Formation of Complex and Unstable Chromosomal Translocations in Yeast

Genome instability, associated with chromosome breakage syndromes and most human cancers, is still poorly understood. In the yeast Saccharomyces cerevisiae, numerous genes with roles in the preservation of genome integrity have been identified. DNA-damage-checkpoint-deficient yeast cells that lack Sgs1, a RecQ-like DNA helicase related to the human Bloom's-syndrome-associated helicase BLM, show an increased rate of genome instability, and we have previously shown that they accumulate recurring chromosomal translocations between three similar genes, CAN1, LYP1 and ALP1. Here, the chromosomal location, copy number and sequence similarity of the translocation targets ALP1 and LYP1 were altered to gain insight into the formation of complex translocations. Among 844 clones with chromosomal rearrangements, 93 with various types of simple and complex translocations involving CAN1, LYP1 and ALP1 were identified. Breakpoint sequencing and mapping showed that the formation of complex translocation types is strictly dependent on the location of the initiating DNA break and revealed that complex translocations arise via a combination of interchromosomal translocation and template-switching, as well as from unstable dicentric intermediates. Template-switching occurred between sequences on the same chromosome, but was inhibited if the genes were transferred to different chromosomes. Unstable dicentric translocations continuously gave rise to clones with multiple translocations in various combinations, reminiscent of intratumor heterogeneity in human cancers. Base substitutions and evidence of DNA slippage near rearrangement breakpoints revealed that translocation formation can be accompanied by point mutations, and their presence in different translocation types within the same clone provides evidence that some of the different translocation types are derived from each other rather than being formed de novo. These findings provide insight into eukaryotic genome instability, especially the formation of translocations and the sources of intraclonal heterogeneity, both of which are often associated with human cancers

Dipeptidyl peptidase-1 inhibition in patients hospitalised with COVID-19: a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial

Background Neutrophil serine proteases are involved in the pathogenesis of COVID-19 and increased serine protease activity has been reported in severe and fatal infection. We investigated whether brensocatib, an inhibitor of dipeptidyl peptidase-1 (DPP-1; an enzyme responsible for the activation of neutrophil serine proteases), would improve outcomes in patients hospitalised with COVID-19. Methods In a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial, across 14 hospitals in the UK, patients aged 16 years and older who were hospitalised with COVID-19 and had at least one risk factor for severe disease were randomly assigned 1:1, within 96 h of hospital admission, to once-daily brensocatib 25 mg or placebo orally for 28 days. Patients were randomly assigned via a central web-based randomisation system (TruST). Randomisation was stratified by site and age (65 years or ≥65 years), and within each stratum, blocks were of random sizes of two, four, or six patients. Participants in both groups continued to receive other therapies required to manage their condition. Participants, study staff, and investigators were masked to the study assignment. The primary outcome was the 7-point WHO ordinal scale for clinical status at day 29 after random assignment. The intention-to-treat population included all patients who were randomly assigned and met the enrolment criteria. The safety population included all participants who received at least one dose of study medication. This study was registered with the ISRCTN registry, ISRCTN30564012. Findings Between June 5, 2020, and Jan 25, 2021, 406 patients were randomly assigned to brensocatib or placebo; 192 (47·3%) to the brensocatib group and 214 (52·7%) to the placebo group. Two participants were excluded after being randomly assigned in the brensocatib group (214 patients included in the placebo group and 190 included in the brensocatib group in the intention-to-treat population). Primary outcome data was unavailable for six patients (three in the brensocatib group and three in the placebo group). Patients in the brensocatib group had worse clinical status at day 29 after being randomly assigned than those in the placebo group (adjusted odds ratio 0·72 [95% CI 0·57–0·92]). Prespecified subgroup analyses of the primary outcome supported the primary results. 185 participants reported at least one adverse event; 99 (46%) in the placebo group and 86 (45%) in the brensocatib group. The most common adverse events were gastrointestinal disorders and infections. One death in the placebo group was judged as possibly related to study drug. Interpretation Brensocatib treatment did not improve clinical status at day 29 in patients hospitalised with COVID-19

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

Modification of Trophic Links between an Omnivore and Macroinfaunal Prey from Sandy Beaches of differing Physical Regimes

Sandy beach ecosystems have been studied worldwide; however, ecological data are sparse for the extensive barrier islands of Florida. Accordingly, I investigated the feeding patterns of the ghost crab (Ocypode quadrata), a dominant omnivore inhabiting beaches along the Floridian coast. Density data was collected for ghost crabs and swash macroinfaunal prey. In addition, I utilized stable isotopes in conjunction with the mixing models IsoSource and SIAR to characterize diets of ghost crabs across three barrier islands in spring and summer 2011. Results showed that ghost crabs at Cayo Costa feed primarily on swash macroinfauna, while those from Anclote Key shifted their diet to one comprised primarily of semi-terrestrial amphipods. However, at Honeymoon Island, ghost crab isotopic signatures were best explained by a mixed diet of both macroinfauna and wrack-associated prey. The unique consumption of wrack fauna at Anclote Key co-occurs with comparatively low infaunal densities and biomass, and modified ghost crab behavior due to trait-mediated effects. My results are novel because they suggest that wrack-associated fauna may be an important food source for ghost crabs in certain beach regimes

Predator Diet and Trophic Position Modified with Altered Habitat Morphology

Empirical patterns that emerge from an examination of food webs over gradients of environmental variation can help to predict the implications of anthropogenic disturbance on ecosystems. This “dynamic food web approach” is rarely applied at the coastal margin where aquatic and terrestrial systems are coupled and human development activities are often concentrated. We propose a simple model of ghost crab (Ocypode quadrata) feeding that predicts changing dominant prey (Emerita talpoida, Talorchestia sp., Donax variablis) along a gradient of beach morphology and test this model using a suite of 16 beaches along the Florida, USA coast. Assessment of beaches included quantification of morphological features (width, sediments, slope), macrophyte wrack, macro-invertebrate prey and active ghost crab burrows. Stable isotope analysis of carbon (13C/12C) and nitrogen (15N/14N) and the SIAR mixing model were used to determine dietary composition of ghost crabs at each beach. The variation in habitat conditions displayed with increasing beach width was accompanied by quantifiable shifts in ghost crab diet and trophic position. Patterns of ghost crab diet were consistent with differences recorded across the beach width gradient with respect to the availability of preferred micro-habitats of principal macro-invertebrate prey. Values obtained for trophic position also suggests that the generalist ghost crab assembles and augments its diet in fundamentally different ways as habitat morphology varies across a highly dynamic ecosystem. Our results offer support for a functional response in the trophic architecture of a common food web compartment (ghost crabs, macro-invertebrate prey) across well-known beach morphologies. More importantly, our “dynamic food web approach” serves as a basis for evaluating how globally wide-spread sandy beach ecosystems should respond to a variety of anthropogenic impacts including beach grooming, beach re-nourishment, introduction of non-native or feral predators and human traffic on beaches

Predator Diet and Trophic Position Modified with Altered Habitat Morphology

Empirical patterns that emerge from an examination of food webs over gradients of environmental variation can help to predict the implications of anthropogenic disturbance on ecosystems. This “dynamic food web approach” is rarely applied at the coastal margin where aquatic and terrestrial systems are coupled and human development activities are often concentrated. We propose a simple model of ghost crab (Ocypode quadrata) feeding that predicts changing dominant prey (Emerita talpoida, Talorchestia sp., Donax variablis) along a gradient of beach morphology and test this model using a suite of 16 beaches along the Florida, USA coast. Assessment of beaches included quantification of morphological features (width, sediments, slope), macrophyte wrack, macro-invertebrate prey and active ghost crab burrows. Stable isotope analysis of carbon (13C/12C) and nitrogen (15N/14N) and the SIAR mixing model were used to determine dietary composition of ghost crabs at each beach. The variation in habitat conditions displayed with increasing beach width was accompanied by quantifiable shifts in ghost crab diet and trophic position. Patterns of ghost crab diet were consistent with differences recorded across the beach width gradient with respect to the availability of preferred micro-habitats of principal macro-invertebrate prey. Values obtained for trophic position also suggests that the generalist ghost crab assembles and augments its diet in fundamentally different ways as habitat morphology varies across a highly dynamic ecosystem. Our results offer support for a functional response in the trophic architecture of a common food web compartment (ghost crabs, macro-invertebrate prey) across well-known beach morphologies. More importantly, our “dynamic food web approach” serves as a basis for evaluating how globally wide-spread sandy beach ecosystems should respond to a variety of anthropogenic impacts including beach grooming, beach re-nourishment, introduction of non-native or feral predators and human traffic on beaches

Predator Diet and Trophic Position Modified with Altered Habitat Morphology

<div><p>Empirical patterns that emerge from an examination of food webs over gradients of environmental variation can help to predict the implications of anthropogenic disturbance on ecosystems. This “dynamic food web approach” is rarely applied at the coastal margin where aquatic and terrestrial systems are coupled and human development activities are often concentrated. We propose a simple model of ghost crab (<i>Ocypode quadrata</i>) feeding that predicts changing dominant prey (<i>Emerita talpoida</i>, <i>Talorchestia</i> sp., <i>Donax variablis</i>) along a gradient of beach morphology and test this model using a suite of 16 beaches along the Florida, USA coast. Assessment of beaches included quantification of morphological features (width, sediments, slope), macrophyte wrack, macro-invertebrate prey and active ghost crab burrows. Stable isotope analysis of carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) and the SIAR mixing model were used to determine dietary composition of ghost crabs at each beach. The variation in habitat conditions displayed with increasing beach width was accompanied by quantifiable shifts in ghost crab diet and trophic position. Patterns of ghost crab diet were consistent with differences recorded across the beach width gradient with respect to the availability of preferred micro-habitats of principal macro-invertebrate prey. Values obtained for trophic position also suggests that the generalist ghost crab assembles and augments its diet in fundamentally different ways as habitat morphology varies across a highly dynamic ecosystem. Our results offer support for a functional response in the trophic architecture of a common food web compartment (ghost crabs, macro-invertebrate prey) across well-known beach morphologies. More importantly, our “dynamic food web approach” serves as a basis for evaluating how globally wide-spread sandy beach ecosystems should respond to a variety of anthropogenic impacts including beach grooming, beach re-nourishment, introduction of non-native or feral predators and human traffic on beaches.</p></div