Early outcome of Mitral Valve replacement: Results from Chordal Preservation at Muhimbiba National Hospital, Tanzania

No Abstrac

Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development

Chemotherapy-Induced Late Transgenerational Effects in Mice

To our knowledge, there is no report on long-term reproductive and developmental side effects in the offspring of mothers treated with a widely used chemotherapeutic drug such as doxorubicin (DXR), and neither is there information on transmission of any detrimental effects to several filial generations. Therefore, the purpose of the present paper was to examine the long-term effects of a single intraperitoneal injection of DXR on the reproductive and behavioral performance of adult female mice and their progeny. C57BL/6 female mice (generation zero; G0) were treated with either a single intraperitoneal injection of DXR (G0-DXR) or saline (G0-CON). Data were collected on multiple reproductive parameters and behavioral analysis for anxiety, despair and depression. In addition, the reproductive capacity and health of the subsequent six generations were evaluated. G0-DXR females developed despair-like behaviors; delivery complications; decreased primordial follicle pool; and early lost of reproductive capacity. Surprisingly, the DXR-induced effects in oocytes were transmitted transgenerationally; the most striking effects being observed in G4 and G6, constituting: increased rates of neonatal death; physical malformations; chromosomal abnormalities (particularly deletions on chromosome 10); and death of mothers due to delivery complications. None of these effects were seen in control females of the same generations. Long-term effects of DXR in female mice and their offspring can be attributed to genetic alterations or cell-killing events in oocytes or, presumably, to toxicosis in non-ovarian tissues. Results from the rodent model emphasize the need for retrospective and long-term prospective studies of survivors of cancer treatment and their offspring

Performance of the CMS High Granularity Calorimeter prototype to charged pion beams of 20−-300 GeV/c

The upgrade of the CMS experiment for the high luminosity operation of the LHC comprises the replacement of the current endcap calorimeter by a high granularity sampling calorimeter (HGCAL). The electromagnetic section of the HGCAL is based on silicon sensors interspersed between lead and copper (or copper tungsten) absorbers. The hadronic section uses layers of stainless steel as an absorbing medium and silicon sensors as an active medium in the regions of high radiation exposure, and scintillator tiles directly readout by silicon photomultipliers in the remaining regions. As part of the development of the detector and its readout electronic components, a section of a silicon-based HGCAL prototype detector along with a section of the CALICE AHCAL prototype was exposed to muons, electrons and charged pions in beam test experiments at the H2 beamline at the CERN SPS in October 2018. The AHCAL uses the same technology as foreseen for the HGCAL but with much finer longitudinal segmentation. The performance of the calorimeters in terms of energy response and resolution, longitudinal and transverse shower profiles is studied using negatively charged pions, and is compared to GEANT4 predictions. This is the first report summarizing results of hadronic showers measured by the HGCAL prototype using beam test data.Comment: To be submitted to JINS

The Drosophila speciation factor HMR localizes to genomic insulator sites

Hybrid incompatibility between Drosophila melanogaster and D. simulans is caused by a lethal interaction of the proteins encoded by the Hmr and Lhr genes. In D. melanogaster the loss of HMR results in mitotic defects, an increase in transcription of transposable elements and a deregulation of heterochromatic genes. To better understand the molecular mechanisms that mediate HMR's function, we measured genome-wide localization of HMR in D. melanogaster tissue culture cells by chromatin immunoprecipitation. Interestingly, we find HMR localizing to genomic insulator sites that can be classified into two groups. One group belongs to gypsy insulators and another one borders HP1a bound regions at active genes. The transcription of the latter group genes is strongly affected in larvae and ovaries of Hmr mutant flies. Our data suggest a novel link between HMR and insulator proteins, a finding that implicates a potential role for genome organization in the formation of species

The <i>Hmr</i> and <i>Lhr</i> Hybrid Incompatibility Genes Suppress a Broad Range of Heterochromatic Repeats

<div><p>Hybrid incompatibilities (HIs) cause reproductive isolation between species and thus contribute to speciation. Several HI genes encode adaptively evolving proteins that localize to or interact with heterochromatin, suggesting that HIs may result from co-evolution with rapidly evolving heterochromatic DNA. Little is known, however, about the intraspecific function of these HI genes, the specific sequences they interact with, or the evolutionary forces that drive their divergence. The genes <i>Hmr</i> and <i>Lhr</i> genetically interact to cause hybrid lethality between <i>Drosophila melanogaster</i> and <i>D. simulans</i>, yet mutations in both genes are viable. Here, we report that <i>Hmr</i> and <i>Lhr</i> encode proteins that form a heterochromatic complex with Heterochromatin Protein 1 (HP1a). Using RNA-Seq analyses we discovered that <i>Hmr</i> and <i>Lhr</i> are required to repress transcripts from satellite DNAs and many families of transposable elements (TEs). By comparing <i>Hmr</i> and <i>Lhr</i> function between <i>D. melanogaster</i> and <i>D. simulans</i> we identify several satellite DNAs and TEs that are differentially regulated between the species. <i>Hmr</i> and <i>Lhr</i> mutations also cause massive overexpression of telomeric TEs and significant telomere lengthening. <i>Hmr</i> and <i>Lhr</i> therefore regulate three types of heterochromatic sequences that are responsible for the significant differences in genome size and structure between <i>D. melanogaster</i> and <i>D. simulans</i> and have high potential to cause genetic conflicts with host fitness. We further find that many TEs are overexpressed in hybrids but that those specifically mis-expressed in lethal hybrids do not closely correlate with <i>Hmr</i> function. Our results therefore argue that adaptive divergence of heterochromatin proteins in response to repetitive DNAs is an important underlying force driving the evolution of hybrid incompatibility genes, but that hybrid lethality likely results from novel epistatic genetic interactions that are distinct to the hybrid background.</p></div

Hmr forms a complex with Lhr and HP1a and is required to stabilize Lhr.

<p>(A) mel-Hmr-HA (green) colocalizes with HP1a (top) and H3k9me2 (middle; both red) in nuclear cycle 14 embryos. The HP1a costain is in a <i>mel-Hmr-HA</i> background, while the H3k9me2 costain is in a <i>Hmr³; mel-Hmr-HA</i> background. A negative control shows no HA signal in <i>w¹¹¹⁸</i> embryos lacking the <i>mel-Hmr-HA</i> transgene (bottom). Scale bars represent 10 µm. (B) mel-Hmr-HA (green) colocalizes with 2L3L, dodeca and GA-rich satellites but not with the 359 bp repeat satellite in <i>mel-Hmr-HA</i> (all FISH probes red). Scale bars represent 5 µm. (C) mel-Hmr-HA (red) colocalizes with the nucleolar marker Fibrillarin (green) in <i>mel-Hmr-HA</i> early embryos. Scale bars represent 10 µm. (D) mel-Lhr-HA and mel-Hmr-FLAG co-immunoprecipitate from <i>D. melanogaster</i> embryo extracts derived from flies expressing both transgenes (left 4 lanes) but not from flies expressing only Lhr-HA (right 4 lanes). Extracts were IP'd with the indicated antibodies, and then probed by Western Blots (WB) with the same or different antibodies. (E) Lhr-HA, Hmr-FLAG and HP1a co-immunoprecipitation from embryo extracts. Specificity is indicated by lack of immunoprecipitation of histone H3. Asterisk indicates the antibody light chain. (F) Lhr and Hmr interact in a yeast-two hybrid assay. Interactions were detected by growth on complete media (CM) lacking histidine (his); growth controls were performed on CM lacking tryptophan (trp) and leucine (leu). The top 4 panels test for interactions between orthologs from the same species; the bottom 4 between heterospecific orthologs. AD, activation domain; BD, DNA binding domain. (G) Lhr-HA is detectable in <i>Hmr³</i> and localizes to heterochromatin, as indicated by co-localization with HP1a. Note that a higher gain was used in the <i>Hmr³</i> panels compared to the <i>Hmr⁺</i> panels in order to detect Lhr-HA, and is reflected in the higher background. Western blots confirm that Lhr-HA levels are reduced in <i>Hmr³</i>. HP1a is used as a loading control. (H) Hmr-HA maintains its localization to DAPI-dense heterochromatin in <i>Lhr^KO; Hmr-HA</i> embryos. Scale bars represent 10 µm.</p