Quantitative analysis of gene expression changes in response to genotoxic compounds

Techniques that quantify molecular endpoints sufficiently sensitive to identify and classify potentially toxic compounds have wide potential for high-throughput in vitro screening. Expression of three genes, RAD51C, TP53 and cystatin A (CSTA), in HEPG2 cells was measured by Q-PCR amplification. In parallel, we developed alternative assays for the same 3 gene signature based on an acridinium-ester chemiluminescent reporter molecule. HEPG2 cells were challenged with eighteen different compounds (n = 18) chosen to represent compounds that are genotoxic (n = 8), non-genotoxic non-carcinogenic (n = 2) or have a less well defined mechanism of action with respect to genotoxicity (n = 8). At least one of the three genes displayed dysregulated expression in the majority of compounds tested by Q-PCR and ten compounds changed the CSTA expression significantly. Acridinium-ester labelled probes for the three genes were synthesised and tested. Analytical sensitivity was characterised and suggested a limit of detection generally better than 0.1 fmol but often 10–50 attomol. A linear amplification step was optimised and this quantitative method detected statistically significant increases in RAD51C and CSTA expression in agreement with the Q-PCR results, demonstrating the potential of this technology. The broad agreement of the amplified chemiluminescent method and Q-PCR in measuring gene expression suggests wider potential application for this chemiluminescent technology

5-Bromo-1-(4-bromophenyl)isatin

In the title compound [systematic name: 5-bromo-1-(4-bromo­phen­yl)-2,3-di­hydro-1H-indole-2,3-dione], C14H7Br2NO2, all of the atoms except the C—H groups in the bromo­benzene ring lie on a (010) crystallographic mirror plane, with the benzene ring completed by reflection. The dihedral angle between the ring systems is constrained to be 90° by symmetry. In the crystal, mol­ecules are linked by weak C—H...Br inter­actions in the [001] direction and paired very weak C—H...O inter­actions to the same acceptor in the [100] direction, generating (010) sheets. Possible extremely weak π–π stacking occurs between the layers

Crystal structure of 2-(bis(4-methoxyphenyl)amino)-2-oxoacetic acid, C16H15NO5

Abstract C16H15NO5, monoclinic, P21/n (no. 14), a = 6.7689(5) Å, b = 45.219(3) Å, c = 10.1102(6) Å, β = 101.360(7)°, V = 3033.9(4) Å3, T = 298(2) K.</jats:p

Sensitivity of the Wound Edge Gene Signature “WD14” in Responding to Clinical Change: A Longitudinal Cohort Study

Introduction: Genetic prognostication of chronic wounds is one recognised method of early identification of clinical wound healing status in order to target rigorous and advanced treatment regimens to hard-to-heal wounds. The WounD14 (WD14) gene signature is a recently developed scoring tool, derived from genetic interrogation of wound edge biopsies. It has been shown to predict the propensity of chronic venous leg ulcers to heal. However, it is unknown how WD14 responds with time and to changes in clinical wound healing status. The aim of this pilot study was therefore to evaluate if changes in the clinical healing status of wounds were identified by WD14 gene signature changes. Methods: WD14 was developed through a process of gene screening, refining and subsequent validation in three separate patient cohorts. Validation was undertaken in 85 consecutive patients referred to a tertiary wound healing unit with chronic venous leg ulcers, who underwent a wound edge biopsy to interrogate for a ‘healing’ or ‘non-healing’ genotype. A smaller cohort of patients (18%) underwent a second biopsy, which comprises this pilot cohort reported herein. 12 weeks after the biopsy wounds were clinically assessed for healing status and compared to WD14 genotype. Results: Sequential biopsies and WD14 scores were obtained from 16 patients. WD14 gene signature predicted clinical wound healing status among this cohort at either visit (total analysis of 32 wound edge biopsies) with a positive predictive value (PPV) of 85.2% (95% CI 74.1% to 92.0%) and negative predictive value (NPV) of 80.0% (95% CI 34.2% to 96.9%). Six wounds altered their clinical status between the two visits; in this cohort WD14 has a PPV of 66.7% (95% CI 47.3% to 81.7%) and NPV of 100%. Conclusion: Although the WD14 gene signature did change with wound healing status, further and larger studies are required to clarify precisely the role of this gene signature and its ability to prognosticate accurately over time with wounds of differing clinical status

Sensitivity of the wound edge gene signature "WD14" in responding to clinical change: a longitudinal cohort study

Introduction: WounD14 (WD14) gene signature is a recently developed tool derived from genetic interrogation of wound edge biopsies of chronic venous leg ulcers to identify heard-to-heal wounds and enable clinicians to target aggressive therapies to promote wound healing. This study aimed to evaluate if changes in wound clinical healing status were detected by the WD14 gene signature over time as this is currently poorly understood. Material and methods: WD14 was developed through gene screening and subsequent validation in 3 patient cohorts involving 85 consecutive patients with chronic venous leg ulcers referred to a tertiary wound healing unit. Patients underwent a wound edge biopsy to interrogate for a “healing” or “non-healing” genotype. A smaller cohort (18%) underwent a second biopsy, which comprised this pilot cohort reported herein. Twelve weeks following biopsy, wounds were clinically assessed for healing status based on reduction in size and compared to WD14 genotype. Results: Sequential biopsies and WD14 scores were derived from 16 patients. WD14 signature predicted wound healing status among this cohort at either visit (32 wound edge biopsies) with a positive predictive value (PPV) of 85.2% (95% CI 74.1%-92.0%) and negative predictive value (NPV) of 80.0% (95% CI 34.2%-96.9%). A total of 6 wounds underwent altered clinical status between the 2 visits. In this cohort, WD14 has a PPV of 66.7% (95% CI 47.3%-81.7%) and NPV of 100%. Conclusion: Although the WD14 gene signature did change with wound healing status, larger studies are required to precisely clarify its role and ability to prognosticate wounds of differing clinical status over time

Investigation of the international comparability of population-based routine hospital data set derived comorbidity scores for patients with lung cancer

Introduction: The International Cancer Benchmarking Partnership (ICBP) identified significant international differences in lung cancer survival. Differing levels of comorbid disease across ICBP countries has been suggested as a potential explanation of this variation but, to date, no studies have quantified its impact. This study investigated whether comparable, robust comorbidity scores can be derived from the different routine population-based cancer data sets available in the ICBP jurisdictions and, if so, use them to quantify international variation in comorbidity and determine its influence on outcome. Methods: Linked population-based lung cancer registry and hospital discharge data sets were acquired from nine ICBP jurisdictions in Australia, Canada, Norway and the UK providing a study population of 233 981 individuals. For each person in this cohort Charlson, Elixhauser and inpatient bed day Comorbidity Scores were derived relating to the 4–36 months prior to their lung cancer diagnosis. The scores were then compared to assess their validity and feasibility of use in international survival comparisons. Results: It was feasible to generate the three comorbidity scores for each jurisdiction, which were found to have good content, face and concurrent validity. Predictive validity was limited and there was evidence that the reliability was questionable. Conclusion: The results presented here indicate that interjurisdictional comparability of recorded comorbidity was limited due to probable differences in coding and hospital admission practices in each area. Before the contribution of comorbidity on international differences in cancer survival can be investigated an internationally harmonised comorbidity index is required

The Role of Whole Blood Impedance Aggregometry and Its Utilisation in the Diagnosis and Prognosis of Patients with Systemic Inflammatory Response Syndrome and Sepsis in Acute Critical Illness

Objective: To assess the prognostic and diagnostic value of whole blood impedance aggregometry in patients with sepsis and SIRS and to compare with whole blood parameters (platelet count, haemoglobin, haematocrit and white cell count). Methods: We performed an observational, prospective study in the acute setting. Platelet function was determined using whole blood impedance aggregometry (multiplate) on admission to the Emergency Department or Intensive Care Unit and at 6 and 24 hours post admission. Platelet count, haemoglobin, haematocrit and white cell count were also determined. Results: 106 adult patients that met SIRS and sepsis criteria were included. Platelet aggregation was significantly reduced in patients with severe sepsis/septic shock when compared to SIRS/uncomplicated sepsis (ADP: 90.7±37.6 vs 61.4±40.6; p<0.001, Arachadonic Acid 99.9±48.3 vs 66.3±50.2; p = 0.001, Collagen 102.6±33.0 vs 79.1±38.8; p = 0.001; SD ± mean)). Furthermore platelet aggregation was significantly reduced in the 28 day mortality group when compared with the survival group (Arachadonic Acid 58.8±47.7 vs 91.1±50.9; p<0.05, Collagen 36.6±36.6 vs 98.0±35.1; p = 0.001; SD ± mean)). However haemoglobin, haematocrit and platelet count were more effective at distinguishing between subgroups and were equally effective indicators of prognosis. Significant positive correlations were observed between whole blood impedance aggregometry and platelet count (ADP 0.588 p<0.0001, Arachadonic Acid 0.611 p<0.0001, Collagen 0.599 p<0.0001 (Pearson correlation)). Conclusions: Reduced platelet aggregometry responses were not only significantly associated with morbidity and mortality in sepsis and SIRS patients, but also correlated with the different pathological groups. Whole blood aggregometry significantly correlated with platelet count, however, when we adjust for the different groups we investigated, the effect of platelet count appears to be non-significant

Human peritoneal mesothelial cells respond to bacterial ligands through a specific subset of Toll-like receptors

Background. Bacterial infection remains a major cause of morbidity and mortality in peritoneal dialysis (PD) patients worldwide. Previous studies have identified a key role for mesothelial cells, lining the peritoneal cavity, in coordinating inflammation and host defense. Toll-like receptor (TLR) involvement in early activation events within the mesothelium, however, remains poorly defined. To investigate the initiation of bacterial peritonitis, we characterized TLR activation by bacterial ligands in human peritoneal mesothelial cells (HPMC)

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease