STEPS: A grid search methodology for optimized peptide identification filtering of MS/MS database search results

For bottom‐up proteomics, there are wide variety of database‐searching algorithms in use for matching peptide sequences to tandem MS spectra. Likewise, there are numerous strategies being employed to produce a confident list of peptide identifications from the different search algorithm outputs. Here we introduce a grid‐search approach for determining optimal database filtering criteria in shotgun proteomics data analyses that is easily adaptable to any search. Systematic Trial and Error Parameter Selection‐–referred to as STEPS‐–utilizes user‐defined parameter ranges to test a wide array of parameter combinations to arrive at an optimal “parameter set” for data filtering, thus maximizing confident identifications. The benefits of this approach in terms of numbers of true‐positive identifications are demonstrated using datasets derived from immunoaffinity‐depleted blood serum and a bacterial cell lysate, two common proteomics sample types.     

Epoxiconazole‐Induced Degeneration in Rat Placenta and the Effects of Estradiol Supplementation

Epoxiconazole (CAS‐No. 133855‐98‐8) was recently shown to cause both a marked depletion of maternal estradiol blood levels and a significantly increased incidence of late fetal mortality when administered to pregnant rats throughout gestation (GD 7–18 or 21); estradiol supplementation prevented this epoxiconazole effect in rats (Stinchcombe et al., 2013), indicating that epoxiconazole‐mediated estradiol depletion is a critical key event for induction of late fetal resorptions in rats. For further elucidation of the mode of action, the placentas from these modified prenatal developmental toxicity experiments with 23 and 50 mg/kg bw/d epoxiconazole were subjected to a detailed histopathological examination. This revealed dose‐dependent placental degeneration characterized by cystic dilation of maternal sinuses in the labyrinth, leading to rupture of the interhemal membrane. Concomitant degeneration occurred in the trophospongium. Both placentas supporting live fetuses and late fetal resorptions were affected; the highest degree of severity was observed in placentas with late resorptions. Placental degeneration correlated with a severe decline in maternal serum estradiol concentration. Supplementation with 0.5 and 1.0 μg of the synthetic estrogen estradiol cyclopentylpropionate per day reduced the severity of the degeneration in placentas with live fetuses. The present study demonstrates that both the placental degeneration and the increased incidence of late fetal resorptions are due to decreased levels of estrogen, since estrogen supplementation ameliorates the former and abolishes the latter. Birth Defects Res (Part B) 98:208–221, 2013. © 2013 Wiley Periodicals, Inc.     

Reconstructing Native American Migrations from Whole-Genome and Whole-Exome Data

There is great scientific and popular interest in understanding the genetic history of populations in the Americas. We wish to understand when different regions of the continent were inhabited, where settlers came from, and how current inhabitants relate genetically to earlier populations. Recent studies unraveled parts of the genetic history of the continent using genotyping arrays and uniparental markers. The 1000 Genomes Project provides a unique opportunity for improving our understanding of population genetic history by providing over a hundred sequenced low coverage genomes and exomes from Colombian (CLM), Mexican-American (MXL), and Puerto Rican (PUR) populations. Here, we explore the genomic contributions of African, European, and especially Native American ancestry to these populations. Estimated Native American ancestry is in MXL, in CLM, and in PUR. Native American ancestry in PUR is most closely related to populations surrounding the Orinoco River basin, confirming the Southern America ancestry of the Taíno people of the Caribbean. We present new methods to estimate the allele frequencies in the Native American fraction of the populations, and model their distribution using a demographic model for three ancestral Native American populations. These ancestral populations likely split in close succession: the most likely scenario, based on a peopling of the Americas thousand years ago (kya), supports that the MXL Ancestors split kya, with a subsequent split of the ancestors to CLM and PUR kya. The model also features effective populations of in Mexico, in Colombia, and in Puerto Rico. Modeling Identity-by-descent (IBD) and ancestry tract length, we show that post-contact populations also differ markedly in their effective sizes and migration patterns, with Puerto Rico showing the smallest effective size and the earlier migration from Europe. Finally, we compare IBD and ancestry assignments to find evidence for relatedness among European founders to the three populations.     

Reconstructing the Population Genetic History of the Caribbean

The Caribbean basin is home to some of the most complex interactions in recent history among previously diverged human populations. Here, we investigate the population genetic history of this region by characterizing patterns of genome-wide variation among 330 individuals from three of the Greater Antilles (Cuba, Puerto Rico, Hispaniola), two mainland (Honduras, Colombia), and three Native South American (Yukpa, Bari, and Warao) populations. We combine these data with a unique database of genomic variation in over 3,000 individuals from diverse European, African, and Native American populations. We use local ancestry inference and tract length distributions to test different demographic scenarios for the pre- and post-colonial history of the region. We develop a novel ancestry-specific PCA (ASPCA) method to reconstruct the sub-continental origin of Native American, European, and African haplotypes from admixed genomes. We find that the most likely source of the indigenous ancestry in Caribbean islanders is a Native South American component shared among inland Amazonian tribes, Central America, and the Yucatan peninsula, suggesting extensive gene flow across the Caribbean in pre-Columbian times. We find evidence of two pulses of African migration. The first pulse—which today is reflected by shorter, older ancestry tracts—consists of a genetic component more similar to coastal West African regions involved in early stages of the trans-Atlantic slave trade. The second pulse—reflected by longer, younger tracts—is more similar to present-day West-Central African populations, supporting historical records of later transatlantic deportation. Surprisingly, we also identify a Latino-specific European component that has significantly diverged from its parental Iberian source populations, presumably as a result of small European founder population size. We demonstrate that the ancestral components in admixed genomes can be traced back to distinct sub-continental source populations with far greater resolution than previously thought, even when limited pre-Columbian Caribbean haplotypes have survived.     

Microbial diversity of Alcyonium digitatum

Marine multi-cellular organisms are described as sources of many newly discovered bioactive compounds. Meanwhile, it has been demonstrated repeatedly for several natural products of reputed multicellular origin that they are, in fact, produced by endophytic unicellular organisms—such as microbial fungi or bacteria. Consequently, while studying compounds isolated from a living organism, it is essential to ensure that the sample integrity is not compromised. To test the diversity of the endobiome from Alcyonium digitatum, a cold water coral found along the Atlantic coasts of the northern hemisphere, we performed a culture dependent surveyed using a phylogenetic approach. A 1 cm³ cube from the interior tissue of A. digitatum was excised under aseptic conditions, homogenized, spread onto agar-based growth medium plates and incubated in 22 °C to promote microbial growth. Colonies were transferred to secondary medium plates, incubated, and after harvesting lysed using sterile water to release DNA. 16S and 23S rDNA regions were amplified using PCR, and sequenced for systematic evaluation using phylogenetic analysis. From this survey we identified a broad selection of bacteria, predominantly of the α-proteobacterial, bacteriodete, actinobacterial and firmicute lineages, demonstrating a significant biodiversity of the coral bacterial endobiome.     

Psychosocial Working Conditions and Cognitive Complaints among Swedish Employees

Background

Cognitive complaints involving problems with concentration, memory, decision-making and thinking are relatively common in the work force. The sensitivity of both subjective and objective cognitive functioning to common psychiatric conditions, stress levels and to cognitive load makes it plausible that psychosocial working conditions play a role in cognitive complaints. Thus, this study aimed to test the associations between psychosocial work factors and cognitive complaints in nationally representative samples of the Swedish work force. Cross-sectional (n = 9751) and prospective (n = 3644; two time points two years apart) sequential multiple regression analyses were run, adjusting for general confounders, depressive- and sleeping problems. Additional prospective analyses were run adjusting for baseline cognitive complaints.

Cross-sectional results

High quantitative demands, information and communication technology (ICT) demands, underqualification and conflicts were positively associated with cognitive complaints, while social support, good resources at work and overqualification were negatively associated with cognitive complaints in all models. Skill discretion and decision authority were weakly associated with cognitive complaints. Conflicts were more strongly associated with cognitive complaints in women than in men, after adjustment for general confounders.

Prospective results

Quantitative job demands, ICT demands and underqualification were positively associated with future cognitive complaints in all models, including when adjusted for baseline cognitive complaints. Decision authority was weakly positively associated with future cognitive complaints, only after adjustment for depressive- and sleeping problems respectively. Social support was negatively associated with future cognitive complaints after adjustment for general confounders and baseline cognitive complaints. Skill discretion and resources were negatively associated with future cognitive complaints after adjustment for general confounders. The associations between quantitative demands and future cognitive complaints were stronger in women.

Discussion/Conclusions

The findings indicate that psychosocial working conditions should be taken into account when considering cognitive complaints among employees.     

Tamoxifen Ameliorates Peritoneal Membrane Damage by Blocking Mesothelial to Mesenchymal Transition in Peritoneal Dialysis

Mesothelial-to-mesenchymal transition (MMT) is an auto-regulated physiological process of tissue repair that in uncontrolled conditions such as peritoneal dialysis (PD) can lead to peritoneal fibrosis. The maximum expression of peritoneal fibrosis induced by PD fluids and other peritoneal processes is the encapsulating peritoneal sclerosis (EPS) for which no specific treatment exists. Tamoxifen, a synthetic estrogen, has successfully been used to treat retroperitoneal fibrosis and EPS associated with PD. Hence, we used in vitro and animal model approaches to evaluate the efficacy of Tamoxifen to inhibit the MMT as a trigger of peritoneal fibrosis. In vitro studies were carried out using omentum-derived mesothelial cells (MCs) and effluent-derived MCs. Tamoxifen blocked the MMT induced by transforming growth factor (TGF)-β1, as it preserved the expression of E-cadherin and reduced the expression of mesenchymal-associated molecules such as snail, fibronectin, collagen-I, α-smooth muscle actin, and matrix metalloproteinse-2. Tamoxifen-treatment preserved the fibrinolytic capacity of MCs treated with TGF-β1 and decreased their migration capacity. Tamoxifen did not reverse the MMT of non-epitheliod MCs from effluents, but it reduced the expression of some mesenchymal molecules. In mice PD model, we demonstrated that MMT progressed in parallel with peritoneal membrane thickness. In addition, we observed that Tamoxifen significantly reduced peritoneal thickness, angiogenesis, invasion of the compact zone by mesenchymal MCs and improved peritoneal function. Tamoxifen also reduced the effluent levels of vascular endothelial growth factor and leptin. These results demonstrate that Tamoxifen is a therapeutic option to treat peritoneal fibrosis, and that its protective effect is mediated via modulation of the MMT process.     

The Bacterial Protein Azurin Impairs Invasion and FAK/Src Signaling in P-Cadherin-Overexpressing Breast Cancer Cell Models

P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells. Three different breast cancer cell lines, MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 were treated with sub-killing doses of azurin. Invasion of these cells was measured using Matrigel Invasion Assays and MTT assays were performed to determine cell viability upon treatment and the effects on cadherins expression was determined by Western blot and Immunofluorescence. Gelatin Zymography was used to determine activity of MMP2 in the conditioned media of azurin treated and untreated cells and the phosphorylation levels of intracellular signaling proteins were determined by Western blot. The invasive phenotype of these breast cancer cells was significantly reduced by azurin. Azurin (50–100 µM) also caused a specific decrease on P-cadherin protein levels from 30–50% in MCF-7/AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins. Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, azurin could possibly be considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin in a wild type E-cadherin context.     

Adenosine-Mediated Enteric Neuromuscular Function Is Affected during Herpes Simplex Virus Type 1 Infection of Rat Enteric Nervous System

Adenosine plays an important role in regulating intestinal motility and inflammatory processes. Previous studies in rodent models have demonstrated that adenosine metabolism and signalling are altered during chronic intestinal inflammatory diseases. However, the involvement of the adenosinergic system in the pathophysiology of gut dysmotility associated to a primary neurodysfunction is still unclear. Recently, we showed that the neurotropic Herpes simplex virus type-1 (HSV-1), orally inoculated to rodents, infects the rat enteric nervous system (ENS) and affects gut motor function without signs of systemic infection. In this study we examined whether changes in purinergic metabolism and signaling occur during permanent HSV-1 infection of rat ENS. Using isolated organ bath assays, we found that contraction mediated by adenosine engagement of A₁ or A_2A receptors was impaired at 1 and 6 weeks post-viral administration. Immunofluorescence studies revealed that viral infection of ENS led to a marked redistribution of adenosine receptors: A₁ and A_2B receptors were confined to the muscle layers whereas A_2A and A₃ receptors were expressed mainly in the myenteric plexus. Viral-induced ENS neurodysfunction influenced adenosine metabolism by increasing adenosine deaminase and CD73 levels in longitudinal muscle-myenteric plexus with no sign of frank inflammation. This study provides the first evidence for involvement of the adenosinergic system during HSV-1 infection of the ENS. As such, this may represent a valid therapeutic target for modulating gut contractility associated to a primary neurodysfunction.     

Removal of Vanadium(III) and Molybdenum(V) from Wastewater Using Posidonia oceanica (Tracheophyta) Biomass

The use of dried and re-hydrated biomass of the seagrass Posidonia oceanica was investigated as an alternative and –low-cost biomaterial for removal of vanadium(III) and molybdenum(V) from wastewaters. Initial characterisation of this biomaterial identified carboxylic groups on the cuticle as potentially responsible for cation sorption, and confirmed the toxic-metal bioaccumulation. The combined effects on biosorption performance of equilibrium pH and metal concentrations were investigated in an ideal single-metal system and in more real-life multicomponent systems. There were either with one metal (vanadium or molybdenum) and sodium nitrate, as representative of high ionic strength systems, or with the two metals (vanadium and molybdenum). For the single-metal solutions, the optimum was at pH 3, where a significant proportion of vanadium was removed (ca. 70%) while there was ca. 40% adsorption of molybdenum. The data obtained from the more real-life multicomponent systems showed that biosorption of one metal was improved both by the presence of the other metal and by high ionic strength, suggesting a synergistic effect on biosorption rather than competition. There data ware used for the development of a simple multi-metal equilibrium model based on the non-competitive Langmuir approach, which was successfully fitted to experimental data and represents a useful support tool for the prediction of biosorption performance in such real-life systems. Overall, the results suggest that biomass of P. oceanica can be used as an efficient biosorbent for removal of vanadium(III) and molybdenum(V) from aqueous solutions. This process thus offers an eco-compatible solution for the reuse of the waste material of leaves that accumulate on the beach due to both human activities and to storms at sea.