Mitotic phosphorylation activates hepatoma-derived growth factor as a mitogen

<p>Abstract</p> <p>Background</p> <p>Hepatoma-derived growth factor (HDGF) is a nuclear protein that is a mitogen for a wide variety of cells. Mass spectrometry based methods have identified HDGF as a phosphoprotein without validation or a functional consequence of this post-translational modification.</p> <p>Results</p> <p>We found that HDGF in primary mouse aortic vascular smooth muscle cells (VSMC) was phosphorylated. Wild type HDGF was phosphorylated in asynchronous cells and substitution of S103, S165 and S202 to alanine each demonstrated a decrease in HDGF phosphorylation. A phospho-S103 HDGF specific antibody was developed and demonstrated mitosis-specific phosphorylation. HDGF-S103A was not mitogenic and FACS analysis demonstrated a G2/M arrest in HDGF-S103A expressing cells, whereas cells expressing HDGF-S103D showed cell cycle progression. Nocodazole arrest increased S103 phosphorylation from 1.6% to 29% (P = 0.037).</p> <p>Conclusions</p> <p>Thus, HDGF is a phosphoprotein and phosphorylation of S103 is mitosis related and required for its function as a mitogen. We speculate that cell cycle regulated phosphorylation of HDGF may play an important role in vascular cell proliferation.</p

Comprehensive Immune Monitoring of Clinical Trials to Advance Human Immunotherapy.

The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates ≥98% of peripheral immune cells with ≥4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery

Overexpression of hepatoma-derived growth factor in melanocytes does not lead to oncogenic transformation

<p>Abstract</p> <p>Background</p> <p>HDGF is a growth factor which is overexpressed in a wide range of tumors. Importantly, expression levels were identified as a prognostic marker in some types of cancer such as melanoma.</p> <p>Methods</p> <p>To investigate the presumed oncogenic/transforming capacity of HDGF, we generated transgenic mice overexpressing HDGF in melanocytes. These mice were bred with mice heterozygous for a defective copy of the Ink4a tumor suppressor gene and were exposed to UV light to increase the risk for tumor development both genetically and physiochemically. Mice were analyzed by immunohistochemistry and Western blotting. Furthermore, primary melanocytes were isolated from different strains created.</p> <p>Results</p> <p>Transgenic animals overexpressed HDGF in hair follicle melanocytes. Interestingly, primary melanocytes isolated from transgenic animals were not able to differentiate <it>in vitro </it>whereas cells isolated from wild type and HDGF-deficient animals were. Although, HDGF^-/-/Ink4a^+/-mice displayed an increased number of epidermoid cysts after exposure to UV light, no melanomas or premelanocytic alterations could be detected in this mouse model.</p> <p>Conclusions</p> <p>The results therefore provide no evidence that HDGF has a transforming capacity in tumor development. Our results in combination with previous findings point to a possible role in cell differentiation and suggest that HDGF promotes tumor progression after secondary upregulation and may represent another protein fitting into the concept of non-oncogene addiction of tumor tissue.</p

Comprehensive Immune Monitoring of Clinical Trials to Advance Human Immunotherapy

The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates &gt;= 98% of peripheral immune cells with &gt;= 4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery

Six-Month Mortality among HIV-Infected Adults Presenting for Antiretroviral Therapy with Unexplained Weight Loss, Chronic Fever or Chronic Diarrhea in Malawi.

In sub-Saharan Africa, early mortality is high following initiation of antiretroviral therapy (ART). We investigated 6-month outcomes and factors associated with mortality in HIV-infected adults being assessed for ART initiation and presenting with weight loss, chronic fever or diarrhea, and with negative TB sputum microscopy

Soft branes in supersymmetry-breaking backgrounds

We revisit the analysis of effective field theories resulting from non-supersymmetric perturbations to supersymmetric flux compactifications of the type-IIB superstring with an eye towards those resulting from the backreaction of a small number of anti-D3-branes. Independently of the background, we show that the low-energy Lagrangian describing the fluctuations of a stack of probe D3-branes exhibits soft supersymmetry breaking, despite perturbations to marginal operators that were not fully considered in some previous treatments. We take this as an indication that the breaking of supersymmetry by anti-D3-branes or other sources may be spontaneous rather than explicit. In support of this, we consider the action of an anti-D3-brane probing an otherwise supersymmetric configuration and identify a candidate for the corresponding goldstino.Comment: 36+5 pages. References added, minor typos correcte

A Role for Cytoplasmic PML in Cellular Resistance to Viral Infection

PML gene was discovered as a fusion partner with retinoic acid receptor (RAR) α in the t(15:17) chromosomal translocation associated with acute promyelocytic leukemia (APL). Nuclear PML protein has been implicated in cell growth, tumor suppression, apoptosis, transcriptional regulation, chromatin remodeling, DNA repair, and anti-viral defense. The localization pattern of promyelocytic leukemia (PML) protein is drastically altered during viral infection. This alteration is traditionally viewed as a viral strategy to promote viral replication. Although multiple PML splice variants exist, we demonstrate that the ratio of a subset of cytoplasmic PML isoforms lacking exons 5 & 6 is enriched in cells exposed to herpes simplex virus-1 (HSV-1). In particular, we demonstrate that a PML isoform lacking exons 5 & 6, called PML Ib, mediates the intrinsic cellular defense against HSV-1 via the cytoplasmic sequestration of the infected cell protein (ICP) 0 of HSV-1. The results herein highlight the importance of cytoplasmic PML and call for an alternative, although not necessarily exclusive, interpretation regarding the redistribution of PML that is seen in virally infected cells

EBV Tegument Protein BNRF1 Disrupts DAXX-ATRX to Activate Viral Early Gene Transcription

Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV) typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs) and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs), suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus