Lutimonas zeaxanthinifaciens sp. nov., a zeaxanthin-producing marine bacterium isolated from coastal sediment.

A Gram-stain-negative, yellow-pigmented, strictly aerobic, non-flagellated, motile by gliding, rod-shaped bacterium, designated strain YSD2104T, was isolated from a coastal sediment sample collected from the southeastern part of the Yellow Sea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain YSD2104T was closely related to three type strains, Lutimonas vermicola IMCC1616T (97.4 %), Lutimonas saemankumensis SMK-142T (96.9 %), and Lutimonas halocynthiae RSS3-C1T (96.8 %). Strain YSD2104T has a single circular chromosome of 3.54 Mbp with a DNA G+C content of 38.3 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain YSD2104T and the three type strains (L. vermicola IMCC1616 T, L. saemankumensis SMK-142T, and L. halocynthiae RSS3-C1T) were 74.0, 86.2 and 73.6 %, and 17.9, 30.3 and 17.8 %, respectively. Growth was observed at 20-30 °C (optimum, 30 °C), at pH 6.5-8.5 (optimum, pH 7.0), and with NaCl concentrations of 1.5-3.5 % (optimum, 2.5 %). The major carotenoid was zeaxanthin, and flexirubin-type pigment was not produced. The major respiratory quinone was menaquinone-6. The major fatty acids (>10 %) were iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), and summed feature 9 (iso-C17 : 1 ω9c and/or 10-methyl C16 : 0). The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, two unidentified aminolipids, and eight unidentified lipids. Conclusively, based on this polyphasic approach, we classified strain YSD2104T (=KCTC 102008T=JCM 36287T) as representing a novel species of the genus Lutimonas and proposed the name Lutimonas zeaxanthinifaciens sp. nov.

Ferrimonas lipolytica sp. nov., a facultatively anaerobic bacterium isolated from seawater.

A Gram-stain-negative, motile, facultatively anaerobic rod-shaped bacterium with a polar flagellum, designated strain S7T was isolated from seawater sample collected at Uljin marina, in the East Sea of the Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain S7T was affiliated with members of genus Ferrimonas, showing the highest sequence similarities to the type strains Ferrimonas senticii P2S11T (95.7 %), Ferrimonas balearica PATT (95.7 %) and Ferrimonas pelagia CBA4601T (95.1 %). The genome was 4.13 Mbp with a DNA G+C content of 49.4 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between S7T and F. senticii P2S11T and F. balearica PATT yielded ANI values of 71.9 and 70.7 %, and dDDH values of 15.1 and 13.9 %, respectively. The genome of S7T was predicted to encode triacylglycerol lipase, phospholipase A1/A2 and lysophospholipase as well as esterase involved in lipolytic processes. Growth was observed at 8-31 °C (optimum 27 °C), at pH 7-9 (optimum pH 7), and with 1-6 % NaCl (optimum 2 %). The respiratory quinones were MK-7 and Q-7 and the major fatty acids (>10 %) were C16 : 0, C16 : 1ω9c, C17 : 1ω8c, and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The major polar lipids were identified as phosphatidylethanolamine, phosphatidylglycerol, two unidentified phospholipids, and three unidentified lipids. On the basis of the results of this polyphasic analysis, it was determined that the strain represents a novel species of the genus Ferrimonas, for which the name Ferrimonas lipolytica sp. nov. is proposed. The type strain is S7T (=KCTC 72490T=JCM 33793T).

Chromatin accessibility and transcription dynamics during in vitro astrocyte differentiation of Huntington's Disease Monkey pluripotent stem cells.

BACKGROUND: Huntington's Disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion, resulting in a mutant huntingtin protein. While it is now clear that astrocytes are affected by HD and significantly contribute to neuronal dysfunction and pathogenesis, the alterations in the transcriptional and epigenetic profiles in HD astrocytes have yet to be characterized. Here, we examine global transcription and chromatin accessibility dynamics during in vitro astrocyte differentiation in a transgenic non-human primate model of HD. RESULTS: We found global changes in accessibility and transcription across different stages of HD pluripotent stem cell differentiation, with distinct trends first observed in neural progenitor cells (NPCs), once cells have committed to a neural lineage. Transcription of p53 signaling and cell cycle pathway genes was highly impacted during differentiation, with depletion in HD NPCs and upregulation in HD astrocytes. E2F target genes also displayed this inverse expression pattern, and strong associations between E2F target gene expression and accessibility at nearby putative enhancers were observed. CONCLUSIONS: The results suggest that chromatin accessibility and transcription are altered throughout in vitro HD astrocyte differentiation and provide evidence that E2F dysregulation contributes to aberrant cell-cycle re-entry and apoptosis throughout the progression from NPCs to astrocytes.

Maintenance of CTCF- and Transcription Factor-Mediated Interactions from the Gametes to the Early Mouse Embryo.

The epigenetic information present in mammalian gametes and whether it is transmitted to the progeny are relatively unknown. We find that many promoters in mouse sperm are occupied by RNA polymerase II (Pol II) and Mediator. The same promoters are accessible in GV and MII oocytes and preimplantation embryos. Sperm distal ATAC-seq sites containing motifs for various transcription factors are conserved in monkeys and humans. ChIP-seq analyses confirm that Foxa1, ERα, and AR occupy distal enhancers in sperm. Accessible sperm enhancers containing H3.3 and H2A.Z are also accessible in oocytes and preimplantation embryos. Furthermore, their interactions with promoters in the gametes persist during early development. Sperm- or oocyte-specific interactions mediated by CTCF and cohesin are only present in the paternal or maternal chromosomes, respectively, in the zygote and 2-cell stages. These interactions converge in both chromosomes by the 8-cell stage. Thus, mammalian gametes contain complex patterns of 3D interactions that can be transmitted to the zygote after fertilization.

Possible involvement of Wnt/ß-catenin signaling pathway in hatching and trophectoderm differentiation of pig blastocysts.

The Wnt/ß-catenin signaling pathway plays essential roles in the regulation of cell fate and polarity during embryonic development of many animal species. This study investigated the possible involvement of Wnt/ß-catenin signaling pathway during hatching and trophectoderm (TE) development in pig blastocysts. Results showed that ß-catenin and DVL3, the key mediators of Wnt/ß-catenin signaling, disappeared from the nucleus after blastocyst hatching. Specific inhibition of Wnt/ß-catenin signaling pathway, by Dickkopf-1, increased the rate of blastocyst hatching, total nuclear number per blastocyst, and reduced the ratio of inner cell mass (ICM):TE (P < 0.05). In contrast, specific activation of the Wnt/ß-catenin signaling pathway, by lithium chloride, reduced the rate of blastocyst hatching, total nuclear number per blastocyst, and increased the ratio of ICM:TE (P < 0.05). The change in the ICM:TE ratio was associated with the change in the number of TE cells but not the ICM cells. Activation or inhibition of Wnt/ß-catenin signaling and ß-catenin nuclear accumulation, by lithium chloride or Dickkopf-1, also altered the expression of CDX2. These data therefore, suggest the possible involvement of Wnt/ß-catenin signaling in regulating hatching and TE fate during the development of pig blastocyst.

Differential genomic imprinting and expression of imprinted microRNAs in testes-derived male germ-line stem cells in mouse.

BACKGROUND: Testis-derived male germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells (SSC), can acquire multipotency under appropriate culture conditions to become multipotent adult germ-line stem (maGS) cells, which upon testicular transplantation, produce teratoma instead of initiating spermatogenesis. Consequently, a molecular marker that can distinguish GS cells from maGS cells would be of potential value in both clinical and experimental research settings. METHODS AND FINDINGS: Using mouse as a model system, here we show that, similar to sperm, expression of imprinted and paternally expressed miRNAs (miR-296-3p, miR-296-5p, miR-483) were consistently higher (P<0.001), while those of imprinted and maternally expressed miRNA (miR-127, miR-127-5p) were consistently lower (P<0.001) in GS cells than in control embryonic stem (ES) cells. DNA methylation analyses of imprinting control regions (ICR), that control the expression of all imprinted miRNAs in respective gene clusters (Gnas-Nespas DMR, Igf2-H19 ICR and Dlk1-Dio3 IG-DMR), confirmed that imprinted miRNAs were androgenetic in GS cells. On the other hand, DNA methylation of imprinted miRNA genes in maGS cells resembled those of ES cells but the expression pattern of the imprinted miRNAs was intermediate between those of GS and ES cells. The expression of imprinted miRNAs in GS and maGS cells were also altered during their in vitro differentiation and varied both with the differentiation stage and the miRNA. CONCLUSIONS: Our data suggest that GS cells have androgenetic DNA methylation and expression of imprinted miRNAs which changes to ES cell-like pattern upon their conversion to maGS cells. Differential genomic imprinting of imprinted miRNAs may thus, serve as epigenetic miRNA signature or molecular marker to distinguish GS cells from maGS cells.

MicroRNA signature in testes-derived male germ-line stem cells.

The testis-derived male germ-line stem (GS) cell, the in vitro counterpart of spermatogonial stem cell (SSC), can initiate donor-derived spermatogenesis in recipient testes and therefore, has been viewed as a future therapeutic modality for treatment of male infertility in azoospermic patients and in cancer patients who are expecting chemotherapy. Upon extended in vitro culture, GS cells also generate a second cell type called multipotent adult germ-line stem (maGS) cell which, upon testicular transplantation, produces teratoma instead of initiating spermatogenesis. Here, we show that expressions of both Let-7a and Let-7d were consistently higher while that of miR-294 (embryonic stem cell-cycle-regulating miRNA; ESCC) was lower in GS cells than in maGS cells. Furthermore, among several putative targets of Let-7 identified by in silico bioinformatics, expressions of Igf2 and H19 mRNA targets significantly differed between GS and maGS cells. However, although the CTCF binding factor (a component of DNA methylation machinery at Igf2-H19 cluster) was also a putative target for Let-7, the difference in expressions of Igf2 and H19 between GS and maGS cells was not mediated through a change in DNA methylation. Both GS and maGS cells maintained androgenetic imprinting at the Igf2-H19 imprinting control region and Peg1 differentially methylated region. In conclusion, our study suggests that high Let-7 expression may be a unique property of GS cells and expressions of Let-7 and ESCC miRNAs may serve as miRNA signatures to distinguish them from maGS cells during clinical transplantation, to avoid the likelihood of teratoma formation due to maGS cells generated during extended in vitro culture of GS cells.

Glial cell line-derived neurotrophic factor alters the growth characteristics and genomic imprinting of mouse multipotent adult germline stem cells.

This study evaluated the essentiality of glial cell line-derived neurotrophic factor (GDNF) for in vitro culture of established mouse multipotent adult germline stem (maGS) cell lines by culturing them in the presence of GDNF, leukemia inhibitory factor (LIF) or both. We show that, in the absence of LIF, GDNF slows the proliferation of maGS cells and result in smaller sized colonies without any change in distribution of cells to different cell-cycle stages, expression of pluripotency genes and in vitro differentiation potential. Furthermore, in the absence of LIF, GDNF increased the expression of male germ-line genes and repopulated the empty seminiferous tubule of W/W(v) mutant mouse without the formation of teratoma. GDNF also altered the genomic imprinting of Igf2, Peg1, and H19 genes but had no effect on DNA methylation of Oct4, Nanog and Stra8 genes. However, these effects of GDNF were masked in the presence of LIF. GDNF also did not interfere with the multipotency of maGS cells if they are cultured in the presence of LIF. In conclusion, our results suggest that, in the absence of LIF, GDNF alters the growth characteristics of maGS cells and partially impart them some of the germline stem (GS) cell-like characteristics.

H19 gene is epigenetically stable in mouse multipotent germline stem cells.

Testis-derived germline stem (GS) cells can undergo re-programming to acquire multipotency when cultured under appropriate culture conditions. These multipotent GS (mGS) cells have been known to differ from GS cells in their DNA methylation pattern. In this study, we examined the DNA methylation status of the H19 imprinting control region (ICR) in multipotent adult germline stem (maGS) cells to elucidate how epigenetic imprints are altered by culture conditions. DNA methylation was analyzed by bisulfite sequencing PCR of established maGS cells cultured in the presence of glial cell line-derived neurotrophic factor (GDNF) alone or both GDNF and leukemia inhibitory factor (LIF). The results showed that the H19 ICR in maGS cells of both groups was hypermethylated and had an androgenetic pattern similar to that of GS cells. In line with these data, the relative abundance of the Igf2 mRNA transcript was two-fold higher and that of H19 was three fold lower than in control embryonic stem cells. The androgenetic DNA methylation pattern of the H19 ICR was maintained even after 54 passages. Furthermore, differentiating maGS cells from retinoic acid-treated embryoid bodies maintained the androgenetic imprinting pattern of the H19 ICR. Taken together these data suggest that our maGS cells are epigenetically stable for the H19 gene during in vitro modifications. Further studies on the epigenetic regulation and chromatin structure of maGS cells are therefore necessary before their full potential can be utilized in regenerative medicine.