Microbial mat compositions and localization patterns explain the virulence of black band disease in corals

Black band disease (BBD) in corals is characterized by a distinctive, band-like microbial mat, which spreads across the tissues and often kills infected colonies. The microbial mat is dominated by cyanobacteria but also commonly contains sulfide-oxidizing bacteria (SOB), sulfate-reducing bacteria (SRB), and other microbes. The migration rate in BBD varies across different environmental conditions, including temperature, light, and pH. However, whether variations in the migration rates reflect differences in the microbial consortium within the BBD mat remains unknown. Here, we show that the micro-scale surface structure, bacterial composition, and spatial distribution differed across BBD lesions with different migration rates. The migration rate was positively correlated with the relative abundance of potential SOBs belonging to Arcobacteraceae localized in the middle layer within the mat and negatively correlated with the relative abundance of other potential SOBs belonging to Rhodobacteraceae. Our study highlights the microbial composition in BBD as an important determinant of virulence

Isolation and characterization of the TIGA genes, whose transcripts are induced by growth arrest

We report here the isolation of 44 genes that are upregulated after serum starvation and/or contact inhibition. These genes have been termed TIGA, after Transcript Induced by Growth Arrest. We found that there are two kinds of G0 phases caused by serum starvation, namely, the shallow G0 (or G0/G1) and the deep G0 phases. The shallow G0 is induced by only a few hours of serum starvation, while deep G0 is generated after 3 days of serum starvation. We propose that mammalian cells enter deep G0 through a G0 gate, which is only opened on the third day of serum starvation. TIGA1, one of the uncharacterized TIGA genes, encodes a homolog of cyanate permease of bacteria and localizes in mitochondria. This suggests that Tiga1 is involved in the inorganic ion transport and metabolism needed to maintain the deep G0 phase. Ectopic expression of TIGA1 inhibited not only tumor cell proliferation but also anchorage-independent growth of cancer cell lines. A microsatellite marker, ENDL-1, allowed us to detect loss of heterozygosity around the TIGA1 gene region (5q21–22). Further analysis of the TIGA genes we have identified here may help us to better understand the mechanisms that regulate the G0 phase

Measurement of serum hepcidin-25 levels as a potential test for diagnosing hemochromatosis and related disorders

石川県立中央病院金沢大学医薬保健研究域医学系Iron overload syndromes include a wide spectrum of genetic and acquired conditions. Recent studies suggest suppressed hepcidin synthesis in the liver to be the molecular basis of hemochromatosis. However, a liver with acquired iron overload synthesizes an adequate amount of hepcidin. Thus, hepcidin could function as a biochemical marker for differential diagnosis of iron overload syndromes. Methods We measured serum iron parameters and hepcidin- 25 levels followed by sequencing HFE, HJV, HAMP, TFR2, and SLC40A1 genes in 13 Japanese patients with iron overload syndromes. In addition, we performed direct measurement of serum hepcidin-25 levels using liquid chromatography-tandem mass spectrometry in 3 Japanese patients with aceruloplasminemia and 4 Italians with HFE hemochromatosis. Results One patient with HJV hemochromatosis, 2 with TFR2 hemochromatosis, and 3 with ferroportin disease were found among the 13 Japanese patients. The remaining 7 Japanese patients showed no evidence for genetic basis of iron overload syndrome. As far as the serum hepcidin-25 was concerned, seven patients with hemochromatosis and 3 with aceruloplasminemia showed markedly decreased serum hepcidin-25 levels. In contrast, 3 patients with ferroportin disease and 7 with secondary iron overload syndromes showed serum hepcidin levels parallel to their hyperferritinemia. Patients with iron overload syndromes were divided into 2 phenotypes presenting as low and high hepcidinemia. These were then associated with their genotypes. Conclusion Determining serum hepcidin-25 levels may aid differential diagnosis of iron overload syndromes prior to genetic analysis. © Springer 2010

Enhanced generation of iPSCs from older adult human cells by a synthetic five-factor self-replicative RNA

<div><p>We previously devised a polycistronic, synthetic self-replicating RNA (srRNA) to generate human induced Pluripotent Stem Cells (iPSCs) that simultaneously expresses four reprogramming factors (4F). However, while the best 4F srRNA efficiently generated iPSCs from young fibroblasts, it was inefficient on adult human fibroblasts (>50 years). To increase the iPSC generation efficiency, we included additional reprogramming factors. We found that a single transfection of a five factor (5F) srRNA, containing <i>OCT4</i>, <i>KLF4</i>, <i>SOX2</i>, <i>GLIS1</i> and <i>c-MYC</i>, robustly generated iPSCs from adult human fibroblasts aged 54 to 77 and from a 24 year old cardiomyopathy patient donor. Interestingly, 5F-srRNA induced <i>LIN28A</i>, which was one of the original reprogramming factors. 5F-srRNA also accelerated the generation of iPSCs by seven days compared to 4F-srRNAs. Further improvements include phosphatase treatment to remove 5' phosphate and use of Lipofectamine MessengerMAX that increased transfection efficiency to ~90%. Together, these improvements enabled us to efficiently generate iPSCs from human fibroblasts using 5F-srRNA while eliminating both puromycin selection and feeder cells.</p></div

5F-srRNA generation of iPSCs by messengerMAX transfection in BJ cells.

<p>5F-srRNA generation of iPSCs by messengerMAX transfection in BJ cells.</p

Comparison of iPSC generation with 4F-srRNA, 5F-srRNA and 6F-srRNA.

<p>A: iPSC generation in BJ cells. Cell images on day 7, 10, and 21. ES culture medium was used starting on day 7. Scale bar, 100 μm. B: iPSC generation in BJ cells. iPSC colonies were stained with Alkaline Phosphatase (AP) on day 21 and 28. TRA-1-60 and SSEA4 staining was performed after AP staining. On day 10, a starting well was passaged to 3 wells for OKS-iM, 2 wells for OKS-iG and 5F-srRNA. C: Day 21 iPSC colonies from adult human fibroblasts, FB#31, FB#32 and FB#33, from 5F-srRNA were stained with AP and TRA-1-60. FB#31, 55 year old healthy male donor, FB#32: 54 year old healthy female donor, FB#33: 24 year old cardiomyopathy male donor. D: Day 28 iPSC colonies from adult human fibroblasts, FB#31, FB#32 and FB#33, were stained with AP and TRA-1-60. A starting well was passaged to 3 wells for OKS-iM, 1 well for OKS-iG and 2 wells for 5F-srRNA. E: iPSC generation in feeder-free conditions. iPSC colonies were generated on Laminin- 521 or Matrigel. BJ cells were co-transfected with 5F-srRNA plus B18R mRNA (1:1 ratio), and selected with puromycin, then passaged onto Laminin-521 or Matrigel. iPSC colonies were stained with AP and TRA-1-60 on day 21. Scale bar, 100 μm. F: iPSC generation without puromycin selection. BJ cells were co-transfected with 5F-srRNA plus B18R mRNA (1:1 ratio), then on day 5, one well was passaged into 6 wells. AP staining and TRA-1-60, TRA-1-80, or SSEA4 staining were performed on day 24. The numbers of AP positive colonies per starting well were counted and are summarized in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182018#pone.0182018.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182018#pone.0182018.t002" target="_blank">2</a>.</p

Construction and expression of srRNA.

<p>A: Scheme of Self-Replicating srRNA constructs. B: Expression of reprogramming factors in OKS-iM 4F-srRNA, OKS-iG 4F-srRNA, OKS-iGM 5F-srRNA, and OKS-iGML 6F-srRNA on day 2 (left) and day 10 (right). BJ cells were co-transfected with srRNAs plus B18R mRNA (1:1 srRNA:B18R mRNA), selected with puromycin and cultured in Advanced DMEM/10% FBS for 10 days. C: qRT-PCR analysis of <i>LIN28A</i> and <i>NANOG</i> from 4F-srRNA and 5F-srRNA transfected BJ cells normalized to GAPDH. Cells were selected with puromycin after the transfection, and cultured in Advanced DMEM/10% FBS (Ad) or ES medium. Cells were collected on day 2, 4, 6, 8 and 10 for 5F-srRNA, and day 10 for 4F-srRNA (OKS-iM and OKS-iG). D: Immunoblot analysis of LIN28A and NANOG in 5F-srRNA transfected BJ cells on day 6 and 10. iPSC: iPSC clone generated with 5F-srRNA from BJ cells.</p

5F-srRNA iPSC generation with messengerMax transfection.

<p>A: Comparison of Lipofectamine 2000 and MessengerMax transfection efficiency. GFP srRNA was co-transfected with B18R mRNA (1: 1 ratio) into BJ cells and GFP expression was measured by flow cytometry on day 2. B: 5F-srRNA was co-transfected with B18R mRNA (1:1 ratio) into BJ cells, and selected for puromycin for 4 days (Day 5). Scale bar, 200 μm. C: Comparison of iPSC generation with Lipofectamine-2000 and MessengerMax transfection. iPSC colonies were generated, and stained with AP, TRA-1-60, SSEA4 and SSEA1, as indicated.</p

Characterization of iPSC clones from 5F-srRNA.

<p>A: qRT-PCR analysis of ES cells markers on 5F-srRNA iPSC clones. Expression of ES marker genes were normalized to GAPDH. B: Immunofluorescence analysis of ES markers in 5F-srRNA iPSC clones from BJ, FB#31, FB#32 and FB#33. Scale bar, 100 mm. C: Teratoma formation of 5F-srRNA iPSC clones. iPSC clones were injected intramuscular or subcutaneous into NRG mice. Teratomas were allowed to form for 4–6 months after injection. Four of five BJ-5F-iPSC clones (#1, 3, 4, 5), three of three FB#31-5F-iPSC clones (#1, 2, 3), and one of two FB#33-5F-iPSC clones (#3) generated teratomas. Cartilage (a, b, c), muscle (d, e, f), neural tissue (g), pigmented epithelium (h), squamous epithelium (i), intestinal epithelium (j, f), and respiratory epithelium (k). Scale bar, 50 μm. D: qRT-PCR for srRNA (nsP1 region) was performed on FB#31, FB#32, and FB #33 -5F-srRNA iPSC clones. Total RNA was isolated from passage 5 (P5) cells. * = no detected amplification.</p