) (24.2+/-7.7%) than extracellular (XC) (67.4+/-5.5%) passage when the bacteria entered new macrophages in small clusters. These effects were inhibited by serum, and were not observed with H37Ra or BCG bacteria (78.9+/-1.6% fused for all conditions). In addition, vacuoles which contained single bacilli were less likely to acquire markers (26.9+/-2.6%) than those that contained multiple bacilli (77.3+/-2.8%). CONCLUSION: These results indicate that phagolysosomal fusion patterns can be modulated by a variety of factors and that virulent Mtb bacteria may express proteins within macrophages that alter their interaction with these host cells.

Chemotherapy for metastatic melanoma during pregnancy.

Melanoma during pregnancy represents a difficult problem. Although surgery is a definitive therapy for early-stage disease, rapidly progressive metastatic disease in pregnancy requires a series of more difficult decisions. We report the use of combination chemotherapy with tamoxifen, carmustine, dacarbazine, and cisplatin in a patient with metastatic melanoma during the second trimester of pregnancy. The patient received 2 cycles of chemotherapy prior to delivery of a healthy 1520-g baby girl at 30 weeks gestation. Placental pathology, however, revealed malignant melanoma in the intervillous space and melanin pigment granules in villous Hofbauer cells and synctial trophoblasts. This report also reviews the literature, assessing the importance of pregnancy as a prognostic factor, the effects of metastasis on the products of conception, and the use of chemotherapy in pregnancy. We conclude that combination chemotherapy can be administered in the second trimester of pregnancy for the treatment of rapidly progressive melanoma without interfering with the successful maturation and delivery of an infant of 30 weeks gestation.