Supernatants derived from chemotherapy-treated cancer cell lines can modify angiogenesis

BACKGROUND: There is evidence that tumours produce substances such as cytokines and microvesicular bodies bearing bioactive molecules, which support the carcinogenic process. Furthermore, chemotherapy has also been shown to modify these exudates and in doing so, neutralise their tumourigenic influence. METHODS: In the current study, we have investigated the effect of chemotherapy agents on modifying the cytokine profile and microvesicular cargo of supernatants derived from cancer cell lines. In addition, we have explored the effect of these tumour-derived supernatants on angiogenesis, and how chemotherapy can alter the supernatants rendering them less pro-angiogenic. RESULTS: Herein, we show that supernatants contain a rich cocktail of cytokines, a number of which are potent modulators of angiogenesis. They also contain microvesicular bodies containing RNA transcripts that code for proteins involved in transcription, immune modulation and angiogenesis. These supernatants altered intracellular signalling molecules in endothelial cells and significantly enhanced their tubulogenic character; however, this was severely compromised when supernatants from tumours treated with chemotherapy was used instead. CONCLUSION: This study suggests tumour exudates and bioactive material from tumours can influence cellular functions, and that treatment with some chemotherapy can serve to negate these pro-tumourigenic processes

Stronger inflammatory/cytotoxic T cell response in women identified by microarray analysis

Women develop chronic inflammatory autoimmune diseases like lupus more often than men. The mechanisms causing the increased susceptibility are incompletely understood, although estrogen is believed to contribute. Chronic immune stimulation characterizes many autoimmune disorders. We hypothesized that repeated stimulation may cause a different T cell immune response in women than men. Microarray approaches were used to compare gene expression in T cells from healthy men and women with and without repeated stimulation. Four days following a single stimulation only 25% of the differentially expressed, gender-biased genes were expressed at higher levels in the women. In contrast, following restimulation 72% were more highly expressed in women. Immune response genes were significantly over-represented among the genes upregulated in women, and among the immune response genes, the inflammatory/cytotoxic effector genes interferon gamma (IFNG), lymphotoxin beta (LTB), granzyme A (GZMA), interleukin-12 receptor beta2 (IL12RB2), and granulysin (GNLY) were among those overexpressed to the greatest degree. In contrast, IL17A was the only effector gene more highly expressed in men. Estrogen response elements were identified in the promoters of half of the overexpressed immune genes in women, and in <10% of the male biased genes. The differential expression of inflammatory/cytotoxic effector molecules in restimulated female T cells may contribute to the differences in autoimmune diseases between women and men

Dissecting the molecular genetics and pathogenesis of Hereditary Dyserythropoietic Anemias

Hereditary anemias (HAs) embrace a heterogeneous group of chronic disorders with a highly variable clinical picture. Within HAs, congenital dyserythropoietic anemias (CDAs) are a large group of hypo-productive anemias that result from various kinds of abnormalities during late stages of erythropoiesis. Among them, CDAI is characterized by relative reticulocytopenia, and congenital anomalies. It is caused by biallelic mutations in CDAN1 and C15orf41. Differential diagnosis, classification, and patient stratification of CDAs and related HAs are often difficult, particularly between CDAI-II and enzymatic defects, such as pyruvate kinase deficiency (PKD). The classical diagnostic workflow for these conditions includes different lines of investigation, in which genetic testing by next generation sequencing (NGS) approaches has become the frontline system. Indeed, the primary aim of this study was to analyze a large cohort of HAs patients (n=244), by our (t)-NGS RedPanel, to identify the proper molecular diagnosis despite their clinical suspicion. Indeed, only 16.3% of patients originally suspected to suffer from CDA (14/86) showed a matched genotype. Conversely, 64% of patients (72/86) initially suspected for CDA were diagnosed as other HAs, mainly PKD. In agreement with this observation, the analysis of the main erythroid markers demonstrated that PKD patients showed a dyserythropoietic component that may underlie the frequent misdiagnosis with CDAI-II. Beyond achieving a definitive diagnosis, knowing the genetic basis of these patients is valuable also for guiding treatment. Indeed, in our cohort of patients, we identified a novel case of syndromic CDA due to a novel variant in CAD gene, leading to a specific treatment with uridine supplementation. Finally, we described three cases of CDAI, identifying two novel variants in the DNA binding domain of C15orf41, Y94S and P20T, and another one in the nuclease domain of the protein, H230P. Functional characterization of these variants showed that the H230P leads to reduced gene expression and protein levels, while Y94S and P20T do not affect C15orf41 expression. Moreover, Y94S and H230P variants accounted for impaired erythroid differentiation in K562 cells, and H230P mutant also exhibits an increased S-phase of the cell cycle. Nowadays, C15orf41 is still an uncharacterized gene, encoding a protein with an unknown function. Thus, we aimed to unravel novel insights on its physiological role. Indeed, we demonstrated that C15orf41 endogenous protein exhibits nuclear and cytosolic localization, being mostly in the nucleus. Our data showed that C15orf41 is a cell-cycle regulated protein, mostly expressed during G1/S phase, and that both the predicted isoforms of the protein are degraded by the ubiquitin-proteasome pathway. Finally, we demonstrated that gene expression of C15orf41 and CDAN1, the other causative gene of CDAI, is tightly correlated, suggesting a shared mechanism of regulation between the two genes. Overall, these studies pointed out the relevance of genetic testing for the achievement of a correct and definitive diagnosis of CDAs and the related HAs, for the treatment of these conditions, and for elucidating the underlying pathogenic mechanisms of such rare disorders

Interrogation of mammalian protein complex structure, function, and membership using genome-scale fitness screens

Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes, as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9-based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks that recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone, and in combination with large-scale biophysical data, to enhance our understanding of mammalian protein complexes in normal and disease states

Genetic variability in adenosine deaminase-like contributes to variation in alcohol preference in mice.

BACKGROUND: A substantial part of the risk for alcohol use disorder (AUD) is determined by genetic factors. We previously used chromosome substitution (CSS) mice, to identify a QTL for alcohol preference on mouse chromosome 2. The aim of this study was to identify candidate genes within this QTL that confer the risk for alcohol preference. METHODS: In order to delineate the neurobiological underpinnings of alcohol consumption, we expanded on the QTL approach to identify candidate genes for high alcohol preference in mice. We narrowed down a QTL for alcohol preference on mouse chromosome 2, that we previously identified using chromosome substitution (CSS) mice, to four candidate genes in silico. Expression levels of these candidate genes in prefrontal cortex, amygdala and nucleus accumbens, brain regions implicated in reward and addiction, were subsequently compared for the CSS-2 and the C57BL/6J host strain. RESULTS: We observed increased expression of adenosine deaminase-like (Adal) in all three regions in CSS-2 mice. Moreover, we found that the adenosine deaminase inhibitor EHNA reduced the difference in alcohol preference between CSS-2 and C57Bl/6J mice. CONCLUSION: The current study identifies Adal as a genetically protective factor against alcohol consumption in mice, in which elevated Adal levels contribute to low alcohol preference. This article is protected by copyright. All rights reserved

A genome-wide association study on liver stiffness changes during hepatitis c virus infection cure

Liver stiffness (LS) at sustained virological response (SVR) after direct-acting antivirals (DAA)-based therapy is a predictor of liver events in hepatitis C virus (HCV)-infected patients. The study aim was to identify genetic factors associated with LS changes from the moment of starting anti-HCV therapy to SVR. This prospective study included HCV-infected patients from the GEHEP 011 cohort who achieved SVR with DAA-based therapy, with LS pre-treatment ≥9.5 kPa and LS measurement available at SVR. Plink and Magma software were used to carry out genome-wide single-nucleotide polymorphism (SNP)-based and gene-based association analyses, respectively. The ShinyGO application was used for exploring enrichment in Gene Ontology (GO) categories for biological processes. Overall, 242 patients were included. Median (quartile 1, quartile 3) LS values at pre-treatment and at SVR were 16.8 (12, 28) kPa and 12.0 (8.5, 19.3) kPa, respectively. Thirty-five SNPs and three genes reached suggestive association with LS changes from the moment of starting anti HCV therapy to SVR. GO categories related to DNA packaging complex, DNA conformation change, chromosome organization and chromatin organization were significantly enriched. Our study reports possible genetic factors associated with LS changes during HCV-infection cure. In addition, our results suggest that processes related to DNA conformation are also involved in these changes

Biochemical characterization of C15ORF41: a novel nuclease

Congenital dyserythropoietic anaemia type I (CDA-I) is a rare autosomal recessive form of anaemia that is characterized by ineffective erythropoiesis and abnormal morphologies of bone marrow erythroblasts. While previous studies have indicated mutations in the CDAN1 gene for a large group of patients with CDA-I, new research has revealed that mutations in the C15ORF41 gene are also linked to patients with the condition. With the recent purification of the C15ORF41 protein by the Structural Genomics Consortium at the University of Oxford, a series of biochemical assays were conducted in order to characterize the catalytic and mechanistic behaviour of the protein. Nuclease assays indicated that the protein, when co-purified with the CDAN1 protein Codanin1, acts as a single-stranded endonuclease that acts on both DNA and RNA substrates. These assays also revealed that the complex incises in 5-6 nucleotide increments in a 3’-to-5’ directionality and exhibits a preference for 3’ flaps. Electrophoretic Mobility Shift Assays indicated that there is likely a 2:1 protein to substrate stoichiometry. Fluorescence anisotropy data showed that the C15ORF41-Codanin complex appeared to have much higher affinity to DNA substrates than the individual constituent proteins. The results of this study provided initial evidence into the predicted endonucleolytic behaviour of the protein and established a solid foundation for further investigations of the mechanistic basis of its activity. This is absolutely critical for the molecular understanding of CDA-I for the formulation of future treatments, and is the first step in examining a novel endonuclease that could further our grasp on DNA damage repair proteins

Movement disorders associated with hemochromatosis

Background: Hereditary hemochromatosis (HH) is a genetic disorder causing pathological iron deposition and functional impairment of various organs, predominantly the liver. We assessed patients with HH for the presence of movement disorders. Methods: We reviewed the charts of 616 patients with HH who attended hemochromatosis clinic at London Health Sciences Centre, London, ON, Canada, from 1988 to 2015. Results: We found three HH patients with movement disorders, without any other major systemic manifestation. One had parkinsonism, another had chorea, and the third had tremor. All three patients had evidence of iron deposition in the brain, affecting the basal ganglia in the first two, and the dentate nucleus, red nucleus, and substantia nigra in the third patient. In addition to the C282Y homozygous mutation in the HFE gene, two of our patients had non-HFE gene mutations. Conclusion: HH should be considered in the differential diagnosis of movement disorders with pathological brain iron deposition. We report for the first time chorea in a patient with HH. Non-HFE gene mutations may predispose HH patients to iron deposition in the brain