Nitrogen and oxygen isotopes in nitrate in the groundwater and surface water discharge from two rural catchments: implications for nitrogen loading to coastal waters

In Prince Edward Island, Canada, widespread intensive potato production has contributed to elevated nitrate concentrations in groundwater and streams, and eutrophic or anoxic conditions occur regularly in several estuarine systems. In this research, the stable isotopes of nitrogen and oxygen in nitrate in intertidal groundwater discharge and stream water were used, in conjunction with water quality and quantity data and land use information, to better understand the characteristics of nitrate delivered to two small estuaries with contrasting land use in their contributory catchments. Most of the water samples collected during the two-year study had isotopic signatures that fell in the range expected for nitrate derived from ammonium-based fertilizers (26.5 % of the samples) or in the overlapping range formed between ammonium-based fertilizers and nitrate derived from soil (64 % of the samples). Overall, isotopic signatures spanned over relatively narrow ranges, and correlations with other water quality parameters, or catchment characteristics, were weak. Nitrate in groundwater discharge and surface water in the Trout River catchment exhibited significantly different isotopic signatures only for the nitrogen isotope, while in the McIntyre Creek catchment groundwater discharge and surface water had similar isotopic signatures. When the isotopic results for the waters from the two catchments were compared, the surface waters were found to be similar, while the isotopic signatures of nitrate in groundwater were distinct only for the nitrogen isotope. Denitrification in the two study catchments was not evident based on the isotopic results for nitrate; however, in the case of the Trout River catchment, where a small freshwater pond exists, an average nitrate load reduction of 14 % was inferred based on a comparison of nitrate loads entering and leaving the pond. Overall, it appears that natural attenuation processes, occurring either in the streams or groundwater flow systems, do not significantly reduce nitrate loading to these estuaries.     

A Simple Method for Encapsulating Single Cells in Alginate Microspheres Allows for Direct PCR and Whole Genome Amplification

Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk reactions, although further work must be done to improve the amplification coverage of single genomes.     

Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines

Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.     

Conformational flexibility of a model protein upon immobilization on self-assembled monolayers

The present study reports on the retention of conformational flexibility of a model allosteric protein upon immobilization on self-assembled monolayers (SAMs) on gold. Organothiolated SAMs of different compositions were utilized for adsorptive and covalent attachment of bovine liver glutamate dehydrogenase (GDH), a well-characterized allosteric enzyme. Sensitive fluorimetric assays were developed to determine immobilization capacity, specific activity, and allosteric properties of the immobilized preparations as well as the potential for repeated use and continuous catalytic transformations. The allosteric response of the free and immobilized forms towards ADP, L-leucine and high concentrations of NAD(+), some of the well-known activators for this enzyme, were determined and compared. The enzyme immobilized by adsorption or chemical binding responded similarly to the activators with a greater degree of activation, as compared to the free form. Also loss of activity involving the two immobilization procedures were similar, suggesting that residues essential for catalytic activity or allosteric properties of GDH remained unchanged in the course of chemical modification. A recently established method was used to predict GDH orientation upon immobilization, which was found to explain some of the experimental results presented. The general significance of these observations in connection with retention of native properties of protein structures upon immobilization on SAMs is discussed.     

First Evaluation after Implementation of a Quality Control System for the Second Line Drug Susceptibility Testing of Mycobacterium tuberculosis Joint Efforts in Low and High Incidence Countries

Three networks/projects involving 27 European countries were established to investigate the quality of second-line drug (SLD) susceptibility testing with conventional and molecular methods. 1. The “Baltic-Nordic TB-Laboratory Network” comprised 11 reference laboratories in the Baltic-Nordic States. They performed SLD testing in the first phase with a panel of 20 Mycobacterium tuberculosis strains. After several laboratories made technical changes a second panel of 10 strains with a higher proportion of resistant strains were tested. Although the concordance for Ofloxacin, Kanamycin, and Capreomycin was consistently high, the largest improvements in performance were achieved for the analysis of Ofloxacin resistant (from 88.9 to 95.0%), and Capreomycin resistant (from 71.0 to 88.9%) strains. 2. Within the FP7 TB PAN-NET project (EU Grant agreement 223681) a quality control panel to standardize the EQA (External Quality Assurance) for first-line drugs (FLD) and SLD testing for phenotypic and molecular methods was established. The strains were characterized by their robustness, unambiguous results when tested, and low proportion of secondary drug resistances. 3. The (European Reference Laboratory Network-TB) ERLN-TB network analyzed four different panels for drug resistance testing using phenotypic and molecular methods; in two rounds in 2010 the 31 participating laboratories began with 5 strains, followed by 10 strains and 6 additional crude DNA extracts in 2011 and 2012 were examined by conventional DST and molecular methods. Overall, we demonstrated the importance of developing inter-laboratory networks to establish quality assurance and improvement of SLD testing of M. tuberculosis.     

Metal accumulation in two contiguous eutrophic peri-urban lakes,Chivero and Manyame,Zimbabwe

Concentrations of aluminium, cadmium, chromium, cobalt, copper, iron, lead, nickel and zinc were determined in surface water, benthic sediments, and the gills, liver and stomach muscle tissues of Oreochromis niloticus and Clarias gariepinus in peri-urban lakes Chivero and Manyame, Zimbabwe. Five sites were sampled in each lake once per month in November 2015, February, May, August and November 2016. Pollution load index detected no metal contamination, whereas the geo-accumulation index reflected heavy to extreme sediment pollution, with Fe, Cd, Zn, Cr, Ni and Cu present in both lakes. Significant spatial temporal variations were detected for Al, Cr, Cu and Pb across sites within and between the two lakes. High Fe, Al and Cr concentrations in water and sediments in lakes Chivero and Manyame derive from geogenic background sources in addition to anthropogenic loads and intensity. Elevated concentrations of Al, Pb, Cu, Cd, Fe and Zn detected in gills, liver and stomach tissue of catfish corroborate concentrations in water and sediments, and pose the highest ecological and health risk for hydrobionts in lakes Chivero and Manyame. Contiguity of peri-urban lakes exposes them to similar threats, necessitating creative water management strategies, which ensure ecological continuity.     

The Frequencies of IFNγ+IL2+TNFα+ PPD-Specific CD4+CD45RO+ T-Cells Correlate with the Magnitude of the QuantiFERON? Gold In-Tube Response in a Prospective Study of Healthy Indian Adolescents

Background

QuantiFERON-TB Gold In-Tube (QFT) is an IFNγ-release assay used in the diagnosis of Mycobacterium tuberculosis (MTB) infection. The risk of TB progression increases with the magnitude of the MTB-specific IFNγ-response. QFT reversion, also associated with low Tuberculin Skin Test responses, may therefore represent a transient immune response with control of M. tuberculosis infection. However, studies at the single cell level have suggested that the quality (polyfunctionality) of the T-cell response is more important than the quantity of cytokines produced.

Objective

To explore the quality and/or magnitude of mycobacteria-specific T-cell responses associated with QFT reversion and persistent QFT-positivity.

Methods

Multi-color flowcytometry on prospectively collected peripheral blood mononuclear cells was applied to assess mycobacteria-specific T-cell responses in 42 QFT positive Indian adolescents of whom 21 became QFT negative (reverters) within one year. Ten QFT consistent negatives were also included as controls.

Results

There was no difference in the qualitative PPD-specific CD4+ T-cell response between QFT consistent positives and reverters. However, compared with QFT consistent positives, reverters displayed lower absolute frequencies of polyfunctional (IFNγ+IL2+TNFα+) CD4+ T-cells at baseline, which were further reduced to the point where they were not different to QFT negative controls one year later. Moreover, absolute frequencies of these cells correlated well with the magnitude of the QFT-response.

Conclusion

Whereas specific polyfunctional CD4+ T-cells have been suggested to protect against TB progression, our data do not support that higher relative or absolute frequencies of PPD-specific polyfunctional CD4+ T-cells in peripheral blood can explain the reduced risk of TB progression observed in QFT reverters. On the contrary, absolute frequencies of these cells correlated with the QFT-response, suggesting that this readout reflects antigenic load.