Fluorinated organic compounds (FOCs), such as perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), and perfluorooctane sulfonylamide (PFOSA), are widely used in the manufacture of plastic, electronics, textile, and construction material in the apparel, leather, and upholstery industries. FOCs have been detected in human blood samples. Studies have indicated that FOCs may be detrimental to rodent development possibly by affecting thyroid hormone levels. In the present study, we determined the concentrations of FOCs in maternal and cord blood samples. Pregnant women 17–37 years of age were enrolled as subjects. FOCs in 15 pairs of maternal and cord blood samples were analyzed by liquid chromatography–electrospray mass spectrometry coupled with online extraction. The limits of quantification of PFOS, PFOA, and PFOSA in human plasma or serum were 0.5, 0.5, and 1.0 ng/mL, respectively. The method enables the precise determination of FOCs and can be applied to the detection of FOCs in human blood samples for monitoring human exposure. PFOS concentrations in maternal samples ranged from 4.9 to 17.6 ng/mL, whereas those in fetal samples ranged from 1.6 to 5.3 ng/mL. In contrast, PFOSA was not detected in fetal or maternal samples, whereas PFOA was detected only in maternal samples (range, < 0.5 to 2.3 ng/mL, 4 of 15). Our results revealed a high correlation between PFOS concentrations in maternal and cord blood (r2 = 0.876). However, we did not find any significant correlations between PFOS concentration in maternal and cord blood samples and age bracket, birth weight, or levels of thyroid-stimulating hormone or free thyroxine. Our study revealed that human fetuses in Japan may be exposed to relatively high levels of FOCs. Further investigation is required to determine the postnatal effects of fetal exposure to FOCs.

A biocleavable polyrotaxane, having a necklace-like structure consisting of many cationic alpha-cyclodextrins (alpha-CDs) and a disulfide-introduced poly(ethylene glycol) (PEG), was synthesized and examined as a nonviral gene carrier. The polyrotaxane formed a stable polyplex having positively charged surface even at low charge ratio. This is likely to be due to structural factors of the polyrotaxane, such as the mobile motion of alpha-CDs in the necklace-like structure. Rapid endosomal escape was observed 90 min after transfection. The positively charged surface and the good buffering capacity are advantageous to show the proton sponge effect. The pDNA decondensation occurred through disulfide cleavage of the polyrotaxane and subsequent supramolecular dissociation of the noncovalent linkages between alpha-CDs and PEG. Transfection of the DMAE-SS-PRX polyplex is independent of the amount of free polycation. Those properties played a key role for delivery of pDNA clusters to the nucleus. Therefore, the polyplex nature and the supramolecular dissociation of the polyrotaxane contributed to the enhanced gene delivery.

BackgroundPerfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are man-made, ubiquitous, and persistent contaminants in the environment, wildlife, and humans. Although recent studies have shown that these chemicals interfere with fetal growth in humans, the results are inconsistent.ObjectivesOur goal was to investigate the correlation between relatively low levels of PFOS and PFOA in maternal serum and birth weight and birth size.MethodsWe conducted a hospital-based prospective cohort study between July 2002 and October 2005 in Sapporo, Japan. A total of 428 women and their infants were involved in the study. We obtained characteristics of the mothers and infants from self-administered questionnaire surveys and from medical records. We analyzed maternal serum samples for PFOS and PFOA by liquid chromatography–tandem mass spectrometry (LC/MS/MS).ResultsAfter adjusting for confounding factors, PFOS levels negatively correlated with birth weight [per log10 unit: β = −148.8 g; 95% confidence interval (CI), −297.0 to −0.5 g]. In addition, analyses stratified by sex revealed that PFOS levels negatively correlated with birth weight only in female infants (per log10 unit: β = −269.4 g; 95% CI, −465.7 to −73.0 g). However, we observed no correlation between PFOA levels and birth weight.ConclusionOur results indicate that in utero exposure to relatively low levels of PFOS was negatively correlated with birth weight.

The precise and conceptual insight of circulating endothelial progenitor cell (EPC) kinetics is hampered by the absence of an assay system capable of evaluating the EPC differentiation cascade. An assay system for EPC colony formation was developed to delineate circulating EPC differentiation. EPC colony-forming assay using semisolid medium and single or bulk CD133؉ cells from umbilical cord blood exhibited the formation of two types of attaching cell colonies made of small or large cells featuring endothelial lineage potential and properties, termed small EPC colony-forming units and large EPC colony-forming units, respectively. In vitro and in vivo assays of each EPC colony-forming unit cell revealed a differentiation hierarchy from small EPC to large EPC colonies, indicating a primitive EPC stage with highly proliferative activity and a definitive EPC stage with vasculogenic properties, respectively. Experimental comparison with a conventional EPC culture assay system disclosed EPC colony-forming unit cells differentiate into noncolony-forming early EPC. The fate analysis of single CD133؉ cells into the endothelial and hematopoietic lineage was achieved by combining this assay system with a hematopoietic progenitor assay and demonstrated the development of colony-forming EPC and hematopoietic progenitor cells from a single hematopoietic stem cell. EPC colony-forming assay permits the determination of circulating EPC kinetics from single or bulk cells, based on the evaluation of hierarchical EPC colony formation. This assay further enables a proper exploration of possible links between the origin of EPC and hematopoietic stem cells, representing a novel and powerful tool to investigate the molecular signaling pathways involved in EPC biology. (Circ Res. 2011;109:20-37.) Key Words: clonogenic assay Ⅲ differentiation Ⅲ endothelial progenitor cell Ⅲ vasculogenesis D espite significant efforts in research and development with respect to endothelial progenitor cell (EPC) biology during the past 10 years after their initial isolation, 1 EPC remain a controversial topic among researchers because there is no definitive delineation of EPC, no clear differentiation hierarchy, or any unambiguously defined isolation protocol.EPC have been quantified and qualified either as cell populations identified by cell surface markers such as CD34, CD133, vascular endothelial growth factor receptor-2 (VEGFR-2), [1][2][3][4][5][6][7][8] or as adhesive cells 6,9,10 and colonies 11 using conventional EPC culture methods to produce spindle-shape adherent cells from peripheral blood (PB), bone marrow (BM), or umbilical cord blood (UCB) mononuclear cells (MNC) with endothelial growth factors and cytokines. These assays using conventional EPC culture protocols were simple and satisfactory to speculate on the vasculogenic properties of EPC-enriched fractions but have recently been criticized. These assays further group heterogeneous EPC into one qualitative category: "adhesive cultured EPC" without any hierarchical discrimination ...

To develop nonviral gene vectors that are sufficient for clinical application, it is necessary to understand why and to what extent nonviral vectors are inferior to viral vectors, which in general show a more efficient transfection activity. This study describes a systematic and quantitative comparison of the cellular uptake and subsequent intracellular distribution (e.g., endosome/lysosome, cytosol, and nucleus) of exogenous DNA transfected by viral and nonviral vectors in living cells, using a combination of TaqMan PCR and a recently developed confocal image-assisted three-dimensionally integrated quantification method. As a model, adenovirus (Ad) and Lipofectamine Plus (LFN) were used for comparison since they are highly potent and widely used viral and nonviral vectors, respectively. The findings indicate that the efficiency of cellular uptake for LFN is significantly higher than that for Ad. Once taken up by a cell, Ad exhibited comparable endosomal escape and slightly higher nuclear transfer efficiency compared with LFN. In contrast, LFN requires 3 orders of magnitude more intranuclear gene copies to exhibit a transgene expression comparable to that of the Ad, suggesting that the difference in transfection efficiency principally arises from differences in nuclear transcription efficiency and not from a difference in intracellular trafficking between Ad and LFN.

Background and Aims Growing evidence suggests that microRNAs coordinate various biological processes in the liver. We describe experiments to address the physiological roles of these new regulators of gene expression in the liver that are as of yet largely undefined. Methods We disrupted Dicer, an enzyme essential for the processing of microRNAs, in hepatocytes using a conditional knockout mouse model to elucidate the consequences of loss of microRNAs. Results The conditional knockout mouse livers showed the efficient disruption of Dicer1 at 3 weeks after birth. This resulted in prominent steatosis and the depletion of glycogen storage. Dicer1-deficient liver exhibited increased growth-promoting gene expression and the robust expression of fetal stage-specific genes. The consequence of Dicer elimination included both increased hepatocyte proliferation as well as overwhelming apoptosis. Over time, Dicer1-expressing wild-type hepatocytes that had escaped Cre-mediated recombination progressively repopulated the entire liver. Unexpectedly, however, two-third of the mutant mice spontaneously developed hepatocellular carcinomas derived from residual Dicer1-deficient hepatocytes at 1 year old. Conclusions Dicer and microRNAs have critical roles in hepatocyte survival, metabolism, developmental gene regulation and tumor suppression in the liver. Loss of Dicer primarily impairs hepatocyte survival, but can promote hepatocarcinogenesis in cooperation with additional oncogenic stimuli.

Since endosomal escape and the nuclear delivery of plasmid DNA (pDNA) constitute major barriers for transgene expression, a quantitative evaluation of intracellular trafficking of pDNA would be highly desirable in terms of optimizing a nonviral gene delivery system. In the present study, a novel strategy is proposed for the quantification of rhodamine-labeled pDNA in endosomes/lysosomes, cytosol, and nucleus. Endosomes/lysosomes and nucleus were stained with LysoSensor DND-189 and Hoechst 33258, respectively, to distinguish them from the cytosol. The pixel areas of the clusters derived from the rhodamine were used as an index for the amount of pDNA. This approach was applied to the analysis of the intracellular trafficking of pDNA transfected by LipofectAMINE PLUS, stearylated octaarginine (STR-R8), and octaarginine (R8). In the case of R8, most of the pDNA was trapped by endosomes/lysosomes. STR-R8 exhibited endosomal escape followed by nuclear translocation in a time-dependent manner. LipofectAMINE PLUS was the most effective in rapidly delivering pDNA to the nucleus as well as the cytosol. These differences in the intracellular trafficking of pDNA correlated well with the transgene expression. Therefore, this method enables the quantitative analysis of the intracellular pharmacokinetics of pDNA and promises to provide useful information for optimizing nonviral gene delivery systems.

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