Specific proteome changes in platelets from individuals with GATA1-, GFI1B-, and RUNX1-linked bleeding disorders.

Mutations in the transcription factors GATA binding factor 1 (GATA1), growth factor independence 1B (GFI1B), and Runt-related transcription factor 1 (RUNX1) cause familial platelet and bleeding disorders. Mutant platelets exhibit common abnormalities including an α-granule reduction resulting in a grayish appearance in blood smears. This suggests that similar pathways are deregulated by different transcription factor mutations. To identify common factors, full platelet proteomes from 11 individuals with mutant GATA1R216Q, GFI1BQ287*, RUNX1Q154Rfs, or RUNX1TD2-6 and 28 healthy controls were examined by label-free quantitative mass spectrometry. In total, 2875 platelet proteins were reliably quantified. Clustering analysis of more than 300 differentially expressed proteins revealed profound differences between cases and controls. Among cases, 44 of 143 significantly downregulated proteins were assigned to platelet function, hemostasis, and granule biology, in line with platelet dysfunction and bleedings. Remarkably, none of these proteins were significantly diminished in all affected cases. Similarly, no proteins were commonly overrepresented in all affected cases compared with controls. These data indicate that the studied transcription factor mutations alter platelet proteomes in distinct largely nonoverlapping manners. This work provides the quantitative landscape of proteins that affect platelet function when deregulated by mutated transcription factors in inherited bleeding disorders.

Let's talk about sex: A qualitative study exploring the experiences of HIV nurses when discussing sexual risk behaviours with HIV-positive men who have sex with men.

BACKGROUND: Despite prevention efforts, the incidence of sexually transmitted infection among HIV-positive men who have sex with men remains high, which is indicative of unchanged sexual risk behaviour. Discussing sexual risk behaviour has been shown to help prevent sexually transmitted infections among HIV-positive men who have sex with men. OBJECTIVES: The aim of this study was to identify factors that influence whether - and how - specialised HIV nurses discuss sexual risk behaviour with HIV-positive men who have sex with men. Identifying these factors could indicate how best to improve the frequency and quality of discussions about sexual risk behaviour, thereby reducing sexual risk behaviour and sexually transmitted infections. DESIGN: Qualitative study, focus groups among HIV nurses. SETTING: Dutch HIV treatment centres. PARTICIPANTS: A purposive sample was taken of 25 out of 87 HIV nurses working in one of the 26 specialised HIV treatment centres in the Netherlands. Of the 25 HIV nurses we approached, 22 participate in our study. METHODS: Three semi-structured focus group interviews were held with 22 HIV nurses from 17 hospitals. Interviews were transcribed verbatim, and thematic analysis was performed. RESULTS: HIV nurses agreed that discussing sexual risk behaviour is important, but barriers were experienced in relation to doing so. In accordance with the theory of planned behaviour, attitudes, perceived norms and perceived behavioural control were all found to be relevant variables. Barriers to discussing sexual risk behaviour were identified as: dealing with embarrassment, the changing professional role of an HIV nurse, time constraints, and the structure of the consultation. CONCLUSIONS: To improve the frequency and quality of discussions about sexual risk behaviour with HIV-positive men who have sex with men, our data suggests it would be beneficial to support HIV nurses by developing tools and guidelines addressing what to discuss and how. Using a related topic as a conversational 'bridge' may help nurses to broach this subject with their patients. This would allow HIV nurses to discuss possible risk reduction strategies, such as pre-exposure prophylaxis for HIV-negative partners, condom use, strategic positioning, or sero-sorting.

MLL2 mutation detection in 86 patients with Kabuki syndrome: a genotype-phenotype study.

Recently, pathogenic variants in the MLL2 gene were identified as the most common cause of Kabuki (Niikawa-Kuroki) syndrome (MIM#147920). To further elucidate the genotype-phenotype correlation, we studied a large cohort of 86 clinically defined patients with Kabuki syndrome (KS) for mutations in MLL2. All patients were assessed using a standardized phenotype list and all were scored using a newly developed clinical score list for KS (MLL2-Kabuki score 0-10). Sequencing of the full coding region and intron-exon boundaries of MLL2 identified a total of 45 likely pathogenic mutations (52%): 31 nonsense, 10 missense and four splice-site mutations, 34 of which were novel. In five additional patients, novel, i.e. non-dbSNP132 variants of clinically unknown relevance, were identified. Patients with likely pathogenic nonsense or missense MLL2 mutations were usually more severely affected (median 'MLL2-Kabuki score' of 6) as compared to the patients without MLL2 mutations (median 'MLL2-Kabuki score' of 5), a significant difference (p < 0.0014). Several typical facial features such as large dysplastic ears, arched eyebrows with sparse lateral third, blue sclerae, a flat nasal tip with a broad nasal root, and a thin upper and a full lower lip were observed more often in mutation positive patients.

Pharmacological characterization of a small-molecule agonist for the chemokine receptor CXCR3.

BACKGROUND AND PURPOSE: The chemokine receptor CXCR3 is a GPCR found predominantly on activated T cells. CXCR3 is activated by three endogenous peptides; CXCL9, CXCL10 and CXCL11. Recently, a small-molecule agonist, VUF10661, has been reported in the literature and synthesized in our laboratory. The aim of the present study was to provide a detailed pharmacological characterization of VUF10661 by comparing its effects with those of CXCL11. EXPERIMENTAL APPROACH: Agonistic properties of VUF10661 were assessed in a chemotaxis assay with murine L1.2 cells transiently transfected with cDNA encoding the human CXCR3 receptor and in binding studies, with [(125)I]-CXCL10 and [(125)I]-CXCL11, on membrane preparations from HEK293 cells stably expressing CXCR3. [(35)S]-GTPγS binding was used to determine its potency to induce CXCR3-mediated G protein activation and BRET-based assays to investigate its effects on intracellular cAMP levels and ß-arrestin recruitment. KEY RESULTS: VUF10661 acted as a partial agonist in CXCR3-mediated chemotaxis, bound to CXCR3 in an allosteric fashion in ligand binding assays and activated G(i) proteins with the same efficacy as CXCL11 in the [(35)S]-GTPγS binding and cAMP assay, while it recruited more ß-arrestin1 and ß-arrestin2 to CXCR3 receptors than the chemokine. CONCLUSIONS AND IMPLICATIONS: VUF10661, like CXCL11, activates both G protein-dependent and -independent signalling via the CXCR3 receptor, but probably exerts its effects from an allosteric binding site that is different from that for CXCL11. It could stabilize different receptor and/or ß-arrestin conformations leading to differences in functional output. Such ligand-biased signalling might offer interesting options for the therapeutic use of CXCR3 agonists.

An 8.35 Mb overlapping interstitial deletion of 8q24 in two patients with coloboma, congenital heart defect, limb abnormalities, psychomotor retardation and convulsions.

Chromosome analysis in two young patients with multiple congenital anomalies revealed a de novo interstitial deletion of 8q that has not been reported before. The deletions were overlapping by 8.35 Mb (8q24.21q24.23). The clinical features shared by our patients were coloboma, VSD, digital abnormalities, congenital dislocation of a hip, feeding problems, psychomotor delay and convulsions. The deletion included the region for Langer-Giedion syndrome (TRPS1 and EXT1) in the girl only. However, she is too young to present features of this syndrome, apart from dysmorphic features like a bulbous nose and notched alae nasi. Several genes are present in the commonly deleted region, including genes with unknown function, and genes for which haploinsufficiency is known to have no phenotypic effect in mice (Wnt1). A gene that might play a role in the convulsions of our patients is KCNQ3.