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The H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG–binding domain protein 1 MBD1 as a physical and functional partner of the H19 long noncoding RNA (lncRNA). The H19 lncRNA–MBD1 complex is required for the control of five genes of the IGN. For three of these genes— Igf2 (insulin-like growth factor 2), Slc38a4 (solute carrier family 38 member 4), and Peg1 (paternally expressed gene 1)—both MBD1 and H3K9me3 binding were detected on their differentially methylated regions. The H19 lncRNA–MBD1 complex, through its interaction with histone lysine methyltransferases, therefore acts by bringing repressive histone marks on the differentially methylated regions of these three direct targets of the H19 gene. Our data suggest that, besides the differential DNA methylation found on the differentially methylated regions of imprinted genes, an additional fine tuning of the expressed allele is achieved by a modulation of the H3K9me3 marks, mediated by the association of the H19 lncRNA with chromatin-modifying complexes, such as MBD1. This results in a precise control of the level of expression of growth factors in the embryo.

Background Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. Results We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. Conclusions We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

The H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG–binding domain protein 1 MBD1 as a physical and functional partner of the H19 long noncoding RNA (IncRNA). The H19 IncRNA–MBD1 complex is required for the control of five genes of the IGN. For three of these genes—Igf2 (insulin-like growth factor 2), Slc38a4 (solute carrier family 38 member 4), and Peg1 (paternally expressed gene 1)—both MBD1 and H3K9me3 binding were detected on their differentially methylated regions. The H19 IncRNA–MBD1 complex, through its interaction with histone lysine methyltransferases, therefore acts by bringing repressive histone marks on the differentially methylated regions of these three direct targets of the H19 gene. Our data suggest that, besides the differential DNA methylation found on the differentially methylated regions of imprinted genes, an additional fine tuning of the expressed allele is achieved by a modulation of the H3K9me3 marks, mediated by the association of the H19 IncRNA with chromatin-modifying complexes, such as MBD1. This results in a precise control of the level of expression of growth factors in the embryo.

Doc number: P54

BackgroundVentilator-associated pneumonia (VAP) caused by Stenotrophomonas maltophilia is poorly described in the literature. However, it has been shown to be associated with increased morbidity and mortality. Probabilistic antibiotic therapy against S. maltophilia is often ineffective as this pathogen is resistant to many antibiotics. There is no consensus at present on the best therapeutic strategy to adopt (class of antibiotics, antibiotic combination, dosage, treatment duration). The aim of this study was to evaluate the effect of antibiotic therapy strategy on the prognosis of patients with VAP caused by S. maltophilia.ResultsThis retrospective study evaluated all consecutive patients who developed VAP caused by S. maltophilia between 2010 and 2018 while hospitalized in the intensive care unit (ICU) of a French university hospital in Reunion Island, in the Indian Ocean region. A total of 130 patients with a median Simplified Acute Physiology Score II of 58 [43–73] had VAP caused by S. maltophilia after a median duration of mechanical ventilation of 12 [5–18] days. Ventilator-associated pneumonia was polymicrobial in 44.6% of cases, and ICU mortality was 50.0%. After multivariate Cox regression analysis, the factors associated with increased ICU mortality were older age (hazard ratio (HR): 1.03; 95% CI 1.01–1.04, p = 0.001) and high Sequential Organ Failure Assessment score on the day of VAP onset (HR: 1.08; 95% CI 1.03–1.14, p = 0.002).Appropriate antibiotic therapy, and in particular trimethoprim–sulfamethoxazole, was associated with decreased ICU mortality (HR: 0.42; 95% CI 0.24–0.74, p = 0.003) and decreased hospital mortality (HR: 0.47; 95% CI 0.28–0.79, p = 0.04).Time to start of appropriate antibiotic therapy, combination therapy, and duration of appropriate antibiotic therapy had no effect on ICU mortality (p > 0.5).ConclusionIn our study, appropriate antibiotic therapy, and in particular trimethoprim–sulfamethoxazole, was associated with decreased ICU and hospital mortality in patients with VAP caused by S. maltophilia.