Polymorphisms in the bradykinin B2 receptor gene and childhood asthma

Bradykinin has been suggested as one of the key mediators of bronchial asthma. Polymorphisms with a potential functional relevance have been described in the B2 bradykinin receptor gene. Study of these polymorphisms in 77 children with asthma and 73 controls revealed no association. However, when comparing the asthmatics according to their age at onset (before and after age 4), the exon 1 allele BE1-2G was significantly associated with late-onset asthma (p <0.05). Since BE1-2G has previously been shown to lead to a higher transcription rate of the B2 receptor, this result warrants further investigation of the role of bradykinin in conferring susceptibility to pediatric asthma

A new insight into MYC action: control of RNA polymerase II methylation and transcription termination

MYC oncoprotein deregulation is a common catastrophic event in human cancer and limiting its activity restrains tumor development and maintenance, as clearly shown via Omomyc, an MYC-interfering 90 amino acid mini-protein. MYC is a multifunctional transcription factor that regulates many aspects of transcription by RNA polymerase II (RNAPII), such as transcription activation, pause release, and elongation. MYC directly associates with Protein Arginine Methyltransferase 5 (PRMT5), a protein that methylates a variety of targets, including RNAPII at the arginine residue R1810 (R1810me2s), crucial for proper transcription termination and splicing of transcripts. Therefore, we asked whether MYC controls termination as well, by affecting R1810me2S. We show that MYC overexpression strongly increases R1810me2s, while Omomyc, an MYC shRNA, or a PRMT5 inhibitor and siRNA counteract this phenomenon. Omomyc also impairs Serine 2 phosphorylation in the RNAPII carboxyterminal domain, a modification that sustains transcription elongation. ChIP-seq experiments show that Omomyc replaces MYC and reshapes RNAPII distribution, increasing occupancy at promoter and termination sites. It is unclear how this may affect gene expression. Transcriptomic analysis shows that transcripts pivotal to key signaling pathways are both up- or down-regulated by Omomyc, whereas genes directly controlled by MYC and belonging to a specific signature are strongly down-regulated. Overall, our data point to an MYC/PRMT5/RNAPII axis that controls termination via RNAPII symmetrical dimethylation and contributes to rewiring the expression of genes altered by MYC overexpression in cancer cells. It remains to be clarified which role this may have in tumor development

The Protein Arginine Methyltransferases 1 and 5 affect Myc properties in glioblastoma stem cells

Protein Arginine (R) methylation is the most common post-translational methylation in mammalian cells. Protein Arginine Methyltransferases (PRMT) 1 and 5 dimethylate their substrates on R residues, asymmetrically and symmetrically, respectively. They are ubiquitously expressed and play fundamental roles in tumour malignancies, including glioblastoma multiforme (GBM) which presents largely deregulated Myc activity. Previously, we demonstrated that PRMT5 associates with Myc in GBM cells, modulating, at least in part, its transcriptional properties. Here we show that Myc/PRMT5 protein complex includes PRMT1, in both HEK293T and glioblastoma stem cells (GSCs). We demonstrate that Myc is both asymmetrically and symmetrically dimethylated by PRMT1 and PRMT5, respectively, and that these modifications differentially regulate its stability. Moreover, we show that the ratio between symmetrically and asymmetrically dimethylated Myc changes in GSCs grown in stem versus differentiating conditions. Finally, both PRMT1 and PRMT5 activity modulate Myc binding at its specific target promoters. To our knowledge, this is the first work reporting R asymmetrical and symmetrical dimethylation as novel Myc post-translational modifications, with different functional properties. This opens a completely unexplored field of investigation in Myc biology and suggests symmetrically dimethylated Myc species as novel diagnostic and prognostic markers and druggable therapeutic targets for GBM

The effects of inspiratory muscle training in older adults

Purpose: Declining inspiratory muscle function and structure and systemic low-level inflammation and oxidative stress may contribute to morbidity and mortality during normal ageing. Therefore, we examined the effects of inspiratory muscle training (IMT) in older adults on inspiratory muscle function and structure and systemic inflammation and oxidative stress, and re-examined the reported positive effects of IMT on respiratory muscle strength, inspiratory muscle endurance, spirometry, exercise performance, physical activity levels (PAL) and quality of life (QoL). Methods: Thirty-four healthy older adults (68 ± 3 years) with normal spirometry, respiratory muscle strength and physical fitness were divided equally into a pressure-threshold IMT or sham-hypoxic placebo group. Before and after an 8 week intervention, measurements were taken for dynamic inspiratory muscle function and inspiratory muscle endurance using a weighted plunger pressure-threshold loading device, diaphragm thickness using B-mode ultrasonography, plasma cytokine concentrations using immunoassays, DNA damage levels in peripheral blood mononuclear cells (PBMC) using Comet Assays, spirometry, maximal mouth pressures, exercise performance using a six minute walk test, PAL using a questionnaire and accelerometry, and QoL using a questionnaire

Novel statistical approaches for non-normal censored immunological data: analysis of cytokine and gene expression data

Background: For several immune-mediated diseases, immunological analysis will become more complex in the future with datasets in which cytokine and gene expression data play a major role. These data have certain characteristics that require sophisticated statistical analysis such as strategies for non-normal distribution and censoring. Additionally, complex and multiple immunological relationships need to be adjusted for potential confounding and interaction effects. Objective: We aimed to introduce and apply different methods for statistical analysis of non-normal censored cytokine and gene expression data. Furthermore, we assessed the performance and accuracy of a novel regression approach in order to allow adjusting for covariates and potential confounding. Methods: For non-normally distributed censored data traditional means such as the Kaplan-Meier method or the generalized Wilcoxon test are described. In order to adjust for covariates the novel approach named Tobit regression on ranks was introduced. Its performance and accuracy for analysis of non-normal censored cytokine/gene expression data was evaluated by a simulation study and a statistical experiment applying permutation and bootstrapping. Results: If adjustment for covariates is not necessary traditional statistical methods are adequate for non-normal censored data. Comparable with these and appropriate if additional adjustment is required, Tobit regression on ranks is a valid method. Its power, type-I error rate and accuracy were comparable to the classical Tobit regression. Conclusion: Non-normally distributed censored immunological data require appropriate statistical methods. Tobit regression on ranks meets these requirements and can be used for adjustment for covariates and potential confounding in large and complex immunological datasets

Myc and Omomyc functionally associate with the Protein Arginine Methyltransferase 5 (PRMT5) in glioblastoma cells

The c-Myc protein is dysregulated in many human cancers and its function has not been fully elucitated yet. The c-Myc inhibitor Omomyc displays potent anticancer properties in animal models. It perturbs the c-Myc protein network, impairs c-Myc binding to the E-boxes, retaining transrepressive properties and inducing histone deacetylation. Here we have employed Omomyc to further analyse c-Myc activity at the epigenetic level. We show that both Myc and Omomyc stimulate histone H4 symmetric dimethylation of arginine (R) 3 (H4R3me2s), in human glioblastoma and HEK293T cells. Consistently, both associated with protein Arginine Methyltransferase 5 (PRMT5)-the catalyst of the reaction-and its co-factor Methylosome Protein 50 (MEP50). Confocal experiments showed that Omomyc co-localized with c-Myc, PRMT5 and H4R3me2s-enriched chromatin domains. Finally, interfering with PRMT5 activity impaired target gene activation by Myc whereas it restrained Omomyc-dependent repression. The identification of a histone-modifying complex associated with Omomyc represents the first demonstration of an active role of this miniprotein in modifying chromatin structure and adds new information regarding its action on c-Myc targets. More importantly, the observation that c-Myc may recruit PRMT5-MEP50, inducing H4R3 symmetric di-methylation, suggests previously unpredictable roles for c-Myc in gene expression regulation and new potential targets for therapy

Early age exposure to moisture damage and systemic inflammation at the age of 6 years

Cross-sectional studies have shown that exposure to indoor moisture damage and mold may be associated with subclinical inflammation. Our aim was to determine whether early age exposure to moisture damage or mold is prospectively associated with subclinical systemic inflammation or with immune responsiveness in later childhood. Home inspections were performed in children's homes in the first year of life. At age 6 years, subclinical systemic inflammation was measured by serum C-reactive protein(CRP) and blood leucocytes and immune responsiveness by ex vivo production of interleukin 1-beta(IL-1beta), IL-6 and tumor necrosis factor-alpha(TNF-alpha) in whole blood cultures without stimulation or after 24h stimulation with phorbol 12-myristate 13-acetate and ionomycin(PI), lipopolysaccharide(LPS) or peptidoglycan(PPG) in 251 to 270 children. Moisture damage in child's main living areas in infancy was not significantly associated with elevated levels of CRP or leucocytes at 6 years. In contrast, there was some suggestion for an effect on immune responsiveness, as moisture damage with visible mold was positively associated with LPS-stimulated production of TNF-alpha and minor moisture damage was inversely associated with PI-stimulated IL-1beta. While early life exposure to mold damage may have some influence on later immune responsiveness, it does not seem to increase subclinical systemic inflammation in later life. This article is protected by copyright. All rights reserved

Agenesis of the putamen and globus pallidus caused by recessive mutations in the homeobox gene GSX2

Basal ganglia are subcortical grey nuclei that play essential roles in controlling voluntary movements, cognition and emotion. While basal ganglia dysfunction is observed in many neurodegenerative or metabolic disorders, congenital malformations are rare. In particular, dysplastic basal ganglia are part of the malformative spectrum of tubulinopathies and X-linked lissencephaly with abnormal genitalia, but neurodevelopmental syndromes characterized by basal ganglia agenesis are not known to date. We ascertained two unrelated children (both female) presenting with spastic tetraparesis, severe generalized dystonia and intellectual impairment, sharing a unique brain malformation characterized by agenesis of putamina and globi pallidi, dysgenesis of the caudate nuclei, olfactory bulbs hypoplasia, and anomaly of the diencephalic-mesencephalic junction with abnormal corticospinal tract course. Whole-exome sequencing identified two novel homozygous variants, c.26C>A; p.(S9*) and c.752A>G; p.(Q251R) in the GSX2 gene, a member of the family of homeobox transcription factors, which are key regulators of embryonic development. GSX2 is highly expressed in neural progenitors of the lateral and median ganglionic eminences, two protrusions of the ventral telencephalon from which the basal ganglia and olfactory tubercles originate, where it promotes neurogenesis while negatively regulating oligodendrogenesis. The truncating variant resulted in complete loss of protein expression, while the missense variant affected a highly conserved residue of the homeobox domain, was consistently predicted as pathogenic by bioinformatic tools, resulted in reduced protein expression and caused impaired structural stability of the homeobox domain and weaker interaction with DNA according to molecular dynamic simulations. Moreover, the nuclear localization of the mutant protein in transfected cells was significantly reduced compared to the wild-type protein. Expression studies on both patients' fibroblasts demonstrated reduced expression of GSX2 itself, likely due to altered transcriptional self-regulation, as well as significant expression changes of related genes such as ASCL1 and PAX6. Whole transcriptome analysis revealed a global deregulation in genes implicated in apoptosis and immunity, two broad pathways known to be involved in brain development. This is the first report of the clinical phenotype and molecular basis associated to basal ganglia agenesis in humans