The Tumor Suppressor Protein P53 and the Antagonizing Oncoprotein E6 from Human Papillomavirus Type 16

The tumor suppressor protein p53 transactivates genes involved in cell cycle arrest and apoptosis in response to DNA damage, cellular stress and some oncogenic proteins. The wild type polypeptide chain of p53 has four distinct domains, including the sequence specific DNA binding core domain and the C terminal tetramerization domain. p53 must retain its ability to oligomerize and bind DNA targets in vivo to fulfill its function. Using X-ray crystallography and a crosslinking strategy, the structure of a wild type tetrameric p53 core domain bound to its consensus sequence was solved. This structure gives insight into DNA bend, core domain-DNA binding cooperativity and surface residue conservation within the tetramer. Many cancers result from cells harboring mutant forms of p53 that are unable to function due to their inability to oligomerize, DNA binding defects or, in the case of human papillomavirus infection, p53 degradation. High risk forms of the human papillomvirus (HPV) infect the basal layers of stratified epithelia and express two oncoproteins, called E6 and E7, which can lead to cell cycle disruption and cervical cancer. E6 mediates its cell transformation, in part, by forming a complex with the cellular E3 ligase E6-Association Protein (E6AP) to target p53 for degradation by the ubiquitin- viii proteasome pathway. A high throughput solution screen was designed to search for small molecule inhibitors of the E6 / E6AP interaction. Of the 80,000 compounds that were screened, 30 inhibitors with IC50 values in the low-micromolar to mid-nanomolar range were confirmed using secondary assays. Two compounds were shown to specifically block p53 degradation and promote apootosis in cell lines infected with high risk forms of HPV. These HPV-E6 inhibitors provide a framework for developing HPV inhibitors with possible therapeutic applications

The impact of stroma and ionising radiation on the plasticity of dendritic cells and macrophages

The basic model of antigen-presenting cells (APC) activation describes a change of state from resting (immature) to activated (mature) upon encounter with a first signal. However, it is apparent that APC maturation is not binary but instead has a spectrum of outcomes. This likely reflects the complex and dynamic crosstalk resulting from their encounter with a wide variety of stromal cells. A deeper knowledge of these interactions, and how they are processed by APC to instruct immunity is required. This study explores the plasticity of the major APC, namely macrophage (Mφ) and dendritic cells (DC). We test the hypothesis that plasticity in APC function depends on the sequence of signals encountered during generation and activation. Human laboratory models of Mφ and DC were generated from monocytes as follows; monocyte-derived Mφ were cultured with M-CSF (M-Mφ) or GM-CSF (GM-Mφ) and DC with IL-4 and GM-CSF. These were characterised and the impact of cellular (e.g. fibroblasts (FB)) or external factors (e.g. ionising radiation (IR)) studied. Changes in microenvironment considerably alter the functional-phenotype of Mφ however it remains to be shown if their initial polarisation restricts responses. Activation of GM-Mφ elicited IL-12 and -23 whilst M-Mφ expressed IL-10. However, the classic pro-inflammatory signal interferon γ (IFNγ) did not elicit M-Mφ that secreted cytokines to the level expected of GM- Mφ. The M-Mφ displayed mixed phenotype with both pro-inflammatory cytokines (albeit at lower levels, 75% less IL-23, 21% less IL-12) on a background of high IL-10. The IL-23 response of Mφ to re-stimulation was reduced or abrogated upon pre-treatment with LPS but enhanced in Mφ conditioned by IFNγ. These findings demonstrate limitations to Mφ plasticity reflecting initial conditioning and suggest the development of innate memory with a degree of specificity in Mφ responses to external factors. We applied ionising radiation (IR) to our models at doses commonly used for radiotherapy (1-20Gy). Irradiation markedly suppressed IL-23 secretion by both Mφ and DC (p<0.01) in a dose-dependent manner without affecting viability. This was selective for IL-23 as there was no change in IL-1β, -6, -10, -27 or TNFα, and only a modest decrease in IL-12 secretion. To investigate the mechanism for IL-23 regulation we showed that IR activated Ataxia telangiectasia mutated kinase (ATM) which in turn inhibited the function of cAMP response element-binding protein (CREB) through phosphorylation of an inhibitory residue (S121). ATM activation suppressed IL-23 as inhibition of ATM with KU55933 increased its expression. Furthermore, the small-molecule CREB inhibitor C-Ci reduced IL-23 levels. As a consequence of modulation of IL-23 in APC, IR indirectly affected the generation of T-cell responses, namely Th-17. Cross-talk between APC and stromal cells was modelled by co-culture of DC with FB. The presence of FB prevented IL-23 downregulation by IR. This was dependent on TNFα and IL-1β secretion by DC which were unaffected by IR. In response, FB secreted PGE2 which resulted in a feed-back loop to enforce IL-23 secretion via activation of cAMP. Therefore, whilst IR could be seen to regulate IL-23 in a single cell culture system, this did not occur in co-cultures that simulated the interaction APC have with stroma. In conclusion, we shown that the order in which APC receive conditioning signals ultimately affects how they respond to re-challenge. This is further influenced by a mixture of micro-environmental and external factors. Understanding the molecular pathways through which these cues manifest will result in in vitro models that better reflect the biology of APC and create opportunity for intervention

Co-expression of functional human Heme Oxygenase 1, Ecto-5'-Nucleotidase and ecto-nucleoside triphosphate diphosphohydrolase-1 by "self-cleaving" 2A peptide system

We developed an F2A-based multicistronic system to evaluate functional effects of co-expression of three proteins important for xenotransplantation: heme oxygenase 1 (HO1), ecto-5'-nucleotidase (E5NT) and ecto-nucleoside triphosphate diphosphohydrolase-1 (ENTPD1). The tricistronic p2A plasmid that we constructed was able to efficiently drive concurrent expression of HO1, E5NT and ENTPD1 in HEK293T cells. All three overexpressed proteins possessed relevant enzymatic activities, while addition of furin site interfered with protein expression and activity. We conclude that our tricistronic p2A construct is effective and optimal to test the combined protective effects of HO1, E5NT and ENTPD1 against xeno-rejection mechanisms

Crop rotations sustain cereal yields under a changing climate

Agriculture is facing the complex challenge of satisfying increasing food demands, despite the current and projected negative impacts of climate change on yields. Increasing crop diversity at a national scale has been suggested as an adaptive measure to better cope with negative climate impacts such as increasing temperatures and drought, but there is little evidence to support this hypothesis at the field scale. Using seven long-term experiments across a wide latitudinal gradient in Europe, we showed that growing multiple crop species in a rotation always provided higher yields for both winter and spring cereals (average +860 and +390 kg ha−1 per year, respectively) compared with a continuous monoculture. In particular, yield gains in diverse rotations were higher in years with high temperatures and scant precipitations, i.e. conditions expected to become more frequent in the future, rendering up to c. 1000 kg ha−1 per year compared to monocultures. Winter cereals yielded more in diverse rotations immediately after initiation of the experiment and kept this advantage constant over time. For spring cereals, the yield gain increased over time since diversification adoption, arriving to a yearly surplus of c. 500 kg ha−1 after 50-60 years with still no sign of plateauing. Diversified rotations emerge as a promising way to adapt temperate cropping systems and contribute to food security under a changing climate. However, novel policies need to be implemented and investments made to give means and opportunities for farmers to adopt diversified crop rotations

Transvenous Lead Extraction in Patients with Cardiac Implantable Device: The Impact of Systemic and Local Infection on Clinical Outcomes. An ESC‐EHRA ELECTRa (European Lead Extraction Controlled) Registry Substudy

Background: Infections of cardiac implantable devices (CIEDI) have poor outcomes despite improvement in lead extraction (TLE) procedures. Methods: To explore the influence of CIEDI on the outcomes of TLE and the differences between patients with systemic (Sy) vs. local (Lo) CIEDI, we performed a sub‐analysis of the EORP ELECTRa (European Lead Extraction ConTRolled) Registry. Results: Among 3555 patients enrolled by 73 centers in 19 Countries, the indication for TLE was CIEDI in 1850: 1170 with Lo‐CIEDI and 680 with Sy‐CIEDI. Patients with CIEDI had a worse in‐hospital prognosis in terms of major complications (3.57% vs. 1.71%; p = 0.0007) and mortality (2.27% vs. 0.49%; p &lt; 0.0001). Sy‐CIEDI was an independent predictor of in‐hospital death (H.R. 2.14; 95%CI 1.06–4.33. p = 0.0345). Patients with Sy‐CIEDI more frequently had an initial CIED implant and a higher prevalence of comorbidities, while subjects with Lo‐CIEDI had a higher prevalence of previous CIED procedures. Time from signs of CIEDI and TLE was longer for Lo‐CIEDI despite a shorter pre‐TLE antibiotic treatment. Conclusions: Patients with CIEDI have a worse in‐hospital prognosis after TLE, especially for patients with Sy‐CIEDI. These results raise the suspicion that in a relevant group of patients CIEDI can be systemic from the beginning without progression from Lo‐CIEDI. Future research is needed to characterize this subgroup of patients

Efficacy of EGFR Inhibition Is Modulated by Model, Sex, Genetic Background and Diet: Implications for Preclinical Cancer Prevention and Therapy Trials

Molecule-targeted therapies are being widely developed and deployed, but they are frequently less effective in clinical trials than predicted based upon preclinical studies. Frequently, only a single model or genetic background is utilized using diets that are not relevant to that consumed by most cancer patients, which may contribute to the lack of predictability of many preclinical therapeutic studies. Inhibition of epidermal growth factor receptor (EGFR) in colorectal cancer was used to investigate potential causes for low predictive values of many preclinical studies. The efficacy of the small molecule EGFR inhibitor AG1478 was evaluated using two mouse models, ApcMin/+ and azoxymethane (AOM), both sexes on three genetic backgrounds, C57BL/6J (B6) and A/J (A) inbred strains and AB6F1 hybrids, and two diets, standard chow (STD) or Western-style diet (WD). AG1478 has significant anti-tumor activity in the B6-ApcMin/+ model with STD but only moderately on the WD and in the AOM model on an A background with a WD but not STD. On the F1 hybrid background AG1478 is effective in the ApcMin/+ model with either STD or WD, but has only moderate efficacy in the AOM model with either diet. Sex differences were also observed. Unexpectedly, the level of liver EGFR phosphorylation inhibition by AG1478 was not positively correlated with inhibition of tumor growth in the AOM model. Model-dependent interactions between genetic background and diet can dramatically impact preclinical results, and indicate that low predictive values of preclinical studies can be attributed to study designs that do not account for the heterogeneous patient population or the diets they consume. Better-designed preclinical studies should lead to more accurate predictions of therapeutic response in the clinic

Impact of p38 mitogen-activated protein kinase inhibition on immunostimulatory properties of human 6-sulfo LacNAc dendritic cells

p38 Mitogen-activated protein kinase (MAPK) plays a crucial role in the induction and regulation of innate and adaptive immunity. Furthermore, p38 MAPK can promote tumor invasion, metastasis, and angiogenesis. Based on these properties, p38 MAPK inhibitors emerged as interesting candidates for the treatment of immune-mediated disorders and cancer. However, the majority of p38 MAPK inhibitor-based clinical trials failed due to poor efficacy or toxicity. Further studies investigating the influence of p38 MAPK inhibitors on immunomodulatory capabilities of human immune cells may improve their therapeutic potential. Here, we explored the impact of the p38 MAPK inhibitor SB203580 on the pro-inflammatory properties of native human 6-sulfo LacNAc dendritic cells (slanDCs). SB203580 did not modulate maturation of slanDCs and their capacity to promote T-cell proliferation. However, SB203580 significantly reduced the production of pro-inflammatory cytokines by activated slanDCs. Moreover, inhibition of p38 MAPK impaired the ability of slanDCs to differentiate naïve CD4(+) T cells into T helper 1 cells and to stimulate interferon-γ secretion by natural killer cells. These results provide evidence that SB203580 significantly inhibits various important immunostimulatory properties of slanDCs. This may have implications for the design of p38 MAPK inhibitor-based treatment strategies for immune-mediated disorders and cancer

The association of hydration status with physical signs, symptoms and survival in advanced cancer-The use of Bioelectrical Impedance Vector Analysis (BIVA) technology to evaluate fluid volume in palliative care: An observational study

Background Hydration in advanced cancer is a controversial area; however, current hydration assessments methods are poorly developed. Bioelectrical impedance vector analysis (BIVA) is an accurate hydration tool; however its application in advanced cancer has not been explored. This study used BIVA to evaluate hydration status in advanced cancer to examine the association of fluid status with symptoms, physical signs, renal biochemical measures and survival. Materials and methods An observational study of 90 adults with advanced cancer receiving care in a UK specialist palliative care inpatient unit was conducted. Hydration status was assessed using BIVA in addition to assessments of symptoms, physical signs, performance status, renal biochemical measures, oral fluid intake and medications. The association of clinical variables with hydration was evaluated using regression analysis. A survival analysis was conducted to examine the influence of hydration status and renal failure. Results The hydration status of participants was normal in 43 (47.8%), 'more hydrated' in 37 (41.1%) and 'less hydrated' in 10 (11.1%). Lower hydration was associated with increased symptom intensity (Beta = -0.29, p = 0.04) and higher scores for physical signs associated with dehydration (Beta = 10.94, p = 0.02). Higher hydration was associated with oedema (Beta = 2.55, p<0.001). Median survival was statistically significantly shorter in 'less hydrated' patients (44 vs. 68 days; p = 0.049) and in pre-renal failure (44 vs. 100 days; p = 0.003). Conclusions In advanced cancer, hydration status was associated with clinical signs and symptoms. Hydration status and pre-renal failure were independent predictors of survival. Further studies can establish the utility of BIVA as a standardised hydration assessment tool and explore its potential research application, in order to inform the clinical management of fluid balance in patients with advanced cancer