A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

http://science.sciencemag.org/content/sci/291/5507/1304.full.pdf

The sequence of the human genome.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

/pdf/biological-properties-of-extracellular-vesicles-and-their-bef7axtqvb.pdf

Biological properties of extracellular vesicles and their physiological functions

The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells. Here we demonstrate that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin(+) MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent 'mesenspheres' that can self-renew and expand in serial transplantations. Nestin(+) MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or beta3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin(+) cells and favours their osteoblastic differentiation, in vivo nestin(+) cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin(+) MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin(+) cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs.

/pdf/mesenchymal-and-haematopoietic-stem-cells-form-a-unique-bone-3477057m0k.pdf

Mesenchymal and haematopoietic stem cells form a unique bone marrow niche

The calpain system originally comprised three molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. Both mu- and m-calpain are heterodimers containing an identical 28-kDa subunit and an 80-kDa subunit that shares 55-65% sequence homology between the two proteases. The crystallographic structure of m-calpain reveals six "domains" in the 80-kDa subunit: 1). a 19-amino acid NH2-terminal sequence; 2). and 3). two domains that constitute the active site, IIa and IIb; 4). domain III; 5). an 18-amino acid extended sequence linking domain III to domain IV; and 6). domain IV, which resembles the penta EF-hand family of polypeptides. The single calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85 kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear, although all bind to three different places on the calpain molecule; binding to at least two of the sites is Ca2+ dependent. Since 1989, cDNA cloning has identified 12 additional mRNAs in mammals that encode polypeptides homologous to domains IIa and IIb of the 80-kDa subunit of mu- and m-calpain, and calpain-like mRNAs have been identified in other organisms. The molecules encoded by these mRNAs have not been isolated, so little is known about their properties. How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke, and brain trauma.

The Calpain System

Peptide sequence analysis using a combination of gas-phase ion/ion chemistry and tandem mass spectrometry (MS/MS) is demonstrated. Singly charged anthracene anions transfer an electron to multiply protonated peptides in a radio frequency quadrupole linear ion trap (QLT) and induce fragmentation of the peptide backbone along pathways that are analogous to those observed in electron capture dissociation. Modifications to the QLT that enable this ion/ion chemistry are presented, and automated acquisition of high-quality, single-scan electron transfer dissociation MS/MS spectra of phosphopeptides separated by nanoflow HPLC is described.

/pdf/peptide-and-protein-sequence-analysis-by-electron-transfer-2sqn15vkex.pdf

Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry

Thrombus formation following vascular injury is an essential component of both hemostasis and pathologic vessel occlusion. This process occurs in a closed, pressurized environment in which blood flows rapidly over the injury site. Thrombus formation must occur quickly to reduce blood loss, but is carefully modulated to limit vessel occlusion. Circulating cells, plasma proteins, vessel wall components, and physical forces such as shear all influence thrombus formation. Historically, thrombus formation has been studied by isolating the separate components of blood involved in clot formation. With improved optical techniques, investigators have increasingly studied thrombus formation under conditions of flow in vitro and in live animals in vivo. Using multichannel fluorescence intravital videomicroscopy, the authors have studied the changes in the kinetics and deposition of platelets, fibrin, and tissue factor at the injury site during thrombosis in transgenic mice, bone marrow transplanted mice, and mice treated with pharmacological agents that modulate thrombosis. The differences in the kinetics of accumulation of the various components of thrombus in these mice have provided new insights about thrombus formation in arterioles. This review discusses the role of platelet intracellular signaling, P-selectin expression on platelets, and tissue factor-bearing microparticles in thrombus formation.

Interactions of platelets, blood-borne tissue factor, and fibrin during arteriolar thrombus formation in vivo.

Crystal structure of the bovine lactadherin C2 domain, a membrane binding motif, shows similarity to the C2 domains of factor V and factor VIII.

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors, and thus bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a new target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond-forming enzymes Escherichia coli DsbB (EcDsbB) or Mycobacterium tuberculosis VKOR (MtbVKOR), which can replace EcDsbB, although the two are not homologs. Initial screening of 51,487 compounds yielded six specifically inhibiting EcDsbB. These compounds share a structural motif and do not inhibit MtbVKOR. A medicinal chemistry approach led us to select related compounds, some of which are much more effective DsbB inhibitors than those found in the screen. These compounds inhibit purified DsbB and prevent anaerobic growth of E. coli. Furthermore, these compounds inhibit all but one of the DsbBs of nine other Gram-negative pathogenic bacteria tested.

/pdf/compounds-targeting-disulfide-bond-forming-enzyme-dsbb-of-43qapiea9c.pdf

Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria

Activation of factor X: Kinetic properties of the reaction☆

Phospholipid membranes play a significant role during the proteolytic activation of blood coagulation proteins. This investigation identifies a role for phosphatidylethanolamine (PE) during the activation of factor X by the tenase complex, an enzymatic complex composed of the serine protease, factor IXa, a protein cofactor, factor VIIIa, a phospholipid membrane, and Ca(2+). Phospholipid vesicles composed of PE, phosphatidylserine (PS), and phosphatidylcholine support factor Xa generation. The K(m) and k(cat) for factor X activation by the tenase complex are independent of PE in the presence of 20% PS. At lower PS concentrations, the presence of 20 or 35% PE lowers the K(m) and increases the k(cat) as compared to those in vesicles without PE. The effect of PE on the k(cat) of the tenase complex is mediated through factor VIIIa. PE also enhances factor Xa generation by facilitating tenase complex formation; PE lowers the K(d(app)) of factor IXa for both phospholipid/Ca(2+) and phospholipid/Ca(2+)/factor VIIIa complexes in the presence of suboptimal PS. In addition, the K(d)s of factor IXa and factor X were lower for phospholipid vesicles containing PE. N-Methyl-PE increased the k(cat) and decreased the K(d(app)), whereas N,N-dimethyl-PE had no effect on either parameter, indicating the importance of headgroup size. Lyso-PE had no effect on kinetic parameters, indicating the sn-2 acyl chain dependence of the PE effect. Together, these results demonstrate a role for PE in the assembly and activity of the tenase complex and further extend the understanding of the importance of PE-containing membranes in hemostasis.

Barbara C. Furie

Papers

Interactions of platelets, blood-borne tissue factor, and fibrin during arteriolar thrombus formation in vivo.

Crystal structure of the bovine lactadherin C2 domain, a membrane binding motif, shows similarity to the C2 domains of factor V and factor VIII.

Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria

Activation of factor X: Kinetic properties of the reaction☆

Role of phosphatidylethanolamine in assembly and function of the factor IXa-factor VIIIa complex on membrane surfaces.