The standard protein mix database: a diverse data set to assist in the production of improved Peptide and protein identification software tools

Tandem mass spectrometry (MS/MS) is frequently used in the identification of peptides and proteins. Typical proteomic experiments rely on algorithms such as SEQUEST and MASCOT to compare thousands of tandem mass spectra against the theoretical fragment ion spectra of peptides in a database. The probabilities that these spectrum-to-sequence assignments are correct can be determined by statistical software such as PeptideProphet or through estimations based on reverse or decoy databases. However, many of the software applications that assign probabilities for MS/MS spectra to sequence matches were developed using training data sets from 3D ion-trap mass spectrometers. Given the variety of types of mass spectrometers that have become commercially available over the last 5 years, we sought to generate a data set of reference data covering multiple instrumentation platforms to facilitate both the refinement of existing computational approaches and the development of novel software tools. We analyzed the proteolytic peptides in a mixture of tryptic digests of 18 proteins, named the "ISB standard protein mix", using 8 different mass spectrometers. These include linear and 3D ion traps, two quadrupole time-of-flight platforms (qq-TOF), and two MALDI-TOF-TOF platforms. The resulting data set, which has been named the Standard Protein Mix Database, consists of over 1.1 million spectra in 150+ replicate runs on the mass spectrometers. The data were inspected for quality of separation and searched using SEQUEST. All data, including the native raw instrument and mzXML formats and the PeptideProphet validated peptide assignments, are available at http://regis-web.systemsbiology.net/PublicDatasets/.     

Semaphorin 3B is a 1,25-Dihydroxyvitamin D3-induced gene in osteoblasts that promotes osteoclastogenesis and induces osteopenia in mice

The vitamin D endocrine system is important for skeletal homeostasis. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] impacts bone indirectly by promoting intestinal absorption of calcium and phosphate and directly by acting on osteoblasts and osteoclasts. Despite the direct actions of 1,25(OH)(2)D(3) in bone, relatively little is known of the mechanisms or target genes that are regulated by 1,25(OH)(2)D(3) in skeletal cells. Here, we identify semaphorin 3B (SEMA3B) as a 1,25(OH)(2)D(3)-stimulated gene in osteoblastic cells. Northern analysis revealed strong induction of SEMA3B mRNA by 1,25(OH)(2)D(3) in MG-63, ST-2, MC3T3, and primary osteoblastic cells. Moreover, differentiation of these osteogenic cells enhanced SEMA3B gene expression. Biological effects of SEMA3B in the skeletal system have not been reported. Here, we show that osteoblast-derived SEMA3B alters global skeletal homeostasis in intact animals and osteoblast function in cell culture. Osteoblast-targeted expression of SEMA3B in mice resulted in reduced bone mineral density and aberrant trabecular structure compared with nontransgenic littermates. Histomorphometry studies indicated that this was likely due to increased osteoclast numbers and activity. Indeed, primary osteoblasts obtained from SEMA3B transgenic mice stimulated osteoclastogenesis to a greater extent than nontransgenic osteoblasts. This study establishes that SEMA3B is a 1,25(OH)(2)D(3)-induced gene in osteoblasts and that osteoblast-derived SEMA3B impacts skeletal biology in vitro and in vivo. Collectively, these studies support a putative role for SEMA3B as an osteoblast protein that regulates bone mass and skeletal homeostasis.     

New tripodal hydroxypyridinone based chelating agents for Fe(III), Al(III) and Ga(III): Synthesis, physico-chemical properties and bioevaluation

Two new tris-hydroxypyridinone based compounds (KEMPPr(3,4-HP)₃ and KEMPBu(3,4-HP)₃) have been developed and studied as strong sequestering agents for iron and the group III of metal ions, aimed as potential pharmacological applications on metal-chelation therapy. Their structure is based on the KEMP acid scaffold to which three 3-hydroxy-4-pyridinone chelating moieties are attached via two different size spacers. After the preparation and characterization of the compounds their physico-chemical properties were studied, in relation with their metal binding affinity and lipophilicity. The KEMPPr(3,4-HP)₃ ligand was also bioassayed to evaluate its in vivo metal sequestering capacity from most organs using an animal model overload with ⁶⁷Ga. These studies showed that, for both in solution and in vivo conditions, the compounds have higher metal chelating efficacy than Deferriprone, the commercially available iron chelator in medical application, thus some perspectives are envisaged as potential pharmaceutical drug candidates for chelating therapy.     

Nyamanini and Midway Viruses Define a Novel Taxon of RNA Viruses in the Order Mononegavirales

Here, we report the sequencing and classification of Nyamanini virus (NYMV) and Midway virus (MIDWV), two antigenically related viruses that were first isolated in 1957 and 1966, respectively. Although these viruses have been cultured multiple times from cattle egrets, seabirds, and their ticks, efforts to classify them taxonomically using conventional serological and electron microscopic approaches have failed completely. We used a random shotgun sequencing strategy to define the genomes of NYMV and MIDWV. Contigs of 11,631 and 11,752 nucleotides, representing the complete genome of NYMV and the near-complete genome of MIDWV, respectively, were assembled. Each virus genome was predicted to carry six open reading frames (ORFs). BLAST analysis indicated that only two of the ORF proteins of each virus, the putative nucleocapsid and polymerase, had detectable sequence similarity to known viral proteins. Phylogenetic analysis of these ORF proteins demonstrated that the closest relatives of NYNV and MIDWV are negative-stranded-RNA viruses in the order Mononegavirales. On the basis of their very limited sequence similarity to known viruses, we propose that NYMV and MIDWV define a novel genus, Nyavirus, in this order.Nyamanini virus (NYMV) was first isolated in 1957 from a cattle egret (Bubulcus ibis) in South Africa (24). It has subsequently been isolated in Nigeria, Egypt, India, and Thailand from cattle egrets and Argas walkerae ticks (14, 16, 24). Although there has been no recognized human infection or disease associated with NYMV, suckling mice succumbed to NYMV infection 7 or 8 days after intracerebral inoculation (14). NYMV has not been definitively characterized or classified to date (10).Midway virus (MIDWV) was first isolated in 1966 from seabird ticks of two species [Ornithodoros (Alectorobius) spp.] collected on the Midway, Kure, and Manana islands in the Central Pacific and from northern Honshu, Japan. In addition, on Aomatsushima Island, nestling seabirds of two seabird species, Larus crassirostris and Nycticorax nycticorax, were found to have antibody to MIDWV. This virus is pathogenic for newborn Swiss mice but not 4-week-old Swiss mice injected intracranially. Also, the virus is cytopathic in BHK-21 cells and produces plaques in Vero cells. Efforts to classify MIDWV have revealed only that MIDWV is antigenically related to NYMV in cross-box complement fixation (CF) assays (22).To date, conventional approaches, such as serological analysis and electron microscopy (EM), have not yielded definitive characterization of MIDWV or NYMV. Heretofore, it was not known whether MIDWV and NYMV are DNA viruses or RNA viruses or to which virus family they belong. In this paper, we describe the application of unbiased high-throughput sequencing to define the genome sequences of NYMV and MIDWV. By analysis of their genomes, NYMV and MIDWV were determined to be negative-stranded RNA viruses highly divergent from all known viruses but most closely related to viruses in the order Mononegavirales. On the basis of the analysis presented herein, we propose that NYMV and MIDWV define a novel taxon within the order Mononegavirales.     

Sex differences in anthropoid mandibular canine lateral enamel formation

Previous research has demonstrated that great ape and macaque males achieve large canine crown sizes primarily through extended canine growth periods. Recent work has suggested, however, that platyrrhine males may achieve larger canine sizes by accelerating rather than prolonging growth. This study tested the hypothesis that the ontogenetic pathway leading to canine sexual dimorphism in catarrhines differs from that of platyrrhines. To test this hypothesis, males and females of several catarrhine genera (Hylobates, Papio, Macaca, Cercopithecus, and Cercocebus) and three platyrrhine genera (Cebus, Ateles, and Callicebus) were compared in the number and spacing of perikymata (enamel growth increments) on their canine crowns. In addition, perikymata periodicities (the number of days of growth perikymata represent) were determined for five genera (Hylobates, Papio, Macaca, Cebus, and Ateles) using previously published as well as original data gathered for this study. The central findings are as follows: 1) males have more perikymata than females for seven of eight genera (in five of the seven, the differences are statistically significant); 2) in general, the greater the degree of sexual dimorphism, the greater the sex difference in male and female perikymata numbers; 3) there is no evidence of a systematic sex difference in primate periodicities; and 4) there is some evidence that sex differences in enamel formation rates may make a minor contribution to canine sexual dimorphism in Papio and Cercopithecus. These findings strongly suggest that in both catarrhines and platyrrhines prolongation of male canine growth is the primary mechanism by which canine crown sexual dimorphism is achieved. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Candida albicans ABG1 gene is involved in endocytosis

The human fungal pathogen Candida albicans undergoes reversible morphogenetic transitions between yeast, hyphal and pseudohyphal forms. The fungal vacuole actively participates in differentiation processes and plays a key role supporting hyphal growth. The ABG1 gene of C. albicans encodes an essential protein located in the vacuolar membranes of both yeast and hyphae. Using fluorescence microscopy of a green fluorescent protein-tagged version of Abg1p, a fraction of the protein was detected in hyphal tips, not associated with vacuolar membranes. Live cell imaging of emerging germ tubes showed that Abg1p migrated to the polarized growth site and colocalized with endocytic vesicles. Phenotypic analysis of a methionine-regulated conditional mutant confirmed that Abg1p is involved in endocytosis.     

DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps

The innate immune response plays a crucial role in satisfactory host resolution of bacterial infection. In response to chemotactic signals, neutrophils are early responding cells that migrate in large numbers to sites of infection. The recent discovery of secreted neutrophil extracellular traps (NETs) composed of DNA and histones opened a novel dimension in our understanding of the microbial killing capacity of these specialized leukocytes. M1 serotype strains of the pathogen Group A Streptococcus (GAS) are associated with invasive infections including necrotizing fasciitis (NF) and express a potent DNase (Sda1). Here we apply a molecular genetic approach of allelic replacement mutagenesis, single gene complementation, and heterologous expression to demonstrate that DNase Sda1 is both necessary and sufficient to promote GAS neutrophil resistance and virulence in a murine model of NF. Live fluorescent microscopic cell imaging and histopathological analysis are used to establish for the first time a direct linkage between NET degradation and bacterial pathogenicity. Inhibition of GAS DNase activity with G-actin enhanced neutrophil clearance of the pathogen in vitro and reduced virulence in vivo. The results demonstrate a significant role for NETs in neutrophil-mediated innate immunity, and at the same time identify a novel therapeutic target against invasive GAS infection.     

Adiposity and age explain most of the association between physical activity and fitness in physically active men

Background

To determine if there is an association between physical activity assessed by the short version of the International Physical Activity Questionnaire (IPAQ) and cardiorespiratory and muscular fitness.

Methodology/Principal Findings

One hundred and eighty-two young males (age range: 20–55 years) completed the short form of the IPAQ to assess physical activity. Body composition (dual-energy X-Ray absorptiometry), muscular fitness (static and dynamic muscle force and power, vertical jump height, running speed [30 m sprint], anaerobic capacity [300 m running test]) and cardiorespiratory fitness (estimated VO₂max: 20 m shuttle run test) were also determined in all subjects.Activity-related energy expenditure of moderate and vigorous intensity (EEPA_moderate and EEPA_vigorous, respectively) was inversely associated with indices of adiposity (r = −0.21 to −0.37, P<0.05). Cardiorespiratory fitness (VO₂max) was positively associated with LogEEPA_moderate (r = 0.26, P<0.05) and LogEEPA_vigorous (r = 0.27). However, no association between VO₂max with LogEEPA_moderate, LogEPPA_vigorous and LogEEPA_total was observed after adjusting for the percentage of body fat. Multiple stepwise regression analysis to predict VO₂max from LogEEPA_walking, LogEEPA_moderate, LogEEPA_vigorous, LogEEPA_total, age and percentage of body fat (%fat) showed that the %fat alone explained 62% of the variance in VO₂max and that the age added another 10%, while the other variables did not add predictive value to the model [VO₂max  = 129.6−(25.1× Log %fat) − (34.0× Log age); SEE: 4.3 ml.kg⁻¹. min⁻¹; R² = 0.72 (P<0.05)]. No positive association between muscular fitness-related variables and physical activity was observed, even after adjusting for body fat or body fat and age.

Conclusions/Significance

Adiposity and age are the strongest predictors of VO₂max in healthy men. The energy expended in moderate and vigorous physical activities is inversely associated with adiposity. Muscular fitness does not appear to be associated with physical activity as assessed by the IPAQ.