How do distinct protein-tyrosine kinases activate specific down-stream events? Src-homology-2 (SH2) domains on tyrosine kinases or targets of tyrosine kinases recognize phosphotyrosine in a specific sequence context and thereby provide some specificity. The role of the catalytic site of tyrosine kinases in determining target specificity has not been fully investigated. Here we use a degenerate peptide library to show that each of nine tyrosine kinases investigated has a unique optimal peptide substrate. We find that the cytosolic tyrosine kinases preferentially phosphorylate peptides recognized by their own SH2 domains or closely related SH2 domains (group I; ref. 3), whereas receptor tyrosine kinases preferentially phosphorylate peptides recognized by subsets of group III SH2 domains. The importance of these findings for human disease is underscored by our observation that a point mutation in the RET receptor-type tyrosine kinase, which causes multiple endocrine neoplasia type 2B, results in a shift in peptide substrate specificity.

Haematopoietic stem cell (HSC) niches, although proposed decades ago, have only recently been identified as separate osteoblastic and vascular microenvironments. Their interrelationships and interactions with HSCs in vivo remain largely unknown. Here we report the use of a newly developed ex vivo real-time imaging technology and immunoassaying to trace the homing of purified green-fluorescent-protein-expressing (GFP(+)) HSCs. We found that transplanted HSCs tended to home to the endosteum (an inner bone surface) in irradiated mice, but were randomly distributed and unstable in non-irradiated mice. Moreover, GFP(+) HSCs were more frequently detected in the trabecular bone area compared with compact bone area, and this was validated by live imaging bioluminescence driven by the stem-cell-leukaemia (Scl) promoter-enhancer. HSCs home to bone marrow through the vascular system. We found that the endosteum is well vascularized and that vasculature is frequently localized near N-cadherin(+) pre-osteoblastic cells, a known niche component. By monitoring individual HSC behaviour using real-time imaging, we found that a portion of the homed HSCs underwent active division in the irradiated mice, coinciding with their expansion as measured by flow assay. Thus, in contrast to central marrow, the endosteum formed a special zone, which normally maintains HSCs but promotes their expansion in response to bone marrow damage.

Records of 642 outbreaks of acute gastroenteritis were reviewed to determine the proportion of outbreaks that were clinically and epidemiologically consistent with Norwalk-like virus infection. Using as our criteria stool cultures negative for bacterial pathogens, mean (or median) duration of illness 12-60 hours, vomiting in 250 per cent of cases, and, if known, mean (or median) incubation period of 24-48 hours, we found that 23 per cent of waterborne outbreaks, 4 per cent of foodborne outbreaks, and 67 per cent, 60 per cent, and 28 per cent of outbreaks in nursing homes, in summer camps, and on cruise ships,

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Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase). The severe forms of GD are associated with neurodegeneration with either rapid (Type 2) or slow progression (Type 3). Although the neurodegenerative process in GD has been linked to lysosomal dysfunction, the mechanisms involved are largely unknown. To identify the lysosomal alterations in GD neurons and uncover the mechanisms involved, we used induced pluripotent stem cells (iPSCs) derived from patients with GD. In GD iPSC-derived neuronal cells (iPSC-NCs), GBA1 mutations caused widespread lysosomal depletion, and a block in autophagic flux due to defective lysosomal clearance of autophagosomes. Autophagy induction by rapamycin treatment in GD iPSC-NCs led to cell death. Further analysis showed that in GD iPSC-NCs, expression of the transcription factor EB (TFEB), the master regulator of lysosomal genes, and lysosomal gene expression, were significantly downregulated. There was also reduced stability of the TFEB protein and altered lysosomal protein biosynthesis. Treatment of mutant iPSC-NCs with recombinant GCase (rGCase) reverted the lysosomal depletion and autophagy block. The effect of rGCase on restoring lysosomal numbers in mutant cells was enhanced in the presence of overexpressed TFEB, but TFEB overexpression alone did not reverse the lysosomal depletion phenotype. Our results suggest that GBA1 mutations interfere with TFEB-mediated lysosomal biogenesis, and that the action of GCase in maintaining a functioning pool of lysosomes is exerted in part through TFEB. The lysosomal alterations described here are likely to be a major determinant in GBA1-associated neurodegeneration.

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Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid β-glucocerebrosidase gene. To model GD, we generated human induced pluripotent stem cells (hiPSC), by reprogramming skin fibroblasts from patients with type 1 (N370S/N370S), type 2 (L444P/RecNciI), and type 3 (L444P/L444P) GD. Pluripotency was demonstrated by the ability of GD hiPSC to differentiate to all three germ layers and to form teratomas in vivo. GD hiPSC differentiated efficiently to the cell types most affected in GD, i.e., macrophages and neuronal cells. GD hiPSC-macrophages expressed macrophage-specific markers, were phagocytic, and were capable of releasing inflammatory mediators in response to LPS. Moreover, GD hiPSC-macrophages recapitulated the phenotypic hallmarks of the disease. They exhibited low glucocerebrosidase (GC) enzymatic activity and accumulated sphingolipids, and their lysosomal functions were severely compromised. GD hiPSC-macrophages had a defect in their ability to clear phagocytosed RBC, a phenotype of tissue-infiltrating GD macrophages. The kinetics of RBC clearance by types 1, 2, and 3 GD hiPSC-macrophages correlated with the severity of the mutations. Incubation with recombinant GC completely reversed the delay in RBC clearance from all three types of GD hiPSC-macrophages, indicating that their functional defects were indeed caused by GC deficiency. However, treatment of induced macrophages with the chaperone isofagomine restored phagocytosed RBC clearance only partially, regardless of genotype. These findings are consistent with the known clinical efficacies of recombinant GC and isofagomine. We conclude that cell types derived from GD hiPSC can effectively recapitulate pathologic hallmarks of the disease.Gaucher model | Gaucher macrophages | lipid storage disease | glucosylsphingolipids | erythrophagocytosis