Precise analysis of human CD34-negative (CD34 ؊ ) hematopoietic stem cells (HSCs) has been hindered by the lack of a simple and reliable assay system of these rare cells. Here, we successfully identify human cord blood-derived CD34 ؊ severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) technique. Lineage-negative (Lin ؊ ) CD34 ؊ cells did not show SRC activity by conventional tail-vein injection, possibly due to their low levels of homing receptor expression and poor SDF-1/CXCR4-mediated homing abilities, while they clearly showed a high SRC activity by IBMI. They generated CD34 ؉ progenies not only in the injected left tibia but also in other bones following migration. Moreover, they showed slower differentiating and reconstituting kinetics than CD34 ؉ cells in vivo. These in vivo-generated CD34 ؉ cells showed a distinct SRC activity after secondary transplantation, clearly indicating the long-term human cell repopulating capacity of our identified CD34 ؊ SRCs in nonobese diabetic (

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Recently, we have identified human cord blood (CB)-derived CD34-negative (CD34 ؊ ) severe combined immunodeficiency (SCID)-repopulating cells (SRCs STEM CELLS 2007;25:1348 -1355 Disclosure of potential conflicts of interest is found at the end of this article.

Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet induces hepatocellular carcinoma formation in rats. Previous studies have shown that promoter CpG islands in various cancer-related genes are aberrantly methylated in this model. Moreover, the global genome in methyl-donor-deficient diet fed rats contains a lesser amount of 5-methylcytosine than control livers. It is speculated that more than 90% of all 5-methylcytosines lie within the CpG islands of the transposons, including the long/short interspersed nucleotide elements (LINE and SINE). It is considered that the 5-methylcytosines in LINE-1 limit the ability of retrotransposons to be activated and transcribed; therefore, the extent of hypomethylation of LINE-1 could be a surrogate marker for aberrant methylation in other tumor-related genes as well as genome instability. Additionally, LINE-1 methylation status has been shown to be a good indicator of genome-wide methylation. In this study, we determined cytosine methylation status in the LINE-1 repetitive sequences of rats fed a choline-deficient (CD) diet for various durations and compared these with rats fed a choline-sufficient (CS) diet. The methylation status of LINE-1 was assessed by the combined bisulfite restriction analysis (COBRA) method, where the amount of bisulfite-modified and RsaI-cleaved DNA was quantified using gel electrophoresis. Progressive hypomethylation was observed in LINE-1 of CD livers as a function of feeding time; that is, the amount of cytosine in total cytosine (methylated and unmethylated) increased from 11.1% (1 week) to 19.3% (56 weeks), whereas in the control CS livers, it increased from 9.2% to 12.9%. Hypomethylation in tumor tissues was slightly higher (6%) than the nontumorous surrounding tissue. The present result also indicates that age is a factor influencing the extent of cytosine methylation.

We determined the DNA cytosine methylation status in the promoter CpG islands of eight cancer-related genes (p16,

BackgroundIdentification of carbapenemase-producing Enterobacteriaceae (CPE) in faecal specimens is challenging. This fact is particularly critical because low-level carbapenem-resistant organisms such as IMP-producing CPE are most prevalent in Japan. We developed a modified selective medium more suitable for IMP-type CPE.MethodsFifteen reference CPE strains producing different types of β-lactamases were used to evaluate the commercially available CHROMagar KPC and chromID CARBA as well as the newly prepared MC-ECC medium (CHROMagar ECC supplemented with meropenem, cloxacillin, and ZnSO4) and M-ECC medium (CHROMagar ECC supplemented with meropenem and ZnSO4). A total of 1035 clinical samples were then examined to detect CPE using chromID CARBA and M-ECC medium.ResultsAll tested strains producing NDM-, KPC-, and OXA-48-carbapenemases were successfully cultured in the media employed. Although most of the IMP-positive strains did not grow in CHROMagar KPC, chromID CARBA, or MC-ECC, all tested strains grew on M-ECC. When faecal samples were applied to the media, M-ECC medium allowed the best growth of IMP-type CPE with a significantly higher sensitivity (99.3%) than that of chromID CARBA (13.9%).ConclusionsM-ECC medium was determined as the most favourable selective medium for the detection of IMP-type CPE as well as other types of CPE.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-017-2312-1) contains supplementary material, which is available to authorized users.

Highlights 8.1% of COVID-19 patients were mechanically ventilated in the first outbreak in Osaka. The 30-day mortality rate of mechanically ventilated COVID-19 patients was 24.0%. Among these, 97% of patients were intubated within 14 days from clinical onset. Age ≥65 years and men were associated with a higher 30-day mortality rate. Among these, the median duration of viral RNA shedding from onset was 35 days.

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