A local translation program regulates centriole amplification in the airway epithelium.

Biogenesis of organelles requires targeting of a subset of proteins to specific subcellular domains by signal peptides or mechanisms controlling mRNA localization and local translation. How local distribution and translation of specific mRNAs for organelle biogenesis is achieved remains elusive and likely to be dependent on the cellular context. Here we identify Trinucleotide repeat containing-6a (Tnrc6a), a component of the miRNA pathway, distinctively localized to apical granules of differentiating airway multiciliated cells (MCCs) adjacent to centrioles. In spite of being enriched in TNRC6A and the miRNA-binding protein AGO2, they lack enzymes for mRNA degradation. Instead, we found these apical granules enriched in components of the mRNA translation machinery and newly synthesized proteins suggesting that they are specific hubs for target mRNA localization and local translation in MCCs. Consistent with this, Tnrc6a loss of function prevented formation of these granules and led to a broad reduction, rather than stabilization of miRNA targets. These included downregulation of key genes involved in ciliogenesis and was associated with defective multicilia formation both in vivo and in primary airway epithelial cultures. Similar analysis of Tnrc6a disruption in yolk sac showed stabilization of miRNA targets, highlighting the potential diversity of these mechanisms across organs.

Transcription Factor GATA4 Regulates Cell Type-Specific Splicing Through Direct Interaction With RNA in Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitors.

BACKGROUND: GATA4 (GATA-binding protein 4), a zinc finger-containing, DNA-binding transcription factor, is essential for normal cardiac development and homeostasis in mice and humans, and mutations in this gene have been reported in human heart defects. Defects in alternative splicing are associated with many heart diseases, yet relatively little is known about how cell type- or cell state-specific alternative splicing is achieved in the heart. Here, we show that GATA4 regulates cell type-specific splicing through direct interaction with RNA and the spliceosome in human induced pluripotent stem cell-derived cardiac progenitors. METHODS: We leveraged a combination of unbiased approaches including affinity purification of GATA4 and mass spectrometry, enhanced cross-linking with immunoprecipitation, electrophoretic mobility shift assays, in vitro splicing assays, and unbiased transcriptomic analysis to uncover GATA4's novel function as a splicing regulator in human induced pluripotent stem cell-derived cardiac progenitors. RESULTS: We found that GATA4 interacts with many members of the spliceosome complex in human induced pluripotent stem cell-derived cardiac progenitors. Enhanced cross-linking with immunoprecipitation demonstrated that GATA4 also directly binds to a large number of mRNAs through defined RNA motifs in a sequence-specific manner. In vitro splicing assays indicated that GATA4 regulates alternative splicing through direct RNA binding, resulting in functionally distinct protein products. Correspondingly, knockdown of GATA4 in human induced pluripotent stem cell-derived cardiac progenitors resulted in differential alternative splicing of genes involved in cytoskeleton organization and calcium ion import, with functional consequences associated with the protein isoforms. CONCLUSIONS: This study shows that in addition to its well described transcriptional function, GATA4 interacts with members of the spliceosome complex and regulates cell type-specific alternative splicing via sequence-specific interactions with RNA. Several genes that have splicing regulated by GATA4 have functional consequences and many are associated with dilated cardiomyopathy, suggesting a novel role for GATA4 in achieving the necessary cardiac proteome in normal and stress-responsive conditions.

A Human Intestinal Infection Caused by a Novel Non-O1/O139 Vibrio cholerae Genotype and Its Dissemination Along the River.

Non-O1/O139 Vibrio cholerae is increasingly reported in the clinical settings. However, intestinal infections via the consumption of non-O1/O139 V. cholerae-carrying seafood are rarely documented in China. In this study, we reported a case of mild watery diarrhea in a young male, caused by non-O1/O139 V. cholerae in the downstream of Liaohe River. Epidemiological investigation showed that this intestinal infection potentially associated with the raw consumption of mollusc. Prior to this finding, we conducted a 6-month pathogen surveillance of three locations along the Liaohe River and identified three environmental non-O1/O139 V. cholerae strains. To confirm the epidemiological links between clinical and environmental strains, high-resolution genomic typing was employed and revealed that V. cholerae isolated from human stool sample was genomically related to the one found in local mollusc and shared a common ancestor with other environmental strains obtained in the upstream sites of the Liaohe River. This fact suggests that the river is a natural reservoir for non-O1/O139 V. cholerae which poses a potential threat to the public health. In summary, our results deepened the insights on the transmission of non-pandemic V. cholerae strains and underscored the significance of genomic surveillance for drinking water along the river sites.

Delineating the origins of the multidrug-resistant pathogens in ornamental fish farms by multilocus sequence typing and identification of a novel multidrug-resistant plasmid.

To evaluate the overuse of antibiotics and to identify the origin of pathogens in the ornamental fish industry, we conducted a field investigation of three representative fish farms in Liaoning province, China. Drug-resistant pathogens in the fishponds and groundwater were isolated and subtyped by multilocus sequence typing (MLST). In total, 33 pathogenic strains, including Aeromonas veronii and five other pathogens, were isolated from diseased fish and from groundwater. MLST revealed that A. veronii obtained from diseased fish in three fish farms can be subtyped into four sequence types, which were also identified in the corresponding groundwater. All of the isolates obtained from diseased fish showed resistance to at least four antibiotics. Notably, Citrobacter freundii JY-17 exhibited resistance to the majority of the antibiotics and was a carrier of a megaplasmid with 15 drug resistance genes. PCR assays targeting ß-lactam, kanamycin, macrolide, phenicol, sulfonamide, and trimethoprim resistance genes in the pathogens from the diseased fish and groundwater were also conducted. The results revealed strong correlations between antibiotic treatment and increased antimicrobial resistance in fish pathogens. The results suggested that groundwater is the origin of the pathogens in ornamental fish. Antibiotic treatment of ornamental fish promoted the emergence of resistant pathogens.

Mitochondrial genome sequencing of notorious scuticociliates (Pseudocohnilembus persalinus) isolated from Turbot (Scophthalmus maximus L.).

Previously, a pathogenic ciliate was isolated from the surface ulcer of a diseased Turbot (Scophthalmus maximus L.) at an aquaculture farm in North China. After morphological and molecular biological identification based on 18rRNA, the ciliated was identified as the notorious scuticociliates (Pseudocohnilembus persalinus). In this study, the whole sequence of the mitochondrial genomic gene of P. persalinus was carried out. The sequencing results showed that the complete sequence of P. persalinus mitogenome was 38,375 bp. There were 2 rRNAs, 4 tRNAs, and 34 protein-coding genes (PCGs), respectively, located on both the heavy strand and the light stand. 15 PCGs were on the heavy strand, and 19 PCGs on the light strand. Besides, phylogenetic trees among 11 ciliates were constructed based on the sequences of 17 PCGs located in mitogenome using BI methods. The results of clustering showed that P. persalinus and Uronema marinum was the first cluster belonging to the order Scuticociliatida. Our research results will further provide primary data for evolution and classified study of scuticociliates.

The mitochondrial protein CHCHD2 primes the differentiation potential of human induced pluripotent stem cells to neuroectodermal lineages.

Human induced pluripotent stem cell (hiPSC) utility is limited by variations in the ability of these cells to undergo lineage-specific differentiation. We have undertaken a transcriptional comparison of human embryonic stem cell (hESC) lines and hiPSC lines and have shown that hiPSCs are inferior in their ability to undergo neuroectodermal differentiation. Among the differentially expressed candidates between hESCs and hiPSCs, we identified a mitochondrial protein, CHCHD2, whose expression seems to correlate with neuroectodermal differentiation potential of pluripotent stem cells. We provide evidence that hiPSC variability with respect to CHCHD2 expression and differentiation potential is caused by clonal variation during the reprogramming process and that CHCHD2 primes neuroectodermal differentiation of hESCs and hiPSCs by binding and sequestering SMAD4 to the mitochondria, resulting in suppression of the activity of the TGFß signaling pathway. Using CHCHD2 as a marker for assessing and comparing the hiPSC clonal and/or line differentiation potential provides a tool for large scale differentiation and hiPSC banking studies.

A Novel Role for miR-1305 in Regulation of Pluripotency-Differentiation Balance, Cell Cycle, and Apoptosis in Human Pluripotent Stem Cells.

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are defined as pluripotent in view of their self-renewal ability and potential to differentiate to cells of all three germ layers. Recent studies have indicated that microRNAs (miRNAs) play an important role in the maintenance of pluripotency and cell cycle regulation. We used a microarray based approach to identify miRNAs that were enriched in hESCs when compared to differentiated cells and at the same time showed significant expression changes between different phases of cell cycle. We identified 34 candidate miRNAs and performed functional studies on one of these, miR-1305, which showed the highest expression change during cell cycle transition. Overexpression of miR-1305 induced differentiation of pluripotent stem cells, increased cell apoptosis and sped up G1/S transition, while its downregulation facilitated the maintenance of pluripotency and increased cell survival. Using target prediction software and luciferase based reporter assays we identified POLR3G as a downstream target by which miR-1305 regulates the fine balance between maintenance of pluripotency and onset of differentiation. Overexpression of POLR3G rescued pluripotent stem cell differentiation induced by miR-1305 overexpression. In contrast, knock-down of POLR3G expression abolished the miR-1305-knockdown mediated enhancement of pluripotency, thus validating its role as miR-1305 target in human pluripotent stem cells. Together our data point to an important role for miR-1305 as a novel regulator of pluripotency, cell survival and cell cycle and uncovers new mechanisms and networks by which these processes are intertwined in human pluripotent stem cells. Stem Cells 2016;34:2306-2317.

Calcineurin-NFAT signaling critically regulates early lineage specification in mouse embryonic stem cells and embryos.

Self-renewal and pluripotency are hallmarks of embryonic stem cells (ESCs). However, the signaling pathways that trigger their transition from self-renewal to differentiation remain elusive. Here, we report that calcineurin-NFAT signaling is both necessary and sufficient to switch ESCs from an undifferentiated state to lineage-specific cells and that the inhibition of this pathway can maintain long-term ESC self-renewal independent of leukemia inhibitory factor. Mechanistically, this pathway converges with the Erk1/2 pathway to regulate Src expression and promote the epithelial-mesenchymal transition (EMT), a process required for lineage specification in response to differentiation stimuli. Furthermore, calcineurin-NFAT signaling is activated when the earliest differentiation event occurs in mouse embryos, and its inhibition disrupts extraembryonic lineage development. Collectively, our results demonstrate that the NFAT and Erk1/2 cascades form a signaling switch for early lineage segregation in mouse ESCs and provide significant insights into the regulation of the balance between ESC self-renewal and early lineage specification.

Germline-competent mouse-induced pluripotent stem cell lines generated on human fibroblasts without exogenous leukemia inhibitory factor.

Induced pluripotent stem (iPS) cells have attracted enormous attention due to their vast potential in regenerative medicine, pharmaceutical screening and basic research. Most prior established iPS cell lines were derived and maintained on mouse embryonic fibroblast (MEF) cells supplemented with exogenous leukemia inhibitory factor (LIF). Drawbacks of MEF cells impede optimization as well as dissection of reprogramming events and limit the usage of iPS cell derivatives in therapeutic applications. In this study, we develop a reproducible protocol for efficient reprogramming mouse neural progenitor cells (NPCs) on human foreskin fibroblast (HFF) cells via retroviral transfer of human transcriptional factors OCT4/SOX2/KLF4/C-MYC. Two independent iPS cell lines are derived without exogenous LIF. They display typical undifferentiated morphology and express pluripotency markers Oct4 and Sox2. Transgenes are inactivated and the endogenous Oct4 promoter is completely demethylated in the established iPS cell lines, indicating a fully reprogrammed state. Moreover, the iPS cells can spontaneously differentiate or be induced into various cell types of three embryonic germ layers in vitro and in vivo when they are injected into immunodeficient mice for teratoma formation. Importantly, iPS cells extensively integrate with various host tissues and contribute to the germline when injected into the blastocysts. Interestingly, these two iPS cell lines, while both pluripotent, exhibit distinctive differentiation tendencies towards different lineages. Taken together, the data describe the first genuine mouse iPS cell lines generated on human feeder cells without exogenous LIF, providing a reliable tool for understanding the molecular mechanisms of nuclear reprogramming.

Pluripotency can be rapidly and efficiently induced in human amniotic fluid-derived cells.

Direct reprogramming of human somatic cells into pluripotency has broad implications in generating patient-specific induced pluripotent stem (iPS) cells for disease modeling and cellular replacement therapies. However, the low efficiency and safety issues associated with generation of human iPS cells have limited their usage in clinical settings. Cell types can significantly influence reprogramming efficiency and kinetics. To date, human iPS cells have been obtained only from a few cell types. Here, we report for the first time rapid and efficient generation of iPS cells from human amniotic fluid-derived cells (hAFDCs) via ectopic expression of four human factors: OCT4/SOX2/KLF4/C-MYC. Significantly, typical single iPS cell colonies can be picked up 6 days after viral infection with high efficiency. Eight iPS cell lines have been derived. They can be continuously propagated in vitro and express pluripotency markers such as AKP, OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81, maintaining the normal karyotype. Transgenes are completely inactivated and the endogenous OCT4 promoter is adequately demethylated in the established iPS cell lines. Moreover, various cells and tissues from all three germ layers are found in embryoid bodies and teratomas, respectively. In addition, microarray analysis demonstrates a high correlation coefficient between hAFDC-iPS cells and human embryonic stem cells, but a low correlation coefficient between hAFDCs and hAFDC-iPS cells. Taken together, these data identify an ideal human somatic cell resource for rapid and efficient generation of iPS cells, allowing us to establish human iPS cells using more advanced approaches and possibly to establish disease- or patient-specific iPS cells.