Determination of the Binding Site of Adenovirus E4 11K on the Cellular Protein DDX6

Adenovirus is a double-stranded DNA virus that is responsible for localized infections, such as upper respiratory tract infections. The virus takes over the target cell through many mechanisms, in particular taking control of host cell gene expression mechanisms as well as controlling host cell protein synthesis machinery. One of the functions of the adenovirus E4 11k protein is in turning off host cell protein synthesis and regulating late viral gene expression. E4 11k from all adenovirus subclasses has been shown to disrupt cellular RNA processing bodies (P-bodies), and adenovirus serotype 5 has a direct interaction with a P-body protein, Ddx6. Our research goal is the identification of the binding site of Ddx6 on the E4 11k protein. Once this site is narrowed down to a single amino acid or sequence of amino acids, we aim to determine whether or not the binding of E4 11k with Ddx6 and later disruption of P-bodies during an adenovirus infection is involved in the control of host cell and late viral protein synthesis

Solid-state-concentration effects on the optical absorption and emission of poly(p-phenylene vinylene)-related materials

We present measurements of the optical absorption and emission properties of poly(p-phenylene vinylene) (PPV)-related materials focusing on the differences between molecules isolated by dispersion in an inert host and concentrated molecular films. Optical absorption spectra, photoluminescence (PL) spectra, PL efficiency, and time-resolved PL spectra of dilute blends of PPV oligomers with 2-5 phenylene-phenyl rings are compared with those of dense oligomer and polymer films. In dilute oligomer-poly(methyl methacrylate) (PMMA) blends with high PL efficiency, the PL decay is exponential, independent of both temperature and oligomer length. This implies that the fundamental radiative lifetime of PPV oligomers is essentially independent of oligomer length. Concentrated spin-cast oligomer films and polymers have a faster and strongly temperature-dependent PL decay that approaches that of the dilute oligomer results at low temperature. The differences in PL decay correspond to changes in PL efficiency. The efficiency of the oligomer-PMMA blend is high and only weakly temperature dependent, whereas that of concentrated films is lower and strongly temperature dependent, decreasing by more than a factor of 3 from 10 to 350 K. The quenching of the PL efficiency in concentrated films is due to migration to extrinsic, impurity related centers as opposed to an intrinsic intermolecular recombination process. The PL spectrum of a dilute oligomer blend redshifts substantially, both as the excitation energy is decreased and as the emission time increases. This spectral redshift is due to disorder-induced site-to-site variation and not to diffusion to lower-energy sites. In contrast, no spectral shift with excitation energy or emission time was observed for dense oligomer films

Regulators of genetic risk of breast cancer identified by integrative network analysis.

Genetic risk for breast cancer is conferred by a combination of multiple variants of small effect. To better understand how risk loci might combine, we examined whether risk-associated genes share regulatory mechanisms. We created a breast cancer gene regulatory network comprising transcription factors and groups of putative target genes (regulons) and asked whether specific regulons are enriched for genes associated with risk loci via expression quantitative trait loci (eQTLs). We identified 36 overlapping regulons that were enriched for risk loci and formed a distinct cluster within the network, suggesting shared biology. The risk transcription factors driving these regulons are frequently mutated in cancer and lie in two opposing subgroups, which relate to estrogen receptor (ER)(+) luminal A or luminal B and ER(-) basal-like cancers and to different luminal epithelial cell populations in the adult mammary gland. Our network approach provides a foundation for determining the regulatory circuits governing breast cancer, to identify targets for intervention, and is transferable to other disease settings.This work was funded by Cancer Research UK and the Breast Cancer Research Foundation. MAAC is funded by the National Research Council (CNPq) of Brazil. TEH held a fellowship from the US DOD Breast Cancer Research Program (W81XWH-11-1-0592) and is currently supported by an RAH Career Development Fellowship (Australia). TEH and WDT are funded by the NHMRC of Australia (NHMRC) (ID: 1008349 WDT; 1084416 WDT, TEH) and Cancer Australia/National Breast Cancer Foundation (ID 627229; WDT, TEH). BAJP is a Gibb Fellow of Cancer Research UK. We would like to acknowledge the support of The University of Cambridge, Cancer Research UK and Hutchison Whampoa Limited.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ng.345

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Investigating the Interaction between Adenoviral Protein E4 11k and Cellular Processing Body Protein Ddx6

Adenovirus is a non-enveloped virus with an icosahedral capsid encompassing a double-stranded DNA genome. In immunocompetent individuals, adenovirus most commonly causes infections that are localized to the upper respiratory and gastrointestinal tracts. However, immunocompromised people experience more serious, generalized infections. Our lab focuses on the adenovirus early region 4 11 k protein (E4 11k), which has been shown to inhibit host cell protein synthesis as well as stimulate viral late protein synthesis. During an adenovirus serotype 5 infection (Ad5), E4 11k has been shown to bind to the cellular processing body (p-body) protein, Ddx6. The two proteins then co-localize in aggresomes, which are juxtanuclear aggregates of misfolded proteins marked by -tubulin. Our lab aims to determine the binding location between the two proteins. In order to determine this site, we had Ad5 and Ad9 chimeras of the E4 ORF3 gene, which encodes the E4 11k protein, synthesized. The E4 11k protein encoded by Ad9 does not bind to Ddx6, only E4 11 k from Ad5 has been shown to bind Ddx6. Therefore, the chimeras will allow us to narrow down the binding site to a particular region on the E4 11k protein. We have begun using the Ad5/Ad9 E4 ORF3 chimeras in transfections to express them in the human cell line, HeLa, to observe cellular responses. We plan to use confocal microscopy to visualize whether aggresomes are able to form using the various chimeras. Once we find the binding region that allows for E4 11k to reorganize Ddx6 to aggresomes, we will perform alanine substitutions to disrupt the binding to observe any changes that result during an infection. These results will contribute to our knowledge of the roles that p-bodies play during viral infections, which may also help to understand their basic role in cell biology

Cranial Variation in a Sample of Gray Squirrels (Sciurus carolinensis) from Putnam County, Georgia

Understanding the range of morphological variation in extant taxa is of vital importance for taxonomists and paleontologists. Variation in extant species is used to determine taxonomic relationships as well as understand the possible range of variation for extinct species. In a previous study, we explored the presence of skeletal sexual dimorphism within a local population of gray squirrels (Sciurus carolinensis) from Putnam County, Georgia. No statistically significant sexual dimorphism was found. Recent studies by other researchers on additional squirrel species indicate the presence of cranial dimorphism in some taxa. Therefore, in this current study, intersexual and intrasexual cranial variation is being analyzed in our sample of gray squirrels from Putnam County. Twelve standard measurements are being recorded on 55 skeletons (30 females, 25 males) housed in the Georgia College Recent Mammal Collection. Measurements include total skull length, condylobasal length, length of upper diastema, length of upper molar row in alveoli, length of lower molar row in alveoli, length of the os palatinum, mandible length, zygomatic breadth, brain-case width, interorbital width, brain-case height per bullae, and mandible height. The potential significance of this study is two-fold: first, which of the cranial measurements is accurately repeatable and useful in morphological studies; and second, does cranial sexual dimorphism exist in this species that does not show skeletal dimorphism

DETERMINGING THE BINDING SITE OF ADENOVIRUS E4 11K TO CELLULAR DDX6**

Adenovirus is a linear double-stranded DNA virus known to cause illnesses such as conjunctivitis and the common cold. The virus uses the host cell’s machinery as a means of its own genome replication and protein synthesis. Early genes often modulate this disruption of the host cell. Our protein of interest is the early viral protein E4 11k and is encoded in early region 4 open reading frame 3 (E4 ORF3) of the genome. E4 11k is known to play key roles in the inhibition of host cell protein synthesis, while facilitating the stimulation of viral protein synthesis. In serotype 5 of adenovirus (Ad5), E4 11k has been observed to localize with cellular processing bodies (P-bodies) and binds to the P-body protein, Ddx6. This interaction is thought to play a role in the reorganization of P-bodies to aggregates of misfolded proteins, termed aggresomes. Our goal is to determine the binding site between these two proteins and to observe the consequences of disrupting that binding on the viral lifecycle. To do this, Ad5 and Ad9 hybrids of the viral E4 ORF3 gene will be transfected into A549 cells, a human lung carcinoma cell line. Our current objective is to optimize our transfection methods. We have used a number of different transfection reagents and methods such as the use of lipofectamine, and various protocols using polyethylenimine (PEI) and calcium phosphate. Following the success of our transfection, we will perform a co-immunoprecipitation assay to narrow down the region of binding between the two proteins. Alanine amino acid substitutions will be used to further narrow the binding site. Once the binding site has been determined, the interaction between E4 11k and Ddx6 can be disrupted and the role E4 11k has on the re-organization of P-bodies and host cell protein shut off can be determined