Public proteomic MS repositories and pipelines: available tools and biological applications

As proteomic MS has increased in throughput, so has the demand to catalogue the increasing number of peptides and proteins observed by the community using this technique. As in other 'omics' fields, this brings obvious scientific benefits such as sharing of results and prevention of unnecessary repetition, but also provides technical insights, such as the ability to compare proteome coverage between different laboratories, or between different proteomic platforms. Journals are also moving towards mandating that proteomics data be submitted to public repositories upon publication. In response to these demands, several web-based repositories have been established to store protein and peptide identifications derived from MS data, and a similar number of peptide identification software pipelines have emerged to deliver identifications to these repositories. This paper reviews the latest developments in public domain peptide and protein identification databases and describes the analysis pipelines that feed them. Recent applications of the tools to pertinent biological problems are examined, and through comparing and contrasting the capabilities of each system, the issues facing research users of web-based repositories are explored. Future developments and mechanisms to enhance system functionality and user-interfacing opportunities are also suggested.     

Distance models as a tool for modelling detection probability and density of native bumblebees

Effective monitoring of native bee populations requires accurate estimates of population size and relative abundance among habitats. Current bee survey methods, such as netting or pan trapping, may be adequate for a variety of study objectives but are limited by a failure to account for imperfect detection. Biases due to imperfect detection could result in inaccurate abundance estimates or erroneous insights about the response of bees to different environments. To gauge the potential biases of currently employed survey methods, we compared abundance estimates of bumblebees (Bombus spp.) derived from hierarchical distance sampling models (HDS) to bumblebee counts collected from fixed‐area net surveys (“net counts”) and fixed‐width transect counts (“transect counts”) at 47 early‐successional forest patches in Pennsylvania. Our HDS models indicated that detection probabilities of Bombus spp. were imperfect and varied with survey‐ and site‐covariates. Despite being conspicuous, Bombus spp. were not reliably detected beyond 5 m. Habitat associations of Bombus spp. density were similar across methods, but the strength of association with shrub cover differed between HDS and net counts. Additionally, net counts suggested sites with more grass hosted higher Bombus spp. densities whereas HDS suggested that grass cover was associated with higher detection probability but not Bombus spp. density. Density estimates generated from net counts and transect counts were 80%–89% lower than estimates generated from distance sampling. Our findings suggest that distance modelling provides a reliable method to assess Bombus spp. density and habitat associations, while accounting for imperfect detection caused by distance from observer, vegetation structure, and survey covariates. However, detection/non‐detection data collected via point‐counts, line‐transects and distance sampling for Bombus spp. are unlikely to yield species‐specific density estimates unless individuals can be identified by sight, without capture. Our results will be useful for informing the design of monitoring programs for Bombus spp. and other pollinators.     

In vitro anticryptosporidial activity of dinitroaniline herbicides

Abstract Despite the evaluation of over 100 antimicrobial drugs, the diarrheal disease cryptosporidiosis has remained refractory to treatment. We report the evaluation of five dinitroaniline herbicides including trifluralin, profluralin, nitralin, pendimethalin, and fluchloralin for anticryptosporidial activity in an in vitro cultivation model of Cryptosporidium parvum . All five compounds exhibited significant anticryptosporidial activities with no corresponding evidence of toxicity. The most active compound was pendimethalin with an IC₅₀ of 0.19 μM while nitralin was the least active with an IC₅₀ of 4.5 μM. These compounds should be evaluated further in an animal model of cryptosporidiosis.     

Exogenous interleukin-12 (IL-12) exacerbates Cryptosporidium parvum infection in gamma interferon knockout mice

Experimental infection of BALB/c- or C57BL/6-gamma-interferon-knockout (GKO) mice with Cryptosporidium parvum results in infection in both strains with different outcomes of disease. The BALB/c-GKO mice recover from infection, whereas the C57BL/6-GKO mice succumb to infection in less than 2 weeks. Differences in cytokine mRNA expression suggested that recovery may involve other cytokines. To determine whether the addition of either a Th1 or Th2 cytokine could alter the outcome of infection, we treated GKO mice with either recombinant (r)IL-4 or rIL-12 1 day before infection (DBI) or daily. No effect on the oocyst shedding patterns in either strain nor an increase in survival of the C57BL/6-GKO mice was observed in the rIL-4-treated mice. Whereas one dose of 0.5 microg rIL-12 given 1 DBI had no effect on oocyst shedding, we found that daily doses of rIL-12 administered intraperitoneally exacerbated C. parvum infection in both animal models. Administration of rIL-12 shortened the survival time in the C57BL/6-GKO mice and prevented BALB/c-GKO mice from recovering from infection. Specific proliferation of T cells to cryptosporidial antigen and Th1 and Th2 mRNA cytokine expression was markedly decreased in rIL-12-treated mice. Nitric oxide (NO) may have played a minor role in the decreased proliferation observed since levels of NO present in the splenocyte cultures from rIL-12-treated mice in response to parasite antigen stimulation were higher than those observed in controls. Thus, we propose that resistance to and recovery from C. parvum infections involves a fine balance in the amount and timing of Th1 and Th2 cytokines.     

Cryptosporidium andersoni n. sp. (Apicomplexa: Cryptosporiidae) from cattle, Bos taurus

A new species of Cryptosporidium is described from the feces of domestic cattle, Bos taurus. Oocysts are structurally similar to those of Cryptosporidium muris described from mice but are larger than those of Cryptosporidium parvum. Oocysts of the new species are ellipsoidal, lack sporocysts, and measure 7.4 x 5.5 microm (range, 6.0-8.1 by 5.0-6.5 microm). The length to width ratio is 1.35 (range, 1.07-1.50). The colorless oocyst wall is < 1 microm thick, lacks a micropyle, and possesses a longitudinal suture at one pole. A polar granule is absent, whereas an oocyst residuum is present. Oocysts were passed fully sporulated and are not infectious to outbred, inbred immunocompetent or immunodeficient mice, chickens or goats. Recent molecular analyses of the rDNA 18S and ITS1 regions and heat-shock protein 70 (HSP-70) genes demonstrate this species to be distinct from C. muris infecting rodents. Based on transmission studies and molecular data, we consider the large form of Cryptosporidium infecting the abomasum of cattle to be a new species and have proposed the name Cryptosporidium andersoni n. sp. for this parasite.     

A 23-kDa recombinant antigen of Cryptosporidium parvum induces a cellular immune response on in vitro stimulated spleen and mesenteric lymph node cells from infected mice

In the present study, we focused on a 23-kDa antigen, Cp23, which has been shown to be a major target of humoral immune responses in Cryptosporidium parvum infections and is present in both the sporozoite and merozoite stages. Recombinant Cp23 antigen was shown to stimulate a specific proliferative response by splenocytes and mesenteric lymph node cells from infected interferon gamma knockout BALB/c mice. Cp23 stimulation also induced TNF-alpha, IL-2, and IL-5 mRNA production by spleen cells from infected animals. In contrast, IL-12 mRNA was decreased by Cp23 stimulation compared with unstimulated splenocytes. These data suggest that, as with humoral responses, Cp23 is an important target of cellular immune responses in experimental C. parvum infections. The potential role of this antigen in conferring protective immunity is also discussed.     

Validated predictive modelling of the environmental resistome

Multi-drug-resistant bacteria pose a significant threat to public health. The role of the environment in the overall rise in antibiotic-resistant infections and risk to humans is largely unknown. This study aimed to evaluate drivers of antibiotic-resistance levels across the River Thames catchment, model key biotic, spatial and chemical variables and produce predictive models for future risk assessment. Sediment samples from 13 sites across the River Thames basin were taken at four time points across 2011 and 2012. Samples were analysed for class 1 integron prevalence and enumeration of third-generation cephalosporin-resistant bacteria. Class 1 integron prevalence was validated as a molecular marker of antibiotic resistance; levels of resistance showed significant geospatial and temporal variation. The main explanatory variables of resistance levels at each sample site were the number, proximity, size and type of surrounding wastewater-treatment plants. Model 1 revealed treatment plants accounted for 49.5% of the variance in resistance levels. Other contributing factors were extent of different surrounding land cover types (for example, Neutral Grassland), temporal patterns and prior rainfall; when modelling all variables the resulting model (Model 2) could explain 82.9% of variations in resistance levels in the whole catchment. Chemical analyses correlated with key indicators of treatment plant effluent and a model (Model 3) was generated based on water quality parameters (contaminant and macro- and micro-nutrient levels). Model 2 was beta tested on independent sites and explained over 78% of the variation in integron prevalence showing a significant predictive ability. We believe all models in this study are highly useful tools for informing and prioritising mitigation strategies to reduce the environmental resistome.