The cellular DNA helicase ChlR1 regulates chromatin and nuclear matrix attachment of the human papillomavirus type 16 E2 protein and high copy viral genome establishment

In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2Y131A) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2Y131A showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2WT), the chromatin-bound pool of E2Y131A was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2Y131A occurred in mid-S phase. A similar alteration between the subcellular pools of the E2WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2Y131A genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes

Mindfulness-Oriented Recovery Enhancement versus CBT for co-occurring substance dependence, traumatic stress, and psychiatric disorders: Proximal outcomes from a pragmatic randomized trial

In clinical settings, there is a high comorbidity between substance use disorders, psychiatric disorders, and traumatic stress. As such, transdiagnostic therapies are needed to address these co-occurring issues efficiently. The aim of the present study was to conduct a pragmatic randomized controlled trial comparing Mindfulness-Oriented Recovery Enhancement (MORE) to group Cognitive-Behavioral Therapy (CBT) and treatment-as-usual (TAU) for previously homeless men residing in a therapeutic community. Men with co-occurring substance use and psychiatric disorders, as well as extensive trauma histories, were randomly assigned to 10 weeks of group treatment with MORE (n=64), CBT (n=64), or TAU (n=52). Study findings indicated that from pre- to post-treatment MORE was associated with modest yet significantly greater improvements in substance craving, post-traumatic stress, and negative affect than CBT, and significantly greater improvements in post-traumatic stress and positive affect than TAU. A significant indirect effect of MORE on decreasing craving and post-traumatic stress by increasing dispositional mindfulness was observed, suggesting that MORE may target these issues via enhancing mindful awareness in everyday life. This pragmatic trial represents the first head-to-head comparison of MORE against an empirically-supported treatment for co-occurring disorders. Results suggest that MORE, as an integrative therapy designed to bolster self-regulatory capacity, may hold promise as a treatment for intersecting clinical conditions

Semicontinuous Bioreactor Production of Recombinant Butyrylcholinesterase in Transgenic Rice Cell Suspension Cultures.

An active and tetrameric form of recombinant butyrylcholinesterase (BChE), a large and complex human enzyme, was produced via semicontinuous operation in a transgenic rice cell suspension culture. After transformation of rice callus and screening of transformants, the cultures were scaled up from culture flask to a lab scale bioreactor. The bioreactor was operated through two phases each of growth and expression. The cells were able to produce BChE during both expression phases, with a maximum yield of 1.6 mg BChE/L of culture during the second expression phase. Cells successfully regrew during a 5-day growth phase. A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE

Oncogenic Transformation of Mammary Epithelial Cells by Transforming Growth Factor Beta Independent of Mammary Stem Cell Regulation

BackgroundTransforming growth factor beta (TGFβ) is transiently increased in the mammary gland during involution and by radiation. While TGFβ normally has a tumour suppressor role, prolonged exposure to TGFβ can induce an oncogenic epithelial to mesenchymal transition (EMT) program in permissive cells and initiate the generation of cancer stem cells. Our objective is to mimic the transient exposure to TGFβ during involution to determine the persistent effects on premalignant mammary epithelium

Activin Limits Progenitor Capability by Promoting Epithelial Cell Differentiation in the Mammary Gland

Transforming growth factor beta (TGF-beta) and activin utilize common signaling pathways, via smad2/3 and smad4, to mediate tumor suppression by effecting cell cycle arrest and apoptosis. Differences in temporal expression patterns suggest that each cytokine has specific roles in mammary gland development. Activin is expressed during pregnancy and lactation and is required for branching and lactogenesis, implying a role in mammary gland maturation. In contrast, TGF-beta is expressed during involution during mammary gland regression and functions to re-organize the mammary epithelial content to the non-lactating state. Previously, we found that TGF-beta and activin do share common signaling pathways allowing both cytokines to restrict the growth of mammary epithelial cells. However, extended exposure to TGF-beta (5ng/ml; 14 days) causes epithelial to mesencymal transition (EMT). The TGF-beta-treated cells were de-differentiated with loss of both luminal and basal markers. Activin treatment (50ng/ml; 14 days) did not activate EMT. Rather, activin promotes luminal epithelial differentiation with increased expression of prolactin receptor and luminal keratins. Therefore, to test the hypothesis that activin-treatment promotes luminal differentiation and decreases the proportion of progenitor cells in the epithelial population, we compared mammosphere forming capability in vitro and performed limiting dilution experiments in vivo by transplanting 50,000 or 500,000 pre-treated cells into cleared mouse mammary fat pads. The mammosphere assay showed that secondary mammospheres were significantly decreased in the activin-treated cells compared to both the control and TGF-beta treated cells. Tumor incidence between activin-treated and control cells were similar for transplants of 50,000 cells, but tumor incidence was significantly greater in TGF-beta-treated transplants. However, the activin-treated cells had poor outgrowth potential at both 50,000 and 500,000 cells relative to control. We conclude that activin may have the potential to reduce the stem cell population by promoting epithelial cell differentiation

Pregnancy Induces Persistent Changes that Potentiate Apoptotic Signaling and Responses to DNA Damage

A full-term pregnancy reduces the lifetime risk of breast cancer by up to 50%. This effect is mediated, in part, by p53-dependent pathways. Gene expression profiling was used to investigate the mechanisms that alter apoptotic responses to DNA damage in the mammary gland. Radiation-induced responses in BALB/c-Trp53+/+ and BALB/c-Trp53-/- mice identified 121 genes that were altered by radiation and p53 status (p53-IR). To determine the effect of parity, mice were mated, force-weaned and mammary glands were allowed to involute for 21 days (parous) and compared with age-matched nulliparous mice. Gene expression profiles were determined in mammary tissues from nulliparous (N), parous (P), irradiated nulliparous (N-IR) and irradiated parous (P-IR) mice. The p53-IR gene signature did not differ among the N-IR and P-IR groups indicating that transcriptional activity of p53 was not altered by parity. However, expression profiles of apoptosis-related genes differed significantly in the parous group. The alterations in parous mammary tissues was accompanied by over-representation of biological processes that included “signal transduction” (e=1.69E-05). Within this set, Wnt signaling was especially pronounced (e Parity-regulated genes collaborate with p53-dependent targets, which act as a “switch”, to elicit apoptosis following ionizing radiation. The epigenetic states of the parity-regulated genes Tgfb2 and Wnt5a provide a mechanism for the persistent alterations in gene expression and apoptosis in parous mammary epithelial cells

Electrical properties of methane hydrate + sediment mixtures

Knowledge of the electrical properties of multicomponent systems with gas hydrate, sediments, and pore water is needed to help relate electromagnetic (EM) measurements to specific gas hydrate concentration and distribution patterns in nature. Toward this goal, we built a pressure cell capable of measuring in situ electrical properties of multicomponent systems such that the effects of individual components and mixing relations can be assessed. We first established the temperature-dependent electrical conductivity (?) of pure, single-phase methane hydrate to be ~5 orders of magnitude lower than seawater, a substantial contrast that can help differentiate hydrate deposits from significantly more conductive water-saturated sediments in EM field surveys. Here we report ? measurements of two-component systems in which methane hydrate is mixed with variable amounts of quartz sand or glass beads. Sand by itself has low ? but is found to increase the overall ? of mixtures with well-connected methane hydrate. Alternatively, the overall ? decreases when sand concentrations are high enough to cause gas hydrate to be poorly connected, indicating that hydrate grains provide the primary conduction path. Our measurements suggest that impurities from sand induce chemical interactions and/or doping effects that result in higher electrical conductivity with lower temperature dependence. These results can be used in the modeling of massive or two-phase gas-hydrate-bearing systems devoid of conductive pore water. Further experiments that include a free water phase are the necessary next steps toward developing complex models relevant to most natural systems

The Role of Pharmacists in Caring for Young People with Chronic Illness

PurposeTo explore the perceived and potential roles of pharmacists in the care of young people aged 10-24 years with chronic illness, through the exemplar of juvenile arthritis, from the perspectives of UK community and hospital pharmacists, health service commissioners, rheumatology health professionals and lay advocates.MethodsA sequential mixed methods study design comprising: focus groups with community and hospital pharmacists; telephone interviews with pharmacy and rheumatology stakeholders and commissioners, and multidisciplinary group discussions to prioritize roles generated by the first two qualitative phases.ResultsThe high priority roles for pharmacists, identified by pharmacists and rheumatology staff, were: developing generic healthcare skills among young people; transferring information effectively across care interfaces; building trusting relationships with young people; helping young people to find credible online health information, and the need to develop specialist expertise. Participants identified associated challenges for pharmacists in supporting young people with chronic illness. These challenges included parents collecting prescription refills alone, thus reducing opportunities to engage, and pharmacist isolation from the wider healthcare team.ConclusionsThis study has led to the identification of specific enhancements to pharmacy services for young people which have received the endorsement of a wide range of stakeholders. These suggestions could inform the next steps in developing the contribution of community and hospital pharmacy to support young people with chronic illness in the optimal use of their medication

Estrogen receptor beta selectively restricts proliferation and favors surveillance in mammary epithelial cells

Estrogen (17β-estradiol) has paradoxical effects in both promoting and preventing breast cancer as estrogen activates proliferation, but also promotes p53-mediated surveillance pathways. Estrogen mediates its effects in target tissues through the activation of estrogen receptor subtypes: ERα and ERβ. To examine the capability of these receptors in mediating surveillance as opposed to proliferation, selective estrogen receptor agonists were compared with 17β-estradiol for induction of proliferation and radiation induced apoptosis in vivo. Transcriptional regulation of estrogen-responsive genes was also compared in mouse mammary epithelium in vivo and in the human mammary MCF7 cell line transduced with a repressible ERβ. Selective activation of ERβ with the agonist diarylpropionitrile (DPN) in vivo enhances p53-mediated apoptosis in the mouse mammary epithelium without stimulating proliferation. In addition, radiation-induced apoptosis is significantly reduced in mice lacking ERβ (βERKO). As expected, 17β-estradiol or selective activation of ERα with pyrazole triol (PPT) induced the expression of estrogen-response genes including progesterone receptor, amphiregulin and trefoil factor 1. DPN and ERβ failed to induce the expression of these genes. Interestingly, the ERβ agonist DPN selectively induced the expression of genes that repress proliferation including TGFβ2 while inhibiting proliferative canonical wnt signaling via beta-catenin by inducing WNT5a and AXIN2. DPN was also more potent in stimulating the expression of EGR1, a modulator of p53 activity. These results suggest that ERα and ERβ have distinct roles in gene regulation. In addition, the ability of DPN and ERβ to potentiate surveillance pathways while limiting proliferation suggests that ERβ agonists may have therapeutic and chemopreventive value in breast cancer

Outflows in Star-forming Galaxies: Stacking Analyses of Resolved Winds and the Relation to Their Hosts' Properties

Outflows form an integral component in regulating the gas cycling in and out of galaxies, although their impact on the galaxy hosts is still poorly understood. Here we present an analysis of 405 high mass (log M$_{*}$/M$_{\odot}\geqslant10$), star-forming galaxies (excluding AGN) with low inclinations at $z\sim$0, using stacking techniques of the NaD $\lambda\lambda$5889,5895 A neutral gas tracer in IFU observations from the MaNGA DR15 survey. We detect outflows in the central regions of 78/405 galaxies and determine their extent and power through the construction of stacked annuli. We find outflows are most powerful in central regions and extend out to $\sim$1R$_{e}$, with declining mass outflow rates and loading factors as a function of radius. The stacking of spaxels over key galaxy quantities reveals outflow detections in regions of high $\Sigma_{\text{SFR}}$ ($\gtrsim$0.01 M$_{\odot}$yr$^{-1}$kpc$^{-2}$) and $\Sigma_{M_{*}}$ ($\gtrsim$10$^{7}$ M$_{\odot}$kpc$^{-2}$) along the resolved main sequence. Clear correlations with $\Sigma_{\text{SFR}}$ suggest it is the main regulator of outflows, with a critical threshold of $\sim$0.01 M$_{\odot}$yr$^{-1}$kpc$^{-2}$ needed to escape the weight of the disk and launch them. Furthermore, measurements of the H$\delta$ and D$_{n}$4000 indices reveal virtually identical star formation histories between galaxies with outflows and those without. Finally, through stacking of HI 21 cm observations for a subset of our sample, we find outflow galaxies show reduced HI gas fractions at central velocities compared to their non-detection control counterparts, suggestive of some removal of HI gas, likely in the central regions of the galaxies, but not enough to completely quench the host.Comment: 20 pages, 11 figures. Accepted for publication in MNRA