BackgroundMicroRNAs (miRNAs) play vital regulatory roles in many cellular processes. The expression of miRNA (miR)-34c is highly enriched in adult mouse testis, but its roles and underlying mechanisms of action are not well understood.Methodology/Principal FindingsIn the present study, we show that miR-34c is detected in mouse pachytene spermatocytes and continues to be highly expressed in spermatids. To explore the specific functions of miR-34c, we have established an in vivo model by transfecting miR-34c inhibitors into primary spermatocytes to study the loss-of-function of miR-34c. The results show that silencing of miR-34c significantly increases the Bcl-2/Bax ratio and prevents germ cell from apoptosis induced by deprivation of testosterone. Moreover, ectopic expression of the miR-34c in GC-2 cell trigger the cell apoptosis with a decreased Bcl-2/Bax ratio and miR-34c inhibition lead to a low spontaneous apoptotic ratio and an increased Bcl-2/Bax ratio. Furthermore, ectopic expression of miR-34c reduces ATF1 protein expression without affecting ATF1 mRNA level via directly binding to ATF1's 3′UTR, indicating that ATF1 is one of miR-34c's target genes. Meanwhile, the knockdown of ATF1 significantly decreases the Bcl-2/Bax ratio and triggers GC-2 cell apoptosis. Inhibition of miR-34c does not decrease the GC-2 cell apoptosis ratio in ATF1 knockdown cells.Conclusions/SignificanceOur study shows for the first time that miR-34c functions, at least partially, by targeting the ATF1 gene in germ cell apoptosis, providing a novel mechanism with involvement of miRNA in the regulation of germ cell apoptosis.

Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important diseases in rice. However, little is known about the pathogenicity mechanisms of Xoc. Here we have investigated the function of three HD-GYP domain regulatory proteins in biofilm formation, the synthesis of virulence factors and virulence of Xoc. Deletion of rpfG resulted in altered production of extracellular polysaccharides (EPS), abolished virulence on rice and enhanced biofilm formation, but had little effect on the secretion of proteases and motility. In contrast, mutational analysis showed that the other two HD-GYP domain proteins had no effect on virulence factor synthesis and tested phenotypes. Mutation of rpfG led to up-regulation of the type III secretion system and altered expression of three putative glycosyltransferase genes gumD, pgaC and xagB, which are part of operons directing the synthesis of different extracellular polysaccharides. The pgaABCD and xagABCD operons were greatly up-regulated in the Xoc ΔrpfG mutant, whereas the expression of the gum genes was unaltered or slightly enhanced. The elevated biofilm formation of the Xoc ΔrpfG mutant was dramatically reduced upon deletion of gumD, xagA and xagB, but not when pgaA and pgaC were deleted. Interestingly, only the ΔgumD mutant, among these single gene mutants, exhibits multiple phenotype alterations including reduced biofilm and EPS production and attenuated virulence on rice. These data indicate that RpfG is a global regulator that controls biofilm formation, EPS production and bacterial virulence in Xoc and that both gumD- and xagB-dependent EPS contribute to biofilm formation under different conditions.

The second messenger c-di-GMP is implicated in regulation of various aspects of the lifestyles and virulence of Gram-negative bacteria. Cyclic di-GMP is formed by diguanylate cyclases with a GGDEF domain and degraded by phosphodiesterases with either an EAL or HD-GYP domain. Proteins with tandem GGDEF-EAL domains occur in many bacteria, where they may be involved in c-di-GMP turnover or act as enzymatically-inactive c-di-GMP effectors. Here, we report a systematic study of the regulatory action of the eleven GGDEF-EAL proteins in Xanthomonas oryzae pv. oryzicola, an important rice pathogen causing bacterial leaf streak. Mutational analysis revealed that XOC_2335 and XOC_2393 positively regulate bacterial swimming motility, while XOC_2102, XOC_2393 and XOC_4190 negatively control sliding motility. The ΔXOC_2335/XOC_2393 mutant that had a higher intracellular c-di-GMP level than the wild type and the ΔXOC_4190 mutant exhibited reduced virulence to rice after pressure inoculation. In vitro purified XOC_4190 and XOC_2102 have little or no diguanylate cyclase or phosphodiesterase activity, which is consistent with unaltered c-di-GMP concentration in ΔXOC_4190. Nevertheless, both proteins can bind to c-di-GMP with high affinity, indicating a potential role as c-di-GMP effectors. Overall our findings advance understanding of c-di-GMP signaling and its links to virulence in an important rice pathogen.

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BackgroundAbnormalities in pyloric development or in contractile function of the pylorus cause reflux of duodenal contents into the stomach and increase the risk of gastric metaplasia and cancer. Abnormalities of the pyloric region are also linked to congenital defects such as the relatively common neonatal hypertrophic pyloric stenosis, and primary duodenogastric reflux. Therefore, understanding pyloric development is of great clinical relevance. Here, we investigated the role of the LIM homeodomain transcription factor Isl1 in pyloric development.ResultsExamination of Isl1 expression in developing mouse stomach by immunohistochemistry, whole mount in situ hybridization and real-time quantitative PCR demonstrated that Isl1 is highly expressed in developing mouse stomach, principally in the smooth muscle layer of the pylorus. Isl1 expression was also examined by immunofluorescence in human hypertrophic pyloric stenosis where the majority of smooth muscle cells were found to express Isl1. Isl1 function in embryonic stomach development was investigated utilizing a tamoxifen-inducible Isl1 knockout mouse model. Isl1 deficiency led to nearly complete absence of the pyloric outer longitudinal muscle layer at embryonic day 18.5, which is consistent with Gata3 null mouse phenotype. Chromatin immunoprecipitation, luciferase assays, and electrophoretic mobility shift assays revealed that Isl1 ensures normal pyloric development by directly targeting Gata3.ConclusionsThis study demonstrates that the Isl1-Gata3 transcription regulatory axis is essential for normal pyloric development. These findings are highly clinically relevant and may help to better understand pathways leading to pyloric disease.