Sorting nexin 24 is required for α-granule biogenesis and cargo delivery in megakaryocytes.

Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterized by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be involved in platelet α-granule biogenesis by undertaking transcriptome analysis of control platelets and platelets harboring a DNA-binding variant of FLI1. Our analysis identified 2,276 transcripts that were differentially expressed in FLI1-deficient platelets. Functional annotation clustering of the coding transcripts revealed significant enrichment for gene annotations relating to protein transport, and identified Sorting nexin 24 (SNX24) as a candidate for further investigation. Using an induced pluripotent stem cell-derived megakaryocyte model, SNX24 expression was found to be increased during the early stages of megakaryocyte differentiation and downregulated during proplatelet formation, indicating tight regulatory control during megakaryopoiesis. CRISPR-Cas9 mediated knockout (KO) of SNX24 led to decreased expression of immature megakaryocyte markers, CD41 and CD61, and increased expression of the mature megakaryocyte marker CD42b (P=0.0001), without affecting megakaryocyte polyploidisation, or proplatelet formation. Electron microscopic analysis revealed an increase in empty membrane-bound organelles in SNX24 KO megakaryocytes, a reduction in α-granules and an absence of immature and mature multivesicular bodies, consistent with a defect in the intermediate stage of α-granule maturation. Co-localization studies showed that SNX24 associates with each compartment of α-granule maturation. Reduced expression of CD62P and VWF was observed in SNX24 KO megakaryocytes. We conclude that SNX24 is required for α-granule biogenesis and intracellular trafficking of α-granule cargo within megakaryocytes.

Genetically variant human pluripotent stem cells selectively eliminate wild-type counterparts through YAP-mediated cell competition.

The appearance of genetic changes in human pluripotent stem cells (hPSCs) presents a concern for their use in research and regenerative medicine. Variant hPSCs that harbor recurrent culture-acquired aneuploidies display growth advantages over wild-type diploid cells, but the mechanisms that yield a drift from predominantly wild-type to variant cell populations remain poorly understood. Here, we show that the dominance of variant clones in mosaic cultures is enhanced through competitive interactions that result in the elimination of wild-type cells. This elimination occurs through corralling and mechanical compression by faster-growing variants, causing a redistribution of F-actin and sequestration of yes-associated protein (YAP) in the cytoplasm that induces apoptosis in wild-type cells. YAP overexpression or promotion of YAP nuclear localization in wild-type cells alleviates their "loser" phenotype. Our results demonstrate that hPSC fate is coupled to mechanical cues imposed by neighboring cells and reveal that hijacking this mechanism allows variants to achieve clonal dominance in cultures.

Intra-ER sorting of the peroxisomal membrane protein Pex3 relies on its luminal domain.

Pex3 is an evolutionarily conserved type III peroxisomal membrane protein required for peroxisome formation. It is inserted into the ER membrane and sorted via an ER subdomain (the peroxisomal ER, or pER) to peroxisomes. By constructing chimeras between Pex3 and the type III ER membrane protein Sec66, we have been able to separate the signals that mediate insertion of Pex3 into the ER from those that mediate sorting within the ER to the pER subdomain. The N-terminal 17-amino acid segment of Pex3 contains two signals that are each sufficient for sorting to the pER: a chimeric protein containing the N-terminal domain of Pex3 fused to the transmembrane and cytoplasmic segments of Sec66 sorts to the pER in wild type cells, and does not colocalise with peroxisomes. Subsequent transport to existing peroxisomes requires the Pex3 transmembrane segment. When expressed in Drosophila S2R+ cells, ScPex3 targeting to peroxisomes is dependent on the intra-ER sorting signals in the N-terminal segment. The N-terminal segments of both human and Drosophila Pex3 contain intra-ER sorting information and can replace that of ScPex3. Our analysis has uncovered the signals within Pex3 required for the various steps of its transport to peroxisomes. Our generation of versions of Pex3 that are blocked at each stage along its transport pathway provides a tool to dissect the mechanism, as well as the molecular machinery required at each step of the pathway.

Cytokeratin 19 expression in normal endometrium and in low-grade endometrioid adenocarcinoma of the endometrium.

The low-molecular-weight cytokeratin (CK) protein CK19 has been shown to have diagnostic use and prognostic significance in some types of human malignancy, but little is known of its distribution in normal endometrial mucosa or in endometrial endometrioid adenocarcinoma. However, we had observed that CK19 appeared to selectively label invasive tumor areas showing microcystic, elongated, and fragmented ("MELF") changes. Therefore, CK19 expression was assessed in 15 hysterectomy specimens showing normal proliferative, secretory, or atrophic endometrial appearances, and in 26 endometrioid adenocarcinoma cases with areas of MELF-type invasion. Normal endometrial glands were usually CK19 positive; however, there was more consistent expression in the functional layer, whereas basal zone epithelium was typically only focally stained. Proliferative epithelium frequently showed basal and apical cytoplasmic accentuation of staining. Endometrial carcinomas were also CK19 positive, but in most cases there was a distinct zonal pattern of expression with strong staining only in the central aspects of the larger tumor glands and weak-to-absent staining in peripheral glandular areas. In contrast, MELF-type tumor epithelium was consistently and strongly CK19 positive even when the adjacent "conventional"-type tumor glands were not stained. Intravascular tumor cells were also highlighted, including 2 cases in which this feature was not identified on hematoxylin and eosin stains. Thus CK19 immunohistochemistry was useful in showing the extent of myometrial invasion and subtle foci of lympho-vascular space invasion. Further studies are required to determine the mechanism and biologic significance of localized alterations in CK19 expression within endometrial neoplasms.

Study of receptor-chaperone interactions using the optical technique of spectroscopic ellipsometry.

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms.

Silent cerebral infarcts occur despite regular blood transfusion therapy after first strokes in children with sickle cell disease.

Children with sickle cell disease (SCD) and strokes receive blood transfusion therapy for secondary stroke prevention; despite this, approximately 20% experience second overt strokes. Given this rate of second overt strokes and the clinical significance of silent cerebral infarcts, we tested the hypothesis that silent cerebral infarcts occur among children with SCD being transfused for secondary stroke prevention. A prospective cohort enrolled children with SCD and overt strokes at 7 academic centers. Magnetic resonance imaging and magnetic resonance angiography of the brain were scheduled approximately every 1 to 2 years; studies were reviewed by a panel of neuroradiologists. Eligibility criteria included regularly scheduled blood transfusion therapy. Forty children were included; mean pretransfusion hemoglobin S concentration was 29%. Progressive cerebral infarcts occurred in 45% (18 of 40 children) while receiving chronic blood transfusion therapy; 7 had second overt strokes and 11 had new silent cerebral infarcts. Worsening cerebral vasculopathy was associated with new cerebral infarction (overt or silent; relative risk = 12.7; 95% confidence interval, 2.65-60.5, P = .001). Children with SCD and overt strokes receiving regular blood transfusion therapy experience silent cerebral infarcts at a higher rate than previously recognized. Additional therapies are needed for secondary stroke prevention in children with SCD.

Alcohol brief interventions: exploring perceptions and training needs.

One of the main thrusts of contemporary alcohol misuse policies is that early intervention can make a real difference to patterns of problem drinking, as long as healthcare professionals are given the right skills. However, healthcare professionals themselves suggest that they are often unsure of how to raise issues, and feel that they lack the skills or knowledge to do this effectively. This study investigates the perceptions and training needs of health visitors, school nurses, nursery nurses and practice nurses in relation to alcohol misuse primary prevention and the delivery of brief interventions in their day-to-day work. The findings indicate a variation in the need for training, which is reflected by the level of knowledge, skills and confidence of the different professional groups. This may help to facilitate the provision of needs-led alcohol training, and promote the effective delivery of support and brief interventions to individuals, families, schools and communities.