The progestin receptor interactome in the female mouse hypothalamus: Interactions with synaptic proteins are isoform specific and ligand dependent

Progestins bind to the progestin receptor (PR) isoforms, PR-A and PR-B, in brain to influence development, female reproduction, anxiety, and stress. Hormone-activated PRs associate with multiple proteins to form functional complexes. In the present study, proteins from female mouse hypothalamus that associate with PR were isolated using affinity pull-down assays with glutathione S-transferase–tagged mouse PR-A and PR-B. Using complementary proteomics approaches, reverse phase protein array (RPPA) and mass spectrometry, we identified hypothalamic proteins that interact with PR in a ligand-dependent and isoform-specific manner and were confirmed by Western blot. Synaptic proteins, including synapsin-I and synapsin-II, interacted with agonist-bound PR isoforms, suggesting that both isoforms function in synaptic plasticity. In further support, synaptogyrin-III and synapsin-III associated with PR-A and PR-B, respectively. PR also interacted with kinases, including c-Src, mTOR, and MAPK1, confirming phosphorylation as an integral process in rapid effects of PR in the brain. Consistent with a role in transcriptional regulation, PR associated with transcription factors and coactivators in a ligand-specific and isoform-dependent manner. Interestingly, both PR isoforms associated with a key regulator of energy homeostasis, FoxO1, suggesting a novel role for PR in energy metabolism. Because many identified proteins in this PR interactome are synaptic proteins, we tested the hypothesis that progestins function in synaptic plasticity. Indeed, progesterone enhanced synaptic density, by increasing synapsin-I–positive synapses, in rat primary cortical neuronal cultures. This novel combination of RPPA and mass spectrometry allowed identification of PR action in synaptic remodeling and energy homeostasis and reveals unique roles for progestins in brain function and disease

Use of ELISpot assay to study HBs-specific B cell responses in vaccinated and HBV infected humans

Hepatitis B surface antibody (HBsAb) plays a critical role in protecting against infection of hepatitis B virus (HBV) and were extensively studied in literature. At the same time, the status of hepatitis B surface antigen (HBs)-specific B cells in both vaccinated and HBV infected people received limited attention. In the current study, we adopted a highly specific B-cell Enzyme Linked ImmunoSpot (ELISpot) assay to analyze HBs-specific B cells in various clinical settings: healthy individuals with the history of HBV vaccination before and after receiving an extra HBV vaccine boost, people chronically infected with HBV (CHB) in various clinical stages, with or without a particular type anti-viral treatment, or whether receiving a dose of HBV vaccine. In all of these cases, B-cell ELISpot assay was used effectively in enumerating the frequency of HBs-specific B cells. While the focus of the current report was to establish the utility of this assay for HBV research, a number of interesting observations were made in this pilot study based on the profiles and dynamics of HBs-specific B cells in various conditions. Such information is useful to guide the future work in designing novel therapeutic strategies against CHB

Changes in gene expression during the development of mammary tumors in MMTV-Wnt-1 transgenic mice

BACKGROUND: In human breast cancer normal mammary cells typically develop into hyperplasia, ductal carcinoma in situ, invasive cancer, and metastasis. The changes in gene expression associated with this stepwise progression are unclear. Mice transgenic for mouse mammary tumor virus (MMTV)-Wnt-1 exhibit discrete steps of mammary tumorigenesis, including hyperplasia, invasive ductal carcinoma, and distant metastasis. These mice might therefore be useful models for discovering changes in gene expression during cancer development. RESULTS: We used cDNA microarrays to determine the expression profiles of five normal mammary glands, seven hyperplastic mammary glands and 23 mammary tumors from MMTV-Wnt-1 transgenic mice, and 12 mammary tumors from MMTV-Neu transgenic mice. Adipose tissues were used to control for fat cells in the vicinity of the mammary glands. In these analyses, we found that the progression of normal virgin mammary glands to hyperplastic tissues and to mammary tumors is accompanied by differences in the expression of several hundred genes at each step. Some of these differences appear to be unique to the effects of Wnt signaling; others seem to be common to tumors induced by both Neu and Wnt-1 oncogenes. CONCLUSION: We described gene-expression patterns associated with breast-cancer development in mice, and identified genes that may be significant targets for oncogenic events. The expression data developed provide a resource for illuminating the molecular mechanisms involved in breast cancer development, especially through the identification of genes that are critical in cancer initiation and progression

Proteomic resistance biomarkers for PI3K inhibitor in triple negative breast cancer patient-derived xenograft models

PI3K pathway activation is frequently observed in triple negative breast cancer (TNBC). However, single agent PI3K inhibitors have shown limited anti-tumor activity. To investigate biomarkers of response and resistance mechanisms, we tested 17 TNBC patient-derived xenograft (PDX) models representing diverse genomic backgrounds and varying degrees of PI3K pathway signaling activities for their tumor growth response to the pan-PI3K inhibitor, BKM120. Baseline and post-treatment PDX tumors were subjected to reverse phase protein array (RPPA) to identify protein markers associated with tumor growth response. While BKM120 consistently reduced PI3K pathway activity, as demonstrated by reduced levels of phosphorylated AKT, percentage tumor growth inhibition (%TGI) ranged from 35% in the least sensitive to 84% in the most sensitive model. Several biomarkers showed significant association with resistance, including elevated baseline levels of growth factor receptors (EGFR, pHER3 Y1197), PI3Kp85 regulatory subunit, anti-apoptotic protein BclXL, EMT (Vimentin, MMP9, IntegrinaV), NFKB pathway (IkappaB, RANKL), and intracellular signaling molecules including Caveolin, CBP, and KLF4, as well as treatment-induced increases in the levels of phosphorylated forms of Aurora kinases. Interestingly, increased AKT phosphorylation or PTEN loss at baseline were not significantly correlated to %TGI. These results provide important insights into biomarker development for PI3K inhibitors in TNBC

A new mild hyperthermia device to treat vascular involvement in cancer surgery

Abstract Surgical margin status in cancer surgery represents an important oncologic parameter affecting overall prognosis. The risk of disease recurrence is minimized and survival often prolonged if margin-negative resection can be accomplished during cancer surgery. Unfortunately, negative margins are not always surgically achievable due to tumor invasion into adjacent tissues or involvement of critical vasculature. Herein, we present a novel intra-operative device created to facilitate a uniform and mild heating profile to cause hyperthermic destruction of vessel-encasing tumors while safeguarding the encased vessel. We use pancreatic ductal adenocarcinoma as an in vitro and an in vivo cancer model for these studies as it is a representative model of a tumor that commonly involves major mesenteric vessels. In vitro data suggests that mild hyperthermia (41–46 °C for ten minutes) is an optimal thermal dose to induce high levels of cancer cell death, alter cancer cell’s proteomic profiles and eliminate cancer stem cells while preserving non-malignant cells. In vivo and in silico data supports the well-known phenomena of a vascular heat sink effect that causes high temperature differentials through tissues undergoing hyperthermia, however temperatures can be predicted and used as a tool for the surgeon to adjust thermal doses delivered for various tumor margins

Baiji genomes reveal low genetic variability and new insights into secondary aquatic adaptations

The baiji, or Yangtze River dolphin (Lipotes vexillifer), is a flagship species for the conservation of aquatic animals and ecosystems in the Yangtze River of China; however, this species has now been recognized as functionally extinct. Here we report a high-quality draft genome and three re-sequenced genomes of L. vexillifer using Illumina short-read sequencing technology. Comparative genomic analyses reveal that cetaceans have a slow molecular clock and molecular adaptations to their aquatic lifestyle. We also find a significantly lower number of heterozygous single nucleotide polymorphisms in the baiji compared to all other mammalian genomes reported thus far. A reconstruction of the demographic history of the baiji indicates that a bottleneck occurred near the end of the last deglaciation, a time coinciding with a rapid decrease in temperature and the rise of eustatic sea level

Dynamic changes of cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and natural killer T (NKT) cells in patients with acute hepatitis B infection

<p>Abstract</p> <p><b>Background</b></p> <p>The goal of this study is to observe changes in HBcAg-specific cytotoxic T lymphocytes (CTLs), natural killer (NK) and natural killer T (NKT) cells from peripheral blood and to relate such changes on viral clearance and liver injury in patients with acute hepatitis B (AHB).</p> <p><b>Methods</b></p> <p>Dynamic profiles on the frequency of HLA-A0201-restricted HBcAg18-27 pentamer complex (MHC-Pentamer)-specific CTLs and lymphocyte subsets in AHB patients were analyzed in addition to liver function tests, HBV serological markers, and HBV DNA levels. ELISPOT was used to detect interferon-gamma (INF-γ) secretion in specific CTLs stimulated with known T cell epitope peptides associated with HBV surface protein, polymerase, and core protein.</p> <p><b>Results</b></p> <p>HBV-specific CTL frequencies in AHB patients were much higher than in patients with chronic hepatitis B (CHB) (p < 0.05). HBeAg and HBV DNA disappeared earlier in AHB patients with a high frequency of HBV-specific CTLs compared with those with a low frequency of HBV-specific CTLs (p = 0.001 and 0.024, respectively). INF-γ spots of effector cells stimulated by Pol575-583, Env348-357, or Core18-27 epitope peptides were significantly greater in AHB patients than in CHB patients (p < 0.01). CD3⁺CD8⁺T cell numbers in AHB patients was more than observed in the healthy control group from the first to the fourth week after admission (p <it>= </it>0.008 and 0.01, respectively); the number of CD3⁺CD8⁺T cells and frequency of HBcAg18-27-specific CTLs in AHB patients reached peak levels at the second week after admission. NK and NKT cell numbers were negatively correlated with the frequency of HBcAg-specific CTLs (<it>r </it>= -0.266, p = 0.05).</p> <p><b>Conclusions</b></p> <p>Patients with AHB possess a higher frequency of HBcAg-specific CTLs than CHB patients. The frequency of specific CTLs in AHB patients is correlated with HBeAg clearance indicating that HBV-specific CTLs play an important role in viral clearance and the self-limited process of the disease. Furthermore, NK and NKT cells are likely involved in the early, non-specific immune response to clear the virus.</p

Hominin occupation of the Chinese Loess Plateau since about 2.1 million years ago

Considerable attention has been paid to dating the earliest appearance of hominins outside Africa. The earliest skeletal and artefactual evidence for the genus Homo in Asia currently comes from Dmanisi, Georgia, and is dated to approximately 1.77-1.85 million years ago (Ma)(1). Two incisors that may belong to Homo erectus come from Yuanmou, south China, and are dated to 1.7 Ma(2); the next-oldest evidence is an H. erectus cranium from Lantian (Gongwangling)-which has recently been dated to 1.63 Ma(3) and the earliest hominin fossils from the Sangiran dome in Java, which are dated to about 1.5-1.6 Ma(4). Artefacts from Majuangou III5 and Shangshazui(6) in the Nihewan basin, north China, have also been dated to 1.6-1.7 Ma. Here we report an Early Pleistocene and largely continuous artefact sequence from Shangchen, which is a newly discovered Palaeolithic locality of the southern Chinese Loess Plateau, near Gongwangling in Lantian county. The site contains 17 artefact layers that extend from palaeosol S15-dated to approximately 1.26 Ma-to loess L28, which we date to about 2.12 Ma. This discovery implies that hominins left Africa earlier than indicated by the evidence from Dmanisi

Polyvalent DNA Vaccines Expressing HA Antigens of H5N1 Influenza Viruses with an Optimized Leader Sequence Elicit Cross-Protective Antibody Responses

Highly pathogenic avian influenza A (HPAI) H5N1 viruses are circulating among poultry populations in parts of Asia, Africa, and the Middle East, and have caused human infections with a high mortality rate. H5 subtype hemagglutinin (HA) has evolved into phylogenetically distinct clades and subclades based on viruses isolated from various avian species. Since 1997, humans have been infected by HPAI H5N1 viruses from several clades. It is, therefore, important to develop strategies to produce protective antibody responses against H5N1 viruses from multiple clades or antigenic groups. In the current study, we optimized the signal peptide design of DNA vaccines expressing HA antigens from H5N1 viruses. Cross reactivity analysis using sera from immunized rabbits showed that antibody responses elicited by a polyvalent formulation, including HA antigens from different clades, was able to elicit broad protective antibody responses against multiple key representative H5N1 viruses across different clades. Data presented in this report support the development of a polyvalent DNA vaccine strategy against the threat of a potential H5N1 influenza pandemic

Generation of an in vitro 3D PDAC stroma rich spheroid model

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic/stromal reaction, which contributes to the poor clinical outcome of this disease. Therefore, greater understanding of the stroma development and tumor-stroma interactions is highly required. Pancreatic stellate cells (PSC) are myofibroblast-like cells that located in exocrine areas of the pancreas, which as a result of inflammation produced by PDAC migrate and accumulate in the tumor mass, secreting extracellular matrix components and producing the dense PDAC stroma. Currently, only a few orthotopic or ectopic animal tumor models, where PDAC cells are injected into the pancreas or subcutaneous tissue layer, or genetically engineered animals offer tumors that encompass some stromal component. Herein, we report generation of a simple 3D PDAC in vitro micro-tumor model without an addition of external extracellular matrix, which encompasses a rich, dense and active stromal compartment. We have achieved this in vitro model by incorporating PSCs into 3D PDAC cell culture using a modified hanging drop method. It is now known that PSCs are the principal source of fibrosis in the stroma and interact closely with cancer cells to create a tumor facilitatory environment that stimulates local and distant tumor growth. The 3D micro-stroma models are highly reproducible with excellent uniformity, which can be used for PDAC-stroma interaction analysis and high throughput automated drug-screening assays. Additionally, the increased expression of collagenous regions means that molecular based perfusion and cytostaticity of gemcitabine is decreased in our Pancreatic adenocarcinoma stroma spheroids (PDAC-SS) model when compared to spheroids grown without PSCs. We believe this model will allow an improved knowledge of PDAC biology and has the potential to provide an insight into pathways that may be therapeutically targeted to inhibit PSC activation, thereby inhibiting the development of fibrosis in PDAC and interrupting PSC-PDAC cell interactions so as to inhibit cancer progression