Application of MALDIs-MS to Identification of Phytoplankton in Ballast Water

Non-native invasive species are increasingly evident in marine and estuarine environments, largely because of the intake and release of ballast water from sea vessels. Innovative methods are needed to quickly and accurately detect and speciate non-native and/or harmful phytoplankton in ballast water. Recent advances in ionization techniques such as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have allowed detection of intact biomolecules within ballast samples. Statistically-based algorithms are used to discern fingerprints of phytoplankton species and to discern individual species from mixtures. MALDI-MS is particularly attractive for field applications because of the speed of analysis, minimal liquids/consumables required, and femtomole (10-15) sensitivity. The objective of this project was to develop a rapid assay technique that was less time-consuming than more traditional methods of microorganism species identification in ballast water samples. Specific objectives included: (1) modifying MALDI-MS bacterial identification techniques for the analysis of phytoplankton, (2) determining the uniqueness of phytoplankton MALDI-MS fingerprints with a limited subset of phytoplankton, and (3) initiating evaluations of known phytoplankton identification in spiked environmental/ballast water samples

Bacterial analysis by MALDI-TOF mass spectrometry: An inter-laboratory comparison

Bacterial analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been demonstrated in numerous laboratories, and a few attempts have been made to compare results from different laboratories on the same organism. It has been difficult to understand the causes behind the observed differences between laboratories when different instruments, matrices, solvents, etc. are used. In order to establish this technique as a useful tool for bacterial identification, additional efforts in standardizing the methods by which MALDI mass spectra are obtained and comparisons of spectra from different instruments with different operators are needed. Presented here is an extension of our previous single-laboratory reproducibility study with three different laboratories in a controlled experiment with aliquots of the same bacterial culture, matrix stock solution, and calibrant standards. Using automated spectral collection of whole-cell bacteria and automated data processing and analysis algorithms, fingerprints from three different laboratories were constructed and compared. Nine of the ions appeared reproducibly within all three laboratories, with additional unique ions observed within each of the laboratories. An initial evaluation of the ability to use a fingerprint generated within one laboratory for bacterial identification of a sample from another laboratory is presented, and strategies for improving identification rates between laboratories is discussed

Notch ligation by Delta1 inhibits peripheral immune responses to transplantation antigens by a CD8⁺ cell–dependent mechanism

Notch signaling plays a fundamental role in determining the outcome of differentiation processes in many tissues. Notch signaling has been implicated in T versus B cell lineage commitment, thymic differentiation, and bone marrow hematopoietic precursor renewal and differentiation. Notch receptors and their ligands are also expressed on the surface of mature lymphocytes and APCs, but the effects of Notch signaling in the peripheral immune system remain poorly defined. The aim of the studies reported here was to investigate the effects of signaling through the Notch receptor using a ligand of the Delta-like family. We show that Notch ligation in the mature immune system markedly decreases responses to transplantation antigens. Constitutive expression of Delta-like 1 on alloantigen-bearing cells renders them nonimmunogenic and able to induce specific unresponsiveness to a challenge with the same alloantigen, even in the form of a cardiac allograft. These effects could be reversed by depletion of CD8⁺ cells at the time of transplantation. Ligation of Notch on splenic CD8⁺ cells results in a dramatic decrease in IFN-γ with a concomitant enhancement of IL-10 production, suggesting that Notch signaling can alter the differentiation potential of CD8⁺ cells. These data implicate Notch signaling in regulation of peripheral immunity and suggest a novel approach for manipulating deleterious immune responses

DNA Methylation Signatures of Chronic Low-Grade Inflammation Are Associated with Complex Diseases

Background: Chronic low-grade inflammation reflects a subclinical immune response implicated in the pathogenesis of complex diseases. Identifying genetic loci where DNA methylation is associated with chronic low-grade inflammation may reveal novel pathways or therapeutic targets for inflammation. Results: We performed a meta-analysis of epigenome-wide association studies (EWAS) of serum C-reactive protein (CRP), which is a sensitive marker of low-grade inflammation, in a large European population (n = 8863) and trans-ethnic replication in African Americans (n = 4111). We found differential methylation at 218 CpG sites to be associated with CRP (P \u3c 1.15 × 10–7) in the discovery panel of European ancestry and replicated (P \u3c 2.29 × 10–4) 58 CpG sites (45 unique loci) among African Americans. To further characterize the molecular and clinical relevance of the findings, we examined the association with gene expression, genetic sequence variants, and clinical outcomes. DNA methylation at nine (16%) CpG sites was associated with whole blood gene expression in cis (P \u3c 8.47 × 10–5), ten (17%) CpG sites were associated with a nearby genetic variant (P \u3c 2.50 × 10–3), and 51 (88%) were also associated with at least one related cardiometabolic entity (P \u3c 9.58 × 10–5). An additive weighted score of replicated CpG sites accounted for up to 6% inter-individual variation (R2) of age-adjusted and sex-adjusted CRP, independent of known CRP-related genetic variants. Conclusion: We have completed an EWAS of chronic low-grade inflammation and identified many novel genetic loci underlying inflammation that may serve as targets for the development of novel therapeutic interventions for inflammation

The pharmacological effect of BGC20-1531, a novel prostanoid EP4 receptor antagonist, in the Prostaglandin E2 human model of headache

Using a human Prostaglandin E2 (PGE2) model of headache, we examined whether a novel potent and selective EP4 receptor antagonist, BGC20-1531, may prevent headache and dilatation of the middle cerebral (MCA) and superficial temporal artery (STA). In a three-way cross-over trial, eight healthy volunteers were randomly allocated to receive 200 and 400 mg BGC20-1531 and placebo, followed by a 25-min infusion of PGE2. We recorded headache intensity on a verbal rating scale, MCA blood flow velocity and STA diameter. There was no difference in headache response or prevention of the dilation of the MCA or the STA (P > 0.05) with either dose of BGC20-1531 relative to placebo, although putative therapeutic exposures were not reached in all volunteers. In conclusion, these data suggest that the other EP receptors may be involved in PGE2 induced headache and dilatation in normal subjects

Drinking behaviours and blood alcohol concentration in four European drinking environments: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Reducing harm in drinking environments is a growing priority for European alcohol policy yet few studies have explored nightlife drinking behaviours. This study examines alcohol consumption and blood alcohol concentration (BAC) in drinking environments in four European cities.</p> <p>Methods</p> <p>A short questionnaire was implemented among 838 drinkers aged 16-35 in drinking environments in four European cities, in the Netherlands, Slovenia, Spain and the UK. Questions included self-reported alcohol use before interview and expected consumption over the remainder of the night. Breathalyser tests were used to measured breath alcohol concentration (converted to BAC) at interview.</p> <p>Results</p> <p>Most participants in the Dutch (56.2%), Spanish (59.6%) and British (61.4%) samples had preloaded (cf Slovenia 34.8%). In those drinking < 3 h at interview, there were no differences in BAC by gender or nationality. In UK participants, BAC increased significantly in those who had been drinking longer, reaching 0.13% (median) in females and 0.17% in males drinking > 5 h. In other nationalities, BAC increases were less pronounced or absent. High BAC (> 0.08%) was associated with being male, aged > 19, British and having consumed spirits. In all cities most participants intended to drink enough alcohol to constitute binge drinking.</p> <p>Conclusions</p> <p>Different models of drinking behaviour are seen in different nightlife settings. Here, the UK sample was typified by continued increases in inebriation compared with steady, more moderate intoxication elsewhere. With the former being associated with higher health risks, European alcohol policy must work to deter this form of nightlife.</p

Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci.

We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease

DNA methylation signatures of chronic low-grade inflammation are associated with complex diseases.

BACKGROUND: Chronic low-grade inflammation reflects a subclinical immune response implicated in the pathogenesis of complex diseases. Identifying genetic loci where DNA methylation is associated with chronic low-grade inflammation may reveal novel pathways or therapeutic targets for inflammation. RESULTS: We performed a meta-analysis of epigenome-wide association studies (EWAS) of serum C-reactive protein (CRP), which is a sensitive marker of low-grade inflammation, in a large European population (n = 8863) and trans-ethnic replication in African Americans (n = 4111). We found differential methylation at 218 CpG sites to be associated with CRP (P < 1.15 × 10-7) in the discovery panel of European ancestry and replicated (P < 2.29 × 10-4) 58 CpG sites (45 unique loci) among African Americans. To further characterize the molecular and clinical relevance of the findings, we examined the association with gene expression, genetic sequence variants, and clinical outcomes. DNA methylation at nine (16%) CpG sites was associated with whole blood gene expression in cis (P < 8.47 × 10-5), ten (17%) CpG sites were associated with a nearby genetic variant (P < 2.50 × 10-3), and 51 (88%) were also associated with at least one related cardiometabolic entity (P < 9.58 × 10-5). An additive weighted score of replicated CpG sites accounted for up to 6% inter-individual variation (R2) of age-adjusted and sex-adjusted CRP, independent of known CRP-related genetic variants. CONCLUSION: We have completed an EWAS of chronic low-grade inflammation and identified many novel genetic loci underlying inflammation that may serve as targets for the development of novel therapeutic interventions for inflammation