Robo4 cooperates with CXCR4 to specify hematopoietic stem cell localization to bone marrow niches

Specific bone marrow (BM) niches are critical for hematopoietic stem cell (HSC) function during both normal hematopoiesis and in stem cell transplantation therapy. We demonstrate that the guidance molecule Robo4 functions to specifically anchor HSCs to BM niches. Robo4-deficient HSCs displayed poor localization to BM niches and drastically reduced long-term reconstitution capability while retaining multilineage potential. Cxcr4, a critical regulator of HSC location, is upregulated in Robo4(-/-) HSCs to compensate for Robo4 loss. Robo4 deletion led to altered HSC mobilization efficiency, revealing that inhibition of both Cxcr4- and Robo4-mediated niche interactions are necessary for efficient HSC mobilization. Surprisingly, we found that WT HSCs express very low levels of Cxcr4 and respond poorly to Cxcr4 manipulation relative to other hematopoietic cells. We conclude that Robo4 cooperates with Cxcr4 to endow HSCs with competitive access to limited stem cell niches, and we propose Robo4 as a therapeutic target in HSC transplantation therapy.     

The STR252 – IVS10nt546 – VNTR7 phenylalanine hydroxylase minihaplotype in five Mediterranean samples

IVS10nt546 (IVS10nt-11g→a) is the most common molecular defect of the phenylalanine hydroxylase gene causing phenylketonuria in Mediterranean populations. Previous studies have proposed various and alternative hypotheses concerning the geographical origin and pattern of diffusion of this mutation in this area. In this study, this issue was re-examined on a large sample (149) of “Mediterranean” IVS10nt546 mutant alleles analysed with multiallelic intragenic polymorphisms. The analysis of intragenic microsatellite (STR) and minisatellite (VNTR) polymorphisms shows allelic heterogeneity of the IVS10nt546 mutation. Eight STR and three VNTR alleles were found in association with the splicing defect. Of the ten detected STR–VNTR combinations (“minihaplotypes”), we identified a predominant allelic association (VNTR7 – STR252) embedded in a RFLP-haplotype 6 background, which seems to correspond to the ancestral gene originating in the Turkey–Israel area. Analysis of both absolute and relative gene frequencies of the STR252 – IVS10nt546 – VNTR7 minihaplotypes, shows statistically significant (P < 0.02) variations and may suggest gene flow from Turkey and/or Israel to Italy and Spain. The associated migratory events need not be unique in time (and people) but seem to suggest they may be traced back to the expansion of the Neolithic culture and people, thus allowing dating of the origin of this mutation to at least 5000–10 000 years ago. Alternative hypotheses are discussed to explain, in light of the available historical and pre-historical evidence, the pattern of diffusion of the IVS10nt546 mutation in the Mediterranean basin. Received: 24 March 1997 / Accepted: 9 April 1997     

Separation and identification of plasma short-chain acylcarnitine isomers by HPLC/MS/MS for the differential diagnosis of fatty acid oxidation defects and organic acidemias

This paper reports a new, high-performance liquid chromatography/tandem mass spectrometry method for the separation and identification of human plasma short-chain acylcarnitine isomers. This simple, rapid procedure involves the use of a single sample previously shown to contain elevated acylcarnitine concentrations by flow injection analysis, and can separate two C4, three C5, two C5:1 and four C5-OH acylcarnitine isomers, thus permitting the differential diagnosis of certain fatty acid oxidation defects and organic acidemias.     

Towards the human intestinal microbiota phylogenetic core

The paradox of a host specificity of the human faecal microbiota otherwise acknowledged as characterized by global functionalities conserved between humans led us to explore the existence of a phylogenetic core. We investigated the presence of a set of bacterial molecular species that would be altogether dominant and prevalent within the faecal microbiota of healthy humans. A total of 10 456 non-chimeric bacterial 16S rRNA sequences were obtained after cloning of PCR-amplified rDNA from 17 human faecal DNA samples. Using alignment or tetranucleotide frequency-based methods, 3180 operational taxonomic units (OTUs) were detected. The 16S rRNA sequences mainly belonged to the phyla Firmicutes (79.4%), Bacteroidetes (16.9%), Actinobacteria (2.5%), Proteobacteria (1%) and Verrumicrobia (0.1%). Interestingly, while most of OTUs appeared individual-specific, 2.1% were present in more than 50% of the samples and accounted for 35.8% of the total sequences. These 66 dominant and prevalent OTUs included members of the genera Faecalibacterium , Ruminococcus , Eubacterium , Dorea , Bacteroides , Alistipes and Bifidobacterium . Furthermore, 24 OTUs had cultured type strains representatives which should be subjected to genome sequence with a high degree of priority. Strikingly, 52 of these 66 OTUs were detected in at least three out of four recently published human faecal microbiota data sets, obtained with very different experimental procedures. A statistical model confirmed these OTUs prevalence. Despite the species richness and a high individual specificity, a limited number of OTUs is shared among individuals and might represent the phylogenetic core of the human intestinal microbiota. Its role in human health deserves further study.     

Functional characterization of PCCA mutations causing propionic acidemia

Propionic acidemia (PA, MIM 232000 and 232050) is caused by a deficiency of mitochondrial biotin-dependent propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a heteropolymeric enzyme composed of alpha and beta subunits, which are encoded by the PCCA and PCCB genes, respectively. The PCCA protein (alpha subunit) is responsible for the formation of carboxybiotin upon hydrolysis of ATP and contains a C-terminal biotin-binding domain and a biotin carboxylase domain, defined by homology with other biotin-dependent carboxylases, some of them characterized structurally. More than 24 mutations have been found in the PCCA gene in patients with PA, among them 14 missense mutations and one in-frame deletion, for which the precise molecular effect is unknown. In this study, we have established the pathogenicity of 11 PCCA mutations (10 missense and an in-frame deletion) by expression studies in deficient fibroblasts and in a cell-free in vitro system, and analyzed the effect of each mutation on PCC activity, protein stability and domain structure. The results show that most mutant proteins show an increased turnover and are functionally deficient, suggesting that the structural alterations they cause are incompatible with normal assembly to produce a stable, functional PCC oligomer. These results are discussed in the context of the genotype-phenotype correlations in PCCA-deficient PA patients.     

Changes in composition of rockweed (Ascophyllum nodosum) beds due to possible recent increase in sea temperature in Eastern Canada

Ascophyllum nodosum (rockweed) is the main economic resource of the seaweed industry in the Atlantic Provinces of Canada. The annual harvest steadily increased since 1995, reaching a historic peak of 37,000 tonnes in 2007. Due to a high demand for fertilizers and animal feed supplements derived from rockweed, this trend seems likely to continue. The current management plan for the sustainable harvest of the A. nodosum resource is considered conservative. The resource has been managed with a precautionary approach since 1995 to protect the integrity of the habitat. Acadian Seaplants Limited (ASL) has been granted approximately 90% of the government-issued licenses to harvest A. nodosum resources in the Maritimes. The Canadian approach is based on an annual harvest from a given bed and not strip-and-return after several years. Since 1995, ASL has proactively undertaken extensive annual surveys and research on biomass productivity of this renewable resource to establish acceptable annual exploitation rates. Historically, the rockweed beds of southwestern Nova Scotia (NS) have been almost 99% pure A. nodosum, with a minor component of Fucus vesiculosus. However, since 2004, a steady increase in F. vesiculosus, with a peak of 4.6% of the total biomass in 2008, was recorded. This coincided with one of the mildest winters on record for the Maritimes. This increase in temperature seemed to be also responsible for an unusual recruitment of the blue mussel Mytilus edulis in rockweed beds in some areas of southern New Brunswick (NB) in 2006, causing the detachment of up to 30% of the seaweed biomass in some harvesting sectors. Another phenomenon observed in southwestern NS during 2003 and 2004 was extensive ice damage on rockweed beds produced by an early melting of the ice, with losses of up to 90% of the rockweed biomass in some areas.