IXPE Mirror Module Assemblies

Expected to launch in 2021 Spring, the Imaging X-ray Polarimetry Explorer (IXPE) is a NASA Astrophysics Small Explorer Mission with significant contributions from the Italian space agency (ASI). The IXPE observatory features three identical x-ray telescopes, each comprised of a 4-m-focal-length mirror module assembly (MMA, provided by NASA Marshall Space Flight Center) that focuses x rays onto a polarization-sensitive, imaging detector (contributed by ASI-funded institutions). This paper summarizes the MMAs design, fabrication, alignment and assembly, expected performance, and calibration plans

RUNX/AML and C/EBP factors regulate CD11a integrin expression in myeloid cells through overlapping regulatory elements

The CD11a/CD18 (leukocyte functionassociated antigen 1 [LFA-1]) integrin mediates critical leukocyte adhesive interactions during immune and inflammatory responses. The CD11a promoter directs CD11a/CD18 integrin expression, and its activity in lymphoid cells depends on a functional RUNX1/AML-1–binding site (AML-110) within the MS7 sequence. We now report that MS7 contains a C/EBPbinding site (C/EBP-100), which overlaps with AML-110 and is bound by C/EBP factors in myeloid cells. C/EBP and RUNX/ AML factors compete for binding to their respective cognate elements and bind to the CD11a promoter MS7 sequence in a cell lineage- and differentiation-dependent manner. In myeloid cells MS7 is primarily recognized by C/EBP factors in proliferating cells whereas RUNX/AMLfactors (especially RUNX3/AML-2) bind to MS7 in differentiated cells. RUNX3/AML-2 binding to the CD11a promoter correlates with increased RUNX3/AML-2 protein levels and enhanced CD11a/CD18 cell surface expression. The relevance of the AML-110 element is underscored by the ability of AML-1/ETO to inhibit CD11a promoter activity, thus explaining the low CD11a/CD18 expression in t(8;21)–containing myeloid leukemia cells. Therefore, the expression of the CD11a/CD18 integrin in myeloid cells is determined through the differential occupancy of the CD11a proximal promoter by transcription factors implicated in the pathogenesis of myeloid leukemia

Otitis media in the Tgif knockout mouse implicates TGFβ signalling in chronic middle ear inflammatory disease

Otitis media with effusion (OME) is the most common cause of hearing loss in children and tympanostomy to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of OM are known to have a very significant genetic component, however, until recently little was known of the underlying genes involved. The identification of mouse models of chronic OM has indicated a role of transforming growth factor beta (TGFβ) signalling and its impact on responses to hypoxia in the inflamed middle ear. We have, therefore, investigated the role of TGFβ signalling and identified and characterized a new model of chronic OM carrying a mutation in the gene for transforming growth interacting factor 1 (Tgif1). Tgif1 homozygous mutant mice have significantly raised auditory thresholds due to a conductive deafness arising from a chronic effusion starting at around 3 weeks of age. The OM is accompanied by a significant thickening of the middle ear mucosa lining, expansion of mucin-secreting goblet cell populations and raised levels of vascular endothelial growth factor, TNF-α and IL-1β in ear fluids. We also identified downstream effects on TGFβ signalling in middle ear epithelia at the time of development of chronic OM. Both phosphorylated SMAD2 and p21 levels were lowered in the homozygous mutant, demonstrating a suppression of the TGFβ pathway. The identification and characterization of the Tgif mutant supports the role of TGFβ signalling in the development of chronic OM and provides an important candidate gene for genetic studies in the human population

Roles of Coactivators in Hypoxic Induction of the Erythropoietin Gene

Hypoxia-inducible expression of the erythropoietin (EPO) gene is mediated principally by hypoxia-inducible factor 2alpha (HIF-2alpha) in Hep3B cells under physiologic conditions. How/whether p300/CBP and the members of p160 coactivator family potentiate hypoxic induction of endogenous EPO and other HIF-2alpha and hypoxia-inducible factor 1alpha (HIF-1alpha) target genes remains unclear.We demonstrate, using chromatin immunoprecipitation (ChIP) analysis, that the histone acetyl transferase (HAT) coactivators p300, SRC-1 and SRC-3 are recruited to the 3' enhancer of the EPO gene upon hypoxic stimulation, and that each associates with the enhancer in a periodic fashion. Hypoxia induced acetylation of the EPO gene 5' promoter at histone 4 and lysine 23 of histone 3. Knocking down SRC-3, but not SRC-1 or SRC-2, using short interfering RNAs (siRNAs), reduced EPO transcriptional activity. Knocking down p300 resulted in dramatic down-regulation of hypoxic stimulation of EPO gene transcription, negated recruitment of RNA polymerase II to the gene's promoter, and eliminated hypoxia-stimulated acetylation at the promoter and recruitments of SRC-1 and SRC-3 to the enhancer. The inhibitory effects of knocking down p300 and the chromatin remodeling coactivator, Brm/Brg-1, on EPO transcription were additive, suggesting that p300 and Brm/Brg-1 act independently. p300 was also required for hypoxia induced transcription of the HIF-1alpha target gene, VEGF, but was dispensable for induction of two other HIF-1alpha target genes, PGK and LDHA. Knocking down CBP, a homolog of p300, augmented hypoxic induction of VEGF, LDHA and PGK. Different HIF target genes also exhibited different requirements for members of the p160 coactivator family.p300 plays a central coactivator role in hypoxic induction of EPO. The coactivators exhibit different specificities for different HIF target genes and each can behave differently in transcriptional regulation of different target genes mediated by the same transcription factor

Targeting the tumor mutanome for personalized vaccination in a TMB low non-small cell lung cancer.

BackgroundCancer is characterized by an accumulation of somatic mutations, of which a significant subset can generate cancer-specific neoepitopes that are recognized by autologous T cells. Such neoepitopes are emerging as important targets for cancer immunotherapy, including personalized cancer vaccination strategies.MethodsWe used whole-exome and RNA sequencing analysis to identify potential neoantigens for a patient with non-small cell lung cancer. Thereafter, we assessed the autologous T-cell reactivity to the candidate neoantigens using a long peptide approach in a cultured interferon gamma ELISpot and tracked the neoantigen-specific T-cells in the tumor by T-cell receptor (TCR) sequencing. In parallel, identified gene variants were incorporated into a Modified Vaccinia Ankara-based vaccine, which was evaluated in the human leucocyte antigen A*0201 transgenic mouse model (HHD).ResultsSequencing revealed a tumor with a low mutational burden: 2219 sequence variants were identified from the primary tumor, of which 23 were expressed in the transcriptome, involving 18 gene products. We could demonstrate spontaneous T-cell responses to 5/18 (28%) mutated gene variants, and further analysis of the TCR repertoire of neoantigen-specific CD4+ and CD8+ T cells revealed TCR clonotypes that were expanded in both blood and tumor tissue. Following vaccination of HHD mice, de novo T-cell responses were generated to 4/18 (22%) mutated gene variants; T cells reactive against two variants were also evident in the autologous setting. Subsequently, we determined the major histocompatibility complex restriction of the T-cell responses and used in silico prediction tools to determine the likely neoepitopes.ConclusionsOur study demonstrates the feasibility of efficiently identifying tumor-specific neoantigens that can be targeted by vaccination in tumors with a low mutational burden, promising successful clinical exploitation, with trials currently underway

Tap63 regulates oncogenic miR-155 to mediate migration and tumour growth

miR-155 is an oncogenic microRNA which is upregulated in many solid cancers. The targets of miR-155 are well established , with over 100 confirmed mRNA targets. However, the regulation of miR-155 and the basis of its upregulation in cancer is not well understood. We have previously shown that miR-155 is regulated by p63, and here we investigate the role of the major p63 isoforms TAp63 and ΔNp63 in this regulation. When the TAp63 isoform was knocked down, or exogenously overexpressed, miR-155 levels were elevated in response to TAp63 knockdown or reduced in response to TAp63 overexpression. The ΔNp63 isoform is shown to directly bind to the p63 response element on the miR-155 host gene, and this binding is enriched when TAp63 is knocked down. This could indicate that TAp63 prevents ΔNp63 from binding to the miR-155 host gene. The knockdown of TAp63, and the subsequent elevation of miR-155, enhances migration and tumour growth similar to that seen when directly overexpressing miR-155. The migratory phenotype is abrogated when miR-155 is inhibited, indicating that miR-155 is responsible for the phenotypic effect of TAp63 knockdown.Sam Mattiske, Kristen Ho, Jacqueline E. Noll, Paul M. Neilsen, David F. Callen and Rachel J. Suetan

Non-Coding RNAs in Retinal Development

Retinal development is dependent on an accurately functioning network of transcriptional and translational regulators. Among the diverse classes of molecules involved, non-coding RNAs (ncRNAs) play a significant role. Members of this family are present in the cell as transcripts, but are not translated into proteins. MicroRNAs (miRNAs) are small ncRNAs that act as post-transcriptional regulators. During the last decade, they have been implicated in a variety of biological processes, including the development of the nervous system. On the other hand, long-ncRNAs (lncRNAs) represent a different class of ncRNAs that act mainly through processes involving chromatin remodeling and epigenetic mechanisms. The visual system is a prominent model to investigate the molecular mechanisms underlying neurogenesis or circuit formation and function, including the differentiation of retinal progenitor cells to generate the seven principal cell classes in the retina, pathfinding decisions of retinal ganglion cell axons in order to establish the correct connectivity from the eye to the brain proper, and activity-dependent mechanisms for the functionality of visual circuits. Recent findings have associated ncRNAs in several of these processes and uncovered a new level of complexity for the existing regulatory mechanisms. This review summarizes and highlights the impact of ncRNAs during the development of the vertebrate visual system, with a specific focus on the role of miRNAs and a synopsis regarding recent findings on lncRNAs in the retina

An integrative genomics approach identifies Hypoxia Inducible Factor-1 (HIF-1)-target genes that form the core response to hypoxia

The transcription factor Hypoxia-inducible factor 1 (HIF-1) plays a central role in the transcriptional response to oxygen flux. To gain insight into the molecular pathways regulated by HIF-1, it is essential to identify the downstream-target genes. We report here a strategy to identify HIF-1-target genes based on an integrative genomic approach combining computational strategies and experimental validation. To identify HIF-1-target genes microarrays data sets were used to rank genes based on their differential response to hypoxia. The proximal promoters of these genes were then analyzed for the presence of conserved HIF-1-binding sites. Genes were scored and ranked based on their response to hypoxia and their HIF-binding site score. Using this strategy we recovered 41% of the previously confirmed HIF-1-target genes that responded to hypoxia in the microarrays and provide a catalogue of predicted HIF-1 targets. We present experimental validation for ANKRD37 as a novel HIF-1-target gene. Together these analyses demonstrate the potential to recover novel HIF-1-target genes and the discovery of mammalian-regulatory elements operative in the context of microarray data sets

Correlation between CD105 expression and postoperative recurrence and metastasis of hepatocellular carcinoma

BACKGROUND: Angiogenesis is one of the mechanisms most critical to the postoperative recurrence and metastasis of hepatocellular carcinoma (HCC). Thus, finding the molecular markers associated with angiogenesis may help identify patients at increased risk for recurrence and metastasis of HCC. This study was designed to investigate whether CD105 or CD34 could serve as a valid prognostic marker in patients with HCC by determining if there is a correlation between CD105 or CD34 expression and postoperative recurrence or metastasis. METHODS: Immunohistochemical staining for the CD105, CD34 and vascular endothelial growth factor (VEGF) antibodies was performed in 113 HCC tissue specimens containing paracarcinomatous tissue and in 14 normal liver tissue specimens. The quantitation of microvessels identified by anti-CD105 and anti-CD34 monoclonal antibodies and the semiquantitation of VEGF expression identified by anti-VEGF monoclonal antibody were analyzed in conjunction with the clinicopathological characteristics of the HCC and any available follow-up information about the patients from whom the specimens were obtained. RESULTS: CD105 was not expressed in the vascular endothelial cells of any normal liver tissue or paracarcinomatous liver tissue but was expressed in the vascular endothelial cells of all HCC tissue. In contrast, CD34 was expressed in the vascular endothelial cells of normal liver tissue, paracarcinomatous tissue, and HCC tissue in the following proportions of specimens: 86.7%, 93.8%, and 100%, respectively. The microvascular densities (MVDs) of HCC determined by using an anti-CD105 mAb (CD105-MVD) and an anti-CD34 mAb (CD34-MVD), were 71.7 ± 8.3 (SD) and 106.3 ± 10.4 (SD), respectively. There was a significant correlation between CD105-MVD and CD34-MVD (r = 0.248, P = 0.021). Although CD34-MVD was significantly correlated with VEGF expression (r = 0.243, P = 0.024), CD105-MVD was more closely correlated (r = 0.300, P= 0.005). The correlation between microscopic venous invasion and CD105-MVD, but not CD34-MVD, was also statistically significant (r = 0.254, P = 0.018). Univariate analysis showed that CD105-MVD was significantly correlated with the 2-year overall survival rate (P = 0.014); CD34-MVD was not (P = 0.601). Multivariate analysis confirmed that CD105-MVD was an independent prognostic factor and that CD34-MVD was not. CONCLUSION: The anti-CD105 mAb is an ideal instrument to quantify new microvessels in HCC as compared with anti-CD34 mAb. CD105-MVD as compared with CD34-MVD is relevant a significant and independent prognostic indicator for recurrence and metastasis in HCC patients